Vaccines

9 pages, 855 KiB

Open AccessArticle

CD19⁺ CD24^hi CD38^hi Regulatory B Cells and Memory B Cells in Periodontitis: Association with Pro-Inflammatory and Anti-Inflammatory Cytokines

by Helal F. Hetta, Ibrahim M. Mwafey, Gaber El-Saber Batiha, Suliman Y. Alomar, Nahed A. Mohamed, Maggie A. Ibrahim, Abeer Elkady, Ahmed Kh. Meshaal, Hani Alrefai, Dina M. Khodeer and Asmaa M. Zahran

Vaccines 2020, 8(2), 340; https://doi.org/10.3390/vaccines8020340 - 26 Jun 2020

Cited by 24 | Viewed by 3924

Abstract

Regulatory B cells (Bregs) are unique subpopulations of B cells with immune-regulating or immune-suppressing properties and play a role in peripheral tolerance. Due to the current limitations of human Breg studies among periodontal diseases, in the present study, we tried to analyze the [...] Read more.

Regulatory B cells (Bregs) are unique subpopulations of B cells with immune-regulating or immune-suppressing properties and play a role in peripheral tolerance. Due to the current limitations of human Breg studies among periodontal diseases, in the present study, we tried to analyze the change in circulating Bregs, pro-inflammatory, and anti-inflammatory cytokines in patients with periodontitis. Peripheral blood from 55 patients with stage 2 periodontitis and 20 healthy controls was analyzed using flow cytometry to evaluate the frequency of CD19⁺CD24⁺CD38⁺ Breg cells. ELISA was used to assess the serum levels of the pro-inflammatory cytokines, including interleukins (IL)-1β, IL-6, TNF-α, and anti-inflammatory cytokines including IL-10, IL-35, and TGF-β. Increased proportions of Breg cells were observed in patients with stage 2 periodontitis compared to controls. Serum levels of cytokines were significantly higher in patients with periodontitis compared to controls. A significant positive correlation was observed between the frequencies of Breg cells and IL35 levels, IL10 levels, and TGF-β. In conclusion, our results suggest that the increase in peripheral Breg cells and serum cytokine levels among periodontitis patients seems to be closely associated with disease progression, a possible link between periodontitis, and systemic inflammatory process. Full article

(This article belongs to the Special Issue B and T Cell-Mediated Immunity)

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13 pages, 1614 KiB

Open AccessArticle

Cross-Sectional Study on the Sero- and Viral Dynamics of Porcine Circovirus Type 2 in the Field

by Chao-Nan Lin, Ni-Jyun Ke and Ming-Tang Chiou

Vaccines 2020, 8(2), 339; https://doi.org/10.3390/vaccines8020339 - 26 Jun 2020

Cited by 10 | Viewed by 3260

Abstract

Porcine circovirus-associated diseases (PCVADs) cause considerable economic losses in industrial pork production in the field. To minimize the economic losses due to PCVAD, porcine circovirus type 2 (PCV2) vaccines have been developed, and there is widespread vaccination worldwide today. However, limited information is [...] Read more.

Porcine circovirus-associated diseases (PCVADs) cause considerable economic losses in industrial pork production in the field. To minimize the economic losses due to PCVAD, porcine circovirus type 2 (PCV2) vaccines have been developed, and there is widespread vaccination worldwide today. However, limited information is available concerning the current status of PCV2 infection in the field on the Asian continent. The present study aimed to assess sero- and viral dynamics of PCV2 from 12 PCV2-contaminated pig herds with vaccination against PCV2 in Southern and Central Taiwan. In particular, the level of PCV2 load during the window period for seroconversion using real-time polymerase chain reaction and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Our results revealed that pig herds showed slight or no seroconversion after three to four weeks post-PCV2 immunization. The presence of PCV2 was observed during the window period for seroconversion in all herds. In conclusion, natural exposure of PCV2 occurs in the growing to fattening period, and viremia can last until slaughter. Additionally, our findings indicate that using ELISA showed the level of antibodies and aided in the understanding and surveillance of the current PCV2 status in the field. Full article

(This article belongs to the Section Veterinary Vaccines)

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Comparison of maternally derived antibodies (MDA) of porcine circovirus type 2 (PCV2) on suckling piglets (only from 3 to 4-week-old piglets without PCV2 vaccination) born from different sow immunization programs against PCV2. A: sows without PCV2 vaccination; B: sows with mass vaccination; C: sow vaccination at 2–4 weeks pre-farrowing. Student’s t-test was used to assess differences in the presence of PCV2 MDA from suckling piglets born from different sow vaccination programs against PCV2. p values <0.05, <0.01, and <0.001 were considered statistically significant, highly significant, and very highly significant, respectively. Full article ">Figure 2
Serodynamic profile of porcine circovirus type 2 (PCV2) measured by BioCheck ELISA in different aged pigs born from different sow immunization programs against PCV2 from different farms. (a) sows without PCV2 vaccination (Farms A, B, C, and D); (b) sows with mass PCV2 vaccination (Farms E, F, G, and H); (c) PCV2 vaccination in sows at 2-4 weeks pre-farrowing (Farms I, J, K, and L). Full article ">Figure 3
Viremia loads (line graph, left Y axis) and detection rate (bar chart, right Y axis) of porcine circovirus type 2 (PCV2) in pigs born from sows without PCV2 vaccination group, Farms A, B, C, and D. The error bars show the standard deviation (SD) of positive samples. Full article ">Figure 4
Viremia loads (line graph, left Y axis) and detection rate (bar chart, right Y axis) of porcine circovirus type 2 (PCV2) in pigs born from the sow mass PCV2 vaccination group, Farms E, F, G, and H. The error bars show the standard deviation (SD) of positive samples. Full article ">Figure 5
Viremia loads (line graph, left Y axis) and detection rate (bar chart, right Y axis) of porcine circovirus type 2 (PCV2) in pigs born from PCV2 vaccination in sows at 2–4 weeks pre-farrowing, Farms I, J, K, and L. The error bars show the standard deviation (SD) of positive samples. Full article ">

24 pages, 1926 KiB

Open AccessArticle

Establishing a Robust Manufacturing Platform for Recombinant Veterinary Vaccines: An Adenovirus-Vector Vaccine to Control Newcastle Disease Virus Infections of Poultry in Sub-Saharan Africa

by Omar Farnós, Esayas Gelaye, Khaled Trabelsi, Alice Bernier, Kumar Subramani, Héla Kallel, Martha Yami and Amine A. Kamen

Vaccines 2020, 8(2), 338; https://doi.org/10.3390/vaccines8020338 - 26 Jun 2020

Cited by 9 | Viewed by 6442

Abstract

Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of [...] Read more.

Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of public health global policies in the context of “one world, one health” principles. Sub-Saharan Africa is frequently impacted by outbreaks of poultry diseases such as avian influenza and Newcastle Disease (ND). Here, an adenovirus-vectored vaccine technology platform is proposed for rapid adaptation to ND or other avian viral threats in the region. Ethiopian isolates of the Newcastle Disease virus (NDV) were subjected to sequence and phylogenetic analyses, enabling the construction of antigenically matched vaccine candidates expressing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. A cost-effective vaccine production process was developed using HEK293 cells in suspension and serum-free medium. Productive infection in bioreactors (1–3 L) at 2 × 10⁶ cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5–6 × 10⁸ TCID₅₀/mL (approximately 10¹¹VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 × 10¹⁰ TCID₅₀ of the vectors, and full protection against a lethal NDV challenge was provided by the vector expressing the F antigen. These results consolidate the basis for a streamlined and scalable-vectored vaccine manufacturing process for deployment in low- and medium-income countries. Full article

(This article belongs to the Special Issue Genomic Medicine and Advances in Vaccine Technology and Development in the Developing and Developed World)

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Nonreplicating chicken codon-optimized recombinant adenovirus constructions as vaccine candidates against Newcastle Disease virus (NDV): The schematic representation of the adenoviral constructs shows the regulatory regions for the control of expression and the fusion and hemagglutinin-neuraminidase proteins of NDV. The expression of the foreign antigens is driven either by a fragment of the human Cytomegalovirus (CMV) enhancer/promoter or by the avian β-actin promoter, as indicated. The adenoviral vectors have been designed to carry one or two (head-to-tail oriented) expression units for the individual expression or the co-expression of the NDV antigens. One of the constructs carries both the hemagglutinin-neuraminidase protein and the green fluorescent protein for monitoring and quantification during the process development steps implemented at different scales. Full article ">Figure 2
Effect of two different culture media and feeding supplements on HEK293SF cell density and viability (in percent) in shake flask experiments: HyCell TransFx-H media (Hyclone Laboratories Inc) and HEK-GM (Xell AG, Bielefeld, Germany) were used in the assay for comparative purposes, including the use of feeding supplements following essentially each manufacturer’s recommendations. Supplements consisted of a bolus addition. The feeding supplement HEK-FS (Xell AG, Bielefeld, Germany) was added starting from day 2 of the culture at 3% (v/v), then increasing to 4, then 5% (v/v), and finally 10% until the end of the culture at day 12. The HyCell TransFx-H medium was supplemented with CellBoost 5 (GE Healthcare, Chicago, IL, USA, USA) every two days by adding the feeding bolus at 5% (v/v). The higher cell densities were achieved with the HEK-GM basal medium and with the HEK-GM + HEK-FS combination, in which 1 × 107 cells/mL and 1.3 × 107 cells/mL were reached, respectively, after 9 days in culture. High cell densities (around 1 × 107 cells/mL) were also reached by day 8 with the combination of HyCell TransFx-H and its feeding supplement. The complete assessment was conducted in duplicate, and each point corresponds to the mean ± standard deviation (bars in both senses are shown in the figure). Full article ">Figure 3
Adenovirus production in shake flasks experiments and evaluation of cell culture parameters and infection conditions for increased virus per cell production yields: Infection experiments were conducted with the recombinant Ad-HN-GFP-CMV adenoviral vector in order to monitor parameters in both phases of the culture. Two different culture media were evaluated: HyCell TransFx-H and Xell AG HEK-GM. In this approach, cells were cultured in batch until reaching densities of 1, 2, 4, and 6 × 106/mL (this last value is only for HEK-GM), and viral infection was initiated at each of these points. The effect of cell density on the specific cell yield was evident when a decrease in specific production was observed at higher cell densities, indicating a metabolic limitation. A feed supplement was added at each cell concentration at the time of infection, using paralleled cultures and following the regimens explained in Materials and Methods. It was demonstrated at every cell density analyzed that feeding significantly increased the specific production yield (IVP/cell) of the cultures compared to the non-supplemented cultures. The highest values of cell specific yields (calculated from fluorescence values detected by flow cytometry) were reached in both media by infection at 2M cells/mL. The combination HEK-GM + HEK FS resulted in the highest specific production and was used either in shake flasks or for the scale-up to bioreactors. The experiment was conducted in duplicate and deviations between replicates are indicated in the figure by standard deviations bars in the positive sense. Full article ">Figure 4
Adenoviral vector production in 1 L controlled bioreactors operated in batch mode: (A) Time course of viable cell growth and cell viability during the production of the three recombinant adenoviral vectors encoding the F, HN, and F-HN antigens from NDV under the regulation of the CMV enhancer/promoter. The culture medium was Xell AG HEK-GM and the operational parameters were described in the Materials and Methods section. The time of infection was set at a cell density of 2 × 106 cells/mL as indicated in the figure, and the cultures were harvested when cell viability reached approximately 60–70%. The cell density data are shown by solid symbols and solid lines, and the percentage of cell viability is shown by empty symbols and dotted lines. The infections were conducted with the different adenoviral vectors; ∆, Ad-F-CMV; —, Ad-HN-CMV; ◊, Ad-F-HN-CMV. (B) Production of infectious viral particles per mL in the cell culture lysate supernatants analyzed at different time points using culture samples taken during the run. Calculations of TCID50/mL values were performed as described. They were in the range of 1.3 to 2.2 × 108 TCID50/mL. Full article ">Figure 5
Adenoviral vector production in a 3 L controlled bioreactor operated in fed-batch mode for the production of one adenoviral vector as vaccine candidate: (A) Time course of the viable and total cell growth in Xell AG HEK-GM medium in which the feed supplement was initiated at a cell concentration of 1 × 106 cells/mL as indicated in the figure. The infection was set at a cell density of 2 × 106 cells/mL, and the final cell density during the virus production phase reached 4.56 × 106 cells/mL. The cultures were strictly monitored and harvested when the cell viability reached approximately 60–70%. At harvest, the infectious viral particles of the Ad-F-HN-CMV produced reached 5.8 × 108 TCID50/mL in the cell culture lysate supernatants. In addition, the curve of the capacitance values (B) measured in-line shows the evident changes registered after infection with the adenoviral vector as the cells undergo the productive phase. The profile of various bioreactor sensors and process parameters are also shown (speed of agitation, pH, dissolved oxygen concentration, cumulative oxygen, and temperature). The onset of feeding supplementation is indicated with an arrow in panel A. The dotted line in both panels indicates the time point of infection and the shift in the culture phase. Full article ">Figure 6
Analysis of the humoral immune response generated in mice against NDV after vaccination with the recombinant adenoviral vectors Ad-F-CMV, Ad-HN-CMV, Ad-F-HN-CMV, and Ad-GFP-CMV (negative control): Enzyme-Linked Immunosorbent Assays (ELISA) and Hemagglutination Inhibition Assay (HIA) assays were conducted for the detection of anti-F IgG antibodies and antibodies with HI activity, respectively, in the serum of vaccinated mice. Mice were immunized via i.m. injections at days 0 and 21 with doses of 107 TCID50. (A) The mean ± standard deviations of anti-F IgG antibody titers measured at days 0 and 60 of the experiment. Specific titers elicited by the Ad-F-CMV and the Ad-F-HN-CMV vectors increased over the indicated cutoff (>993) for existence of protective immunity (dotted line). Both groups of animals, injected with the Ad-F-CMV or Ad-F-HN-CMV viruses, showed statistically significant differences compared to the negative controls. (B) The generation of antibodies with hemagglutination inhibition capacity in vitro is shown. Mean titers were calculated from values of antibodies with HI activity detected in serum of animals using the logarithm base 2 of the titer. In animals vaccinated with the Ad-HN-CMV and Ad-F-HN-CMV, this response was statistically superior to measurements in the negative control group. According to the routine practices at the NVI, different degrees of protection against NDV are achieved in chickens when HI titers over Log2(3) (dotted line). Statistically significant differences (p < 0.05) are represented with one asterisk in the figure and two for p < 0.01. Full article ">Figure 7
Analysis of the humoral immune response generated in chickens against NDV-encoded antigens and assessment of the protective efficacy: The level of anti-NDV antibodies after immunization with the recombinant adenoviral vectors Ad-F-β-actin, Ad-F-CMV, and Ad-F-HN-CMV were evaluated by ELISA (A) for the detection of IgY-specific antibodies in the serum of chickens vaccinated via i.m. with two doses of 1010TCID50. Mean optical values were calculated from serum of individual animals. The figure shows the means ± standard deviations of optical density values representing the S/P ratio calculated as previously described. Specific antibodies were detected until the last determination, prior to the lethal challenge with NDV and were found to be over the S/P cutoff value of 0.3, one indicative of protective efficacy. Specific antibodies were not detected in the negative control groups. Statistically significant differences (p < 0.05) are represented in the figure with an asterisk. (B) Eight weeks after the primary immunization, all chickens were intramuscularly challenged with 0.5 × 106.5 ELD50 of the NDV isolate Debre/zeit/2018 (MN909678). A positive control group vaccinated with NDV live vaccine according to the manufacturer’s instructions as well as one group of non-vaccinated animals were included in the challenge. All chickens were monitored for 15 days to record the appearance of clinical signs of the disease and the total number of deaths. After this period, the percent survival was 100% for the groups vaccinated with the Ad-F-CMV and the NDV live vaccine, 80% for the group of chickens receiving the Ad-F-βactin vector, and 10% in the group vaccinated with the Ad-F-HN-CMV. One hundred percent of mortality and the typical signs of the disease were recorded in the group of non-immunized animals. Full article ">

21 pages, 3021 KiB

Open AccessArticle

Features of the Human Antibody Response against the Respiratory Syncytial Virus Surface Glycoprotein G

by Kristina Borochova, Katarzyna Niespodziana, Katarina Stenberg Hammar, Marianne van Hage, Gunilla Hedlin, Cilla Söderhäll, Margarete Focke-Tejkl and Rudolf Valenta

Vaccines 2020, 8(2), 337; https://doi.org/10.3390/vaccines8020337 - 25 Jun 2020

Cited by 5 | Viewed by 3547

Abstract

Respiratory syncytial virus (RSV) infections are a major cause of serious respiratory disease in infants. RSV occurs as two major subgroups A and B, which mainly differ regarding the surface glycoprotein G. The G protein is important for virus attachment and G-specific antibodies [...] Read more.

