WO2019035972A1 - Sequential immunizations with hiv-1 env virus-like particles - Google Patents

Abstract

Disclosed are novel sequential immunization strategy and kits for performing the same.

Description

SEQUENTIAL IMMUNIZATIONS WITH HIV-1 ENV VIRUS-LIKE PARTICLES

This study was supported by grant number All 16361 awarded by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH. The government has certain rights in the invention.

This application claims the benefit of U.S. Provisional Application No. 62/546,350, filed on August 16, 2017, which is incorporated herein by reference in its entirety.

I. BACKGROUND

1. Almost 37 million people are currently living with human immunodeficiency virus type-1 (HIV-1), with another 2.1 million newly contracting the virus every year. A successful

HIV vaccine would have a massive influence in curtailing new infections, but a potentially licensable vaccine candidate remains elusive. Though some of the key milestones have been achieved in the development of an HIV-1 vaccine, including the RV144 trial which showed an unprecedented 31.2% reduction in HIV incidence, the prospects for a highly effective and affordable HIV-1 vaccine seem remote. Recently, a further endeavor, the HVTN 100 trial has been carried out to evaluate the adapted versions of the RV144 trial designed specifically for the population of South Africa. If several key immune response targets are met, it will set the stage for a far larger Phase III efficacy trial (HVTN 702) with the potential to lead to licensure.

2. HIV-1 evolves rapidly within the host, resulting in the accumulation of diverse HIV- 1 quasi-species. The envelope gene, the most variable in the genome, encodes a 160-kDa glycoprotein designated HIV-1 envelope (Env) that hides conserved CD4 and co-receptor binding sites with an evolving shield of glycans, variable immunodominant loops, and conformational masking. HIV-1 Env, embedded in the viral membrane, is the only target of neutralizing antibodies, thus presenting a moving target to the host immune system. What is needed are new vaccines or immunization strategies that can more effectively adapt to the rapid mutation and evolution of HIV-1.

II. SUMMARY

3. Disclosed are methods related to generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a virus.

4. In one aspect, disclosed herein are methods of generating broadly neutralizing antibodies against a virus and/or method of immunizing a subject against a viral infection the methods comprising sequentially administering to a subject one or more viral antigens from a virus or viruses for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.

5. Also disclosed are methods of any preceding aspect, wherein the virus is selected from the group of viruses consisting of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Zika virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type- 1 , and Human Immunodeficiency virus type-2.

6. In one aspect, disclosed herein are methods of any preceding aspect, wherein the viral anti en is a virus like particle (VLP).

7. Also disclosed herein are methods of any preceding aspect, wherein the antigen of the VLP is selected from the HIV antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat), regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif), for example, an HIV envelope (env) VLP such as a chimeric trimeric HIV env VLP.

8. In one aspect, disclosed herein are methods of any preceding aspect, wherein the order of administration of each chimeric trimeric HIV-1 envelope VLP from first to last VLP is HIV-1 subtype C, HIV-1 subtype B, HIV-1 subtype D, HIV-i subtype A, and HIV-1 subtype E.

9. Also disclosed are methods of any preceding aspect, wherein each antigen

administration occurs one month after the previous administration.

10. In one aspect, disclosed herein are viral administration strategies comprising the sequential administration of one or more viral antigens to a subject over two or more

administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.

11. Also disclosed herein are kits immunizing a subject against a virus comprising two or more clade variants each of one or more viral antigens. III. BRIEF DESCRIPTION OF THE DRAWINGS

12. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

13. Figures 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J and IK show the chimeric HIV Env gene construct and characterization of VLPs. (la) Schematic representation of HIV- 1 Env gene. The signal peptide-encoding sequences of HIV Env genes from subtypes A, B, C, D, and E were replaced by the honeybee melittin signal peptide sequence using overlapping PCR, to increase Env glycoprotein expression in SF9 insect cells. Conformation-stabilized trimeric Env proteins were made by the addition to C-terminus of a trimeric form of a leucine zipper sequence,

GCN4pii to stabilize Env trimers. HIV Env gene containing melittin and GCN4pii were cloned into the pFastBac-1 transfer vector; (lb) Surface expression of Env glycoprotein. The surface expression of Env glycoproteins on SF9 cells was determined by cell-based ELISA using polyclonal goat anti-HIV-1 Env followed by HRP-conjugated antibodies. The figure showed the data of three independent experiments and represented as mean ± SD; (lc) FACS analysis of glycoprotein-CD4 binding. The amount of bound CD4 was analyzed by FACS using APC-Cy7 labeled goat anti-human CD4 antibody. An unstained and antibody isotype control cells stained with APC-Cy7 labeled anti-human CD4 antibody were used as negative control groups. Results in figure (c) showed one of the representative experiment, (Id) showed the data of three independent experiments and represented as mean ± SD (n = 5); (le) SDS-PAGE analysis. Gag and Env glycoprotein profiles in VLPs were analyzed by SDS-PAGE in reducing conditions (in the presence of 1% BME). Unrelated (influenza) VLPs were used as a negative control group; (If) BS3 crosslinking assay. Prepared VLPs were crosslinked with BS3 at various concentration to confirm the oligomeric state of the HIV Envs as described in the materials and methods, (lg) Total Env content in VLPs. A quantitative ELISA was done to determine the Env glycoprotein content in VLPs, using recombinant HIV- 1 S 162 Env as a calibration standard. Unrelated (influenza) VLPs were used as negative control group. Results showed the data of three independent experiments and represented as mean ± SD; (lh) PGT145 monoclonal antibody binding assay. The assay was performed as mentioned in material and methods. Unrelated (influenza) VLPs were used as negative control group. Results showed the data of three independent experiments and represented as mean ± SD; (li) HIV-1 Env VLPs binding to glycan-dependent PGT126 and PGT128. Unrelated antibody (anti-histidine) and Gag VLP were used as negative control groups in the assay. Results showed the data of three independent experiments and represented as mean ± SD; (lj) TEM pictures of prepared VLPs; and (lk) Zeta potential of the representative VLPs.

14. Figure 2 demonstrates the results of cell-based ELISA to determine the endpoint titers against respective HIV Env (autologous) antigens as described in materials and methods. At the 3rd week of last vaccination, the IgG endpoint titers were measured against HIV-1 Env of subtype A, subtype B, subtype C, subtype D, or subtype E expressed on HE 293T cells. Cells infected with unrelated plasmid and pre-immune sera have been used as negative control group. Rabbits were immunized with PBS, Gag VLPs, single (subtype B) Env VLPs, a mixture of various Env VLPs, and sequential immunization of HIV Env VLPs through i.m. route of vaccination. The highest dilution which gave an OD450 two-fold higher than that of the naive group without dilution was designated as the antibody endpoint titer. Results showed the data of three independent experiments and results were expressed as the mean ± SD (n = 5).

15. Figure 3a and 3b show Con-S Env-specific antibody titers and IgGASCs. The figure represents antigen-specific (3a) serum IgG endpoint titers; and (3b) IgGASCs, at the 3rd week of last vaccination using the recombinant Con-S Env protein as coating antigen. Rabbits were immunized with PBS, Gag VLPs, single (subtype B) Env VLPs, a mixture of various Env VLPs, and sequential immunization of HIV Env VLPs through i.m. route of vaccination. The highest dilution which gave an OD450 two-fold higher than that of the naive group without dilution was designated as the antibody endpoint titer. Results showed the data of three independent experiments and results were expressed as the mean ± SD (n = 5). For ELISPOT, PBMCs from heparinized blood were collected and added to plates (1 ^χ 10⁶ cells/well). Spots were developed as mentioned in materials and methods using capture/ detection IgG antibody pair. Results were expressed as the mean ± SD (n = 5).

16. Figures 4A, 4B and 4C show antigen-specific serum IgG subtype levels. Figure represents Env-specific IgG subtype endpoint titers in serum samples (4a) IgG2a; (4b) IgG2b; and (4c) IgGl subtypes. Rabbits were immunized with PBS, Gag VLPs, single (HIV subtype B) Env VLPs, mixture of various Env VLPs, and sequential immunization of HIV Env VLPs through i.m. and in. routes of vaccination. Animals were primed at week 0; and further repetitive and sequential doses were given at every 4^th week. Blood and mucosal washes were collected at every 3 ^rd week post- vaccination and antibody titers were investigated using the recombinant Con-S Env gpl20 protein as coating antigen. The highest dilution which gave an OD₄₅o two-fold higher than that of the naive group without dilution, was designated as the antibody endpoint titer. Results showed the data of three independent experiments and results were expressed as the mean ± SD (n = 5). 17. Figures 5 A, 5B, 5C, and 5D represent the avidity indices of immune serum against various pseudoviruses. Antibody avidity assays were conducted with immune sera collected at the 3rd week of last vaccination from the animals immunized with (5a) Gag VLPs; (5b) single (subtype B) Env VLPs; (5c) a mixture of various HIV Env VLPs; and (5d) sequential immunization of HIV Env VLPs. The avidity index values were determined by measuring the resistance of antibody-envelope glycoprotein complexes in the ELISA by treatment with 1.5 M NaSCN. The avidity indices were calculated from the ratio of the absorbance value obtained with NaSCN treatment to that observed with PBS treatment multiplied by 100. Results were expressed as the mean ± SD (n = 5).

18. Figure 6 shows sera neutralization assay. Heat map of ID50 values obtained with the sera tested individually against a panel of 32 pseudoviruses from tier 1, 2, and 3 of various HIV subtypes. The figure shows the ID50 values in the animal groups vaccinated with PBS, Gag VLPs, single (subtype B) Env VLPs, a mixture of various VLPs, and sequential immunization of Env VLPs. Results were compared with the ID50 values of the sera collected before vaccination (pre-immune sera), Gag only (non-Env) VLP, and unrelated (influenza H7N9) pseudovirus. The reciprocal of sera dilution necessary to achieve 50% neutralization was reported as the ID50 value. All values were calculated with respect to virus-only wells with the following formula: [(value for virus only minus value for cells only) minus (value for serum minus value for cells only)] divided by (value for virus minus value for cells only). Results showed the data of three independent experiments and results were expressed as the mean±SD (n=5).

19. Figures 7A, 7B, 7C, 7D, 7E, and 7F show ADCC assay results. ADCC was assessed using a standard 51Cr release assay. Figure showed ADCC in serum samples collected from immunized animals with (7a) single (HIV subtype B) Env VLPs; (7b) mixture of various VLPs; (7c) sequential i.m. immunization; (7d) sequential i.n. immunization; while (7e) and (7f) showed ADCC in nasal and vaginal wash samples, respectively, of animals immunized using sequential i.n. immunization approach. Freshly isolated PBMCs from naive animals and 293 T-cells infected with Env-expressing plasmid of different subclades were used as effector and target cells, respectively at an E:T ratio of 10:1. After co-culturing of 51Cr labelled target and effector cells, 51Cr activity was quantitated using liquid scintillation counter. Each test was performed in triplicate. The results were expressed as the percentage of lysis, which was calculated as per the following equation, where experimental release represents the mean counts per minute (cpm) for the target cells in the presence of effector cells; spontaneous release represents the mean cpm for target cells incubated without effector cells; and maximal release represents the mean cpm for target cells incubated with 1% Triton X 100. Results were expressed as the mean ± SD (n = 5). 20. Figures 8A, -8B, 8C, and 8D show serum and mucosal antibody levels. Rabbits were immunized repetitively with PBS, Gag (non-Env) VLPs, single Env (clade B) VLPs, or sequentially with different HIV-1 Env VLPs. Sera and mucosal washes were collected at one- week pre- (preimmune samples) and 3-week after the last vaccination. Serum (IgG, IgGl, IgG2a, and IgG2b) and mucosal IgA (nasal and vaginal) levels were determined against Con-S Env (consensus) and various Env (autologous) antigens using indirect ELISA and cell-based ELISA, respectively. The HIV Con-S Env protein (200ng/well) and the transfected HEK293T cells (5 xlO⁴ cells/well) were used as coating antigens. HEK293T cells were transfected with various HIV-1 Env clones and negative control plasmids as mentioned in the methods. The transfected cells were seeded at a density of 5xl0⁴ cells/well and later fixed by 80% acetone prior to sample inoculation. After washing and blocking, serially diluted sera/mucosal washes were added, and the color was developed. The figure represents antigen-specific endpoint titers of (8 A) serum IgG; (8B) nasal IgA; (8C) vaginal IgA; and OD₄₅₀ values at 1 :100 fixed dilution of (8D) serum IgG subtype. The highest dilution which gave an OD₄₅o 2-fold higher than that of the naive group without dilution was designated as the antibody endpoint titer. Results show the data of three independent experiments and results were expressed as the mean ± SD.

