IntroductionRecent vaccine and infectious disease studies have highlighted the importance of antibodies that activate cellular Fc functions, including antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), which are mediated by different Fc gamma Receptors (FcγRs). Activation of these functions requires complex overlapping interactions between IgG antibodies, FcγRs, and antigens that can be challenging to deconvolve experimentally.MethodsHere we created an ordinary differential equation model that simultaneously predicted FcγRIIIa immune complexes upstream of ADCC and FcγRIIa immune complexes upstream of ADCP as a function of antigen, IgG, and FcγR concentration and binding properties. We then used the model to dissect mechanisms driving immune complex formation.ResultsModel results suggested that the maximum formation of immune complexes would not occur at highest total IgG titers. Instead, higher IgG titers have the potential to decrease FcγRIIIa (ADCC) and/or FcγRIIa (ADCP) immune complexes, due to competition between antibody subclasses for antigen and FcγR binding. We used the model to simulate vaccine boosts of IgG1 or IgG3 in 105 participants from an HIV vaccine trial, and found that boosting IgG1 and IgG3 in combination was not predicted to result in significant changes in either FcγRIIIa (ADCC) or FcγRIIa (ADCP) immune complexes. Surprisingly, simulated boosting of IgG3 alone had the potential to significantly decrease ADCP (p<0.00001), though it would increase ADCC responses. We also illustrated how the model could be used to assess how variability in viral load, FcγR expression, FcγR polymorphisms, and IgG titers across different tissue compartments can lead to differences in FcγRIIIa and FcγRIIa complexes.DiscussionAltogether, these results illustrate how a computational framework provides new quantitative insights into activation of Fc effector functions that could be used to guide future rational design of therapeutic and prophylactic interventions.

Solid tumor malignancy (STM) patients experience increased risk of breakthrough SARS-CoV-2 infection owing to reduced COVID-19 vaccine immunogenicity. However, the underlying immunological causes of impaired neutralization remain poorly characterized. Furthermore, non-neutralizing antibody functions can contribute to reduced disease severity but remain understudied within high-risk populations. We dissected polyfunctional antibody responses in STM patients and age-matched controls who received adenoviral vector- or mRNA-based COVID-19 vaccine regimens. Elevated inflammatory biomarkers, including agalactosylated IgG, interleukin (IL)-6, IL-18, and an expanded population of CD11c−CD21− double negative 3 (DN3) B cells were observed in STM patients and were associated with impaired neutralization. In contrast, mRNA vaccination induced Fc effector functions that were comparable in patients and controls and were cross-reactive against SARS-CoV-2 variants. These data highlight the resilience of Fc functional antibodies and identify systemic inflammatory biomarkers that may underpin impaired neutralizing antibody responses, suggesting potential avenues for immunomodulation via rational vaccine design.

Proportional increases in anti-Spike (S) IgG4 associated with decreased Fc effector functions have been reported following repeated mRNA, but not recombinant protein-based (rS) (NVX-CoV2373, Novavax, Inc.), SARS-CoV-2 vaccination. We demonstrate the first evidence of a negative correlation between anti-S IgG4 and neutralizing antibody (nAb), as well as antibody-dependent surrogate Fc effector functions. Priming with two NVX-CoV2373 vaccines followed by a third dose was associated with higher IgG1 and IgG3, lower IgG4, higher nAb titers and surrogate Fc effector functions versus mRNA. Immune imprinting of anti-S IgG4 and nAbs, and Fc effector function imprinting after mRNA priming was observed. This effect was partially overcome by updated XBB.1.5 protein subunit vaccination, but not by ancestral vaccine strains. We establish correlation of anti-S IgG4 responses to reduced nAbs and surrogate Fc effector functions and demonstrate the impact of additional booster vaccination on subsequent immune response and Fc effector functions in the context of ancestral and XBB.1.5 strains.

BACKGROUND. The immunogenicity of current influenza vaccines need improvement. Inactivated influenza and COVID-19 mRNA vaccines can be co-administered but randomized controlled trial data is lacking on whether the two vaccines are more immunogenic if given in the same or opposite arms. Murine studies suggest mRNA vaccines can adjuvant influenza vaccines when co-formulated and delivered together.

Introduction Substantial population-level variation in vaccine-specific antibody responses has been observed following global coronavirus disease 2019 (COVID-19) vaccination efforts. Beyond the influence of clinical and demographic features, immunogenetic variation is suggested to underlie divergent serological responses following COVID-19 vaccination of distinct populations. Methods Immunoglobulin G1 (IgG1) allotypic markers (G1m) for 121 COVID-19 vaccinated healthy adults were genotyped via Sanger sequencing. Vaccine-specific IgG and Fc gamma receptor (FcγR) engagement were characterised via bead-based multiplex array. Results Following two COVID-19 vaccine doses, G1m1,17+/+ compared to G1m-1,3+/+ vaccinees had increased IgG and FcγR engagement specific for the antigenically conserved SARS-CoV-2 Spike 2 (S2) domain. IgG targeting antigenically novel SARS-CoV-2 receptor binding domain (RBD) trended higher in G1m1,17+/+ vaccinees, facilitating increased RBD-specific FcγR2a-R131 and FcγR2b binding. Conclusion Primary COVID-19 vaccination induced increased S2-specific IgG in G1m1,17+/+ vaccinees, facilitating enhanced anti-viral FcγR engagement and suggesting immunogenetics may be a valuble consideration for next-generation vaccine design.

