Inactivated Antigen Research Articles

Field evaluation of a novel oral reservoir-targeted vaccine against Borrelia burgdorferi utilizing an inactivated whole-cell bacterial antigen expression vehicle.

Blacklegged ticks (Ixodes scapularis) are the principal vector for Borrelia burgdorferi, among other infectious agents, in the northeastern, mid-Atlantic, and upper midwestern USA. White-footed mice (Peromyscus leucopus) are the primary and most competent reservoir host of B. burgdorferi in the Northeast. Live reservoir-targeted vaccines (RTVs) to limit enzootic transmission of B. burgdorferi were previously developed and successfully evaluated in laboratory and controlled field trials. A novel, inactivated RTV was developed to minimize regulatory and market challenges facing previous RTVs based on live bacterial or viral vehicles. Thirty-two residential properties in Redding, Connecticut, participated in a field trial of an orally delivered, inactivated RTV efficacy study (2015-2016). During the two-year vaccination period, a significant decrease in the percentage of B. burgdorferi-infected I. scapularis larvae parasitizing P. leucopus was observed, as was a significant reduction in the percentage of infected P. leucopus on RTV-treated properties when compared to control properties. This novel inactivated RTV was effective in reducing numbers of B. burgdorferi-infected I. scapularis and B. burgdorferi-infected P. leucopus on properties where it was distributed.

Evaluation of Safety and Immunogenicity of an Adjuvanted, TH-1 Skewed, Whole Virion Inactivated SARS-CoV-2 Vaccine - BBV152

We report the development and evaluation of safety and immunogenicity of a whole virion inactivated SARS-COV-2 vaccine (BBV152), adjuvanted with aluminium hydroxide gel (Algel), or a novel TLR7/8 agonist chemisorbed Algel. We used a well-characterizedSARS-CoV-2 strain and an established vero cell platform to produce large-scale GMP grade highly purified inactivated antigen, BBV152. Product development and manufacturing were carried out in a BSL-3 facility. Immunogenicity was determined at two antigen concentrations (3µgand 6µg), with two different adjuvants, in mice, rats, and rabbits. Our results show that BBV152 vaccine formulations generated significantly high antigen-binding and neutralizing antibody titers, at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation containing TLR7/8 agonist adjuvant-induced Th1 biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2 specific IFN-Υ+ CD4 T lymphocyte response. Our results support further development for Phase I/II clinical trials in humans.Funding: This work was supported and funded by Bharat Biotech International Limited and the Indian Council of Medical Research.Conflict of Interest: All authors have no personal financial or non-financial interests to disclose.Ethical Approval: All animal experiments were performed after obtaining necessary approvals from the Institutional Animal Ethics Committee (IAEC). Maintenance (housing and care) of all animals (mice, rats & rabbits) involved in the study was adhered to guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 2003). All studies were performed following both national and international guidelines in compliance with OECD (Organization for Economic Co-operation and Development) Principles of Good Laboratory Practice (1997)(“OECD Guidelines for Testing of Chemicals, Section 4, No. 471: ‘Bacterial Reverse Mutation Test’, adopted July 21st, 1997,” n.d.).

LTA1 is a safe, intranasal enterotoxin-based adjuvant that improves vaccine protection against influenza in young, old and B-cell-depleted (\u03bcMT) mice

Enterotoxin-based adjuvants including cholera toxin and heat-labile toxin (LT) are powerful manipulators of mucosal immunity; however, past clinical trials identified unacceptable neurological toxicity when LT or mutant AB5 adjuvant proteins were added to intranasal vaccines. Here, we examined the isolated enzymatic A1 domain of LT (LTA1) for intranasal safety and efficacy in combination with influenza (flu) vaccination. LTA1-treated mice exhibited no neurotoxicity, as measured by olfactory system testing and H&E staining of nasal tissue in contrast with cholera toxin. In vaccination studies, intranasal LTA1 enhanced immune responses to inactivated virus antigen and subsequent protection against H1N1 flu challenge in mice (8-week or 24-months). In addition, lung H1N1 viral titers post-challenge correlated to serum antibody responses; however, enhanced protection was also observed in μMT mice lacking B-cells while activation and recruitment of CD4 T-cells into the lung was apparent. Thus, we report that LTA1 protein is a novel, safe and effective enterotoxin adjuvant that improves protection of an intranasal flu vaccination by a mechanism that does not appear to require B-cells.