21. Figures 9A, 9B, 9C, and 9D show antibody avidity indices and neutralization titers towards different pseudoviruses. Antibody avidity and neutralization assays were conducted with immune sera and mucosal lavages collected from the ammals immunized with various vaccine formulations. The avidity indices were determined by measuring the strength of antibody-envelope glycoprotein complexes in ELISA by treatment with 1.5M NaSCN. The avidity indices were calculated from the ratio of the absorbance value obtained with 1.5M NaSCN treatment to that observed with PBS treatment multiplied by 100. The figures 9A, 9B, and 9C represent the antibody avidities of the animals immunized sequentially with various HIV Env VLPs towards various pseudoviruses (tier 1, 2, and 3) in the (9 A) sera; (9B) nasal wash; and (9C) vaginal wash samples. Results showed the data of three independent experiments and results were expressed as the mean ± SD. The figure 9D shows the heat map of ID₅₀ obtained with the nasal wash tested against a panel of 32 pseudoviruses using TZM-bl neutralization assay in the animal groups vaccinated with various vaccine formulations. Results were compared with the ID₅o of the samples collected before vaccination (preimmune nasal wash) and unrelated (influenza H7N9) control pseudovirus. The reciprocal of sample dilution necessary to achieve 50% neutralization was reported as the ID₅₀. All values were calculated with respect to virus-only wells with the following formula: [(value for virus only minus value for cells only) minus (value for serum minus value for cells only)] divided by (value for virus minus value for cells only). Results showed the data of single representative experiment.

22. Figures 10A and 10B show CD4 and CD8 T cell proliferation. PBMCs were collected from the individual animals of each vaccination group after the last immunization and stimulated in vitro with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin

(500ng/ml), or media alone and cultured for 6 days. The figure shows the FACS analysis of (10A) CD3⁺CD4⁺ and (10B) CD3⁺CD8⁺ double positive T cell populations in various groups i.e. Gag VLPs, single Env VLPs, and sequential immunization group. The results were shown as one of the representative experiment.

23. Figures 11 A, 1 IB, 11C, 1 ID, 1 IE, and 1 IF show Thl/Th2 cytokine secretions and evaluation of cytokine-secreting cells. The levels of Thl/Th2 cytokines such as (11 A) IFN-γ; (1 IB) IL-2; (11C) TNF-a; and (1 ID) IL-6, were estimated from the cultured PBMCs' supernatant using quantitative ELISA. PBMCs were in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone. Cytokine

concentrations were calculated from their respective standard curves and data were presented as mean ± SD. The concentration of each cytokine was represented in pg/ml for each group of rabbits from three independent experiments. The figure 1 IE shows IFN-y-producing cell number in the in vitro stimulated PBMCs' culture. The ELISPOT assay was used for quantitating IFN-γ- producing cell spots as described in the methods. Figure 1 IF represents the results of antigen- specific IFN-γ producing CD4 and CD8 T cells, quantified by an ICS assay as mentioned in the methods. CD4/CD8 T cells were gated with IFN-γ expression at specific antigen concentration for measuring IFN-γ producing CD4 or CD8 T cell populations. Results were expressed as the mean ± SD of each group of animals from three independent experiments.

24. Figures 12A and 12B show flow cytometric detection of CD4 and CD8 T cell degranulation. The upper half of the figure (12 A) represents CD4⁺CD107a⁺ T cell population in the group of animals immumzed with Gag VLPs, single Env LPs, or sequentially immunized group of animals. The lower half of the figure (12B) shows CD8⁺CD107a⁺ T cell subset in the group of animals immunized with various vaccine formulations. Freshly isolated PBMCs from each group after the last vaccination were cultured and in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone and later stained with appropriate fluorescent antibodies as described in the methods. The results were expressed as a percentage of CD4⁺/CD8⁺CD107a⁺ cells and the results represented one of the

representative experiment. 25. Figures 13 A, 13B, and 13C show cytokine polyfunctionality and CD8 TCR avidity. The figure shows the cytokine polyfunctionality or cytokines' response profile of (13 A) CD4 and (13B) CD8 T cells from each vaccination group. For determining the polyfunctionality, CD4 or CD8 T cell populations were gated and single, double or triple cytokine expressing cell percentage enumerated. The percentage cytokine expression of each animal was averaged within the group and is expressed as a pie-chart of relative proportions of the total cytokine expressing cell population. The figure (13C) demonstrates the CD8 TCR avidities or the IFN-γ secretions by CD8 T cells in various vaccine formulations using ICS protocol as described in the methods. To estimate the effector CD8 TCR avidity, CD8 T cells were gated with IFN-γ expression and was measured as percent (%) maximum response at different antigen concentrations. All the results were expressed as the mean ± SD from three independent experiments.

IV. DETAILED DESCRIPTION

26. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

27. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

28. Ranges can be expressed herein as from "about" one particular value, and/or to

"about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

29. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

30. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

31. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

B. Methods of immunizing against HIV

32. Because HIV mutates frequently to evade host's immune responses, a vaccine which is able to stimulate B-cell populations to produce broadly neutralizing antibodies (bnAbs) that can target diverse mutant strains is vital. These bnAbs are formed through successive cycles of antibody mutation, selection and virus escape in some of HIV- 1 infected individuals. This process usually takes at least a couple of years, too long to offer any natural resistance to infection. New technologies have contributed to the identification of many bnAbs capable of potently inhibiting multiple HIV-1 isolates from different clades. Because endogenous neutralizing antibodies are so slow to arise and do not always have a sufficient repertoire of recognized epitopes to keep pace with viral mutations, these antibodies do not help infected individuals to control the virus. However, they can prevent infection in animal models and are now being tested in preclinical and clinical trials. The bnAbs can provide protection when they are in the host immune system prior to an exposure, such as being induced by a vaccination approach. Unfortunately, many attempts to generate bnAbs have uniformly failed. The methods disclosed herein succeed where others have failed. Accordingly, in one aspect, disclosed herein are methods of generating broadly neutralizing antibodies against a virus. It is understood and herein contemplated that the disclosed methods of generating broadly neutralizing antibodies against a virus can be used to immunize a subject against infection with a virus. Thus, in one aspect, also disclosed herein are methods of immunizing a subject against a viral infection.

33. The lack of neutralization typically observed by prior methods can arise from the use of monomeric proteins which present epitopes that are not exposed on the native viral spike or the vaccine regimen cannot successfully display some conserved, weakly-immunogenic, but critical, determinants of bnAbs to the host immune system. As disclosed herein, it was found that sequential immunizations over time can induce maturation of the effector and memory B cell populations to produce neutralizing Abs. Accordingly, in one aspect, disclosed herein are methods of generating broadly neutralizing antibodies against a virus and methods of

immunizing a subject against a virus comprising sequentially administering to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more viral antigens from one or more viruses against which neutralizing antibodies or immunological protection are sought.

34. In one aspect, it is contemplated herein that the route of administration of the VLP can affect the immune response generated. Routes such as intramuscular, intraperitoneal, intravenous, and subcutaneous, can have greater effect on systemic immunity, whereas mucosal routes of administration such as intranasal, oral, rectal, or vaginal can be used to elicit an more mucosal immune response. In one aspect, disclosed herein are methods of generating mucosal antibodies against a virus comprising sequentially administering to a subject one or more viral antigens from a virus for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round, and wherein each

administration occurs intranasally, orally, vaginally, and/or rectally.

35. As used herein "Antigen" means any native or foreign substance that is capable of eliciting an immune response. Preferably, the one or more antigen will elicit an antibody, plasma cell, plasmablast, or B-cell response (including, but not limited to memory B cells and activated B cells). Such antigens can include but are not limited to peptides, polypeptides, and/or proteins from a virus (i.e., viral antigens). Viral antigens can include any peptide, polypeptide, or protein from a virus and can be part of a live virus, a live attenuated virus, an inactivated or killed virus, a virus-like particle (VLP). In one embodiment the antigen can be an antigen from a virus selected from the group consisting of Herpes Simplex virus- 1 , Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Zika virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, - Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2. For example, the antigen can comprise HIV antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat), regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif). Accordingly, in one aspect, disclosed herein are methods of generating broadly neutralizing antibodies against a virus and or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from a virus against which immunological protection or broadly neutralizing antibodies is sought; wherein the virus is HIV (for example HIV-1). In one aspect, also disclosed herein are methods of generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from a virus against which immunological protection or broadly neutralizing antibodies is sought; wherein the virus is HIV (for example HIV-1); and wherein the antigen of the VLP is selected from the HIV (including HIV-1) antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat),regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif).

36. The disclosed antigens can be presented as part of a composition, such as a vaccine, In the context of this document the term "vaccine" refers to an agent, including but not limited to a peptide or modified peptide, a protein or modified protein, a live virus, a live attenuated virus, an inactivated or killed virus, a virus-like particle (VLP), or any combination thereof, that is used to stimulate the immune system of an animal or human in order to provide protection against e.g., an infectious agent. For example, the vaccine can comprise an HIV-1 env viruslike particle. Vaccines frequently act by stimulating the production of an antibody, an antibody- like molecule, or a cellular immune response in the subject(s) that receive such treatments. As used herein, vaccines can be administered in a pharmaceutically acceptable carrier either prophylactically or therapeutically.

37. It is evident that even the trimers assumed conformations that were different from the membrane-bound viral spike and therefore they too failed to elicit bnAb responses. Many animal research studies indicate that the HIV-1 Env proteins that more closely mimic the natural conformation can induce antibodies with improved neutralization capacity. Thus, the

administration of different variants of functional "native-like" Env that closely resemble the natural conformation of the viral spike represents the best opportunity in inducing bnAb responses. In one aspect, the VLPs can assume a more "native like" conformation of a viral antigen (such as, for example, HIV-1 env which natively forms a trimeric form). For example, wherein the conformation of the antigen is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or combination thereof such as a dimer of dimers, a trimer of dimers, a dimer of trimers or other combination, the VLP can assume said conformation including homologous or heterologous permutations. Thus, in one aspect, the VLP's can be constructed comprising trimeric HIV-env. Moreover, said trimeric HIV-env can be single (i.e, homologous) or chimeric (either heterologous including monomers in the timer form different clades or strains or monomers that comprise components from two or more clades, variants, or strains) constructs of HIV env. In one aspect, to stabilize the trimeric form of the env, a trimeric form of a leucine zipper sequence (such as, for example GCN4pii) can be used. In such instances the leucine zipper sequence can be operationally linked to C-termini of the HIV antigen (for example, a trimeric Env).

38. The most effective antibodies target only a few sites of HIV-1 Env, and this selective recognition has led to the development of vaccines that use epitope immunogens and stimulate immune responses to these highly conserved and functionally important domains. As the development of bnAbs usually requires extensive antigen exposure over a long-period, a vaccination strategy can start with an immunogen that presents a specific conserved epitope and then boosts the response with the same epitope on a different immunogen to achieve high- affinity recognition of the epitope in the context of the native viral spike is desirable. Also, clonal bnAb producing B-cell development in virus infected individuals co-evolves with the diversification of the virus and germline B-cell receptors are unable to bind native Env. Thus, sequential immunizations with several Env variants can shape the B-cell maturation and germinal centers (GCs) towards bnAb responses. In one aspect, the sequential immunizations can occur, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more times. Thus, disclosed herein are methods of generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from one or more viruses against which immunological protection or broadly neutralizing antibodies is sought sequentially over two or more administration rounds (for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more rounds.

