KR20240137110A - Omicron coronavirus vaccine structure and method of making and using the same - Google Patents

Abstract

Summary
Provided herein are adenoviral vectors encoding a spike protein variant of the SARS-CoV2 coronavirus, and compositions comprising the vectors. Methods of preventing and treating SARS-CoV2 using the provided compositions are also provided.

Description

Omicron coronavirus vaccine structure and method of making and using the same

Government support

This invention was made with government support under CA211096 provided by the National Institutes of Health. The government has certain rights in the invention.

Field of technology

The present disclosure relates generally to the fields of biotechnology and medicine, and more specifically to nucleic acid constructs, polypeptides and vectors that can be used in vaccines for enhanced therapy against respiratory viral infections and methods of using the same.

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Application No. 63/434,815, filed December 22, 2022, which claims the benefit of U.S. Provisional Application No. 63/384,075, filed November 16, 2022, which claims the benefit of U.S. Provisional Application No. 63/375,803, filed September 15, 2022, and which claims the benefit of U.S. Provisional Application No. 63/305,979, filed February 2, 2022, the contents of which are incorporated herein by reference in their entirety.

Background

Hundreds of thousands of people die each year from viral infections. However, for many viruses, treatment options are limited. In addition, since carriers of the virus may be asymptomatic, the rate of transmission may be increased from infected but asymptomatic individuals. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19) syndrome, which can rapidly progress to pneumonia, respiratory failure, and systemic inflammatory disease. SARS-CoV-2 is a benign single-stranded RNA virus that was first isolated from a patient with severe respiratory illness in Wuhan, China, in late 2019. SARS-CoV-2, a betacoronavirus, is related to two other highly pathogenic respiratory viruses, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). SARS-CoV-2 infection causes a clinical syndrome that can progress to respiratory failure, and may also present with cardiac pathology, gastrointestinal disease, coagulopathy, and hyperinflammatory syndrome. Older people, immunocompromised people, and people with underlying medical conditions ( such as obesity, diabetes, and high blood pressure) are at highest risk of death from COVID-19. Since the pandemic began, more than 669 million infections and more than 6.8 million deaths have been recorded worldwide.

The widespread morbidity, mortality, and destabilizing socioeconomic consequences of COVID-19 highlight the urgent need for the deployment of an effective SARS-CoV-2 vaccine to mitigate the severity of infection, suppress transmission, end the pandemic, and prevent resurgence of the pandemic.

summation

Aspects of the present technology encompass an adenoviral vector comprising the genome of a non-human adenovirus. The genome of the adenovirus is modified to lack the native E1 and optionally the E3 or E3B locus in the vector, and comprises a nucleic acid sequence encoding the SARS-CoV-2 spike (S) protein having an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 10, 12, 20, 21, or an immunogenic portion thereof or a fragment thereof.

Another aspect of the present invention comprises an adenoviral vector comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to any of sequence identification numbers 10-12, 20, 21, or an immunogenic portion thereof, or a fragment thereof.

Additional aspects of the present technology encompass adenoviral vectors comprising or consisting of a nucleic acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to any one of the sequences identified by SEQ ID NOs: 13-19.

Still another aspect of the present technology encompasses an adenovirus vector comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises at least 10 selected from the list consisting of T19I, L24S, del25-27, 69-70del, G142D, V213G, G339D, S371F, S373P, S375F, T376A, K417N, N440K, S477N, T478K, E484A, Q493R, L452R, F486V, Q498R, N501Y, D614G, H655Y, N679K, P681H, R682G, R683S, R685S, N764K, D796Y, A942P Q954H, N969K, K988P and V989P. Or encodes an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to sequence identification number: 3, which comprises at least 15, or at least 20, or at least 25, or at least 30 mutations.

Another aspect encompasses a pharmaceutical composition comprising an adenovirus vector described herein. Similarly, one aspect encompasses an immunogenic composition comprising an adenovirus vector described herein.

Certain aspects of the present technology encompass host cells transduced with the adenoviral vectors described herein, or packaging cell lines that produce the adenoviral vectors described herein.

Other aspects of the present technology encompass kits. Generally speaking, the kit comprises: (i) one or more of a host cell described herein, a packaging cell line described herein, an adenoviral vector described herein, a pharmaceutical composition described herein, or an immunogenic composition described herein, and (ii) instructions for use.

Certain aspects of the present technology encompass a coronavirus vaccine comprising an adenovirus vector as described in detail herein. In certain aspects, the technology encompasses a composition comprising serum from a first subject to whom the adenovirus vector as described herein, the pharmaceutical composition as described herein, or the immunogenic composition as described herein has been previously administered. The technology further encompasses a method of treating a second subject infected with a coronavirus, comprising administering to the second subject an immunogenically effective amount of a composition comprising serum from the first subject.

In yet another aspect, the present technology encompasses a method of inducing an immune response against a coronavirus in a subject in need thereof. The method comprises administering to the subject an immunologically effective amount of a composition comprising an adenovirus vector described herein, a pharmaceutical composition described herein, or an immunogenic composition described herein.

Other aspects and descriptions are explained in more detail below.

Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
This application file contains at least one drawing executed in color. Copies of this patent application publication with color drawings are available from the Office upon request and payment of the required fee.
Figure 1A shows a diagram of the transgene cassettes: ChAd-Control has no transgene insert; ChAd-SARS-CoV-2-S encodes a SARS-CoV-2 S protein with the two proline mutations indicated.
Figure 1B shows the binding of anti-S mAbs to ChAd-SARS-CoV-2-S transduced 293 cells. (Left) Summary: +, ++, +++, - indicate <25%, 25-50%, >50%, and no binding, respectively. (Right) Representative flow cytometry histograms from two experiments.
Figure 1C shows that 4-week-old female BALB/c mice were immunized intramuscularly with ChAd-control or ChAd-SARS-CoV-2-S, followed by a booster injection 4 weeks later. Antibody responses in sera from immunized mice were assessed 21 days post-priming or post-boost.
Figure 1D shows anti-S and RBD IgG levels measured by ELISA.
Figure 1E shows the FRNT-determined neutralizing activity. Data are pooled from two experiments (n = 15 to 30; Mann-Whitney test: ****, P < 0.0001).
Figure 1F shows that cell-mediated responses were analyzed 7 days post-booster immunization following re-stimulation with a pool of S protein peptides. Splenocytes were analyzed for IFNγ and granzyme B expression in CD8+ T cells, while granzyme B expression was analyzed exclusively in CD4 ⁺ T cells by flow cytometry.
Figure 1G shows a summary of the frequency and number of positive cell populations (n = 5; Mann-Whitney test: *, P <0.05; **, P <0.01; ***, P < 0.001). Bars represent medians, and the dotted line represents the limit of detection (LOD) of the assay.
Figure 1H shows that spleens were harvested on day 7 post-boost, and SARS-CoV-2 spike-specific IgG+ antibody-secreting cell (ASC) frequencies were measured by ELISPOT (Mann-Whitney test: ****, P < 0.0001). Bars and columns represent medians, and the dashed line represents the limit of detection (LOD) of the assay.
Figure 2A shows that sera from ChAd-control immunized mice collected 21 days after priming or boosting (described in Figure 1 ) were analyzed for S-specific IgG responses by ELISA. Four-week-old female BALB/c mice were primed or booster-vaccinated intramuscularly with ChAd-control or ChAd-SARS-CoV-2-S.
Figure 2B shows that serum samples from ChAd-control or ChAd-SARS-CoV-2 vaccinated mice were collected on day 21 post-priming. Four-week-old female BALB/c mice were primed or booster-vaccinated with ChAd-control or ChAd-SARS-CoV-2-S via the intramuscular route.
Figure 2C shows that serum samples from ChAd-control or ChAd-SARS-CoV-2 vaccinated mice were collected 21 days post-booster vaccination and analyzed for neutralizing activity by FRNT. Serum neutralization curves corresponding to individual mice are shown for the indicated vaccines (n = 15-30 per group). Each point represents the mean of two technical replicates with error bars representing the standard deviation (SD). ChAd-SARS-CoV-2-S vaccine induces neutralizing antibodies as measured by the focus reduction neutralization test (FRNT).
Figure 2D shows anti-SARS-CoV-2 NP IgG responses measured by ELISA in paired sera obtained 5 days before and 8 days after SARS-CoV-2 challenge from ChAd-control or ChAd-SARS-CoV-2-S mice vaccinated via the intramuscular route (n = 5: ** P <0.01; *** P <0.001; paired t test). The dotted line represents the mean IgG titer of naïve sera.
Figure 3 shows the gating strategy for analyzing T cell responses. 4-week-old female BALB/c mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S, and boostered 4 weeks later. T cell responses were analyzed in splenocytes at day 7 post-boost. Cells were gated on lymphocytes (FSC-A/SSC-A), singlets (SSC-W/SSC-H), live cells (Aqua-), CD45+, CD19-, and then CD4+ or CD8+ cell populations expressing IFNγ or granzyme B.
Figure 4A shows the neutralizing activity of Hu-AdV5-hACE2 as determined by FRNT only after prime in sera of the indicated vaccine groups. Four-week-old female BALB/c mice were primed or primed and booster vaccinated. Serum samples were collected one day prior to Hu-AdV5-hACE2 transduction.
Figure 4B shows the neutralizing activity of Hu-AdV5-hACE2 as determined by FRNT in sera from the indicated vaccine groups after prime and booster vaccination. Each symbol represents a single animal; each dot represents two technical replicates, and the bars represent the range. A positive control (anti-Hu-Adv5 serum) is included as a reference frame.
Figure 5A is a scheme of vaccination and challenge to study the protective efficacy of ChAd-SARS-CoV-2-S delivered intramuscularly against SARS-CoV-2 infection. Four-week-old BALB/c female mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S. Some mice were given a booster dose of the homologous vaccine. On day 35 post-immunization, mice were challenged with SARS-CoV-2 as follows: Animals were treated with anti-Ifnar1 mAb and one day later were transduced with Hu-AdV5-hACE2 via the intranasal route. Five days later, mice were challenged with SARS-CoV-2 via the intranasal route at 4 × 10 ⁵ focus-forming units (FFU).
Figure 5B shows that tissues were harvested at 4 and 8 dpi for analysis. Infectious virus in the lungs was measured by plaque assay.
Figure 5C shows that viral RNA levels were measured in the lung, spleen, and heart at 4 and 8 dpi by RT-qPCR (C) (n = 3-7, Mann-Whitney test: *** P < 0.001).
Figure 5D shows viral RNA in situ hybridization using a SARS-CoV-2 probe (brown) in lungs harvested at 4 dpi. Images show low- (top, scale bar, 100 μm) and intermediate- (middle, scale bar, 100 μm) magnifications with high-magnification insets (representative images from n = 3 per group).
Figure 5E shows fold changes in gene expression of indicated cytokines and chemokines in lung homogenates at 4 dpi, as determined by RT-qPCR after normalization to Gapdh levels and comparison with unvaccinated, unchallenged, naïve controls (n = 7; Mann-Whitney test: ***, P < 0.001).
Figure 5F : Mice that received prime-boost immunization were challenged 35 days post-booster immunization. Tissues were collected at 4 dpi for analysis. Infectious virus in the lungs was determined by plaque assay.
Figure 5G shows that mice that received prime-boost immunization were infected 35 days after booster immunization, and viral RNA was measured in the lung, spleen, and heart using RT-qPCR (G) (n = 6-7; Mann-Whitney test: **, P < 0.01). (BC and EG) Columns represent medians, and dotted lines represent LOD of the assay.
Figure 6 shows that a single dose of intramuscular vaccination with ChAd-SARS-CoV-2-S protects mice from SARS-CoV-2-induced lung inflammation. Four-week-old female BALB/c mice were immunized with ChAd-control and ChAd-SARS-CoV-2-S and challenged as described in Figure 5 . Lungs were harvested at 8 dpi. Sections were stained with hematoxylin and eosin and imaged at 40x (left, scale bar, 250 μm), 200x (middle, scale bar, 50 μm), and 400x (right, scale bar, 25 μm). Each image is representative of a group of three mice.
Figure 7A shows the experimental scheme for studying immune responses following intranasal immunization with ChAd-SARS-CoV-2-S. Five-week-old BALB/c female mice were immunized via the intranasal route with ChAd-control or ChAd-SARS-CoV-2-S.
Figure 7B shows antibody responses in sera of mice vaccinated 1 month after priming. SARS-CoV-2 S- and RBD-specific IgG were measured by ELISA.
Figure 7C shows an ELISA measuring SARS-CoV-2 S- and RBD-specific IgA levels.
Figure 7D shows the FRNT-determined neutralizing activity. Data are pooled from two experiments with n = 10-25 mice per group (Mann-Whitney test: ****, P < 0.0001).
Figure 7E shows that mice that received the booster dose were sacrificed one week later to assess mucosal and cell-mediated immune responses. SARS-CoV-2 S- and RBD-specific IgG.
Figure 7F shows that SARS-CoV-2 S- and RBD-specific IgA levels in BAL fluid were determined by ELISA.
Figure 7G shows the neutralizing activity of BAL fluid against SARS-CoV-2 as measured by FRNT.
Figure 7H shows that CD8+ T cells from the lung were restimulated with the S protein peptide pool and then analyzed for IFNγ and granzyme B expression by flow cytometry.
Figure 7I shows that CD8+ T cells in the lung were also phenotyped for expression of CD103 and CD69.
Figure 7J shows that the frequency of SARS-CoV-2 spike-specific IgG+ and IgA+ antibody-secreting cells (ASCs) in spleens harvested 1 week after an additional challenge was measured by ELISPOT. Data for mucosal and cell-mediated responses were pooled from two experiments (EI: n = 7–9 per group; Mann-Whitney test: ***, P <0.001); J: n = 5 per group; Mann-Whitney test: **, P <0.01; ***, P < 0.001). (B-J) Bars and columns represent medians, and dashed lines represent LOD of the assay.
Figure 8A shows that serum samples from ChAd-control or ChAd-SARS-CoV-2-S vaccinated mice were tested for neutralizing activity against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 (n = 8-10 per group). 5-week-old female BALB/c mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via the intranasal route. Serum samples collected 1 month post-immunization were analyzed for neutralizing activity by FRNT. Mice were booster-vaccinated 30 days post-priming and sacrificed 1 week later to assess immune responses.
Figure 8B shows neutralization of recombinant luciferase-expressing SARS-CoV-2 viruses (wild type (left) and D614G variant (middle)) in serum samples from ChAd-SARS-CoV-2-S vaccinated mice. (Right) Corresponding EC50 values are shown (n = 5; ns not significant, paired t test).
Figure 8C shows that BAL fluid was collected from ChAd-control or ChAd-SARS-CoV-2-S vaccinated mice, and neutralization of SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was measured using the FRNT assay (n = 8–10 per group). Each point represents the mean of two technical replicates, with error bars representing SD.
Figure 9A shows that tissues and nasal washings were collected for analysis at 4 and 8 dpi. Infectious virus in the lungs was measured by plaque assay. 5-week-old BALB/c female mice were immunized intranasally with ChAd-control or ChAd-SARS-CoV-2-S. On day 35 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the intranasal route. Five days later, mice were challenged intranasally with 4 x 105 FFU of SARS-CoV-2.
Figure 9B shows that viral RNA levels in lung, spleen, heart, nasal conchae, and nasal lavage were measured by RT-qPCR at 4 and 8 dpi. 5-week-old BALB/c female mice were immunized intranasally with ChAd-control or ChAd-SARS-CoV-2-S. On day 35 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the intranasal route. Five days later, mice were challenged intranasally with 4 × 105 FFU of SARS-CoV-2.
Figure 9C shows fold changes in gene expression of the indicated cytokines and chemokines, as determined by RT-qPCR, normalized to Gapdh, and compared to naïve controls in lung homogenates at 4 dpi (two experiments, n = 6-9; shown as median: *, P < 0.05, ** P < 0.01, *** P < 0.001, **** P <0.0001; Mann-Whitney test). Columns represent medians, and the dotted line represents the LOD of the analysis. 5-week-old BALB/c female mice were immunized via the intranasal route with ChAd-control or ChAd-SARS-CoV-2-S. On day 35 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the intranasal route. Five days later, mice were challenged intranasally with 4 x 105 FFU of SARS-CoV-2.
Figure 9D shows lungs harvested at 8 dpi. Sections were stained with hematoxylin and eosin and imaged at 40x (left, scale bar, 250 μm), 200x (middle, scale bar, 50 μm), and 400x (right, scale bar, 25 μm). Each image is representative of one group of three mice. 5-week-old BALB/c female mice were immunized intranasally with ChAd-control or ChAd-SARS-CoV-2-S. On day 35 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the intranasal route. Five days later, mice were challenged intranasally with 4 × 105 FFU of SARS-CoV-2.
Figure 9E shows anti-SARS-CoV-2 NP IgM (left) and IgG (right) antibody responses measured by ELISA in paired sera obtained at days 5 pre-challenge and 8 post-challenge from hAd-control or ChAd-SARS-CoV-2-S mice vaccinated by the intranasal route (n = 6: ns; not significant; ** P < 0.01, **** P <0.0001; paired t test). Dashed lines represent mean IgM and IgG titers of naive sera (n = 6). 5-week-old BALB/c female mice were immunized by the intranasal route with ChAd-control or ChAd-SARS-CoV-2-S. On day 35 post-vaccination, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the intranasal route. Five days later, mice were challenged intranasally with 4 × 105 FFU of SARS-CoV-2.
Figure 10A shows the immunization scheme. 5-week-old female BALB/c mice were vaccinated via the IN or IM route with ChAd-control virus particles (10 ^{10 )} or decreasing doses (10 ¹⁰ , 10 ^{9 ,} and 10 ⁸ vp) of ChAd-SARS-CoV-2-S.
In Figure 10B, humoral responses were assessed in the serum of vaccinated mice (n = 6-14). ELISA measuring anti-S and RBD IgG levels in IN-vaccinated mice at 100 μg/ml is provided. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
In Figure 10C, humoral responses were assessed in the serum of immunized mice (n = 6-14). ELISA measuring IgA levels in IN-vaccinated mice at 100 is provided. One-way ANOVA with Dunnett's post-test comparing vaccine groups to controls: ns, not significant; **, P <0.01; ****, P < 0.0001). One-way ANOVA with Dunnett's post-test comparing vaccine groups to controls: ns, not significant; **, P <0.01; ****, P < 0.0001). One-way ANOVA with Dunnett's post-test comparing vaccine groups to controls: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10D shows the neutralizing activity of serum from IN-vaccinated mice at 100 days post-vaccination as measured by FRNT. One-way ANOVA with Dunnett's post-test comparing vaccinated and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10E shows anti-S and RBD IgG levels measured by ELISA from IN-vaccinated mice at day 100 post-vaccination or IM-vaccinated mice at day 100. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10F shows anti-S and RBD IgA levels measured by ELISA from IN-vaccinated mice at day 100 post-vaccination or IM-vaccinated mice at day 100. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10G shows the neutralizing activity of serum from IM-vaccinated mice at 100 days post-vaccination as measured by FRNT. One-way ANOVA with Dunnett's post-test comparing vaccinated and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
In Figure 10H, humoral responses were assessed in the serum of vaccinated mice (n = 6-14). ELISA measuring anti-S and RBD IgG levels in IN-vaccinated mice at 200 is presented. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
In Figure 10I, humoral responses were assessed in the serum of vaccinated mice (n = 6-14). ELISA measuring anti-S and RBD IgA levels in IN-vaccinated mice at 200 is provided. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10J shows the neutralizing activity of serum from IN-vaccinated mice at 200 days post-vaccination as measured by FRNT. One-way ANOVA with Dunnett's post-test comparing vaccinated and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10K shows anti-S and RBD IgG levels measured by ELISA from IN-vaccinated mice at day 200 post-vaccination or IM-vaccinated mice at day 100. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10L shows anti-S and RBD IgA levels measured by ELISA from IN-vaccinated mice at day 200 post-vaccination or IM-vaccinated mice at day 100. One-way ANOVA with Dunnett's post-test comparing vaccine and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10M shows the neutralizing activity of serum from IM-vaccinated mice at 200 days post-vaccination as measured by FRNT. One-way ANOVA with Dunnett's post-test comparing vaccinated and control groups: ns, not significant; **, P <0.01; ****, P < 0.0001).
Figure 10N shows the frequency of S-specific IgG or IgA-producing LLPCs in the bone marrow as measured by ELISPOT analysis (n = 4). Mann-Whitney test: *, P < 0.05. BN, bars represent median, dotted line represents limit of detection (LOD) of the assay.
Figure 11A shows serum samples collected from ChAd-SARS-CoV-2-S vaccinated mice on day 100. 5-week-old female BALB/c mice were immunized with a single 10 ¹⁰ , 10 ⁹ , or 10 ⁸ dose of ChAd-SARS-CoV-2-S via the IN or IM route.
Figure 11B shows that serum samples from ChAd-SARS-CoV-2-S vaccinated mice were collected at day 200 post-immunization and analyzed for neutralizing activity by FRNT. 5-week-old female BALB/c mice were immunized IN or IM with a single 10 ¹⁰ , 10 ⁹ , or 10 ⁸ dose of ChAd-SARS-CoV-2-S. Serum neutralization curves for individual mice are shown for the indicated vaccines (n = 6-14 per group). Each point represents the mean of two technical replicates with SD.
Figure 12A shows the serum from mice intranasally challenged with ChAd-SARS-CoV-2-S analyzed on the Luminex platform to quantify the amount of anti-SARS-CoV-2 (WA1/2020 D614G) Spike and RBD IgG1. Bars represent mean values.
Figure 12B shows that serum was analyzed by luminex to quantify the amount of anti-SARS-CoV-2 IgG1 for various SARS-CoV-2 protein variants. Polar plots represent median IgG1 percentiles for each SARS-CoV-2 protein and variant.
Figure 12C shows a heatmap showing the IgG titers and FcγR binding titers of each vaccine regimen against SARS-CoV-2 spike or RBD proteins. Each square represents the within-group mean z-score for that condition.
Figure 12D shows that serum was incubated with primary mouse neutrophils (mADNP) or J774A.1 cells (mADCP) and SARS-CoV-2 spike-coated beads, and phagocytosis was measured 1 h later. Bars represent means, and error bars represent standard deviations.
Figure 12E shows that serum was incubated with primary mouse neutrophils (mADNP) or J774A.1 cells (mADCP) and WA1/2020 D614G, B.1.1.7, or B1.351 spike-coated beads, and phagocytosis was measured 1 h later. Polar plots represent median mADNP or mADCP percentiles for each SARS-CoV-2 protein and variant. For (A and D): One-way ANOVA with Dunnett’s post-test comparing vaccine to control: **, P <0.01; ***, P <0.001; ****, P < 0.0001). (A and D): Bars represent medians.
Figure 13A shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were obtained IN with ChAd-control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 100, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced IN with Hu-AdV5-hACE2. Five days later, mice were challenged IN with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured by RT-qPCR in mice challenged 100 days post-immunization (n = 6–14; Kruskal Wallis and Dunn's post-hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13B shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IN route with ChAd control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 100 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 IN route. Five days later, mice were challenged IN route with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured by RT-qPCR in mice challenged 100 days post-immunization (n = 6–14; Kruskal Wallis and Dunn's post-hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13C shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IN route with ChAd control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 100 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 IN route. Five days later, mice were challenged IN route with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured by RT-qPCR in mice challenged 100 days post-immunization (n = 6–14; Kruskal Wallis and Dunn's post-hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13D shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were immunized via the IM route with ChAd-control 10 ¹⁰ vp or ChAd-control 10 ¹⁰ vp. On day 100, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the IN route. Five days later, mice were challenged via the intranasal route with 5 x 104 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured by RT-qPCR in mice challenged 100 days post-immunization (n = 6–14; Kruskal Wallis and Dunn's post-hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13E shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IM route with ChAd control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 100 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via IN route. Five days later, mice were challenged intranasally with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured by RT-qPCR in mice challenged 100 days post-immunization (n = 6–14; Kruskal Wallis and Dunn's post-hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13F shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IM route with ChAd-control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 100 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via IN route. Five days later, mice were challenged intranasally with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured by RT-qPCR in mice challenged 100 days post-immunization (n = 6–14; Kruskal Wallis and Dunn's post-hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13G shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were obtained IN with ChAd-control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 200, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced IN with Hu-AdV5-hACE2. Five days later, mice were challenged IN with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured in mice challenged 200 days postimmunization via RT-qPCR (n = 6–14; Kruskal Wallis and Dunn's post hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13H shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IN route with ChAd control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 200 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 IN route. Five days later, mice were challenged IN route with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured in mice challenged 200 days postimmunization via RT-qPCR (n = 6–14; Kruskal Wallis and Dunn's post hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13I shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IN route with ChAd control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 200 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 IN route. Five days later, mice were challenged IN route with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured in mice challenged 200 days postimmunization via RT-qPCR (n = 6–14; Kruskal Wallis and Dunn's post hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13J shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were immunized via the IM route with ChAd-control 10 ¹⁰ vp or ChAd-control 10 ¹⁰ vp. On day 200, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via the IN route. Five days later, mice were challenged via the intranasal route with 5 x 104 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured in mice challenged 200 days postimmunization via RT-qPCR (n = 6–14; Kruskal Wallis and Dunn's post hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13K shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IM route with ChAd control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 200 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via IN route. Five days later, mice were challenged intranasally with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured in mice challenged 200 days postimmunization via RT-qPCR (n = 6–14; Kruskal Wallis and Dunn's post hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 13L shows the persistence of protective efficacy of ChAd-SARS-CoV-2-S against SARS-CoV-2 infection in BALB/c mice. 5-week-old female BALB/c mice were administered IM route with ChAd-control 10 ¹⁰ vp or ChAd-SARS-CoV-2-S 10 ¹⁰ , 10 ⁹ , and 10 ⁸ . On day 200 post-immunization, mice were challenged as follows: Animals were treated with anti-Ifnar1 mAb and one day later transduced with Hu-AdV5-hACE2 via IN route. Five days later, mice were challenged intranasally with 5 x 10 4 FFU of SARS-CoV-2 WA1/2020. Tissues shown in the figure were harvested 4 dpi, and viral RNA levels were measured in mice challenged 200 days postimmunization via RT-qPCR (n = 6–14; Kruskal Wallis and Dunn's post hoc test: ns, not significant; **, P <0.01; *, P <0.1; ***, P <0.001; ****, P < 0.0001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 14A shows ELISA measuring SARS-CoV-2 S- and RBD-specific IgG levels. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Antibody responses in mouse sera were assessed 6 weeks post-immunization. Mann-Whitney test: ***, P <0.001; ****, P < 0.0001.
Figure 14B shows ELISA measuring SARS-CoV-2 S- and RBD-specific IgA levels. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Antibody responses in mouse sera were assessed 6 weeks post-immunization. Mann-Whitney test: ***, P <0.001; ****, P < 0.0001.
Figure 14C shows the neutralizing activity determined by FRNT in mouse sera at week 6. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Pairwise analysis of neutralizing activity in sera of immunized mice collected at week 6 for WA1/2020 and Wash-B.1.351 is provided. Two-tailed Wilcoxon matched-pairwise signed-rank test: *, P <0.05; ****, P < 0.0001.
Figure 14D shows the neutralizing activity determined by FRNT in mouse sera at week 6. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Pairwise analysis of neutralizing activity in sera of immunized mice collected at week 6 for WA1/2020 and Wash-B.1.1.28 is provided. Two-tailed Wilcoxon matched-pairwise signed-rank test: *, P <0.05; ****, P < 0.0001.
Figure 14E shows ELISA measuring SARS-CoV-2 S- and RBD-specific IgG levels. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Antibody responses in mouse sera were assessed 9 weeks post-immunization. Mann-Whitney test: ***, P <0.001; ****, P < 0.0001.
Figure 14F shows ELISA measuring SARS-CoV-2 S- and RBD-specific IgA levels. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Antibody responses in mouse sera were assessed 9 weeks post-immunization. Mann-Whitney test: ***, P <0.001; ****, P < 0.0001.
Figure 14G shows the neutralizing activity determined by FRNT in mouse sera at week 6. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Pairwise analysis of neutralizing activity in sera of immunized mice collected at week 9 for WA1/2020 and Wash-B.1.351 is provided. Two-tailed Wilcoxon matched-pairwise signed-rank test: *, P <0.05; ****, P < 0.0001.
Figure 14H shows the neutralizing activity determined by FRNT in mouse sera at week 6. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S. Pairwise analysis of neutralizing activity in sera of immunized mice collected at week 9 for WA1/2020 and Wash-B.1.1.28 is presented. Two-tailed Wilcoxon matched-pairwise signed-rank test: *, P <0.05; ****, P < 0.0001.
Figure 15A shows the experimental scheme. 5-week-old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S.
Figure 15B Five-week-old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At week 6 post-immunization, mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. A plot of body weight change over time is shown. Data are means ± SEM comparing vaccine and control groups (n = 6–9 in each group; area under the curve is de-paired t test, **** P < 0.0001).
Figure 15C Five-week-old K18-hACE2 female mice were immunized IN with 10 ¹⁰ vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Lung viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, and dashed lines represent LOD of the assay.
Figure 15D Five-week-old K18-hACE2 female mice were immunized IN with 10 ¹⁰ vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Heart viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent median, dotted lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with 10 ¹⁰ vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Viral RNA levels in nasal wash fluid were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, dotted lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. At 6 weeks post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Viral RNA levels in the brain were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, dotted lines represent LOD of the assays.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At week 6 post-immunization, the mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from Wash-B.1.1.28. A plot of body weight change over time is shown. Data are means ± SEM comparing vaccine and control groups (n = 6–9 in each group; area under the curve is de-paired t test, **** P < 0.0001).
Five- week- old K18-hACE2 female mice were immunized IN with 10 ¹⁰ vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from Wash-B.1.1.28. Lung viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, dotted lines represent LOD of the assay.
Figure 15I Five-week-old K18-hACE2 female mice were immunized IN with 10 ¹⁰ vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from Wash-B.1.1.28. Heart viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent median, dotted lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from Wash-B.1.1.28. Viral RNA levels in nasal wash fluid were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent median, dotted lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from Wash-B.1.1.28. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from Wash-B.1.351. Brain viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, dotted lines represent LOD of the assays.
15L 5-week-old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, the mice were challenged with ¹⁰⁴ FFU of SARS-CoV-2 from WA1/2020. A plot of body weight change over time is shown. Data are means ± SEM comparing vaccine and control groups (n = 6–9 in each group; area under the curve is de-paired t test, ****P < 0.0001).
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, the mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from WA1/2020. Lung viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, and dotted lines represent LOD of the assay.
Five-week-old K18-hACE2 female mice (n = 15N) were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from WA1/2020. Heart viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent median, dotted lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, the mice were challenged with 10 ⁴ FFU of SARS-CoV-2 from WA1/2020. Viral RNA levels in nasal wash fluid were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, and dotted lines represent LOD of the assays.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. Nine months post-immunization, the mice were challenged with ¹⁰⁴ FFU of Wash-B.1.351 SARS-CoV-2. A plot of body weight change over time is shown. Data are means ± SEM comparing vaccine and control groups (n = 6–9 in each group; area under the curve is de-paired t test, ****P < 0.0001).
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. At 6 weeks post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Lung viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, and dashed lines represent LOD of the assays.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. Nine months post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Heart viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, and dashed lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. Nine months post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Viral RNA levels in nasal wash fluid were measured at 6 dpi by RT-qPCR (n = 6–9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, dotted lines represent LOD of the assay.
Five- week- old K18-hACE2 female mice were immunized IN with ^10–10 vp ChAd-control or ChAd-SARS-CoV-2-S. Nine months post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Nine months post-immunization, the mice were challenged with 10 ⁴ FFU of Wash-B.1.351 SARS-CoV-2. Brain viral RNA levels were measured at 6 dpi by RT-qPCR (n = 6-9; Mann-Whitney test: ** P < 0.01, *** P < 0.001). Bars represent medians, dotted lines represent LOD of the assays.
Figure 16A shows that ChAd-SARS-CoV-2-S vaccine induced neutralizing activity against WA1/2020, Wash-B.1.351, or Wash-B.1.1.28 as measured by FRNT. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S, and serum samples were collected at week 6. Serum neutralization curves for individual mice are shown for the indicated vaccines (n = 7-20 per group). Each point represents the mean of two technical replicates with SD.
Figure 16B shows that ChAd-SARS-CoV-2-S vaccine induced neutralizing activity against WA1/2020, Wash-B.1.351, or Wash-B.1.1.28 as measured by FRNT. 5-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S, and serum samples were collected at 9 months. Serum neutralization curves corresponding to individual mice for the indicated vaccines are shown (n = 7-20 per group). Each point represents the mean of two technical replicates with SD.
Figure 17 shows the sequence alignment of the stabilized variant of SARS-Cov2-Omicron S protein and SARS-CoV2-Wuhan S protein.
Figure 18 shows the evaluation of SARS-CoV-2 spike gene expression mediated by ChAd vector derivatives containing Omicron BA.5.
Figure 19A shows the schedule and timing of vaccination, blood sampling, viral infection, and autopsy.
Figure 19B shows the binding of anti-SARS-CoV-2 IgG to SARS-CoV-2 Wuhan-1 S protein. Kruskal Wallis and Dunn’s post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 19C shows the binding of anti-SARS-CoV-2 IgA to SARS-CoV-2 Wuhan-1 S protein. Kruskal Wallis and Dunn's post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 19D shows the binding of anti-SARS-CoV-2 IgG to BA.5 S protein. Kruskal Wallis and Dunn's post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 19E shows the binding of anti-SARS-CoV-2 IgA to BA.5 S protein. Kruskal Wallis and Dunn's post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 19F shows the binding of anti-SARS-CoV-2 IgG to SARS-CoV-2 BQ.1.1 S protein. Kruskal Wallis and Dunn's post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 19G shows the binding of anti-SARS-CoV-2 IgA to SARS-CoV-2 BQ.1.1 S protein. Kruskal Wallis and Dunn's post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 20A shows the neutralizing activity against WA1/2020 by FRNT. 7-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S vaccine, and sera were collected 28 days later. Each point represents data from an individual mouse and is the mean of two technical replicates.
Figure 20B shows the neutralizing activity against BA.5 by FRNT. 7-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S vaccine, and sera were collected 28 days later. Each point represents data from an individual mouse and is the mean of two technical replicates.
Figure 20C shows the neutralizing activity against BF.7 by FRNT. 7-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S vaccine, and sera were collected 28 days later. Each point represents data from an individual mouse and is the mean of two technical replicates.
Figure 20D shows the neutralizing activity against BQ.1.1 by FRNT. 7-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S vaccine, and sera were collected 28 days later. Each point represents data from an individual mouse and is the mean of two technical replicates.
Figure 20E shows the neutralizing activity against XBB.1.1 by FRNT. 7-week-old K18-hACE2 female mice were immunized IN with 10 ⁹ vp ChAd-control or ChAd-SARS-CoV-2-S vaccine, and sera were collected 28 days later. Each point represents data from an individual mouse and is the mean of two technical replicates.
Figure 20F shows neutralization data presented as a direct comparison of a given vaccine (ChAd-SARS-CoV-2 S (Wuhan-1)) and the indicated SARS-CoV-2 strains used for infection.
Figure 20G shows neutralization data presented as a direct comparison of the given vaccine ChAd-SARS-CoV-2-S (BA.5) with the indicated SARS-CoV-2 strains used for infection.
Figure 20H shows neutralization data presented as a direct comparison of the given vaccine ChAd-SARS-CoV-2-S (IgA) with the indicated SARS-CoV-2 strains used for infection.
Figure 21A shows viral RNA levels in the lung at 6 dpi post-challenge with SARS-CoV-2 WA1/2020 D614G (left) or Omicron BQ.1.1 (right). Animals were immunized via the intranasal route with either unvaccinated or a single dose (total of 109 virus particles) of ChAd-control, ChAd-SARS-CoV-2 S (Wuhan-1), ChAd-SARS-CoV-2-S (BA.5) or bivalent (ChAd-SARS-CoV-2 S (Wuhan- ¹ ) + ChAd-SARS-CoV-2-S (BA.5)). Kruskal Wallis and Dunn’s post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 21B shows viral RNA levels in the nasal conchae ( Figure 21B ) and nasal washes at 6 dpi post-challenge with SARS-CoV-2 WA1/2020 D614G (left) or Omicron BQ.1.1 (right). Animals were immunized intranasally with either unvaccinated or a single dose (total of 10 9 virus particles) of ChAd-control, ChAd-SARS-CoV-2 S (Wuhan-1), ChAd-SARS-CoV-2-S (BA.5) or bivalent (ChAd-SARS-CoV-2 S (Wuhan- ¹ ) + ChAd-SARS-CoV-2-S (BA.5)). Kruskal Wallis and Dunn’s post-test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 21C shows viral RNA levels in nasal wash fluid at 6 dpi after challenge with SARS-CoV-2 WA1/2020 D614G (left) or Omicron BQ.1.1 (right) or at 6 dpi after challenge with SARS-CoV-2 WA1/2020 D614G (left) or Omicron BQ.1.1 (right). Animals were immunized via the intranasal route with a single dose (total of 10 9 virus particles) of unvaccinated, ChAd-control, ChAd-SARS-CoV-2 S (Wuhan-1), ChAd-SARS-CoV-2-S (BA.5), or bivalent (ChAd-SARS-CoV-2 S (Wuhan- ¹ ) + ChAd-SARS-CoV-2-S (BA.5)). Kruskal Wallis and Dunn's post-hoc test: ns, not significant; *, P < 0.05, **, P <0.01; ***, P < 0.001 ****, P < 0.0001).
Figure 22 shows heatmaps of cytokine induction in vaccinated mice at 6 dpi post-challenge with SARS-CoV-2 WA1/2020 D614G (left) or Omicron BQ.1.1 (right). Vaccination status of mice is indicated at the bottom (unvaccinated, ChAd-control, ChAd-SARS-CoV-2 S, ChAd-SARS-CoV-2-S, or both (ChAd-SARS-CoV-2 S (Wuhan-1) + ChAd-SARS-CoV-2-S (BA.5). Colors in the maps reflect log2-fold changes compared to naïve mice.
Figure 23 shows the sequence alignment of stabilized mutants of the BA.5 S protein with respect to the Wuhan strain.