Respiratory syncytial virus (RSV) infections are a major cause of serious respiratory disease in infants. RSV occurs as two major subgroups A and B, which mainly differ regarding the surface glycoprotein G. The G protein is important for virus attachment and G-specific antibodies can protect against infection. We expressed the surface-exposed part of A2 strain-derived G (A2-G) in baculovirus-infected insect cells and synthesized overlapping peptides spanning complete A2-G. The investigation of the natural IgG response of adult subjects during a period of one year showed that IgG antibodies (i) recognize G significantly stronger than the fusion protein F0, (ii) target mainly non-conformational, sequential peptide epitopes from the exposed conserved region but also buried peptides, and (iii) exhibit a scattered but constant recognition profile during the observation period. The IgG subclass reactivity profile (IgG₁ > IgG₂ > IgG₄ = IgG₃) was indicative of a mixed Th1/Th2 response. Two strongly RSV-neutralizing sera including the 1st WHO standard contained high IgG anti-G levels. G-specific IgG increased strongly in children after wheezing attacks suggesting RSV as trigger factor. Our study shows that RSV G and G-derived peptides are useful for serological diagnosis of RSV-triggered exacerbations of respiratory diseases and underlines the importance of G for development of RSV-neutralizing vaccines. Full article

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14 pages, 1682 KiB

Open AccessArticle

How to Demonstrate Freedom from African Swine Fever in Wild Boar—Estonia as an Example

by Katja Schulz, Christoph Staubach, Sandra Blome, Imbi Nurmoja, Arvo Viltrop, Franz J. Conraths, Maarja Kristian and Carola Sauter-Louis

Vaccines 2020, 8(2), 336; https://doi.org/10.3390/vaccines8020336 - 25 Jun 2020

Cited by 23 | Viewed by 4043

Abstract

Estonia has been combatting African swine fever (ASF) for six years now. Since October 2017, the disease has only been detected in the wild boar population, but trade restrictions had to remain in place due to international regulations. Yet, the epidemiological course of [...] Read more.

Estonia has been combatting African swine fever (ASF) for six years now. Since October 2017, the disease has only been detected in the wild boar population, but trade restrictions had to remain in place due to international regulations. Yet, the epidemiological course of the disease has changed within the last few years. The prevalence of ASF virus (ASFV)-positive wild boar decreased steadily towards 0%. In February 2019, the last ASFV-positive wild boar was detected. Since then, positive wild boar samples have exclusively been positive for ASFV-specific antibodies, suggesting the possible absence of circulating ASFV in the Estonian wild boar population. However, as the role of seropositive animals is controversially discussed and the presence of antibody-carriers is regarded as an indication of virus circulation at EU and OIE level, Estonia remains under trade restrictions. To make the disease status of a country reliable for trading partners and to facilitate the process of declaration of disease freedom, we suggest to monitor the prevalence of seropositive wild boar in absence of ASFV-positive animals. The possibility to include ASF in the list of diseases, for which an official pathway for recognition of disease status is defined by the OIE should be evaluated. Full article

(This article belongs to the Special Issue African Swine Fever Virus Prevention and Control)

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33 pages, 3655 KiB

Open AccessReview

SARS-CoV-2: An Update on Potential Antivirals in Light of SARS-CoV Antiviral Drug Discoveries

by Hatem A. Elshabrawy

Vaccines 2020, 8(2), 335; https://doi.org/10.3390/vaccines8020335 - 23 Jun 2020

Cited by 36 | Viewed by 8859

Abstract

Coronaviruses (CoVs) are a group of RNA viruses that are associated with different diseases in animals, birds, and humans. Human CoVs (HCoVs) have long been known to be the causative agents of mild respiratory illnesses. However, two HCoVs associated with severe respiratory diseases [...] Read more.

Coronaviruses (CoVs) are a group of RNA viruses that are associated with different diseases in animals, birds, and humans. Human CoVs (HCoVs) have long been known to be the causative agents of mild respiratory illnesses. However, two HCoVs associated with severe respiratory diseases are Severe Acute Respiratory Syndrome-CoV (SARS-CoV) and Middle East Respiratory Syndrome-CoV (MERS-CoV). Both viruses resulted in hundreds of deaths after spreading to several countries. Most recently, SARS-CoV-2 has emerged as the third HCoV causing severe respiratory distress syndrome and viral pneumonia (known as COVID-19) in patients from Wuhan, China, in December 2019. Soon after its discovery, SARS-CoV-2 spread to all countries, resulting in millions of cases and thousands of deaths. Since the emergence of SARS-CoV, many research groups have dedicated their resources to discovering effective antivirals that can treat such life-threatening infections. The rapid spread and high fatality rate of SARS-CoV-2 necessitate the quick discovery of effective antivirals to control this outbreak. Since SARS-CoV-2 shares 79% sequence identity with SARS-CoV, several anti-SARS-CoV drugs have shown promise in limiting SARS-CoV-2 replication in vitro and in vivo. In this review, we discuss antivirals described for SARS-CoV and provide an update on therapeutic strategies and antivirals against SARS-CoV-2. The control of the current outbreak will strongly depend on the discovery of effective and safe anti-SARS-CoV-2 drugs. Full article

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Genomic organization of (A) Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV) and (B) SARS-CoV-2. The structure of SARS-CoV and SARS-CoV-2 genomes includes ORF1a and ORF1b occupying two-thirds of the genomes at the 5′ end. ORF1a and ORF1b are translated through a ribosomal frame shift sequence into two polyproteins 1a and 1b (pp1a and pp1ab) which are processed by 3CLpro and PLpro proteases to produce nonstructural proteins, including RdRp, that are important for viral replication. The other one-third of the genomes is comprised of ORFs that code for structural (S, M, E, and N) and nonstructural proteins. ORF: open reading frame; RdRp: RNA-dependent RNA polymerase; 3CLpro: picornavirus 3-chymotrypsin-like protease; PLpro: papain-like protease 2. Full article ">Figure 2
Chemical structures of SARS-CoV and SARS-CoV-2 viral entry inhibitors: (A) emodin, (B) imatinib, (C) tetra-O-galloyl-beta-D-glucose (TGG), (D) luteolin, (E) N-(2-aminoethyl)-1-aziridineethanamine (NAAE), (F) chloroquine, (G) chlorpromazine, (H) E-64D, (I) 5705213, (J) camostat mesylate, and (K) nafamostat. Full article ">Figure 3
Chemical structures of protease inhibitors: (A) lopinavir, (B) ritonavir, (C) nelfinavir, (D) cinanserin, (E) betulinic acid, (F) savinin, (G) dieckol, (H) herbacetin, (I) 6-mercaptopurine, (J) 6-thioguanine, (K) disulfiram, (L) danoprevir, and (M) darunavir. Full article ">Figure 4
Chemical structures of SARS-CoV and SARS-CoV-2 helicase and RdRp inhibitors: (A) myricetin, (B) scutellarein, (C) SSYA10-001, (D) β-D-N4-hydroxycitidine, (E) galidesivir, (F) 6-azauridine, (G) pyrazofurin, (H) ribavirin, (I) remdesivir, and (J) favipiravir. Full article ">Figure 5
Chemical structures of miscellaneous drugs against SARS-CoV and SARS-CoV-2: (A) hexmethylene amiloride, (B) aurintricarboxylic acid, (C) rimantadine, (D) niclosamide, (E) amiodarone, (F) DETA NONOate, (G) S-nitroso-N-acetylpenicillamine (SNAP), (H) cyclosporine A, (I) alisporivir, and (J) geldanamycin. Full article ">Figure 6
Chemical structures of miscellaneous drugs against SARS-CoV and SARS-CoV-2: (A) glycyrrhizin, (B) umifenovir, (C) nitazoxanide, (D) ruxolitinib, (E) baricitinib, (F) valsartan, and (G) telmisartan. Full article ">Figure 7
Potential therapeutics targeting different steps of the SARS-CoV-2 life cycle. The steps of the SARS-CoV-2 life cycle are (1) attachment to angiotensin-converting enzyme 2 (ACE2) on target cells, (2) entering the cell by endocytosis (cathepsin L-mediated cleavage of S protein) or through the plasma membrane (TMPRSS2-mediated cleavage of S protein), (3) uncoating and release of viral RNA, (4) translation of viral RNA 5’ end (ORF1a and ORF1b), (5) proteolysis of pp1a and pp1ab by 3CLpro and PLpro into nonstructural proteins, (6) formation of replicase complex, (7) replication of viral RNA, (8) transcription of viral genome into subgenomic mRNAs, (9) translation of subgenomic mRNAs into structural and nonstructural proteins, (10) assembly and budding of new viral particles through Golgi apparatus, and (11) exocytosis and exit of new viral particles out of the cell. Antivirals against SARS-CoV-2 include: entry inhibitors such as convalescent plasma and monoclonal antibodies (e.g., 47D11, HA001, B38, H4, and CR3022), chloroquine, camostat mesylate and nafamostat, cathepsin L inhibitors, and soluble ACE2; 3CLpro inhibitors such as HIV protease inhibitors (lopinavir, ritonavir, and darunavir) and the hepatitis C virus (HCV) protease inhibitor danoprevir; and RdRp inhibitors such as remdesivir, favipiravir, and galidesivir. ER; endoplasmic reticulum, ERGIC; endoplasmic reticulum–Golgi intermediate compartment, and ORF; open reading frame. Full article ">

15 pages, 758 KiB

Open AccessArticle

Characterization of Physicians That Might Be Reluctant to Propose HIV Cure-Related Clinical Trials with Treatment Interruption to Their Patients? The ANRS-APSEC Study

by Christel Protiere, Lisa Fressard, Marion Mora, Laurence Meyer, Marie Préau, Marie Suzan-Monti, Jean-Daniel Lelièvre, Olivier Lambotte, Bruno Spire and the APSEC Study Group

Vaccines 2020, 8(2), 334; https://doi.org/10.3390/vaccines8020334 - 23 Jun 2020

Cited by 5 | Viewed by 3178

Abstract

HIV cure-related clinical trials (HCRCT) with analytical antiretroviral treatment interruptions (ATIs) have become unavoidable. However, the limited benefits for participants and the risk of HIV transmission during ATI might negatively impact physicians’ motivations to propose HCRCT to patients. Between October 2016 and March [...] Read more.

HIV cure-related clinical trials (HCRCT) with analytical antiretroviral treatment interruptions (ATIs) have become unavoidable. However, the limited benefits for participants and the risk of HIV transmission during ATI might negatively impact physicians’ motivations to propose HCRCT to patients. Between October 2016 and March 2017, 164 French HIV physicians were asked about their level of agreement with four viewpoints regarding HCRCT. A reluctance score was derived from their answers and factors associated with reluctance identified. Results showed the highest reluctance to propose HCRCT was among physicians with a less research-orientated professional activity, those not informing themselves about cure trials through scientific literature, and those who participated in trials because their department head asked them. Physicians’ perceptions of the impact of HIV on their patients’ lives were also associated with their motivation to propose HCRCT: those who considered that living with HIV means living with a secret were more motivated, while those worrying about the negative impact on person living with HIV’s professional lives were more reluctant. Our study highlighted the need to design a HCRCT that minimizes constraints for participants and for continuous training programs to help physicians keep up-to-date with recent advances in HIV cure research. Full article

(This article belongs to the Special Issue Therapeutic Vaccination of HIV-infected Patients)

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19 pages, 8891 KiB

Open AccessArticle

Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

by José Manuel Rojas, Alí Alejo, Jose Miguel Avia, Daniel Rodríguez-Martín, Carolina Sánchez, Antonio Alcamí, Noemí Sevilla and Verónica Martín

Vaccines 2020, 8(2), 333; https://doi.org/10.3390/vaccines8020333 - 22 Jun 2020

Cited by 4 | Viewed by 3515

Abstract

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned [...] Read more.

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, OaOX40L and OaCD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of OaOX40L and OaCD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-κB signalling pathway through their cognate receptor. These adenovirus-secreted OaOX40L and OaCD70 forms could also activate ovine T cell proliferation and enhance IFN-γ production in CD4⁺ and CD8⁺ T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants. Full article

(This article belongs to the Section Cellular/Molecular Immunology)

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16 pages, 1678 KiB

Open AccessArticle

Nitric Oxide Production and Fc Receptor-Mediated Phagocytosis as Functional Readouts of Macrophage Activity upon Stimulation with Inactivated Poultry Vaccines In Vitro

by Robin H.G.A. van den Biggelaar, Willem van Eden, Victor P.M.G. Rutten and Christine A. Jansen

Vaccines 2020, 8(2), 332; https://doi.org/10.3390/vaccines8020332 - 22 Jun 2020

Cited by 7 | Viewed by 4873

Abstract

Vaccine batches must pass routine quality control to confirm that their ability to induce protection against disease is consistent with batches of proven efficacy from development studies. For poultry vaccines, these tests are often performed in laboratory chickens by vaccination-challenge trials or serological [...] Read more.