SPECIFIC ASPECTS OF AVIBACTERIUM PARAGALLINARUM INACTIVATION WITH FORMALDEHYDE AND THIOMERSAL

The paper demonstrates results of testing different modes of Avibacterium paragallinarum inactivation with formaldehyde and thiomersal. The bacterium destruction by 0.20% and 0.10% formaldehyde proceeds at the constant rate thus indicating exponential dependence of the microorganism inactivation processes. This fact allows for calculation of the inactivation rate constant that amounts to 2.94 ± 0.37 h-1 for 0.10% formaldehyde and 5.86 ± 0.72 h-1 for 0.20% formaldehyde. Inactivation using formaldehyde at final concentration of 0.10% at 37 °С and continuous stirring (60 rpm) produces 7.0 dm3 of bacterin at concentration of 9.5 ± 0.2 lg microbial cells (mc)/cm3 in 4.3 ± 0.1 h. Thiomersal demonstrated bactericidal action against Avibacterium paragallinarum at concentration of 0.04% (1:2500) or higher. Herewith, inactivation process is specified by linearity and the inactivation rate constant amounts to 7.92±1.12 h-1. Under thiomersal sublethal concentration of 0.2% (1:5000) the survival curve is of irregular shape. However, the process of the microorganism death is not exponential, and under continuous decrease, the inactivation rate is going to zero thus making impossible the calculation of the inactivation rate constant. Inactivation mode involving use of 0.04% thiomersal at 37 °С allows production of 7.0 dm3 of bacterin at 9.5 ± 0.2 lg mc/cm3 concentration in 5.8 ± 0.1 h. Right after production, the hemagglutination activity of the thiomersal inactivated antigen was higher as compared to formaldehyde inactivated antigen (Р 0.05). The antigen produced using formaldehyde maintains high hemagglutination activity during storage that is critical for high quality vaccine production.

Antibody Response to Feline Calicivirus Vaccination in Healthy Adult Cats.

This study evaluated the prevalence of feline calicivirus (FCV) antibodies and response to vaccination in healthy adult cats. Cats >1 year (n = 111) that had not been vaccinated within 12 months of enrollment in the study received a vaccine containing inactivated FCV antigen strains 431 and G1. Antibodies were determined on Days 0, 7, and 28 by virus neutralization (VN) using FCV isolate KS20, and by broad spectrum blocking FCV enzyme-linked immunosorbent assay (ELISA). Factors associated with the presence of antibodies and vaccine response were determined by uni- and multivariate analysis. Pre-vaccination antibodies were detected in 62.2% of cats (CI95%: 52.9–70.1) by VN and in 77.2% (CI95%: 67.5–84.6) by ELISA. A ≥4-fold titer increase after vaccination was observed in 13.6% (CI95%: 8.3–21.4) of cats with VN and 33.7% (CI95%: 24.5–44.5) with ELISA. Factors associated with the presence of pre-vaccination VN antibodies were age (≥2 years; OR: 7.091; p = 0.022) and lack of previous vaccination (OR: 3.472; p = 0.014). The presence of pre-vaccination ELISA antibodies was associated with time since last vaccination (OR: 5.672; p = 0.043). Outdoor cats were more likely to have a ≥4-fold ELISA titer increase (OR: 5.556; p = 0.005). Many cats had pre-vaccination FCV antibodies, and their presence depended on previous vaccinations and increases with age. A ≥4-fold titer increase was rarely observed and was influenced by the lifestyle of the cat.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 μg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

TIR-TLR7 as a Molecular Adjuvant: Simultaneous Enhancing Humoral and Cell-Mediated Immune Responses Against Inactivated Infectious Bursal Disease Virus.

