Antigen-specific Serum Antibody Research Articles

Vaccine efficacy induced by virus-like particles containing Leishmania donovani surface glycoprotein GP63.

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.

Vaccination with Mincle agonist UM-1098 and mycobacterial antigens induces protective Th1 and Th17 responses

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the top infectious killers in the world. The only licensed vaccine against TB, Bacille Calmette-Guérin (BCG), provides variable protection against pulmonary TB, especially in adults. Hence, novel TB vaccine approaches are urgently needed. Both Th1 and Th17 responses are necessary for protection against TB, yet effective adjuvants and vaccine delivery systems for inducing robust Th1 and Th17 immunity are lacking. Herein we describe a synthetic Mincle agonist, UM-1098, and a silica nanoparticle delivery system that drives Th1/Th17 responses to Mtb antigens. Stimulation of human peripheral blood mononuclear cells (hPBMCs) with UM-1098 induced high levels of Th17 polarizing cytokines IL-6, IL-1β, IL-23 as well as IL-12p70, IL-4 and TNF-α in vitro. PBMCs from both C57BL/6 and BALB/c mice responded with a similar cytokine pattern in vitro and in vivo. Importantly, intramuscular (I.M.) vaccination with UM-1098-adjuvanted TB antigen M72 resulted in significantly higher antigen-specific IFN-γ and IL-17A levels in C57BL/6 wt mice than Mincle KO mice. Vaccination of C57BL/6 wt mice with immunodominant Mtb antigens ESAT6/Ag85B or M72 resulted in predominantly Th1 and Th17 responses and induced antigen-specific serum antibodies. Notably, in a virulent Mtb challenge model, vaccination with UM-1098 adjuvanted ESAT6/Ag85B or M72 significantly reduced lung bacterial burden when compared with unvaccinated mice and protection occurred in the absence of pulmonary inflammation. These data demonstrate that the synthetic Mincle agonist UM-1098 induces strong Th1 and Th17 immunity after vaccination with Mtb antigens and provides protection against Mtb infection in mice.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

Effects of different immunomodulating liposome-based adjuvants and injection sites on immunogenicity in pigs

Vaccine adjuvants, such as liposome-based cationic adjuvant formulations (CAFs), are able to boost immune responses and, by incorporation of distinct immunomodulators, steer immunity towards a desired direction in mice, non-human primates and humans, while less studied in pigs. Here we used commercial pigs to investigate polarizing adjuvant effects of CAFs with immunomodulators: C-type lectin receptor ligands trehalose-6,6ʼ-dibehenate and monomycolyl glycerol, toll-like receptor 3 ligand Poly(I:C) or retinoic acid. Vaccines were formulated with a recombinant Chlamydia model protein antigen and administered via three injection routes. All adjuvants significantly increased antigen-specific IgG in serum, compared to non-adjuvanted antigen. Administering the vaccines through intramuscular and intraperitoneal routes induced significantly higher antigen-specific IgG and IgA serum antibodies, than the perirectal route. Although immunizations triggered cell-mediated immunity, no significant differences between adjuvants or injection sites were detected. Genes depicting T cell subtypes revealed only minor differences. Our findings suggest that specific signatures of the tested adjuvant immunomodulation do not translate well from mice to pigs in standard two-dose immunizations. This study provides new insights into immune responses to CAFs in pigs, and highlights that adjuvant development should ideally be carried out in the intended species of interest or in models with high predictive validity/translational value.

