Robust IgG Response Research Articles

229. IDSA FEATURED ORAL ABSTRACT: MVA-BN Induces a Low Avidity, Non-Durable Humoral Response Against Mpox Virus

Abstract Background The 2022 global outbreak of clade IIb mpox, the first significant occurrence outside Africa, led to vaccination campaigns using the third-generation orthopoxvirus vaccine modified vaccinia Ankara, Bavarian Nordic (MVA-BN or JYENNEOS vaccine). In the US, a one-fifth dose of an intradermal regimen of the MVA-BN was authorized as a dose-sparing alternative to the two-dose subcutaneous regimen licensed in 2019. During the vaccination campaign, we launched the New York City Observational Study of Mpox Immunity (NYC OSMI) which enrolled participants who received the MVA-BN vaccine during the outbreak in NYC. Here we report the antibody data at one-year post vaccination. Methods We initiated the New York City Observational Study of Mpox Immunity (NYC OSMI), a longitudinal study involving 171 MVA-BN vaccinees, some with prior smallpox vaccination and mpox convalescents. Study participants had blood drawn prior to vaccination, after one dose, and after two doses. We performed Immunofluorescence-based Mpox virus (MPXV) clade IIb microneutralization assays, Multiplexed immunoassay for binding antibodies and avidity, and Anti-MPXV H3 Enzyme-Linked Immunosorbent Assay (ELISA). Paired measurements in figures were analyzed by Wilcoxon matched-pairs signed rank test. Comparisons of multiple sample groups in figures were conducted by Kruskal-Wallis test with Dunn’s method for multiple comparisons. Linear mixed-effects regressions (LMER) were used to model neutralizing titer durability with repeated measurements. Results Our findings indicate that neutralizing titers in naïve individuals return to baseline in less than a year, while those with prior smallpox vaccination maintain elevated titers. Both groups generate robust IgG responses against MPXV H3 and A35, but the response in naïve vaccinees, characterized by lower avidity, is short-lived. Conclusion These results highlight the necessity for further investigation into MVA-BN's long-term protection, potential need for booster doses, and the development of next-generation orthopoxvirus vaccines. Disclosures Mark J. Mulligan, M.D., HilleVax: Advisor/Consultant|Meissa Vaccines: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi: Grant/Research Support

Proteomic and serologic assessments of responses to mRNA-1273 and BNT162b2 vaccines in human recipient sera

IntroductionThe first vaccines approved against SARS-CoV-2, mRNA-1273 and BNT162b2, utilized mRNA platforms. However, little is known about the proteomic markers and pathways associated with host immune responses to mRNA vaccination. In this proof-of-concept study, sera from male and female vaccine recipients were evaluated for proteomic and immunologic responses 1-month and 6-months following homologous third vaccination.MethodsAn aptamer-based (7,289 marker) proteomic assay coupled with traditional serology was leveraged to generate a comprehensive evaluation of systemic responsiveness in 64 and 68 healthy recipients of mRNA-1273 and BNT162b2 vaccines, respectively.ResultsSera from female recipients of mRNA-1273 showed upregulated indicators of inflammatory and immunological responses at 1-month post-third vaccination, and sera from female recipients of BNT162b2 demonstrated upregulated negative regulators of RNA sensors at 1-month. Sera from male recipients of mRNA-1273 showed no significant upregulation of pathways at 1-month post-third vaccination, though there were multiple significantly upregulated proteomic markers. Sera from male recipients of BNT162b2 demonstrated upregulated markers of immune response to doublestranded RNA and cell-cycle G(2)/M transition at 1-month. Random Forest analysis of proteomic data from pre-third-dose sera identified 85 markers used to develop a model predictive of robust or weaker IgG responses and antibody levels to SARS-CoV-2 spike protein at 6-months following boost; no specific markers were individually predictive of 6-month IgG response. Thirty markers that contributed most to the model were associated with complement cascade and activation; IL-17, TNFR pro-apoptotic, and PI3K signaling; and cell cycle progression.DiscussionThese results demonstrate the utility of proteomics to evaluate correlates or predictors of serological responses to SARS-CoV-2 vaccination.

Evaluating small extracellular vesicle-based vaccination across heterologous Salmonella strains isolated from wastewater.