Despite the robust induction of humoral immune responses, a limitation of many adjuvants is their weak stimulation of cellular immunity. The development of synthetic gene-encoding adjuvants for simultaneous induction of both humoral and cell-mediated immune responses is under study. In this study, we examined the impact of toll/interleukin-1 receptor (TIR) domain of toll-like receptor 7 (TLR7) as molecular adjuvants on potency of inactivated infectious bursal disease (IBD) vaccines. A total of 60 specific pathogen-free week-old chicks were randomized grouped to receive either TIR-TLR7-adjuvanted IBD-inactivated vaccine or inactivated IBD antigen along with an unvaccinated control. Serum antibody titers were measured to estimate the humoral immunity, as well as lymphocyte proliferation activity for cellular immune responses. The protection was estimated after challenge with a very virulent IBD virus (IBDV) strain at 4 weeks postvaccination. The results indicated that one dose of IBD/TIR-TLR7 vaccine induced specific antibody responses, whereas a lower response after administration of inactivated IBD antigen was observed. The stimulation of splenocytes results indicated that the TIR-TLR7 adjuvanted IBD vaccine is capable of modulating cell-mediated immune response in treated chickens. A full protection against IBDV infection was achieved by injection of one dose IBD/TIR-TLR7 vaccine in the challenge trial. This study demonstrated that codelivery of TIR-TLR7 with inactivated IBD antigen resulted in simultaneous enhancing immune responses against IBD.

Influence of aging on germinal centre reaction and antibody response to inactivated influenza virus antigens in mice: sex-based differences.

The study examined sex-specificities in age-related changes in BALB/c mice IgG antibody responses to immunisation with trivalent inactivated split-virus influenza bulk. Aging diminished the total serum IgG antibody responses to H1N1 and H3N2 and B influenza virus antigens in mice of both sexes, but they remained greater in aged females. This sex difference in aged mice correlated with the greater post-immunisation increase in the frequency of spleen germinal centre (GC) B cells and more favourable T follicular regulatory (Tfr)/GC B cell ratio, as Tfr cells are suggested to control antibody production through suppression of glycolysis. The greater post-immunisation GC B cell response in aged females compared with males correlated with the greater proliferation of B cells and CD4+ cells in splenocyte cultures from aged females restimulated with inactivated split-virus influenza from the bulk. To support the greater post-immunisation increase in the frequency GC B cell in aged females was more favourable Tfr/T follicular helper (Tfh) cell ratio. Additionally, compared with aged males, in age-matched females the greater avidity of serum IgG antibodies was found. However, in aged females IgG2a/IgG1 antibody ratio, reflecting spleen Th1/Th2 cytokine balance, was shifted towards IgG1 when compared with age-matched male mice. This shift was ascribed to a more prominent decline in the titres of functionally important IgG2a antibodies in females with aging. The study suggest that biological sex should be considered as a variable in designing strategies to manipulate with immune outcome of immunisation in aged animals, and possibly, at very long distance, humans.

Immunomodulatory capability of VCG in Chlamydia immunity

Abstract Immunization with UV-inactivated Chlamydia trachomatis elementary bodies (UV-EB), the infectious form of Chlamydia, does not protect against infection with live Chlamydia. Vibrio cholerae ghosts (VCG), which are empty V. cholerae cell envelopes derived by genetic inactivation, constitute an effective delivery vehicle for cloned vaccine antigens and promotes the induction of protective immunity in the absence of added adjuvants. In this study, we sought to investigate the ability of VCG to enhance the innate and adaptive immune responsesinduced by UV-EB. Thus, bone marrow-derived dendritic cells (BMDC) were pulsed with UV-EB in the presence or absence of VCG and the induction of innate immune responses, T cell proliferation and ability to afford protection against Chlamydia following adoptive transfer into susceptible mice was evaluated. The results show that VCG efficiently modulated the immunostimulatory properties of DCs as indicated by increased secretion of proinflammatory cytokines and expression of toll-like receptors and co-stimulatory molecules associated with DC maturation. Also, UV-EB-pulsed DCs that were exposed to VCG and adoptively transferred into mice resulted in effective chlamydial antigen presentation and protective immunity. The results demonstrate that VCG activate the maturation of DCs leading to enhancement of innate and adaptive immunity to an otherwise non-protective antigen, suggesting their utility as adjuvants for enhancement of protective immunity to inactivated vaccine antigens.