Vaccine batches must pass routine quality control to confirm that their ability to induce protection against disease is consistent with batches of proven efficacy from development studies. For poultry vaccines, these tests are often performed in laboratory chickens by vaccination-challenge trials or serological assays. The aim of this study was to investigate innate immune responses against inactivated poultry vaccines and identify candidate immune parameters for in vitro quality tests as alternatives for animal-based quality tests. For this purpose, we set up assays to measure nitric oxide production and phagocytosis by the macrophage-like cell line HD11, upon stimulation with inactivated poultry vaccines for infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and egg drop syndrome virus (EDSV). In both assays, macrophages became activated after stimulation with various toll-like receptor agonists. Inactivated poultry vaccines stimulated HD11 cells to produce nitric oxide due to the presence of mineral oil adjuvant. Moreover, inactivated poultry vaccines were found to enhance Fc receptor-mediated phagocytosis due to the presence of allantoic fluid in the vaccine antigen preparations. We showed that inactivated poultry vaccines stimulated nitric oxide production and Fc receptor-mediated phagocytosis by chicken macrophages. Similar to antigen quantification methods, the cell-based assays described here can be used for future assessment of vaccine batch-to-batch consistency. The ability of the assays to determine the immunopotentiating properties of inactivated poultry vaccines provides an additional step in the replacement of current in vivo batch-release quality tests. Full article

(This article belongs to the Special Issue Poultry Vaccines)

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Graphical abstract

Graphical abstract
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Inactivated poultry vaccines induced nitric oxide production by HD11 cells, whereas inactivated IBV and NDV antigens did not. (a) Nitric oxide production by HD11 cells was assessed upon stimulation with TLR agonists, i.e., 100 ng/mL LPS, 100 ng/mL CpG, and 10 μg/mL R848. (b) In addition, HD11 cells were exposed to inactivated IBV and NDV antigens (companies A and B) in doses ranging from 0.1–10 μL/mL. (c) Finally, HD11 cells were exposed to vaccines, an “empty vaccine” containing allantoic fluid without inactivated viruses, and mineral oil in doses ranging from 1–100 μL/mL. Three independent experiments were performed, and the experimental conditions of each independent experiment were tested in triplicate. Error bars represent the standard error of the mean (SEM). The experimental groups were tested for statistically significant increases in nitric oxide production as compared to unstimulated HD11 cells using a Kruskal–Wallis test and Dunn’s multiple comparisons test. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001. Full article ">Figure 2
TLR agonists stimulated the uptake of IgY-opsonized beads by HD11 cells. (a) Confocal microscopy confirmed the uptake of IgY-opsonized beads by HD11 cells. The surface of unstimulated HD11 cells was made visible by WGA-Alexa Fluor 488 shown in green and IgY-opsonized beads are shown in red. A corresponding video showing the 3-D model of this composition can be found in <a href="#app1-vaccines-08-00332" class="html-app">Supplementary Materials Video S1</a>. (b) Bead uptake by HD11 cells was quantified by flow cytometry. HD11 cells were gated for their scatter profile (FSC/SSC) and viability (zombie aqua live/dead staining). Moreover, HD11 cells with 1 bead/cell were gated to determine the fluorescence of a single bead, from which the average beads/cell for all HD11 cells could be calculated. (c) HD11 cells were stimulated with 300 ng/mL LPS, 500 ng/mL CpG, 10 µg/mL R848, 10 ng/mL Pam3CSK4 (Pam), 5 µg/mL zymosan (Zymo), or left unstimulated (Unst). The results are expressed as fold changes in bead uptake after stimulation in comparison to unstimulated controls. Three independent experiments were performed, and the experimental conditions of each independent experiment were tested in triplicate. Error bars represent the standard error of the mean (SEM). The experimental groups were tested for statistically significant differences in bead uptake between stimulated and unstimulated groups using a one-way ANOVA and Holm–Sidak’s multiple comparisons test. Statistical significance is indicated by *** p < 0.001. Full article ">Figure 3
Phagocytosis of IgY-opsonized beads by HD11 cells is increased upon exposure to inactivated IBV and NDV antigens. (a) Differences in bead uptake upon exposure to IBV and NDV antigens are expressed as fold changes compared to unstimulated controls. (b) The effects of IBV and NDV antigens on HD11 cell viability, as determined by Zombie Aqua Fixable Viability Dye, is expressed as the percentage of living cells (% alive). Inactivated IBV antigens were provided by three different companies (A–C) and inactivated NDV antigen was provided by one company (B). In addition, the effects of allantoic fluid without virus (provided by company A) on HD11 cell phagocytosis capacity (c) and cell viability (d) were determined. The x-axis shows the titrated doses at which IBV antigens, NDV antigens, or allantoic fluid without antigens were added, expressed as μL dose, added to 1 mL of cell culture medium. Three independent experiments were performed, and the experimental conditions of each independent experiment were tested in duplicate. Error bars represent the SEM. The experimental groups were tested for statistically significant differences in bead uptake and viability between stimulated and unstimulated groups using Kruskal–Wallis tests and Dunn’s multiple comparisons tests. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001. Full article ">Figure 4
Phagocytosis capacity of HD11 cells can be increased upon exposure to inactivated viral w/o vaccines. (a) The fold change in bead uptake by HD11 cells upon stimulation with vaccines is compared to unstimulated controls. The x-axis shows the graded doses at which the vaccines have been added, expressed as μL dose, added to 1 mL of cell culture medium. † indicates that datapoints were missing because the threshold of ≥100 viable cells was not reached. (b) Light microscopy photos show unstimulated HD11 cells (top), HD11 cells exposed to 10 μL/mL inactivated bivalent vaccine B (middle), and 100 μL/mL inactivated bivalent vaccine B (top). (c) A linear correlation curve shows the relationship between the average flow cytometric SSC and number of IgY-opsonized beads/cell for HD11 cells containing 0–4 beads/cell. (d) A non-linear saturation curve shows the relationship between the average SSC and different doses of empty vaccine (without viral antigens) from company B. (e) The flow cytometric SSC of HD11 cells is shown for graded doses of the different vaccines. Three independent experiments were performed, and the experimental conditions of each independent experiment were tested in duplicate. Error bars represent the SEM. The experimental groups were tested for statistically significant differences in bead uptake and SSC between stimulated and unstimulated groups using Kruskal–Wallis tests and Dunn’s multiple comparisons tests. Statistical significance is indicated by * p < 0.05. For figure (e), all data was found to be statistically different from the unstimulated sample with p < 0.001. Full article ">Figure 5
The induction of phagocytosis after exposure to inactivated IBV antigen is dependent on the IgY Fc receptor CHIR-AB1. (a) Representative histograms show CHIR-AB1 expression by HD11 cells after 24 h without stimulation, stimulation with 100 ng/mL LPS, or stimulation with 10 μL/mL IBV antigen. (b,c) CHIR-AB1 surface expression by HD11 cells was quantified and expressed as the geometric mean fluorescent intensity (gMFI) after 24 h stimulation with different concentrations of LPS (b) and IBV antigen (c). (d) Unstimulated HD11 cells and HD11 cells stimulated with LPS or inactivated IBV antigen for 24 h received the blocking antibody 8D12 specific for chicken CHIR-AB1 10 min before the addition of IgY-opsonized beads. The average number of phagocytosed beads per HD11 cell is shown for different concentrations of blocking antibody. Three independent experiments were performed, and the experimental conditions of each independent experiment were tested in duplicate. Error bars represent the SEM. The experimental groups were tested for statistically significant differences in CHIR-AB1 expression or bead uptake between stimulated and unstimulated groups using one-way ANOVA tests and Holm–Sidak’s multiple comparisons tests. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001. Full article ">

6 pages, 622 KiB

Open AccessCommunication

Risk Assessment and Virological Monitoring Following an Accidental Exposure to Concentrated Sabin Poliovirus Type 3 in France, November 2018

by Marion Jeannoël, Denise Antona, Clément Lazarus, Bruno Lina and Isabelle Schuffenecker

Vaccines 2020, 8(2), 331; https://doi.org/10.3390/vaccines8020331 - 22 Jun 2020

Cited by 3 | Viewed by 2830

Abstract

The safe and secure containment of infectious poliovirus (PV) in facilities where live PV are handled is the condition to achieve and maintain poliomyelitis eradication. Despite precautions to minimize the risk of release of PV from such facilities to the environment, breaches of [...] Read more.

The safe and secure containment of infectious poliovirus (PV) in facilities where live PV are handled is the condition to achieve and maintain poliomyelitis eradication. Despite precautions to minimize the risk of release of PV from such facilities to the environment, breaches of containment have already been documented. Here, we report the management of an incident that occurred on 30 November 2018 in a French vaccine manufacturing plant. Five adequately vaccinated operators were exposed to a Sabin poliovirus type 3 (PV3) spill. A microbiological risk assessment was conducted and the operators were monitored for PV shedding. On day 5 after exposure, Sabin PV3 was detected only in the stool sample of the most exposed worker. Shedding of Sabin PV3 (as detected by viral culture) was restricted to a very short period (less than 15 days). Monitoring of this incident was an opportunity to assess the relevance of our national response plan. We concluded that the measures undertaken and reported here were appropriate and proportional. Full article

(This article belongs to the Special Issue Vaccination and Public Health: Optimizing Vaccine Uptake through the Application of Social and Behavioral Science Theory, Principles, and Strategies)

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19 pages, 2291 KiB

Open AccessArticle

Limited Cross-Protection against Infectious Bronchitis Provided by Recombinant Infectious Bronchitis Viruses Expressing Heterologous Spike Glycoproteins

by Sarah Keep, Samantha Sives, Phoebe Stevenson-Leggett, Paul Britton, Lonneke Vervelde and Erica Bickerton

Vaccines 2020, 8(2), 330; https://doi.org/10.3390/vaccines8020330 - 22 Jun 2020

Cited by 10 | Viewed by 4773

Abstract

Gammacoronavirus infectious bronchitis virus (IBV) causes an economically important respiratory disease of poultry. Protective immunity is associated with the major structural protein, spike (S) glycoprotein, which induces neutralising antibodies and defines the serotype. Cross-protective immunity between serotypes is limited and can be difficult [...] Read more.

Gammacoronavirus infectious bronchitis virus (IBV) causes an economically important respiratory disease of poultry. Protective immunity is associated with the major structural protein, spike (S) glycoprotein, which induces neutralising antibodies and defines the serotype. Cross-protective immunity between serotypes is limited and can be difficult to predict. In this study, the ability of two recombinant IBV vaccine candidates, BeauR-M41(S) and BeauR-4/91(S), to induce cross-protection against a third serotype, QX, was assessed. Both rIBVs are genetically based on the Beaudette genome with only the S gene derived from either M41 or 4/91, two unrelated serotypes. The use of these rIBVs allowed for the assessment of the potential of M41 and 4/91 S glycoproteins to induce cross-protective immunity against a heterologous QX challenge. The impact of the order of vaccination was also assessed. Homologous primary and secondary vaccination with BeauR-M41(S) or BeauR-4/91(S) resulted in a significant reduction of infectious QX load in the trachea at four days post-challenge, whereas heterologous primary and secondary vaccination with BeauR-M41(S) and BeauR-4/91(S) reduced viral RNA load in the conjunctiva-associated lymphoid tissue (CALT). Both homologous and heterologous vaccination regimes reduced clinical signs and birds recovered more rapidly as compared with an unvaccinated/challenge control group. Despite both rIBV BeauR-M41(S) and BeauR-4/91(S) displaying limited replication in vivo, serum titres in these vaccinated groups were higher as compared with the unvaccinated/challenge control group. This suggests that vaccination with rIBV primed the birds for a boosted humoral response to heterologous QX challenge. Collectively, vaccination with the rIBV elicited limited protection against challenge, with failure to protect against tracheal ciliostasis, clinical manifestations, and viral replication. The use of a less attenuated recombinant vector that replicates throughout the respiratory tract could be required to elicit a stronger and prolonged protective immune response. Full article

(This article belongs to the Special Issue Development of Cross-Protective Vaccines)

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Figure 1
Schematic detailing protocol for the in vivo heterologous vaccine-challenge experiment. Groups of eight-day-old specific-pathogen-free (SPF) Rhode Island Red (RIR) chickens received a primary vaccination of BeauR-M41(S), BeauR-4/91(S), or phosphate buffered saline (PBS). Two weeks (14 days) later, birds received a second vaccination of either BeauR-M41(S), BeauR-4/91(S), or PBS. Nine days post-secondary vaccination (dpsv), birds were challenged with QX or mock challenged with PBS. Clinical signs were assessed for both post-vaccination and post-challenge birds. At defined intervals, randomly chosen birds were culled from each group and a variety of tissues harvested. Serum was collected pre-vaccination, post-vaccination (pre-challenge), and post-challenge. Tracheal ciliary activity was assessed at 4 days post-primary vaccination (dppv) and 4 days post-challenge (dpc). The experiment ended at 14 dpc with all remaining birds culled. Full article ">Figure 2
The S gene from a pathogenic strain does not confer virulence to a non-pathogenic strain. SPF birds, at eight days of age, were vaccinated with either BeauR-M41(S), BeauR-4/91(S), or PBS for mock vaccination. (a) The number of snicks in each group was assessed from day three to seven days post-primary vaccination (dppv). The numbers of snicks were independently counted over a two-minute period by two or three persons with the average (mean) of these scores presented; (b) Trachea was harvested from five randomly sampled birds four dppv. Each trachea was sectioned in 10 × 1 mm rings and the ciliary activity of each ring was assessed by light microscopy and the percentage activity calculated. Plotted points represent individual birds and the mean activity of the 10 rings assessed. Error bars represent standard error of the mean (SEM). Statistical differences were evaluated using a Kruskal–Wallis test followed by a post hoc Mann–Whitney test corrected for multiple comparisons; no differences were identified; (c) Fourteen days post-primary vaccination, birds received a secondary vaccination of either BeauR-M41(S), BeauR-4/91(S), or PBS for mock vaccination. The numbers of snicks in each group were independently counted over a two-minute period by two or three persons from day three to seven post-secondary vaccination (dpsv) with the average (mean) of these scores presented. Snicking post-secondary vaccination was comparable between the vaccinated groups and the mock vaccinated groups. Full article ">Figure 3
Vaccination with BeauR-M41(S) or BeauR-4/91(S) reduced the clinical signs observed after challenge with QX but was not able to protect against tracheal ciliostasis. Nine days post-secondary vaccination, birds were challenged with QX or mock challenged with PBS. (a) The numbers of snicks in each group were assessed from 2 to 7 dpc. Snicks were independently counted by two or three persons over a two-minute period with the average of these scores presented; (b) Birds were individually assessed for tracheal rales from 2 to 7 dpc. The percentage of birds per group positive for rales was calculated; (c) Trachea was harvested from 5 or 10 randomly sampled birds at 4 dpc. Each trachea was sectioned in 10 × 1 mm rings and the ciliary activity of each ring was assessed by light microscopy and the percentage activity calculated. Plotted points represent individual birds and the mean activity of the 10 rings assessed. Error bars represent SEM. Statistical differences were evaluated using a Kruskal–Wallis test followed by a post Hoc Mann–Whitney test corrected for multiple comparisons and are highlighted by * (p < 0.0001). Ciliary activity in all groups except BeauR-M41(S)/BeauR-4/91(S) was significantly reduced as compared with the mock vaccinated/mock challenged (Mock/Mock/Mock) group; ciliary activity in all vaccinated groups was comparable to the mock vaccinated/challenged (Mock/Mock/QX) control group. Full article ">Figure 4
Vaccination with BeauR-M41(S)/BeauR-M41(S) and BeauR-4/91(S)/BeauR-4/91(S) resulted in a reduction in infectious viral load in response to the QX challenge within the trachea 4 dpc. Tissue-derived supernatant prepared from tracheas harvested at 4 dpc were titrated in ex vivo tracheal organ cultures (TOCs). Data points represent individual birds, with lines representing the mean and standard deviation (SD). Statistical differences between vaccinated groups and mock vaccinated/challenged control group (Mock/Mock/QX) highlighted by * (p < 0.05) were evaluated using a one–way ANOVA with a Tukey test for multiple comparisons. Full article ">Figure 5
Vaccination with BeauR/M41(S)/BeauR-4/91(S) and BeauR-4/91(S)/BeauR-M41(S) resulted in a reduction in viral load in response to a QX challenge in the conjunctiva-associated lymphoid tissue (CALT) at 4 dpc. Relative viral RNA loads (expressed as corrected 40 cycle threshold) were assessed at specific time-points post-challenge. (a) Harderian gland at 2 dpc; (b) CALT at 2 dpc; (c) Harderian gland at 4 dpc; (d) CALT at 4 dpc. Data points are shown as the mean of three technical replicates per individual bird. Lines represent group mean and SEM. Statistical differences between the groups were evaluated using a one-way ANOVA with a TUKEY test for multiple comparisons. Significant differences between vaccinated groups and the mock vaccinated/challenged control group (Mock/Mock/QX) are highlighted by * (p < 0.05). Full article ">Figure 6
Vaccination with recombinant infectious bronchitis virus (rIBV) BeauR-M41(S) and BeauR-4/91(S) induced the production of IBV-specific antibodies and primed chickens for a boosted humoral response to challenge. Serum anti-IBV antibody titres were assessed by commercial ELISA (BioCheck, Reeuwijk, The Netherlands). (a) Pre-challenge serum samples were diluted 1:80 and dilutions 1:80 through to 1:2560 were investigated for (b) 4 dpc serum samples and (c) 14 dpc serum samples. The mean S/P of four technical replicates of each bird from each group is presented. The cut-off threshold for positive samples is S/P ratio = 0.2. The error bars represent SD. Statistical differences between the group antibody titre means at (a) pre-challenge were assessed using Kruskal–Wallis and Dunn’s multiple comparison test. Post-challenge (b) and (c), antibody titres were assessed using a one-way ANOVA with a Friedman test and Dunn’s multiple comparison test. Significant differences between vaccinated groups and the mock vaccinated/challenged (Mock/Mock/QX) control group are highlighted by * (p < 0.05), ** (p < 0.01), and **** (p < 0.0001). Full article ">