Abstract Objectives Bacille Calmette–Guérin (BCG) vaccination has off‐target effects on disease risk for unrelated infections and immune responses to vaccines. This study aimed to determine the immunomodulatory effects of BCG vaccination on immune responses to vaccines against SARS‐CoV‐2. Methods Blood samples, from a subset of 275 SARS‐CoV‐2‐naïve healthcare workers randomised to BCG vaccination (BCG group) or no BCG vaccination (Control group) in the BRACE trial, were collected before and 28 days after the primary course (two doses) of ChAdOx1‐S (Oxford‐AstraZeneca) or BNT162b2 (Pfizer‐BioNTech) vaccination. SARS‐CoV‐2‐specific antibodies were measured using ELISA and multiplex bead array, whole blood cytokine responses to γ‐irradiated SARS‐CoV‐2 (iSARS) stimulation were measured by multiplex bead array, and SARS‐CoV‐2‐specific T‐cell responses were measured by activation‐induced marker and intracellular cytokine staining assays. Results After randomisation (mean 11 months) but prior to COVID‐19 vaccination, the BCG group had lower cytokine responses to iSARS stimulation than the Control group. After two doses of ChAdOx1‐S, differences in iSARS‐induced cytokine responses between the BCG group and Control group were found for three cytokines (CTACK, TRAIL and VEGF). No differences were found between the groups after BNT162b2 vaccination. There were also no differences between the BCG and Control groups in COVID‐19 vaccine‐induced antigen‐specific antibody responses, T‐cell activation or T‐cell cytokine production. Conclusion BCG vaccination induced a broad and persistent reduction in ex vivo cytokine responses to SARS‐CoV‐2. Following COVID‐19 vaccination, this effect was abrogated, and BCG vaccination did not influence adaptive immune responses to COVID‐19 vaccine antigens.

The WHO has given a permissive recommendation for an off-label one-dose human papillomavirus (HPV) vaccine schedule to prevent cervical cancer, based on evidence of comparable protection to two or three doses of vaccine. While neutralizing antibodies are thought to be the primary mechanism of protection, the persistence of immunity and whether other antibody-mediated mechanisms of protection are involved is unclear. Using systems serology, we investigated HPV antibody responses in serum from Fijian girls who were unvaccinated or received one, two or three doses of quadrivalent HPV vaccine six years earlier. We also evaluated their HPV antibody responses 28 days following a dose of bivalent HPV vaccine. After six years, one dose induced lower antibody concentrations but similar antibody profiles and phagocytic function as two or three doses. Following bivalent vaccine, antibody concentrations, particularly IgG1/IgG3, antibody profiles and phagocytic function were similar between previously vaccinated girls, indicating immune memory after one dose. Cross-reactive antibody responses against non-vaccine genotypes (HPV31/33/45/52/58) were lower following one dose than two or three doses. These findings provide novel insights into serological immunity and recall responses following one-dose HPV vaccination.

BACKGROUND The immunogenicity of current influenza vaccines needs improvement. Inactivated influenza and COVID-19 mRNA vaccines can be coadministered, but randomized controlled trial data are lacking on whether the 2 vaccines are more immunogenic if given in the same arm or opposite arms. Murine studies suggest mRNA vaccines can adjuvant influenza vaccines when coformulated and codelivered.METHODS We randomly assigned 56 adults to receive the Afluria quadrivalent inactivated influenza and Moderna monovalent SARS-CoV-2 XBB.1.5 mRNA vaccines, either in opposite arms or both in the same arm at the same site. The primary endpoint was the difference in median combined serum hemagglutination inhibition titer to the H1, H3, and B-Vic vaccine influenza strains after vaccination.RESULTS We found no significant difference in hemagglutination inhibition antibody levels between the groups (P = 0.30), with the same-arm group having a 1.26-fold higher titer than the opposite-arm group. There were no differences in analyses of antibodies against individual influenza strains or in nasal or saliva antibody levels. While both binding and neutralizing antibody titers against SARS-CoV-2 were not significantly different between groups postvaccination, there was a higher fold-change in BA.5 and ancestral strain neutralizing antibodies in the opposite-arm group.CONCLUSION Influenza vaccination is equivalently immunogenic if given in the same arm or opposite arms as the SARS-CoV-2 vaccine, but it may be preferable to administer the SARS-CoV-2 vaccine at a different site from influenza vaccines.TRIAL REGISTRATION Australian New Zealand Clinical Trials Registry ACTRN12624000445572.FUNDING Australian National Health and Medical Research Council, Australian Medical Research Future Fund, and National Institutes of Health (UH2AI176172).