details

The present disclosure is based at least in part on the development of recombinant non-human adenovirus vector compositions and immunogenic compositions thereof for treating or preventing coronavirus infections. The present disclosure also provides methods of administering the compositions disclosed herein to provide sustained cell-mediated immunity and humoral-mediated immunity against coronavirus infections.

Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), which caused the SARS epidemic in 2002–2004, Middle East respiratory syndrome coronavirus (MERS-CoV), which caused MERS first reported in 2012, and SARS-CoV-2, which is responsible for the more recent coronavirus disease 2019 (Covid-19) pandemic, all infect cells by binding to angiotensin-converting enzyme 2 (ACE2) on the cell surface. Essentially, ACE-2 is a functional receptor for SARS-CoV-1, SARS-CoV-2, MERS-COV, and future SARS-COV variants. ACE-2 is a critical component of the renin-angiotensin-aldosterone system (RAAS). ACE-2 converts angiotensin 2 to angiotensin 1–7. Elevated angiotensin 2 has been associated with vasoconstriction, inflammation, and acute lung injury. ACE2 is expressed in various organs, including the lungs, heart, kidneys, liver, intestines, and other tissues. The SARS-CoV virus enters these cells by binding to ACE-2.

The SARS-CoV-2 RNA genome is approximately 30,000 nucleotides in length. The 5' two-thirds encode nonstructural proteins that enable genome replication and viral RNA synthesis. The remaining one-third encodes structural proteins such as spike (S), envelope, membrane, and nucleoprotein (NP), which form the globular virion, and accessory proteins that regulate cellular responses. The S protein forms a homotrimeric spike on the virion and binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2), facilitating the entry of the coronavirus into human cells. The SARS-CoV and SARS-CoV-2 S proteins are sequentially cleaved during the entry process, generating the S1 and S2 fragments, which are then further processed to generate the smaller S2' protein (Hoffmann et al., 2020). The S1 protein contains a receptor binding domain (RBD), while the S2 protein promotes membrane fusion. The structure of the soluble, stabilized pre-fusion conformation of the SARS-CoV-2 S protein was analyzed by cryo-electron microscopy and shows significant similarity to the SARS-CoV-2 S protein. This form of the S protein is recognized by potent neutralizing monoclonal antibodies and may be a promising vaccine target.

The release of the SARS-CoV-2 genome sequence prompted academic, government, and industry groups to immediately begin developing vaccine candidates, primarily targeting the viral S protein. Improved genome sequencing capabilities have also provided a wealth of information about the SARS-CoV-2 variants circulating at any given time. SARS-CoV-2 mutates over time, resulting in the emergence of new variants that differ in sequence, transmissibility, and severity of infection. A number of SARS-CoV-2 variants have emerged: The original form of SARS-CoV-2 identified in December 2019; often referred to as the “original” or “Wuhan” variant; subsequent variants emerged between December 2020 and April 2021, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2 and AY sublineages). However, the currently dominant variants are the Omicron variants (B.1.1.529 and BA sublineages) that emerged around November 2021. As used herein, the term "variant" in relation to a virus is the viral genome (genetic code) that may contain one or more mutations. In some cases, groups of variants with similar genetic changes may emerge, such as lineages or groups of lines. As used herein, a "lineage" is a group of viruses that are closely related to a common ancestor. As used herein, the terms "sublineage" or "subvariant" are groups of similar viruses within a lineage. Sublineages or subvariants of Omicron and other variants continue to emerge.

A variety of platforms have been developed to deliver the SARS-CoV-2 S protein, including DNA plasmids, lipid nanoparticle-encapsulated mRNA, inactivated virions, and virus-vectored vaccines. Several vaccines have entered clinical trials to assess safety, while others have advanced to trials assessing immunogenicity and efficacy. Given the urgency of the pandemic, most vaccines have progressed to human testing without substantial efficacy data in animals. This is in part because vaccine design and development have outpaced the generation of accessible preclinical disease models for SARS-CoV-2 infection and disease.

Adenovirus (Ad)-based vaccines against betacoronaviruses have been previously evaluated. A single dose of a chimpanzee Ad-vectored vaccine encoding the full-length S protein of MERS-CoV protected human dipeptidyl peptidase 4 (hDPP4) transgenic mice from infection, reduced viral shedding, and improved survival in camels, and was safe and immunogenic in humans in a phase 1 clinical trial. A human Ad-based vaccine expressing the MERS S1-CD40L fusion protein also showed protection in transgenic hDPP4 mice. An Ad-based SARS-CoV vaccine expressing the full-length S protein prevented pneumonia in ferrets following challenge and was highly immunogenic in rhesus macaques. A chimpanzee Ad vector (Y25, simian Ad-23) encoding the wild-type SARS-CoV-2 S protein (ChAdOx1 nCoV-19) is currently being evaluated in humans as a single intramuscular injection (NCT04324606). Primary pre-print analysis showed that the vaccine prevented lung infection and pneumonia, but not upper respiratory tract infection or nasal viral shedding (doi.org/10.1101/2020.05.13.093195). However, the vaccine failed as an intranasal vaccine.

Disclosed herein are compositions, methods, and treatment regimens for treating individuals at risk for respiratory viral infections, having mild symptoms of respiratory viral infections, or having severe symptoms of respiratory viral infections. The compositions of the present disclosure can be used to treat, prevent, or reduce respiratory viral infections. The treatment regimen can include administering a composition of the present disclosure to an individual at risk for viral infections or having a viral infection, thereby preventing or treating the viral infection. In some embodiments, viral transmission can be prevented or reduced by reducing viral infections of the upper respiratory tract. The compositions and methods of the present disclosure provide potent antigen-specific antibodies, neutralizing antibodies, and B and T cell responses. This significantly reduces viral production, inflammation, and pathology in the lungs, thereby protecting against infection. The compositions and methods of the present disclosure produce potent mucosal immunity, including high levels of neutralizing and anti-RBD IgA and IgG in the serum and lungs, and SARS-CoV-2-specific resident memory T cells in the lungs. The disclosed compositions and methods provide complete protection against SARS-CoV-2 infection in the nasal cavity, upper respiratory tract, lung tissue, and all other possible sites of transmission. Based on measurements of anti-NP and anti-ORF8 responses, a single intranasal dose of the disclosed compositions confers significantly less sterilizing immunity than a single dose, which has not been previously described for any COVID-19 vaccine.

The compositions of the present disclosure can be formulated topically, for example, intranasally (e.g., as a nasal spray or inhalation) or systemically (e.g., intravenously or intraperitoneally), and can be administered to treat or prevent a respiratory viral infection (e.g., a coronavirus infection, such as SARS-CoV-2). The compositions of the present disclosure (e.g., compositions formulated for nasal delivery or inhalation) can be administered to a subject at risk for a viral infection ( e.g., SARS-CoV-2). For example, the compositions of the present disclosure can be administered to individuals in high-risk settings ( e.g., health care workers), individuals who have been exposed to or are suspected of being exposed to a virus (e.g. , SARS-CoV-2), or individuals who have tested positive for a viral infection. The compositions of the present disclosure can be administered to individuals who exhibit symptoms of a respiratory infection ( e.g., SARS-CoV-2 infection) or who are asymptomatic at the time of administration. In some embodiments, the compositions of the present disclosure can be self-administered by the subject ( e.g., as a nasal spray or inhalation) and administered outside of a medical facility ( e.g., at home).

The methods and compositions disclosed herein may be used to treat, prevent, or reduce respiratory viral infections. In some embodiments, the viral infection may be a coronavirus infection. Pathogens with long incubation periods, such as SARS-CoV-2, which has an average incubation period of about 5 days, pose a high risk of transmission, as many infected individuals may not be aware that they are infected. In addition, carriers of coronaviruses are often asymptomatic or have mild symptoms, which may lead to unknowing contact between the viral host and other members of the population. A subject at risk for coronavirus infection may unknowingly contract a coronavirus infection by contacting an asymptomatic carrier of a coronavirus infection. There is a need for methods and compositions for preventing coronavirus infection in at-risk individuals ( e.g., individuals who have been in contact with a coronavirus carrier or who are likely to be in contact with a coronavirus carrier). In some embodiments, the compositions, methods, or treatment regimens disclosed herein may treat or prevent SARS-CoV-2 infection ( e.g., COVID-19).

The components used to make the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein, are discussed below. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, each is specifically contemplated and described herein, while specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed. For example, when a particular compound is disclosed and discussed and various modifications that can be applied to various molecules of that compound are discussed, unless specifically stated otherwise, all of the possible modifications and each and every combination and permutation of the compounds are specifically contemplated. Thus, if a class of molecules A, B, and C, as well as a class of molecules D, E, and F, and a combination molecule, A-D, are disclosed, then the combinations of A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are individually and collectively contemplated, even if each is not individually mentioned. Likewise, any subset or combination thereof is disclosed. Thus, for example, sub-groups A-E, B-F and C-E are considered disclosed. This concept applies to all aspects of the present application, including but not limited to the steps of the method of making and using the disclosed composition. Accordingly, it is understood that if there are various additional steps that can be performed, each of these additional steps can be performed in any specific embodiment or combination of embodiments of the disclosed method.

Various aspects of the present invention are further described in detail in the following paragraphs.

I. Definition

In order that the present invention may be more readily understood, certain terms are first defined. Unless explicitly stated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of embodiments of the present invention without undue experimentation, and the preferred materials and methods are described herein. In describing and claiming embodiments of the present invention, the following terms will be used in accordance with the following definitions.

The phraseology and terminology used herein is for the purpose of description and should not be considered limiting. For example, the use of singular terms such as "a" is not intended to limit the number of items represented. Additionally, relational terms, including but not limited to "above," "below," "left," "right," "top," "bottom," "downward," "upward," and "side," are used to refer to specific items in the drawings, but are not intended to limit the scope of the appended claims or the inventive concepts.

Terms such as (but not limited to) “substantially” as used in the description and the appended claims should be understood to encompass configurations that are exact or nearly, but not exactly, planar. For example, a “substantially planar surface” means having a truly planar surface or a nearly planar surface that is not exactly planar. Similarly, the terms “about” or “approximately” as used in the description and the appended claims should be understood to encompass the recited value, or a value that is three times or one-third greater than the recited value. For example, about 3 mm includes all values from 1 mm to 9 mm, and about 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, it may refer to ± 5% or less, such as ± 2% or less, such as ± 1% or less, such as ± 0.5% or less, such as ± 0.2% or less, such as ± 0.1% or less, such as ± 0.05% or less.

The terms "including," "included with," and "having" are used interchangeably throughout this disclosure. The terms "including," "included with," and "having" mean that what is so described is included, but is not necessarily limited to.

The terms "or" and "and/or" as used herein are to be interpreted to encompass or mean any one or any combination thereof. Thus, "A, B or C" or "A, B and/or C" means any one of the following: "A," "B" or "C"; "A and B"; "A and C"; "B and C"; "A, B and C." An exception to this definition only occurs where combinations of elements, functions, steps or acts are in any way inherently mutually exclusive.

Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of ordinary skill in the art with general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), all of which are incorporated herein by reference. As used herein, the following terms have the meanings herein unless otherwise specified.

The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or preferred aspects(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that additional elements may be present other than the listed elements. Whenever the terms "comprising," "including," are used, it should be understood that the term "consisting of" elements also disclosed herein explicitly contemplates and encompasses additional aspects that do not include the listed elements.

The terms “about” or “approximately,” as used herein, mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how such value is measured or determined, such as limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation from a given value, depending on the practice in the art. When particular values are described in the application and claims, unless otherwise specified, the term “about” can mean an acceptable error range for the particular value, such as 10% of the value corrected by the term “about.” As used herein, the term "about" can mean ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to the cited value, such as an amount, dose, temperature, time, percentage, etc.

