Murine Cross-Reactive Nonneutralizing Polyclonal IgG1 Antibodies Induced by Influenza Vaccine Inhibit the Cross-Protective Effect of IgG2 against Heterologous Virus in Mice

Abstract

Annual vaccination against influenza viruses is the most reliable and efficient way to prevent and control annual epidemics and protect from severe influenza disease. However, current split influenza vaccines are generally not effective against antigenically mismatched (heterologous) strains. To broaden the protective spectrum of influenza vaccines, adjuvants that can induce cross-reactive antibodies with cross-protection via Fc-mediated effector functions are urgently sought. Although IgG2 antibodies are generally more efficient than IgG1 antibodies in Fc-mediated effector functions, it is not yet clear which IgG isotypes show superior cross-protection against heterologous strains. It also remains unclear whether these IgG isotypes interfere with each other’s protective effects. Here, we found that influenza split vaccine adjuvanted with aluminum salts, which predominantly induce cross-reactive IgG1, did not confer cross-protection against heterologous virus challenge in mice. In contrast, split vaccine adjuvanted with CpG oligodeoxynucleotides, which predominantly induce cross-reactive IgG2, showed cross-protection through the interaction of cross-reactive nonneutralizing IgG2 and alveolar macrophages, indicating the importance of cross-reactive nonneutralizing IgG2 for cross-protection. Furthermore, by using serum samples from immunized mice and isolated polyclonal antibodies, we show that vaccine-induced cross-reactive nonneutralizing IgG1 suppress the cross-protective effects of IgG2 by competitively inhibiting the binding of IgG2 to virus. Thus, we demonstrate the new concept that cross-reactive IgG1 may interfere with the potential for cross-protection of influenza vaccine. We propose that adjuvants that selectively induce virus-specific IgG2 in mice, such as CpG oligodeoxynucleotides, are optimal for heterologous protection. IMPORTANCE Current influenza vaccines are generally effective against highly similar virus strains by inducing neutralizing antibodies. However, these antibodies fail to neutralize antigenically mismatched (heterologous) strains and therefore provide limited protection against them. Efforts are being made to develop vaccines with cross-protective ability that would protect broadly against heterologous strains, because the mismatch between predicted and epidemic strains cannot always be avoided, resulting in low vaccine efficacy. Here, we show that nonneutralizing IgG2 antibodies induced by an optimal adjuvant play a crucial role in cross-protection against heterologous virus challenge in mice. Furthermore, nonneutralizing polyclonal IgG1 suppressed the cross-protective effects of nonneutralizing polyclonal IgG2 by competitively blocking the binding of IgG2 to its antigen. These data shed new light on the importance of IgG isotypes and the selection of appropriate adjuvants for the development of universal influenza vaccines. Furthermore, our findings are applicable to the rational design of vaccines against other pathogens.

Synergistic effect of non-neutralizing antibodies and interferon-γ for cross-protection against influenza

“…Mice were immunized with SV alone, SV plus alum, SV plus CpG–ODN, or SV plus CpG/alum and the levels of SV-specific total mIgG, mIgG1, mIgG2b, and mIgG2c were measured in the plasma after the last immunization ( Figure 1 A). As reported previously ( Shibuya et al., 2020 ), SV plus CpG–ODN induced significantly higher levels of SV-specific mIgG2b and mIgG2c than SV alone and SV plus alum, whereas the level of SV-specific mIgG1 in SV plus alum-immunized mice was significantly higher than that in SV alone-immunized mice and SV plus CpG–ODN-immunized mice ( Figure 1 A). Mice immunized with SV plus CpG/alum showed significantly higher levels of SV-specific total mIgG, mIgG1, mIgG2b, and mIgG2c than those immunized with SV plus alum and SV plus CpG–ODN ( Figure 1 A).…”

“…To examine the contribution of cross-reactive non-neutralizing antibodies for conferring a strong cross-protection by the CpG/alum-adjuvanted vaccine, serum samples from immunized or PBS-treated control mice were mixed with PR8 in vitro and transferred into naive mice intranasally ( Figures 3 A and 3B (low virus titer) and Figure S3 (high virus titer)). As reported previously ( Shibuya et al., 2020 ), the serum obtained from mice immunized with SV plus CpG–ODN suppressed body weight loss and improved the survival upon transfer than that obtained from PBS-treated control mice, while the serum obtained from mice immunized with SV plus alum did not improve body weight loss and survival ( Figures 3 A, 3B and S3 ). Furthermore, the serum obtained from mice immunized with SV plus CpG/alum did not show an improvement in body weight loss and survival upon transfer ( Figures 3 A, 3B and S3 ).…”

“…We recently demonstrated the importance of the use of rational adjuvants in providing cross-protective activity in an influenza vaccine by using oligodeoxynucleotides (ODN) with unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG–ODN), which are toll-like receptor 9 (TLR9) agonists ( Shirota and Klinman, 2014 ) and aluminum salts (alum), which are commonly used globally ( HogenEsch et al., 2018 ). We showed that CpG–ODN-adjuvanted influenza vaccine, which induces predominantly viral-specific mIgG2b and mIgG2c, confers stronger cross-protection against heterologous virus challenge via ADCC or ADCP than alum-adjuvanted vaccine, which predominantly induces viral-specific mIgG1 ( Shibuya et al., 2020 ). Furthermore, we demonstrated a new concept that vaccine-induced cross-reactive non-neutralizing mIgG1 anti-viral antibodies suppressed the cross-protective effects of mIgG2b/c against a heterologous virus challenge ( Shibuya et al., 2020 ).…”

“…We showed that CpG–ODN-adjuvanted influenza vaccine, which induces predominantly viral-specific mIgG2b and mIgG2c, confers stronger cross-protection against heterologous virus challenge via ADCC or ADCP than alum-adjuvanted vaccine, which predominantly induces viral-specific mIgG1 ( Shibuya et al., 2020 ). Furthermore, we demonstrated a new concept that vaccine-induced cross-reactive non-neutralizing mIgG1 anti-viral antibodies suppressed the cross-protective effects of mIgG2b/c against a heterologous virus challenge ( Shibuya et al., 2020 ). These results suggest that an adjuvant that induces viral-specific mIgG2b/c selectively, such as CpG–ODN, is optimal for heterologous protection.…”

Understanding the relationship between norovirus diversity and immunity

“… 100 In contrast, IgG1 antibodies against influenza virus were shown to inhibit the cross-protective effect of IgG2 antibodies after vaccination in mice, suggesting that antibody isotypes play a major role in pathogen control. 101 Other mechanisms by which non-neutralizing antibodies enhance or antagonize the immune response have been reviewed. 102 Vaccine design should focus on targeting conserved, protective epitopes and inducing the proper Ig subtype, perhaps by manipulating the presentation of the antigen and/or by selecting adjuvants that will enhance the antibody repertoire as shown for influenza and human papilloma virus.…”