17 pages, 986 KiB

Open AccessReview

Modulation of Anti-Tumour Immune Responses by Probiotic Bacteria

by Georgios Aindelis and Katerina Chlichlia

Vaccines 2020, 8(2), 329; https://doi.org/10.3390/vaccines8020329 - 21 Jun 2020

Cited by 21 | Viewed by 6001

Abstract

There is a growing amount of evidence to support the beneficial role of a balanced intestinal microbiota, or distinct members thereof, in the manifestation and progression of malignant tumours, not only in the gastrointestinal tract but also in distant tissues as well. Intriguingly, [...] Read more.

There is a growing amount of evidence to support the beneficial role of a balanced intestinal microbiota, or distinct members thereof, in the manifestation and progression of malignant tumours, not only in the gastrointestinal tract but also in distant tissues as well. Intriguingly, bacterial species have been demonstrated to be indispensable modulatory agents of widely-used immunotherapeutic or chemotherapeutic regiments. However, the exact contribution of commensal bacteria to immunity, as well as to neoplasia formation and response to treatment, has not been fully elucidated, and most of the current knowledge acquired from animal models has yet to be translated to human subjects. Here, recent advances in understanding the interaction of gut microbes with the immune system and the modulation of protective immune responses to cancer, either naturally or in the context of widely-used treatments, are reviewed, along with the implications of these observations for future therapeutic approaches. In this regard, bacterial species capable of facilitating optimal immune responses against cancer have been surveyed. According to the findings summarized here, we suggest that strategies incorporating probiotic bacteria and/or modulation of the intestinal microbiota can be used as immune adjuvants, aiming to optimize the efficacy of cancer immunotherapies and conventional anti-tumour treatments. Full article

(This article belongs to the Section Cancer Vaccines and Immunotherapy)

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9 pages, 1295 KiB

Open AccessArticle

Seroprevalence of Neutralizing Antibodies against Japanese Encephalitis Virus among Adolescents and Adults in Korea: A Prospective Multicenter Study

by Byung Ok Kwak, Young Se Kwon, Young Jin Hong, Chung Hyun Nahm, Woori Jang, Young Uh, Yong Gon Cho, Jimyung Kim, Myungshin Kim and Dong Hyun Kim

Vaccines 2020, 8(2), 328; https://doi.org/10.3390/vaccines8020328 - 19 Jun 2020

Cited by 8 | Viewed by 3400

Abstract

The immunization schedule for the Japanese encephalitis (JE) vaccine in Korea is a two-dose primary series at 12–24 months of age, followed by booster doses 12 months after the second dose and at the ages of 6 and 12 years. Although the number [...] Read more.

The immunization schedule for the Japanese encephalitis (JE) vaccine in Korea is a two-dose primary series at 12–24 months of age, followed by booster doses 12 months after the second dose and at the ages of 6 and 12 years. Although the number of JE cases has markedly decreased after the universal vaccination program, JE predominantly occurs in adults. The aim of this study was to assess the age-specific prevalence of the JE-neutralizing antibody (NTAb) among adolescents and adults in Korea. A total of 1603 specimens were collected from a healthy Korean population above 15 years old in five provinces. The JE-NTAb titers were measured with the pseudotyped virus assay and considered to be positive at ≥ 1:50. The seropositivity of JE-NTAb was the highest in the 15–29 years category (>95%) and gradually began to decrease in the age group of 30–44 years (89.42%). The lowest and second lowest JE-NTAb seropositive rates were observed among those aged 70 years or older (59.77%) and those aged 55–59 years (75.24%), respectively. Subjects from Seoul exhibited the highest JE-NTAb titer in all age groups compared to other provinces. In conclusion, the JE-NTAb seropositive rates and titers have maintained appropriate levels in the general Korean population. We propose that adult immunization and boosters at 12 years of age against JE are not strongly recommended in Korea. Full article

(This article belongs to the Section Attenuated/Inactivated/Live and Vectored Vaccines)

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18 pages, 640 KiB

Open AccessArticle

Trends, Coverage and Influencing Determinants of Influenza Vaccination in the Elderly: A Population-Based National Survey in Spain (2006–2017)

by Silvia Portero de la Cruz and Jesús Cebrino

Vaccines 2020, 8(2), 327; https://doi.org/10.3390/vaccines8020327 - 19 Jun 2020

Cited by 17 | Viewed by 3852

Abstract

Influenza is a significant public health problem and the elderly are at a greater risk of contracting the disease. The vaccination coverage of the elderly is below the Spanish target of 65% for each influenza season. The aims of this study were to [...] Read more.

Influenza is a significant public health problem and the elderly are at a greater risk of contracting the disease. The vaccination coverage of the elderly is below the Spanish target of 65% for each influenza season. The aims of this study were to report the coverage of influenza vaccination in Spain among the population aged ≥65 years and high-risk groups for suffering chronic diseases, to analyze the time trends from 2006 to 2017 and to identify the factors which affect vaccination coverage. A nationwide cross-sectional study was conducted including 20,753 non-institutionalized individuals aged ≥65 years who had participated in the Spanish National Health Surveys in 2006, 2011/2012, and 2017. Sociodemographic, health-related variables, and influenza vaccination data were used. A logistic regression analysis was performed to determine the variables associated with influenza vaccination. Influenza vaccination coverage was 60%. By chronic condition, older people with high cholesterol levels and cancer had the lowest vaccination coverage (62.41% and 60.73%, respectively). This coverage declined from 2006 to 2017 in both groups. Higher influenza vaccination was associated with males, Spanish nationality, normal social support perceived, polypharmacy, worse perceived health, participation in other preventive measures, and increasing age and the number of chronic diseases. Full article

(This article belongs to the Special Issue Vaccination and Public Health: Optimizing Vaccine Uptake through the Application of Social and Behavioral Science Theory, Principles, and Strategies)

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21 pages, 5934 KiB

Open AccessArticle

IFN-I Independent Antiviral Immune Response to Vesicular Stomatitis Virus Challenge in Mouse Brain

by Anurag R. Mishra, Siddappa N. Byrareddy and Debasis Nayak

Vaccines 2020, 8(2), 326; https://doi.org/10.3390/vaccines8020326 - 19 Jun 2020

Cited by 7 | Viewed by 5194

Abstract

Type I interferon (IFN-I) plays a pivotal role during viral infection response in the central nervous system (CNS). The IFN-I can orchestrate and regulate most of the innate immune gene expression and myeloid cell dynamics following a noncytopathic virus infection. However, the role [...] Read more.

Type I interferon (IFN-I) plays a pivotal role during viral infection response in the central nervous system (CNS). The IFN-I can orchestrate and regulate most of the innate immune gene expression and myeloid cell dynamics following a noncytopathic virus infection. However, the role of IFN-I in the CNS against viral encephalitis is not entirely clear. Here we have implemented the combination of global differential gene expression profiling followed by bioinformatics analysis to decipher the CNS immune response in the presence and absence of the IFN-I signaling. We observed that vesicular stomatitis virus (VSV) infection induced 281 gene changes in wild-type (WT) mice primarily associated with IFN-I signaling. This was accompanied by an increase in antiviral response through leukocyte vascular patrolling and leukocyte influx along with the expression of potent antiviral factors. Surprisingly, in the absence of the IFN-I signaling (IFNAR^−/− mice), a significantly higher (1357) number of genes showed differential expression compared to the WT mice. Critical candidates such as IFN-γ, CCL5, CXCL10, and IRF1, which are responsible for the recruitment of the patrolling leukocytes, are also upregulated in the absence of IFN-I signaling. The computational network analysis suggests the presence of the IFN-I independent pathway that compensates for the lack of IFN-I signaling in the brain. The analysis shows that TNF-α is connected maximally to the networked candidates, thus emerging as a key regulator of gene expression and recruitment of myeloid cells to mount antiviral action. This pathway could potentiate IFN-γ release; thereby, synergistically activating IRF1-dependent ISG expression and antiviral response. Full article

(This article belongs to the Special Issue Virus Immune Escape and Host Immune System)

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Vesicular stomatitis virus (VSV) infection in the central nervous system (CNS) induces fatal encephalitis in wild type mice. (A) B6 mice (n = 4 per group) were injected intracerebrally with the designated doses of VSV to induce encephalitis. Intracardially perfused mouse brain was collected after 72 hpi to estimate brain viral load and to collect total cellular RNA. Total cellular RNA was later used for gene microarray analysis as well as for validation of real-time PCR assays. (B) Kaplan–Meier survival curve of animals challenged with the varying doses of VSV (doses represented in plaque-forming units (PFU) of VSV are shown in the panel to the right of the graph). (C) In this experiment, all animals were intracerebrally injected with 104 PFU of VSV, and brain viral loads were determined by plaque assay technique conducted in Vero cells at said time points and reported as PFU of VSV per gram of tissue. Full article ">Figure 2
VSV infection in CNS induces microglia proliferation and myeloid cell recruitment. Mice were either infected with 104 PFU of VSV or mock-infected. At day two post-infection, the total CNS myeloid cell populations were analyzed by flow cytometry. (A) Representative dot plot showing the percentage increase in the number of microglia (CD45low, Thy1.2−, Gr1, and CD11b+) population in VSV-infected mice brain (n = 4 per group). An asterisk denotes statistical significance, *** denotes p value < 0.001. (B) The bar graph represents the absolute count of myeloid cells obtained from mice brain (n = 4): microglia, neutrophils (CD45low, Thy.12−, CD11b+ Gr1+, and Ly6Cmed), monocytes, and macrophages (CD45low, Thy.12−, Gr1−, CD11b+, and Ly6Chigh) in the CNS of mice (n = 4 per group) following VSV infection. Note an increase in the CNS myeloid cell number in VSV-infected animals compared to the mock-infected animals (p > 0.05). (C) The two-photon light scanning microscopy (TPLSM) was performed through a surgically thinned skull window in mock-infected LysM-GFP reporter mice (upper row) and compared to VSV-infected mice at day three post-infection (lower row). Representative figure showing an increase in the number of cells infiltrated in the brain parenchyma (see corresponding <a href="#app1-vaccines-08-00326" class="html-app">movies Videos S1 and S2</a>). Blood vessels are represented in red, and macrophages, monocyte, and neutrophils are represented in green. Please note the presence of prominent vascular leakage (damaged blood vessels in red) in the VSV-infected brain. Surrounding this, a cluster of infiltrating cells are observed at the beginning of the movie; these cells later shifted to elsewhere over a time period of 4 h. Full article ">Figure 3
VSV infection in the brain induces IFN-I-driven antiviral response. Microarray analysis was performed on the total RNA extracted from the mice brain (n = 4) tissue at day three post-infection and compared to the mock-infected mice. (A) A representative bar graph showing the top 10 differentially expressed gene candidates (blue represent upregulation while red represent downregulation). (B) Few selected genes falling to the category of antiviral response (STAT1, IRF-1, CXCL10, IFN-γ, and Viperin) were validated by RT-PCR experiments. The fold change in gene expression was calculated by normalizing the data with the housekeeping gene (β-actin) as an internal control from the same sample using ∆∆Ct method. * denotes p-value < 0.05. (C) Ingenuity pathway analysis tool was used to predict the top biological canonical pathways corresponding to the gene expression profile and are represented here in the bar graph. The negative log p values are plotted on the graph X-axis. Full article ">Figure 4
VSV infection induces a massive change in the gene expression in the absence of type I interferon. Ifnar−/− mice (n = 4 per group) were intracerebrally infected with the VSV to induce encephalitis, and comparative CNS gene microarray analysis was performed along with the wild type mice. (A) The upper subset represents a Kaplan–Meier survival curve of animals, which shows a marginal delay in onset death in Ifnar−/− mice. While the lower subset represents the brain viral titers determined by plaque assay, which shows a significant increase (p < 0.05) in brain viral load in Ifnar−/− mice. * denotes p-value < 0.05 (B) Global heat map depicting the change in gene expression patterns and clustered based on functional relatedness (see corresponding <a href="#app1-vaccines-08-00326" class="html-app">Table S1</a>). The change in expression level is depicted by blue (upregulation pattern), red (downregulation pattern), and gray (no change in the pattern). Full article ">Figure 5
VSV infection in the brain induces type I IFN signaling-independent innate immune pathways. Microarray analysis was performed on the total RNA extracted from the mice brains at day three post-infection along with mock-infected control. (A) A representative bar graph showing the top 10 candidates differentially (upregulated in blue and downregulated gene in red). (B) Ingenuity pathway analysis tool was used to find the top biological pathway in the VSV-infected mice brains and are represented in the bar graph. The negative log p values are plotted on the graph X-axis. (C) A few selected genes were subjected to qRT-PCR analysis to validate the temporal change in the expression observed in microarray analysis, e.g., CXCL10, CCL-5 IRFs, BST-2, Viperin, etc. The fold change in these genes was calculated by normalizing the data with housekeeping gene (β-actin) as an internal control from the same sample using ∆∆Ct method. Full article ">Figure 6
Massive gene expression in the absence of IFN-I signaling during VSV infection. Representative heat maps are shown to differentially regulate genes (p < 0.05, fold expression >1.5) in the absence of the IFN-I signaling associated with the (A) IFN signaling, (B), nervous system, (C), innate immune response, and (D) olfactory related signaling genes at day two post-infection. The aforementioned function was assigned to a gene using the Ingenuity Pathway Analysis (IPA) analysis tool. Blue nodes = upregulated genes, red nodes = downregulated genes, gray nodes = no change. Full article ">Figure 7
Network analysis of the up-regulated gene in Ifnar−/− mice reveals a TNF to be a crucial regulator for antiviral immune response. The figure depicts a modular network formed using the upregulated genes. (A) protein-to-protein interaction (PPI) network was developed using the STRING (Search Tool for the Retrieval of Interacting Gene) database. The interaction was divided into four groups using the K-means clustering tool from the STRING database. Each group defines the essential unique function: group 1 includes metabolic pathway genes, group 2 comprises olfactory related gene, group 3 represents the protein translation gene, and group 4 contains antiviral response-related genes. (B) Later, group 4 genes representing the antiviral response were used for the interactome generation by using the STRING database. The network shows the maximum connectivity patterns where Jun was found to have the highest number of connective nodes, followed by that of TNF-α. Full article ">Figure 7 Cont.
Network analysis of the up-regulated gene in Ifnar−/− mice reveals a TNF to be a crucial regulator for antiviral immune response. The figure depicts a modular network formed using the upregulated genes. (A) protein-to-protein interaction (PPI) network was developed using the STRING (Search Tool for the Retrieval of Interacting Gene) database. The interaction was divided into four groups using the K-means clustering tool from the STRING database. Each group defines the essential unique function: group 1 includes metabolic pathway genes, group 2 comprises olfactory related gene, group 3 represents the protein translation gene, and group 4 contains antiviral response-related genes. (B) Later, group 4 genes representing the antiviral response were used for the interactome generation by using the STRING database. The network shows the maximum connectivity patterns where Jun was found to have the highest number of connective nodes, followed by that of TNF-α. Full article ">