BACKGROUND:Susceptibility to malaria can be influenced by host genetic factors, including immune response genes. Antibodies against Plasmodium antigens are known to play an important role in protection from clinical disease. Polymorphisms in these antibodies may result in different functional properties that could provide protection from malaria. METHODS:Immunoglobulin G1 (IgG1) and immunoglobulin G3 (IgG3) alleles and IgG3 hinge region were investigated by polymerase chain reaction and Sanger sequencing in a longitudinal cohort of children aged 1-3 years (N = 203) from the East Sepik region of Papua New Guinea. Linear regression was used to investigate associations between immunoglobulin alleles and Plasmodium infections. RESULTS:Seventy-eight percent of the children were either heterozygous (n = 82 [40%]) or homozygous (n = 77 [38%]) for IGHG3*30 (G3m29), a novel IgG3 allele. G3m29 has a long hinge region of 4 exons. Significantly fewer Plasmodium spp infections were observed in children with the IGHG3*30 allele compared to children without the allele (β = -1.736 [95% confidence interval {CI}, -3.39, -.079]; P = .038). This effect was most noticeable for Plasmodium vivax asymptomatic infections as IGHG3*30 carriers had on average 1 fewer infection in the 18-month follow-up period compared with non-IGHG3*30 allele carriers (β = -1.06 [95% CI, -2.01, -.12]; P = .028). Additionally, IGHG3*30 allele carriers had significantly lower levels of IgG to P vivax vaccine candidate proteins compared to non-IGHG3*30 allele carriers. CONCLUSIONS:The IGHG3*30 allele is highly prevalent in the East Sepik region and is associated with fewer Plasmodium spp infections.

Abstract Background IgG4, the least abundant human IgG subtype, increases after repetitive exposure to some antigens, and may induce immune tolerance. Studies have found that repeat mRNA SARS-CoV-2 vaccination leads to large proportional increases in spike (S)-specific IgG4. By contrast, increased IgG4 has not been observed following repeat vaccination with recombinant SARS-CoV-2 S (rS) protein (NVX-CoV2373, Novavax). Whether vaccine-induced anti-S IgG4 might impair SARS-CoV-2 immunity remains unknown.Figure:Serum anti-Spike IgG1 (A), IgG3 (B), and IgG4 (C) concentrations (ng/mL) measured by ELISA were correlated with neutralizing antibody titers (MN50) measured by infectious virus microneutralization assay. Methods Sera were collected from study participants in 2019nCoV-307 (NCT05463068) and 2019nCoV-301 (NCT04611802), including 2 groups from 2019nCoV-307 who received 3 homologous mRNA doses followed by 1 NVX-CoV2373 dose (mRNA-1273, Moderna, n=10; or BNT162b2, Pfizer, n=10), and a 3rd group from 2019nCoV-301 who received 4 homologous NVX-CoV2373 doses (n=18). Sera collected ≥6 months after the last dose in the first 2 groups and ∼4 weeks after the last dose in the 3rd group were assessed for neutralizing antibodies (nAb), IgG subclass profiles (anti-S total IgG, IgG1, IgG2, IgG3, and IgG4), and Fc effector activities (cell-based ADCP assay; surrogate ADCC by FcγRIIIa binding; and ADCD by C1q binding) after repeated NVX-CoV2373 or a single NVX-CoV2373 dose following repeated mRNA vaccine.Table:Summary of correlations between IgG1, IgG3, and IgG4 with neutralizing antibody titers (ancestral) and Fc effector functions in 2019nCoV-301 and 2019nCoV-307 studies. Results Increased nAb titers after NVX-CoV2373 were observed irrespective of the prior vaccine background, whereas increased IgG4 levels were only observed in recipients of mRNA vaccines. Spearman's rank correlation between nAb and Fc functions found significant positive correlations with anti-S IgG1 and IgG3 (Figure A, B), and significant negative correlation with anti-S IgG4 (Figure C) antibody responses (Table). Conclusion These are the first data to demonstrate the negative correlation of increased SARS-CoV-2 vaccine–induced anti-S IgG4 levels and nAb titers and Fc effector functions. The limitations of our data include small sample sizes, and potential differences attributable to vaccination and serum collection intervals. IgG4-mediated suboptimal nAb and reduced Fc effector function could reduce the vaccine effectiveness of the SARS-CoV-2 response, though more work is needed to understand its impact on the optimization of vaccine platform choice. Disclosures Raj Kalkeri, PhD, Novavax: Employee|Novavax: Stocks/Bonds (Public Company) Mingzhu Zhu, n/a, Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company) Shane Cloney-Clark, n/a, Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company) Anand Parekh, M.Sc., Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company) Drew Gorinson, B.S., Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company) Rongman Cai, PhD, Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company) Soham Mahato, PhD, Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company) Anthony M. Marchese, PhD, Novavax Inc: Employee|Novavax Inc: Stocks/Bonds (Public Company) Louis F. Fries, III, MD, Novavax, Inc.: contractor for Novavax|Novavax, Inc.: Stocks/Bonds (Public Company) Lisa M. Dunkle, MD, Novavax, Inc: Employee|Novavax, Inc: Ownership Interest|Novavax, Inc: Stocks/Bonds (Public Company) Amy W. Chung, PhD, Novavax: Grant/Research Support Joyce S. Plested, n/a, Novavax, Inc.: employee|Novavax, Inc.: Stocks/Bonds (Public Company)