39. There is now an intense focus on creating strategies capable of coaxing B-cells into generating bnAbs using sequential immunizations with HIV-1 antigens specifically designed for this purpose. Continuous exposure to the constantly mutating virus can stimulate multiple processes which eventually give rise to potent antibodies to neutralize a wide swath of HIV-1 variants. A sequential immunization regimen allows the antibodies to gradually evolve to improve their recognition to conserved components that are essential for viral function shared by diverse HIV-1 strains. Thus, the sequential immunization approach should be much more successful than exposing the immune cells to either all the antigen variants at once or one antigen at various time-points. Moreover, by providing exposure to clade variants over the course of the immunization regimen, the ability to cover escape variants and other mutations increases as does the breadth of the neutralization coverage. Accordingly, in one aspect, are methods of generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from a virus against which immunological protection or broadly neutralizing antibodies is sought over two or more administration rounds wherein each successive

administration round provides to the subject a clade variant of the one or more antigens that is different than any clade variant of any preceding administration round. For example, with a method of generating bnAb and/or immunizing a subject against HIV, each successive round can comprise one or more HIV antigens to a different HIV clade variant (for example, HIV subtype A (such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5), HIV subtype B (such as, for example, SF162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58), HIV subtype C (such as, for example, ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12), HIV subtype D (such as, for example, 92UG021.16, 92UG013.70, 93ZR001.3, 92UG021.9, 92UG024.2, and/or 92UG021.16), HIV subtype E (such as, for example, 93TH976.17 and/or 93TH966.8), HIV subtype F (such as, for example, 93BR019.4, 93BR019.10, 93BR020.17, and/or 93BR029.2), and/or HIV subtype G (such as, for example, 92RU131.16 and/or

92RU131.9)). For example, the method of generating bnAb and/or immunizing a subject against HIV, each successive round can comprise one or more HIV antigens to a different HIV clade variant, wherein the HIV clade variants comprise 92UG037.8 (HIV subtype A), SF162 (HIV subtype B), ZM53M.PB12 (HIV subtype C), 92UG021.16 (HIV subtype D), 93TH976.17 (HIV subtype E).

40. Herein it is shown that the sequential immunization of modified Env-enriched VLPs from various HIV-1 subclades generates bnAb responses with a high breadth and potency of neutralization in rabbits. In another aspect one or more subsequent rounds can provide to the subject a clade variant of the one or more antigens that is the same as at least one preceding administration round. In one aspect, it is understood and herein contemplated that the antigen from the subsequent administration round that is of the same clade variant than one or more preceding administration rounds can be the same or a different antigen than previously administered.

41. In one aspect, the order of the immunization can be based on any rationale deemed appropriate by the administering physician. For example, for HIV the order of clades administered can be A,R,C,D,E; C,B,D,A,E; B,A,C,D,E; C,A,B,D,E ; A,C,B,D,E; B,C,A,D,E;

B,E,C,D,A; or E,B,C,D,A. For example, for HIV envelope VLP, the order of clades for each round can be HIV subtype C, HIV subtype B, HIV subtype D, HIV subtype A, and then HIV subtype E. In one aspect, the immunization order can further comprise HIV subtype F and HIV subtype G. It is understood and herein contemplated that the order for one antigen clade variants that is administered concurrently with a second antigen does not have to be the same order.

42. In one aspect, it is understood and herein contemplated that where a particular antigen (for example, an HIV Env from a particular subtype (for example, Clade C) is used, the VLP vaccine being administered can comprise strain variants from multiple different strains of the clade subtype. For example, where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade A is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade A strains including, but not limited to 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade B is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade B strains including, but not limited to SF162, 6535.3, WIT0416.33,

RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade C is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade C strains including, but not limited to ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade D is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade D strains including, but not limited to 92UG021.16, 92UG013.70, 93ZR001.3, 92UG021.9, 92UG024.2, and/or 92UG021.16; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade E is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade E strains including, but not limited to

93TH976.17 and/or 93TH966.8; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade F is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade F strains including, but not limited to 93BR019.4, 93BR019.10, 93BR020.17, and/or 93BR029.2; and/or where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade G is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade G strains including, but not limited to 92RU131.16 and/or 92RU131.9. Thus, in one aspect disclosed herein are VLPs and methods of generating broadly neutralizing antibodies, methods of generating mucosal antibodies, and/or methods of immunizing a subject against a virus wherein the VLPs and/or VLPs being administered comprises strain variants of each viral antigen for one or more successive subtype immunizations.

43. Chimeric antigens are genes or proteins comprising regions, domains, and/or features from two or more different strain or subtype (i.e., clade) variants of the same genes or protein or a domains of different genes or proteins of the same or different virus, strain, or subtype. Thus, in one aspect, it is understood and herein contemplated that the viral antigens of the VLPs disclosed herein and used in the disclosed methods of generating broadly neutralizing

antibodies, methods of generating mucosal antibodies, and/or methods of immunizing a subject against a virus can be chimeric antigens. It is understood and herein contemplated that the disclosed chimeric antigens can comprise any combination of strains and/or subtypes of gpl20, gp41 or the variable or constant domains of gpl20 of any of the viral strains or subtypes (i.e., clades) disclosed herein. For example, the chimeric antigen can be a chimeric Env comprising at least one variable domain from a first HIV strain variant (such as, for example, HIV subtype A (such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5), HIV subtype B (such as, for example, SF162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58), HIV subtype C (such as, for example, ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12), HIV subtype D (such as, for example, 92UG021.16, 92UG013.70, 93ZR001.3, 92UG021.9, 92UG024.2, and/or 92UG021.16), HIV subtype E (such as, for example, 93TH976.17 and/or 93TH966.8), HIV subtype F (such as, for example, 93BR019.4, 93BR019.10, 93BR020.17, and/or 93BR029.2), and/or HIV subtype G (such as, for example, 92RU131.16 and/or

92RU131.9)) and a constant domain from a second HIV strain variant, but both the strains for the constant and variable domains being form the same subtype. Alternatively, the chimeric Env can comprise at least one variable domain from a first strain variant and a constant domain from a second strain variant wherein the strain variants for the constant and variable domains are from different subtypes.

44. In one aspect, it is understood and herein contemplated that multiple chimeric antigens can be used in a single immunization. Thus, where a VLP comprises a chimeric antigen in a dimeric, trimeric, tetrameric, pentameric, hexameric, or heptameric form, one or more of the monomeric components of the multimeric chimeric antigen can be derived from a different virus, strain, or subtype. For example, in one aspect the chimeric Env can be a trimeric Enc wherein one, two, or three of the monomer Envs forming the trimer comprises a gpl20 variable domain that is from a different strain of HIV, but the same subtype relative to the variable domains of the other Envs in the trimer, but the gpl20 constant domains are from the same strain of HIV. Alternatively, the chimeric Env can be a trimeric Enc wherein one, two, or three of the monomer Envs forming the trimer comprises a gpl20 variable domain that is from a different strain and subtype of HIV relative to the variable domains of the other Envs in the trimer, but the gpl20 constant domains are from the same strain of HIV.

45. In one aspect, it is understood and herein contemplated that the HIV Env gene, gpl60 protein product is proteolytically cleaved in the endoplasmic reticulum into two subunits, gpl20 and gp41. In one aspect, it is understood and herein contemplated that a multimeric chimeric antigen can comprise at least one, two, three, four, five, six, or seven gpl20 subunits from Envs of different strains of the same or different subtypes relative to the other gpl20 Envs in the multimer while the gp41 is from Envs of the same strain. For example, the chimeric Env can be a trimeric Env comprising at least one gpl20 from HIV a first HIV subtype (such as, for example ZM53M.PB12 of subtype C) and at least one gpl20 from a second HIV subtype (such as, for example SF162 or subtype B) and potentially at least one gpl20 from a third HIV subtype (such as, for example, 92UG021.16 of subtype D); wherein the gp41 subunits of each monomer are from the same HIV strain (such as, for example, ZM53M.PB12). In another aspect, one or more of the gpl20 Envs can be from different strains of the same subtype. For example, the chimeric Env can be a trimeric Env comprising at least one gpl20 from HIV a first HIV strain of an HIV subtype (such as, for example, subtype C) and at least one gpl20 from a second HIV strain of the same subtype (such as, for example, ZM197M.PB7 of subtype C) and potentially at least one gpl20 from a third HIV strain of the same subtype (such as, for example, DU172.17 of subtype C); wherein the gp41 subunits of each monomer are from the same HIV strain (such as, for example, ZM53M.PB12).

46. Successive rounds of administration can be accomplished at any rate sufficient to allow promote affinity maturation of B cells and antibodies and ultimately provide broadly neutralizing antibodies. Accordingly, in one aspect disclosed herein are methods of generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a viral infection comprising wherein each antigen administration (i.e., each successive round) occurs about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 weeks, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the preceding round . In one aspect, it is contemplated herein that the period between immunization rounds can be the same or different between rounds. For example, the time period between successive rounds can increase or decrease. In one aspect, the clade variants of the immunizing antigen (such as, for example, an HIV env VLP) can be administered every 4 weeks.

47. The disclosed immunization methods can be used prophylactically and

therapeutically. Accordingly, administration of the antigen (such as, for example, an HIV env VLP) can occur prior to exposure to the virus against which protection is sought. Nevertheless, where the immunization methods disclosed herein are being used in a therapeutic manner, the first VLP is administered after exposure to the virus against which protection is sought.

48. In one aspect, it is contemplated herein that the generation of broadly neutralizing antibodies against a viral antigen can be an active immunization of the subject in which protection against the virus is sought with the broadly neutralizing antibodies developing in the subject. However, also contemplated herein is the immunization of a subject with broadly neutralizing antibodies developed outside of the subject being immunized (i.e., a passive immunization). Thus, in one aspect, the methods of immunization can include generating broadly neutralizing antibodies against an antigen in a first subject such as a human, non-human primate, rodent (such as, for example, a mouse, rat, guinea pig, rabbit, hamster, or gerbil), canine, bovine, feline, or equine; obtaining antibodies from the first subject; and administering the broadly neutralizing antibodies from the first subject to a second subject desiring/needing protection against the virus form which the antigen is derived. In one aspect, the first subject can be from the same or different species than the second subject. In another aspect, the first subject can be a genetically modified animal that produces human or humanized antibodies.

49. The above methods of generating broadly neutralizing antibodies and methods of immunizing a subject against a viral infection utilize virus-like particles (VLPs) as a viral antigen against which the immune response is raised. It is understood and herein contemplated that the VLPs disclosed herein are unique and novel. In particular, disclosed herein are VLPs comprising an HIV (including HIV-1) antigens selected from the consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, egative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat),regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif). It is understood and herein contemplated tat the HIV specific antigen can be any Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif derived from any of the HIV clades or strains disclosed herein, including, but not limited to HIV subtype A (such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5), HIV subtype B (such as, for example, SF162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58), HIV subtype C (such as, for example, ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12), HIV subtype D (such as, for example, 92UG021.16, 92UG013.70, 93ZR001.3, 92UG021.9, 92UG024.2, and/or 92UG021.16), HIV subtype E (such as, for example, 93TH976.17 and/or 93TH966.8), HIV subtype F (such as, for example, 93BR019.4, 93BR019.10, 93BR020.17, and/or 93BR029.2), and/or HIV subtype G (such as, for example, 92RU131.16 and/or

92RU131.9). As noted above, in some aspects, the VLPs can assume a more "native like" conformation of a viral antigen (such as, for example, HIV-1 env which natively forms a trimeric form). For example, wherein the conformation of the antigen is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or combination thereof such as a dimer of dimers, a trimer of dimers, a dimer of trimers or other combination, the VLP can assume said

conformation including homologous or heterologous permutations. To assist in the formation of native conformations, the VLP can further comprise a dimeric, trimeric, tetrameric, pentameric, hexameric, or heptameric form of a leucine zipper sequence such as, for example GCN4pii, to the C-termini of an HIV protein (for example, Env) cytoplasmic sequences to produce conformation-stabilized multimeric (such as, for example trimeric Env) proteins. Thus, in one aspect, disclosed herein are VLP constructs comprising HIV Env gene and GCN4pii, wherein the GCN4pii is functionally linked to the C-termini of Env. The VLP can also be further modified to increase expression in a particular cell-type such as, for example, removing the native signal peptide encoding sequences of the antigen and replacing said sequence with signal peptide sequence that will be more readily expressed in the infected cell (for example, honeybee melittin signal peptide for expression in SF9 cells). Thus, in one aspect, disclosed herein are

VLP constructs comprising the signal peptide sequence of honeybee melittin, the HIV env gene, and GCN4pii, , wherein the GCN4pii is functionally linked to the C-termini of Env, for example, as depicted in Figure 1 A.