14 pages, 265 KiB

Open AccessArticle

Immunogenicity Measures of Influenza Vaccines: A Study of 1164 Registered Clinical Trials

by Alexander Domnich, Ilaria Manini, Donatella Panatto, Giovanna Elisa Calabrò and Emanuele Montomoli

Vaccines 2020, 8(2), 325; https://doi.org/10.3390/vaccines8020325 - 19 Jun 2020

Cited by 13 | Viewed by 3359

Abstract

Influenza carries an enormous burden each year. Annual influenza vaccination is the best means of reducing this burden. To be clinically effective, influenza vaccines must be immunogenic, and several immunological assays to test their immunogenicity have been developed. This study aimed to describe [...] Read more.

Influenza carries an enormous burden each year. Annual influenza vaccination is the best means of reducing this burden. To be clinically effective, influenza vaccines must be immunogenic, and several immunological assays to test their immunogenicity have been developed. This study aimed to describe the patterns of use of the various immunological assays available to measure the influenza vaccine-induced adaptive immune response and to determine its correlates of protection. A total of 76.5% of the studies included in our analysis measured only the humoral immune response. Among these, the hemagglutination-inhibition assay was by far the most widely used. Other, less common, humoral immune response assays were: virus neutralization (21.7%), enzyme-linked immunosorbent (10.1%), single radial hemolysis (4.6%), and assays able to quantify anti-neuraminidase antibodies (1.7%). By contrast, cell-mediated immunity was quantified in only 23.5% of studies. Several variables were significantly associated with the use of single assays. Specifically, some influenza vaccine types (e.g., adjuvanted, live attenuated and cell culture-derived or recombinant), study phase and study sponsorship pattern were usually found to be statistically significant predictors. We discuss the principal findings and make some suggestions from the point of view of the various stakeholders. Full article

(This article belongs to the Special Issue Progress on Seasonal and Pandemic Influenza Vaccines)

12 pages, 2239 KiB

Open AccessArticle

The Effect of Vaccination with Live Attenuated Neethling Lumpy Skin Disease Vaccine on Milk Production and Mortality—An Analysis of 77 Dairy Farms in Israel

by Michal Morgenstern and Eyal Klement

Vaccines 2020, 8(2), 324; https://doi.org/10.3390/vaccines8020324 - 19 Jun 2020

Cited by 14 | Viewed by 5206

Abstract

Lumpy skin disease (LSD) is an economically important, arthropod borne viral disease of cattle. Vaccination by the live attenuated homologous Neethling vaccine was shown as the most efficient measure for controlling LSD. However, adverse effects due to vaccination were never quantified in a [...] Read more.

Lumpy skin disease (LSD) is an economically important, arthropod borne viral disease of cattle. Vaccination by the live attenuated homologous Neethling vaccine was shown as the most efficient measure for controlling LSD. However, adverse effects due to vaccination were never quantified in a controlled field study. The aim of this study was to quantify the milk production loss and mortality due to vaccination against LSD. Daily milk production, as well as culling and mortality, were retrieved for 21,844 cows accommodated in 77 dairy cattle farms in Israel. Adjusted milk production was calculated for each day during the 30 days post vaccination. This was compared to the preceding month by fitting mixed effects linear models. Culling and mortality rates were compared between the 60 days periods prior and post vaccination, by survival analysis. The results of the models indicate no significant change in milk production during the 30 days post vaccination period. No difference was observed between the pre- and post-vaccination periods in routine culling, as well as in immediate culling and in-farm mortality. We conclude that adverse effects due to Neethling vaccination are negligible. Full article

(This article belongs to the Special Issue Controlled Clinical Evaluation of Veterinary Vaccines)

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Overall number of observations and cows available for milk production analysis in each lactation group in 77 farms. Full article ">Figure 2
Overall number of observations and cows available for survival analysis in each lactation group in 73 farms. Full article ">Figure 3
The estimated daily milk change (Kg) per cow in 73 dairy cattle farms in Israel, during a 30-day period after vaccination. Linear mixed effects model was fitted to the milk production gaps (MPG) for each cow with distance from vaccination day (DIV) as a fixed variable and the farm as a random variable. This was performed separately for lactation group 1 (Lac 1), lactation group 2 (Lac 2), lactation group >2 (Lac > 2), and all lactation groups combined (All lactations). For specific details on the model, see text. Full article ">Figure 4
The estimated daily milk change (Kg) per cow in 4 dairy cattle farms in Israel, during a 30-day period after vaccination. Linear mixed effects model was fitted to the milk production gaps (MPG) for each cow with distance from vaccination day (DIV) as a fixed variable and the farm as a random variable. This was performed separately for lactation group 1 (Lac 1-naive), lactation group 2 (Lac 2-mixed: naïve and vaccinated), lactation group >2 (Lac > 2-vaccinated), and all lactations groups combined (All lactations). For specific details on the model, see text. Full article ">Figure 5
Kaplan-Meier survival curves as a function of vaccination status in 73 dairy cattle farms in Israel. (a) Event defined as in-farm death or urgent culling. (b) Event defined as in-farm death, urgent culling, or routine culling. Full article ">

14 pages, 5253 KiB

Open AccessArticle

Liver Stiffness Hinders Normalization of Systemic Inflammation and Endothelial Activation after Hepatitis C Virus (HCV) Eradication in HIV/HCV Coinfected Patients

by Beatriz Álvarez, Clara Restrepo, Marcial García, María A. Navarrete-Muñoz, María A. Jiménez-Sousa, Laura Prieto, Alfonso Cabello, Sara Nistal, Salvador Resino, Miguel Górgolas, Norma Rallón and José M. Benito

Vaccines 2020, 8(2), 323; https://doi.org/10.3390/vaccines8020323 - 19 Jun 2020

Cited by 6 | Viewed by 3037

Abstract

Systemic inflammation, endothelial dysfunction and coagulopathy are of high clinical relevance in the management of people living with HIV (PLWH), and even more in patients coinfected with hepatitis C virus (HCV). It has been suggested a significant impact of HCV coinfection on these [...] Read more.

Systemic inflammation, endothelial dysfunction and coagulopathy are of high clinical relevance in the management of people living with HIV (PLWH), and even more in patients coinfected with hepatitis C virus (HCV). It has been suggested a significant impact of HCV coinfection on these conditions. However, HCV can be eradicated in most patients with the new direct-acting antivirals (DAAs) therapy. We have analyzed the effect of HCV on systemic inflammation, endothelial activation and coagulopathy in PLWH and its evolution after HCV eradication with DAAs. Twenty-five HIV/HCV coinfected (HIV/HCV group), 25 HIV monoinfected (HIV group) and 20 healthy controls (HC) were included in the study. All patients were on ART and HIV suppressed. Levels of fourteen markers of systemic inflammation, endothelial activation and coagulopathy (IL-1ß, IL-6, IL-12p70, IL-8, TNFα, D-dimer, Eotaxin, IL-18, IP-10, monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1), TNFα receptor 1 (TNFR1), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1)) were measured on plasma at baseline and after DAAs-mediated HCV eradication. Non-parametric tests were used to establish inter/intra-group differences. At baseline, the HIV/HCV group showed increased levels of IL-18 (p = 0.028), IP-10 (p < 0.0001), VCAM-1 (p < 0.0001) and ICAM-1 (p = 0.045), compared to the HC and HIV groups, with the highest levels for IL18 and IP10 observed in HIV/HCV patients with increased liver stiffness (≥7.1 KPa). Eradication of HCV with DAAs-based therapy restored some but not all the evaluated parameters. VCAM-1 remained significantly increased compared to HC (p = 0.001), regardless of the level of basal liver stiffness in the HIV/HCV group, and IP-10 remained significantly increased only in the HIV/HCV group, with increased level of basal liver stiffness compared to the HC and to the HIV groups (p = 0.006 and p = 0.049, respectively). These data indicate that DAAs therapy in HIV/HCV co-infected patients and HCV eradication does not always lead to the normalization of systemic inflammation and endothelial dysfunction conditions, especially in cases with increased liver stiffness. Full article

(This article belongs to the Special Issue Research on Innate Immunity and Inflammation)

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Box-plots graphs showing the levels of IL-18 (A), IP-10 (B), vascular cell adhesion molecule 1 (VCAM-1) (C) and intercellular adhesion molecule 1 (ICAM-1) (D) in healthy controls (HC) donors, HIV monoinfected (HIV), and in HIV/ hepatitis C virus (HCV) coinfected patients before direct-acting antivirals (DAAs) treatment (pre-DAAs HIV/HCV). p-values for global comparison between the groups (Kruskal-Wallis test, K-W) are shown. (*): p < 0.05 with respect to HC donors (Mann-Whitney U test). (¶): p < 0.05 with respect to the HIV group (Mann-Whitney U test). Full article ">Figure 2
Box-plots graphs showing the levels of IL-18 (A), IP-10 (B), VCAM-1 (C) and ICAM-1 (D) in HC donors, HIV monoinfected, and HIV/HCV coinfected patients at baseline (before DAAs treatment) stratified according to level of liver stiffness (LS). p-values for global comparison between the groups (Kruskal-Wallis test, K-W) and for the comparison between the two groups of HIV/HCV patients (Mann-Whitney U test) are shown. (*): p < 0.05 with respect to HC donors (Mann-Whitney U test). (¶): p < 0.05 with respect to the HIV group (Mann-Whitney U test). Full article ">Figure 3
Box-plots graphs showing the levels of IL-18 (A), IP-10 (B), VCAM-1 (C) and ICAM-1 (D) in HC donors, HIV mono-infected, and HIV/HCV co-infected patients at 12 weeks after the end of DAAs therapy (post-DAAs HIV/HCV). p-values for the global comparison between the groups (Kruskal-Wallis test; K-W) are shown. (*): p < 0.05 with respect to the HC group (Mann-Whitney U test). Full article ">Figure 4
Box-plots graphs showing the levels of IP-10 (A), and VCAM-1 (B), in HC donors (HC), HIV monoinfected (HIV), and HIV/HCV coinfected patients at 12 weeks after the end of DAAs therapy (post-DAAs HIV/HCV), stratified according to the level of liver stiffness (LS) at baseline (before DAAs treatment). p-values for the global comparison between the groups (Kruskal-Wallis test; K-W) are shown. (*): p < 0.05 with respect to HC donors (Mann-Whitney U test). (¶): p < 0.05 with respect to the HIV group (Mann-Whitney U test). Full article ">

9 pages, 348 KiB

Open AccessReview

Livestock and Poultry Production in Nepal and Current Status of Vaccine Development

by Uddab Poudel, Umesh Dahal, Nabin Upadhyaya, Saroj Chaudhari and Santosh Dhakal

Vaccines 2020, 8(2), 322; https://doi.org/10.3390/vaccines8020322 - 19 Jun 2020

Cited by 20 | Viewed by 15833

Abstract

The livestock and poultry sectors are an integral part of Nepalese economy and lifestyle. Livestock and poultry populations have continuously been increasing in the last decade in Nepal and are likely to follow that trend as the interests in this field is growing. [...] Read more.

The livestock and poultry sectors are an integral part of Nepalese economy and lifestyle. Livestock and poultry populations have continuously been increasing in the last decade in Nepal and are likely to follow that trend as the interests in this field is growing. Infectious diseases such as Foot and Mouth Disease (FMD), Peste des Petits Ruminants (PPR), hemorrhagic septicemia (HS), black quarter (BQ), swine fever, avian influenza, and Newcastle disease (ND) constitute one of the major health challenges to the Nepalese livestock and poultry industry. Vaccinations are an efficient means of preventing the occurrence and spread of several diseases in animals and birds. Considering this fact, the government of Nepal began the production of veterinary vaccines in the 1960s. Nepal is self-reliant in producing several vaccines for cattle and buffaloes, sheep and goats, pigs, and poultry. Despite these efforts, the demand for vaccines is not met, especially in the commercial poultry sector, as Nepal spends billions of rupees in vaccine imports each year. There is a need of strengthening laboratory facilities for the isolation and characterization of field strains of pathogens and capacity building for the production of different types of vaccines using the latest technologies to be self-reliant in veterinary vaccine production in the future in Nepal. Full article

(This article belongs to the Section Veterinary Vaccines)

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17 pages, 4355 KiB

Open AccessArticle

Overcoming Tumor Resistance to Oncolyticvaccinia Virus with Anti-PD-1-Based Combination Therapy by Inducing Antitumor Immunity in the Tumor Microenvironment

by So Young Yoo, Narayanasamy Badrinath, Su-Nam Jeong, Hyun Young Woo and Jeong Heo

Vaccines 2020, 8(2), 321; https://doi.org/10.3390/vaccines8020321 - 19 Jun 2020

Cited by 20 | Viewed by 4365

Abstract

The tumor microenvironment (TME) comprises different types of immune cells, which limit the therapeutic efficacy of most drugs. Although oncolytic virotherapy (OVT) boosts antitumor immunity via enhanced infiltration of tumor-infiltrated lymphocytes (TILs), immune checkpoints on the surface of tumors and TILs protect tumor [...] Read more.