Furthermore, since the inventive concept is susceptible to various forms of "aspects" or "embodiments," this disclosure is intended to be considered as an example of the principles of the inventive concept, and not to limit the inventive concept to the specific aspects illustrated and described. Any one of the features of the invention may be used separately or in combination with other features. Reference in the detailed description to "aspect," "aspects," and/or the like means that the mentioned feature and/or feature is included in at least one aspect of the description. Separate references in the detailed description to "aspect," "aspects," and/or the like do not necessarily refer to the same aspect, and are not mutually exclusive unless so stated, and/or unless readily apparent to one of ordinary skill in the art from the description. For example, features, structures, processes, steps, operations, and the like described in one aspect may, but are not necessarily, included in another aspect. Accordingly, the present invention may include various combinations and/or integrations of the aspects described herein. Additionally, not all aspects of the present invention described herein are essential to its implementation. Likewise, other systems, methods, features, and advantages of the present invention will become apparent to those skilled in the art upon review of the drawings and description. All such additional systems, methods, features, and advantages are intended to be included within this description, within the scope of the present invention, and within the scope of the claims.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in single or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in the same manner as naturally occurring nucleotides. Unless explicitly indicated, a particular nucleic acid sequence encompasses not only the specified sequence but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. See, e.g., Batzer et al., Nucleic Acid Res. See 19:5081 (1991), the contents of which are incorporated herein in their entirety. In some aspects, the nucleic acid sequence encodes a polypeptide sequence.

As used herein, the term "encodes" broadly refers to any process that uses information from a macromolecular macromolecule to direct the production of a second molecule that is different from the first molecule. The second molecule may have a chemical structure that is different from the chemical properties of the first molecule. For example, in some aspects, the term "encodes" describes a semi-conservative DNA replication process, in which one strand of a double-stranded DNA molecule is used as a template for encoding a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule may encode an RNA molecule (e.g., by a transcription process using a DNA-dependent RNA polymerase). An RNA molecule may also encode a polypeptide, such as in a translation process. When used to describe a translation process, the term "encodes" extends to a triplet codon encoding an amino acid. In some aspects, an RNA molecule may encode a DNA molecule, for example, by a reverse transcription process that incorporates an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, and the term "encode" as used herein is understood to encompass both transcription and translation processes.

A nucleic acid is "operably linked" when it is placed in a structural or functional relationship with another nucleic acid sequence. For example, a segment of DNA can be operably linked to another segment of DNA if they are positioned relative to each other on the same adjacent DNA molecule and have a structural or functional relationship; for example, a promoter or enhancer positioned relative to a coding sequence to facilitate transcription of the coding sequence; a ribosome binding site positioned relative to a coding sequence to facilitate translation; or a pre-sequence or secretory leader positioned relative to a coding sequence to facilitate expression of a pre-protein (e.g., a pre-protein that participates in the secretion of an encoded polypeptide). In other embodiments, operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they are functionally related to each other as nucleic acids or as proteins expressed thereby. For example, enhancers need not be contiguous. Linking can be accomplished by linking to convenient restriction sites, or by using synthetic oligonucleotide adaptors or linkers.

In some aspects, a nucleic acid encoding a polypeptide can be operably linked to an expression control sequence.

An expression control sequence is a nucleic acid sequence that controls the expression of an operably linked heterologous nucleic acid sequence. An expression control sequence is operably linked to a nucleic acid sequence when the expression control sequence controls and regulates transcription, and, where appropriate, transcription and translation of the nucleic acid sequence. Thus, an expression control sequence may include a suitable promoter, an enhancer, a transcription terminator, an initiation codon (ATG) preceding a protein-coding gene, a splicing signal for an intron, a maintenance of the correct reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The term "control sequences" includes at least all components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence. An expression control sequence may include a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Promoter elements sufficient to allow promoter-dependent gene expression to be controlled inducibly by cell-type specific, tissue-specific, or external signals or agents are also included; these elements may be located 5' or 3' of the gene of interest. Both constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloned from a bacterial system, inducible promoters may be used, such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like of bacteriophage lambda. In one embodiment, when cloned in a mammalian cell system, promoters derived from the mammalian cell genome (e.g., the metallothionein promoter) or promoters derived from mammalian viruses (e.g., the retroviral long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of a nucleic acid sequence. Expression vector: A vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to the nucleotide sequence so that it is expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in vitro expression system. Expression vectors include all those known in the art into which the recombinant polynucleotide can be incorporated, including cosmids, plasmids (e.g., naked or contained within liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

The term "heterologous" refers to deriving from a different genetic source. A nucleic acid molecule that is heterologous to a cell is derived from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a recombinant coronavirus S protein is expressed in a cell, such as a mammalian cell. Methods for introducing heterologous nucleic acid molecules into cells or organisms are well known in the art, including, for example, transformation using nucleic acids, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

The terms “peptide", “polypeptide", and “protein" are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can make up a protein sequence or peptide sequence. Polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chain peptides (also commonly referred to in the art as peptides, oligopeptides, and oligomers) and longer chains (generally referred to as various types of proteins). “Polypeptide” includes, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. The polypeptide may include a natural peptide, a recombinant peptide, or a combination thereof.

In the context of the present application, a protein is represented by an amino acid sequence, and correspondingly, a polynucleotide represented by a nucleic acid molecule or a nucleic acid sequence. Throughout the present application, identity and similarity between sequences refers to a specific amino acid sequence sequence identification number (e.g., when referring to SEQ ID NO: Y): This may be replaced by a polypeptide represented by an amino acid sequence, a sequence having at least 60% sequence identity or similarity to SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another desirable level of sequence identity or similarity is 99%.

Each amino acid sequence described herein further preferably has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity or similarity. The terms "homology", "sequence identity" and the like are used interchangeably herein. As used herein, "sequence identity" refers to the relationship between two or more amino acid (polypeptide or protein) sequences, or between two or more nucleic acid (polynucleotide) sequences when compared. In a preferred aspect, the sequence identity is calculated based on two given sequence identifiers or a portion thereof. By a portion thereof, preferably, 50%, 60%, 70%, 80%, 90% or 100% or more of the two sequence identifiers. In the art, "identity" also refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences as determined by the correspondence between the strings of such sequences. The degree of sequence identity between two sequences can be determined by comparing the two sequences using computer programs commonly used for this purpose, such as, for example, global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT or any other suitable method or algorithm. The two sequences can be aligned over the full length or a portion thereof (wherein the portion can mean at least 50%, 60%, 70%, 80%, 90% of the sequence length) while maximizing the number of matches and minimizing the number of gaps, using the Needleman and Wunsch global alignment algorithm. Default settings can be used, with the preferred programs being Needle for pairwise alignments (on one hand, EMBOSS Needle 6.6.0.0, Gap open penalty: 10, Gap size penalty: 0.5, End gap penalty: false, End gap open penalty: 10, End gap size penalty: 0.5 are used) and MAFFT for multiple sequence alignments (on the other hand, MAFFT v7 defaults are: BLOSUM62 [bl62], Gap opening: 1.53, Gap extension: 0.123, Order: Sorted, Number of tree re-buildings: 2, Guide tree output: ON [true], Max iterations: 2, Perform FFTS: unused).

The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide with the conserved amino acid substitutions thereof in the sequence of the second polypeptide. Similar algorithms used for determining sequence identity can be used for determining sequence similarity. Optionally, when determining the degree of amino acid similarity, one of ordinary skill in the art can also consider so-called "conservative" amino acid substitutions. As used herein, a "conservative" amino acid substitution refers to the interchangeability of residues having similar side chains.

For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequences described herein have at least one residue deleted from the disclosed sequence and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are: Ala for Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser or Ala; Gln for Asn; Glu for Asp; Gly for Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg; Gln or Glu; Met for Leu or Ile; Phe for Met, Leu or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp or Phe; and Val for Ile or Leu.

An adjuvant refers to a vehicle used to enhance antigenicity. In some embodiments, the adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) to which the antigen is adsorbed; or a water-in-oil emulsion, e.g., an antigen solution emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including killed mycobacteria to further enhance antigenicity (by inhibiting antigen degradation or inducing the recruitment of macrophages) (Freund's complete adjuvant). In some embodiments, the adjuvant used in the disclosed immunogenic compositions is a combination of lecithin and a carbomer homopolymer (see, e.g., ADJUPLEX™ ADJUVANT from Advanced Bioadjuvants, LLC, Wegmann, Clin Vaccine Immunol, 22(9): 1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include QS21 purified plant extract, Matrix M, AS01, MF59 and ALFQ adjuvants. Immunostimulating oligonucleotides (e.g., those comprising a CpG motif) may also be used as adjuvants. Adjuvants include biological molecules (“biological adjuvants”), such as co-stimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. Further description of adjuvants is found in, for example, Singh (ed.) Vaccine adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed compositions.

The term "antibody" refers to an immunoglobulin, antigen-binding fragment, or derivative thereof that specifically binds and recognizes an analyte (antigen), such as a coronavirus S protein, an antigen fragment thereof, or a dimer or multimer of the antigen. The term antibody is used herein in a broad sense to encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( e.g. , bispecific antibodies), and antibody fragments that exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2;diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments contain antigen-binding fragments that are either produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press, 2010).

Coronaviruses are a family of benign, single-stranded RNA viruses known to cause serious respiratory diseases. Currently, viruses in the coronavirus family known to infect humans belong to the genera Alphacoronavirus and Betacoronavirus. Additionally, it is believed that the genera Gammacoronavirus and Deltacoronavirus may infect humans in the future.

Non-limiting examples of betacoronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus HKU1 (HKU1-CoV), human coronavirus OC43 (OC43-CoV), murine hepatitis virus (MHV-CoV), bat SARS-like coronavirus WIV1 (WIV1-CoV), and human coronavirus HKU9 (HKU9-CoV). Non-limiting examples of alphacoronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV). A non-limiting example of a deltacoronavirus is porcine delta coronavirus (SDCV).

The viral genome is capped, polyadenylated, and covered by nucleocapsid proteins. Coronavirus virions contain a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a common genome organization with the replicase gene embedded in the 5'-portion of the genome and the structural genes embedded in the 3'-portion of the genome.

Coronavirus Spike (S) Protein: A class I fusion glycoprotein initially synthesized as a precursor protein. Individual precursor S polypeptides form a homotrimer, undergo glycosylation within the Golgi apparatus, and undergo processing to remove the signal peptide and cleavage by cellular proteases to produce separate SI and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer, resulting in a heterodimeric trimer. The S1 subunit is distal to the viral membrane and contains a receptor binding domain (RBD) that mediates viral attachment to its host receptor. The S2 subunit contains the fusion protein mechanism, i.e., the fusion peptide, two heptad-repeat sequences (HR1 and HR2), a central helix typical of fusion glycoproteins, a transmembrane domain, and a cytoplasmic tail domain.

The coronavirus spike (S) protein pre-fusion conformation is the structural conformation adopted by the ectodomain of the coronavirus S protein after it has been processed into the mature coronavirus S protein in the secretory system, but before the fusion event in which coronavirus S converts to the post-fusion conformation. The three-dimensional structure of an exemplary coronavirus S protein (HKU1-CoV) in the pre-fusion conformation is disclosed herein and is provided in Kirchdoerfer et al., "Pre-fusion structure of a human coronavirus spike protein," Nature, 531: 118-121, 2016 (incorporated herein by reference).

A "stabilized pre-fusion conformation" coronavirus ectodomain trimer comprises one or more amino acid substitutions, deletions or insertions, as compared to a native coronavirus S sequence, which results in increased maintenance of the pre-fusion conformation as compared to a coronavirus S ectodomain trimer formed from a corresponding native coronavirus S sequence. The "stabilization" of the pre-fusion conformation by the one or more amino acid substitutions, deletions or insertions can be, for example, energetic stabilization (e.g., decreasing the energy of the pre-fusion conformation relative to the post-fusion open conformation) and/or dynamic stabilization (e.g., decreasing the rate of transition from the pre-fusion conformation to the post-fusion conformation). Additionally, stabilization of the coronavirus S ectodomain trimer in the pre-fusion conformation can imply increased resistance to denaturation as compared to the corresponding native coronavirus S sequence. Methods for determining whether a coronavirus S ectodomain trimer is in a pre-fusion conformation are provided herein, including but not limited to, negative-stain electron microscopy and antibody binding assays using pre-fusion conformation specific antibodies.

Degenerate Variant: In the context of the present disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide that contains a degenerate sequence as a result of the genetic code. There are 20 natural amino acids, most of which are specialized by one or more codons. Accordingly, any degenerate nucleotide sequence encoding a peptide is contained, so long as the amino acid sequence of the peptide encoded by the nucleotide sequence remains unchanged.

In one example, the desired response is to inhibit, reduce, or prevent a CoV (e.g., SARS-CoV-2) infection. The method need not be effective so as to completely eliminate, reduce, or prevent a CoV infection. For example, administration of an effective amount of an immunogen can induce an immune response that reduces a CoV infection to a desired level, e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CoV infection), as compared to a suitable control (e.g., as measured by the number or percentage of cells infected or subjects infected with the CoV). Epitope: An antigenic determinant. These are specific chemical groups or peptide sequences of molecules that have antigenic properties that elicit a specific immune response, for example, an epitope is an antigenic region to which B and/or T cells respond. Antibodies can bind to specific antigenic epitopes, such as epitopes of a coronavirus S ectodomain, such as the SARS-CoV S ectodomain. Epitopes can be formed from both contiguous amino acids and non-contiguous amino acids that are juxtaposed by tertiary folding of the protein.

Expression refers to the transcription or translation of a nucleic acid sequence. For example, a gene is expressed when DNA is transcribed into RNA or RNA fragments, which in some instances are processed into mRNA. The gene may also be expressed and the mRNA translated into an amino acid sequence, such as a protein or protein fragment. In a specific example, a heterologous gene is expressed when it is transcribed into RNA. In another example, a heterologous gene is expressed when the RNA is translated into an amino acid sequence. The term "expression" is used herein to refer to transcription or translation. Regulation of expression can include regulation of transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or regulation through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been produced.

The term "host cell" means a cell into which an exogenous nucleic acid has been introduced, and includes progeny of such a cell. Host cells include "transformants" and "transformed cells", which include a primary transformed cell and progeny derived therefrom, regardless of the number of passages. Host cells include cells within the body of a subject (e.g., a mammalian subject (e.g., a human)) into which an exogenous nucleic acid has been introduced.

"Immunogen" means any polypeptide that, when administered, is capable of inducing an immune response in a subject. In some embodiments, the immunogen is encoded by a nucleic acid molecule that can be incorporated into, for example, a polynucleotide or vector, for subsequent expression of the immunogen (e.g., a gene product of interest or a fragment thereof (e.g., a polypeptide)).

The term "immunogenic composition" as used herein is defined as a substance used to induce an immune response, and is capable of conferring immunity to a subject after administration of the immunogenic composition to the subject.

The term "immunostimulant" refers to a substance (e.g., a drug or nutrient) that stimulates the immune system by inducing activation or increasing the activity of any component. Immunostimulants include cytokines (e.g., granulocyte-macrophage colony-stimulating factor) and interferons (e.g., IFN-α and/or IFN-γ).

"Pharmaceutical composition" preferably means any composition suitable for administration to a subject, such as an immunogenic composition or vaccine containing a nucleotide sequence encoding an antigenic gene product of interest or a fragment thereof, and containing a therapeutic or biologically active agent for treating or preventing a disease (e.g., a CoV infection) or reducing or ameliorating one or more symptoms of the disease (e.g., CoV virus titer, virus spread, infection and/or cell fusion). For the purposes of the present invention, a pharmaceutical composition includes a vaccine, and a pharmaceutical composition suitable for delivering a therapeutic or biologically active agent can include, for example, a tablet, gelcap, capsule, pill, powder, granule, suspension, emulsion, solution, gel, hydrogel, oral gel, paste, eye drop, ointment, cream, plaster, infusion, delivery device, suppository, enema, injection, implant, spray or aerosol. Any of these formulations can be prepared by well-known and accepted technical methods. See, e.g., Remington: The Science and Practice of Pharmacy (21st ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is incorporated herein by reference. An immune response is a response of an immune system cell, such as a B cell, a T cell, or a monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, which results in the production of a particular antibody.

Prime-boost vaccination is an immunotherapy that includes administering to a subject a first immunogenic composition (primary vaccine) followed by administration of a second immunogenic composition (booster vaccine) to induce an immune response. The priming vaccine and/or the booster vaccine contain a vector (e.g., a viral vector, an RNA or DNA vector) expressing an antigen to which the immune response is directed. The booster vaccine is administered to the subject after administration of the priming vaccine; examples of appropriate intervals between administration of the priming vaccine and the booster vaccine, and such periods, are disclosed herein. In some embodiments, the priming vaccine, the booster vaccine, or both the primer vaccine and the booster vaccine further contain an adjuvant.

A vaccine is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response to an antigen of a pathogen, e.g., a viral pathogen, or a cellular component associated with a pathological condition. A vaccine may contain a polynucleotide (e.g., a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In a non-limiting example, the vaccine induces an immune response that reduces the severity of symptoms associated with a coronavirus infection (e.g., a SARS-CoV or MERS-CoV infection), or reduces the viral load, as compared to a control. In another non-limiting example, the vaccine induces an immune response that reduces and/or prevents a coronavirus infection (e.g., a SARS-CoV or MERS-CoV infection), as compared to a control. In some aspects, the vaccines herein may be referred to as “bivalent” or “multivalent” because they are a single vector capable of simultaneously inducing immune responses to two or more viral pathogens, such as two coronavirus isolates.

A vector is an entity comprising a DNA or RNA molecule operably linked to a coding sequence of an antigen(s) of interest and having a promoter capable of expressing said coding sequence. Non-limiting examples include naked or packaged (lipid and/or protein) DNA, naked or packaged RNA, or sub-components thereof of a replication-incompetent virus or bacterium or other microorganism, or a replication-competent virus or bacterium or other microorganism. A vector is sometimes also referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector may include a nucleic acid sequence that permits replication in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.

Virus-like particles (VLPs) are non-replicating viral envelopes derived from one of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can be formed spontaneously upon recombinant expression of the proteins in a suitable expression system. Methods for making VLPs are known in the art. Following recombinant expression of viral proteins, the presence of VLPs can be detected using conventional techniques known in the art, such as electron microscopy, biophysical characterization, and the like. Furthermore, VLPs can be separated by known techniques, such as density gradient centrifugation, and identified by characteristic density banding. See, e.g., Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) /. Virol. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol, 354: 53073, 2012).

II. Composition

The compositions of the present disclosure may comprise one or more active agents. In some embodiments, the active agent may be an agent that prevents, treats, or reduces the infectivity of a viral infection. In some embodiments, treating a viral infection may comprise reducing the infectivity and/or transmission of the virus. In some embodiments, preventing a viral infection may comprise reducing the infectivity and/or transmission of the virus. The compositions of the present disclosure may comprise an active agent for preventing a viral infection, an active agent for treating a viral infection, an active agent for reducing the infectivity of a viral infection, or a combination thereof. The compositions of the present disclosure may further comprise a pharmaceutically acceptable excipient, carrier, or diluent. Furthermore, the compositions of the present disclosure may contain a preservative, a solubilizer, a stabilizer, a wetting agent, an emulsifier, a sweetener, a colorant, an odorant, a salt (wherein the material of the present disclosure itself may be provided in the form of a pharmaceutically acceptable salt), a buffer, a coating agent, or an antioxidant.

The present disclosure relates to non-human adenoviral vector compositions and methods of using the immunogenic compositions comprising the adenoviral vector and optionally one or more additional active ingredients, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, to treat or prevent respiratory viral infections. Applicants have discovered that the use of simian adenoviral vectors overcomes the heterologous vector cross-immunity problem seen with the human adenoviral vector platform (PMID:32450106). Applicants have constructed chimpanzee adenoviral vectors expressing a SARS-CoV-2 antigen, such as a stabilized form of the SARS-CoV-2 "S" protein, and have shown that this can protect against COVID-19 in an animal model of the disease. The SARS-CoV-2 antigen or immunogenic portion thereof, when expressed in an adenoviral vector, can stimulate an immune response upon human infection, thereby conferring immunity against COVID-19 disease. The Applicants also discovered that this adenovirus vector platform can be successfully used to deliver SARS-CoV-2 antigenic variants that are effective in generating immune responses against sub-lineages or sub-variants of SARS-CoV-2.

Other aspects of the present invention are described in more detail below.

a) Adenovirus vector

The present disclosure provides adenoviral vectors for delivery of DNA encoding a SARS-CoV-2 antigen, such as a stabilized form of the SARS-CoV-2 "S" protein. The nucleotide sequence of a transgene encoding SARS-CoV2-Wuhan S protein is provided as SEQ ID NO: 1, and the nucleotide sequence of an exemplary adenoviral vector comprising a transgene encoding SARS-CoV2-Wuhan S protein is provided as SEQ ID NO: 2. The protein coding sequence for SARS-CoV2-Wuhan S protein is provided as SEQ ID NO: 3.

Adenoviruses are nonenveloped viruses, approximately 90-100 nm in diameter, consisting of a nucleocapsid and a linear double-stranded DNA genome. The viral nucleocapsid is composed of pentons and hexameric capsomers. A unique fiber is associated with each penton base, which helps the virus attach to the host cell via the Coxsackie-adenovirus receptor on the host cell surface. More than 50 serotypes of adenovirus have been identified, most of which cause respiratory infections, conjunctivitis, and gastroenteritis in humans. Adenoviruses typically replicate as episomal elements in the host cell nucleus rather than being integrated into the host genome. The genome of adenoviruses contains four early transcription units (E1, E2, E3, and E4), which primarily have regulatory functions and prepare the host cell for viral replication. The genome also contains five late transcription units (L1, L2, L3, L4 and L5) encoding structural proteins, including penton (L2), hexon (L3), scaffolding protein (L4) and fiber protein (L5), which are under the control of a single promoter. Each terminus of the genome contains inverted terminal repeats (ITRs) required for viral replication.

Recombinant adenoviruses were originally developed for gene therapy, but the strong and persistent transgene-specific immune responses elicited by these gene transfer agents have prompted their use as vaccine vectors. In addition to their high immunogenicity, adenoviruses offer many other advantages for clinical vaccine development. The adenovirus genome is relatively small (26–45 kbp), well characterized, and easy to manipulate. Deletion of a single transcription unit, E1, renders the virus replication-impossible, increasing predictability and reducing adverse effects in clinical applications. Recombinant adenoviruses can accommodate relatively large transgenes (up to 8 kb in some cases), allowing flexibility in subunit design and having a relatively broad tropism that facilitates transgene delivery to a variety of cells and tissues. Crucial to clinical applications is the well-established method of scaling up and purifying recombinant adenoviruses to high titers. To date, subgroup C serotypes AdHu2 or AdHu5 have been primarily used as vectors. However, first-generation vaccine vectors based on the typical human adenovirus AdHu5 showed low efficacy in clinical trials despite encouraging preclinical data. Subsequently, as a result of natural infection, a large proportion of human adults were found to have significant titers of neutralizing antibodies against common human serotypes such as AdHu2 and AdHu5. Neutralizing antibodies can reduce the efficacy of viral vector vaccines by blocking viral entry into host cells and thus also blocking the delivery of the target transgene.

The compositions described herein include vectors that deliver heterologous molecules to cells for therapeutic or vaccine purposes. As used herein, the vectors can contain any genetic element, including but not limited to, naked DNA, phage, transposons, cosmids, episomes, plasmids, or viruses. In some embodiments, these vectors contain a transgene or nucleic acid sequence encoding a simian adenovirus DNA (e.g., SAdV-36) and a protein (e.g., S-protein). As used herein, the term "transgene" refers to a combination of a selected heterologous gene (a nucleic acid sequence encoding a heterologous polypeptide) and other regulatory elements or expression control sequences necessary to induce translation, transcription, and/or expression of the gene product in a host cell. In some embodiments, the viral vector can include an adenovirus vector expressing a transgene encoding a coronavirus S protein. Adenoviruses from various origins, subtypes, or mixtures of subtypes can be used as the source of the viral genome for the adenoviral vector. Non-human adenoviruses ( e.g., adenoviruses of simian, chimpanzee, gorilla, avian, canine, ovine, or bovine origin) can be used to make the adenoviral vector. For example, a simian adenovirus can be used as the source of the viral genome for the adenoviral vector. The simian adenovirus can be serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 36, 38, 39, 48, 49, 50, or any other simian adenovirus serotype. The simian adenovirus can be referred to by any suitable abbreviation known in the art, such as, for example, SV, SAdV, SAV, or sAV. In some examples, the simian adenovirus vector is a simian adenovirus vector of serotype 36.

Typically, SAdV-derived adenoviral vectors are designed such that the transgene is positioned in the nucleic acid molecule such that it contains other adenoviral sequences within a region unique to the selected adenoviral gene. If desired, the transgene can be inserted into an existing gene region to disrupt the function of that region. Alternatively, the transgene can be inserted into a site of an adenoviral gene that is partially or completely deleted. For example, the transgene can be positioned at a site such as a functional E1 deletion and/or a functional E3 deletion (or E3B) and/or a complete E3 deletion region, which can be particularly selected. The term "functionally deleted" or "functionally deleted" means that a sufficient portion of the gene region is removed or impaired, for example by mutation or modification, such that the gene region can no longer produce a functional product of gene expression. If desired, the entire gene region can also be removed. In one specific embodiment, an adenoviral vector useful according to the present disclosure is a simian adenovirus SAd36, which has deletions in the E1 and E3B genes. In one aspect, the vector comprises a complete E3 deletion. In one aspect, the adenoviral vector has the nucleic acid sequence of SEQ ID NO: 4. In another aspect, the adenovirus vector has a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 4.