Enhancement of antibody-dependent cellular cytotoxicity is a promising adjunct approach to achieve HIV control in the absence of antiretroviral therapy but requires the development of potent antibody-dependent cellular cytotoxicity-eliciting antibodies that can recognize diverse HIV-infected cell types. A panel of broadly neutralizing antibodies targeting the HIV envelope was identified that specifically binds both HIV-infected CD4+ T cells and monocyte-derived macrophages. Afucosylated versions of these broadly neutralizing antibodies containing ≈30% less core fucose were generated and elicited a significant increase in antibody-dependent cellular cytotoxicity responses from natural killer cells against HIV-infected T-cell and monocyte-derived macrophage targets. Afucosylation did not alter virus neutralization or cell-binding activity of these broadly neutralizing antibodies. Afucosylation modification of broadly neutralizing antibody Fc regions is thus a promising strategy to enhance Fc-mediated activity against both T-cell and macrophage targets in vivo, which may be employed to heighten the therapeutic potential of antibody-based immunotherapy approaches for drug-free HIV control.

Purpose:To characterize corneal immune cell morphodynamics and nerve features, and define the in vivo immune landscape in older adults with human immunodeficiency virus (HIV) receiving antiretroviral therapy (ART), relative to healthy age-matched adults. Methods:In this cross-sectional study, 16 HIV-positive individuals receiving ART and 15 age-matched controls underwent ocular surface examinations and functional in vivo confocal microscopy (Fun-IVCM). Time-lapsed videos were created to analyze corneal immune cells (T cells, dendritic cells [DCs], macrophages). Subclinical indicators of corneal health (sensory nerve and endothelial cell features), clinical ocular surface findings, and tear cytokines (analyzed using multiplex bead-based immunoassay) were compared between groups. Results:Participants comprised mostly males (HIV 71 ± 5 years; male:female 15:1; controls 67 ± 6 years; 14:1). The HIV-positive group showed less T-cell motility at the corneal whorl relative to the control group (P = 0.01), and region-dependent differences in T-cell speed (P = 0.001) and DC area (P < 0.001). The HIV-positive group showed greater central corneal nerve fiber width (P = 0.004) and larger endothelial cells (P = 0.02). Clinical findings, corneal immune cell densities, and tear cytokine profiles were similar between groups. Conclusions:Among older individuals with well-controlled HIV infection and clinically-normal ocular surface health, this study identifies subclinical group differences in corneal immune cells (potentially indicative of a heightened, pro-inflammatory activation state in the peripheral cornea) and corneal endothelial cell morphology that parallel those in chronic inflammatory disease. This study demonstrates the utility of Fun-IVCM to evaluate subclinical immune cell features in systemic disease, which could inform the future identification of biomarkers in immune-related conditions.

Repeated mRNA COVID-19 vaccination increases spike-specific immunoglobulin G4 (IgG4) titers. Here, we characterized the influence of increased IgG4 titers on a range of Fc-mediated responses. Elevated spike-specific IgG4 reduced binding to FcγRIIIa and decreased antibody-dependent cellular cytotoxicity. However, in individuals with lower total spike-specific IgG, IgG4 acted in synergy with other IgG subclasses to improve FcγRI and FcγRIIa binding and consequently antibody-dependent cellular phagocytosis. Furthermore, this trend was more pronounced with more recent SARS-CoV-2 variants where vaccination induced comparably lower total spike-specific titers. These observations were further confirmed by in silico modeling where antibody subclass concentrations and FcγR polymorphisms were modulated. Collectively, we illustrate that the impact of elevated IgG4 titers upon Fc functions is dependent on multiple interconnected antibody and antigen factors, which should be taken into consideration when dissecting the mechanisms driving an effective Fc-mediated response following vaccination.

Vaccine-induced immunoglobulin G (IgG) profiles can vary with respect to the predominant subclasses that characterize the response. Among IgG subclasses, IgG4 is reported to have anti-inflammatory properties, but can also exhibit reduced capacity for virus neutralization and activation of Fc-dependent effector functions. Here, we review evidence that IgG4 subclass responses can be disproportionately increased in response to some types of vaccines targeting an array of diseases, including pertussis, HIV, malaria, and COVID-19. The basis for enhanced IgG4 induction by vaccines is poorly understood but may be associated with platform- or dose regimen–specific differences in antigen exposure and/or cytokine stimulation. The clinical implications of vaccine-induced IgG4 responses remain uncertain, though collective evidence suggests that proportional increases in IgG4 might reduce vaccine antigen-specific immunity. Additional work is needed to determine underlying mechanisms and to elucidate what role IgG4 may play in modifications of vaccine-induced immunity to disease.

Repeated mRNA SARS-CoV-2 vaccination has been associated with increases in the proportion of IgG4 in spike-specific antibody responses and concurrent reductions in Fcγ-mediated effector functions that may limit control of viral infection. Here, we assessed anti-Spike total IgG, IgG1, IgG2, IgG3 and IgG4, and surrogate markers for antibody-dependent cellular phagocytosis (ADCP, FcγRIIa binding), antibody-dependent cellular cytotoxicity (ADCC, FcγRIIIa binding), and antibody-dependent complement deposition (ADCD, C1q binding) associated with repeated SARS-CoV-2 vaccination with NVX-CoV2373 (Novavax Inc., Gaithersburg, MD). The NVX-CoV2373 protein vaccine did not induce notable increases in spike-specific IgG4 or negatively impact surrogates for Fcγ effector responses. Conversely, repeated NVX-CoV2373 vaccination uniquely enhanced IgG3 responses which are known to exhibit strong affinity for FcγRIIIa and have previously been linked to potent neutralization of SARS-CoV-2. Subsequent investigations will help to understand the immunological diversity generated by different SARS-CoV-2 vaccine types and have the potential to reshape public health strategies.