Field testing of recombinant subunit vaccines against Teladorsagia circumcincta in lambing ewes demonstrates a lack of efficacy in the face of a multi-species parasite challenge

IntroductionWe previously demonstrated efficacy of an 8-antigen recombinant subunit vaccine against a single species homologous Teladorsagia circumcincta challenge in lambs and in lambing ewes in pen trials. We subsequently demonstrated efficacy of a simplified, 2-antigen, version of this vaccine in lambs in pen trials. Here, we test both vaccines in lambing ewes in a field setting.MethodsIn the work presented here, 12 adjacent plots were seeded with a mixed infection of several common species of parasitic nematodes of sheep in temperate regions, including T. circumcincta. Ewes (n = 144), in groups of 12, grazed for 2 years on these plots and, in the first year, six of these groups of ewes were vaccinated with a 2-antigen prototype vaccine against T. circumcincta prior to mating and then again prior to lambing. In the following year these ewes were immunised again, this time with the 8-antigen prototype vaccine against T. circumcincta prior to mating and then prior to lambing. Throughout both seasons antigen-specific serum antibody levels in ewes and faecal worm egg counts (FEC) in ewes and their lambs were monitored, along with nematode species diversity at lambing.ResultsImmunised ewes produced elevated serum antibody levels to each of the vaccine antigens following immunisation but their FEC levels were not statistically significantly impacted by vaccination with either vaccine. FEC levels were also not impacted in lambs co-grazing the pastures with these immunised ewes. Nematode species diversity was not significantly impacted by vaccination in either year.DiscussionThe immunosuppressive effects of co-infecting gastrointestinal nematodes, the absence of vaccine cross-protection against co-infecting species and the influence of the periparturient relaxation in immunity probably all contributed to the inability of either vaccine to protect against T. circumcincta infection in field trials in the work presented here.

Bone marrow plasma cells require P2RX4 to sense extracellular ATP.

Plasma cells produce large quantities ofantibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells.

Characterizing the cellular and humoral immune responses to Tdap during pregnancy compared to non-pregnant controls

Abstract Maternal Tdap vaccination during pregnancy is recommended to protect infants from pertussis, pre-vaccination. However, it is unclear whether maternal immune responses improve following Tdap booster. We present analysis of immune responses pre- and post-Tdap booster, of pregnant women (Pw) primed by whole cell vaccines, who received Tdap booster during pregnancy, compared to Tdap boosted non-pregnant women (NPw). Tdap antigen specific serum antibody reactivity and avidity increased following booster in both groups. Reactivity and avidity were higher in NPw compared to Pw. Activation-induced marker assay of peripheral blood revealed that pertussis specific Tmem cells increased following Tdap booster in Pw and NPw. After Tdap booster, the percentage of CD4+CD25+OX40+ T cells was approximately 2.5-fold higher in NPw. In contrast, the proportion of CD4+CD25+PDL1+ T cells post-booster were 3-fold higher in Pw compared to NPw, suggesting that Tdap booster may elevate Tregs in Pw. Interim analysis suggests that booster vaccination increases immune responses, although the magnitude and phenotype differ between Pw and NPW. Antibody function, race related immune profiles, and detailed phenotyping of T cell responses are in progress.

Unmasking Arterial-resident Autoreactive B cells involved in Atherosclerosis and Peripheral Artery Disease Progression

Abstract Atherosclerosis activates the immune system, including B cells which secrete antibodies that bind antigens released by necrotic cells within plaques and may promote inflammation. To identify these antibodies, we isolated activated B cells from atherosclerotic ApoE-deficient mice aortas and human tibial arteries with peripheral artery disease post-amputation. Single-cell sequencing of the B cell receptors identified 35 mouse and 48 human overrepresented disease-relevant heavy and light chain pairings, which were sequentially expressed as antibody. Epitope determination was performed by human proteome array, and 14 mouse and 21 human protein targets were found with significant affinity and specificity. Mouse antigens were scrutinized by immunohistochemistry to confirm presence in inflamed plaques, and expression of these proteins were used to interrogate autoreactive antibody titers in diseased sera by ELISA. ApoE-deficient mice were immunized with the 6 most promising antigens. Serum antibody measurement showed strong, rapid, and early onset of antigen-specific antibody responses in the immunized mice compared to an adjuvant only control group. Strikingly, we saw decreased plaque accumulation in the immunized animals, suggesting antigen responses may clear pathogenic antigens before disease develops. Likewise, human antigen ELISAs comparing antigen-specific serum antibody titers to patient Framingham score for cardiovascular risk supported a role for two antigens in disease. To confirm these antigens as potential biomarkers, we will characterize the emergence of their antibody counterparts using a historical library of serum samples from individuals enrolled in the Baltimore Longitudinal Study on Aging. This work is directly funded by the National Institutes of Health – National Institute on Aging’s Intramural Research Program in the Lab of Molecular Biology and Immunology