Salmonella infections pose significant public health challenges worldwide. The diversity of Salmonella strains, particularly those isolated from environmental and clinical sources, necessitates innovative approaches to prevention and treatment. Previous research has shown that small extracellular vesicles (sEVs) produced by macrophages during Salmonella Typhimurium infection can induce robust immune responses when used as a vaccine, offering complete protection in systemic infection models. In this study, we isolated 120 Salmonella strains from qPCR invA-positive wastewater samples collected in Gainesville, FL. These strains underwent enrichment, selection, and biochemical confirmation, followed by serotyping and whole genome sequencing. Two isolates, Salmonella enterica subsp. diarizonae (Diarizonae) and S. enterica serovar Enteritidis, were selected for further analysis based on community prevalence and clinical severity. We also assessed the ability of sEVs produced by S. Typhimurium-infected macrophages to induce immune responses against these heterologous and circulating strains in mice. Immunization with sEVs induced robust antigen-specific SIgA and IgG responses against S. Typhimurium, Enteritidis, and Diarizonae, with high titers observed in sera and fecal samples. Proteomic analysis revealed differential expression of proteins in these strains, including antigenic proteins present in sEVs such as OmpA, FliC, or OmpD. Moreover, this study highlights the role of wastewater-based epidemiology (WBE) as a tool for environmental surveillance, offering a complementary perspective on Salmonella dynamics within a population. Integrating WBE with traditional surveillance methods, along with the promising results of sEV-based vaccination, provides a pragmatic strategy for developing effective preventative measures against Salmonella infections, addressing the diversity of non-typhoidal Salmonella strains.

Immunity and Protective Efficacy of a Plant-Based Tobacco Mosaic Virus-like Nanoparticle Vaccine against Influenza a Virus in Mice.

The rapid production of influenza vaccines is crucial to meet increasing pandemic response demands. Here, we developed plant-made vaccines comprising centralized consensus influenza hemagglutinin (HA-con) proteins (H1 and H3 subtypes) conjugated to a modified plant virus, tobacco mosaic virus (TMV) nanoparticle (TMV-HA-con). We compared immune responses and protective efficacy against historical H1 or H3 influenza A virus infections among TMV-HA-con, HA-con protein combined with AddaVax™ adjuvant, and whole-inactivated virus vaccine (Fluzone®). Immunogenicity studies demonstrated robust IgG, IgM, and IgA responses in the TMV-HA-con and HA-con protein vaccinated groups, with relatively low induction of interferon (IFN)-γ+ T-cell responses across all vaccinated groups. The TMV-HA-con and HA-con protein groups displayed partial protection (100% and 80% survival) with minimal weight loss following challenge with two H1N1 strains. The HA-con protein group exhibited 80% and 100% survival against two H3 strains, whereas the TMV-HA-con groups showed reduced protection (20% survival). The Fluzone® group conferred 20-100% survival against two H1N1 strains and one H3N1 strain, but did not protect against H3N2 infection. Our findings indicate that TMV-HA and HA-con protein vaccines with adjuvant induce protective immune responses against influenza A virus infections. Furthermore, our results underscore the potential of plant-based production using TMV-like nanoparticles for developing influenza A virus candidate vaccines.

Production and Characterization of Self-Assembled Virus-like Particles Comprising Capsid Proteins from Genotypes 3 and 4 Hepatitis E Virus (HEV) and Rabbit HEV Expressed in Escherichia coli.

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7-95.3%), as assessed by bioanalyzer, with yields of 13.9-89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development.

Scalable bioprocess for high-yield production of SARS-CoV-2 trimeric spike protein-based immunogen (IMT-CVAX) using suspension CHO cells

The COVID-19 pandemic has brought global attention towards the readiness of biotechnology platforms for rapid development of immunogens to be used as vaccine antigens, in diagnostics and in fundamental research. In the present study we provide a bioprocess that is both robust and industry-compatible for high level expression, efficient purification, and scale-up of IMT-CVAX, a prefusion-stabilized trimeric spike protein of SARS-CoV-2. The recombinant IMT-CVAX protein was produced using stable cell pool of Chinese Hamster Ovary (CHO-S) cells that were banked in compliance with cGMP (Current Good Manufacturing Practices) regulations. High quantity expression of IMT-CVAX was achieved through optimization of the shake flask-based process with regards to media, nutrient feeding regimen, incubation temperature, viable cell density and cell viability. We employed refined expression procedures to scale up IMT-CVAX using bioreactor, resulting in a significantly higher yield of around 500 mg/L. Subsequently, a straightforward and simple purification process was developed to produce high-quality IMT-CVAX protein. This procedure consisted of centrifugation, tangential flow filtration (TFF), and either one- or two-step liquid chromatography. A robust anti-spike IgG response was observed in mice after immunization with purified adjuvanted IMT-CVAX. In pseudoviral neutralization assay, mice-generated anti-IMT-CVAX sera could neutralize various SARS-CoV-2 variants. The human convalescent sera from COVID-19-recovered patients recognized IMT-CVAX effectively, which confirms the conformational integrity of IMT-CVAX epitopes. The bioprocess demonstrated here is able to produce sufficient quantities of biopharmaceutical-quality spike protein, which can be used as immunogen against emerging SARS-CoV-2 variants, and can aid in the rapid response to any other pandemic-potential pathogens.