The tumor microenvironment (TME) comprises different types of immune cells, which limit the therapeutic efficacy of most drugs. Although oncolytic virotherapy (OVT) boosts antitumor immunity via enhanced infiltration of tumor-infiltrated lymphocytes (TILs), immune checkpoints on the surface of tumors and TILs protect tumor cells from TIL recognition and apoptosis. OVT and immune checkpoint blockade (ICB)-based combination therapy might overcome this issue. Therefore, combination immunotherapies to modify the immunosuppressive nature of TME and block immune checkpoints of immune cells and tumors are considered. In this study, cancer-favoring oncolytic vaccinia virus (CVV) and anti–programmed cell death protein-1 (anti-PD-1) were used to treat mouse colorectal cancer. Weekly-based intratumoral CVV and intraperitoneal anti-PD-1 injections were performed on Balb/c mice with subcutaneous CT26 tumors. Tumor volume, survival curve, and immunohistochemistry-based analysis demonstrated the benefit of co-treatment, especially simultaneous treatment with CVV and anti-PD-1. Infiltration of CD8⁺PD-1⁺ T-cells showed correlation with these results. Splenocytes enumeration also suggested CD4⁺ and CD8⁺ T-cell upregulation. In addition, upregulated CD8, PD-1, and CD86 messenger RNA expression was observed in this combination therapy. Therefore, CVV+anti-PD-1 combination therapy induces antitumor immunity in the TME, overcoming the rigidity and resistance of the TME in refractory cancers. Full article

(This article belongs to the Special Issue Cancer Vaccine)

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4 pages, 192 KiB

Open AccessViewpoint

Merit of an Ursodeoxycholic Acid Clinical Trial in COVID-19 Patients

by Subbaya Subramanian, Tinen Iles, Sayeed Ikramuddin and Clifford J. Steer

Vaccines 2020, 8(2), 320; https://doi.org/10.3390/vaccines8020320 - 19 Jun 2020

Cited by 33 | Viewed by 8127

Abstract

Corona Virus Disease 2019 (COVID-19) has affected over 8 million people worldwide. We underscore the potential benefits of conducting a randomized open-label unblinded clinical trial to evaluate the role of ursodeoxycholic acid (UDCA) in the treatment of COVID-19. Some COVID-19 patients are characterized [...] Read more.

Corona Virus Disease 2019 (COVID-19) has affected over 8 million people worldwide. We underscore the potential benefits of conducting a randomized open-label unblinded clinical trial to evaluate the role of ursodeoxycholic acid (UDCA) in the treatment of COVID-19. Some COVID-19 patients are characterized with cytokine storm syndrome that can cause severe and irreversible damage to organs leading to multi-organ failure and death. Therefore, it is critical to control both programmed cell death (apoptosis) and the hyper-immune inflammatory response in COVID-19 patients to reduce the rising morbidity and mortality. UDCA is an existing drug with proven safety profiles that can reduce inflammation and prevent cell death. National Geographic reported that, “China Promotes Bear Bile as Coronavirus Treatment”. Bear bile is rich in UDCA, comprising up to 40–50% of the total bile acid. UDCA is a logical and attainable replacement for bear bile that is available in pill form and merits clinical trial consideration. Full article

17 pages, 3169 KiB

Open AccessArticle

Vaccination with Recombinant Subolesin Antigens Provides Cross-Tick Species Protection in Bos indicus and Crossbred Cattle in Uganda

by Paul D. Kasaija, Marinela Contreras, Fredrick Kabi, Swidiq Mugerwa and José de la Fuente

Vaccines 2020, 8(2), 319; https://doi.org/10.3390/vaccines8020319 - 18 Jun 2020

Cited by 33 | Viewed by 4278

Abstract

Cattle tick infestations and transmitted pathogens affect animal health, production and welfare with an impact on cattle industry in tropical and subtropical countries. Anti-tick vaccines constitute an effective and sustainable alternative to the traditional methods for the control of tick infestations. Subolesin (SUB)-based [...] Read more.

Cattle tick infestations and transmitted pathogens affect animal health, production and welfare with an impact on cattle industry in tropical and subtropical countries. Anti-tick vaccines constitute an effective and sustainable alternative to the traditional methods for the control of tick infestations. Subolesin (SUB)-based vaccines have shown efficacy for the control of multiple tick species, but several factors affect the development of new and more effective vaccines for the control of tick infestations. To address this challenge, herein we used a regional and host/tick species driven approach for vaccine design and implementation. The objective of the study was to develop SUB-based vaccines for the control of the most important tick species (Rhipicephalus appendiculatus, R. decoloratus and Amblyomma variegatum) affecting production of common cattle breeds (Bos indicus and B. indicus x B. taurus crossbred) in Uganda. In this way, we addressed the development of anti-tick vaccines as an intervention to prevent the economic losses caused by ticks and tick-borne diseases in the cattle industry in Uganda. The results showed the possibility of using SUB antigens for the control of multiple tick species in B. indicus and crossbred cattle and suggested the use of R. appendiculatus SUB to continue research on vaccine design and formulation for the control of cattle ticks in Uganda. Future directions would include quantum vaccinology approaches based on the characterization of the SUB protective epitopes, modeling of the vaccine E under Ugandan ecological and epidemiological conditions and optimization of vaccine formulation including the possibility of oral administration. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Experimental design. The study design included the cloning and analysis of subolesin (SUB)-coding genes in Ugandan strains of R. appendiculatus, R. decoloratus and A. variegatum, followed by production of recombinant proteins and vaccine formulations with single and all combined antigens. Vaccination trials were conducted in the most common cattle breeds (Bos indicus and B. indicus × B. taurus crossbred) in Uganda using 4 calves/group and infested with R. appendiculatus and A. variegatum larvae, nymphs and adults and R. decoloratus larvae in crossbred cattle only. The effect of vaccination on cattle antibody response and on different tick developmental stages (number of engorged larvae (DL), nymphs (DN) and adult female ticks (DA), molting of tick larvae (DMn) and nymphs (DMa), oviposition (DO) and fertility (DF) was used to evaluate vaccine E. Full article ">Figure 2
Phylogenetic analysis of SUB protein sequences. The evolutionary analysis was performed using the maximum likelihood method and JTT matrix-based model in Mega X. (A) Rhipicephalus spp.: The tree with the highest log likelihood (−1075.01) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories; +G, parameter = 1.7321). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 34 amino acid sequences with a total of 192 positions in the final dataset. (B) Amblyomma spp.: The tree with the highest log likelihood (−722.52) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 8 amino acid sequences with a total of 184 positions in the final dataset. Sequences from Ugandan tick strains are marked with a red arrow. Full article ">Figure 3
Production of recombinant R. appendiculatus, R. decoloratus and A. variegatum SUB in E. coli. (A) alignment of SUB amino acid protein sequences. Distinctive amino acid residues are highlighted (blue: Identical only between R. appendiculatus and R. decolortus, green: Identical only between A. variegatum and R. appendiculatus and pink: Identical only between A. variegatum and R. decoloratus). Conserved residues between all sequences are indicated with asterisks (*). (B) ten μg per well of purified recombinant proteins were loaded into an SDS-12% polyacrylamide gel. Gels were stained with Coomassie Brilliant Blue or used for Western blot analysis. For Western blot analysis, the gel was transferred to a nitrocellulose membrane and the membrane was incubated with pooled sera collected from vaccinated cattle at day 60. The positions of the monomer and dimer recombinant proteins is indicated with black and grey arrows, respectively. Abbreviation: MW, molecular weight markers (Spectra multicolor broad range protein ladder; Thermo Scientific). Full article ">Figure 4
Antibody response in SUB-vaccinated and control cattle. (A) B. indicus cattle. (B) crossbred cattle. Blood samples were collected before each vaccination (days 0, 30 and 60; violet arrows), at day 45 between second and third vaccinations and at the end of the experiment (days 180 or 195 for B. indicus and crossbred cattle, respectively). Serum IgG antibody titers were determined using an indirect antigen-specific ELISA. Antibody titers were expressed as the OD450 nm values and compared between vaccinated and control groups using a one-way ANOVA test (<a href="https://www.socscistatistics.com/tests/anova/default2.aspx" target="_blank">https://www.socscistatistics.com/tests/anova/default2.aspx</a>) (* p < 0.05, ** p < 0.005; n = 4 biological replicates). Full article ">Figure 5
Effect of vaccination with SUB on different tick developmental stages. (A) tick developmental stages included in the analysis. (B) vaccination with R. appendiculatus SUB. (C) vaccination with A. variegatum SUB. (D) vaccination with R. decoloratus SUB. (E) vaccination with all combined SUB formulation. The tick developmental stages included DL, DN, DA, DMn, DMa, DO and DF. Only parameters with statistically significant differences (Chi-square test; p < 0.05, n = 4 biological replicates; <a href="#app1-vaccines-08-00319" class="html-app">Data S1</a>) are shown here and were included in the vaccine E calculation. Full article ">Figure 6
Analysis of vaccine E. Vaccine E (%) was calculated as 100 (1−(DL × DMn × DN × DMa × DA × DO × DF)), and only parameters with statistically significant differences (Chi-square test; p < 0.05, n = 4 biological replicates; <a href="#app1-vaccines-08-00319" class="html-app">Data S1</a>) were included in the analysis. (A) results of the vaccination trials in B. indicus cattle. (B) results of the vaccination trials in crossbred cattle. (C) total vaccine E for each antigen against all tick species was compared between B. indicus and crossbred cattle by a Student’s t-test with unequal variance (p = 0.05; n = 2–3, i.e., three tick species in crossbred cattle or two tick species in B. indicus). (D) a Spearman’s Rho correlation analysis was performed between total vaccine E values for each antigen in all cattle breeds and tick species (f-ratio = 0.31, p = 0.82; n = 5, i.e., three tick species in crossbred cattle plus two tick species in B. indicus). Full article ">Figure 7
Correlation analysis between anti-SUB antibody titers and vaccination effect on different tick developmental stages. (A) B. indicus cattle infested with R. appendiculatus. (B) crossbred cattle infested with R. appendiculatus. (C) crossbred cattle infested with R. decoloratus. (D) values (average + SD) of the parameters of the tick developmental stages used in the analysis (in red are shown the parameters with significant negative correlation with anti-SUB antibody titers and the threshold value line for observing significant differences). The correlation analyses were conducted between the values of the parameters of Rhipicephalus spp. tick life stages used for the calculation of vaccine E that showed significant differences with at least three of the SUB vaccine formulations (<a href="#vaccines-08-00319-t001" class="html-table">Table 1</a>) and the anti-R. appendiculatus SUB antibody titers at day 60 before tick challenge. All animals in both vaccinated and control groups were included in the analysis. Data was analyzed using a Spearman’s Rho correlation analysis (p ≤ 0.05; n = 20, i.e., 4 animals for each of the 5 vaccine formulations including the control). Full article ">

42 pages, 4917 KiB

Open AccessArticle

Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

by Juris Jansons, Ekaterina Bayurova, Dace Skrastina, Alisa Kurlanda, Ilze Fridrihsone, Dmitry Kostyushev, Anastasia Kostyusheva, Alexander Artyuhov, Erdem Dashinimaev, Darya Avdoshina, Alla Kondrashova, Vladimir Valuev-Elliston, Oleg Latyshev, Olesja Eliseeva, Stefan Petkov, Maxim Abakumov, Laura Hippe, Irina Kholodnyuk, Elizaveta Starodubova, Tatiana Gorodnicheva, Alexander Ivanov, Ilya Gordeychuk and Maria Isaguliants Show full author list Hide full author list

Vaccines 2020, 8(2), 318; https://doi.org/10.3390/vaccines8020318 - 18 Jun 2020

Cited by 2 | Viewed by 4796

Abstract

Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune [...] Read more.

Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-γ/IL-2/TNF-α producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer. Full article

(This article belongs to the Special Issue Cancer Immunotherapy: Advances and Future Prospects)

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13 pages, 2847 KiB

Open AccessArticle

PD-1 Immune Checkpoint Blockade Promotes Therapeutic Cancer Vaccine to Eradicate Lung Cancer

by Pournima Kadam and Sherven Sharma

Vaccines 2020, 8(2), 317; https://doi.org/10.3390/vaccines8020317 - 18 Jun 2020

Cited by 20 | Viewed by 3937

Abstract

(1) Background: Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor antigens (Ags), is an attractive option. (2) Methods: Utilizing a K-RasG12Dp53null murine [...] Read more.

(1) Background: Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor antigens (Ags), is an attractive option. (2) Methods: Utilizing a K-RasG12Dp53null murine lung cancer model we determined tumor burden, tumor-infiltrating T cell (TIL) cytolysis, immunohistochemistry, flow cytometry, and CD4 and CD8 depletion to evaluate the efficacy of PD-1 blockade combined with CCL21-DC tumor lysate vaccine. (3) Results: Anti-PD-1 plus CCL21-DC tumor lysate vaccine administered to mice bearing established tumors (150 mm³) increased expression of perforin and granzyme B in the tumor microenvironment (TME), increased tumor-infiltrating T cell (TIL) activity, and caused 80% tumor eradication. Mice with treatment-induced tumor eradication developed immunological memory, enabling tumor rejection upon challenge and cancer-recurrence-free survival. The depletion of CD4 or CD8 abrogated the antitumor activity of combined therapy. PD-1 blockade or CCL21-DC tumor lysate vaccine monotherapy reduced tumor burden without tumor eradication. (4) Conclusion: Immune checkpoint blockade promotes the activity of the therapeutic cancer vaccine. PD-1 blockade plus CCL21-DC tumor lysate vaccine therapy could benefit lung cancer patients. Full article

(This article belongs to the Special Issue Cancer Vaccine)