Method of manufacturing CAF®09 liposomes affects immune responses induced by adjuvanted subunit proteins

The ability to induce antigen-specific CD4+ and CD8+T-cell responses is one of the fundamental requirements when developing new efficacious vaccines against challenging infectious diseases and cancer. However, no adjuvants are currently approved for human subunit vaccines that induce T-cell immunity. Here, we incorporated a Toll-like receptor 4 agonist, i.e., the ionizable lipidoid L5N12, in the liposomal cationic adjuvant formulation 09 (CAF®09), and found that modified CAF®09 liposomes possess preserved adjuvant function as compared to unmodified CAF®09. CAF®09 consists of the cationic lipid dimethyldioctadecylammonium (DDA), monomycoloyl glycerol analogue 1 (MMG-1), and polyinosinic:polycytidylic acid [poly(I:C)]. By using the microfluidic mixing technology for liposome preparation, we gradually replaced DDA with L5N12, while keeping the molar ratios of MMG-1 and poly(I:C) constant. We found that this type of modification resulted in colloidally stable liposomes, which were significantly smaller and displayed reduced surface charge as compared to unmodified CAF®09, prepared by using the conventional thin film method. We showed that incorporation of L5N12 decreases the membrane rigidity of CAF®09 liposomes. Furthermore, vaccination with antigen adjuvanted with L5N12-modified CAF®09 or antigen adjuvanted with unmodified CAF®09, respectively, induced comparable antigen-specific serum antibody titers. We found that antigen adjuvanted with L5N12-modified CAF®09 induced antigen-specific effector and memory CD4+ and CD8+T-cell responses in the spleen comparable to those induced when unmodified CAF®09 was used as adjuvant. However, incorporating L5N12 did not have a synergistic immunopotentiating effect on the antibody and T-cell responses induced by CAF®09. Moreover, vaccination with antigen adjuvanted with unmodified CAF®09, which was manufactured by using microfluidic mixing, induced significantly lower antigen-specific CD4+ and CD8+T-cell responses than vaccination with antigen adjuvanted with unmodified CAF®09, which was prepared by using the thin film method. These results show that the method of manufacturing affects CAF®09 liposome adjuvanted antigen-specific immune responses, which should be taken into consideration when evaluating immunogenicity of subunit protein vaccines.

Safety and immunogenicity of a thermostable ID93 + GLA-SE tuberculosis vaccine candidate in healthy adults

Adjuvant-containing subunit vaccines represent a promising approach for protection against tuberculosis (TB), but current candidates require refrigerated storage. Here we present results from a randomized, double-blinded Phase 1 clinical trial (NCT03722472) evaluating the safety, tolerability, and immunogenicity of a thermostable lyophilized single-vial presentation of the ID93 + GLA-SE vaccine candidate compared to the non-thermostable two-vial vaccine presentation in healthy adults. Participants were monitored for primary, secondary, and exploratory endpoints following intramuscular administration of two vaccine doses 56 days apart. Primary endpoints included local and systemic reactogenicity and adverse events. Secondary endpoints included antigen-specific antibody (IgG) and cellular immune responses (cytokine-producing peripheral blood mononuclear cells and T cells). Both vaccine presentations are safe and well tolerated and elicit robust antigen-specific serum antibody and Th1-type cellular immune responses. Compared to the non-thermostable presentation, the thermostable vaccine formulation generates greater serum antibody responses (p < 0.05) and more antibody-secreting cells (p < 0.05). In this work, we show the thermostable ID93 + GLA-SE vaccine candidate is safe and immunogenic in healthy adults.