1. Pharmaceutical carriers/Delivery of pharmaceutical products

50. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

51. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.

Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

52. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

53. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, .D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al, Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.

Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research; 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

54. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

55. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

56. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

57. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiindElarnmatory agents, anesthetics, and the like.

58. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

59. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

60. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

61. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

62. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryi amines and substituted ethanolamines.

b) Therapeutic Uses

63. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

64. It is understood and herein contemplated that disclosed methods of generating a broadly neutralizing antibody or immunizing a subject against a virus are based upon an immunization regimen that, is designed to generate broadly neutralizing antibodies.

Accordingly, in one aspect, disclosed herein are viral immunization strategies for generating broadly neutralizing antibodies and/or developing protective immunity against a virus in a subject comprising the sequential administration of one or more viral antigens to a subject over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding

administration round.

2. Kits

65. Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include one or more antigens and the clade variants for each antigen. In one aspect, disclosed herein arc kits for generating broadly neutralizing antibodies against HIV, mucosal antibodies against HIV, and/or immunizing a subject against HIV comprising VLPs of two or more clade variants each of one or more HIV antigens (for example, HIV-1 Env VLP for HIV-1 subtype C, HIV-1 subtype B, HIV-1 subtype D, HIV-1 subtype A, HIV-1 subtype E, HIV-1 Env VLP for HIV subtype F, and/or HIV-1 Env VLP for HIV subtype G) and/or chimeric HIV antigens. In one aspect, the kit can further comprise instructions to administer clade variants of the one or more viral antigens sequentially. Thus, for example, the kit can comprise HIV-1 Env VLP for HIV-1 subtype A, HIV-1 subtype B, HIV-1 subtype C, HIV-1 subtype D, HIV-1 subtype E, and instructions to administer the VLP to a subject in the order of subtype C, subtype B, subtype D, subtype A, and subtype E.

C. Examples

66. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1: Sequential immunizations with a panel of HIV-1 Env viruslike particles coach immune system to make broadly neutralizing antibodies a) Results:

(1) HIV-1 Env incorporated VLPs showed high levels of Env content and retained their physical and functional properties:

Modified Env gene constructs were generated by replacing the original signal peptide encoding sequences with the honeybee melittin sequence and adding a trimeric form of leucine zipper sequence, GCN4pii to the C-termini of Env cytoplasmic sequences to increase Env glycoprotein production and to express conformation-stabilized trimeric Env proteins, respectively (Fig. la). In the current study, we have used Bacto-Bac protein expression system in Spodoptera frugiperda (SF9) insect cells for the expression of various Env glycoproteins. An insect cell system is easy to handle, requires less maintenance, and economic without many of the potential bio-hazards associated with mammalian system. Insect cells have been used since the early years of HIV-1 vaccines and because these cells carry out many post-translational modifications including high- mannose type N- and O-linked glycosylations, resulting in glycoproteins with similar antigenicity and functionality as of mammalian system. Cell-based ELISA results demonstrated that Env glycoproteins were expressed at the surfaces of recombinant baculovirus (rBV)-infected SF9 insect cells, as indicated by the enhanced OD450 value when compared to the control cells infected with unrelated (influenza) rBVs (Fig. lb).

We further tested the CD4 binding ability of expressed Envs on the SF9 cells using FACS. An increase in the mean fluorescent intensities (MFIs) was found with cells infected with rBVs expressing specific Env versus unrelated (influenza) rBV infected control cells (Fig. lc). The MFIs were 20-40% higher with cells infected by rBVs expressing various Envs than with control cells. We observed comparable CD4 binding efficiency with Env of different subtypes (Fig. Id). The VLPs containing these Env and Gag were produced by co-infection of SF9 cells with rBVs expressing different Env (subtype A-E) and Gag at an optimal multiplicity of infection (MOI). The protein composition of VLPs was characterized by SDS-PAGE in the reducing conditions.

Characteristic bands with molecular weights of 125-130 KD and 55-60 D were detected for HIV-1 Env and Gag proteins, respectively. Comparable levels of Env and Gag proteins in three independent VLP preparations were demonstrated (Fig. le). Bis [sulfosuccinimidyl] suberate (BS3) cross-linking analysis showed that HIV Env VLPs primarily express the trimeric Env form (Fig. If). Quantitative ELIS A. results showed that the prepared VLPs contained a high level of Env (10-15 μg) per 100 μg of VLPs (Fig. lg), which was comparable to previously observed incorporation levels. In the PGT145 monoclonal antibody binding assay, prepared HIV-1 Env VLPs of various subtypes showed higher binding towards PGT145 antibody than the unrelated (influenza) VLP group. These results showed that Env in VLPs retained their conserved

conformational structure (Fig. lh). Insect cells and mammalian cells produce proteins with different glycosylation patterns. We analyzed the binding of PGT126 and PGT128 to HIV-1 Env VLPs produced in insect cells to assess if the Env retained the antigenicity as Env produced in a mammalian system. PGT126 and PGT128 antibodies bind specifically to the V3 glycan of HIV-1 gpl20. The binding results demonstrate that the insect cell prepared VLPs retain the Env antigenicity for these antibodies, which is implying the capability of the Env to induce antibody responses, specific to these glycan epitopes (Fig. li). Transmission electron micrograph (TEM) and Zetasizer data showed that the prepared VLPs were pure, spherical in morphology, and 180-200 nm in diameter (Fig. lj and k). The data demonstrated that these stabilized Envs are incorporated into

VLPs with their existing physical and functional properties. In the current study, we made five different HIV Env VLPs from each of the HIV-1 clades A, B, C, D, or E for sequential immunizations.

(2) Sequentially i.m. immunized animals showed higher serum reactivity towards the respective HIV-1 subtype Env

(autologous) antigens.

67. An animal group was sequentially immunized with different HIV-1 Env VLPs through an intramuscular (i.m.) route at four-week intervals, and the results were compared with that of groups of animals immunized by repetitive immunizations of a mixture of various VLPs or a single type (subtype B) of Env VLPs. To provide additional insight into factors that might improve immunogenicity, we assessed serum reactivity towards the various HIV-1 subtype Envs after the last vaccination using cell-based ELISA. Results in Fig. 2 show IgG endpoint titers against Env antigens (autologous) from various HIV-1 sub- types, expressed on transfected cells. HKE293T cells transfected with an unrelated plasmid (control cells) and pre-immune sera were used as negative controls for the assay. In the sequential-immunization group, we observed that subtype-specific IgG endpoint titers were at its maximum. The sequential immunization group developed IgG with endpoint titers an order of magnitude greater (p < 0.001) than the mixture of various VLPs and other control groups. Simultaneously, single Env VLPs group demonstrated significantly (p < 0.01) higher sera reactivity towards subtype B Env antigen. On the other hand, the mixture of various VLPs group showed moderate levels of antibody levels towards each of the HIV-1 Env antigens than other negative control groups.

(3) Animals sequentially i.m. vaccinated with HIV-1 Env VLPs showed an increase in serum antibody levels towards the Con- S Env antigen.

68. As the recent discovery of potent bnAbs from HIV infected individuals has galvanized interest in protective or therapeutic interventions23, it is important to acknowledge the role of antibodies specific to conserved epitopes in Env24-26. At the 3rd week after the last vaccination, we determined serum IgG endpoint titers against HIV-1 Con-S Env protein by ELISA as described previously27,28. As shown in Fig. 3a, sequential immunizations with various VLPs induced significantly (p < 0.01) higher endpoint titers than those induced by other control treatments, when HIV-1 Con-S Env protein was used as the coating antigen.

(4) Sequential i.m. immunizations enhanced Con-S-speciflc IgG antibody-secreting cells (ASCs).

69. During the 3rd week of the last vaccination, we evaluated IgGASCs specific to HIV-

1 Con-S Env antigen using freslily pre- pared PBMCs from vaccinated animals by ELISPOT. We observed that the results were well correlated with antibody responses generated. ELISPOT data showed that antigen-specific IgGASCs were significantly (p < 0.01) higher in sequentially immunized animals than that of other animal groups. We observed 40-50 immunospots in the sequentially immunized group while the other groups developed only 20-25 spots. The number of detected spots was significantly (p < 0.01) higher (two-fold) in the sequential immunization groups than the mixture of various VLPs and single Env VLPs groups (Fig. 3b). (5) Mixture and sequential in. immunized animals promoted

Thl and Th2 type of immunity, respectively:

70. An antibody response can result in changes in the distribution of IgG subclasses, and depend on the nature of the antigen, frequency and duration of the antigenic stimulation, and routes of immunization. Like serum IgG, single Env VLPs or physical mixture of various VLPs dramatically increased antigen-specific IgG subclasses when compared to other control groups. Rabbits vaccinated with single Env VLPs or mixture of various VLPs showed much higher IgG2a (Fig. 4a) and IgG2b (Fig. 4b) levels when compared to IgGl responses, indicating that these groups induce Thl -biased antibody responses. Also, a significant (p<0.01) increase was observed in IgG2a/2b levels with animals immunized sequentially through i.m. route. Sequential i.n. immunized animals showed a remarkable increase in IgGl levels when compared with other vaccine formulations (Fig. 4c). Thus, single Env VLPs, mixture of VLPs or sequential i.m. immunized groups showed IgG2-dominant immune responses, defining Th2 type of immunity while sequential i.n. immunized group enhanced IgGl subclass production, demonstrating a Thl antibody profile.

(6) Sequentially i.m. immunized animals showed antibodies with high avidity.

71. Several recent studies have shown a direct correlation between the avidity of neutralizing antibodies and HIV protective efficacy29,30. The results shown in Fig. 5

demonstrate that serum antibodies in the sequentially immunized group exhibited increased avidity compared to other immunization groups to the majority of pseudoviruses tested. Gag only VLP-immunized animals did not display an increase in avidity towards any of these pseudoviruses (Fig. 5a). Rabbits vaccinated with subtype B VLPs showed higher avidity towards the subtype B pseudoviruses especially B3, B5, and B6 (Fig. 5b). Intermediate enhancement of avidity indices to some of the strains, especially Al, Bl, B5, and C6, were observed in the mixture of various VLP-immunized group (Fig. 5c). Serum antibodies from the sequentially immunized group showed increased avidity indices towards Al, A3, A5, A6, Bl, B4, CI, C4, Dl, and D3 HIV-1 pseudostrains (Fig. 5d). These results demonstrate that sequential- immunizations induced antibody responses with higher avidity indices to most of the

pseudotyped virions tested including some of the tier 3 pseudostrains and might also indicate that sequential exposure of different Env sharing conserved epitopes to the immune system is important in triggering bnAb responses. (7) Sequential i.m. immunizations with various HTV-1 Env

VLPs enhanced bnAb responses.

72. Next, we evaluated neutralizing activity of the resulting immune sera against a panel of 32 HIV-1 pseudoviruses. In Fig. 6, the heat map shows the 50% inhibitory dilution (ID50), the reciprocal of sera dilution necessary to achieve 50% neutralization, from the various vaccine groups. Sera from the Gag VLP-immunized group did not show detecta- ble ID50 (<10) against any of the pseudoviruses (data not shown). In the single Env VLP group, we observed ID50 of 150-200 especially against Bl, B4, and B6. In the mixture of various VLPs immunization group, we observed ID50 in a range of 200^00 against Bl, B3, B5, C5, C6, and D2

pseudoviruses, which represents a 22% (7 out of 32 pseudoviruses) neutralization potency. Low levels of ID50 (<20) against subtype E and F pseudoviruses were found while no detectable ID50 was observed against subtype G pseudoviruses. In sequentially immunized animals, we found significantly (p < 0.001) higher and broader ID50 values. Immune sera showed a significantly (p < 0.001) higher ID50 levels against all the pseudoviruses belonging to the subtypes A, B, and C (except A2, A4, B2, C5, and C6 pseudoviruses), especially Al, A3, A5, A6, Bl, B4, B5, B6, and CI. Results showed that immune sera of the sequential immunization group neutralized -70% of the pseudoviruses (23 out of 32 pseudoviruses). Interestingly, we have observed that the sequentially immunized group has shown greater ID50 values against some of the tier 3 HIV pseudostrains also such as A5, A6, and B6. Animals in this group showed ID50 in the range of 100-150 against subtype F and G pseudoviruses also, even though the vaccine formulation did not contain sub- type F and G HIV-1 Env antigens. The negative control groups including pre-immune sera, non-Env (Gag) VLPs, and unrelated (influenza H7N9) pseudovirus showed no background neutralization. These data demonstrate the potential of sequential immunizations with various Env VLPs in inducing antibody responses with broad . neutral- izing activity when compared to the pre-immune sera or non-Env (Gag only VLPs) groups.