For example, for the production of a vector useful for producing a recombinant virus, the vector can contain a transgene at the 5' end of the adenovirus genome, or at the 3' end of the adenovirus genome, or at both the 5' end and the 3' end of the adenovirus genome. The 5' end of the adenovirus genome contains 5' cis-elements necessary for packaging and replication, such as, for example, a 5' inverted terminal repeat (ITR) sequence (which functions as an origin of replication) and a native 5' packaging enhancer domain (which includes sequences necessary for linear Ad genome packaging and enhancer elements for the E1 promoter). The 3' end of the adenovirus genome contains 3' cis-elements (including ITRs) necessary for packaging and encapsidation. Suitably, the recombinant adenovirus contains both 5' and 3' adenoviral cis-elements, and the transgene is located between the 5' and 3' adenoviral sequences. Simian-based adenoviral vectors (e.g., SAdV-36) may also contain additional adenoviral sequences.

Suitably, these simian-based adenoviral vectors contain one or more adenoviral elements derived from an adenovirus genome. In one embodiment, the vectors contain adenoviral sequences derived from an adenovirus different from the adenovirus serotype that provides the ITRs. As defined herein, a pseudotyped adenovirus refers to an adenovirus whose capsid protein is derived from an adenovirus different from the adenovirus that provides the ITRs. Chimeric or hybrid adenoviruses can be constructed using the adenoviruses described herein using techniques known to those of skill in the art. See, e.g., US 7,291,498.

The transgene is a nucleic acid sequence heterologous to the vector sequence encoding the polypeptide, protein or other product of interest. The nucleic acid coding sequence is operably linked to a regulatory element in a manner that allows transcription, translation and/or expression of the transgene in a host cell.

In some embodiments, the introduced gene encodes a stabilized form of a SARS-CoV-2 antigen, such as the SARS-CoV-2 "S" protein. In some embodiments, the transgene comprises a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a coronavirus spike protein portion of SEQ ID NO: 3 (or an immunogenic portion thereof or a variant thereof); or SEQ ID NO: 3 having the K986P and V987P mutations; Or encodes at least a portion or immunogenic portion of the coronavirus spike protein (S) protein, or an immunogenic fragment thereof or a variant thereof, having a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 3 having a D614G mutation. In some specific embodiments, the transgene encodes a spike protein of a SARS-CoV-2 variant, including but not limited to, a spike protein having a D80G, 144del, F157S, L5F, T95I, A67V, S477N, 144del, Q677H, A701V, F888L, T791I, T859N, D950H, E484Q, D614G, E484K, N501Y, Δ69-70, L452R, or K417N or RBD E484K mutation as compared to SEQ ID NO: 3. In some embodiments, the transgene encodes a spike protein from a WA1/2020, B.1.1.7, B.1.351, B.1.1.28, P.1, B.1.427, B.1.526, B.1.526.1, B.1.525, P.2, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.429, B.1.429, B.1.529 variant. In each of these embodiments, the spike protein is further modified to be in a stabilized form prior to fusion (e.g., having a double proline substitution between residues 1050 to 1069, or between residues 981 to 999).

A variant with multiple spike protein mutations, Omicron (B.1.1.529), was first identified in Botswana. In particular, the genomes of some omicron mutants encode S proteins with the following mutations: A67V, Δ69-70, T95I, G142D/A143-145, A211/L212I, 214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. In some embodiments, these exemplary strains and other emerging strains (e.g., of concern for additional variants) are candidates for the methods and formulations disclosed herein. Thus, in some embodiments, the present disclosure also encompasses recombinant adenoviral vectors comprising a transgenic stabilized spike protein sequence derived from an Omicron B.1.1.529 strain or a variant thereof. Non-limiting examples of subvariants of the Omicron strains include BA1.1, BA.2, BA.3, BA.4, and BA.5. In exemplary aspects, the BA.5 variant may have the following mutations: T19I, L24S, del25-27, 69-70del, G142D, V213G, G339D, S371F, S373P, S375F, T376A, K417N, N440K, S477N, T478K, E484A, Q493R, L452R, F486V, Q498R, N501Y, D614G, H655Y, N679K, P681H, R682G, R683S, R685S, N764K, D796Y, Q954H, N969K.

Thus, in some embodiments, the introduced gene nucleic acid encodes a stabilized SARS-CoV2-Omicron-Spike protein (SARS-CoV2-Omicron-S2P) provided in SEQ ID NO: 10; Or a nucleic acid sequence encoding a protein having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 10 (SARS-CoV-2-Omicron-S2P).

In some specific embodiments, the introduced gene nucleic acid encodes the SARS-Cov2-Omicron-Spike protein (SARS-CoV-2-Omicron-S6P) of SEQ ID NO: 11; Or a nucleic acid sequence encoding a protein having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 11 (SARS-CoV2-Omicron-S6P).

In some specific embodiments, the introduced gene nucleic acid encodes the SARS-Cov2-Omicron-Spike protein (SARS-CoV-2-Omicron-S6PδF) of SEQ ID NO: 12; Or a nucleic acid sequence encoding a protein having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 12 (SARS-CoV-2-Omicron-S6PdF).

In some specific embodiments, the introduced gene nucleic acid encodes the SARS-Cov2-Omicron-Spike protein (SARS-CoV-2-Omicron-S6PdF) of SEQ ID NO: 20; Or a nucleic acid sequence encoding a protein having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 20 (BA.5-S6PdF).

In some specific embodiments, the introduced gene nucleic acid encodes the SARS-Cov2-Omicron-Spike protein (SARS-CoV-2-Omicron-S6P) of SEQ ID NO: 21; Or a nucleic acid sequence encoding a protein having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 21 (BA.5-S6P).

In some aspects, the adenovirus vector comprises a nucleic acid sequence (transgene), wherein the nucleic acid sequence encodes an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 3, or the following mutations T19I, L24S, comprising at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30 or more mutations among del25-27, 69-70del, G142D, V213G, G339D, S371F, S373P, S375F, T376A, K417N, N440K, S477N, T478K, E484A, Q493R, L452R, F486V, Q498R, N501Y, D614G, H655Y, N679K, P681H, R682G, R683S, R685S, N764K, D796Y, A942P Q954H, N969K, K988P and V989P Encoding a variant thereof: F819P, A894P, A901P, A944P, K988P and V989P, further comprising at least two, or at least three, or at least four, or at least five, or six stabilizing mutations. In some aspects, the adenovirus vector comprises a protein that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 3, or at least as provided by F819P, A894P, A901P, A944P, K988P and V989P. Encodes omicron variants as provided in SEQ ID NOs: 10-11 and 20-21, comprising two, or at least three, or at least four, or at least five, or six stabilizing mutations. Sequence alignments of SEQ ID NOs: 10, 11, 20, 21 to the Wuhan sequence are provided in FIG. 23 .

In some embodiments, the present disclosure encompasses an adenoviral vector comprising a nucleic acid sequence encoding one or more coronavirus spike protein variants or immunogenic fragments thereof. In some embodiments, the present disclosure also encompasses an adenoviral vector comprising a nucleic acid sequence encoding at least a portion or immunogenic fragment of a coronavirus spike protein (S) protein, or an immunogenic fragment or variant thereof having a sequence at least 80% identical to SEQ ID NO: 3, 10, 11, 20, or 21, and a nucleic acid sequence encoding at least a portion or immunogenic fragment or variant thereof having a sequence at least about 80% identical to one or more additional proteins derived from SARS-CoV2. In some aspects, the one or more additional proteins can be selected from non-structural proteins 1-16 (nsp1-16), S protein, structural proteins including envelope (E), membrane (M) and nucleocapsid (N), and 11 accessory proteins: ORF3a, ORF3b, ORF3c, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10. In some embodiments, the present disclosure also encompasses an adenovirus vector comprising a nucleic acid sequence encoding at least a portion or an immunogenic fragment thereof of a coronavirus spike protein (S) protein, or an immunogenic fragment thereof having a sequence at least 80% identical to SEQ ID NO: 3, 10, 11, 20, or 21, or a variant thereof, and a nucleic acid sequence encoding at least a portion or an immunogenic fragment thereof or a variant thereof of one or more additional proteins derived from a virus, e.g., rabies, measles, RSV, or influenza. Examples of suitable proteins include, but are not limited to, influenza hemagglutinin, influenza nucleoprotein, influenza M2, tetanus toxin C fragment, anthrax protective antigen, anthrax lethal factor, anthrax germination factor, rabies glycoprotein, HBV surface antigen, HIV gp120, HIV gp160, human carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg, botulinum toxin A, and Mycobacterium tuberculosis HSP. In some embodiments, the adenoviral vector is said to express a bivalent, trivalent or multivalent immunogenic protein/proteins, wherein the protein induces an immune response against one or more viruses, one or more SARS-CoV2 lineages, variants, sublineages or subvariants.

In some embodiments, the present disclosure also encompasses plasmids and adenoviral vectors (vectors) comprising a nucleic acid sequence encoding an S protein sequence as disclosed herein. In some aspects, the nucleic acid is at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the adenoviral vector or plasmid comprises or consists of a nucleic acid sequence that is at least about 80% identical to SEQ ID NO: 2, 5 or 6. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 2, 5 or 6.

In one embodiment, the vector comprises or consists of a nucleic acid sequence provided in SEQ ID NO: 13 (ChAd-S6PdF529). In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 13.

In some embodiments, the vector comprises and/or consists of a nucleic acid sequence as provided in SEQ ID NO: 14 (ChAd-S2P529). In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 14.

In some embodiments, the vector comprises and/or consists of a nucleic acid sequence as provided in SEQ ID NO: 15 (ChAd-S6P529). In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 15.

In some embodiments, the plasmid comprises or consists of the nucleic acid sequence provided in SEQ ID NO: 16 (pSAd36E3E1stS), wherein the entire E3 gene is deleted. In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 16.

In some embodiments, the vector comprises or consists of the nucleic acid sequence provided in SEQ ID NO: 17 (SAd36E3E1stS), wherein the entire E3 gene is deleted. In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 17. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 17.

In some embodiments, the vector comprises and/or consists of a nucleic acid sequence as provided in SEQ ID NO: 18 (ChAd.BA.5-S6PdF). In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 18. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 18.

In some embodiments, the vector comprises and/or consists of a nucleic acid sequence as provided in SEQ ID NO: 19 (ChAd.BA.5-S6P). In some embodiments, the vector comprises or consists of a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 19. In some embodiments, the nucleic acid sequence has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to SEQ ID NO: 19.

In addition to the major elements identified above for the transgene, the vector may also contain conventional control elements operably linked to the transgene in a manner that allows transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences that are adjacent to the gene of interest, and expression control sequences that are trans to, or at a distance from, the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product.

A significant number of expression control sequences are known and available in the art, including native, constitutive, inducible, and/or tissue-specific promoters. Examples of constitutive promoters include, but are not limited to, the retroviruses Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al. , Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF-Ia promoter [Invitrogen].

Inducible promoters allow for the regulation of gene expression and may be regulated by exogenously supplied compounds, environmental factors such as temperature, or by the presence of specific physiological conditions (e.g., acute phase, the presence of a specific differentiation state of these cells, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and are readily available to those skilled in the art. For example, inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter and the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter. Other inducible systems include the T7 polymerase promoter system [WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)], tetracycline-inducible system [Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], tetracycline-inducible system [Gossen et al, Science, 268:1766-1769 (1995), Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998) also see]. Other systems include FK506 dimer, castradiol VP 16 or p65, diphenol murisrelon, and RU486-inducible systems [Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and rapamycin-inducible systems [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)] are included. The effectiveness of some inducible promoters increases over time. In such cases, the efficiency of the system can be improved by inserting multiple repressors, for example, TetR linked to TetR by IRES. Alternatively, one can wait at least 3 days before screening for the desired function. To improve the efficiency of this system, one can enhance the expression of the desired protein by known means. For example, using the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In another embodiment, a native promoter for the transgene may be utilized. A native promoter may be preferred where it is desired that expression of the transgene mimic native expression. The native promoter may be used where expression of the transgene is to be regulated temporally or developmentally, in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may be used to mimic such native expression. Another embodiment of the transgene comprises the transgene operably linked to a tissue-specific promoter. For example, if expression is desired in skeletal muscle, a promoter that is active in muscle should be used. These include promoters of the genes encoding skeletal β-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, as well as synthetic muscle promoters with higher activity than naturally occurring promoters (see Li et al, Nat. Biotech., 17:241-245 (1999)). Examples of tissue-specific promoters are known for liver (albumin, Miyatake et al, J. Virol., 71:5124-32 (1997); inter alia, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al, Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al, MoI. Biol Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al, J. Bone Miner. Res., 11:654-64 (1996)), lymphocyte (CD2, Hansal et al, J. Immunol, 161: 1063-8 (1998); immunoglobulin heavy chain; T cell receptor chain), neuronal such as the neuron-specific enolase (NSE) promoter (Andersen et al, Cell. MoI Neurobiol, 13:503-15 (1993)), neurofilament light-chain gene (Piccioli et al, Proc. Natl Acad. ScL USA, 88:561 1-5 (1991)), and neuron-specific vgf gene (Piccioli et al, Neuron, 15:373-84 (1995)). Optionally, the vector carrying the transgene encoding the therapeutically useful or immunogenic product may include a selectable marker, and the reporter gene may include sequences encoding, among others, geneticin, hygromycin or purimycin resistance. Such selectable reporter or marker genes (preferably located outside the viral genome that will be packaged into the viral particle) may be used to signal the presence of the plasmid in the bacterial cell, such as ampicillin resistance. Other components of the vector may include an origin of replication. The choice of these and other promoters and vector elements is routine, and many such sequences are available (see, e.g., Sambrook et al, and the materials cited herein). These vectors are generated using techniques and sequences provided herein, together with techniques known to those of skill in the art. These techniques may include conventional cloning techniques of cDNA, such as those described in the text [Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY], using overlapping oligonucleotide sequences of the adenovirus genome, the polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence.

In some embodiments, the present disclosure provides virus-like particles (VLPs) comprising the disclosed recombinant adenoviral vectors. VLPs lack the viral components necessary for viral replication, and thus represent a highly attenuated and replication-incompetent form of the virus. Virus-like particles and methods for their production are known and familiar to those skilled in the art and include human papillomavirus, HIV (Kang et al., Biol. Chem. 380: 353-64 (1999)), Semliki-Forest virus (Notka et al., Biol. Chem. 380: 341-52 (1999)), human polyomavirus (Goldmann et al., J. Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al , Vaccine 17: 1005-13 (1999)), parvovirus (Casal, Biotechnology and Applied Biochemistry, Vol 29, Part 2, pp 141-150 (1999)), feline parvovirus (Hurtado et al., J. Virol. 70: 5422-9 (1996)), hepatitis E virus (Li et al, J. Virol. 71: 7207-13 (1997)), and Newcastle disease virus are known to form VLPs. The formation of such VLPs can be detected by any suitable technique. Examples of suitable techniques known in the art for detecting VLPs in media include, for example, electron microscopy techniques, dynamic light scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction, and/or size exclusion chromatographic separation of VLPs), and density gradient centrifugation.

b) Components of the composition

The present disclosure also provides a pharmaceutical composition, which comprises an adenovirus composition of the present disclosure as an active ingredient, and at least one pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients may be diluents, binders, fillers, buffers, pH regulators, disintegrants, dispersants, preservatives, lubricants, taste-masking agents, flavoring agents or coloring agents. The amount and type of excipients used to form the pharmaceutical composition can be selected according to known pharmaceutical science principles.

In each of the embodiments described herein, the composition of the present invention may optionally comprise one or more additional drugs or therapeutically active agents in addition to the adenovirus composition of the present disclosure. Thus, in addition to the therapies described herein, other therapies known to be effective in treating viral infections may also be provided to the subject. In some embodiments, the secondary agent is selected from a corticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), an intravenous immunoglobulin, a kinase inhibitor, a fusion or recombinant protein, a monoclonal antibody, or a combination thereof. In some embodiments, agents suitable for combination therapy include, but are not limited to, an inhaled bronchodilator and an inhaled steroid.

(i) Diluent

In one specific embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or susceptible to wear. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphorylated corn starch, gelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, mallitol, sorbitol, xylitol, maltodextrin, trehalose. Non-limiting examples of suitable abrasive brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), tribasic calcium phosphate, calcium carbonate and magnesium carbonate.

(ii) binder

In another specific embodiment, the excipient can be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, sugars, oligosaccharides, polypeptides, oligopeptides and combinations thereof.

(iii) Filler

In another specific embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be dibasic and tribasic calcium sulfate starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

(iv) Buffer

In yet another embodiment, the excipient can be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphate, carbonate, citrate, Tris buffer, and buffered saline (e.g., Tris buffered saline or phosphate buffered saline).

(v) pH modifier

In various specific embodiments, the excipient may be a pH modifier. By way of non-limiting example, the pH modifier may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

(vi) disintegrant

In a further embodiment, the excipient can be a disintegrant. The disintegrant can be non-effervescent or effervescent. Examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate complexed with citric acid and sodium bicarbonate complexed with tartaric acid.

(vii) Dispersant

In another specific embodiment, the excipient may be a dispersant or a dispersion enhancing agent. Suitable dispersants include, but are not limited to, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isocrystalline silicates, and microcrystalline cellulose.

(viii) Siblings

In another alternative embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants such as BHA, BHT, vitamin A, vitamin C, vitamin E or retinyl palmitate, citric acid, sodium citrate; chelates such as EDTA or EGTA; and antimicrobials such as parabens, chlorobutanol or phenol.

(ix) Lubricants

In a further specific embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica and fats such as vegetable stearin, magnesium stearate, stearic acid.

(x) Taste-masking agent

In yet another embodiment, the excipient can be a taste-masking agent. Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohols; copolymers of polyvinyl alcohol and polyethylene glycols; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers and cellulose ethers; cellulose acetate phthalates; and combinations thereof.

(xi) flavoring agent

In an alternative embodiment, the excipient may be a flavoring agent. The flavoring agent may be selected from synthetic fragrance oils and fragrance aromatics and/or natural oils; plant, leaf, flower and fruit extracts; and combinations thereof.

(xii) Color developer

In yet a further specific embodiment, the excipient may be a colorant. Suitable colorants include, but are not limited to, food, pharmaceutical and cosmetic colorants (FD&C), pharmaceutical and cosmetic colorants (D&C), or topical and cosmetic colorants (Ext. D&C).

(xiii) Adjuvant

In yet a further embodiment, the formulation may comprise an adjuvant. Adjuvants are known in the art to further increase the immune response to the applied antigenic determinant, and pharmaceutical compositions comprising adenovirus and a suitable adjuvant are disclosed, for example, in WO 2007/110409 (incorporated herein by reference). Examples of suitable adjuvants include, but are not limited to, aluminium salts, such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90/14837); saponin formulations, such as, for example, QS21 and immunostimulating complexes (ISCOMS); Bacterial or microbial derivatives, examples of which include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing a CpG motif, ADP-ribosylated bacterial toxins or mutants thereof, such as Escherichia coli heat labile enterotoxin LT, cholera toxin CT, and the like. It is also possible to use vector-encoded adjuvants, for example, by using a heterologous nucleic acid encoding a fusion construct wherein the oligomerization domain of C4-binding protein (C4 bp) is fused to an antigen of interest. In certain embodiments, the compositions of the invention comprise aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or combinations thereof, at a concentration of from 0.05 to 5 mg, for example, from 0.075 to 1.0 mg per dose.

The weight fraction of the excipient or combination of excipients in the composition can be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

The formulations and compositions described herein can be formulated in any conventional manner using one or more pharmaceutically acceptable carriers or excipients, for example, as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which may be in purified form, together with a suitable amount of carrier to provide a form for proper administration to a subject.

The term "formulation" means preparing a drug in a form suitable for administration to a subject, such as a human. Thus, "formulation" may include pharmaceutically acceptable excipients, including diluents or carriers.

The term "pharmaceutically acceptable" as used herein refers to a substance or ingredient that does not cause unacceptable loss of pharmacological activity or unacceptable adverse effects. Examples of pharmaceutically acceptable ingredients include those having monographs in the United States Pharmacopeia (USP 29) and the National Formulary (NF 24), United States Pharmacopeial Convention, Inc., Rockville, Maryland, 2005 ("USP/NF"), or more recent versions thereof, and those ingredients listed in the FDA's continuously updated Inactive Ingredient Search online database. Other useful ingredients not described in the USP/NF, etc., may also be used.

The term "pharmaceutically acceptable excipient" as used herein can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic or absorption delaying agents. The use of such media and agents with pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except for any conventional media or agent that is incompatible with the active ingredient, use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

A "stable" formulation or composition can refer to a composition that is sufficiently stable to allow storage at a convenient temperature, such as from about 0° C. to about 60° C., for a commercially reasonable period of time, such as at least about 1 day, about 1 week or more, about 1 month or more, about 3 months or more, about 6 months or more, about 1 year or more, or about 2 years or more.

The above formulations must be suitable for the mode of administration. The formulations used in conjunction with the present disclosure can be formulated by known methods for administration to a subject using various routes, including but not limited to parenteral, pulmonary, oral, topical, mucosal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual formulations can also be administered together with one or more additional agents or together with other biologically active or biologically inactive agents. Such biologically active or inactive agents can be in fluidic or mechanical communication with the agent(s) or can be attached to the agent(s) by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

In some aspects, the present disclosure encompasses a formulation for nasal delivery. A formulation comprising a composition for intranasal delivery can have a pH corresponding to a physiologically acidic nasal pH. The physiologically acidic nasal pH can vary depending on whether nasal mucosal function is impaired. The pH of the composition can be about 6.5 ± 0.5 (5.9 to 7.3) or about 6.7 ± 0.6 (5.3 to 7.6). The pH of the composition can be about 3.8-7.7 (mean ± SD 5.7 ± 0.9). The composition for nasal delivery can be in the slightly acidic range. The average pH can be acidic, with a pH of about 5.7.

Effective delivery of therapeutics via intranasal administration must take into account not only drug loss due to binding to glycoproteins in the mucus layer, but also the reduced rate of transport through the protective mucus lining of the nasal mucosa. Normal mucus is a viscoelastic, gel-like substance composed of water, electrolytes, mucus, macromolecules, and denuded epithelial cells. It primarily acts as a lubricating covering that protects the underlying mucosal tissue. Mucus is secreted by randomly distributed secretory cells located in the nasal epithelium and other mucosal epithelia. The structural unit of mucus is mucin. This glycoprotein is primarily responsible for the viscoelastic properties of mucus, but other macromolecules can also contribute to these properties. In airway mucus, these macromolecules include locally produced secretory IgA, IgM, IgE, lysozyme, and bronchial transferrin, which also play an important role in host defense mechanisms.

The integrated administration methods of the present disclosure optionally incorporate effective mucolytics or mucus-removing agents that act to break down and thin or clear mucus on the nasal mucosal surfaces to facilitate absorption and/or adsorption of the intranasally administered biotherapeutic agent. Within these methods, the mucolytics or mucus-removing agents are co-administered as adjuvants to enhance intranasal delivery of the biologically active agent. Alternatively, an effective amount of the mucolytics or mucus-removing agents is incorporated as a processing agent within the multi-processing methods of the invention, or as an additive within the combination formulations of the invention, to provide improved formulations that reduce the barrier effect of intranasal mucus to enhance intranasal delivery of the biotherapeutic compound.

A variety of mucolytic or mucus-removing agents can be incorporated into the methods and compositions of the present invention. Depending on their mechanism of action, mucolytic and mucus-removing agents can often be classified into the following groups: proteases ( e.g., pronase, papain) that cleave the protein core of mucin glycoproteins; sulfhydryl compounds that cleave mucoprotein disulfide linkages; and detergents ( e.g., Triton X-100, Tween 20) that break down non-covalent bonds within mucus. Additional compounds in this context include, but are not limited to, bile salts and surfactants, such as sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate, and lysophosphatidylcholine.

The effect of bile salts in causing structural decomposition of mucus is in the order deoxycholate > taurocholate > glycocholate. Other effective agents that improve intranasal delivery by reducing mucus viscosity or adhesion according to the method of the present invention include, for example, short-chain fatty acids, and mucolytics with chelating action, such as N-acyl collagen peptides, bile acids and saponins (the latter functioning in part by chelating Ca ²⁺ and/or Mg ²⁺ , which play an important role in maintaining the mucus layer structure).

Additional mucolytics for use in the methods and compositions of the present invention include N-acetyl-L-cysteine (ACS), a potent mucolytic that reduces both the viscosity and adhesion of bronchopulmonary mucus, and has been reported to slightly increase the nasal bioavailability of human growth hormone in anesthetized mice (from 7.5 to 12.2%). These and other mucolytics or mucus removers are typically contacted with the nasal mucosa at concentrations ranging from about 0.2 to 20 mM, in conjunction with administration of a biologically active agent to reduce the polar viscosity and/or elasticity of nasal mucus.

Other mucolytic or mucus-removing agents may be selected from the range of glycosidase enzymes, which are capable of cleaving glycosidic bonds within mucus glycoproteins, notably α-amylase and β-amylase, although their mucolytic effect may be limited. In contrast, bacterial glycosidases, which allow these microorganisms to penetrate the host's mucus layer.

For use in combination with the adenovirus compositions herein, non-ionic detergents are generally useful as mucolytic or mucus-removing agents. These agents generally do not modify or substantially impair the activity of the adenovirus composition.

c) Administration

(i) Dosage form

The compositions may be formulated in a variety of dosage forms and may be administered by a number of different means to deliver a therapeutically effective amount of the active ingredient. Such compositions may be administered in unit dosage form containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles, as desired, by any suitable route, for example, oral, parenteral, pulmonary, topical, mucosal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ocular, buccal and rectally. Topical administration may also involve the use of a transdermal delivery device, such as a transdermal patch or iontophoretic device. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intra-articular or intracolonic injection or infusion techniques. Drug formulations are described, for example, in Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980). In certain embodiments, the composition can be a food supplement or the composition can be a cosmetic.

