Altered IgG4 Antibody Response to Repeated Mrna Versus Recombinant Protein SARS-CoV-2 Vaccines

•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.