(8) Induction of antibodies with high ADCC (antibody dependent cellular cytotoxicity) activity was found in sequential in. immunized groups:

73. Most anti-HIV antibodies generated by infected individuals do not display potent neutralizing activities. These non-neutralizing antibodies (nnAbs) with ADCC have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. Considering the significance of nnAbs with ADCC, serum and mucosal samples were analyzed for their ADCC activity using 51Cr release assay. The mixture of various VLPs group (Fig. 7b) showed serum antibody responses with significantly (p<0.05) higher ADCC than single Env VLP (Fig. 7a) or other control groups while the sequential i.m. immunized animals showed very low serum ADCC activity (Fig. 7c). Animals immunized sequentially through i.n. route produced antibodies in nasal cavity with significantly (p<0.01) higher ADCC than other antibodies in the systemic circulations (Fig. 7d and 7e) to a number of pseudoviruses. Notably, high ADCC were observed in these animals at vaginal sites also (Fig. 7f). The results indicate that sequential i.n. immunizations with different Env VLPs elicited high ADCC mucosal antibodies. b) Discussion:

74. Despite many efforts since HIV-1 first identified in 1983, and even after encouraging results from the RV144 trial, HIV-1 continues to infect almost one million individuals each year, a potentially licensable vaccine candidate remains a decade away. Major challenges for an HIV preventing vaccine that can elicit protective bnAb responses are the genetic diversity, mutability of HIV-1 target epitopes and structural properties of a viral Env that hides conserved CD4 and co-receptor binding sites by modulating signature glycan motifs. These challenges were overcome by the design of novel Env immunogens that resemble the natural viral Env spikes and can trigger the selection and expansion of germline precursor and intermediate memory B- cells to recapitulate B-cell ontogenies associated with the generation of a bnAb response.

Equally important for vaccine development is the identification of innovative vaccination strategies that can mimic the natural process of infection to drive somatic hypermutation and B- cell maturation against heterologous primary virus envelopes.

75. In the past few years, major advancement in the understanding of how bnAbs evolve has illuminated the mechanics of decades of past vaccine failures and has created the

opportunity to start anew by engineering a better Env immunogen. Prime-boost strategies delivering HIV Env as plasmid DNA, replication incompetent viral vectors, adjuvanted recombinant proteins, and stabilized soluble protein trimers have all failed to induce efficient bnAb responses. A practical vaccine, can elicit antibodies recognizing conserved epitopes of Env protein. As these antibodies act by recognizing native Env trimers on the HIV-1 surface, immunogens intended to induce bnAbs can mimic the native structure as closely as possible. Thus, it is fair to display Env in situ, such as VLPs, in the optimal approach to induce bnAb responses. On the other hand, a vaccination regimen, recapitulate the dynamic process of antigenic changes of an HIV infection to induce bnAbs is needed. 76. As shown herein, bnAb responses to HIV Env-enriched VLPs were evaluated in rabbits when immunized through a sequential immunization pattern. Sequential i.m.

immunization induced enhanced and broad sera neutralizing activity even though mean antibody endpoint titers were lower when compared to repetitive immunizations of single Env VLP, or mixture of various VLPs. Antibodies induced by mixture of various VLPs were different and reactive to only some of the variants. However, in animals vaccinated sequentially, antibodies generated were focused on the target patch of conserved elements, were able to respond to most of the protein variants, and showed broader neutralization. With the single Env VLP-vaccinated group, there was none of the antigenic diversity for the induction of bnAb responses. Many longitudinal studies of HIV-infected patients and SIV-infected macaques have demonstrated that the immune system gradually recognizes viral variants that emerge over time. During the course of an infection, antibodies directed to Env undergo immunological maturation, increasing in avidity, conformation dependence, and neutralizing capacity. Env VLPs were used from various HIV-1 subclades, using this diversity to generate bnAb responses both by presenting new epitopes as escape variants and by fostering the response against more conserved epitopes. The findings indicate that sequential administrations of several Env VLPs can stimulate a stronger bnAb response than repetitive deliveries of a cocktail of these VLPs or single Env VLPs. A properly designed sequential vaccination scheme with different variants of Envs offers hope to manipulate antibody development which can more efficiently produce bnAbs. As the

development of bnAbs requires extensive antigen exposure over a long period of time, sequential immunizations included booster immunizations with one or more Env variants can shape the B-cell immunity toward bnAbs responses. The clonal bnAb producing B-cell development in HIV-infected individuals co-evolves with the diversification of the virus. Thus, this new analysis of optimal immunogen designs and the successful sequential vaccination scheme provides some important novel insights into how immune responses to antigens develop and clues for creating a vaccine in the future. Low endpoint titers of such antibodies, however, emphasizes the need for more research elaborating vaccination strategies via appropriate routes with suitable or advanced adjuvants, clever depot/release formulations, optimized immunogen cocktails and prime/boost regimens.

77. Additionally, sequential i.m. immunized animals also showed an increase in antibody avidity. Antibody avidity has been used as a measure of functional maturation of the humoral immune response and represents the combined binding affinities of a variety of antibodies and their multivalent antigen. Class switching, affinity maturation, and somatic hypermutation that occur during B-cell maturation generate high-affinity antibodies of different subclasses. Simultaneously, as most antigens have a diversity of antigenic determinants per protein molecule, an increased avidity can be a consequence of progressive appearance and accumulation of classes of antibodies, each specific for a distinctly different antigenic determinant.

78. In the finding, HIV-1 Env VLPs administered through sequential i.n. immunization enhanced antigen-specific cellular immunity. Interestingly, sequential i.n. immunized rabbits showed sustainable and significant increases in lymphocytes proliferation and CD8⁺ T-cell cytotoxicity. During cytokine profile evaluation, some high levels of IFN-γ, IL-2, and IL-6 in the culture supernatant showed a Thl type of immune response. Preliminary evidence and different non-human primate studies indicate that a vigorous T-cell response, including IFN-γ production, is an immune correlation of protection from HIV-1/SIV infection. The sequential i.n.

immunization can involve activating macrophages and lymphocytes, for the enhancement of antigen-specific T-cell functions. Collaboratively, sequential i.n. immunization preferentially augmented Thl -cell response resulting in high levels of IFN-γ and IL-2. Another possible explanation is that mucosally administered Env VLPs can be presented to T-cells and stimulate the transcription of cytokine genes in activated T-cells or the enhancing cytokine levels can be promoted by local innate inflammatory and systemic adaptive immune responses.

79. The role of different routes of immunization cannot be denied for the difference in the elicited predominant IgG subclasses. In the current study, a considerable difference in IgG subclass profiles were observed between two routes of immunizations. While i.m. inoculation elicited more IgG2a/2b than IgGl antibodies, i.n. immunizations resulted in the reverse scenario. Thus, data demonstrate that IgGl and IgG2a/2b subclass ratios for an antigen are still influenced by the route or method of antigen delivery. Also, cellular immunity results were comparable with the IgG subtype patterns. Two major explanations were considered for the preferential helper T-cell effect on IgG2 production. In normal animals, T-cells can exist that can interact with IgG2a positive B-cells, perhaps by recognizing B-cell surface IgG2a. Alternatively, T-cells can induce greater Ig heavy chain gene switching in virgin B-cell clones responding to antigen exposure. Thus, additional activation signals given by T-cells at the time of B-cell triggering may promote the process of gene switching in such a way as to make it more likely that distal constant region genes, such as IgG2a, are expressed.

80. From the above results, it was concluded that although the serum antibody endpoint titers and total ASCs were found lower in the sequential i.m. immunized animals, a higher sera neutralization and avidity indices against several HIV pseudotyped virions were observed. In the findings, mixture of various VLPs group did not show bnAb responses but these animals demonstrated significantly higher serum antibody titers and ADCC against various HIV pseudoviruses than sequential immunized groups. Sequential in. immunizations are more effective in enhancing mucosal antibody levels, higher ADCC, and elevated cellular immunity. Thus, sequential immunizations with a panel of trimeric Env-enriched VLPs, either as a component applied to a heterologous priming/boosting vaccination strategy synergistically combined with other vaccine vectors or as a standalone vaccine approach, have the potential to be further studies for an HIV vaccine. c) Methods:

(1) Cells, reagents, and pseudoviruses:

81. SF9 insect cells (ATCC, Manassas, VA, USA) (CRL-1711), and 293 T-cells (ATCC, Manassas, VA, USA) (CRL-3216) were maintained in Sf-900 II media containing 1%

Penicillin/Streptomycin and supplemented Dulbecco's Modified Eagle's Medium (DMEM), respectively. TZM-bl cells, HIV-1 Env clones of various strains, soluble human CD4, HIV-1 SF162 gpl20, Gag recombinant protein pr55, HIV Con-S Env peptide pool, HIV Con-S Env protein, goat anti-HlV-1 Env polyclonal antibody, monoclonal antibodies e.g. VRCOl, PGT126, PGT145, F425 B4al, and bl2 were acquired from the NIH AIDS Research and Reference Reagent Program.

(2) HIV-1 Env gene construct and VLPs preparation:

82. Gene constructs and rBVs of HIV-1 Env (subtype A-E) proteins were generated. Five different rBVs using Env clones were made from each of the HIV-1 subclades for sequential immunizations: 92UG037.8 (subtype A), SF162 (subtype B), ZM53M.PB12 (subtype C), 92UG021.16 (subtype D), and 93TH976.17 (subtype E). The rBVs expressing HIV-1 Env glycoproteins from different subtypes or Gag protein were generated by using the Bac-to-Bac insect cell protein expression system (Life Technologies, Carlsbad, CA, USA). HIV-1 Env (Env/Gag) VLPs were produced by co-infection of SF9 cells with rBVs expressing trimeric Env and Gag protein at the optimum MOI of 4: 1. Gag VLPs produced by infection of SF9 cells with rBVs expressing Gag protein alone were used as a control. At 60 h post-infection, VLPs were concentrated from the cell culture supernatant by porous fiber filtration using AKTA Flux (GE Healthcare, Uppsala, Sweden) and purified using sucrose density gradient centrifugation.

(3) Physical and functional characterization of prepared VLPs:

83. To confirm the surface expression of Env glycoproteins, SF9 cells were infected with rBVs expressing Env at the MOI of 4 PFU/cell. After fixing the cells, Env surface expression was determined by ELISA using polyclonal goat anti-HIV-1 Env followed by horseradish peroxidase (HRP)-conjugated antibodies. The OD₄₅₀nm was read with an ELISA reader (BioTek, Winooski, VT, USA), which was proportional to the surface expression of Env glycoproteins. Furthermore, the glycoprotein-CD4 binding capability was measured to examine whether the Env glycoprotein expressed on the cell surface were folded correctly. Briefly, SF9 cells were infected with rBVs expressing Env at optimum MOIs and later incubated with soluble human CD4 (5 μ^ιηΐ). The amount of bound CD4 was analyzed by FACS with FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using FITC labeled goat anti-human CD4 antibody. The VLP protein concentration was determined by Micro BCA protein assay and contamination of endotoxins in VLPs were excluded by Limulus amebocyte lysate (LAL) assay (Thermo Fisher Scientific, Waltham, MA, USA). Gag and Env glycoprotein profiles in VLPs were analyzed by SDS-PAGE followed by Western blot using anti-Gag and anti-HIV-1 Env polyclonal antibodies, respectively. A quantitative sandwich ELISA was also done to determine total Env glycoprotein contents in VLPs, using recombinant Con-S Env gp 120 as a calibration standard. The morphology, size distribution, and zeta potential of prepared VLPs were determined by TEM (Zeiss, Oberkochen, Germany) and zetasizer (Malvern, Massachusetts, MA, USA).