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K-RasG12Dp53null tumor cells (106) were inoculated in the supra scapular region of 129-E mice. Mice bearing 12-day established tumors were treated with (i) diluent, (ii) anti-PD-1, (iii) CCL21-DC lysate vaccine, (iv) CCL21-DC lysate vaccine plus anti-PD-1, (v) CCL21-DC lysate vaccine plus anti-PD-1 plus anti-CD4, and (vi) CCL21-DC lysate vaccine plus anti-PD-1 plus anti-CD8. In comparison to monotherapy, combined therapy was more effective at inhibiting tumor growth (A). Depletion of CD4 T or CD8 T cells abrogated antitumor activity of combined therapy (A). Combined therapy reduced the weight of tumors in comparison to monotherapy and control (B,C); ** p < 0.01 in comparison to diluent control, * p < 0.05 in comparison to monotherapy. Results are representative of an independent experiment. The experiment was repeated twice (n = 10 mice/group). Full article ">Figure 2
Tumor-infiltrating T cells (TILs) were incubated with K-RasG12Dp53null tumor cells at ratio of 5:1 overnight followed by addition of almar blue for 4 h. TILs from mice treated with combined therapy were most effective at lysing K-RasG12Dp53null tumor cells; ** p < 0.01 in comparison to diluent control, * p < 0.05 compared to monotherapy. Results are representative of an independent experiment (A). The experiment was repeated twice (n = 4 mice/group). Flow cytometric analyses of single-cell suspensions of the tumor microenvironment (TME) following therapy showed enhancement of CD8 T cells expressing granzyme B (p < 0.05) compared to monotherapy. P98 is dumped cell population, P99 is dead cell population and P100 is CD8 T cell population. Results are representative of an independent experiment. The experiment was repeated twice (n = 4 mice/group) (B). Full article ">Figure 3
H&E revealed enhanced immune infiltrates and reduced tumor staining following combined therapy. (A) IHC of tumor sections revealed enhanced (i) CD3 T cell staining (brown), (ii) tumor cell apoptosis (brown), (iii) perforin, (iv) and granzyme B staining (brown) following combined therapy. (B) Stained areas of tumors were quantified by microscopy of IHC-stained paraffin-embedded sections. Anti-PD-1 plus CCL21-DC lysate vaccine treatment led to the highest levels of CD3 T cells, perforin, and granzyme B and greatest reduction in tumor burden, as denoted by enhanced caspase-3-stained apoptotic tumor cells compared with diluent control and monotherapy. Bars represent SE; p < 0.01 compared with diluent-treated control; p < 0.05 compared with monotherapy. Results are representative of a single experiment (n = 4 mice/group). Full article ">Figure 3 Cont.
H&E revealed enhanced immune infiltrates and reduced tumor staining following combined therapy. (A) IHC of tumor sections revealed enhanced (i) CD3 T cell staining (brown), (ii) tumor cell apoptosis (brown), (iii) perforin, (iv) and granzyme B staining (brown) following combined therapy. (B) Stained areas of tumors were quantified by microscopy of IHC-stained paraffin-embedded sections. Anti-PD-1 plus CCL21-DC lysate vaccine treatment led to the highest levels of CD3 T cells, perforin, and granzyme B and greatest reduction in tumor burden, as denoted by enhanced caspase-3-stained apoptotic tumor cells compared with diluent control and monotherapy. Bars represent SE; p < 0.01 compared with diluent-treated control; p < 0.05 compared with monotherapy. Results are representative of a single experiment (n = 4 mice/group). Full article ">Figure 4
Anti-PD-1 plus CCL21-DC tumor antigen (Ag) vaccine therapy enhances dendritic cell (DC) activity in the TME. CD4 and CD8 T cells were purified from the spleens of mice following vaccination with DC tumor lysate vaccine. DCs from TME of (i) diluent control, (ii) anti-PD-1, (iii) CCL21-DC tumor lysate vaccine, and (iv) anti-PD-1 plus CCL21-DC tumor lysate vaccine were pulsed with MHC Class I K-Ras peptide (LVVVGADGV) or MHC Class II (MTEYKLVVVGADGVG) and co-cultured with splenic CD8 or CD4 T cells of the vaccinated mice at a ratio of 1:5 for 24 h. IFNγ secreted by T cells in the co-culture was determined by IFNγ-specific ELISA. In comparison to diluent control or monotherapy, anti-PD-1 plus CCL21-DC tumor lysate Ag induced the highest DC activity of presenting MHC Class I and MHC Class II K-RasG12D tumor peptides to T cells. Control peptide (FECNTAQAC)-pulsed DCs did not stimulate T cell IFNγ production (data not shown). Bars represent SE; p < 0.01 compared with diluent-treated control, p < 0.05 compared with monotherapy. Results are representative of a single experiment (n = 4 mice/group). Full article ">Figure 5
(A,B) Tregs and NK cells from CCL21-DC lysate vaccine plus anti-PD-1 did not have a change in activity. Purified Tregs (1:5) from combined therapy did not alter the proliferation of anti-CD3/anti-CD28 (0.2/2 µg/mL) stimulated T cell proliferation. Almar blue (20 µL) was added for 4 h on day 3, and fluorescence was read at excitation/emission (530nm/595nm) in a Wallac Fluorescence reader. Full article ">

17 pages, 1888 KiB

Open AccessArticle

Defining Elimination of Genital Warts—A Modified Delphi Study

by Laila Khawar, Dorothy A. Machalek, David G. Regan, Basil Donovan, Skye McGregor and Rebecca J. Guy

Vaccines 2020, 8(2), 316; https://doi.org/10.3390/vaccines8020316 - 18 Jun 2020

Cited by 1 | Viewed by 3993

Abstract

Background: Substantial declines in genital warts (GW) have been observed in countries with quadrivalent HPV vaccination programmes, with Australia showing the highest reductions due to early commencement and high vaccination coverage. There is a real potential to achieve GW elimination; however, no GW [...] Read more.

Background: Substantial declines in genital warts (GW) have been observed in countries with quadrivalent HPV vaccination programmes, with Australia showing the highest reductions due to early commencement and high vaccination coverage. There is a real potential to achieve GW elimination; however, no GW elimination definition exists. Taking Australia as a case study, we aimed to reach expert consensus on a proposed GW elimination definition using a modified Delphi process. Method: We used modelling and epidemiological data to estimate the expected number of new GW cases, from pre-vaccination (baseline) in 2006 to the year 2060 in Australian heterosexuals, men who have sex with men (MSM), and newly arrived international travellers and migrants. We used these data and the literature, to develop a questionnaire containing ten elimination-related items, each with 9-point Likert scales (1—strongly disagree; 9—strongly agree). The survey was completed by 18 experts who participated in a full day face-to-face modified Delphi study, in which individuals and then small groups discussed and scored each item. The process was repeated online for items where consensus (≥70% agreement) was not initially achieved. Median and coefficient of variation (COV) were used to describe the central tendency and variability of responses, respectively. Findings: There was a 95% participation rate in the face-to-face session, and 84% response rate in the final online round. The median item score ranged between 7.0 and 9.0 and the COV was ≤0.30 on all items. Consensus was reached that at ≥80% HPV vaccination coverage, GW will be eliminated as a public health problem in Australia by 2060. During this time period there will be a 95% reduction in population-level incidence compared with baseline, equivalent to <1 GW case per 10,000 population. The reductions will occur most rapidly in Australian heterosexuals, with 73%, 90% and 97% relative reductions by years 2021, 2030 and 2060, respectively. The proportion of new GW cases attributable to importation will increase from 3.6% in 2006 to ~49% in 2060. Interpretation: Our results indicate that the vaccination programme will minimise new GW cases in the Australian population, but importation of cases will continue. This is the first study to define GW elimination at a national level. The framework developed could be used to define GW elimination in other countries, with thresholds particularly valuable for vaccination programme impact evaluation. Funding: LK supported through an Australian Government Research Training Programme Scholarship; unconditional funding from Seqirus to support the Delphi Workshop. Full article

(This article belongs to the Special Issue Vaccination and Public Health: Optimizing Vaccine Uptake through the Application of Social and Behavioral Science Theory, Principles, and Strategies)

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18 pages, 3400 KiB

Open AccessArticle

ELISA-Based Assay for Studying Major and Minor Group Rhinovirus–Receptor Interactions

by Petra Pazderova, Eva E. Waltl, Verena Niederberger-Leppin, Sabine Flicker, Rudolf Valenta and Katarzyna Niespodziana

Vaccines 2020, 8(2), 315; https://doi.org/10.3390/vaccines8020315 - 18 Jun 2020

Cited by 4 | Viewed by 4041

Abstract

Rhinovirus (RV) infections are a major cause of recurrent common colds and trigger severe exacerbations of chronic respiratory diseases. Major challenges for the development of vaccines for RV include the virus occurring in the form of approximately 160 different serotypes, using different receptors, [...] Read more.

Rhinovirus (RV) infections are a major cause of recurrent common colds and trigger severe exacerbations of chronic respiratory diseases. Major challenges for the development of vaccines for RV include the virus occurring in the form of approximately 160 different serotypes, using different receptors, and the need for preclinical models for the screening of vaccine candidates and antiviral compounds. We report the establishment and characterization of an ELISA-based assay for studying major and minor group RV–receptor interactions. This assay is based on the interaction of purified virus with plate-bound human receptor proteins, intercellular adhesion molecule 1 (ICAM-1), and low density lipoprotein receptor (LDLR). Using RV strain-specific antibodies, we demonstrate the specific binding of a panel of major and minor RV group types including RV-A and RV-B strains to ICAM-1 and LDLR, respectively. We show that the RV–receptor interaction can be blocked with receptor-specific antibodies as well as with soluble receptors and neutralizing RV-specific antibodies. The assay is more sensitive than a cell culture-based virus neutralization test. The ELISA assay will therefore be useful for the preclinical evaluation for preventive and therapeutic strategies targeting the RV–receptor interaction, such as vaccines, antibodies, and anti-viral compounds. Full article

(This article belongs to the Special Issue Development of Cross-Protective Vaccines)

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Flow chart summarizing the experimental design and reagents for the ELISA-based virus–receptor interaction assay. (top) The preparation of viral strains and the confirmation of their identity by reverse transcription (RT)-PCR. (bottom) The scheme for the ELISA detection of virus–receptor interactions. Full article ">Figure 2
Characterization of recombinant human ICAM-1 and LDLR. (a) Coomassie-blue stained SDS-PAGE with recombinant human ICAM-1 (lane 1) and LDLR (lane 2). A molecular-weight marker (kDa) is indicated on the left. (b) Mean residue ellipticities (θ, y-axis) of ICAM-1 (red) and LDLR (blue) recorded at different wavelengths (nm, x-axis) by circular dichroism (CD) spectroscopy. Detection of (c) ICAM-1 or (d) LDLR with serial dilutions of anti-ICAM-1 antibodies (c = 1 µg/mL; left), anti-LDLR antibodies (c = 0.2 µg/mL; right) or bovine serum albumin (BSA)-containing buffer alone (BSA) by ELISA. Shown are means of optical density (O.D.) values corresponding to bound antibodies (y-axes) measured at different time points (x-axes). The variations of individual duplicate results of ELISAs were less than 5%. The antibody titration in (c) and (d) was done once. Full article ">Figure 3
Binding of major group and minor group RVs to ICAM-1and LDLR. Shown are O.D. values (y-axes) corresponding to receptor (ICAM-1 or LDLR)-bound (a) major or (b) minor group RVs (x-axes) as determined by ELISA. Tested major group RV-A or RV-B species (a) and minor group RV-A species (b) are indicated on the x-axes. The analyses were performed with virus (50 µg/mL RV) and, for control purposes, without virus (−RV), without virus and detection antibodies (−RV, −anti-RV) or with HSA- instead of receptor-coated plates (HSA coated). The results are means of duplicate determinations with a variation of less than 5%. RV–receptor binding experiments were repeated two times. Full article ">Figure 4
Inhibition of RV89 and RV14 binding to plate-bound receptors by receptor-specific antibodies. Binding of RV89 (upper part) (a) and RV14 (lower part) (b) to plate-bound ICAM-1 (left panels) or plate-bound LDLR (right panels) is reported as mean O.D. values (y-axes) in the presence (+) or absence (−) of virus, anti-ICAM-1 antibodies, non-specific antibodies, anti-RV detection antibodies, and secondary detection antibodies (anti-GP) (below x-axes). The percentage inhibitions of RV89 binding and RV14 binding obtained with anti-ICAM-1 antibodies versus non-specific antibodies are indicated in red. The results are means of duplicate determinations with a variation of less than 10%. Inhibition experiments were repeated three times. Full article ">Figure 5
Inhibition of RV89 infection by anti-ICAM-1 antibodies determined by cell culture-based virus inhibition assay. (a) Crystal violet-stained viable cells incubated only with anti-ICAM-1 or non-specific antibodies (top), different dilutions of antibodies (1:100–1:100,000); anti-ICAM-1 and non-specific antibodies separated by red vertical line) in the presence of virus (100 TCID50), 1:100 diluted antibodies without virus, cell incubated only with virus (100 TCID50), or only with medium (bottom). (b) Mean O.D. values corresponding to viable cells in triplicate wells ±SDs (y-axis) of the above experiment (x-axis: conditions applied). Neutralization tests were repeated two times. Full article ">Figure 6
Inhibition of major group RV89 and minor group RV2 binding to plate-bound receptors by receptor-specific antibodies. Binding of RV89 (upper part) (a) and RV2 (lower part) (b) to plate-bound ICAM-1 (left panels) or plate-bound LDLR (right panels) reported as mean O.D. values (y-axes) in the presence (+) or absence (−) of virus, commercial anti-ICAM-1 antibodies, commercial anti-LDLR antibodies, anti-RV detection antibodies, and secondary detection antibodies (anti-GP) (see below x-axes). The percentage inhibitions of RV89 binding and RV2 binding obtained with commercial anti-receptor antibodies compared to without anti-receptor antibodies are indicated in red. The results are means of duplicate determinations with a variation of less than 10%. Inhibition experiments were performed once. Full article ">Figure 7
Auto-inhibition of the binding of major group RV89 and minor group RV2 to their receptors. Binding of RV89 to ICAM-1 (left) and of RV2 to LDLR (right) without (first column) or with (second column) pre-incubation with the receptor plus controls in which virus and receptor (third column) or virus, receptor and detection antibodies (fourth column) were omitted, reported as mean optical density (i.e., O.D.) values (y-axes). The percentage inhibitions of RV89 and RV2 binding produced by pre-incubation with receptors are indicated in red. The results are means of duplicate determinations with a variation of less than 10%. Inhibition experiments with soluble receptors were performed two times. Full article ">Figure 8
Inhibition of RV89 binding to ICAM-1 by antibodies specific for each of the four viral capsid proteins (VP). Binding of RV89 to ICAM-1 after pre-incubation of virus with antibodies against VP1, VP2, VP3, and VP4 compared to pre-incubation with buffer containing BSA only reported as O.D. (y-axes). The inhibitions of RV89 binding by anti-VP1 and anti-VP2 antibodies are indicated by red arrows. Omission of RV89 served as a negative control. The results are means of duplicate determinations with a variation of less than 10%. Inhibition experiments were performed three times. Full article ">

14 pages, 2348 KiB

Open AccessArticle

Search of Potential Vaccine Candidates against Trueperella pyogenes Infections through Proteomic and Bioinformatic Analysis

by Ángela Galán-Relaño, Lidia Gómez-Gascón, Antonio Rodríguez-Franco, Inmaculada Luque, Belén Huerta, Carmen Tarradas and Manuel J. Rodríguez-Ortega

Vaccines 2020, 8(2), 314; https://doi.org/10.3390/vaccines8020314 - 17 Jun 2020

Cited by 7 | Viewed by 2766

Abstract

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis [...] Read more.

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis of 15 T. pyogenes clinical isolates, by “shaving” live cells, followed by LC-MS/MS, aiming at the identification of the whole set of surface proteins (i.e., the “pan-surfome”) as a source of antigens to be tested in further studies as putative vaccine candidates, or used in diagnostic tools. A total of 140 surface proteins were detected, comprising 25 cell wall proteins, 10 secreted proteins, 23 lipoproteins and 82 membrane proteins. After describing the “pan-surfome”, the identified proteins were ranked in three different groups based on the following criteria: to be (i) surface-exposed, (ii) highly conserved and (iii) widely distributed among different isolates. Two cell wall proteins, three lipoproteins, four secreted and seven membrane proteins were identified in more than 70% of the studied strains, were highly expressed and highly conserved. These proteins are potential candidates, alone or in combination, to obtain effective vaccines against T. pyogenes or to be used in the diagnosis of this pathogen. Full article

(This article belongs to the Special Issue Omics and Bioinformatics Approaches to Identify Novel Antigens for Vaccine Investigation and Development)

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24 pages, 696 KiB

Open AccessReview

Innate Immune Response against Hepatitis C Virus: Targets for Vaccine Adjuvants

by Daniel Sepulveda-Crespo, Salvador Resino and Isidoro Martinez

Vaccines 2020, 8(2), 313; https://doi.org/10.3390/vaccines8020313 - 17 Jun 2020

Cited by 15 | Viewed by 4128

Abstract

Despite successful treatments, hepatitis C virus (HCV) infections continue to be a significant world health problem. High treatment costs, the high number of undiagnosed individuals, and the difficulty to access to treatment, particularly in marginalized susceptible populations, make it improbable to achieve the [...] Read more.