For parenteral administration (including subcutaneous, intraocular, intradermal, intravenous, intramuscular, intraarticular and intraperitoneal), such preparations may be aqueous or oily solutions. The aqueous solutions may contain sterile diluents such as water, saline, pharmaceutically acceptable polyols such as glycerol, propylene glycol or other synthetic solvents; antibacterial and/or antifungal agents such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and/or tonicity regulators such as sodium chloride, glucose or polyalcohols such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The oily solution or suspension may additionally contain sesame, peanut, olive or mineral oil. The composition may be presented in unit-dose or multi-dose quantities, for example, in sealed containers, for example, ampoules and vials, which may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

If nasal or respiratory (mucosal) administration is required, the composition may be in the form of a squeeze spray dispenser, a pump dispenser or an aerosol dispenser. Such dispensers may also be used to deliver the composition to the oral or oral (e.g., buccal or lingual) mucosa, the nasal cavity. Aerosols are generally under pressure by hydrocarbons. Pump dispensers are preferably capable of dispensing metered doses or doses having a specific particle size.

In certain embodiments, the vector compositions disclosed herein are formulated as a single dosage composition, e.g., together with a pharmaceutically acceptable buffer, carrier, excipient, and/or adjuvant, and are administered to a subject simultaneously, e.g., mixed. In other embodiments, the vector and the protein are formulated as separate compositions, e.g., together with a pharmaceutically acceptable buffer, carrier, excipient, and/or adjuvant, and are administered to a subject as separate compositions within 24 hours, such as within 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour or less.

In general, a safe and effective amount of an adenovirus composition is an amount that can cause a desired therapeutic effect in a subject, for example, while minimizing undesirable side effects. In various embodiments, an effective amount of an adenovirus composition described herein can substantially reduce viral infectivity in a subject suffering from a viral infection. In some embodiments, an effective amount is an amount capable of treating a respiratory viral infection. In some embodiments, an effective amount is an amount capable of treating one or more symptoms associated with a respiratory viral infection.

The amount of the compositions described herein that can be combined with a pharmaceutically acceptable carrier to form a single dosage form will vary depending upon the host being treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit quantity of the formulation contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, since the required therapeutically effective amount can be achieved by multiple administrations.

The dosage of the adenoviral vector will vary depending primarily on factors such as the condition being treated, the age, weight and health of the patient, and therefore will vary from patient to patient. For example, a therapeutically effective adult human or adult animal dosage of the viral vector typically ranges from about 100 μL to about 100 mL of carrier containing a virus concentration of about 1 x 10 ⁶ to about 1 x 10 ¹⁵ particles, about 1 x 10 ⁷ to 1 x 10 ¹³ particles, or about 1 x 10 ⁹ to 1 x 10 ¹² particles. The dosage will vary depending on the body size of the animal and the route of administration. For example, a suitable human or animal dosage for intramuscular injection (for an animal weighing about 80 kg) is from about 1 x 10 ⁹ to about 5 x 10 ¹² particles per mL for administration at a single site. Optionally, multiple sites of administration may be administered. In another example, a suitable human or animal dose may be from about 1 x 10 ¹¹ to about 1 x 10 ¹⁵ particles for an oral formulation. Those skilled in the art can adjust these doses depending on the route of administration and the therapeutic or vaccine application for which the recombinant vector is being used. The frequency of dosing can be determined by monitoring the level of expression of the transgene, or in the case of an immunogen, the level of circulating antibodies. Other methods for determining the timing of dosing frequency will be readily apparent to those skilled in the art.

An optional method step involves co-administering to the patient an appropriate amount of a short-acting immunomodulatory agent concurrently with, or prior to, or subsequent to, administration of the viral vector. The selected immunomodulatory agent is defined herein as an agent capable of inhibiting the formation of neutralizing antibodies to the recombinant vector of the invention or inhibiting cytolytic T lymphocyte (CTL) clearance of the vector. The immunomodulatory agent can inhibit the interaction between T helper subsets (T _H i or T^) and B cells, thereby inhibiting neutralizing antibody formation. Alternatively, the immunomodulatory agent can inhibit the interaction between T _H i cells and CTLs, thereby reducing the incidence of CTL clearance of the vector. Various useful immunomodulatory agents and dosages for their use are described, for example, in Yang et al., J. Virol., 70(9) (Sept., 1996); International Patent Application Publication No. WO 96/12406 (published May 2, 1996); and International Patent Application No. PCT/US96/03035 (all of which are incorporated herein by reference).

The specific therapeutic effect level for any particular subject will depend on a number of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of treatment; and drugs used in combination or concurrently with the specific compound employed. And will vary depending on a variety of factors, including similar factors known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, ^4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, one of ordinary skill in the art may initiate dosage of the composition at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple dosages for administration purposes. Consequently, a single dose composition may contain such amounts or multiples thereof to constitute a daily dose. However, as disclosed herein, it will be understood that the total daily usage of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

Administration of the viral composition may be performed as a single event, or may be administered over a period of treatment. For example, one or more nanoparticle compositions may be administered daily, weekly, biweekly, or monthly. For treatment of acute conditions, the treatment period may typically be as short as a few days. Certain conditions may extend treatment from several days to several weeks. For example, treatment may extend over one week, two weeks, or three weeks. For more chronic conditions, treatment may extend over several weeks to several months or even a year or more.

Treatment according to the methods described herein may be performed before, after, or concurrently with conventional treatment methods for respiratory viruses.

The present disclosure encompasses pharmaceutical compositions comprising the compounds described above to facilitate administration of the active agent and to promote stability. For example, the compounds of the present disclosure may be mixed with at least one pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition capable of being administered (provided) effectively and efficaciously to a suitable subject, such as a living subject (i.e., a "subject in need of treatment" or a "subject in need thereof"). For purposes of aspects and embodiments of the present invention, the subject may be a human or any other animal.

III. Method

The present disclosure encompasses methods of treating, preventing, or reducing the infectivity or transmission of a virus in a subject in need thereof. In some embodiments, the methods prevent or reduce the infectivity of a viral infection by preventing internalization of the virus into a cell of the subject, or by preventing internalization of the viral genome into a cell of the subject. In some embodiments, administering a composition provided herein for use as described in Section II can produce an immune response in the subject that interferes with or prevents the interaction between a viral surface protein ( e.g., spike protein or envelope protein) and a host receptor protein ( e.g., epithelial angiotensin converting enzyme (ACE)). Administering a composition of the present disclosure to a subject at risk for a viral infection can reduce the risk of coronavirus infection in the subject.

The disclosed composition can be administered to a subject to induce an immune response to the corresponding coronavirus spike protein in the subject. In certain embodiments, the subject is a human. The immune response can be a protective immune response, for example, a response that inhibits subsequent infection by the corresponding coronavirus. The induction of an immune response can be used to treat or inhibit infections and diseases associated with the corresponding coronavirus.

For example, a subject who is infected with, or at risk for infection with, a coronavirus corresponding to the S protein of the immunogen, due to exposure or potential exposure to a coronavirus, can be selected for treatment. Following administration of the disclosed immunogen, the subject can be monitored for infection or symptoms associated with the coronavirus, or both.

Typical subjects to be treated with the therapeutics and methods of the present disclosure include humans as well as non-human primates and other animals. To identify subjects for prevention or treatment according to the methods of the present disclosure, accepted screening methods are used to determine risk factors associated with the targeted or suspected disease or condition, or to determine the status of the subject's pre-existing disease or condition. Such screening methods include, for example, conventional screening methods for identifying environmental, familial, occupational, and other risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods such as various ELISAs and other immunoassay methods for detecting and/or characterizing coronavirus infection. These and other conventional methods allow clinicians to select patients in need of treatment using the methods and pharmaceutical compositions of the present disclosure. According to these methods and principles, the compositions may be administered according to the teachings herein, or other conventional methods as a stand-alone prevention or treatment program, or as adjuvant or modulatory therapy to other treatments as a follow-up.

Administration of the disclosed compositions may be for prophylactic or therapeutic purposes. When provided prophylactically, the disclosed therapeutic is provided prior to the onset of any symptoms, for example, prior to the onset of an infection. Prophylactic administration of the disclosed therapeutic serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the described therapeutic is provided at or after the onset of the disease or infection, for example, after the development of symptoms of infection with a coronavirus corresponding to the S protein in the composition, or after a diagnosis of a coronavirus infection. Thus, the therapeutic may be provided prior to an anticipated exposure to a coronavirus, upon exposure or suspected exposure to the virus, or after the onset of an actual infection, to attenuate the severity, duration or extent of the anticipated infection and/or associated disease symptoms.

The compositions described herein are provided to a subject in an amount effective to induce or enhance an immune response to a coronavirus S protein in a subject, preferably a human. The actual dosage of the disclosed composition will vary depending on factors such as the disease manifestations and the particular condition of the subject (e.g., the subject's own age, physique, physical fitness, degree of symptoms, susceptibility factors, and the like), the time and route of administration, other drugs or treatments administered concurrently, as well as the particular pharmacology of the composition to induce the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide the optimal prophylactic or therapeutic response.

The compositions according to the present disclosure may be used in a combined vaccination protocol or combination formulation. In certain embodiments, the compositions and combined vaccination protocol utilize separate transgenes or formulations that induce an anti-viral immune response, such as an immune response to a coronavirus S protein. The separate immunogenic compositions that induce an anti-viral immune response may be combined into a multivalent immunogenic composition that is administered to a subject in a single immunization step, or they may be administered separately (as a monovalent immunogenic composition) in a combined (or prime-boost) immunization protocol.

There may be multiple boosts, each boost can be a different transgene. In some examples, the boost can be another boost, or the same transgene as the prime. The prime and boost can be administered as a single dose or multiple doses, for example, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses or more can be administered to the subject over a period of days, weeks or months. Multiple boosts can be given, from 1 to 5 (e.g., 1, 2, 3, 4 or 5 boosts) or more. A series of sequential immunizations can use different doses. For example, a relatively large dose is administered as a primary immunization, followed by a relatively smaller dose as a booster.

In some embodiments, the booster may be administered about 2 weeks, about 3 to 8 weeks, or about 4 weeks, or about several months after the prime vaccination. In some embodiments, the booster may be administered about 5 months, about 6 months, about 7 months, about 8 months, about 10 months, about 12 months, about 18 months, about 24 months, or more or less after the prime vaccination. Periodic additional boosts may also be used at appropriate times to enhance the "immune memory" of the subject. The adequacy of the selected vaccination parameters, such as formulation, dosage, regimen, and the like, may be determined by taking an aliquot of serum from the subject and analyzing the antibody titer over the course of the immunization program. In addition, the clinical status of the subject may be monitored for a desired effect, such as prevention of infection or improvement in disease state (e.g., reduction in viral load). If such monitoring indicates that vaccination is less than optimal, the subject can be given a booster dose of the immunogenic composition and the vaccination parameters can be modified in a manner expected to enhance the immune response.

In some embodiments, the compositions of the present disclosure are administered in a single-dose dose.

Upon administration of a composition disclosed herein, the immune system of the subject typically responds to the composition by producing antibodies specific for the coronavirus S protein contained within the composition. Such a response indicates that an immunologically effective dose has been delivered to the subject.

In some embodiments, the antibody response of the subject will be determined in the context of evaluating an effective dose/immunization protocol. In most cases, assessing antibody titers in serum or plasma obtained from the subject will be sufficient. The decision as to whether to administer a booster dose and/or to modify the amount of therapeutic administered to the individual may be based, at least in part, on the level of antibody titers. The level of antibody titers may be based on an immunobinding assay that measures the concentration of antibodies in serum that bind to an antigen comprising, for example, a recombinant coronavirus S protein included in the immunogen.

The method need not be effective to completely eliminate, reduce, or prevent a coronavirus infection. For example, by inducing an immune response to a coronavirus with one or more of the disclosed compositions, infection with the coronavirus can be reduced or inhibited to a desired level, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (detectable infected cells are eliminated or prevented), as compared to infection with the coronavirus in the absence of the composition. In a further example, coronavirus replication can be reduced or inhibited by the disclosed methods. It is not necessary for the method to completely eliminate coronavirus replication for it to be effective. For example, an immune response induced using one or more of the compositions described herein can reduce replication of a corresponding coronavirus to a desired level, e.g., by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable replication of the coronavirus) compared to replication of the coronavirus in the absence of the composition.

In some embodiments, the disclosed composition is administered to the subject together with administration of an adjuvant. Adjuvants are known in the art to further enhance the immune response to the applied antigenic determinant, and pharmaceutical compositions comprising adenovirus and a suitable adjuvant are disclosed, for example, in WO 2007/110409 (incorporated herein by reference). Examples of suitable adjuvants are provided herein.

In some embodiments, administering a therapeutically effective amount of one or more of the disclosed compositions to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum can be collected from the subject at an appropriate time point following immunization of the subject, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity are known to those of skill in the art and are further described herein, including but not limited to plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, the serum neutralizing activity can be assayed using a panel of coronavirus pseudoviruses. For example, a pseudotyped reporter virus neutralization assay similar to that previously developed for SARS-CoV (Martin et al, Vaccine 26, 6338, 2008; Yang et al, Nature 428, 561, 2004; Naldini et al, PNAS 93, 11382, 1996; Yang et al, PNAS 102, 797, 2005) was previously developed to test the immunogenicity of vaccine candidates against multiple MERS-CoV strains without requiring a Biosafety Level 3 facility (Wand et al., Nat Commun, 6:7712, 2015).

In other embodiments, the present disclosure provides methods for treating, preventing, or reducing the infectivity of a respiratory viral infection. In some embodiments, the viral infection can be a coronavirus infection. The coronavirus can be SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1, OC43, or 229E. The coronavirus can be a beta-coronavirus. A subject at risk for a coronavirus infection can unknowingly contract a coronavirus infection by coming into contact with an asymptomatic carrier of the coronavirus infection.

Certain methods of the present disclosure include methods of producing an adenovirus as described herein. Such methods may include the steps of transfecting a cell with an adenovirus vector as described herein; culturing the cell under conditions that allow the cell to produce a recombinant adenovirus; and collecting the recombinant adenovirus. Suitable cells are known in the art. In some embodiments, the cell may be a HEK, Vero, or PER cell.

The present disclosure also encompasses methods of treating a second subject with serum from a first subject. In particular, the present disclosure encompasses compositions comprising serum from a first subject who has previously been administered an adenoviral vector as detailed herein, a pharmaceutical composition as detailed herein, or an immunogenic composition as detailed herein. Methods for collecting such serum are known in the art. Methods of treating a second subject with serum from a first subject generally comprise administering to the second subject an immunogenically effective amount of a composition comprising serum from the first subject. Methods for administering serum to a subject are known, and determining the dose of serum is within the skill of the art.

In general, the methods described herein comprise administering to a subject a therapeutically effective amount of a nanoparticle composition of the present disclosure. The methods described herein are generally performed on a subject in need thereof. The subject can be a rodent, a human, a domestic animal, a companion animal, or a zoo animal. In one embodiment, the subject can be a rodent, such as a mouse, a rat, a guinea pig, or the like. In another embodiment, the subject can be a domestic animal. Non-limiting examples of suitable domestic animals include pigs, cows, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject can be a companion animal. Non-limiting examples of companion animals include pets such as dogs, cats, rabbits, birds, and the like. In yet another embodiment, the subject can be a zoo animal. As used herein, “zoo animal” means an animal that can be found in a zoo. Such animals can include non-human primates, big cats, wolves, and bears. In a preferred embodiment, the subject is a human.

IV. Kit

Kits are also provided herein. Such kits may include a formulation or composition described herein, and in certain embodiments, instructions for administration. The components include, but are not limited to, one or more of the following: (i) one or more of a host cell described herein, a packaging cell described herein, an adenoviral vector described herein, a pharmaceutical composition described herein, or an immunogenic composition described herein, and (ii) instructions for use. In some embodiments, the kits of the present disclosure may include a composition as described in detail in Section II above. Such kits may facilitate the performance of the methods described herein. When provided as a kit, the various components of the composition may be packaged in separate containers and mixed immediately prior to use. If individual packaging of the components is desired, they may be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the composition. The pack may include, for example, a metal or plastic foil, such as a blister pack. In certain cases, if the above components are packaged separately, the components can be stored for long periods of time without losing their activity.

The kit may contain reagents, such as sterile water or saline, in separate containers in addition to the separately packaged lyophilized active ingredients. For example, sealed glass ampoules may contain the lyophilized ingredients, the separate ampoules containing sterile water, sterile saline or sterile, each packaged under a neutral, non-reactive gas such as nitrogen. The ampoules may be constructed of any suitable material, such as glass, polycarbonate, polystyrene, ceramic, metal or other materials commonly used to contain reagents. Other examples of suitable containers include bottles, which may be constructed of a similar material to the ampoules, and envelopes, which may be constructed with a foil-lined interior of aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes and the like. The container may have a sterile access port, such as a vial having a stopper pierceable by a hypodermic needle. Other containers may have two compartments separated by a readily removable membrane, the removal of which allows the components to be mixed. The removable membrane may be made of glass, plastic, rubber, or the like.

In certain embodiments, the kit may be provided with instructional materials. The instructions may be printed on paper or other printed matter and/or may be provided on electronically-readable media, such as a floppy disk, mini CD-ROM, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, and the like. The detailed instructions may not be physically associated with the kit; instead, the user may be directed to an Internet website designated by the kit manufacturer or distributor.

The compositions and methods described herein utilizing molecular biology protocols can follow a variety of standard techniques known in the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Specific embodiments disclosed herein may be further limited in the claims by use of language such as "consisting of" or "consisting essentially of," rather than "comprising." When used in the claims, the transition term "consisting of" excludes any elements, steps, or ingredients that are not specified in the claim, whether added by filing or amendment. The transition term "consisting essentially of" is limited to those materials or steps that are basic to the scope of the claim and do not materially affect the novel characteristic(s). Embodiments of the invention so claimed are essentially or explicitly described and enabled by this specification.

As various changes may be made in the materials and methods described above without departing from the scope of the present invention, all materials included in the above description and the examples given below should be construed as illustrative and not limiting.

Examples

The following examples are included to demonstrate various embodiments of the present disclosure. The techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the present invention and therefore are readily apparent to those skilled in the art and can therefore be considered to constitute preferred modes for practicing the invention. However, those skilled in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments disclosed and still obtain similar or like results without departing from the spirit and scope of the invention.

Example 1: A single-dose chimpanzee adenovirus vectored vaccine protects against SARS-CoV-2 infection and pneumonia in mice expressing the human ACE2 receptor.

The present invention provides a chimpanzee Ad (simian AdV-36)-based SARS-CoV-2 vaccine (ChAd-SARS-CoV-2-S) encoding a pre-fusion stabilized spike (S) protein following introduction of two proline substitutions in the S2 subunit. Intramuscular administration of ChAd-SARS-CoV-2-S induced robust systemic humoral and cell-mediated immune responses to the S protein. Single or two doses of the vaccine prevented lung infection, inflammation, and pathology following challenge with SARS-CoV-2 in mice transiently expressing the human ACE2 (hACE2) receptor. Despite high levels of neutralizing antibodies induced in the serum, none of the dosing regimens completely prevented SARS-CoV-2 infection, as significant levels of viral RNA were still detected in the lungs. In comparison, a single dose of ChAd-SARS-CoV-2-S administered intranasally resulted in high levels of neutralizing antibodies and anti-SARS-CoV-2 IgA, indicating complete protection against infection in both the upper and lower respiratory tracts. Furthermore, unlike the control ChAd vaccine, no serum antibody responses to the SARS-CoV-2 NP protein were observed in animals immunized with ChAd-SARS-CoV-2-S via intranasal delivery at day 8 post-challenge with SARS-CoV-2. Thus, ChAd-SARS-CoV-2-S has the potential to confer bactericidal immunity at the site of vaccination, thereby preventing both virus-induced disease and transmission.

result

Chimpanzee Ad-vectored vaccine induces potent antibody responses against SARS-CoV-2: Two replication-deficient ChAd vectors based on the simian Ad-36 virus were constructed. The ChAd-SARS-CoV-2-S vector encodes the full-length sequence of the SARS-CoV-2 S protein as a transgene containing the ectodomain, transmembrane domain, and cytoplasmic domain (GenBank: QJQ84760.1), stabilized in the pre-fusion conformation by two proline substitutions at residues K986 and V987. The ChAd-control group does not contain the transgene. The S protein transgene is transcriptionally regulated by the cytomegalovirus promoter. To render the vector replication-deficient and improve its packaging ability, the E1A/B genes were replaced and a deletion of the E3B gene was introduced, respectively ( Figure 1A ). To confirm that the S protein was expressed and antigenically intact, 293T cells were transduced, and binding of a panel of 22 neutralizing monoclonal antibodies against the S protein was confirmed by flow cytometry ( Figure 1B ).

To assess the immunogenicity of ChAd-SARS-CoV-2-S, groups of 4-week-old BALB/c mice were immunized intramuscularly with ^10–10 virus particles of ChAd-SARS-CoV-2-S or ChAd-control. Some mice received a booster vaccination 4 weeks later. Serum samples were collected 21 days after primary or booster vaccination ( Fig. 1C ), and IgG responses to purified S protein and RBD proteins were assessed by ELISA. ChAd-SARS-CoV-2-S induced high levels of S-specific and RBD-specific IgG, whereas levels were low, if detectable, in ChAd control-vaccinated mice ( Figs. 1D and 2A ). Serum samples were assayed in vitro for neutralization of infectious SARS-CoV-2 using the focus-reduction neutralization test (FRNT). As expected, sera from ChAd-control-immunized mice failed to inhibit SARS-CoV-2 infection following primary or booster immunization. In contrast, sera from animals vaccinated with ChAd-SARS-CoV-2-S potently neutralized SARS-CoV-2 infection, and potentiation enhanced this inhibitory activity ( Figure 1E and Figures 2B-2C ).

Vaccine-induced memory CD8+ T cell and antigen-specific B cell responses: Since optimal vaccine immunity often consists of humoral and cellular responses, we measured SARS-CoV-2-specific CD4+ and CD8+ T cell levels post-vaccination. Four-week-old BALB/c mice were immunized with ChAd-SARS-CoV-2-S or ChAd-control, and boosted 3 weeks later. To assess vaccine-induced SARS-CoV-2-specific CD4+ and CD8+ T cell responses, splenocytes were harvested 1 week post-boost and stimulated ex vivo with a pool of 253 overlapping 15-mer S peptides. Intracellular IFNγ and granzyme B expression was then quantified by flow cytometry. After peptide restimulation in vitro, splenic CD8+ T cells from mice immunized with ChAd-SARS-CoV-2-S expressed IFNγ, and both splenic CD4+ and CD8+ T cells expressed granzyme B, but not the ChAd control vector ( Figures 1F-1G and Figure 3 ). To assess antigen-specific B cell responses, splenocytes were harvested and subjected to ELISPOT analysis using S protein. ChAd-SARS-CoV-2-S vaccine induced S protein-specific IgG antibody-secreting cells in the spleen, whereas the control vaccine did not ( Figure 1H ).

Intramuscular vaccination with ChAd-SARS-CoV-2-S vaccine protects against pulmonary SARS-CoV-2 infection: We tested the protective activity of ChAd vaccine in a recently developed SARS-CoV-2 infection model in which BALB/c mice express hACE2 in the lungs after intranasal delivery of vectored human Ad (Hu-Ad5-hACE2). Endogenous mouse ACE2 does not support virus entry, and this system enables productive infection with SARS-CoV-2 in the mouse lungs. Four-week-old BALB/c mice were first immunized via the intramuscular route with ChAd-control or ChAd-SARS-CoV-2-S vaccine. Approximately 30 days later, mice were challenged intranasally and intraperitoneally with 10 ⁸ plaque-forming units (PFU) of Hu-Ad5-hACE2 and anti-Ifnar1 monoclonal antibody (mAb), respectively. A single dose of anti-Ifnar1 mAb was also administered to enhance lung virulence in this model. No cross-immunity was confirmed between ChAd and Hu-Ad5 vectors. Serum from ChAd-immunized mice did not neutralize Hu-Ad5 infection ( Figures 4A-4B ).

Five days post-Hu-Ad5-hACE2 transduction, mice were infected intranasally with 4 × 10 ⁵ focus-forming units (FFU) of SARS-CoV-2 ( Fig. 2A ). Mice were euthanized on day 4 post-infection (dpi), the peak viral load in this model, and lungs, spleen, and heart were harvested for viral load and cytokine analysis. Notably, there was no detectable infectious virus in the lungs of mice vaccinated with ChAd-SARS-CoV-2-S, as measured by plaque assay, whereas high levels of virus were present in mice vaccinated with ChAd-control ( Fig. 5B ). Consistent with these results, reduced viral RNA levels were observed in the lungs, hearts, and spleens of ChAd-SARS-CoV-2-S vaccinated animals compared to mice receiving the ChAd-control vector ( Fig. 5C ). In situ hybridization staining for viral RNA from lungs harvested at 4 dpi showed a significant reduction in SARS-CoV-2 RNA in lung cells from animals immunized with ChAd-SARS-CoV-2-S compared to ChAd control ( Figure 5D ). A subset of vaccinated animals were euthanized at 8 dpi and tissues were harvested for evaluation. At this time point, viral RNA levels were again lower or absent in the lungs and spleen of ChAd-SARS-CoV-2-S immunized mice compared to control ChAd vector ( Figure 5C ). Collectively, these data indicate that a single intramuscular immunization with ChAd-SARS-CoV-2-S significantly reduces, but does not completely eliminate, SARS-CoV-2 infection in the challenged lungs of mice.