Role of innate lymphoid cells and dendritic cells in intradermal immunization of the enterovirus antigen

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand, foot, and mouth disease (HFMD). In our previous study, intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity, while immunization with the inactivated CA16 vaccine did not. In this report, we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different, but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-κB pathway signaling molecules, which were identified as being capable of activating DCs, ILCs, and T cells, were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly, ILC colocalization decreased over time, while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore, neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100% protected against wild-type EV71 or CA16 viral challenge. Together, our results provide new insights into the development of vaccines for HFMD.

SPRi-based hemagglutinin quantitative assay for influenza vaccine production monitoring

Influenza vaccine manufacturers lack tools, whatever the involved production bioprocess (egg or cell-based), to precisely and accurately evaluate vaccine antigen content from samples. Indeed, the gold standard single-radial immunodiffusion (SRID) assay, which remains the only validated assay for the evaluation of influenza vaccine potency, is criticized by the scientific community and regulatory agencies since a decade for its high variability, lack of flexibility and low sensitivity. We hereby report an imaging surface plasmon resonance (SPRi) assay for the quantification of both inactivated vaccine influenza antigens and viral particles derived from egg- and cell-based production samples, respectively. The assay, based on fetuin-hemagglutinin interactions, presents higher reproducibility (<3%) and a greater analytical range (0.03–20 µg/mL) than SRID for bulk monovalent and trivalent vaccine and its limit of detection was evaluated to be 100 times lower than the SRID’s one. Finally, viral particles production through cell culture-based bioprocess was also successfully monitored using our SPRi-based assay and a clear correlation was found between the biosensor response and total virus particle content.

PLGA encapsulated inactivated-viral vaccine: Formulation and evaluation of its protective efficacy against viral haemorrhagic septicaemia virus (VHSV) infection in olive flounder (Paralichthys olivaceus) vaccinated by mucosal delivery routes

Viral haemorrhagic septicaemia virus (VHSV), an OIE listed viral pathogen, is the etiological agent of a contagious disease, causing huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. In continuation with our previous work, where injection vaccination with inactivated VHSV mixed with squalene (as adjuvant) conferred higher protective immunity to olive flounder, the present study focused on replacing the injection route of vaccine delivery by immersion/oral route to overcome the limitations of the parenteral immunization method. Here, we encapsulated the inactivated VHSV vaccine with PLGA (poly lactic-co-glycolic acid) nanoparticles (PNPs-IV) and evaluated its ability to induce protective immunity in olive flounder (12.5 ± 1.5 g) by initially immunizing the fishes by immersion route followed by a booster with the same dose two weeks later with half of the fish through immersion route and other half through oral route (incorporated into fish feed). Cumulative mortalities post-challenge (1 × 106 TCID50 virus/fish) with virulent VHSV-isolate, were lower in vaccinated fish and RPS of 60% and 73.3% were obtained for PNPs-IV (immersion/immersion) and PNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, PNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to controls, in both the systemic (kidney) and mucosal (skin and intestine) immune compartment of the host post immunization as well as post challenge. Thus it can be inferred that the adopted immunization strategy efficiently protected and transported the inactivated viral antigen to target immune organs and positively stimulated the protective immune response against VHSV in olive flounder.