(4) Animals and immunization strategy:

84. Sequential immunization regimen containing a panel of VLPs of various Envs as mentioned below (groups 5 and 6) were compared with repetitive homologous-immunizations of individual (subtype B) Env VLPs (group 3) and a mixture of various VLPs of various Envs (group 4). PBS (group 1) and Gag VLPs (group 2) were used as negative control groups. The order of Env VLPs in the sequential immunizations (1. HIV-1 subtype C, 2. HIV-1 subtype B, 3. HIV-1 subtype D, 4. HIV-1 subtype A, and 5. HIV-1 subtype E), was determined by the phylogenetic homology of the HIV-1 Env proteins in between different HIV-1 subtypes. For immunizations, 5 doses of VLPs containing 100 μg of Env and 25 μg of Gag proteins were administered at week 0, 4, 8, 12, and 16. In sequential immunization groups, doses of VLPs were given through both i.m. and In. routes for comparison (groups 5 and 6, respectively) (Table

1). Table 1

Groups lmm-1 lmm-2 lmm-3 lmm-4 lmm-5 Route

1 PBS PBS PBS PBS PBS intramuscular

2 Gag Gag Gag Gag Gag intramuscular

3 Sub B-SF162 Sub B-SF162 Sub B-SF162 Sub B-SF162 Sub B-SF162 intramuscular

4 Mix of A - E Mix of A - E Mix of A - E Mix of A— E Mix of A - E intramuscular

5 A-92UG037.8 B-SF162 C-ZM53M.PB12 D-92UG021.16 E-93TH976.17 intramuscular

6 A-92UG037.8 B-SF162 C-ZM53M.PB12 D-92UG021.16 E-93TH976.17 intranasal (5) Samples collection and PBMCs isolation:

85. Immune serum and mucosal wash samples (nasal and vaginal lavages) were collected for immune response assessments at 3 ^rd week after each vaccination. Blood samples were collected from the marginal ear vein of anesthetized animals for sera separation. Nasal and vaginal washes were collected by repeated flushing of the respective cavities with ice cold lavage medium (PBS,lx containing 150 mM PMSF, and 50 mM EDTA). PBMCs were isolated using Ficoll-paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, USA) density gradient method as described earlier.

(6) Serum and mucosal antibody endpoint titers:

86. Env-specific antibody endpoint titers, including serum IgG and its subtypes (IgGl, IgG2a, and IgG2b) and mucosal IgA, were determined by ELISA, using recombinant Con-S Env gpl20 protein (2 μg/ml) as the coating antigen. The OD₄₅₀ was read with an ELISA reader (BioTek, Winooski, VT, USA) and data were compared with appropriate controls.

(7) ELISPOT assay:

87. For estimating antigen-specific IgG and IgA ASCs, multiscreen 96-well filtration plates (Millipore, Bedford, MA, USA) were coated with recombinant Con-S Env gpl20 protein. After washing and blocking, freshly prepared PBMCs at a concentration of 1 ^χ 10⁶ cells/ml were added to each well (100 μΐ/well). Spots were developed and counted by an ELISPOT reader (Biosys, Miami, FL, USA).

(8) Antibody avidity assay:

88. The avidity index values of serum antibodies to Env proteins were determined by discriminating the weak binding in ELISA in the presence of 1.5 M sodium thiocyanate

(NaSCN). To determine whether HIV Env VLPs induce antibody responses with enhanced avidity, binding to 32 Env-pseudotyped viruses of various HIV subclades were tested. Because pseudotyped virus-based neutralizing assays have been extensively used to evaluate an antibody's capacity for blocking HIV infection, antibody avidity to pseudo virus Env reflect the antibody binding to HIV VLPs. Pseudotype virions purified by filtration, pelleted and disrupted with 1% Triton X-100 were used as coating antigens.

(9) HIV pseudoviruses and antibody neutralization assay:

89. Antibody neutralization breadth and potency of immune serum and mucosal wash samples were evaluated using a highly sensitive, single-round pseudotype virus infectivity assay system. A total of 32 HIV-1 Env-pseudotyped virions from tier 1, 2, and 3 of various isolates were generated for comparing the data of the neutralizing antibody assay, using the Fugene 6 transfection method (Promega, Madison, WI, USA) (Table 2). Pseudoviruses were produced by co-transfection of 293T-cells with an Env-expressing plasmid of different subclades and Env- deficient HIV-1 genomic backbone plasmid, pSG3AEnv for TMZ-bl neutralization assays. These pseudoviruses exhibit a neutralization phenotype that is typical of most primary HIV-1 isolates. Neutralization activity was measured as the reduction of viral infectivity in comparison to that in control wells infected with virus alone.

Table 2

HIV-1 Env-pseudotyped virions from various subclades. Pseudoviruses, generated for comparing the data of neutralizing antibody assay were produced by co-transfection of 293T cells with an Env-expressing plasmid of different subclades and Env-deficient HIV-1 g backbone plasmid, pSG3AEnv using TMZ-bl neutralization assay. (10) ADCC Assay:

90. ADCC was assessed by a standard ⁵¹Cr release assay using 100 μθϊ (3.7 MBq) Na⁵¹Cr0₇ (Perkin Elmer, Downers Grove, IL, USA) for labeling. Freshly isolated PBMCs from naive animals and 293 T-cells transfected with Env-expressing plasmid of different subclades were used as effector and target cells, respectively at an E:T ratio of 10: 1. ⁵¹Cr labelled target cells were incubated with 2-fold diluted serum samples and later co-cultured with effector cells. ⁵¹Cr activity was quantitated using liquid scintillation counter (Beckman Coulter, Atlanta, GA, USA) as described earlier⁴⁷ and the results were expressed as the percentage of lysis of target cells.

(11) Measurement of lymphocyte proliferation:

91. Total lymphocyte proliferation was measured using the Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) staining method. After in vitro stimulation, CFSE (final

concentration 5 μΜ) stained PBMCs were cultured in supplemented DMEM media for 6 days with appropriate negative and positive control groups. After fixing the cells, total proliferation was analyzed by FACS with FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

(12) Flow cytometric detection of degranulation:

92. Further characterization of the T-cell responses were carried out by the identification of degranulating CD8⁺ T-lymphocytes in vaccinated animals. Freshly isolated PBMCs from each group, were cultured and in vitro stimulated with appropriate antigens including negative and positive controls. After stimulation, PBMCs were stained with PE labelled anti-rabbit CD 107a antibody (0.5 mg/ml) along with monensin at the beginning of stimulation. At the end of the stimulation, cells were washed, and surface stained with APC-Cy7 labelled anti-rabbit CD8 antibody (0.5 mg/ml) (Biolegend, San Diego, CA, USA). The results were expressed as a percentage of CD8⁺CD107a⁺ T-cells and the data expressed was the one of the representative experiment.

(13) Cytokine measurement:

93. Using quantitative ELISA, the levels of Thl/Th2 cytokine e.g. IFN-γ, IL-2, IL-6, and TNF-a were analyzed from the culture supernatant which was collected from the in vitro stimulated PBMCs at 48 and 72 h of incubation. In brief, 96- well microtiter plates (Nunc- Immuno Plate Maxisorp; Nunc Life Technologies, Basel, Switzerland) were coated with 100 ng/well of specific coating/purified antibody in a carbonate coating buffer (pH 9.2). After washing and blocking with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA), culture supernatants were added in each well (100 μΐ/well). The detection-biotin- conjugated-paired antibody was added and color was developed as described earlier.

(14) Statistical analysis:

94. The data for antigen-specific IgG/IgA levels, IgG subclasses, ASCs, antibody avidity indices, and percent neutralization were analyzed by paired student t-test and compared by parametric one-way ANOVA analysis of variance by ranks. Comparison of neutralization activity was performed using the Wilkoxon Rank Sum Test. Statistical analysis of FACS data was done using SPSS version 12.0.1 for Windows and by paired student t-test or one-way ANOVA test, n = 5 rabbits per group and the results were expressed as mean ± standard deviation (SD). Levels of significance (p value) were compared between the sequential immunization group and other control groups. Tests were performed using GraphPad Prism 7 software (San Diego, California), p values of O.05 (p < 0.05) were considered to be statistically significant. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

2. Example 2: Intranasal sequential immunization of HIV- 1 Env virus-like particles promotes mucosal TgA and high avidity effector CD8 T cell responses

95. Mucosal IgA antibodies are the first line of immune protection for many viral infections at mucosal surfaces, including HIV. The mechanisms by which these antibodies can inhibit HIV-1 movement across the mucosal barrier include direct virus neutralization, viral aggregation, inhibition of transcytosis, intra-epithelial neutralization, phagocytosis, inhibition through mucus, and Fc receptor-mediated neutralization (antibody-dependent cellular cytotoxicity; ADCC). Non-human primate (NHP) studies indicate that mucosal antibodies to gp41 have important functional roles in protection for HIV infection. Thus, there is a resurgence of interest in the role of mucosal IgA antibodies in protection against HIV-1.

96. HIV-specific T cell responses confer protection against the clinical progression of disease after vims infection and help in controlling, if not clearing, HIV infection . The role of CD4 T cells is a major part of the induction and maintenance of memory CD8 T cell and B cell responses. HIV-specific CD4 T cell responses play an imperative role in controlling viral replication during infection. Even more strikingly than CD4 T cells, HIV-specific CD8 T cell or cytotoxic T lymphocyte (CTL) responses also play a key role in controlling viral replication. Furthermore, high avidity CD8 T cell responses are correlated with slow AIDS disease progression and have generally been considered a favorable qualitative property of antiviral CTLs, associated with the greater elimination of target cells and viral clearance. 97. Sequential immunizations with various antigens trigger broader and more robust antigen-specific T cell responses. The effect of sequential immunizations with HIV-1 Env viruslike particles (VLPs) on mucosal antibody and T cell responses has not been studied yet. Other studies have attempted to elicit mucosal immunity against HIV using heterologous prime-boost immunizations, but none have attempted to simulate HIV evolution/escape through a panel of "native-like" Env VLPs. In this study, HIV-specific mucosal antibody and T cell immune responses were induced in rabbits with intranasal (i.n.) sequential immunizations of a panel of Env-enriched VLPs from HIV-1 clades A-E.

a) RESULTS:

(1) Animals vaccinated sequentially with various Env VLPs induced higher mucosal IgA levels and IgG2 dominant systemic antibody immune responses.

98. The sequential immunization group slightly, but non-significantly, increased serum IgG endpoint titers compared with the control groups (Figure 8A). The sequential i.n.

immunization group significantly increased nasal (p<0.01) (Figure 8B) and vaginal (p<0.01) mucosal IgA (Figure 8C) levels versus the control groups. Sequentially vaccinated rabbits increased IgG2a and IgG2b levels than IgGl in immune sera (Figure 8D), indicating that this vaccine protocol induced Thl -biased antibody responses. Overall, the sequentially immunized group enhanced mucosal IgA levels at both local and distant mucosal compartments with IgG2- dominant systemic antibody responses.

(2) Sequentially i.n. immunized animals showed antibodies with low avidity and neutralization.

99. The sequentially immunized group did not enhance serum antibody avidity indices compared with other immunization groups towards any of the pseudoviruses tested (Figure 9A). Mucosal IgA— nasal (Figure 9B) or vaginal (Figure 9C)— generated in sequentially vaccinated rabbits showed no significant change in the antibody avidities towards most of the

pseudoviruses. But, these animals had a slight but non-significant increase towards some of the pseudoviruses. PBS, Gag, or single Env VLPs immunized animals did not show any increase in their antibody avidities towards any of the pseudoviruses tested.

100. The neutralizing activities of the resulting immune sera and mucosal lavages were evaluated against a panel of 32 HIV-1 pseudoviruses. Sera and vaginal lavage collected from all the animal groups did not show detectable ID₅₀ (<10) against any of the pseudoviruses. The sequentially immunized animals had a slight but non-significant increase in the ID₅₀ values of nasal wash samples compared with the other vaccination groups. The negative control groups showed no background neutralization (Figure 9D). These data demonstrate that sequential immunizations were effective in inducing systemic and mucosal antibody responses, but not with broadly neutralizing activities when compared with repeated immunization of other control groups.

(3) Sequential i.n. immunizations promoted CD4 and CD8 T cell proliferation.