Next, we evaluated the effect of the vaccine on lung inflammation and disease. Compared to the ChAd-control, animals vaccinated with ChAd-SARS-CoV-2-S had lower mRNA levels of several pro-inflammatory cytokines and chemokines, including CXCL10, IL1β, IL-6, CCL5, IFNβ, and IFNγ , in lung tissue ( Figure 5E ). Furthermore, mice vaccinated with the ChAd-control vaccine and challenged with SARS-CoV-2 showed evidence of viral pneumonia characterized by perivascular and alveolar immune cell accumulation, vascular congestion, and interstitial edema. In contrast, animals vaccinated with ChAd-SARS-CoV-2-S showed a marked attenuation of the lung inflammatory response developed in ChAd-control-vaccinated mice ( Figure 6 ). Thus, Ch-Ad-SARS-CoV-2 vaccination reduces both viral infection and subsequent lung inflammation and damage associated with SARS-CoV-2 infection.

We then evaluated enhanced protection using a prime-boost vaccination regimen. BALB/c mice were immunized intramuscularly with ChAd-control or ChAd-SARS-CoV-2-S, followed by an allogeneic booster 4 weeks later. On day 29 post-booster vaccination, mice were administered a single dose of anti-Ifnar1 antibody followed by Hu-Ad5-hACE2, and challenged with SARS-CoV-2 5 days later. As expected, the prime-boost regimen protected against SARS-CoV-2 challenge, with no detectable infectious virus in the lungs ( Figure 5F ). A marked reduction in viral RNA in the lungs, spleen, and heart was detected at 4 dpi, although residual levels of viral RNA were still present, indicating that protection was not complete after the booster vaccination ( Figure 5G ).

A single intranasal vaccination with ChAd-SARS-CoV-2-S induces bactericidal immunity against SARS-CoV-2: Mucosal immunization via the nasopharyngeal route can induce local immune responses, including secretory IgA antibodies, that provide protection at or near the site of respiratory pathogen challenge. To evaluate the immunogenicity and protective efficacy of mucosal vaccination, 5-week-old BALB/c mice were challenged via the intranasal route with 10 ¹⁰ virus particles of ChAd-control or ChAd-SARS-CoV-2-S ( Figure 7A ). Serum samples were collected at week 4 post-vaccination to assess humoral immune responses. Intranasal immunization with ChAd-SARS-CoV-2-S, but not ChAd-control, induced high levels of S- and RBD-specific IgG and IgA ( Figures 7B-7C ) and SARS-CoV-2 neutralizing antibodies (geometric mean titer 1/1,574) ( Figures 7D and 8A ). Serum antibodies from mice immunized with ChAd-SARS-CoV-2-S were comparable to neutralizing a recombinant luciferase-expressing SARS-CoV-2 variant encoding the D614G mutation in the S protein ( Figure 8B ); this finding is important because many circulating viruses contain this substitution and are associated with greater infectivity in cell culture (doi.org/10.1101/2020.06.12.148726 ). We also assessed SARS-CoV-2-specific antibody responses in bronchoalveolar lavage (BAL) fluid from immunized mice. BAL fluid from mice vaccinated with ChAd-SARS-CoV-2-S showed high levels of S- and RBD-specific IgG and IgA antibodies, including antibodies with neutralizing activity ( Figures 7G and 8C ), but not from mice vaccinated with ChAd-control ( Figures 7E-7F ).

To assess activated T cell responses following mucosal vaccination, mice were vaccinated intranasally with ChAd-SARS-CoV-2-S or ChAd-control, followed by a booster vaccination 4 weeks later. Lungs were harvested 1 week post-boost, and T cells were analyzed by flow cytometry. Ex vivo restimulation with S peptide pools resulted in a significant increase in CD8+ T cells producing IIFNγ and granzyme B in the lungs of mice that had received the ChAd-SARS-CoV-2-S vaccine ( Figure 7H ). Specifically, a population of IFNγ-secreting, antigen-specific CD103 ⁺ CD69 ⁺ CD8 ⁺ T cells was identified in the lungs ( Figure 7I ), which was phenotypically consistent with vaccine-induced resident memory T cells. In the spleen, antibody-secreting plasma cells producing IgA or IgG against the S protein were detected after intranasal immunization with ChAd-SARS-CoV-2-S ( Figure 7J ). Of note, the frequency of B cells secreting anti-S IgA was observed to be approximately five times higher than that of IgG.

The protective efficacy of the ChAd vaccine was assessed following a single-dose intranasal immunization. At day 30 post-vaccination, mice were challenged with 10 ⁸ PFU of Hu-Ad5-hACE2 and anti-Ifnar1 mAb as described above. Five days later, mice were challenged with 4 x 10 ⁵ FFU of SARS-CoV-2. At 4 and 8 dpi, lungs, spleen, heart, nasal turbinates, and nasal wash were collected and assessed for viral load. Intranasal administration of the ChAd-SARS-CoV-2-S vaccine demonstrated remarkable protective efficacy as judged by the absence of infectious virus in the lungs ( Figure 9A ) and minimal or no measurable viral RNA in the lungs, spleen, heart, nasal saline, or nasal wash ( Figure 9B ). The very low viral RNA levels in the lungs and nasal conchae at 4 dpi are likely due to the entry of non-replicating virus, as similar levels were measured in C57BL/6 mice lacking hACE2 receptor expression at this time point. Cytokine and chemokine mRNA levels in lung homogenates were also significantly lower in mice immunized with ChAd-SARS-CoV-2-S than in the ChAd-control vaccine ( Fig. 9C ), suggesting that the residual expression is likely due to the human Ad vector hACE2 delivery system. Finally, histopathological analysis of lung tissue from animals vaccinated with ChAd-SARS-CoV-2-S by the intranasal route and challenged with SARS-CoV-2 showed minimal or no perivascular and alveolar infiltrates at 8 dpi, compared to the extensive inflammation observed in the ChAd-control vaccinated animals ( Fig. 9D ).

To determine whether bactericidal immunity was achieved when ChAd-SARS-CoV-2-S was administered intranasally, anti-NP antibodies were measured at 8 dpi and compared to responses 5 days prior to SARS-CoV-2 infection. Since the vaccine vector lacks the NP gene, the induction of a humoral immune response against NP following SARS-CoV-2 exposure suggests viral protein translation and active infection. After SARS-CoV-2 challenge, anti-NP antibody responses were detected in ChAd-control mice vaccinated by the intranasal route or in both ChAd-control mice and ChAd-SARS-CoV-2-S mice vaccinated by the intramuscular route ( Figure 9E and Figure 2D ). Of note, none of the mice vaccinated with ChAd-SARS-CoV-2-S via the intranasal route had a significant increase in anti-NP antibody responses following SARS-CoV-2 infection. Combining these data with our virological analyses, we suggest that a single intranasal immunization with ChAd-SARS-CoV-2-S induces potent, bactericidal mucosal immunity, which prevents SARS-CoV-2 infection in the upper and lower respiratory tracts of mice expressing the hACE2 receptor.

argument

In this case, we evaluated intramuscular and intranasal administration of a replication-deficient ChAd vector as a vaccine platform for SARS-CoV-2. Single-dose immunization with a stabilized S protein-based vaccine intramuscularly induced S- and RBD-specific binding and neutralizing antibodies. Vaccination with one or two doses protected mice expressing human ACE2 against SARS-CoV-2 challenge, as evidenced by clearance of infectious virus in the lungs and significant reductions in viral RNA levels in the lungs and other organs. Mice immunized with ChAd-SARS-CoV-2-S had significantly reduced or no evidence of lung pathology, lung inflammation, and pneumonia compared to control ChAd vaccines. However, intramuscular vaccination with ChAd-SARS-CoV-2-S did not confer bactericidal immunity, as evidenced by detectable viral RNA levels in multiple tissues, including the lungs, and by induction of anti-NP antibody responses. Mice vaccinated with a single dose of ChAd-SARS-CoV-2-S via the intranasal route were also protected against SARS-CoV-2 challenge. However, intranasal vaccination generated robust IgA and neutralizing antibody responses that prevented upper and lower respiratory tract SARS-CoV-2 infection and inhibited infection by both wild-type and D614G mutant viruses. The very low viral RNA in upper respiratory tract tissues and the absence of serological responses to NP in the challenge setting strongly suggest that most animals achieve sterilizing immunity after a single intranasal dose of ChAd-SARS-CoV-2-S.

Although several vaccine candidates ( e.g., lipid-encapsulated mRNA, DNA, inactivated, and viral vectors) have been rapidly advanced into human clinical trials in the rapid effort to control the pandemic, few studies have demonstrated efficacy in preclinical models. Immunization of rhesus macaques with 2–3 doses of a DNA plasmid vaccine encoding the full-length SARS-CoV-2 S protein induced neutralizing antibodies in the serum and reduced viral loads in BAL and nasal fluids. Furthermore, three doses of purified, inactivated SARS-CoV-2 administered over 2 weeks induced neutralizing antibodies and, depending on the dose administered, provided partial or complete protection against infection and viral pneumonia in rhesus macaques. One limitation of these challenge models is that rhesus macaques develop milder interstitial pneumonia following SARS-CoV-2 infection compared to some humans and other non-human primate species. This study in hACE2-expressing mice demonstrated that a single dose of ChAd-SARS-CoV-2-S vaccine administered intramuscularly or intranasally could provide substantial and complete protection against viral replication, inflammation, and lung disease.

This specification supports the use of non-human Ad vector vaccines against emerging RNA viruses, including SARS-CoV-2. Previous studies have demonstrated efficacy of single- or double-dose regimens of gorilla Ad encoding the prM-E gene of Zika virus (ZIKV) in several mouse challenge models, including pregnancy. Other investigators have evaluated ChAd or rhesus monkey Ad vaccine candidates against ZIKV, showing efficacy in mice and non-human primates. Another ChAd encoding the wild-type SARS-CoV-2 S protein (ChAdOx1) is currently in human clinical trials (NCT04324606). While no human data have yet been reported, a study in rhesus monkeys showed that a single intramuscular dose protected against pulmonary infection, but not upper respiratory tract infection (biorxiv.org/content/10.1101/2020.05.13.093195v1). None of the vaccines evaluated against SARS-CoV-2 have shown evidence of immune enhancement in preclinical or clinical studies, a theoretical risk based on studies of other human and animal coronaviruses. In fact, in contrast to data for SARS-CoV vaccines or antibodies, no increase in infection, immunopathology, or disease was observed in animals immunized with ChAd encoding the SARS-CoV-2 S protein or in animals administered passively delivered monoclonal antibodies.

ChAd-SARS-CoV-2-S induced SARS-CoV-2-specific CD8 ⁺ T cell responses, including high proportions and numbers of cells expressing IFNγ and granzymes, following ex vivo S peptide restimulation. The induction of robust CD8+ T cell responses by the ChAd-SARS-CoV-2-S vaccine is consistent with reports for other simian Ad vectors. The ChAd vaccine vector has immunological advantages as it overcomes the existing immunological challenges against human adenoviruses and does not induce exhausted T cell responses.

A single dose of ChAd-SARS-CoV-2-S administered intranasally confers superior immunity against SARS-CoV-2 infection compared to single or double immunization with the same vaccine and dose. Given the similar serum neutralizing antibody responses, we hypothesized that the greater protection observed after intranasal delivery was due to mucosal immune responses. Indeed, high levels of anti-SARS-CoV-2 IgA were detected in serum and lung, and IgA-secreting B cells were detected in the spleen. Furthermore, intranasal vaccination induced SARS-CoV-2-specific CD8+ T cells in the lung, namely CD103 ⁺ CD69 ⁺ cells, which are likely to be of a resident memory phenotype. To our knowledge, no SARS-CoV-2 vaccine platform currently in clinical trials utilizes an intranasal delivery route. There has been significant interest in intranasal delivery of influenza A virus vaccines because of their potential to elicit local humoral and cellular immune responses. In fact, sterilizing immunity against influenza A virus reinfection requires a local adaptive immune response in the lung, which is optimally induced by intranasal rather than intramuscular administration. While there are concerns about administering live-attenuated virus vaccines via the intranasal route, subunit-based or replication-deficient vector vaccines show promise in generating mucosal immunity in a safer manner, especially with advances in formulation.

In summary, this example demonstrates that immunization with ChAd-SARS-CoV-2-S induces both neutralizing antibodies and antigen-specific CD8+ T cell responses. A single intramuscular immunization with ChAd-SARS-CoV-2-S provides protection against SARS-CoV-2 infection and inflammation in the lung, while intranasal administration of ChAd-SARS-CoV-2-S appears to induce mucosal immunity, provide superior protection, and enhance bactericidal immunity, at least transiently in mice expressing the hACE2 receptor. Thus, this example supports intranasal delivery of ChAd-SARS-CoV-2-S as a platform to control SARS-CoV-2 infection, disease, and transmission.

method

Viruses and Cells: Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero CCL81 (ATCC), and HEK293 cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1X non-essential amino acids, and 100 U/ml penicillin-streptomycin.

SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was obtained from the Centers for Disease Control and Prevention (a gift from Natalie Thornburg). The virus was passaged once in Vero CCL81 cells and titered by focus forming assay (FFA) in Vero E6 cells. A recombinant luciferase-expressing full-length SARS-CoV-2 reporter virus (strain 2019 n-CoV/USA_WA1/2020) has been previously described (Zost et al., 2020), and the D614G variant will be described elsewhere. All work with infectious SARS-CoV-2 was performed in a BSL3 and A-BSL3 facility approved by the institutional biosafety committee using appropriate positive-pressure respirators and protective equipment.

Mouse experiments: Animal studies were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Washington University School of Medicine (assignment number A3381-01). Viral inoculation was performed under anesthesia induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.

Female BALB/c mice were purchased from Jackson Laboratory (catalog 000651). At 4–5 weeks of age, animals were immunized with 1010 virus particles (vp) of ChAdV-empty or ChAd-SARS-CoV-2-S in 50 μl phosphate-buffered saline (PBS) via hindlimb muscle injection or intranasal inoculation. Some of the vaccinated animals received a booster vaccination 4 weeks after the primary immunization using the same route used for the primary immunization. Vaccinated mice (10–11 weeks of age) were administered a single intraperitoneal injection of 2 mg of anti-Ifnar1 mAb (MAR1-5A3, Leinco) the day before intranasal challenge with 10 ⁸ PFU of Hu-AdV5-hACE2. On day 5 after introduction of Hu-AdV5, mice were challenged intranasally with 4 × 10 ⁵ FFU of SARS-CoV-2. Animals were euthanized at 4 or 8 dpi, and tissues were harvested for virological, immunological, and pathological analyses.

Construction of chimpanzee adenovirus vectors: Simian Ad36 vector (ChAd) was obtained from the Penn Vector Core of the University of Pennsylvania. The ChAd genome was engineered to have deletions in the E1 and E3B regions (GenBank: FJ025917.1; nucleotides 455-3026 and 30072-31869, respectively). A modified human cytomegalovirus major immediate early promoter sequence was integrated counterclockwise in place of the E1 gene on the complementary DNA strand. The CMV variant contained two copies of the tet operator 2 (TetO2) simultaneously inserted (5'-TCT CTA TCA CTG ATA GGG AGA TCT CTA TCA CTG ATA GG GA-3') (SEQ ID NO: 7) between the TATA box and the start of mRNA (GenBank: MN920393, nucleotides 174211-174212). SARS-CoV-2 S (encoding a pre-fusion stabilized mutant with two proline substitutions at residues K986 and V987, stabilizing the pre-fusion conformation of S) was cloned into the pSAd36 genomic plasmid at a unique PmeI site under the control of the CMV-tetO2 promoter, generating pSAd36-S. Similarly, mutant SAR-CoV-2S proteins designated SEQ ID NOs: 10, 11, 20, and 21 were cloned similarly. In parallel, pSAd36-control containing an empty CMV-tetO2 cassette without the transgene was also generated. pSAd36-S and pSAd-control plasmids were linearized with PacI restriction enzyme, releasing the viral genomes that were capable of transfecting T-Rex 293-HEK cells (Invitrogen). Replication-deficient ChAd-SARS-CoV-2-S and ChAd-control vectors rescued were expanded in 293 cells and purified by CsCl density-gradient ultracentrifugation. The viral particle concentration of each vector preparation was determined spectrophotometrically at 260 nm.

Construction of human ACE2 expressing human adenovirus vector: The codon-optimized hACE2 sequence was cloned into the shuttle vector (pShuttle-CMV, Addgene 240007) to generate pShuttle-hACE2. pShuttle-hACE2 was linearized using PmeI and subsequently co-transformed into E. coli strain BJ5183 with the HuAdv5 backbone plasmid (pAdEasy-1 vector; Addgene 240005) to generate pAdV5-ACE2 by homologous recombination. The pAdEasy-1 plasmid containing the HuAdV5 genome has deletions in the E1 and E3 genes. hACE2 is under the transcriptional control of the cytomegalovirus promoter and is flanked at the 3' end by the SV40 polyadenylation signal. pAd-hACE2 was linearized with PacI restriction enzyme before transfection into T-Rex 293 HEK cells (Invitrogen) to generate HuAdv5-hACE2. Recombinant HuAdv5-hACE2 was produced in 293-HEK cells and purified by CsCl density-gradient ultracentrifugation. The virus titer was determined by plaque assay in 293-HEK cells.

In Situ RNA Hybridization and Histology : RNA in situ hybridization was performed using RNAscope 2.5 HD (Brown) (Advanced Cell Diagnostics) according to the manufacturer’s instructions. Left lung tissue was collected at autopsy, expanded in 10% neutral buffered formalin (NBF), and fixed in 10% NBF for 7 days prior to processing. Paraffin-embedded lung sections were deparaffinized by incubation at 60°C for 1 h, and endogenous peroxidase was removed by treatment with H ₂ O ₂ for 10 min at room temperature. Slides were boiled for 15 min in RNAscope Target Retrieval Reagents and then incubated for 30 min in RNAscope Protease Plus reagents prior to hybridization with a SARS-CoV2 RNA probe (Advanced Cell Diagnostics 848561) and signal amplification. Sections were counterstained with Gill's hematoxylin and visualized under brightfield microscopy. Some lung sections were stained with hematoxylin-eosin and then processed for histological examination.

SARS-CoV-2 Neutralization Assay : Heat-inactivated serum samples were serially diluted and incubated with ¹⁰² FFU of SARS-CoV-2 for 1 h at 37°C. The virus-serum mixture was added to Vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. The cells were then overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. The plates were incubated for 1 h at room temperature with 4% PFA in PBS for 30 h prior to fixation. Cells were washed and sequentially incubated with anti-SARS-CoV-2 CR3022 antibody (Yuan et al., 2020) (1 μg/mL) and HRP-conjugated goat anti-human IgG (Sigma) in PBS supplemented with 0.1% (w/v) saponin (Sigma) and 0.1% BSA. Plates were developed prior to focusing on a BioSpot analyzer (Cellular Technology Limited) using TrueBlue peroxidase substrate (KPL). For neutralization experiments against luciferase-expressing SARS-CoV-2, serum samples were diluted threefold starting at 1:50 and then mixed with 85 PFU of each recombinant virus (wild type and D614G). The serum-virus mixtures were inoculated onto Vero E6 cells plated in clear-bottom, black-walled 96-well plates (Corning), and the cells were incubated at 37°C for 48 h. Cells were then lysed, and luciferase activity was measured using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer's specifications.

Hu-AdV5 neutralization assay : One day prior to Hu-AdV5-hACE2 transduction, serum samples were collected from mice immunized intramuscularly with ChAd-control or ChAd-SARS-CoV-2-S. Serum was heat-inactivated and serially diluted prior to incubation with 10 ² FFU of HuAdV5 for 1 h at 37°C. The virus-serum mixture was added to HEK293 cell monolayers in 96-well plates and incubated for 1 h at 37°C. The cells were then overlaid with 1% (w/v) methylcellulose in MEM supplemented with 5% FBS. Prior to fixation, the plates were incubated at 37°C for 48 h and then fixed with 2% PFA in PBS for 1 h at room temperature. The plates were then washed with PBS and incubated overnight at 4°C with biotinylated anti-HuAdV5-hexon antibody (2 μg/mL; Novus Biologicals NB600413) diluted in permeabilization buffer (PBS supplemented with 0.1% (w/v) saponin and 0.1% BSA). The plates were washed again and incubated for 30 min at room temperature with streptavidin-HRP (1:3000; Vector Laboratories SA-5004) in permeabilization buffer. After a final series of washes, the plates were developed using TrueBlue peroxidase substrate (KPL), and foci were counted on a BioSpot analyzer (Cellular Technology Limited).

Protein Expression and Purification: Purified RNA from 2019-nCoV/USA-WA1/2020 SARS-CoV-2 strains was reverse transcribed into cDNA, which served as a template for recombinant gene cloning. Full-length SARS-CoV-2 NP (NP-FL) was cloned into pET21a with a hexahistidine tag and recombinantly expressed using BL21(DE3)-RIL E. coli in Terrific Broth (bioWORLD). After overnight induction with isopropyl β-d-1-thiogalactopyranoside (Goldbio) at 25°C, cells were lysed in 20 mM Tris-HCl pH 8.5, 1 M NaCl, 5 mM β-mercaptoethanol, and 5 mM imidazole for nickel affinity purification. After elution in pre-buffer supplemented with 500 mM imidazole, proteins were purified to homogeneity using size exclusion and, in some cases, cation exchange chromatography. SARS-CoV-2 RBD and S ectodomain (S1/S2 furin cleavage site disrupted, double proline mutations introduced into the S2 subunit, and foldon trimerization motif integrated) were cloned into pFM1.2 with a C-terminal hexahistidine or octahistidine tag, transiently transformed into Expi293F cells, and purified by cobalt-charged resin chromatography (G-Biosciences) as described previously (Alsoussi et al., 2020).

ELISA: Purified antigen (S, RBD, or NP) was coated onto 96-well Maxisorp clear plates at a concentration of 2 μg/mL in 50 mM Na ₂ CO ₃ pH 9.6 (70 μL) and stored overnight at 4°C. The coating buffer was aspirated, and the wells were blocked with 200 μL of 1X PBS + 0.05% Tween-20 + 1% BSA + 0.02% NaN ₃ (Blocking buffer, PBSTBA) for 1 h at 37°C or overnight at 4°C. Heat-inactivated serum samples were diluted in PBSTBA in separate 96-well polypropylene plates. The plates were then washed three times with 1X PBS + 0.05% Tween-20 (PBST) and 50 μL of each serum dilution was added. Serum was incubated in the blocked ELISA plates for at least 1 h at room temperature. The ELISA plates were again washed three times with PBST, and 50 μL of 1:2000 anti-mouse IgG-HRP (Southern Biotech Cat. #1030-05) in PBST or 1:10000 biotinylated anti-mouse IgG, anti-mouse IgM, or anti-mouse IgA in PBSTBA (SouthernBiotech) was added. The plates were incubated for 1 h at room temperature, washed three times with PBST, and 1:5000 diluted streptavidin-HRP (ThermoFisher) solution was added to the wells. After 1 h at room temperature, the plates were washed three times with PBST, and 50 μL of 1-Step Ultra TMB-ELISA was added (ThermoFisher Cat. #34028). After incubation for 12–15 min, the reaction was stopped with 50 μL of 2 M sulfuric acid. The absorbance of each well was read at 450 nm within 2 min after the addition of sulfuric acid (Synergy H1). Optical density measurements (450 nm) were determined using a microplate reader (Bio-Rad).

ELISpot assay: MultiScreen-HA filter 96-well plates (Millipore) were pre-coated with 3 μg/ml SARS-CoV-2 S protein overnight at 4°C. After washing with PBST, the plates were incubated with culture medium (RPMI, 10% FBS, penicillin-streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES, and 50 mM β-mercaptoethanol) at 37 ^°C for 4 h. Single-cell suspensions of splenocytes in the culture medium were added to the S protein-coated plates and incubated at 37°C in a humidified 5% _{CO2 atmosphere} for 4 h. After washing with PBS and PBST, the plates were incubated with biotinylated anti-IgG or anti-IgA (Southern Biotech) followed by streptavidin-conjugated horseradish peroxidase (1 h each at room temperature). After additional washing with PBS, spot development was performed by adding 3-amino-9-ethylcarbazole (Sigma) substrate solution. The reaction was stopped by washing with water. Spots were counted using a Biospot plate reader (Cellular Technology).