Recombinant full-length Bacillus Anthracis protective antigen and its 63 kDa form elicits protective response in formulation with addavax.

Bacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential. We have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores. We have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice. We have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

Monobiotinylated Proteins Tethered to Microspheres for Detection of Antigen-Specific Serum Antibodies.

Surface modified microspheres have been leveraged as a useful way to immobilize antigen for serological studies. The use of carboxyl modified microspheres for this purpose is well-established, but commonly associated with technical challenges. Streptavidin modified microspheres require little technical expertise and thus address some of the shortcomings of carboxyl microspheres. An additional feature of streptavidin microspheres is the use of mono-biotinylated proteins, which contain a single biotinylation motif at the C-terminus. However, the relative performance of streptavidin and carboxyl microspheres is unknown. Here, we performed a head-to-head comparison of streptavidin and carboxyl microspheres. We compared antigen binding, orientation, and staining quality and found that both microspheres perform similarly based on these defined parameters. We also evaluated the utility of streptavidin microspheres bound to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD), to reliably detect RBD-specific IgG1, IgG3, and IgA1 produced in individuals recently immunized with Pfizer/BioNTech mRNA coronavirus disease (COVID) vaccine as 'proof-of-concept'. We provide evidence that each of the antibody targets are detectable in serum using RBD-coated microspheres, Ig-specific 'detector' monoclonal antibodies (mAbs), and flow cytometry. We found that cross-reactivity of the detector mAbs can be minimized by antibody titration to improve differentiation between IgG1 and IgG3. We also coated streptavidin microspheres with SARS-CoV-2 delta variant RBD to determine if the streptavidin microsphere approach revealed any differences in binding of immune serum antibodies to wild-type (Wuhan) versus variant RBD (Delta). Overall, our results show that streptavidin microspheres loaded with mono-biotinylated antigen is a robust alternative to chemically cross-linking antigen to carboxyl microspheres for use in serological assays.

Inactivated rabies-vectored SARS-CoV-2 vaccine provides long-term immune response unaffected by vector immunity

The objective of this study is to further analyze recombinant rabies virus-vectored SARS-CoV-2 vaccine, CORAVAX, as an effective COVID-19 vaccine strategy. CORAVAX has proven immunogenic and protective against SARS-CoV-2 in animal models. Here, we have screened adjuvants for the highest quality antibody titers, negated the concern of pre-existing rabies-vector immunity, and established its potential as a long-term COVID-19 vaccine. We have tested toll-like receptor 4 (TLR4) agonists, inflammasome activators, and alum adjuvants in CORAVAX and found TLR4-activating MPLA-AddaVax to have the greatest potential. We followed the humoral immune response to CORAVAX in mice with pre-existing rabies virus immunity and saw no significant differences compared to naive mice. We then followed the immune response to CORAVAX over several months and 1-year post-immunization. Mice maintained high antigen-specific serum antibody titers as well as long-lived antibody-secreting cells in the spleen and bone marrow. We believe this rabies-vector strategy combats the problem of waning immunity of other COVID-19 vaccines. These results together support CORAVAX’s potential during the ongoing COVID-19 pandemic.

P120 Vaccine induced protection by secreted aspartyl proteinase 2 from Candida parapsilosis in Candida tropicalis mediated murine systemic candidiasis: a role of B-cells and antibodies