101. The sequential immunization regimen enhanced the CD3⁺CD4⁺ T cell population compared with the Gag or single Env VLPs- vaccinated groups of animals (Figure 10A). Also, sequentially immunized animals had increased CD3⁺CD8⁺ T cell proliferation when compared with the Gag or single Env VLPs-vaccinated animals (Figure 10B). Thus, sequentially immunized animals increased both the CD4 and CD8 T cell proliferation when compared with the other animal groups.

(4) Sequential i.n. immunizations enhanced Thl cytokine levels and enhanced IFN-γ production by CD4 and CD8 T cells.

102. The IFN-γ levels were significantly (p<0.001) greater in sequentially immunized animals compared with the other groups (Figure 11 A). Sequentially immunized rabbits showed significantly (p<0.05) enhanced IL-2 levels while other groups had undetectable levels (Figure

1 IB). TNF-a levels were significantly (p<0.01) increased in the sequentially immunized group compared with the other groups (Figure 11C). IL-6 levels were undetectable in all the groups (Figure 1 ID). Cytokine ELISA results showed that sequential immunizations induced Thl -type cytokines in the circulation.

103. The sequential immunization group also showed a significantly (p<0.05) higher number (5-10-fold) of IFN-y-producing cells than the Gag VLPs or single Env VLPs-immunized groups (Figure 1 IE). The IFN-γ ELISPOT data were comparable with the IFN-γ ELISA results.

104. The sequential immunization group had a significantly (p<0.01) higher percentage of CD8⁺IFN-y-producing population compared with other control groups. The Gag VLPs group did not show any increase in the CD8⁺IFN-y-producing cell percentage, but the single Env VLPs group had a modest level of enhancement in this percentage. FACS data also revealed that sequential immunization group had an increase in CD4⁺IFN-y-producing population compared with other control groups (Figure 1 IF). These results show that sequential immunization of various Env VLPs induced antigen-specific IFN-y-producing CD4 and CD8 T cells in rabbits. (5) Sequential i.n. immunizations encouraged CD8 cytolytic responses.

105. To further characterize the cellular immune responses, the levels of degranulation was determined by analyzing the frequencies of double positive CD4⁺CD107a⁺ and

CD8⁺CD 107a⁺ T-subsets. A slight increase in the percentage of CD4⁺CD 107a⁺ cells were observed in the sequentially immunized group versus other vaccination groups (Figure 12A). The CD8⁺CD107a⁺ T cells in the sequential immunization group of animals were increased when compared with the other groups (Figure 12B). These data support that sequential immunizations with various Env VLPs induced CD8 T cell cytolytic immune responses.

(6) Sequential i.n. immunizations triggered cytokine polyfunctionality and high avidity effector CD8 T cell responses.

106. Animals immunized sequentially showed more polyfunctionality compared with other control groups. The sequentially immunized group demonstrated the greatest diversity of cytokine expression and contained the largest populations of double- and triple-cytokine expressing CD4 (Figure 13 A) and CD8 T cells (Figure 13B). Sequential immunization regimen broadened the cellular immune responses and elicited a more divergent antigen-specific T cell population.

107. To further evaluate the CD8 T cell responses, the CD8 TCR avidity was tested using a FACS method after gating CD8 T cell populations with the IFN-γ expression at different antigen concentrations. Sequentially immunized animals showed an increase in the TCR avidities compared with the repetitive-immunizations of Gag only or single Env VLPs (Figure 13C). The increases induced by sequential immunization regimen in CD8 TCR avidities and cytokine polyfunctionality underlies the improved antiviral immune responses of the group.

b) DISCUSSION:

108. In Lhe current study, an i.n. sequential immunization regimen was designed and characterized with a phylogenetically-ordered panel of HIV- 1 Env VLPs from different HIV-1 clades (A-E) to elicit robust mucosal IgA and T cell immunity in rabbits. Sequential

immunizations effectively enhanced: IgA levels at local and distal mucosal compartments, IgG2- dominant serum antibody titers, T cell proliferation, Thl cytokine production (especially IFN-γ), cytokine polyfunctionality, and high avidity CD8 T cell responses.

109. The sequential immunization group significantly enhanced the mucosal IgA levels in nasal and vaginal secretions. Given that the majority of HIV transmission occurs via mucosal surfaces, there is a great interest in strengthening the innate and adaptive mucosal environment which can shape the outcome of HIV infection. Anti-HIV-1 mucosal IgA has not been observed only in HIV-1 infected individuals, but also in some HIV-exposed seronegative individuals (HESNs). IgA isolated from cervicovaginal secretions of these individuals are capable of inhibiting viral infection. Because of mucosal IgA's importance in HIV prevention, vaccine procedures capable of eliciting a strong mucosal IgA response are beneficial by contributing to the containment of HIV-1 infection.

110. Sequential immunizations elicited more IgG2a/2b than IgGl antibodies compared with the single Env VLPs group or other control groups, defining a Thl type of immunity. The self-reinforced polarization of Th differentiation has a profound effect on the outcome of an immune response. This differentiation directs Thl cell immune responses to promote cell- mediated immunity and Th2 cells to develop humoral immune responses.

111. Sequential immunizations enhanced antigen-specific T lymphocyte proliferation. As shown herein sequential immunization regimen generated a broad-spectrum T cell-based immunity with enhanced T lymphocyte proliferation. Because circulating primed T cells have a lower activation threshold than naive T cells, they preferentially activate when presented with conserved epitopes during the sequential immunization with Env VLPs. The finding supports that sequential immunizations can be designed to stimulate cellular immune responses.

112. The sequential immunization group exhibited a Thl type immune responses, demonstrated by the IgG2-dominant antibody responses and by high levels of Thl cytokines (IFN-γ, IL-2, and TNF-a) in PBMCs' culture. The results of ELISPOT and ICS demonstrate that the animals vaccinated through sequential immunizations had enhanced IFN-γ production, especially by CD8 T cells. Preliminary evidence and different NHP studies have shown a vigorous T cell response (including IFN-γ production) is an immune correlate of protection against HIV-1/SIV infection. The sequential immunizations with various Env VLPs induced CD8 T cell cytolytic immune responses. A crucial role of CD8 T lymphocytes in protection from intracellular pathogens such as viruses like HIV-1 supports the results.

113. The increased CD8 functionalities, including TCR avidities and cytokine polyfunctionality, reflected the efficacy of the sequential immunization strategy. These results show that CD8 functionalities are the key determinant in antiviral immunity. More recently, studies have shown that the exclusive determinant of HIV viremic control in progressors was the amount of killing of HIV-infected targets by high avidity CD8 T cells indicating that CD8 T cell avidity with cytokine polyfunctionality can underlie HIV control.

114. The immune responses can result in two important outcomes during HIV exposure; the elevated levels of IFN-γ secretion by CD8 T cells can activate antiviral functions and the antigen-specific CD8 T cells can destroy virus-infected cells. IFN-γ affects a wide range of target cells and induces the host defense against infectious agents by up-regulating MHC class I and II proteins on a variety of cells, like macrophages and epithelial cells; acting on CD4 Thl differentiation; regulating the production of a variety of other pro -inflammatory cytokines, including IL-2 and TNF-a; and stabilizing inflammatory T cell responses.

c) CONCLUSION:

115. From the study, it was concluded that a sequential i.n. immunization regimen of phylogenetically-ordered HIV-1 Env VLPs is effective at enhancing antigen-specific mucosal IgA levels and high avidity effector CD8 T cell immune responses with cytokine

polyfunctionality, in rabbits. The increased IgA titers, high CD8 TCR avidities, high CD8 T cell populations, increased Thl cytokines' levels, elevated CD8 T cell cytolytic responses, and polyfunctional cytokine responses strengthen our sequential i.n. immunization regimen approach. The sequential immunization regimen, either as a component applied to a

heterologous prime/boost vaccination strategy with other vaccine vectors or as a standalone vaccine approach, can significantly improve HIV vaccines.

d) METHODS:

(1) Cells, reagents, and pscudoviruses.

116. Spodoptera frugiperda (SF9) insect cells and HEK293T cells (ATCC) were maintained in Sf-900 II media containing 1% Penicillin/Streptomycin and supplemented Dulbecco's Modified Eagle's Medium (DMEM), respectively. The NIH AIDS Research and Reference Reagent Program provided the HIV-1 Env clones of various strains, soluble human CD4, HIV-1 SF162 Env, Gag recombinant protein pr55, HIV Con-S Env protein and peptide pool, goat anti-HIV-1 Env polyclonal antibody, and monoclonal antibodies (e.g. PGT126, PGT128, and PGT145).

(2) VLPs preparation and characterization.

117. Gene constructs and recombinant bacuioviruses (rBVs) expressing various HIV- 1 Env (clades A-E) were generated. Modified Env gene constructs were generated by replacing the original signal peptide encoding sequences with the honeybee melittin sequence and adding a trimeric form of leucine zipper sequence (GCN4pii) to the C-termini of Env cytoplasmic sequences to increase Env glycoprotein production in insect cells and to express conformation-stabilized trimeric Env proteins, respectively. These Env were from strains; 92UG037.8 (A), SF162 (B), ZM53M.PB12 (C), 92UG021.16 (D), and 93TH976.17 (E). Env based was chosen on the similarity in their amino acid sequences and phylogenetic homology of the HIV-1 Env proteins between different HIV-1 subtypes. HIV-1 Env (Env/Gag) VLPs were produced by co-infection of SF9 cells with rBVs expressing trimeric Env and Gag at the multiplicity of infections (MOIs) of 2:1. Gag VLPs produced by infection of SF9 cells with rBVs expressing Gag alone were used as a control group. VLPs were concentrated by porous fiber filtration using a AKTA Flux (GE Healthcare) and further purified using a sucrose density gradient centrifugation method.

118. Various physical and functional properties of prepared VLPs were evaluated: surface expression of Env glycoproteins, glycoprotein-CD4-binding capability, Env binding to PGT145, PGT126, or PGT128 monoclonal antibodies, protein profiles (Env and Gag), oligomeric status of Env glycoproteins, Env glycoprotein contents in VLPs, morphology, size, and zeta-potential of prepared VLPs, as described earlier. Results demonstrate that high levels of Env content were incorporated into the VLPs and these Env retained their natural confirmation with other physical and functional properties. Transmission electron microscopy (TEM) (Zeiss) and Zetasizer (Malvern) data showed that the prepared VLPs were spherical in morphology and sized in between 180-200 urn.

(3) Ethics statement.

119. This study was carried out with the recommendations found in the Guide of the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Georgia State University. 6-8-week-old, healthy female New Zealand white rabbits were purchased from Charles-River Laboratory and housed in the University's animal facility.

Immunization and sample collection were performed under a mild anesthesia that was induced and maintained with Acetylpromazine. All efforts were made to minimize pain.

(4) Animals and Immunization.

120. 5-6 healthy female New Zealand white rabbits were used per group for immunizations. Sequential immunization regimen containing a panel of VLPs expressing various Env was compared to repetitive-homologous immunizations of a single (clade B) Env or Gag (non-Env) VLPs. Phosphate-buffered saline (PBS) was used as a negative control group (Table 3).

Table 3 Immunization Groups

Groups Immunization 1 Immunization 2 Immunization 3 Immunization 4 Immunization 5

1 PBS PBS PBS PBS PBS

2 Gag (Non-Env) Gag (Non-Env) Gag (Non-Env) Gag (Non-Env) Gag (Non-Env)

3 Clade 'B' Clade Έ' Clade 'B' Clade 'B' Clade Έ'

(SF162) (SF162) (SF162) (SF162) (SF162)

4 Clade 'C Clade 'B' Clade 'D' Clade 'A' Clade Έ'

(ZM53M.PB12) (SF162) (92UG021.16) (92UG037.8) (93TH976.17) 121. For immunizations, the initial dose was given at week 0 and the 4 consecutive booster doses were given at 4- week intervals. The order of Env VLPs in sequential

immunization regimen was determined based on the ability to maximize genetic divergence, which can increase the possibility of generating broader immune responses. Five doses of VLPs containing lOC^g of Env or/and 25μg of Gag proteins in total per rabbit were administered through i.n. route.

(5) Immune sera, mucosal lavages, and PBMCs collection.