A synthetic TLR4 agonist significantly increases humoral immune responses and the protective ability of an MDCK-cell-derived inactivated H7N9 vaccine in mice.

Antigenically divergent H7N9 viruses pose a potential threat to public health, with the poor immunogenicity of candidate H7N9 vaccines demonstrated in clinical trials underscoring the urgent need for more-effective H7N9 vaccines. In the present study, mice were immunized with various doses of a suspended-MDCK-cell-derived inactivated H7N9 vaccine, which was based on a low-pathogenic H7N9 virus, to assess cross-reactive immunity and cross-protection against antigenically divergent H7N9 viruses. We found that the CRX-527 adjuvant, a synthetic TLR4 agonist, significantly enhanced the humoral immune responses of the suspended-MDCK-cell-derived H7N9 vaccine, with significant antigen-sparing and immune-enhancing effects, including robust virus-specific IgG, hemagglutination-inhibiting (HI), neuraminidase-inhibiting (NI), and virus-neutralizing (VN) antibody responses, which are crucial for protection against influenza virus infection. Moreover, the CRX-527-adjuvanted H7N9 vaccine also elicited cross-protective immunity and cross-protection against a highly pathogenic H7N9 virus with a single vaccination. Notably, NI and VN antibodies might play an important role in cross-protection against lethal influenza virus infections. This study showed that a synthetic TLR4 agonist adjuvant has a potent immunopotentiating effect, which might be considered worth further development as a means of increasing vaccine effectiveness.

Insights into the Pathogenesis and Development of Recombinant Japanese Encephalitis Virus Genotype 3 as a Vaccine.

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development.

Intranasal respiratory syncytial virus vaccine attenuated by codon-pair deoptimization of seven open reading frames is genetically stable and elicits mucosal and systemic immunity and protection against challenge virus replication in hamsters.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory illness worldwide, but there is no approved pediatric vaccine. Here, we describe the development of the live-attenuated RSV vaccine candidate Min AL as well as engineered derivatives. Min AL was attenuated by codon-pair deoptimization (CPD) of seven of the 11 RSV open reading frames (ORFs) (NS1, NS2, N, P, M, SH and L; 2,073 silent nucleotide substitutions in total). Min AL replicated efficiently in vitro at the permissive temperature of 32°C but was highly temperature sensitive (shut-off temperature of 36°C). When serially passaged at increasing temperatures, Min AL retained greater temperature sensitivity compared to previous candidates with fewer CPD ORFs. However, whole-genome deep-sequencing of passaged Min AL revealed mutations throughout its genome, most commonly missense mutations in the polymerase cofactor P and anti-termination transcription factor M2-1 (the latter was not CPD). Reintroduction of selected mutations into Min AL partially rescued its replication in vitro at temperatures up to 40°C, confirming their compensatory effect. These mutations restored the accumulation of positive-sense RNAs to wild-type (wt) RSV levels, suggesting increased activity by the viral transcriptase, whereas viral protein expression, RNA replication, and virus production were only partly rescued. In hamsters, Min AL and derivatives remained highly restricted in replication in the upper and lower airways, but induced serum IgG and IgA responses to the prefusion form of F (pre F) that were comparable to those induced by wt RSV, as well as robust mucosal and systemic IgG and IgA responses against RSV G. Min AL and derivatives were fully protective against challenge virus replication. The derivatives had increased genetic stability compared to Min AL. Thus, Min AL and derivatives with selected mutations are stable, attenuated, yet highly-immunogenic RSV vaccine candidates that are available for further evaluation.