Recent progress in vaccines against foot-and-mouth disease

Foot-and-mouth disease (FMD) is the most highly contagious disease affecting livestock resulting in a significant adverse economic impact worldwide. Disease outbreaks in previously FMD-free countries are initially controlled by the culling of infected and in-contact animals, restriction of susceptible animal movement, and vaccination with an inactivated whole-virus antigen preparation. With increasing trade, commerce globalization, and people migration, it is likely that more inter-pool viral exchanges and spillovers will occur posing a greater threat to both, endemic and non-endemic regions, mostly due to the limited antigenic coverage of current vaccines. Currently available inactivated FMD vaccines have a number of disadvantages, including incomplete inactivation of the virus, they require multiple vaccination to maintain good levels of immunity and periodic inclusion of new viral strains into the vaccine formulation to cover new FMDV subtypes against which existing vaccines no longer protect and lack differentiation of infected from vaccinated animals (DIVA). The essential aim of DIVA strategy is realization of the so-called “vaccinate-to-live” policy, which is based on the principles that vaccinated animals exposed to FMDV will not transmit the virus. To address the shortcomings of inactivated vaccines, many efforts are currently devoted to developing novel FMD vaccines, including attenuated and marker inactivated vaccines, recombinant protein vaccines, synthetic peptide vaccines and empty capsid vaccines. Novel vaccine platforms offer promising alternatives for effective FMD control. It is likely that in the near future, multiple FMD vaccine approaches will compete for diverse markets, providing fit-for purpose solutions to evolving challenges in preventing and controlling FMD worldwide.

Th1-biased immunoadjuvant effect of the recombinant B subunit of an Escherichia coli heat-labile enterotoxin on an inactivated porcine reproductive and respiratory syndrome virus antigen via intranasal immunization in mice.

Porcine reproductive and respiratory syndrome (PRRS) is one of the major swine diseases responsible for a significant challenge in the global swine industry. The current PRRS inactivated vaccine only confers limited protection against PRRSV. Thus, using an appropriate adjuvant via a suitable administration route may help improve vaccine efficacy. In this study, the recombinant B subunit of the Escherichia coli heat-labile enterotoxin rLTB, was highly expressed in Pichia pastoris, through high-density fermentation. rLTB intranasal adjuvant properties were evaluated on an inactivated PRRS antigen in mice. Compared to the group immunized with solely PRRS antigen, a dose of 50 µg rLTB remarkably raised antigen-specific IgA antibodies at mucosal sites, and increased serum IgG antibodies, preferentially the IgG2a and IgG2b subclasses. Further, rLTB induced increases in Th1- (IFN-γ and IL-12) and Th17 (IL-6) cytokine profiles, but had little effect on Th2 cytokine profiles (IL-4 and IL-10). Moreover, there were no overt toxicities associated with intranasal rLTB administration. Our data provide evidence that the rLTB produced by P. pastoris fermentation portrays low toxicity, and its intranasal adjuvant effect involves immune system modulation to a Th1 profile.

Immunostimulation of Cyprinus carpio using phage lysate of Aeromonas hydrophila

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1β and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Inactivated H5 Antigens of H5N8 Protect Chickens from Lethal Infections by the Highly Pathogenic H5N8 and H5N6 Avian Influenza Viruses.

IntroductionHighly pathogenic Asian H5-subtype avian influenza viruses have been found in poultry and wild birds worldwide since they were first detected in southern China in 1996. Extensive control efforts have not eradicated them. Vaccination prevents such viruses infecting poultry and reduces the number lost to compulsory slaughter. The study showed the efficacy of inactivated H5 vaccine from the H5N8 virus against highly pathogenic H5N8 and H5N6 avian influenza viruses in chickens.Material and MethodsReverse genetics constructed an H5 vaccine virus using the HA gene of the 2014 H5N8 avian influenza virus and the rest of the genes from A/PR/8/34 (H1N1). The vaccine viruses were grown in fertilised eggs, partially purified through a sucrose gradient, and inactivated with formalin. Chickens were immunised i.m. with 1 μg of oil-adjuvanted inactivated H5 antigens.ResultsSingle dose H5 vaccine recipients were completely protected from lethal infections by homologous H5N8 avian influenza virus and shed no virus from the respiratory or intestinal tracts but were not protected from lethal infections by heterologous H5N6. When chickens were immunised with two doses and challenged with homologous H5N8 or heterologous H5N6, all survived and shed no virus.ConclusionOur results indicate that two-dose immunisations of chickens with H5 antigens with oil adjuvant are needed to provide broad protection against different highly pathogenic H5 avian influenza viruses.

Characterization of immunogenicity of avian influenza antigens encapsulated in PLGA nanoparticles following mucosal and subcutaneous delivery in chickens.