122. Blood and mucosal lavages (nasal and vaginal) were collected at one- week pre- (preimmune) and 3 -week after the last vaccination. Blood samples were collected from the marginal ear vein of anesthetized animals using vacuette blood collection tubes containing sodium heparin (Greiner Bio-One) for peripheral blood mononuclear cells (PBMCs) and sera separation. Sera were isolated from the clotted blood and PBMCs were harvested using the Ficoll Paque PLUS (GE Healthcare) density gradient method (Miltenyi Biotec). Sterile cotton tipped swabs were used to collect nasal/vaginal cavity material. Each individual swab was placed into ice-cold lavage buffer (0.9%,w/v, NaCl; 0.05%,v/v, Tween 20; 0.1%,w/v, NaN³; and Imol/dm³ PMSF). The swabs were vortexed in the buffer and all collection tubes per rabbit were pooled, mixed, aliquoted, and stored at -80°C.

(6) Estimation of IgG/IgA levels.

123. Serum (IgG, IgGl, IgG2a, and IgG2b) and mucosal IgA (nasal and vaginal) levels were determined against Con-S Env (consensus) and various Env (autologous) antigens using indirect ELISA and cell-based ELISA, respectively. For cell-based ELISA, HEK293T cells were transfected with various HIV-1 Env clones (the same HIV-1 Env plasmids that were used in the Env VLPs), influenza full-length (FL) HA, or Gag (non-Env) with Lipofectamine 2000 (Invitrogen). Influenza FL HA and Gag transfected cells were used as control groups in the assay. The HIV Con-S Env protein (200ng/well) and the fixed transfected HEK293T cells (5 xlO⁴ cells/well) were used as coating antigens and the color was developed as described earlier using anti-rabbit HRP-conjugated IgG/IgA antibodies (ThermoFisher). The optical density at 450nm (OD₄₅o) was read with an ELISA reader (BioTek).

(7) HIV-1 pseudoviruses.

124. A total of 32 HIV-1 Env-pseudotyped virions from tier 1, 2, and 3 were generated using the Fugene6 transfection method (Promega) (Table 2). Pseudoviruses were produced by co-transfection of HEK293T cells with an Env-expressing plasmid of different clades and Env-deficient HIV-1 genomic backbone plasmid, pSG3AEnv as mentioned earlier. (8) Avidity Indices.

125. The avidity indices of systemic and mucosal antibodies to Env proteins were determined by ELISA in the presence of 1.5M sodium thiocyanate (NaSCN). Pseudotype virions purified by filtration, pelleted, and disrupted with 1% Triton X-100 were used as coating antigens.

(9) Neutralization assay.

126. The TZM-bl neutralization assay was used with minor modifications. Two-fold serial dilutions of sera/mucosal wash samples from individual animals were plated and

200xTCID₅₀ of each pseudovirus were added to the wells. Later, TZM-bl cells were added

(l xl0⁴/well) in 10% DMEM growth medium containing DEAE-dextran. Recombinant influenza H7N9 pseudovirus was used as a negative viral control in the neutralization assay.

(10) CD4 and CD8 T cells proliferation.

127. CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell proliferation were measured by a FACS method using PBMCs, harvested from the vaccinated animals. PBMCs were in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone. Stimulated PBMCs were stained with anti-rabbit CD3 antibody (Biorad), coupled with anti-rabbit IgG-Pacific-blue secondary antibody (Thermofisher). After washing, these cells were stained for anti-rabbit CD4 or CD8ct antibodies (Biorad), coupled with anti-rabbit IgG-APC or FITC secondary antibodies (Abeam), respectively. After fixing the cells, CD3⁺CD47CD8⁺ T cell proliferation was analyzed with a BD FACSCanto II flow cytometer (BD Biosciences) using FlowJo Software (Treestar Inc).

(11) Cytokine measurement.

128. Using quantitative ELISA, the levels of Thl/Th2 cytokines (IFN-γ, IL-2, TNF-a, and IL-6) were analyzed from PBMCs' culture supernatant at 48 and 72h. PBMCs were in vitro stimulated with HIV-1 Con-S Env peptides, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone. Cytokine concentrations were calculated from their respective standard curves and data were represented in pg/ml for each group of rabbits from three independent experiments.

(12) IFN-γ ELISPOT assay.

129. IFN-γ ELISPOT assays were performed. 96-well filtration plates (Millipore) were coated with 2μg/ml of purified anti-rabbit IFN-γ antibody (Mabtech). After washing and blocking, freshly prepared PBMCs (l xlO⁶ cells/well) were incubated in triplicate with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone for 18- 20h. Plates were washed, incubated with biotinylated anti-rabbit IFN-γ antibody (Mabtech) and further with anti-rabbit streptavidin-HRP IgG secondary antibody (Jackson ImmunoResearch). Spots were developed using 3,3'-diaminobenzidine (DAB) (Pierce) and counted by an ELISPOT reader (Biosys).

(13) Flow cytometric detection of degranulation.

130. Further characterization of the T cell responses was carried out by the

identification of degranulating CD4 and CD8 T lymphocytes in the vaccinated animals. After the last vaccination, freshly isolated PBMCs from each group were cultured and in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone. At the beginning of stimulation, PBMCs were stained with anti-rabbit CD 107a antibody (Abeam), coupled with anti -rabbit IgG-PE secondary antibody (Abeam) along with monensin (BD Biosciences). At the end of the stimulation period, cells were washed, surface stained with anti-rabbit CD4 or CD8 antibodies (Biorad), coupled with anti-rabbit IgG-Per-CP or APC-Cy7 secondary antibodies (Santa cruz), respectively. Data were acquired on a BD FACSCanto II flow cytometer and analyzed with FlowJo Software (Treestar Inc). The results were expressed as a percentage of CD4⁺/CD8⁺CD107a⁺ cells and the result represent one of the representative experiment.

(14) Intracellular cytokine staining.

131. Antigen-specific cytokine-producing cells were quantified by an intracellular cytokine staining (ICS) assay. PBMCs (l xlO⁶ cells/well) were seeded in 96 well plates for 5-6h containing HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone, in the presence of 5 μΐ/ml of brefeldin A (Golgi Plug) (BD Biosciences). Following washing and blocking, cells were surface stained with anti-rabbit CD4 and CD8 antibodies (Biorad), coupled with Per-CP and APC-Cy7 conjugated secondary antibodies, respectively. After fixation, cells were stained with anti-rabbit IFN-γ, IL-2,'and TNF-a antibodies (Abeam), coupled with anti-rabbit IgG-PE, FITC, and APC secondary antibodies (Abeam), respectively after each specific wash. CD4/CD8 T cells were gated with IFN-γ expression at specific antigen concentration for measuring IFN-γ producing CD4 or CD8 T cells. For determining the polyfunctionality, CD4 or CD8 T cell populations were gated and single, double or triple cytokine expressing cell percentages were enumerated. The percentage cytokine expression of each animal was averaged within the group and is expressed as a pie-chart of relative

proportions of the total cytokine expressing cell population. To estimate the effector CD8 T cell receptor (TCR) avidity, CD8 T cells were gated with IFN-γ expression and was measured as percent (%) maximum response at different antigen concentrations. (15) Statistical analysis.

132. The data for CD4/CD8 T cells proliferation, ELISPOT, cell-based ELISA, indirect ELISA, and quantitative ELISA were analyzed by paired student t-test and compared by parametric one-way ANOVA analysis of variance by ranks. Statistical analysis of FACS data was done using SPSS version 12.0.1 for Windows and by paired student t-test or one-way ANOVA test, n = 5 rabbits per group and the results were expressed as the mean ± standard deviation (SD). Cytokine polyfunctionality was analyzed by two-way ANOVA test. TCR avidity measurements were analyzed by nonlinear regression and comparisons were made using the exact sum-of-squares F test. Nonlinear regression plots depict the regression results (line) with 95% confidence intervals (cloud) computed from results calculated from multiple independent experiments. Levels of significance (p-value) were compared between sequential immunization group and other control groups. Tests were performed using Graph Pad Prism 7 software, p- values of <0.05 (p<0.05) were considered to be statistically significant.

*p<0.05;**p<0.01,***p<0.001,****p<0.0001.

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Claims

V. CLAIMS What is claimed is:

1. A method of generating broadly neutralizing antibodies against a virus comprising sequentially administering to a subject one or more viral antigens from a virus for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.

2. The method of claim 1, wherein the virus is selected from the group of viruses consisting of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus,

Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reo virus, Yellow fever virus, Ebola virus, Marburg virus, Zika virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian

Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human

Immunodeficiency virus type-2.

3. The method of claim 3, wherein the virus is HIV-1.

4. The method of any of claims 1-3, wherein the viral antigen comprises a VLP.

5. The method of claim 4, wherein the antigen of the VLP is selected from the HIV antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat),regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif)

6. The method of claim 5, wherein the VLP comprises a trimeric HIV-1 envelope (Env) VLP.

7. The method of claim 6, wherein the VLP comprises a chimeric trimeric HIV Env VLP comprising one or more monomers from different HIV strains relative to the remaining monomers.

8. The method of claim 7, wherein one or more of the strains of the chimeric trimeric HIV Env VLP from one or more of the monomers is from a different HIV subtype relative to the remaining monomers.

9. The method of claim 6, wherein the VLP further comprises a trimeric leucine zipper.

10. The method of claim 7, wherein the trimeric leucine zipper comprise GNC4pii.

11. The method of claim 4, wherein the VLP further comprises a substitution of the viral antigen signal peptide for a signal peptide from a different protein to increase expression of the VLP in a cell or cell line.

12. The method of claim 11, wherein the signal peptide comprises honeybee melittin.

13. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype A.

14. The method of claim 13, wherein at least one HIV Env is from strain 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, or RW92020.5.

15. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype B.

16. The method of claim 15, wherein at least one HIV Env is from strain SF 162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, or TRJ04551.58.

17. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype C.

18. The method of claim 17, wherein at least one HIV Env is from strain ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, or DU156.12.

19. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype D.

20. The method of claim 19, wherein at least one HIV Env is from strain 92UG021.16, 92UG013.70, 93ZR001.3, 92UG021.9, 92UG024.2, or 92UG021.16.

21. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype E.

22. The method of claim 21, wherein at least one HIV Env is from strain 93TH976.17 or 93TH966.8,

23. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype F.

24. The method of claim 23, wherein at least one HIV Env is from strain 93BR019.4, 93BR019.10, 93BR020.17, or 93BR029.2.

25. The method of claims 5 or 6, wherein the HIV-1 Env VLP comprises at least one Env from HIV subtype G.

26. The method of claim 25 , wherein at least one HIV Env is from strain 92RU 131.16 or 92RU131.9.

27. The method of claims 5 or 6, wherein the order of administration of each chimeric trimeric HIV-1 envelope VLP from first to last VLP is HIV-l subtype C, HIV-1 subtype B, HIV-1 subtype D, HIV-1 subtype A, and HIV-1 subtype E.

28. The method of claim 27, wherein the trimeric HIV-1 envelope VLP for HIV-1 subtype C comprises an Env from HIV strain ZM53M.PB12, wherein the trimeric HIV-1 envelope VLP for HIV-1 subtype B comprises an Env from HIV strain SF162, wherein the trimeric HIV-1 envelope VLP for IIIV-1 subtype D comprises an Env from HIV strain 92UG021.16, wherein the trimeric HIV-1 envelope VLP for HIV-1 subtype A comprises an Env from HIV strain 92UG037.8, and wherein the trimeric HIV-1 envelope VLP for HIV-1 subtype E comprises an Env from HIV strain 93TH976.17.

29. The method of claim 1, wherein each antigen administration occurs at least 4 weeks after the prior admimstration.

30. The method of claim 1, wherein the one or more viral antigens are administered to the subject intramuscularly or intranasally.

31. A method of immunizing a subject against a viral infection comprising sequentially administering to the subject one or more viral antigens from a virus for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.

32. A method of generating mucosal antibodies against a virus comprising sequentially administering to a subject one or more viral antigens from a virus for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding

administration round.

33. The method of claim 32, wherein the one or more viral antigens are administered intranasally, rectally, vaginally, or orally.

34. A viral immunization strategy comprising the sequential administration of one or more viral antigens to a subject over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.

35. A kit for immunizing a subject against HIV comprising two or more clade variants of one or more HIV antigens.

36. The kit of claim 22, further comprising instructions to administer clade variants of the one or more viral antigens sequentially.