HIV-1 infection leads to chronic disease requiring life-long treatment and therefore alternative therapeutics, a cure and/or a protective vaccine are needed. Antibody-mediated effector functions could have a role in the fight against HIV-1. However, the properties underlying the potential beneficial effects of antibodies during HIV-1 infection are poorly understood. To identify a specific profile of antibody features associated with delayed disease progression, we studied antibody polyfunctionality during untreated HIV-1 infection in the well-documented Amsterdam Cohort Studies. Serum samples were analyzed from untreated individuals with HIV-1 at approximately 6 months (n = 166) and 3 years (n = 382) post-seroconversion (post-SC). A Luminex antibody Fc array was used to profile 15 different Fc features for serum antibodies against 20 different HIV-1 envelope glycoprotein antigens and the resulting data was also compared with data on neutralization breadth. We found that high HIV-1 specific IgG1 levels and low IgG2 and IgG4 levels at 3 years post-SC were associated with delayed disease progression. Moreover, delayed disease progression was associated with a broad and polyfunctional antibody response. Specifically, the capacity to interact with all Fc γ receptors (FcγRs) and C1q, and in particular with FcγRIIa, correlated positively with delayed disease progression. There were strong correlations between antibody Fc features and neutralization breadth and several antibody features that were associated with delayed disease progression were also associated with the development of broad and potent antibody neutralization. In summary, we identified a strong association between broad, polyfunctional antibodies and delayed disease progression. These findings contribute new information for the fight against HIV-1, especially for new antibody-based therapy and cure strategies.

Abstract Objectives Amino acid variations across more than 30 immunoglobulin (Ig) allotypes may introduce structural changes that influence recognition by anti‐Ig detection reagents, consequently confounding interpretation of antibody responses, particularly in genetically diverse cohorts. Here, we assessed a panel of commercial monoclonal anti‐IgG1 clones for capacity to universally recognise two dominant IgG1 haplotypes (G1m‐1,3 and G1m1,17). Methods Four commercial monoclonal anti‐human IgG1 clones were assessed via ELISAs and multiplex bead‐based assays for their ability to bind G1m‐1,3 and G1m1,17 IgG1 variants. Detection antibodies were validated against monoclonal IgG1 allotype standards and tested for capacity to recognise antigen‐specific plasma IgG1 from G1m‐1,3 and G1m1,17 homozygous and heterozygous SARS‐CoV‐2 BNT162b2 vaccinated (n = 28) and COVID‐19 convalescent (n = 44) individuals. An Fc‐specific pan‐IgG detection antibody corroborated differences between hinge‐ and Fc‐specific anti‐IgG1 responses. Results Hinge‐specific anti‐IgG1 clone 4E3 preferentially bound G1m1,17 compared to G1m‐1,3 IgG1. Consequently, SARS‐CoV‐2 Spike‐specific IgG1 levels detected in G1m1,17/G1m1,17 BNT162b2 vaccinees appeared 9‐ to 17‐fold higher than in G1m‐1,3/G1m‐1,3 vaccinees. Fc‐specific IgG1 and pan‐IgG detection antibodies equivalently bound G1m‐1,3 and G1m1,17 IgG1 variants, and detected comparable Spike‐specific IgG1 levels between haplotypes. IgG1 responses against other human coronaviruses and influenza were similarly poorly detected by 4E3 anti‐IgG1 in G1m‐1,3/G1m‐1,3 subjects. Conclusion Anti‐IgG1 clone 4E3 confounds assessment of antibody responses in clinical cohorts owing to bias towards detection of G1m1,17 IgG1 variants. Validation of anti‐Ig clones should include evaluation of binding to relevant antibody variants, particularly as the role of immunogenetics upon humoral immunity is increasingly explored in diverse populations.

The immunoglobulin heavy constant gamma (IGHG) gene cluster encoding immunoglobulin G (IgG) subclasses is highly polymorphic, resulting in amino acid variation along the antibody constant heavy chain referred to as allotypes. IGHG1 and IGHG3 are the two most polymorphic IgG subclasses in humans, with 4 classical IgG1 allotypes and 13 allotypes described for IgG3, though recent studies suggest greater allelic diversity, especially in underrepresented ethnic populations. Polymerase chain reaction (PCR) and Sanger sequencing of IGHG amplicons allow for the identification of the single nucleotide polymorphisms (SNPs) responsible for the observed amino acid substitutions. Here, we provide a detailed protocol for the amplification of IGHG1 and IGHG3 segments by PCR, sample preparation for Sanger sequencing, and analysis of sequencing data to identify SNPs associated with different IgG1 and IgG3 allotypes.