A TLR4 ligand-based adjuvant for promoting the immunogenicity of typhoid subunit vaccines.

None of the typhoid Vi Polysaccharide (ViPS) subunit vaccines incorporate adjuvants, and the immunogenicity of ViPS vaccines (e.g. Typbar TCV® and Typhim Vi®) is in part due to associated TLR4 ligands such as endotoxin present in these vaccines. Since endotoxin content in vaccines is variable and kept very low due to inherent toxicity, it was hypothesized that incorporating a defined amount of a non-toxic TLR4-ligand such as monophosphoryl lipid A in ViPS vaccines would improve their immunogenicity. To test this hypothesis, a monophosphoryl lipid A-based adjuvant formulation named Turbo was developed. Admixing Turbo with Typbar TCV® (ViPS-conjugated to tetanus toxoid) increased the levels of anti-ViPS IgM, IgG1, IgG2b, IgG2a/c, and IgG3 in inbred and outbred mice. In infant mice, a single immunization with Turbo adjuvanted Typbar TCV® resulted in a significantly increased and durable IgG response and improved the control of bacterial burden compared to mice immunized without Turbo. Similarly, when adjuvanted with Turbo, the antibody response and control of bacteremia were also improved in mice immunized with Typhim Vi®, an unconjugated vaccine. The immunogenicity of unconjugated ViPS is inefficient in young mice and is lost in adult mice when immunostimulatory ligands in ViPS are removed. Nevertheless, when adjuvanted with Turbo, poorly immunogenic ViPS induced a robust IgG response in young and adult mice, and this was observed even under antigen-limiting conditions. These data suggest that incorporation of Turbo as an adjuvant will make typhoid vaccines more immunogenic regardless of their intrinsic immunogenicity or conjugation status and maximize the efficacy across all ages.

Mucosal adjuvanticity and mucosal booster effect of colibactin-depleted probiotic Escherichia coli membrane vesicles

ABSTRACT There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (ΔflhDΔclbP). ΔflhDΔclbP-derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (ΔflhD-MVs). In addition, glycoengineered ΔflhDΔclbP-MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14+MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14+MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g. an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of ΔflhDΔclbP-MVs in the context of vaccine safety.

Monophosphoryl Lipid A-based Adjuvant to Promote the Immunogenicity of Multivalent Meningococcal Polysaccharide Conjugate Vaccines.

Activation of the adaptive immune system requires the engagement of costimulatory pathways in addition to B and T cell Ag receptor signaling, and adjuvants play a central role in this process. Many Gram-negative bacterial polysaccharide vaccines, including the tetravalent meningococcal conjugate vaccines (MCV4) and typhoid Vi polysaccharide vaccines, do not incorporate adjuvants. The immunogenicity of typhoid vaccines is due to the presence of associated TLR4 ligands in these vaccines. Because the immunogenicity of MCV4 is poor and requires boosters, I hypothesized that TLR4 ligands are absent in MCV4 and that incorporation of a TLR4 ligand-based adjuvant would improve their immunogenicity. Consistent with this hypothesis, two Food and Drug Administration-approved MCV4 vaccines, MENVEO and MenQuadfi, lack TLR4 ligands. Admixing monophosphoryl lipid A, a TLR4 ligand-based adjuvant formulation named "Turbo" with MCV4 induced significantly improved IgM and IgG responses to all four meningococcal serogroup polysaccharides in adult and aged mice after a single immunization. Furthermore, in infant mice, a single booster was sufficient to promote a robust IgG response and 100% seroconversion when MCV4 was adjuvanted with Turbo. Turbo upregulated the expression of the costimulatory molecules CD40 and CD86 on B cells, and Turbo-driven adjuvanticity is lost in mice deficient in CD40 and CD86. These data suggest that Turbo induces the required costimulatory molecules for its adjuvant activity and that incorporation of Turbo could make bacterial polysaccharide vaccines more immunogenic, minimize booster requirements, and be cost-effective, particularly for those individuals in low- and middle-income and disease-endemic countries.

A Longitudinal Study in Tunisia to Assess the Anti-RBD IgG and IgA Responses Induced by Three Different COVID-19 Vaccine Platforms.