Despite successful treatments, hepatitis C virus (HCV) infections continue to be a significant world health problem. High treatment costs, the high number of undiagnosed individuals, and the difficulty to access to treatment, particularly in marginalized susceptible populations, make it improbable to achieve the global control of the virus in the absence of an effective preventive vaccine. Current vaccine development is mostly focused on weakly immunogenic subunits, such as surface glycoproteins or non-structural proteins, in the case of HCV. Adjuvants are critical components of vaccine formulations that increase immunogenic performance. As we learn more information about how adjuvants work, it is becoming clear that proper stimulation of innate immunity is crucial to achieving a successful immunization. Several hepatic cell types participate in the early innate immune response and the subsequent inflammation and activation of the adaptive response, principally hepatocytes, and antigen-presenting cells (Kupffer cells, and dendritic cells). Innate pattern recognition receptors on these cells, mainly toll-like receptors, are targets for new promising adjuvants. Moreover, complex adjuvants that stimulate different components of the innate immunity are showing encouraging results and are being incorporated in current vaccines. Recent studies on HCV-vaccine adjuvants have shown that the induction of a strong T- and B-cell immune response might be enhanced by choosing the right adjuvant. Full article

(This article belongs to the Special Issue Research on Innate Immunity and Inflammation)

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16 pages, 447 KiB

Open AccessReview

The African Swine Fever Virus (ASFV) Topoisomerase II as a Target for Viral Prevention and Control

by João Coelho and Alexandre Leitão

Vaccines 2020, 8(2), 312; https://doi.org/10.3390/vaccines8020312 - 17 Jun 2020

Cited by 15 | Viewed by 4678

Abstract

African swine fever (ASF) is, once more, spreading throughout the world. After its recent reintroduction in Georgia, it quickly reached many neighboring countries in Eastern Europe. It was also detected in Asia, infecting China, the world’s biggest pig producer, and spreading to many [...] Read more.

African swine fever (ASF) is, once more, spreading throughout the world. After its recent reintroduction in Georgia, it quickly reached many neighboring countries in Eastern Europe. It was also detected in Asia, infecting China, the world’s biggest pig producer, and spreading to many of the surrounding countries. Without any vaccine or effective treatment currently available, new strategies for the control of the disease are mandatory. Its etiological agent, the African swine fever virus (ASFV), has been shown to code for a type II DNA topoisomerase. These are enzymes capable of modulating the topology of DNA molecules, known to be essential in unicellular and multicellular organisms, and constitute targets in antibacterial and anti-cancer treatments. In this review, we summarize most of what is known about this viral enzyme, pP1192R, and discuss about its possible role(s) during infection. Given the essential role of type II topoisomerases in cells, the data so far suggest that pP1192R is likely to be equally essential for the virus and thus a promising target for the elaboration of a replication-defective virus, which could provide the basis for an effective vaccine. Furthermore, the use of inhibitors could be considered to control the spread of the infection during outbreaks and therefore limit the spreading of the disease. Full article

(This article belongs to the Special Issue African Swine Fever Virus Prevention and Control)

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26 pages, 5576 KiB

Open AccessArticle

Innate Lymphocyte Th1 and Th17 Responses in Elderly Hospitalised Patients with Infection and Sepsis

by John Davis Coakley, Eamon P. Breen, Ana Moreno-Olivera, Alhanouf I. Al-Harbi, Ashanty M. Melo, Brian O’Connell, Ross McManus, Derek G. Doherty and Thomas Ryan

Vaccines 2020, 8(2), 311; https://doi.org/10.3390/vaccines8020311 - 17 Jun 2020

Cited by 7 | Viewed by 3697

Abstract

Background: the role of innate immunity in human sepsis must be fully clarified to identify potential avenues for novel immune adjuvant sepsis therapies. Methods: A prospective observational study was performed including patients with sepsis (septic group), infection without sepsis (infection group), and healthy [...] Read more.

Background: the role of innate immunity in human sepsis must be fully clarified to identify potential avenues for novel immune adjuvant sepsis therapies. Methods: A prospective observational study was performed including patients with sepsis (septic group), infection without sepsis (infection group), and healthy controls (control group) in the setting of acute medical wards and intensive care units in a 1000-bed university hospital. A total of 42 patients with sepsis, 30 patients with infection, and 30 healthy controls were studied. The differentiation states of circulating mucosal associated invariant T (MAIT) cells and Natural Killer T (NKT) cells were characterised as naive (CD45RA⁺, CD197⁺), central memory (CD45RA⁻, CD197⁺), effector memory (CD45RA⁻, CD197⁻), or terminally differentiated (CD45RA⁺, CD197⁻). The differentiation states of circulating gamma-delta T lymphocytes were characterised as naive (CD45RA⁺, CD27⁺), central memory (CD45RA⁻, CD27⁺), effector memory (CD45RA⁻, CD27⁻), or terminally differentiated (CD45RA⁺, CD27⁻). The expression of IL-12 and IL-23 receptors, the transcription factors T-Bet and RORγt, and interferon-γ and IL-17a were analysed. Results: MAIT cell counts were lower in the septic group (p = 0.002) and the infection group (p < 0.001) than in the control group. The MAIT cell T-Bet expression in the infection group was greater than in the septic group (p = 0.012). The MAIT RORγt expression in the septic group was lower than in the control group (p = 0.003). The NK cell counts differed in the three groups (p < 0.001), with lower Natural Killer (NK) cell counts in the septic group (p < 0.001) and in the infection group (p = 0.001) than in the control group. The NK cell counts increased in the septic group in the 3 weeks following the onset of sepsis (p = 0.028). In lymphocyte stimulation experiments, fewer NK cells expressed T-Bet in the septic group than in the infection group (p = 0.002), and fewer NK cells expressed IFN-γ in the septic group than in the control group (p = 0.002). The NKT cell counts were lower in the septic group than both the control group (p = 0.05) and the infection group (p = 0.04). Fewer NKT cells expressed T-Bet in the septic group than in the infection group (p = 0.004). Fewer NKT cells expressed RORγt in the septic group than in the control group (p = 0.003). Fewer NKT cells expressed IFN-γ in the septic group than in both the control group (p = 0.002) and the infection group (p = 0.036). Conclusion: The clinical presentation of infection and or sepsis in patients is linked with a mosaic of changes in the innate lymphocyte Th1 and Th17 phenotypes. The manipulation of the innate lymphocyte phenotype offers a potential avenue for immune modulation in patients with sepsis. Full article

(This article belongs to the Special Issue Research on Innate Immunity and Inflammation)

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Flow cytometry analysis. Lymphocyte gating on PBMCs (A), with exclusion of dead cells (B) and exclusion of doublets (C). Gating on CD3+ cells (D) to show MAIT cells (E). MAIT cells were gated on showing interferon-γ (IFNγ) expression in unstimulated (F) and stimulated (G) cells. Stimulation was performed with phorbol myristate acetate and ionomycin (PMA/I). DCS is Dead Cell Stain. Full article ">Figure 2
MAIT cells phenotypes. (A) Flow cytometry plot showing MAIT cell population (CD3+CD161+Vα7.2+) on gated CD3+ lymphocytes. (B) Frequency of MAIT cells as a percentage of T cells (CD3+ lymphocytes). (C) Frequency of MAIT CD8+ cells as a % of MAIT cells. (D) Total MAIT cell count in circulating blood. (E) Total MAIT cell count over time in the septic group. (F) Frequencies of naive, central memory, effector memory, and terminally differentiated (N, CM, EM, TD, respectively) MAIT cells in circulating blood. Control group (n = 20), infection group (n = 19), and septic group (n = 32). Graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01; ** = p ≤ 0.001. Full article ">Figure 3
MAIT cell expression of IL-12Rβ2 and IL-23R. (A,B) Percentage of MAIT cells in circulating blood expressing IL-12 receptor (IL-12Rβ2) and IL-23 receptor (IL-23R). (C) MAIT cell count in circulating blood expressing IL-12Rβ2 and IL-23R. (D) MAIT cell count expressing IL-23R over time. (E) Flow cytometry plot on gated MAIT cells showing IL-12Rβ2. (F) Flow cytometry plot on gated MAIT cells showing IL-23 receptor. Control group (n = 20), infection group (n = 19), and septic group (n = 32). Graphs are plotted with bars representing the median. * = p ≤ 0.01. Full article ">Figure 4
Frequency of RORγt, T-Bet, IL17A, and IFNγ expression in MAIT cells. (A) MAIT cell expression of T-Bet when stimulated with the medium alone or PMA/I. (B) MAIT cell expression of RORγt when stimulated with the medium alone or PMA/I. (C) Expression of transcription factor T-Bet in unstimulated naive, central memory, effector memory, and terminally differentiated (N, CM, EM, TD, respectively) MAIT cells. (D) Transcription factor RORγt expression in PMA/I-stimulated N, CM, EM, and TD MAIT cells. (E) MAIT cell expression of IL-17a and IFN-γ when stimulated with PMA/I. Control group (n = 10), infection group (n = 10), and septic group (n = 10). Graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01. Full article ">Figure 5
CD3+CD161+ lymphocytes. (A) Frequency of CD3+CD161+ cells as a percentage of lymphocytes. (B) Differentiation status of CD3+CD161+ lymphocytes in the circulating blood. Naive, central memory, effector memory, and terminally differentiated (N, CM, EM, TD, respectively) differentiation states shown. (C) Total number of CD3+CD161+ lymphocytes and the number of CD3+CD161+ lymphocytes in their differentiated states in patients with sepsis over time. (E) Frequency of lymphocytes that are CD3+CD161+ in patients who survived compare to those that died. Control group (n = 20), infection group (n = 19), and septic group (n = 32). (D) RORγt and T-Bet expression in unstimulated CD161+ T cells and cytokines IL17A and IFN-γ in stimulated CD161+ T cells with phorbol myristate acetate and ionomycin. Control group (n = 10), infection group (n = 10), and septic group (n = 10). (F) Flow cytometry plot of gated lymphocytes showing CD3+CD161+ cells. (A,B,D,E) Graphs are plotted with bars representing the median. (C) Data represented as mean with standard deviation. # = p < 0.05; * = p ≤ 0.01; ** = p ≤ 0.001. Full article ">Figure 6
Natural Killer (NK) cells. (A) Flow cytometry plot on gated lymphocytes showing NK cells (CD3−CD56+) and NKT Cells (CD3+CD56+). (B) NK cells as a percentage of lymphocytes. (C) NK cell count in the three patient groups on admission. (D) Frequency of NK cells as a percentage of lymphocytes in septic patients over time. (E) NK cell count in septic patients over time. Control group (n = 20), infection group (n = 19), and septic group (n = 32). Graphs are plotted with bars representing the median. # p < 0.05; * p ≤ 0.01; ** p ≤ 0.001. Full article ">Figure 7
NK cell phenotype and stimulation. (A) Frequency of NK cells expressing IL-12 receptor (IL-12Rβ2) and IL-23 receptor (IL-23R). (B) Absolute numbers of NK cells expressing IL-12Rβ2 and IL-23R. (A,B) Control group (n = 20), infection group (n = 19), and septic group (n = 32). (C) Frequency of NK cells expressing the transcription factors RORγt and T-Bet when stimulated with phorbol myristate acetate and ionomycin (PMA/I). (D) Frequency of NK cells expressing cytokines IL17A and IFN-γ when stimulated with PMA/I. C-D, control group (n = 10), infection group (n = 10), and septic group (n = 10). (E) Flow cytometry plot on gated NK cells showing T-Bet expression. All the graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01. Full article ">Figure 8
NKT cell phenotype. (A) Frequencies of Natural Killer T (NKT) cells as a percentage of lymphocytes. (B) Absolute numbers of NKT cells in the three patient groups. (C) Total number of NKT cells in septic patients over time. (D) Frequency of NKT cells that express the IL-12 receptor (IL-12Rβ2) and IL-23 receptor (IL-23R). (E) Differentiation status of NKT cells in circulating blood. Naive, central memory, effector memory, and terminally differentiated (N, CM, EM, TD, respectively) differentiation states shown. (F) NKT cell count by differentiation status. Control group (n = 20), infection group (n = 19), and septic group (n = 32). Graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01; ** = p ≤ 0.001. Full article ">Figure 9
NKT cell stimulation. (A) Frequencies of unstimulated Natural Killer T (NKT) cells and stimulated NKT cells with phorbol myristate acetate and ionomycin (PMA/I) expressing T-Bet. (B) Frequency of unstimulated NKT cells and stimulated NKT cells with PMA/I expressing RORγt. (C) Frequency of unstimulated NKT cells and stimulated NKT cells with PMA/I expressing IFN-γ. (D) Frequency of unstimulated NKT cells and stimulated NKT cells with PMA/I expressing IL17A. (E) Flow cytometry plot on gated NKT Cells expressing RORγt. Control group (n = 10), infection group (n = 10), and septic group (n = 10). Graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01. Full article ">Figure 10
Vδ1 cell phenotype. (A), Flow cytometry plot on gated T Cells showing Vδ1 and Vδ2 cells. (B) Frequency of Vδ1 T cells expressing IL-12 receptor (IL-12Rβ2) and IL-23 receptor (IL-23R). (C) Frequency of T cells that express Vδ1 TCRs. (D) Vδ1 T cell count in the three patient groups. (E) Differentiation status of Vδ1 T cells. Naive, central memory, effector memory, and terminally differentiated (N, CM, EM, TD, respectively) differentiation states shown. (F) Differentiation status of Vδ1 T cells expressed as the total cell count. Control group (n = 20), infection group (n = 19), and septic group (n = 32). Graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01; ** = p ≤ 0.001. Full article ">Figure 11
Vδ1 cell stimulation. (A) Frequency of unstimulated and stimulated Vδ1 cells expressing RORγt. (B) Frequency of unstimulated and stimulated Vδ1 cells expressing T-Bet. (C) Frequency of unstimulated and stimulated Vδ1 cells expressing IFN-γ. All were stimulated with phorbol myristate acetate and ionomycin (PMA/I). Control group (n = 10), infection group (n = 10), and septic group (n = 10). Graphs are plotted with bars representing the median. # = p < 0.05. Full article ">Figure 12
Vδ2 cell phenotype. (A) Frequency of T lymphocytes that are Vδ2 Cells. (B) Vδ2 T cell count in the three patient groups. (C) Differentiation status of Vδ2 T cells. Naive, central memory, effector memory, and terminally differentiated (N, CM, EM, TD, respectively) differentiation states shown. (D) Differentiation status of Vδ2 T cells expressed as the total cell count. (E) Flow cytometry plot on gated Vδ2 T cells showing differentiation status. Control group (n = 20), infection group (n = 19), and septic group (n = 32). Graphs are plotted with bars representing the median. # = p < 0.05; * = p ≤ 0.01; ** = p ≤ 0.001. Full article ">Figure 13
Vδ2 cell stimulation. (A) Frequency of unstimulated and stimulated Vδ2 T cells expressing T-Bet. (B) Frequency of unstimulated and stimulated Vδ2 T cells expressing RORγt. (C) Frequency of unstimulated and stimulated Vδ2 T cells expressing interferon gamma (IFN-γ). Stimulated with phorbol myristate acetate and ionomycin (PMA/I). Control group (n = 10), infection group (n = 10), and septic group (n = 10). Graphs are plotted with bars representing the median. # = p < 0.05. Full article ">

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Vaccines, Volume 8, Issue 2 (June 2020) – 196 articles

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