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesNon-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. These challenges merit the need for developing novel immune therapies against Candida infections. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading Candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. Moreover, while the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. As such, we also investigated the role of humoral immunity in vaccine-mediated protection during murine systemic candidiasis.MethodsThe Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis strains prevalent in India. Groups of wild-type BALb/c mice were vaccinated with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with a lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Histopathological assessment was performed using H&E and PAS staining. Serum cytokine levels and antigen-specific antibody titers were measured by ELISA. Cellular responses were analyzed in detail using flow cytometry. Functional evaluation of antibody role was performed using in vitro (biofilm inhibition and neutrophil-mediated killing) and in vivo (passive transfer) studies. B-cell epitope analysis was carried out using immunoinformatics approaches.ResultsMice vaccinated with rSap2 cloned from C. parapsilosis (Sap2-parapsilosis) showed highest increase in survival time (P = .02) and maximum reduction in organ fungal burden (spleen, kidney, lungs, brain) (P <.05); compared with sham immunized controls. Vaccination with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ∼60% homology across species. Mice vaccinated with rSap2-parapsilosis also exhibited significantly higher levels of IFN gamma, IL-17, and IL-4 cytokine levels just before infection, which correlated with protection. In addition, rSap2-parapsilosis vaccination induced high titers of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (which were predominantly of IgM isotype). Notably, sera from rSap2-parapsilosis vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19 + B-cells, plasma cells, and Candida-binding B-cells in rSap2-parapsilosis vaccinated mice indicate a role of B-cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B-cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multi-epitope anti-Candida vaccine/s.ConclusionMice immunized with recombinant Sap2 cloned from C. parapsilosis exhibited increased survival during C. tropicalis mediated systemic candidiasis. Both Th1/Th17 mediated cellular immunity and humoral immunity contribute toward protection. Our results establish a role of B-cells and antibodies in generation of protective immune responses against Sap2 vaccine antigen during non-albicans Candida mediated murine systemic candidiasis.

Kinetic of the Antibody Response Following AddaVax-Adjuvanted Immunization with Recombinant Influenza Antigens.

Notwithstanding the current SARS-CoV-2 pandemic, influenza virus infection still represents a global health concern in terms of hospitalizations and possible pandemic threats. The objective of next-generation influenza vaccines is not only to increase the breadth of response but also to improve the elicitation of an effective and robust immune response, especially in high-risk populations. To achieve this second objective, the administration of adjuvanted influenza vaccines has been considered. In this regard, the monitoring and characterization of the antibody response associated with the administration of adjuvanted vaccines has been evaluated in this study in order to shed light on the kinetic, magnitude and subclass usage of antibody secreting cells (ASCs) as well as of circulating antigen-specific serum antibodies. Specifically, we utilized the DBA/2J mouse model to assess the kinetic, magnitude and IgG subclass usage of the antibody response following an intramuscular (IM) or intraperitoneal (IP) immunization regimen with AddaVax-adjuvanted bivalent H1N1 and H3N2 computationally optimized broadly reactive antigen (COBRA) influenza recombinant hemagglutinins (rHAs). While the serological evaluation revealed a homogeneous kinetic of the antibody response, the detection of the ASCs through a FluoroSpot platform revealed a different magnitude, subclass usage and kinetic of the antigen-specific IgG secreting cells peaking at day 5 and day 9 following the IP and IM immunization, respectively.

Oral Administration of a Shigella 2aT32-Based Vaccine Expressing UreB-HspA Fusion Antigen With and Without Parenteral rUreB-HspA Boost Confers Protection Against Helicobacter pylori in Mice Model.

Helicobacter pylori (H. pylori) is a gram-negative pathogen classified as a class I carcinogen. The H. pylori urease B subunit (UreB) and heat shock protein A (HspA) are two important vaccine candidate antigens. In this study, we evaluated the immunogenicity and immunoprotective effect of the attenuated Shigella vector vaccine SH02 expressing the UreB-HspA fusion protein of H. pylori in a mouse model. Oral SH02 with or without subcutaneous injection of rUreB-HspA induced antigen-specific serum IgG, mucosal sIgA, and T cells immune response. Subcutaneous injection of the candidate antigen rUreB-HspA enhanced the level of serum antigen-specific IgG antibodies (p < 0.0001) and the levels of IgG1/IgG2a/IgG2b subtypes. In addition, injection boost also increased the proportion of spleen antigen-specific CD4+CD154+ T cells (p < 0.001), and the proportion of CD4+CD154+ T cells that secrete IFN-γ and IL-17A. Following the H. pylori challenge, the levels of H. pylori colonization in the two experimental groups (Groups A and B) significantly reduced compared with the control group (p < 0.001), indicating that the candidate vaccine yielded a preventive effect of anti-H.pylori infection. Compared with the non-subcutaneous booster injection group (Group A), the subcutaneous booster injection group (Group B) exhibited less gastric inflammation, but there was no significant difference in the level of colonization (p > 0.05). These results lay a foundation for the development of a vaccine against H. pylori and the optimization of immunization methods and procedures to prevent H. pylori infection.