Vaccination constitutes the best strategy against COVID-19. In Tunisia, seven vaccines standing for the three main platforms, namely RNA, viral vector, and inactivated vaccines, have been used to vaccinate the population at a large scale. This study aimed to assess, in our setting, the kinetics of vaccine-induced anti-RBD IgG and IgA antibody responses. Using in-house developed and validated ELISA assays, we measured anti-RBD IgG and IgA serum antibodies in 186 vaccinated workers at the Institut Pasteur de Tunis over 12 months. We showed that RNA vaccines were the most immunogenic vaccines, as compared to alum-adjuvanted inactivated and viral-vector vaccines, either in SARS-CoV-2-naïve or in SARS-CoV-2-experienced individuals. In addition to the IgG antibodies, the vaccination elicited RBD-specific IgAs. Vaccinated individuals with prior SARS-CoV-2 infection exhibited more robust IgG and IgA antibody responses, as compared to SARS-CoV-2-naïve individuals. After following up for 12 months post-immunization, we concluded that the hierarchy between the platforms for anti-RBD antibody-titer dynamics was RNA vaccines, followed by viral-vector and alum-adjuvanted inactivated vaccines.

Long-term immunogenicity and safety of heterologous boosting with a SARS-CoV-2 mRNA vaccine (SYS6006) in Chinese participants who had received two or three doses of inactivated vaccine.

The emerging new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) needs booster vaccination. We evaluated the long-term safety and immunogenicity of heterologous boosting with a SARS-CoV-2 messenger RNA vaccine SYS6006. A total of 1000 participants aged 18 years or more who had received two (Group A) or three (Group B) doses of SARS-CoV-2 inactivated vaccine were enrolled and vaccinated with one dose of SYS6006 which was designed based on the prototype spike protein and introduced mutation sites. Adverse events (AEs) through 30 days and serious AEs during the study were collected. Live-virus and pseudovirus neutralizing antibody (Nab), binding antibody (immunoglobulin G [IgG]) and cellular immunity were tested through 180 days. Solicited all, injection-site and systemic AEs were reported by 618 (61.8%), 498 (49.8%), and 386 (38.6%) participants, respectively. Most AEs were grade 1. The two groups had similar safety profile. No vaccination-related SAEs were reported. Robust wild-type (WT) live-virus Nab response was elicited with peak geometric mean titers (GMTs) of 3769.5 (Group A) and 5994.7 (Group B) on day 14, corresponding to 1602.5- and 290.8-fold increase versus baseline, respectively. The BA.5 live-virus Nab GMTs were 87.7 (Group A) and 93.2 (Group B) on day 14. All participants seroconverted for WT live-virus Nab. Robust pseudovirus Nab and IgG responses to wild type and BA.5 were also elicited. ELISpot assay showed robust cellular immune response, which was not obviously affected by virus variation. In conclusion, SYS6006 heterologous boosting demonstrated long-term good safety and immunogenicity in participants who had received two or three doses of SARS-CoV-2 inactivated vaccine.

Development of NHAcGD2/NHAcGD3 conjugates of bacteriophage MX1 virus-like particles as anticancer vaccines.

The successful development of an anticancer vaccine will be a giant leap forward in cancer prevention and treatment. Herein, the bacteriophage MX1 coat protein virus-like particles (MX1 VLPs) have been conjugated with 9NHAc-GD2 (NHAcGD2) to obtain a MX1-NHAcGD2 conjugate. Intriguingly, vaccinating against this conjugate produced a robust anti-NHAcGD2 IgG response in mice, with an average IgG titer of over 3 million. More interestingly, antibodies induced by the MX1-NHAcGD2 conjugate bound well to IMR-32 neuroblastoma cells and had potent complement-dependent cytotoxic (CDC) effects on IMR-32 cells. Inspired by the superiority of the 9NHAc-GD2 antigen, we also designed another 9NHAc-modified ganglioside antigen, 9NHAc-GD3 (NHAcGD3), to overcome the hydrolytic instability of 9-O-acetylated-GD3. By coupling NHAcGD3 with MX1 VLP, the MX1-NHAcGD3 conjugate was constructed. Strikingly, vaccination of MX1-NHAcGD3 elicited high anti-NHAcGD3 IgG antibodies, which effectively recognized human malignant melanoma SK-MEL-28 cells and had a significant CDC effect against this cell line. This study provides novel MX1-NHAcGD2 and MX1-NHAcGD3 conjugates with broad clinical translational prospects as promising anticancer vaccines.