Mucosal vaccine delivery systems have paramount importance for the induction of mucosal antibody responses. Two studies were conducted to evaluate immunogenicity of inactivated AIV antigens encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). In the first study, seven groups of specific pathogen free (SPF) layer-type chickens were immunized subcutaneously at 7-days of age with different vaccine formulations followed by booster vaccinations two weeks later. Immune responses were profiled by measuring antibody (Ab) responses in sera and lachrymal secretions of vaccinated chickens. The results indicated that inactivated AIV and CpG ODN co-encapsulated in PLGA NPs (2x NanoAI+CpG) produced higher amounts of hemagglutination inhibiting antibodies compared to a group vaccinated with non-adjuvanted AIV encapsulated in PLGA NPs (NanoAI). The tested adjuvanted NPs-based vaccine (2x NanoAI+CpG) resulted in higher IgG responses in the sera and lachrymal secretions at weeks 3, 4 and 5 post-vaccination when immunized subcutaneously. The incorporation of CpG ODN led to an increase in Ab-mediated responses and was found useful to be included both in the prime and booster vaccinations. In the second study, the ability of chitosan and mannan coated PLGA NPs that encapsulated AIV and CpG ODN was evaluated for inducing antibody responses when delivered via nasal and ocular routes in one-week-old SPF layer-type chickens. These PLGA NPs-based and surface modified formulations induced robust AIV-specific antibody responses in sera and lachrymal secretions. Chitosan coated PLGA NPs resulted in the production of large quantities of lachrymal IgA and IgG compared to mannan coated NPs, which also induced detectable amounts of IgA in addition to the induction of IgG in lachrymal secretions. In both mucosal and subcutaneous vaccination approaches, although NPs delivery enhanced Ab-mediated immunity, one booster vaccination was required to generate significant amount of Abs. These results highlight the potential of NPs-based AIV antigens for promoting the induction of both systemic and mucosal immune responses against respiratory pathogens.

Immune Responses in Mice Immunized with Mastitis Multiple Vaccines Using Different Adjuvants

Background: Bovine mastitis, a serious disease associated with both high incidence and significant economic losses, posing a major challenge to the global dairy industry. The development of vaccines for protection from new infections by mastitis pathogens is of considerable interest to the milk production industry. Vaccination is a common and easy strategy for the control of infectious diseases, and the adjuvants used in the formulation is a critical factor for vaccine efficacy improvement. The main objective of the present study was to evaluate three different adjuvants for their ability to enhance immune responses of mice that vaccinated with Bovine Mastitis Multiple Vaccine.Materials, Methods &amp; Results: The thymus and spleen index, the phagocytic ability of macrophage and the serum antibody levels of mice were detected after vaccination, respectively. The results showed that the thymus index, spleen index, and the phagocytic ability of macrophage of mice in Aluminum group exhibited a significant higher level (P &lt; 0.05) compared with those in the control groups. The difference of the serum antibody levels was significant (P &lt; 0.05) between experimental groups and control group after vaccination. The serum antibody concentration of mice in FIA group was higher compared with other groups and had a longer duration. The antibody concentration of mice in France 206 oil group can not increase as fast as the antibody concentration of Aluminum group, but it can last a longer time at a high level. In conclusion, multiple vaccines mixed with three different adjuvants could enhance the immunity of mice and Freund’s incomplete adjuvant is the best choice for this vaccine.Discussion: Adjuvants play an important role in increasing the efficacy of a number of different vaccines. In this study, three kinds of adjuvants (Aluminum hydroxide, France 206 oil and FIA) were evaluated for their adjuvant effects for multiple vaccine of bovine mastitis in mice and aluminum hydroxide did best as the vaccine adjuvant from the results. Aluminum hydroxide is a universally accepted adjuvant for both human and veterinary vaccines. The goal of vaccination is to generate strong immune response providing protection against infection for a time. Different protective effects will usually obtained by different adjuvants even use same antigen. In this work, FIA, Alum and 206 oil were chosen as adjuvants for inactivated antigens of Streptococcus agalactiae, Streptococcus dysgalactiae and Staphylococcus aureus. The results showed that there was a significantly higher antibody levels in vaccinated mice compared with those in control group. In addition, the mice in France 206 oil and FIA group performed a higher antibody levels and stronger immunity than mice in Aluminum hydroxide groups. These findings suggest that Freund’s incomplete adjuvant (FIA) would be the best candidate as the adjuvant for mastitis multiple vaccines investigated in this study.