Absolute Quantitation of Serum Antibody Reactivity Using the Richards Growth Model for Antigen Microspot Titration.

In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen-specific serum antibodies. We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease-specific antigens were treated with different serum dilutions and developed for IgG and IgA binding. By fitting the binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen. By providing universal chemical units, this approach may improve the standardization of serological testing, the quality control of antibodies and the quantitative mapping of the antibody–antigen interaction space.

Comparative analysis of the biological characteristics of three CV-A10 clones adaptively cultured on Vero cells.

Coxsackievirus A10 (CV-A10) is a major pathogen that causes hand, foot, and mouth disease. There are no effective therapeutic drugs for CV-A10 infection; therefore, CV-A10 vaccines should be developed. Previously, we isolated a CV-A10 strain (N25) that can be cultured on Vero cells. In this study, the N25 strain was plaque-purified three times from Vero cells, and three clones were selected for adaptive culture. The three clones of the 5th, 12th, and 19th generations were compared and analyzed in terms of viral titers, plaque morphology, pathogenicity in suckling mice, and nucleotide and amino acid sequences of the complete genome. The infectivity titers of the three clones (P2-P22) were maintained at 6.5-7.0 lgCCID50 /ml. The three clones began to proliferate at 6 h and peaked at 36 h; the corresponding CCID50 was in the range of 106.5 -106.875 /ml, which gradually decreased. The suckling mice in the challenged group exhibited clinical symptoms such as paralysis of the limbs, which gradually worsened until death. The inactivated vaccines prepared using the three clones efficiently induced antigen-specific serum antibodies in mice. There were eight nucleotide mutations in the three clones, which resulted in two and four amino acid substitutions in the VP3 and VP1 coding regions, respectively. The nucleotide and amino acid sequence homology between the three clones and N25 were 99.92%-100% and 99.78%-100%, respectively, indicating high genetic stability. Our findings provide a theoretical basis for screening CV-A10 vaccine candidate clones.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

Engineering aluminum hydroxyphosphate nanoparticles with well-controlled surface property to enhance humoral immune responses as vaccine adjuvants

Aluminum phosphate adjuvants play a critical role in human inactivated and subunit prophylactic vaccines. However, a major challenge is that the underlying mechanism of immune stimulation remains poorly understood, which impedes the further optimal design and application of more effective adjuvants in vaccine formulations. To address this, a library of amorphous aluminum hydroxyphosphate nanoparticles (AAHPs) is engineered with defined surface properties to explore the specific mechanism of adjuvanticity at the nano-bio interface. The results demonstrate that AAHPs could induce cell membrane perturbation and downstream inflammatory responses, with positively-charged particles showing the most significantly enhanced immunostimulation potentials compared to the neutral or negatively-charged particles. In a vaccine using Staphylococcus aureus (S. aureus) recombinant protein as antigens, the positively-charged particles elicit long-lasting and enhanced humoral immunity, and provide protection in S. aureus sepsis mice models. In addition, when formulated with human papillomavirus type 18 virus-like particles, it is demonstrated that particles with positive charges outperform in promoting serum antigen-specific antibody productions. This study shows that engineering AAHPs with well-controlled physicochemical properties enable the establishment of a structure-activity relationship that is critical to instruct the design of suitable engineered nanomaterial-based adjuvants within vaccine formulations for the benefits of human health.