Antigen-specific T helper cells and cytokine profiles predict intensity and longevity of cellular and humoral responses to SARS-CoV-2 booster vaccination.

Pre-existent pools of coronavirus-specific or cross-reactive T cells were shown to shape the development of cellular and humoral immune responses after primary mRNA vaccination against SARS-CoV-2. However, the cellular determinants of responses to booster vaccination remain incompletely understood. Therefore, we phenotypically and functionally characterized spike antigen-specific T helper (Th) cells in healthy, immunocompetent individuals and correlated the results with cellular and humoral immune responses to BNT162b2 booster vaccination over a six-month period. Blood of 30 healthy healthcare workers was collected before, 1, 3, and 6 months after their 3rd BNT162b2 vaccination. Whole blood was stimulated with spike peptides and analyzed using flow cytometry, a 13-plex cytokine assay, and nCounter-based transcriptomics. Spike-specific IgG levels at 1 month after booster vaccination correlated with pre-existing CD154+CD69+IFN-γ+CD4+ effector memory cells as well as spike-induced IL-2 and IL-17A secretion. Early post-booster (1-month) spike IgG levels (r=0.49), spike-induced IL‑2 (r=0.58), and spike-induced IFN‑γ release (r=0.43) correlated moderately with their respective long-term (6-month) responses. Sustained robust IgG responses were significantly associated with S-specific (CD69+±CD154+±IFN-γ+) Th-cell frequencies before booster vaccination (p=0.038), especially double/triple-positive type-1 Th cells. Furthermore, spike IgG levels, spike-induced IL‑2 release, and spike-induced IFN‑γ release after 6 months were significantly associated with increased IL‑2 & IL‑4, IP‑10 & MCP1, and IFN‑γ & IP‑10 levels at 1 month post-booster, respectively. On the transcriptional level, induction of pathways associated with both T-cell proliferation and antigen presentation was indicative of sustained spike-induced cytokine release and spike-specific IgG production 6 months post-booster. Using support vector machine models, pre-booster spike-specific T-cell frequencies and early post-booster cytokine responses predicted sustained (6-month) responses with F1 scores of 0.80-1.00. In summary, spike-specific Th cells and T-cellular cytokine signatures present before BNT162b2 booster vaccination shape sustained adaptive cellular and humoral responses post-booster. Functional T-cell assays might facilitate early identification of potential non-responders.

Appearance of tolerance-induction and non-inflammatory SARS-CoV-2 spike-specific IgG4 antibodies after COVID-19 booster vaccinations.

Understanding the characteristics of the humoral immune responses following COVID-19 vaccinations is crucial for refining vaccination strategies and predicting immune responses to emerging SARS-CoV-2 variants. A longitudinal analysis of SARS-CoV-2 spike receptor binding domain (RBD) specific IgG antibody responses, encompassing IgG subclasses IgG1, IgG2, IgG3, and IgG4 was performed. Participants received four mRNA vaccine doses (group 1; n=10) or two ChAdOx1 nCoV-19 and two mRNA booster doses (group 2; n=19) in Bangladesh over two years. Findings demonstrate robust IgG responses after primary Covishield or mRNA doses; declining to baseline within six months. First mRNA booster restored and surpassed primary IgG responses but waned after six months. Surprisingly, a second mRNA booster did not increase IgG levels further. Comprehensive IgG subclass analysis showed primary Covishield/mRNA vaccination generated predominantly IgG1 responses with limited IgG2/IgG3, Remarkably, IgG4 responses exhibited a distinct pattern. IgG4 remained undetectable initially but increased extensively six months after the second mRNA dose, eventually replacing IgG1 after the 3rd/4th mRNA doses. Conversely, initial Covishield recipients lack IgG4, surged post-second mRNA booster. Notably, mRNA-vaccinated individuals displayed earlier, robust IgG4 levels post first mRNA booster versus Covishield counterparts. IgG1 to IgG4 ratios decreased with increasing doses, most pronounced with four mRNA doses. This study highlights IgG response kinetics, influenced by vaccine type and doses, impacting immunological tolerance and IgG4 induction, shaping future vaccination strategies. This study highlights the dynamics of IgG responses dependent on vaccine type and number of doses, leading to immunological tolerance and IgG4 induction, and shaping future vaccination strategies.