Viral load measurement: Mice infected with SARS-CoV-2 were euthanized using a ketamine and xylazine cocktail, and organs were harvested. Tissues were weighed and bead-homogenized using a MAgNA Lyser (Roche) in 1 ml Dulbecco’s modified Eagle medium (DMEM) containing 2% fetal calf serum (FBS). RNA was extracted from clarified tissue homogenates using the MagMax mirVana total RNA isolation kit (Thermo Scientific) and a Kingfisher Duo Prime Extractor (Thermo Scientific). SARS-CoV-2 RNA levels were measured using a one-step quantitative reverse transcriptase PCR (qRT-PCR) TaqMan assay as previously described (Hassan et al., 2020). SARS-CoV-2 nucleocapsid (N)-specific primer and probe sets were used: (L primer: ATGCTGCAATCGTGCTACAA (SEQ ID NO: 8); R primer: GACTGCCGCCTCTGCTC (SEQ ID NO: 9). Viral RNA was expressed as gene copies per milligram (N) on a log10 scale. For some samples, virus titers were confirmed by plaque assay on Vero E6 cells. Cytokine and chemokine mRNA measurements. RNA extracted from lung homogenates After DNAse treatment of lung homogenates, cDNA was synthesized using a high-performance cDNA reverse transcription kit (Thermo Scientific) with RNase inhibitor according to the manufacturer's protocol. Cytokine and chemokine expression was measured using TaqMan Fast Universal PCR Master Mix (Thermo Scientific) and commercially available primer/probe sets specific for: IFN-γ (IDT: Mm.PT.58.41769240), IL-6 (Mm.PT.58.10005566), IL- 1β (Mm.PT.58.41616450), TNF-α (Mm.PT.58.12575861), CXCL10 (Mm.PT.58.43575827), CCL2 (Mm.PT.58.42151692), CCL5 (Mm.PT.58.43548565), CXCL11 (Mm.PT.58.10773148.g), IFN-β (Mm.PT.58.30132453.g), and IFNγ-2/3 (Thermo Scientific Mm04204156_gH), and the results were normalized to GAPDH (Mm.PT.39a.1) levels. The fold change was 2 comparing treated mice to naïve controls. - It was measured using the ^ΔΔCt method.

Peptide restimulation and intracellular cytokine staining: Splenocytes from intramuscularly vaccinated mice were incubated with 253 pools of overlapping 15-mer SARS-COV-2 S peptides for 12 h at 37°C, prior to 4 h treatment with brefeldin A (Biolegend, 420601). After blocking with FcγR antibody (BioLegend, clone 93), cells were stained on ice with CD45 BUV395 (BD BioSciences clone 30-F11); CD44 PE-Cy7, CD4 PE-Cy5, CD8b PreCP-Cy5.5, and CD19 APC-Cy7 (BioLegend clones, IM7, GK1.5, YTS156.7.7, and 6D5, respectively), and Fixable Aqua Dead Cell Stain (Invitrogen, L34966). Stained cells were fixed and permeabilized with Foxp3/transcription factor staining buffer set (eBioscience, 00-5523). Subsequent intracellular staining was performed with anti-IFN-γAlexa 647 (BD Biosciences, clone XMG1.2), anti-TNFαBV605 (BioLegend, clone MP6-XT22), and anti-GrB PE (Invitrogen, GRB04). Lungs from nasal immunized mice were harvested and digested in digestion buffer consisting of RPMI medium supplemented with (167 μg/ml) Liberase DH (Sigma) and (100 μg/ml) DNase I (Sigma) for 1 h at 37°C. Lung cells were incubated for 5 h at 37°C in the presence of brefeldin A with the 253 overlapping 15-mer SARS-CoV-2 S peptide pools described above. Lung cells were then stained as described above, except CD4-BV421 (BioLegend clone GK1.5) was substituted for CD4-PE-Cy5, CD19 staining was not included, and CD103-FITC and CD69-BV711 (BioLegend clones, 2E7 and H1.2F3, respectively) were added. Analysis was performed on a BD LSRFortessa X-20 cytometer using FlowJo X 10.0 software.

Flow cytometry-based antigen characterization: HEK-293T cells were seeded at ¹⁰⁶ cells/well in 6-well plates 24 h prior to transfection with ChAd-SARS-CoV-2-S (MOI 5). After 20 hours, cells were harvested, fixed, permeabilized using Foxp3 transcription factor staining buffer set (Thermo Fisher), incubated with the following anti-SARS-CoV-2 neutralizing mouse mAbs, and stained for viral antigens: SARS2-01, SARS2-02, SARS2-07, SARS2-11, SARS2-12, SARS2-16, SARS2-18, SARS2-20, SARS2-21, SARS2-22, SARS2-23, SARS2-29, SARS2-31, SARS2-32, SARS2-34, SARS2-38, SARS2-39, SARS2-50, SARS2-55, SARS2-58, SARS2-66, and SARS2-71 (L. VanBlargan and M. Diamond, unpublished results). An isotype-matched anti-HCV E2 mAb, H77.39, was used as a negative control. Cells were washed, incubated with Alexa Fluor 647-conjugated goat anti-mouse IgG (Thermo Fisher), and analyzed by flow cytometry using a MACSQuant Analyzer 10 (Miltenyi Biotec). The percentage of cells that tested positive for a given mAb was compared to cells stained at a saturation level with an oligoclonal mixture of anti-SARS-CoV-2 mAbs.

Example 2 : Intranasal vaccine provides durable protection against SARS-CoV-2 variants in mice

SARS-CoV-2 variants that attenuate antibody neutralization could threaten vaccine efficacy and the end of the COVID-19 pandemic. Example 1 demonstrates the protective activity of a single-dose intranasal spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has been advanced to human trials. The present example provides durability, dose-response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced consistently high neutralizing antibody and Fc effector antibody responses in serum, and long-lived plasma cells secreting S-specific IgG and IgA in the bone marrow. Protection against epidemiological SARS-CoV-2 strains was observed over a 100-fold vaccine dose range and for a 200-day period. At 6 weeks or 9 months post-vaccination, serum antibodies neutralized SARS-CoV-2 strains using the B.1.351 and B.1.1.28 spike proteins, providing near-complete protection in the upper and lower respiratory tracts post-challenge. Thus, intranasal vaccination with ChAd-SARS-CoV-2-S in mice appears to provide sustained protection against both established and novel SARS-CoV-2 strains.

The spike (S) protein of the SARS-CoV-2 virus is a major target of antibody-based and vaccine responses. The S protein serves as the primary attachment and entry factor of the virus, facilitating SARS-CoV-2 entry into human cells by binding to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). The SARS-CoV-2 S protein is cleaved to produce the S1 and S2 fragments, where the S1 protein contains the receptor binding domain (RBD), and the S2 protein facilitates membrane fusion and viral entry into the cytoplasm. The pre-fusion form of the SARS-CoV-2 S protein is recognized by potent neutralizing monoclonal antibodies or protein inhibitors.

Many vaccine candidates targeting the SARS-CoV-2 S protein have been developed using DNA plasmids, mRNA encapsulated in lipid nanoparticles, inactivated virus particles, protein subunits, or viral vector vaccine platforms. Several vaccines administered by intramuscular (IM) injection ( e.g., Pfizer/BioNTech BNT162b2; Moderna 1273 mRNA; Johnson & Johnson Ad26.COV2, and AstraZeneca ChAdOx1 nCoV-19 adenovirus platforms) have received emergency use authorization in many countries, where hundreds of millions of doses have been administered worldwide (covid19.who.int).

Although vaccines administered intramuscularly induce robust systemic immunity that prevents severe disease and death, questions remain about their ability to suppress SARS-CoV-2 transmission, particularly in the absence of a reduction in upper respiratory tract infection. Indeed, many IM-administered vaccines have shown variable protection against upper respiratory tract infection and transmission in preclinical studies and have failed to induce substantial mucosal immunity (IgA). This issue is significant given the emergence of more transmissible SARS-CoV-2 variants with substitutions in the spike protein, including B.1.1.7, B.1.351, and B.1.1.28. Experiments with pseudoviruses and genuine SARS-CoV-2 strains have shown that vaccine-induced sera are less potent than variants with spike gene mutations at L452, E484, and other sites. In addition to the potential negative impact on protection, weakened immunity to certain variants, combined with naturally lower levels of anti-S IgG in the respiratory mucosa, could create conditions for further selection of resistance in the upper respiratory tract and possible transmission to the general population.

As described herein, the single-dose, intranasal (IN)-delivered chimpanzee adenovirus (simian Ad-36)-based SARS-CoV-2 vaccine (ChAd-SARS-CoV-2-S) encodes a pre-fusion stabilized S protein that induces robust humoral, cellular, and mucosal immune responses, limiting upper and lower respiratory tract infections in K18-hACE2 transgenic mice, hamsters, and non-human primates. This vaccine, which has already entered human clinical trials (BBV154, Clinical Trial NCT04751682), differs from the chimpanzee Ad-23-based SARS-CoV-2 vaccine ChAdOx1 nCoV-19, which is currently authorized for emergency use in some countries. Here, as an additional step to evaluate the potential utility of ChAd-SARS-CoV-2-S, the dose-response, persistence, cross-protective activity, and efficacy against upper respiratory tract infections in mice were evaluated. Approximately 9 months after IN immunization, the sera of ChAd-SARS-CoV-2-S vaccinated animals maintained high levels of neutralizing antibodies and anti-S protein IgA, and inhibited infection by SARS-CoV-2 strains containing B.1.351 and B.1.1.28 spike proteins. Currently, infectious K18-hACE2 transgenic mice were completely protected from upper respiratory tract infection and lower respiratory tract infection after challenge with a SARS-CoV-2 virus expressing the B.1.351 spike protein.

result

Single ChAd-SARS-CoV-2-S immunization induces sustained anti-spike and neutralizing responses at various doses: The durability of humoral immune responses in BALB/c mice following IM or IN immunization with increasing doses of ChAd-SARS-CoV-2-S (10 ⁸ , 10 ⁹ , and 10 ¹⁰ virus particles [vp]) or 10 ¹⁰ vp of ChAd-control vaccine was assessed at days 100 or 200 ( Figure 10A ). First, anti-S and anti-RBD IgG and IgA levels were measured by ELISA. Consistent with previous results at 1 month, 30 days, 100 days, or 200 days post-vaccination, IN immunization with ChAd-SARS-CoV-2-S induced superior antibody responses than IM immunization or vaccination with ChAd-control ( Figures 10B-10M and Figure 11 ). Serum anti-S and anti-RBD specific binding IgG levels were higher after IN immunization than after IM immunization at 100 or 200 days. At day 100 after IN vaccination with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S, the geometric mean titers (GMTs) of S-specific IgG responses were 1.1 × 10 ⁶ , 4.8 × 10 ⁵ , and 2.6 × 10 ⁵ , and those of RBD-specific IgG were 3.2 × 10 ⁵ , 1.8 × 10 ⁵ , and 8.7 × 10 ⁴ ( Fig. 10B ). In comparison, S- and RBD-specific IgG responses were 4- to 6-fold lower at day 100 after IM immunization with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S (P < 0.0001), with S-specific IgG titers of 2.1 × 10 ⁵ , 1.1 × 10 ⁵ , and 4.5 × 10 ⁴ , and RBD-specific IgG titers of 5.1 × 10 ⁴ , 2.9 × 10 ⁴ , and 2.3 × 10 ⁴ ( Fig. 10E ). Similar dose-responses were observed in S- and RBD-specific IgG titers at day 200 after IN or IM immunization ( Figs. 10H and 10K ). At day 200 after IN vaccination with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S, the GMTs of S-specific IgG were 2.8 × 10 ⁶ , 2.4 × 10 ⁶ , and 1.2 × 10 ⁶ , and those of RBD-specific IgG were 1.1 × 10 ⁶ , 6.1 × 10 ⁵ , and 3.2 × 10 ⁵ ( Fig. 10H ). At day 200 after IM immunization with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S, the GMTs of S-specific IgG were 8.1 × 10 ⁵ , 6.9 × 10 ⁵ , and 2.6 × 10 ⁵ , and the GMTs of RBD-specific IgG were 1.4 × 10 ⁵ , 1.3 × 10 ⁵ , and 8.0 × 10 ⁴ ( Fig. 10K ). Thus, anti-S and anti-RBD IgG levels were higher after IN immunization than after IM immunization, and the increases in serum persisted even months after single-dose vaccination.

We next assessed the induction and persistence of serum IgA responses. IM immunization did not induce S- or RBD-specific IgA (Fig. 1F and L), but significant levels of anti-S- and RBD-IgA were detected at days 100 or 200 post-IN immunization (Fig. 1C and I). At day 100 post-IN immunization with 1010, 109, and 108 vp of ChAd-SARS-CoV-2-S, the GMTs of S-specific IgA were 4.8 × 10 ³ , 1.2 × 10 ³ , and 8.4 × 10 ² , and those of RBD-specific IgA were 2.2 × 10 ³ , 4.6 × 10 ² , and 2.9 × 10 ² (Fig. 1C). As seen for IgG, IgA levels continued to increase over time, with GMTs of S-specific IgA of 1.1 × 10 ⁴ , 7.4 × 10 ³ , and 5.4 × 10 ³ , and of RBD-specific IgA of 5.2 × 10 ³ , 3.8 × 10 3 , and 9.8 × 10 ² at day 200 after IN immunization with 1010, 109, and 108 vp of ChAd-SARS-CoV-2- ^S (Fig. 1I).

Next, the functional correlates of the serological responses were assessed by assaying neutralizing activity using the focus-reduction neutralization test (FRNT) ( Figures 10D , 10G , 10J , 10M , and 11 ). As expected, no neutralizing activity was detected in the sera of mice treated with the ChAd-control. The mean effective half-maximal inhibitory titers [EC50] at day 100 after IN immunization with 10 ¹⁰ , 10 ^{9 ,} and 10 ⁸ vp of ChAd-SARS-CoV-2-S were 39,449, 9,989, and 7,270, respectively ( Figure 10D ). In comparison, after IM immunization with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S, the EC50 values at these time points were 8- to 20-fold lower, at 4,988, 2,017, and 391, respectively (P < 0.0001) ( Fig. 10G ). Consistent with the higher anti-S and RBD titers observed at day 200 after IN immunization with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S, the EC50 values were 45,591, 22,769, and 23,433, respectively ( Fig. 10J ). In comparison, the EC50 values at day 200 after IM vaccination with 10 ¹⁰ , 10 ⁹ , and 10 ⁸ vp of ChAd-SARS-CoV-2-S were much lower, at 2,524, 940, and 716, respectively ( Figure 10M ).

Long-lived plasma cells (LLPCs) reside in the bone marrow and constitutively secrete high levels of antibodies that correlate with serum levels. To assess the levels of antigen-specific LLPCs at day 200 after IM or IN immunization with ChAd-SARS-CoV-2-S 1010 vp, CD138 ⁺ cells were isolated from the bone marrow and assessed for S-specific IgG or IgA production using ELISPOT analysis. A ∼4-fold higher frequency of LLPCs secreting S-specific IgG was observed after IN immunization than after IM immunization ( Figure 10N ). Additionally, a greater number of LLPCs producing S-specific IgA were detected after IN immunization than after IM immunization ( Figure 10N ). Taken together, these data demonstrate that: (a) single-dose IN immunization promotes superior humoral immunity than IM immunization; (b) A 100-fold lower dose of ChAd-SARS-CoV-2-S induces a robust neutralizing antibody response in mice; (c) IN immunization induces serum IgA responses and IgA-specific LLPCs against the SARS-CoV-2 S protein; and (d) humoral immunity induced by ChAd-SARS-CoV-2-S is persistent and increases for 6 months post-vaccination.

IN vaccination with ChAd-SARS-CoV-2-S induces broad antibody responses via Fc effector function capacity: To further characterize the humoral response, antibody binding and Fc effector function to SARS-CoV-2 variant proteins were analyzed at day 90 post IN or IM vaccination using sera derived from BALB/c mice. Our panel of SARS-CoV-2 proteins included Spike (D614G, E484K, N501Y, Δ69-70, K417N) and RBD (E484K) antigens corresponding to WA1/2020, B.1.1.7, B.1.351, and B.1.1.28 strains. First, we measured anti-SARS-CoV-2-specific antibody responses to multiple isotypes (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) and the ability to bind to Fcγ receptors (mouse FcγRIIB, FcγRIII, FcγRIV) using the Luminex platform. Consistent with the data obtained by ELISA ( Figures 10B and 10E ), IN vaccination with ChAd-SARS-CoV-2-S elicited higher IgG1 levels against the D614G spike and WA1/2020 RBD proteins than IM vaccination, and as expected, lower antibody titers were observed with decreasing vaccine dose ( Figure 12A ). Anti-SARS-CoV-2 IgG1 titers were higher after IN vaccination than after IM vaccination against all spike and RBD variants, and titers decreased with vaccine dose ( Figure 12B ). As shown in the heatmap, this trend was observed across all anti-SARS-CoV-2 specific antibody isotypes and correlated with FcγR binding patterns ( Figure 12C ). These data suggest that IN vaccination elicited larger and broader antibody subtype responses to SARS-CoV-2 than IM vaccination.

Antibody effector functions, i.e., opsonization, are mediated in part through Fcγ receptor binding. To determine whether the observed differences in antibody titers and FcγR binding titers were driven by differences in effector function, antibody-dependent neutrophil phagocytosis (ADNP) and cellular phagocytosis (ADCP) assays were performed ( Figures 12D-12E ). Serum from IN vaccinated mice stimulated ADNP substantially more than sera from IM vaccinated mice. However, minimal differences in ADCP were observed in antibodies derived following IN and IM vaccination ( Figures 12D-12E ). These data demonstrate that IN vaccination with ChAd-SARS-CoV-2-S induces larger and more functional antibody responses than IM vaccination.

Intranasally administered ChAd-SARS-CoV-2-S induces persistent protection against SARS-CoV-2 challenge in BALB/c mice: To evaluate the efficacy of the ChAd-SARS-CoV-2-S vaccine, immunized BALB/c mice receiving the dosing regimen described in Figure 10A were challenged with SARS-CoV-2. Viral challenge was preceded by introduction of Hu-Ad5-hACE2, which allows ectopic expression of hACE2 in BALB/c mice and productive infection with SARS-CoV-2 by historical SARS-CoV-2 strains. Animals received a single immunization via the IN or IM route with 10 ¹⁰ vp of ChAd-control or 10 ⁸ , 10 ⁹ , or 10 ¹⁰ vp of ChAd-SARS-CoV-2-S. At days 95 or 195 post-vaccination, mice were challenged with 10 ⁸ plaque-forming units (PFU) of Hu-Ad5-hACE2 and anti-Ifnar1 mAb; the latter dampens innate immunity and enhances virulence in this model. Five days later, BALB/c mice were challenged intranasally with 5 x 10 ⁴ focus-forming units (FFU) of SARS-CoV-2 (strain WA1/2020). At day 4 post-infection (dpi), lungs, spleen, and heart were collected from challenged mice at day 100 post-vaccination, and nasal saline and nasal washes from a second cohort challenged at day 200 post-vaccination. Tissues were assessed for viral load by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using primers against subgenomic RNA (N gene). IN immunization with the three doses induced a striking protective effect as evidenced by the near absence of viral RNA in the lungs, spleen and heart at day 100 post-vaccination compared to animals vaccinated with the ChAd-control vaccine ( Figures 13A-13C ). At day 200 post-immunization, the protective effect of IN-delivered ChAd-SARS-CoV-2-S remained robust in the upper and lower respiratory tract compared to mice immunized with the ChAd-control. Nevertheless, limited infection breakthrough was observed in the lungs and nasal passages of animals vaccinated with the lowest dose of 10 ⁸ vp of ChAd-SARS-CoV-2-S ( Figures 13G and 13I ). Comparatively, the protection at day 100 post-IM immunization was lower than that observed after IN immunization at the same challenge time point. Although no viral RNA was detected in the heart and spleen ( Figures 13E-13F ), at least 1,000- to 30,000- ^fold (P < 0.0001) higher levels were measured in the lungs of mice immunized with ChAd-SARS-CoV-2-S via the IM route compared to the IN route ( Figures 13A and 13D ). The greater effect of IM route administration was also observed, as the reduction in viral RNA load in the lungs was no longer different from mice vaccinated with ChAd-control at a dose of 10 8 vp ( Figure 13D ). At day 200 after IM immunization, lower protection against SARS-CoV-2 infection was observed in the lungs, nasal wash, and nasal saline compared to IN immunization ( Figures 13G-13L ).

ChAd-SARS-CoV-2-S induces persistent immunity in hACE2 transgenic mice: Next, we evaluated the immunogenicity of ChAd-SARS-CoV-2-S delivered intravitally in K18-hACE2 C57BL/6 mice, which are more susceptible to SARS-CoV-2 infection than BALB/c mice. Five-week-old K18-hACE2 mice were vaccinated IN with 10 ⁹ vp of ChAd-control or ChAd-SARS-CoV-2-S. Serum samples were collected 6 weeks later to assess the humoral immune response. ChAd-SARS-CoV-2-S vaccination induced high levels of S- and RBD-specific IgG and IgA, but not ChAd-control ( Figures 14A-14B ). Neutralizing antibody titers against WA1/2020 and two other SARS-CoV-2 strains harboring spike proteins of the B.1.351 and B.1.1.28 variants were measured by FRNT assay ( Figures 14C-14D and Figure 16 ). Neutralizing antibodies against WA1/2020 were induced at levels comparable to those observed after a single IN dose of ChAd-SARS-CoV-2-S (EC50 9,591). As seen in vaccine-induced human sera, decreased neutralizing antibody titers against Wash-B.1.351 (∼5-fold, P <0.0001; Figure 14C ) and Wash-B.1.1.28 (∼3-fold, P <0.0001; Figure 4D ) SARS-CoV-2 strains were observed compared to WA1/2020. To assess the durability of the humoral response, a separate cohort of K18-hACE2 mice were immunized via the IN route and serum samples were collected 9 months later. ChAd-SARS-CoV-2-S induced high levels of S- and RBD-specific IgG and IgA at this time point, as well as neutralizing antibodies against WA1/2020 (EC50 of 12,550) ( Figures 14E-14H and Figure 16 ). When tested against Wash-B.1.351 and Wash-B.1.1.28 viruses, a decrease in neutralizing titers was observed compared to WA1/2020 (∼6- to 8-fold, P <0.05; Figures 14G-14H ), but high levels were still maintained (EC50 of 1,627 and 1,918, respectively).

ChAd-SARS-CoV-2-S confers cross-protection against Wash B.1.351 and Wash-B.1.1.28 challenge in hACE2 transgenic mice: The protective efficacy of ChAd-SARS-CoV-2-S against WA1/2020 and two chimeric viruses (Wash-B.1.351 and Wash-B.1.1.28) carrying the spike gene corresponding to the variants of interest was tested ( Figure 15A ). Five-week-old K18-hACE2 mice were immunized IN with a single 10 ⁹ vp dose of ChAd-control or ChAd-SARS-CoV-2-S. Six weeks later, mice were challenged IN with 10 ⁴ FFU of Wash-B.1.351, Wash B.1.1.28, or WA1/2020. None of the mice immunized with ChAd-SARS-CoV-2 showed weight loss, whereas most mice vaccinated with ChAd-control experienced significant weight loss at 3–6 dpi ( Figures 15B , 15G , and 15L ). Surprisingly, at 6 dpi after ChAd-SARS-CoV-2-S vaccination, virtually no SARS-CoV-2 RNA was detected in the upper and lower respiratory tract, heart, and brain ( Figures 15C-15F , 15H-15K , and 15M-15O ). As a further test of the durability of the cross-protection response, 5-week-old K18-hACE2 mice were immunized IN with a single ^10–10 vp dose of ChAd-control or ChAd-SARS-CoV-2-S. Nine months later, mice were challenged with 10 ⁴ FFU of Wash-B.1.351 via the IN route. ChAd-SARS-CoV-2-S vaccinated mice maintained their body weight, unlike mice administered the ChAd control ( Fig. 15P ). Moreover, substantial virus protection was observed, as only very low amounts of Wash-B.1.351 SARS-CoV-2 RNA were detected in the upper and lower respiratory tracts, heart, and brain of some mice ( Figs. 15Q-15T ).

argument

The durability of vaccine-induced immune responses is critical to provide sustained protection against SARS-CoV-2 infection and to contain the current pandemic. In this study, a single IN immunization with ChAd-SARS-CoV-2-S induced S- and RBD-specific binding and neutralizing antibodies that persisted for several months, suggesting a sustained germinal center response. At 6 months post-IN vaccination, LLPCs were detected in the bone marrow and secreted SARS-CoV-2-specific IgG and IgA, which likely contributed to maintaining persistently high levels of antiviral antibodies in the circulation. In comparison, IM immunization with ChAd-SARS-CoV-2-S induced lower serum neutralizing antibody levels, reduced spike-specific IgG-secreting LLPCs, and virtually no serum or cellular IgA responses. At least in mice, a single IN dose immunization with ChAd-SARS-CoV-2-S was observed to produce persistent humoral immunity over the 100-dose range. These preclinical immunogenicity results compare favorably with human studies using mRNA vaccines against SARS-CoV-2, which have demonstrated humoral immune responses that persist for at least several months. In comparison, the persistence of antibody responses following natural infection with SARS-CoV-2 may be quite different.