•In agreement with recently published studies, repeated mRNA SARS-CoV-2 vaccination was associated with an increase in Spike-protein specific IgG4.•By contrast, IgG4 class switch was not observed following four doses of Novavax protein-based SARS-CoV-2 vaccine.•SARS-CoV-2 specific IgG3, an IgG subclass known to induce potent neutralization and Fc functions, was higher after Novavax homologous vaccination (>10x vs. mRNA).•In contrast to the mRNA homologous regimens, Novavax homologous and heterologous vaccinations evoked stronger Fcγ-dependent effector activities. Dear Editor, The four unique subclasses of human IgG, numbered in order of relative abundance, include IgG1, IgG2, IgG3, and IgG41,2. IgG1 and IgG3 are the major contributors to rapid IgG responses to protein and membrane antigens, and are active in viral neutralization, although some differences in IgG subclass responses have been identified in the context of SARS-CoV-2 infection and vaccination. Indeed, IgG3 response to SARS-CoV-2 infection has been highlighted by an analysis of convalescent plasma, showing that while IgG3 accounted for 12% of total anti-Spike (anti-S) protein IgG, it contributed to approximately 80% of total live SARS-CoV-2 neutralizing activity3. In addition, IgG1, and especially IgG3, bind FcγRIIa, FcγRIIIa and C1q, thereby supporting antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cytotoxicity (ADCC), and antibody-dependent complement deposition (ADCD)2. In contrast to the findings regarding IgG3 in convalescent sera, repeated mRNA SARS-CoV-2 vaccination has been associated with significant increases in the proportion of immunoglobulin G4 (IgG4) in Spike-specific responses and reductions in Fc-mediated ADCP and ADCD that may limit control of viral infection4Irrgang P. Gerling J. Kocher K. et al.Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination.Sci Immunol. 2023; 8eade2798https://doi.org/10.1126/sciimmunol.ade2798Crossref Scopus (49) Google Scholar, 5Yoshimura M. Sakamoto A. Ozuru R. et al.The appearance of anti-spike receptor binding domain immunoglobulin G4 responses after repetitive immunization with messenger RNA-based COVID-19 vaccines.Int J Infect Dis. 2023; 139: 1-5https://doi.org/10.1016/j.ijid.2023.11.028Abstract Full Text Full Text PDF Scopus (0) Google Scholar, 6Routhu N.K. Stampfer S.D. Lai L. et al.Efficacy of mRNA-1273 and Novavax ancestral or BA.1 spike booster vaccines against SARS-CoV-2 BA.5 infection in non-human primates..Sci Immunol. 2023; eadg7015https://doi.org/10.1126/sciimmunol.adg7015Crossref Scopus (5) Google Scholar, 7Buhre J.S. Pongracz T. Kunsting I. et al.mRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine.Front Immunol. 2022; 131020844https://doi.org/10.3389/fimmu.2022.1020844Crossref Scopus (17) Google Scholar, 8Uversky V.N. Redwan E.M. Makis W. Rubio-Casillas A. IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein.Vaccines (Basel). 2023; 11https://doi.org/10.3390/vaccines11050991Crossref Scopus (10) Google Scholar. After repeated mRNA SARS-CoV-2 vaccination, IgG3 was observed to reach peak titers after a second dose, and steadily declined thereafter to significantly lower levels after the third and fourth dose vaccinations, while IgG4 increased5Yoshimura M. Sakamoto A. Ozuru R. et al.The appearance of anti-spike receptor binding domain immunoglobulin G4 responses after repetitive immunization with messenger RNA-based COVID-19 vaccines.Int J Infect Dis. 2023; 139: 1-5https://doi.org/10.1016/j.ijid.2023.11.028Abstract Full Text Full Text PDF Scopus (0) Google Scholar. IgG4 is generally in low abundance (0–5% of total IgG) but may slowly increase over time due to repeated or excessive exposure to some antigens1Rispens T. Huijbers M.G. The unique properties of IgG4 and its roles in health and disease.Nat Rev Immunol. 2023; 23: 763-778https://doi.org/10.1038/s41577-023-00871-zCrossref Scopus (9) Google Scholar, 2Vidarsson G. Dekkers G. Rispens T. IgG subclasses and allotypes: from structure to effector functions.Front Immunol. 2014; 5: 520https://doi.org/10.3389/fimmu.2014.00520Crossref PubMed Scopus (1637) Google Scholar, 8Uversky V.N. Redwan E.M. Makis W. Rubio-Casillas A. IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein.Vaccines (Basel). 2023; 11https://doi.org/10.3390/vaccines11050991Crossref Scopus (10) Google Scholar. Although repeated antigen exposure seems necessary, it is insufficient to induce IgG4, as prolonged activation of IL-10–expressing CD4+ T-cells or expression of other anti-inflammatory cytokines is also required1Rispens T. Huijbers M.G. The unique properties of IgG4 and its roles in health and disease.Nat Rev Immunol. 2023; 23: 763-778https://doi.org/10.1038/s41577-023-00871-zCrossref Scopus (9) Google Scholar. Increased concentrations of IgG4 have been associated with immunosuppression and poor clinical outcomes of COVID-19, and while generally regarded as anti-inflammatory, may contribute to some autoimmune disorders and inflammatory IgG4-related diseases1Rispens T. Huijbers M.G. The unique properties of IgG4 and its roles in health and disease.Nat Rev Immunol. 