Safety and immunogenicity of primary vaccination with a SARS-CoV-2 mRNA vaccine (SYS6006) in Chinese participants aged 18 years or more: Two randomized, observer-blinded, placebo-controlled and dose-escalation phase 1 clinical trials

ABSTRACT Vaccination plays a key role in preventing morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We aimed to evaluate the safety and immunogenicity of a SARS-CoV-2 messenger ribonucleic acid (mRNA) vaccine SYS6006. In the two randomized, observer-blinded, placebo-controlled phase 1 trials, 40 adult participants aged 18–59 years and 40 elderly participants aged 60 years or more were randomized to receive two doses of SYS6006 or placebo (saline). Adverse events (AEs) were collected through 30 days post the second vaccination. Immunogenicity was assessed by live-virus neutralizing antibody (Nab), spike protein (S1) binding antibody (S1-IgG), and cellular immunity. The result showed that 7/15, 9/15 and 4/10 adult participants, and 9/15, 8/15 and 4/10 elderly participants reported at least one AE in the 20-µg, 30-µg and placebo groups, respectively. Most AEs were grade 1. Injection-site pain was the most common AE. Two adults and one elder reported fever. No vaccination-related serious AE was reported. SYS6006 elicited wild-type Nab response with a peak geometric mean titer of 232.1 and 130.6 (adults), and 48.7 and 66.7 (elders), in the 20-µg and 30-µg groups, respectively. SYS6006 induced moderate-to-robust Nab response against Delta, and slight Nab response against Omicron BA.2 and BA.5. Robust IgG response against wild type and BA.2 was observed. Cellular immune response was induced. In conclusion, two-dose primary vaccination with SYS6006 demonstrated good safety and immunogenicity during a follow-up period of 51 days in immunologically naive population aged 18 years or more. (Trial registry: Chictr.org.cn ChiCTR2200059103 and ChiCTR2200059104)

The gift of preexisting immunity for developing an alternative vaccine strategy.

Despite the worldwide application of vaccination and other antiviral interventions, pulmonary viral infections remain a persistent threat to human health. The 1918 influenza pandemic killed more than 40 million people in just one year, and the SARS-CoV-2 pandemic has killed more than 6.9 million people since 2019. While the current approved COVID-19 vaccines are administered parenterally and induce systemic immunity, they only prevent the progression to severe disease. Thus, other vaccine platforms are still needed for completely preventing the disease and subsequent transmission. In this issue of the JCI, Kawai et al. present an adjuvant-free subunit (RBD-HA) fusion vaccine, which produces robust IgG and IgA antibody responses against SARS-CoV-2, enriched within the nasal cavity, by using the host's preexisting immunity to influenza infection. This preclinical study has tremendous implications for future mucosal vaccine design and provides a roadmap for generating a safer and effective intranasal vaccine against pulmonary infections.

A Virus-like Particle-Based F4 Enterotoxigenic Escherichia coli Vaccine Is Inhibited by Maternally Derived Antibodies in Piglets but Generates Robust Responses in Sows.

F4-positive enterotoxigenic Escherichia coli is associated with diarrhea and poor growth outcomes in neonatal and newly weaned piglets and is thus a major economic and welfare burden in the swine industry. Vaccination of sows with F4 fimbriae protects against the neonatal disease via passive transfer of maternal immunity. However, this strategy does not protect against infection post-weaning. Consequently, prevention and treatment methods in weaner pigs heavily rely on the use of antimicrobials. Therefore, in order to reduce antimicrobial consumption, more effective prophylactic alternatives are needed. In this study, we describe the development of a capsid virus-like particle (cVLP)-based vaccine targeting the major F4 fimbriae subunit and adhesion molecule, FaeG, and evaluate its immunogenicity in mice, piglets, and sows. cVLP-display significantly increased systemic and mucosal antibody responses towards the recombinant FaeG antigen in mice models. However, in piglets, the presence of anti-F4 maternally derived antibodies severely inhibited the induction of active humoral responses towards the FaeG antigen. This inhibition could not be overcome, even with the enhanced immunogenicity achieved via cVLP display. However, in sows, intramuscular vaccination with the FaeG.cVLP vaccine was able to generate robust IgG and IgA responses that were comparable with a commercial fimbriae-based vaccine, and which were effectively transferred to piglets via colostrum intake. These results demonstrate that cVLP display has the potential to improve the systemic humoral responses elicited against low-immunogenic antigens in pigs; however, this effect is dependent on the use of antigens, which are not the targets of pre-existing maternal immunity.