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While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies., Author summary Some HIV-infected individuals develop antibodies that are capable of neutralizing the majority of HIV strains, a highly desirable function mediated by the antibody Fab portion. While antibodies elicited by current vaccines have failed to recreate this activity, the partial protection seen in the RV144 vaccine trial has been attributed to antibody Fc-mediated effector functions such as cell killing. In this study, we found that HIV-infected individuals who show a diversified and potent Fc response early in infection were more likely to develop broadly neutralizing antibodies later on. Examination of B cell functions associated with good germinal center activity, provided evidence for a common mechanistic link between the regulation of the Fc and Fab mediated activities in these individuals. Our finding of an Fc effector function profile that arises early and predicts neutralization breadth could be used in the evaluation of vaccine candidates designed to generate neutralizing antibodies. Common immune determinants associated with both Fab and Fc function could furthermore be exploited for vaccine design to harness the full potential of HIV-specific antibodies.

Broadly neutralizing antibodies (bNAbs) protect against HIV infection in non-human primates and their efficacy may be enhanced through interaction with Fc receptors on immune cells. Antibody isotype is a modulator of this binding with the IgG3 subclass mediating potent Fc effector function and is associated with HIV vaccine efficacy and HIV control. BNAb functions are typically assessed independently of the constant region with which they are naturally expressed. To examine the role of natural isotype in the context of a bNAb lineage we studied CAP256, an HIV-infected individual that mounted a potent V2-specific bNAb response. CAP256 expressed persistently high levels of plasma IgG3 which we found mediated both broad neutralizing activity and potent Fc function. Sequencing of germline DNA and the constant regions of V2-directed bNAbs from this donor revealed the expression of a novel IGHG3 allele as well as IGHG3*17, an allele that produces IgG3 antibodies with increased plasma half-life. Both allelic variants were used to generate CAP256-VRC26.25 and CAP256-VRC26.29 IgG3 bNAbs and these were compared to IgG1 versions. IgG3 variants were shown to have significantly higher phagocytosis and trogocytosis compared to IgG1 versions, which corresponded to increased affinity for FcγRIIa. Neutralization potency was also significantly higher for IgG3 bNAbs, particularly against viruses lacking the N160 glycan. By exchanging hinge regions between subclass variants, we showed that hinge length modulated both neutralization potency and Fc function. This study showed that co-operation between the variable and natural IgG3 constant regions enhanced the polyfunctionality of antibodies, indicating the value of leveraging genetic variation which could be exploited for passive immunity., Author summary In the only partially effective HIV vaccine trial to date, reduced risk of infection correlated with IgG3 antibodies that bound to the V2 region of the HIV envelope. IgG3 antibodies potently mediate the broadest range of Fc cytotoxic functions such as complement deposition, cellular lysis, engulfment and membrane nibbling. The relevance of different IgG3 alleles in antibody function is not typically examined, nor are antibodies usually assessed in the context of their natural isotype. Here we show that IgG3 V2-specific broadly neutralizing antibodies from donor CAP256 mediated both enhanced cytotoxic functions and neutralization potency compared to an IgG1 variant. We demonstrate that the long hinge region of IgG3 facilitated this increased functionality. These data suggest that isotype may be tuned to enhance both neutralization and cytotoxic activity of broadly neutralizing antibodies that can be used to improve the efficacy of passive immunization strategies for HIV prevention.