An Improved Inactivated Influenza Vaccine with Enhanced Cross Protection.

Current inactivated influenza vaccines are strain-specific and poorly effective against variant or mismatched viruses. They are standardized based on their hemagglutinin (HA) or ability to induce strain-specific hemagglutination inhibition (HAI) antibodies. The HA is known to undergo major conformational changes when exposed to the low pH environment of endosomes (pH 5.0 and 37°C), which are required for membrane fusion during virus cell entry. In an effort to improve these vaccines, influenza antigens treated under various low pH conditions were evaluated for increased cross-reactive antibody response and cross protection. It was found that a full range of structural and antigenic changes in HA could be induced by varying low pH treatment conditions from the mild (low pH at ≤25°C) to the strong (low pH at ≥37°C) as determined by analysis of potency, HA morphology, protease sensitivity, and reactivity with an anti-HA2 domain (CD) antibody. Inactivated antigens of both H1N1 and H3N2 strains treated at mild low pH conditions (0–25°C) exhibited only moderate HA structural and antigenic changes and markedly increased antibody response against HA2, the highly conserved part of HA, and cross protection against heterologous challenge in mice by up to 30% in survival. By contrast, antigen treated with low pH at 37°C showed more extensive structural and antigenic changes, and induced much less of an increase in antibody response against HA2, but a greater increase with response against HA1, and did not provide any increased cross protection. These results suggest that the increased response against HA2 obtained with the mild low pH treatment is associated with the increased cross protection. These antigens treated at the mild low pH conditions remained capable of inducing a high level of strain-specific HAI antibodies. Thus, they could readily be formulated as an inactivated influenza vaccine which not only provides the same strain-specific protection but also an increased cross protection against heterologous viruses. Such a vaccine could be particularly beneficial in cases of vaccine mismatch.

Chemokine CCL20 plasmid improves protective efficacy of the Montanide ISA™ 206 adjuvanted foot-and-mouth disease vaccine in mice model

This study aimed to investigate the chemokine CCL20, a macrophage inflammatory protein-3 alpha, for adjuvant potential in inactivated foot-and-mouth disease (FMD) vaccine. Groups of mice were injected intramuscularly with either murine CCL20 DNA or CCL20 protein two days ahead of the immunization with Montanide ISA206 adjuvanted inactivated FMD vaccine and humoral and cellular immune responses were measured in post-vaccinal sera. We demonstrated that the mice immunized with CCL20 plasmid plus FMD vaccine showed earlier and significantly (p < 0.05) higher neutralizing antibody responses compared to the mice vaccinated with CCL20 protein plus FMD vaccine. In fact, CCL20 as a protein did not show any adjuvant effect and the immune responses induced in this group were comparable to that of the mice vaccinated with FMD vaccine alone. All the vaccination groups showed serum IgG1 and IgG2 antibody responses; however, the mice vaccinated with CCL20 plasmid plus FMD vaccine showed significantly (p < 0.05) higher IgG1 and IgG2 responses and the responses remained high at all-time points post vaccination, although not always statistically significant. Upon restimulation of the vaccinated splenocytes with the inactivated FMD viral antigen, significantly (p < 0.05) higher IFN-γ and IL-2 levels in culture supernatants were found in animals vaccinated with the CCL20 plasmid plus FMD vaccine, which is indicative of the TH1 type of cellular immunity. On challenge with the homologous FMD virus on 28th day post immunization, CCL20 plasmid plus FMD vaccine showed complete protection (100%) while animals immunized with CCL20 protein plus FMD vaccine or FMD vaccine alone showed 66% protection. In summary, we show that prior injection of CCL20 plasmid improved protective efficacy of the inactivated FMD vaccine and thus offers a valuable strategy to modulate the efficacy and polarization of specific immunity against inactivated vaccines.