2023; 23: 763-778https://doi.org/10.1038/s41577-023-00871-zCrossref Scopus (9) Google Scholar, 8Uversky V.N. Redwan E.M. Makis W. Rubio-Casillas A. IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein.Vaccines (Basel). 2023; 11https://doi.org/10.3390/vaccines11050991Crossref Scopus (10) Google Scholar. Following repeated mRNA vaccination, IgG4 was observed to increase from 0.04% of total SARS-CoV-2 spike–specific IgG after two doses to 19.27% after three doses4Irrgang P. Gerling J. Kocher K. et al.Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination.Sci Immunol. 2023; 8eade2798https://doi.org/10.1126/sciimmunol.ade2798Crossref Scopus (49) Google Scholar. We assessed IgG subclass and Fc effector function profiles following repeated vaccination with the recombinant Spike (rS) protein SARS-CoV-2 vaccine (NVX-CoV2373, Novavax Inc.) in comparison to a single rS vaccination following repeated mRNA vaccinations. Serum concentrations of anti–ancestral (Wuhan-Hu-1) rS–specific total IgG, IgG1, IgG2, IgG3, and IgG4, and surrogate ADCP (FcγRIIa binding), surrogate ADCC (FcγRIIIa binding), and ADCD (C1q binding) were measured. Participant sera from studies 2019nCoV-307 (ClinicalTrials.gov: NCT05463068) and 2019nCoV-301 (ClinicalTrials.gov: NCT04611802) were included in the analysis; two groups from 2019nCoV-307 who received three homologous doses of Moderna (mRNA-1273, n = 10) or Pfizer (BNT162b2, n = 10) mRNA vaccine followed by one dose of NVX-CoV2373, and a third group from 2019nCoV-301 who received four homologous doses of Novavax (n = 18). Serum was collected ≥6 months after the last dose for the homologous three-dose series and ~4 weeks after the fourth dose for the four-dose series. Anti-S proteins IgG1, IgG2, IgG3, and IgG4, and total anti-S IgG were measured for each serum sample by IgG subclass quantitative enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 rS proteins (produced at Novavax, Inc., Gaithersburg, MD, USA) were used to coat ELISA plates. The reference standards for ELISA were as follows; SARS-CoV-2 Spike RBD human IgG monoclonal antibody (ACROBiosystems, Cat# RAS009-C02) for total IgG, anti-SARS-CoV-2 Spike RBD human IgG1 monoclonal antibody (ACROBiosystems, Cat# SPD-M265) for IgG1, anti-SARS-CoV-2 Spike RBD human IgG2 monoclonal antibody (ACROBiosystems, Cat# SPD-M400a), anti-SARS-CoV-2 Spike RBD human IgG3 monoclonal antibody (ACROBiosystems, Cat# SPD-M401a), and anti-SARS-CoV-2 Spike RBD human IgG4 monoclonal antibody (ACROBiosystems, Cat# SPD-M402a) for IgG4. To determine ADCP, ADCC, and ADCD, surrogate SARS-CoV-2–specific antibody Fc functional assays were performed as previously described9Selva K.J. van de Sandt C.E. Lemke M.M. et al.Systems serology detects functionally distinct coronavirus antibody features in children and elderly.Nat Commun. 2021; 12: 2037https://doi.org/10.1038/s41467-021-22236-7Crossref PubMed Scopus (87) Google Scholar. The surrogate Fc multiplex assays have been shown to correlate strongly with cell-based ADCP and ADCC functional assays9Selva K.J. van de Sandt C.E. Lemke M.M. et al.Systems serology detects functionally distinct coronavirus antibody features in children and elderly.Nat Commun. 2021; 12: 2037https://doi.org/10.1038/s41467-021-22236-7Crossref PubMed Scopus (87) Google Scholar. Briefly, SARS-Cov-2 Spike trimer (Sino Biological Cat# 40589) coupled multiplex beads, were used to profile antigen-specific FcγR activation or C1q (complement) deposition as a high-throughput surrogate to assess ADCP by soluble FcγRIIa dimer binding, ADCC by soluble FcγRIIIa dimer binding (both expressed in house at the University of Melbourne) or ADCD by C1q binding (MP Biomedicals, Irvine, CA, USA). Total anti-S IgG and IgG1 levels following three homologous doses of mRNA or NVX-CoV2373 were similar, although NVX-CoV2373 induced somewhat higher levels, and the fourth dose of NVX-CoV2373 led to increased responses in each group (Fig. 1A). Compared with recipients of prior mRNA vaccine, anti-S IgG3 levels were markedly higher (>10-fold) after three or four homologous doses of NVX-CoV2373. By contrast, much higher anti-S IgG4 levels (>75-fold) were observed following repeated mRNA vaccination, but not after three or four homologous doses of NVX-CoV2373 (Fig. 1A). The fourth dose of NVX-CoV2373 also appeared to enhance surrogate signals for ADCP, ADCC, and ADCD activities in recipients of prior mRNA vaccine, though the effect was greater after a fourth homologous dose of NVX-CoV2373 (Fig. 1B). The clinical importance of SARS-CoV-2–specific Fc-mediated responses (i.e., ADCP, ADCD, and ADCC), which are poorly engaged by IgG4, is rapidly gaining appreciation4Irrgang P. Gerling J. Kocher K. et al.Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination.Sci Immunol. 