A single immunization with ChAd-SARS-CoV-2-S provided sustained protection against SARS-CoV-2 (WA1/2020 strain) infection in hACE2-transgenic BALB/c mice or K18-hACE2 transgenic C57BL/6 mice at multiple time points over 6 months. In particular, IN immunization provided virtually complete virological protection against upper and lower respiratory tract infections, with only limited breakthrough infection observed at a 100-fold lower vaccine dose. The resolution of upper respiratory tract infections suggests that IN vaccination can prevent transmission. In comparison, IM immunization reduced viral RNA levels in the lungs but showed significantly lower protection against the homologous WA1/2020 strain in upper respiratory samples. Although many SARS-CoV-2 vaccine candidates from various platforms have demonstrated immunogenicity and protective efficacy in animal models, to our knowledge, none have demonstrated durability or protection against variant viruses. Long-term protection conferred by IN vaccination is promising even at 100-fold lower doses. If the results in mice are reproducible, dose sparing strategies could be used to produce large quantities of vaccine to suppress infection and transmission of SARS-CoV-2.

The emergence of SARS-CoV-2 S variants with amino acid mutations in the receptor binding motif ( e.g., B.1.351 and B.1.1.28) is of concern because they exhibit resistance to the inhibitory activity of many neutralizing antibodies. Indeed, human sera from subjects vaccinated with BNT162b2 mRNA or ChAdOx1 nCoV-19 (AZD1222) showed reduced neutralizing efficacy against B.1.351. Concernatively, ChAdOx1 nCoV-19 (AZD1222) administered IM showed reduced protection against mild to moderate B.1.351 infection in humans. In K18-hACE2 transgenic mice, when the immunogenicity of IN-delivered ChAd-SARS-CoV-2-S was compared to chimeric SARS-CoV-2 strains expressing WA1/2020 and B.1.1.28 or B.1.351 spike proteins, the neutralization rate of the variant viruses was reduced 3- to 8-fold, but titers were maintained at >1,000. At 6 weeks post-IN immunization with ChAd-SARS-CoV-2-S, K18-hACE2 mice were completely protected from weight loss and upper respiratory, lower respiratory, and brain infections by WA1/2020, Wash-B.1.351, and Wash-B.1.1.28. Of note, in a separate cohort of K18-hACE2 mice, animals were fully protected from challenge with Wash-B.1.351 at 9 months post-single IN immunization. Although the protective correlates have not been fully established for SARS-CoV-2 vaccines, high levels of cross-neutralizing antibodies against the variant virus, combined with robust virus-specific systemic and mucosal CD8 ⁺ T cell responses, likely contribute to protection. In addition, antibody effector functions may aid in the prevention of SARS-CoV-2 infection and disease. Indeed, enhanced Fc effector functions against SARS-CoV-2 variant proteins were observed in sera derived from IN-delivered ChAd-SARS-CoV-2-S, including the induction of robust ADNP and ADCP responses.

In summary, this study demonstrates that IN immunization with ChAd-SARS-CoV-2-S induced potent and sustained IgG and IgA antibody binding, neutralizing antibodies, Fc effector function, and LLPC responses to SARS-CoV-2. In mice, a single IN immunization with ChAd-SARS-CoV-2-S provided cross-protection against SARS-CoV-2 strains expressing spike proteins corresponding to the B.1.351 and B.1.1.28 variants, which was maintained at 9 months post-vaccination. Given the preclinical efficacy in various animal models30,31,32 and the sustained protective immunity against variants of concern, IN delivery of ChAd-SARS-CoV-2-S is a promising platform to prevent SARS-CoV-2 infection and suppress transmission.

method

Viruses and Cells: Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero-TMPRSS2 57, Vero (CCL-81, ATCC), and HEK293 (CRL-1573, ATCC) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1X non-essential amino acids, and 100 U/mL penicillin-streptomycin at 37°C. Vero-TMPRSS2 cells were also supplemented with 5 μg/mL blasticidin.

SARS-CoV-2 strain 2019n-CoV/USA_WA1/2020 (WA1/2020) was obtained from the Centers for Disease Control and Prevention. The virus was passaged once in Vero CCL-81 cells and titered by focus-forming assay (FFA) on ero E6 cells. Wash-B.1.351 and Wash-B.1.1.28 chimeric viruses with mutant spike genes have been described previously28,58. All viruses were passaged once in Vero-TMPRSS2 cells, and introduction and stability of substitutions were confirmed by next-generation sequencing. All virus experiments were performed in an approved biosafety level 3 (BSL-3) facility.

Mouse experiments: Animal studies were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Washington University School of Medicine (assignment number A3381-01). Viral inoculation was performed under anesthesia induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.

Female BALB/c (catalog 000651) and K18-hACE2 C57BL/6 (catalog 034860) mice were purchased from Jackson Laboratory. Four- to 5-week-old animals were immunized IM (hind leg) or IN with 1010 vp of ChAdV-control or ¹⁰⁸ , ¹⁰⁹ , or ¹⁰¹⁰ vp of ChAd-SARS-CoV-2-S in 50 μl of PBS. Vaccinated BALB/c mice (10- to 11-week-old) were given a single intraperitoneal injection of 2 mg of anti-Ifnar1 mAb (MAR1-5A3 59 (Leinco)) the day before IN challenge with 37108 PFU of Hu-Ad5-hACE2. At day 5 post-Hu-Ad5--hACE2 conversion, mice were challenged IN with 4 x 10 ⁵ FFU of WA1/2020 SARS-CoV-2. K18-hACE2 mice were challenged IN with 10 4 FFU of SARS-CoV-2 (WA1/2020, Wash-B.1.351, or Wash-B.1.1.28) on the indicated days post-immunization. Animals were euthanized at 6 dpi, and tissues were harvested for viral analysis.

Chimpanzee and human adenovirus vectors: ChAd-SARS-CoV-2 and ChAd-control vaccine vectors were derived from the simian Ad36 backbone 60 and constructed and validated as described herein. The rescued replication-deficient ChAd-SARS-CoV-2-S and ChAd-control vectors were expanded in HEK293 cells and purified by CsCl density-gradient ultracentrifugation. The viral particle concentration of each vector preparation was determined spectrophotometrically at 260 nm. The Hu-AdV5-hACE2 vector was also produced in HEK293 cells as described above. Viral titers were determined by plaque assay on HEK293 cells.

SARS-CoV-2 neutralization assay: Heat-inactivated serum samples were serially diluted and incubated with 10 ² FFU of various SARS-CoV-2 strains at 37°C for 1 h. The virus-serum mixtures were added to Vero cell monolayers in 96-well plates and incubated at 37°C for 1 h. The cells were then overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. The plates were incubated for 1 h at room temperature with 4% PFA in PBS for 30 h prior to fixation. Cells were washed and sequentially incubated with oligoclonal pools of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 62 anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12-349) in phosphate-buffered saline (PBS) supplemented with 0.1% saponin and 0.1% bovine serum albumin. Plates were developed prior to focusing on a BioSpot analyzer (Cellular Technology Limited) using TrueBlue peroxidase substrate (KPL).

Protein Expression and Purification: Cloning and production of purified S and RBD proteins corresponding to the WA1/2020 SARS-CoV-2 strain have been described previously. Briefly, pre-fusion stabilized S 64 and RBD were cloned into the pCAGGS mammalian expression vector containing a hexahistidine tag and transiently transfected into Expi293F cells. Proteins were purified by cobalt-charged resin chromatography (G-Biosciences).

ELISA: Purified antigen (S or RBD) was coated onto 96-well Maxisorp clear plates at a concentration of 2 μg/mL in 50 mM Na2CO3 pH 9.6 (70 μL) and stored overnight at 4°C. After aspirating the coating buffer, the wells were blocked overnight at 4°C with 200 μL of 1X PBS + 0.05% Tween-20 + 1% BSA + 0.02% NaN3 (blocking buffer, PBSTBA). Heat-inactivated serum samples were diluted in PBSTBA in separate 96-well polypropylene plates. The plates were then washed three times with 1X PBS + 0.05% Tween-20 (PBST) and 50 μL of each serum dilution was added. Serum was incubated in the blocked ELISA plates at room temperature for at least 1 h. The ELISA plates were washed three times with PBST, and 50 μL of 1:1,000 anti-mouse IgG-HRP (Southern Biotech Cat. #1030-05) in PBST or 1:1000 anti-mouse IgA-HRP in PBSTBA (SouthernBiotech) was added. The plates were incubated at room temperature for 1 h, washed three times with PBST, and 100 μL of 1-Step Ultra TMB-ELISA (ThermoFisher Cat. #34028) was added. After incubation for 10–12 min, the reaction was stopped with 50 μL of 2 M sulfuric acid. Optical density measurements (450 nm) were determined using a microplate reader (Bio-Rad).

ELISPOT assay: To quantify S-specific plasma cells in bone marrow, femurs and tibiae were mashed in RPMI 1640 using lysate and lysate, filtered through a 100 μm strainer, and used for ACK lysis. CD138 ⁺ cells were positively selected and enriched with magnetic beads according to the manufacturer’s instructions (EasySep Mouse CD138 Positive Selection, STEMCELL). Enriched CD138 ⁺ cells were incubated overnight in RPMI 1640 supplemented with 10% FBS on MultiScreen-HA filter plates (Millipore) pre-coated with SARS-CoV-2 S protein. Foci were developed using TruBlue substrate (KPL) following sequential incubation with anti-mouse IgG-biotin or anti-mouse IgA-biotin and streptavidin-HRP. Plates were imaged using the BioSpot instrument, and foci were counted manually.

Viral load measurement: Mice infected with SARS-CoV-2 were euthanized using a ketamine and xylazine cocktail, and organs were harvested. Tissues were weighed and homogenized with beads using a MagNA Lyser (Roche) in 1 ml Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal bovine serum (FBS). RNA was extracted from clarified tissue homogenates using the MagMax mirVana total RNA isolation kit (Thermo Scientific) and the KingFisher Flex extraction system (Thermo Scientific). SARS-CoV-2 RNA levels were measured using a stepwise quantitative reverse transcription-PCR (qRT-PCR) TaqMan assay as described previously (37). SARS-CoV-2 nucleocapsid (N)-specific primers and probe sets were used as described above. Viral RNA was expressed as gene copies per milligram (N) on a log10 scale.

Luminex analysis: Luminex analysis was performed as previously described. Briefly, proteins (spike: D614G, E484K, N501Δ69-70, K417N, B.1.1.7, B.1.351; receptor receptor binding domain (RBD) (ImmuneTech): WT, E484K, B.1.1.7, B.1.351, B.1.128) were carboxy-coupled to magnetic Luminex microplex carboxylated beads using an NHS-ester linker with Sulfo-NHS and EDC (Thermo Fisher), then incubated with sera (IgG1, FcγRIIb, FcγRIII 1:3000; IgG2a, G2b, G3, A, FcγRIV 1:1000, IgM 1:500) for 2 h at 37°C. Isotype analysis was performed by incubating immune complexes with secondary goat anti-mouse PE antibodies for each isotype (IgG1 1070-09, IgG2a 1080-09S, IgG2b 1090-09S, IgG3 1100-09, IgM 1020-09, IgA 1040-09 Southern Biotech). FcγR binding was quantified by incubating immune complexes with biotinylated FcγRs (FcγRIIB, FcγRIII, and FcγRIV, provided by Duke Protein Production Facility) conjugated to streptavidin-PE (Prozyme). Flow cytometry was performed using IQue (Intellicyt), and analysis was performed on IntelliCyt ForeCyt (v8.1).

Antibody-Dependent Neutrophil or Cell Phagocytosis: Antibody-dependent neutrophil phagocytosis (ADNP) and cell phagocytosis (ADCP) assays were performed as previously described. Briefly, spike protein was carboxyl-coupled to blue, yellow-green, or red FluoSphere™Carboxylate-modified microspheres 0.2 μm (ThermoFisher) using an NHS-ester linkage with Sulfo-NHS and EDC (Thermo Fisher). Spike-coated beads were incubated with diluted serum (1:150 ADNP, 1:100 ADCP) for 2 h at 37°C. For the ADNP assay, bone marrow cells were collected from BALB/c mice and erythrocytes underwent ACK lysis. Remaining cells were washed with PBS and seeded into 96-well plates (5 × ¹⁰⁴ cells per well). Bead-antibody complexes were added to the cells and incubated at 37°C for 1 h. After washing, cells were stained with the following antibodies: CD11b APC (BioLegend 101212), CD11c A700 (BioLegend 117320), Ly6G Pacific Blue (127628), Ly6C BV605 (BioLegend 128036), Fcblock (BD Bioscience 553142), and CD3 PE/Cy7 (BioLegend 100320). Cells were fixed with 4% PFA and processed on a BD LSRFortessa (BD Biosciences). Neutrophils were characterized as CD3 ^- , CD11b ⁺ , and Ly6G ⁺ . Neutrophil phagocytosis score was calculated as (% FITC+) x (geometric mean fluorescence intensity of FITC)/10000. For ADCP analysis, J774A.1 (ATCC TIB-67) mouse monocytes were incubated with spike-coated bead-antibody complexes at 37°C for 1 h. Cells were washed with 5 mM EDTA PBS, fixed with 4% PFA, and analyzed on a BD LSRFortessa (BD Biosciences). The phagocytosis score was calculated as (% FITC+) x (geometric mean fluorescence intensity of FITC)/10000.

Example 3 : Expression of SARS-CO-V2 Spike GENE (BA. 5) Variants from FROM CHAD Vector

Adenoviral vectors containing the S6P and S6PdF variants of SARS-CoV2-Omicron BA.5 (bivalent) were constructed and tested for expression as previously discussed for construction of chimpanzee adenoviral vectors. These constructs are referred to as ChAd.BA.5-S6P and ChAd.BA.5-S6PdF, respectively. Monolayers of human A549 cells were infected with 3 × 10 ³ vp/cell of freshly generated ChAd.BA.5-S6P (derived from Omicron variant BA.5 and containing S6P) and ChAd.BA.5-S6PdF vector or ChAd-SARS-CoV-2-S (WA1/2020-S2P) and ChAd.BA.1-S6PdF vectors, or ChAd vectors carrying no transgene. Expression of the SARS-CoV-2 Spike gene was detected 48 hours post-infection by flow cytometry using a cocktail of primary mAbs that bind to different epitopes on the Spike glycoprotein. As provided in Fig. 18 , infection with ChAd.BA.5-S6P and ChAd.BA.5-S6PdF vectors resulted in 90% and 92% of cells expressing the Spike gene, respectively, which are very similar to the expression levels achieved by ChAd-SARS-CoV-2-S(WA1/2020-S2P) (93%) and ChAd.BA.1-S6PdF (96%). Infection with the ChAd negative control resulted in 5% background expression, similar to control uninfected cells cultured with primary and secondary Alexa Fluor 594-conjugated F(ab')2 fragments (donkey anti-mouse IgG, 4.2%). These data suggest that both ChAd.BA.5-S6P and ChAd.BA.5-S6PdF vectors can induce efficient spike expression, resulting in robust immune responses comparable to those achieved by ChAd-SARS-CoV-2-S and ChAd.BA.1-S6PdF vaccine vectors.

Example 4 : Intranasal vaccination with CHAD-WUHAN-S and CHAD.BA.5-S or CHAD-BIVALT provides sustained protection against SARS-CoV-2 variants in mice

The efficacy of intranasal vaccines containing Ch-AD-Control, ChAD-Wuhand-S, ChAd-BA.5, and ChAD-2 was tested in vivo using the methods provided herein. Figure 23 provides a sequence alignment of the relevant S proteins. Figure 19A provides a schematic of the timing of vaccination, blood sampling, viral infection, and necrosis assays. In initial experiments, binding of anti-SARS-CoV-2 IgG ( Figures 19B, 19D, 19F ) or IgA ( Figures 19C , 19E, 19G ) to the SARS-CoV-2 Wuhan-1 ( Figures 19B-C ), BA.5 ( Figures 19D-E ), or BQ.1.1 ( Figures 21F-G ) S proteins was tested. When intranasally immunized with two-valent ChAd-CoV-2 S (Wuhan) and ChAd-CoV-2-S (BA.5), the IgA and IgG responses to the Wuhan-1, BA.5, and BQ.1.1 spike proteins were broader in ELISA tests using sera obtained at day 28 post-vaccination compared to the individual monovalent vaccine components.

Next, the neutralizing activity of intranasal immunization with Ch-AD-Control, ChAD-Wuhand-S, ChAd-BA.5 and ChAD-2 against WA1/2020 was tested using FRNT. Figure 20A shows the neutralizing activity against WA1/2020 by FRNT. Figure 20B shows the neutralizing activity against BA.5 by FRNT. Figure 20C shows the neutralizing activity against BF.7 by FRNT. Figure 20D shows the neutralizing activity against BQ.1.1 by FRNT. Figure 20E shows the neutralizing activity against XBB.1.1 by FRNT. Each point represents data from an individual mouse and is the average of two technical replicates. Figures 20F-H show neutralization data plotted as a direct comparison for a given vaccine against the indicated SARS-CoV-2 variants used in infection (ChAd-SARS-CoV-2 S (Wuhan-1, Figure 20F ), ChAd-SARS-CoV-2-S (BA.5, Figure 20G ) or bivalent (ChAd-SARS-CoV-2 S (Wuhan-1) + ChAd-SARS-CoV-2-S (BA.5), Figure 20H ).

According to the presented data, intranasal immunization with bivalent ChAd-CoV-2 S (Wuhan) and ChAd-CoV-2-S (BA.5) induced stronger serum neutralizing antibody responses against Wuhan-1, BA.5, and BQ.1.1 viruses than the respective monovalent vaccines.

Finally, we analyzed viral RNA levels in the lungs ( Figure 21A ), nasal turbinates ( Figure 21B ), and nasal lavage fluid ( Figure 21C ) at 6 dpi after challenge with SARS-CoV-2 WA1/2020 D614G (left) or Omicron BQ.1.1 (right). Intranasal vaccination with bivalent ChAd-CoV-2 S (Wuhan) and ChAd-CoV-2-S (BA.5) provided greater protection against lung infection by WA1/2020 D614G and BQ.1.1 viruses than the individual monovalent vaccines. This was consistent with enhanced protection against lung inflammation as measured by cytokine levels ( Figure 22 ).

Equivalent

While several inventive embodiments have been illustrated and described herein, those skilled in the art will readily conceive of various other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each such modification and/or variation is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary and that the actual parameters, dimensions, materials, and/or configurations may vary depending upon the particular application for which the invention is used. Those skilled in the art will recognize or be able to ascertain, through mere experimentation, many embodiments equivalent to the specific inventive embodiments described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of this disclosure relate to each individual feature, system, article, material, kit, and/or method described herein. Additionally, any combination of two or more of these features, systems, products, materials, kits and/or methods, provided that such features, systems, products, materials, kits and/or methods are not mutually inconsistent, is encompassed within the creative scope of this disclosure.

All references, patents and patent applications disclosed herein are incorporated by reference in their entirety with respect to the subject matter cited, and in some cases may encompass the entire document.

The phrase "and/or" as used in this specification and claims should be understood to mean "one or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed as "and/or" should be construed in the same manner, i.e., "one or more" of the elements conjoined. In addition to the elements specifically identified in the "and/or" clause, other elements may optionally be present, which may or may not be related to the elements specifically identified. Thus, as a non-limiting example, reference to "A and/or B" when used in conjunction with open-ended language such as "comprising" could, in one embodiment, refer to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in still another embodiment, to both A and B (optionally including other elements); etc.

As used herein and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be interpreted as inclusive, meaning that at least one element or list is included, but also optionally includes additional unlisted elements. Only the expressly opposite terms, such as "a single" or "exactly one," or, when used in the claims, "consisting of," indicate that exactly one element of multiple elements or a list of elements is included. In general, the term "or" as used herein should be interpreted to indicate exclusive alternatives (i.e., "either but not both") only when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." The phrase "consisting essentially of" when used in the claims has its ordinary meaning in the art of patent law.

The phrase "at least one," as used in this specification and claims, when referring to a list of one or more elements, should be understood to mean at least one element selected from one or more of the elements in the list of elements, but does not include at least one of each of the elements specifically listed in the list of elements, and does not exclude combinations of elements in the list of elements. By this definition, it is understood that in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, other elements may optionally be present, whether or not associated with the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and/or B") means, in one embodiment, at least one optionally including one or more A, without B (and, optionally, including elements other than B); in another embodiment, at least one optionally including one or more B, without A (and, optionally, including elements other than A); In yet another specific embodiment, at least one optionally including one or more A's, and at least one optionally including one or more B's (and optionally including other elements); etc.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. All references herein to "or" are intended to include "and/or" unless otherwise specified.

Attach electronic file of sequence list

Claims (35)

An adenoviral vector comprising the genome of a non-human adenovirus, wherein the genome of the adenovirus has been modified to lack the native E1 and optionally the E3 or E3B locus in the vector, and wherein the adenoviral vector comprises a nucleic acid sequence encoding the SARS-CoV-2 spike (S) protein having an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 10, 12, 20, 21, or an immunogenic portion thereof or a fragment thereof. An adenovirus vector according to claim 1, wherein the non-human adenovirus is simian adenovirus (SAdV). An adenoviral vector according to any one of claims 1 or 2, wherein the nucleic acid sequence encodes an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to one or more of SEQ ID NOs: 10-12, 20 or 21. An adenoviral vector according to claim 2, comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to any one of SEQ ID NOs: 13-19. An adenovirus vector comprising a nucleic acid sequence encoding an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity to any one of SEQ ID NOs: 10-12, 20, 21, or an immunogenic portion thereof, or a fragment thereof. An adenoviral vector comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to any one of SEQ ID NOs: 13-19. In an adenovirus vector comprising a nucleic acid sequence, wherein the nucleic acid sequence comprises at least 10, or at least 15, selected from the list consisting of T19I, L24S, del25-27, 69-70del, G142D, V213G, G339D, S371F, S373P, S375F, T376A, K417N, N440K, S477N, T478K, E484A, Q493R, L452R, F486V, Q498R, N501Y, D614G, H655Y, N679K, P681H, R682G, R683S, R685S, N764K, D796Y, A942P Q954H, N969K, K988P and V989P. An adenovirus vector encoding an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 3, or having at least 20, or at least 25, or at least 30 mutations. An adenovirus vector comprising at least two, or at least three, or at least four, or at least five, or six stabilizing mutations, as provided in claim 7 as F819P, A894P, A901P, A944P, K988P and V989P. An adenovirus vector according to any one of claims 5 to 8, wherein the adenovirus vector has a functional deletion in E1, and optionally a functional deletion in the E3 or E3B gene. An adenovirus vector according to any one of claims 5 to 9, wherein the adenovirus vector is a simian adenovirus vector. In claim 10, the adenovirus vector is a chimpanzee adenovirus vector 36. A pharmaceutical composition comprising an adenovirus vector according to any one of claims 1-11, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. An immunogenic composition comprising an adenovirus vector according to any one of claims 1-11, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. A pharmaceutical composition or immunogenic composition according to claim 12 or claim 10, further comprising one or more active ingredients. A pharmaceutical composition or immunogenic composition according to claim 12 or claim 13, wherein the composition is formulated for intranasal or intramuscular administration. A host cell transduced with an adenovirus vector or composition according to any one of the preceding claims. A packaging cell line producing a composition or viral vector according to any one of the preceding claims. A packaging cell line according to claim 17, wherein the cell comprises a complement of an adenovirus gene functionally deleted from the adenovirus vector of any one of the preceding claims. A packaging cell line according to claim 17, wherein the complement of the functionally deleted adenovirus genes comprises one or more of the E1, E3 or E3B genes. A kit comprising: (i) one or more of a host cell according to claim 16, a packaging cell line according to claim 18, an adenoviral vector according to any one of claims 1-11, a pharmaceutical composition according to claim 12 or an immunogenic composition according to claim 13, and (ii) instructions for use. A coronavirus vaccine comprising an adenovirus vector according to any one of claims 1-11. A composition comprising serum from a first subject who has already been administered an adenoviral vector according to any one of claims 1-11, a pharmaceutical composition according to claim 9, or an immunogenic composition according to claim 13. A method of treating a second subject having a coronavirus infection, comprising administering to the second subject an immunogenic effective amount of a composition comprising a serum according to claim 22. A method for inducing an immune response against a coronavirus in a subject in need thereof, comprising administering to the second subject an immunogenic effective amount of a composition comprising an adenovirus vector according to any one of claims 1-11, a pharmaceutical composition according to claim 12, or an immunogenic composition according to claim 13. A method for inducing treatment or prevention of a coronavirus infection in a subject in need thereof, comprising administering to said second subject an immunogenic effective amount of a composition comprising an adenovirus vector according to any one of claims 1-11, a pharmaceutical composition according to claim 9, an immunogenic composition according to claim 13, or a composition comprising serum according to claim 22. A method according to any one of claims 23-25, wherein the composition is administered nasally. A method according to any one of claims 23-25, wherein the composition is administered intramuscularly. A method according to any one of claims 23-27, further comprising the step of administering the composition one or more additional times. A method according to claim 24 or 25, wherein the coronavirus is a SARS-CoV-2 virus or a variant thereof. A method according to claim 29, wherein the coronavirus is an alpha, beta, gamma, delta or omicron variant or a subvariant thereof. A method according to claim 29, wherein the coronavirus is an Omicron strain or a subvariant thereof. A method according to claim 24 or 25, wherein the subject is a human. A method according to claim 32, wherein the subject is suspected of having a coronavirus infection or is at risk of developing an infection thereof. A method for producing an adenovirus, comprising the steps of: transfecting a cell with an adenovirus vector according to any one of claims 1-21; culturing the cell under conditions that allow the cell to produce the recombinant adenovirus; and collecting the recombinant adenovirus. A method according to claim 34, wherein the cell is a HEK, Vero or PER cell.