2023; 8eade2798https://doi.org/10.1126/sciimmunol.ade2798Crossref Scopus (49) Google Scholar, 6Routhu N.K. Stampfer S.D. Lai L. et al.Efficacy of mRNA-1273 and Novavax ancestral or BA.1 spike booster vaccines against SARS-CoV-2 BA.5 infection in non-human primates..Sci Immunol. 2023; eadg7015https://doi.org/10.1126/sciimmunol.adg7015Crossref Scopus (5) Google Scholar, 7Buhre J.S. Pongracz T. Kunsting I. et al.mRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine.Front Immunol. 2022; 131020844https://doi.org/10.3389/fimmu.2022.1020844Crossref Scopus (17) Google Scholar, 8Uversky V.N. Redwan E.M. Makis W. Rubio-Casillas A. IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein.Vaccines (Basel). 2023; 11https://doi.org/10.3390/vaccines11050991Crossref Scopus (10) Google Scholar, 10Goldblatt D. Alter G. Crotty S. Plotkin S.A. Correlates of protection against SARS-CoV-2 infection and COVID-19 disease.Immunol Rev. 2022; 310: 6-26https://doi.org/10.1111/imr.13091Crossref PubMed Scopus (90) Google Scholar. Fcγ-dependent effector functions can provide additional mechanisms for virus control that may be complementary to neutralization, and these may be important for the promotion of vaccine-mediated cross-protection to evolving SARS-CoV-2 variants10Goldblatt D. Alter G. Crotty S. Plotkin S.A. Correlates of protection against SARS-CoV-2 infection and COVID-19 disease.Immunol Rev. 2022; 310: 6-26https://doi.org/10.1111/imr.13091Crossref PubMed Scopus (90) Google Scholar. Here, we report that the NVX-CoV2373 rS protein vaccine does not appear to induce notable increases in IgG4, even after multiple exposures, or to impair Fcγ-dependent effector responses as observed with mRNA vaccines. Instead, NVX-CoV2373 drove proportional increases in IgG3, perhaps the most potent SARS-CoV-2 neutralizing antibody subclass3Kober C. Manni S. Wolff S. et al.IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma Pools.PLoS One. 2022; 17e0262162https://doi.org/10.1371/journal.pone.0262162Crossref Scopus (15) Google Scholar, and enhanced surrogate ADCP, ADCD, and ADCC activity. The impact of additional doses with updated mRNA and protein-based XBB.1.5 formulation vaccines is currently under study and represents a potentially important area for future SARS-CoV-2 vaccine research. Ongoing investigations of these effects on IgG subclasses and cellular functions will help to elucidate the immunological diversity generated by different SARS-CoV-2 vaccine platforms. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Raj Kalkeri, Mingzhu Zhu, Shane Cloney-Clark, Joyce S. Plested, Anand Parekh, Drew Gorinson, Rongman Cai, Soham Mahato, Anthony M. Marchese, Louis Fries, and Lisa M. Dunkle are the employees and stockholders of Novavax, Inc. Pradhipa Ramanathan (P.R.), L. Carissa Aurelia (L.C.A.), Kevin John Selva (K.J.S.), and Amy W. Chung (A.W.C.) are the employees of the Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity. A.W.C. received grant funding from NHMRC, MRFF, and NIH. P.R., L.C.A., and K.J.S. declare no conflicts/disclosure information. The Sponsor acknowledges the contributions and participation of all 2019nCoV-301 and 2019nCoV-307 study volunteers, principal investigators, and investigative site personnel who contributed to the success of the studies. We thank Bruce D. Wines and P. Mark Hogarth, Burnet Institute, Melbourne, for sharing their Fcγ Receptor dimer plasmids. The authors would also like to thank Hadi Beyhaghi and Muruga Vadivale for scientific contributions to the development of the letter, and Seth Toback, Matt Rousculp, and Brandy Warren for cross-functional assistance in project coordination. The graphical abstract was created using Canva (https://www.canva.com/). This study was funded by Novavax, Inc.

Understanding the immune profile of acute rheumatic fever (ARF), a serious post-infectious sequelae of Streptococcal pyogenes (group A Streptococcus [GAS]), could inform disease pathogenesis and management. Circulating cytokines, immunoglobulins, and complement were analyzed in participants with first- episode ARF, swab-positive GAS pharyngitis and matched healthy controls. A striking elevation of total IgG3 was observed in ARF (90%> clinical reference range for normal). ARF was also associated with an inflammatory triad with significant correlations between interleukin-6, C-reactive protein, and complement C4 absent in controls. Quantification of GAS-specific antibody responses revealed that subclass polarization was remarkably consistent across the disease spectrum; conserved protein antigens polarized to IgG1, while M-protein responses polarized to IgG3 in all groups. However, the magnitude of responses was significantly higher in ARF. Taken together, these findings emphasize the association of exaggerated GAS antibody responses, IgG3, and inflammatory cytokines in ARF and suggest IgG3 testing could beneficially augment clinical diagnosis.