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Broadly neutralizing antibodies (bNAbs), when administered through passive immunization, are protective against HIV-1 infection. Current HIV-1 vaccine strategies are aimed at guiding the immune system to make bNAbs by mimicking their development during infection. Somatic hypermutation of the variable region is known to be crucial for the development of bNAbs. More recently, however, studies have shown how class-switch recombination (CSR) resulting in the generation of different antibody isotypes may serve as an additional mechanism through which antibodies can gain neutralization breadth and potency. In this review, we discuss the importance of different antibody isotypes for HIV-1 neutralization breadth and potency and how this information can be leveraged to improve passive and active immunization against HIV-1.

Influenza virus hemagglutinin (HA) stalk-specific antibodies have been shown to potently induce Fc-mediated effector functions which are important in protection from disease. In placebo-controlled maternal influenza (MatFlu) vaccination trials of pregnant women living with or without HIV, reduced risk of influenza illness was associated with high HA stalk antibody titers following trivalent inactivated vaccination (TIV). However, the mechanisms of immunity conferred by the HA stalk antibodies were not well understood. Here, we investigated HA stalk-specific Fc effector functions including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent complement deposition (ADCD), and FcγRIIa and FcγRIIIa binding in response to seasonal influenza vaccination. These were measured pre- and 1-month post-vaccination in 141 HIV-uninfected women (67 TIV and 74 placebo recipients) and 119 women living with HIV (WLWH; 66 TIV and 53 placebo recipients). In contrast to HIV-uninfected women, where HA stalk-specific ADCP and FcγRIIa binding were significantly boosted, WLWH showed no increase in response to vaccination. HA stalk-specific ADCC potential and FcγRIIIa binding were not boosted regardless of HIV status but were higher in WLWH compared with HIV-uninfected women prior to vaccination. HA stalk-specific ADCD was significantly increased by vaccination in all women, but was significantly lower in the WLWH both pre- and post- vaccination. Co-ordination between HA stalk-specific ADCP and ADCD in WLWH was improved by vaccination. Fc polyfunctionality was enhanced by vaccination in HIV-uninfected women and driven by the HA stalk antibody titers. However, in the WLWH, higher pre-vaccination Fc polyfunctionality was maintained post-vaccination but was decoupled from titer. Overall, we showed differential regulation of Fc effector HA stalk responses, suggesting that HIV infection results in unique humoral immunity in response to influenza vaccination, with relevance for future strategies that aim to target the HA stalk in this population.

Antibodies with the same variable region can exist as multiple isotypes with varying neutralization potencies, though the mechanism for this is not fully defined. We previously isolated an HIV-directed IgA1 monoclonal antibody (mAb), CAP88-CH06, and showed that IgA1 and IgG3 isotypes of this antibody demonstrated enhanced neutralization compared to IgG1. To explore the mechanism behind this, hinge region and constant heavy chain (CH1) chimeras were constructed between the IgA1, IgG3 and IgG1 mAbs and assessed for neutralization activity, antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). Hinge chimeras revealed that the increased neutralization potency and phagocytosis of the IgG3 isotype was attributed to its longer hinge region. In contrast, for IgA1, CH1 chimeras showed that this region was responsible both for enhanced neutralization potency and decreased ADCP, though ADCC was not affected. Overall, these data show that the enhanced neutralization potency of CAP88-CH06 IgG3 and IgA1, compared to IgG1, is achieved through distinct mechanisms. Understanding the influence of the hinge and CH1 regions on Fab domain function may provide insights into the engineering of therapeutic antibodies with increased neutralization potency.

PURPOSE OF REVIEW: Although the goal of preventive HIV vaccine design is primarily the induction of broadly neutralizing antibodies (bNAbs), recent evidence suggests that a protective response will also benefit from Fc effector functions. Here, we provide an update on the antibody response to HIV infection, including both Fab and Fc-mediated antibody responses. We also highlight recent studies showing the interplay between these functions, focusing primarily on studies published in the last year. RECENT FINDINGS: Identification and characterization of bNAb donors continues to provide insights into viral factors that are potentially translatable to vaccine design. Improved and more diverse measures of Fc effector function, and modulators thereof, are enabling a deeper understanding of their role in infection. New data providing mechanistic links between the innate and adaptive humoral immune responses are creating exciting opportunities for vaccine strategies, with the aim of eliciting a polyfunctional protective response. SUMMARY: New insights into the overall humoral response to HIV infection are defining diverse and synergistic mechanisms required for antibody protection from HIV through vaccination.

To protect future generations against HIV, a vaccine will need to induce immunity by the time of sexual debut and hence requires immunization during childhood. Current strategies for a prophylactic HIV vaccine include the induction of a broadly neutralizing antibody response and the recruitment of potent effector functions of immune cells via the constant antibody Fc region., A prophylactic HIV vaccine would ideally induce protective immunity prior to sexual debut. Children develop broadly neutralizing antibody (bnAb) responses faster and at higher frequencies than adults, but little is known about the underlying mechanisms or the potential role of Fc-mediated effector functions in disease progression. We therefore performed systems immunology, with immunoglobulin profiling, on HIV-infected children with progressive and nonprogressive disease. Pediatric nonprogressors (PNPs) showed distinct immunoglobulin profiles with an increased ability to elicit potent Fc-mediated natural killer (NK)-cell effector functions. In contrast to previous reports in adults, both groups of children showed high levels of gp120-specific IgG Fc glycan sialylation compared to bulk IgG. Importantly, higher levels of Fc glycan sialylation were associated with increased bnAb breadth, providing the first evidence that Fc sialylation may drive affinity maturation of HIV-specific antibodies in children, a mechanism that could be exploited for vaccination strategies. IMPORTANCE To protect future generations against HIV, a vaccine will need to induce immunity by the time of sexual debut and hence requires immunization during childhood. Current strategies for a prophylactic HIV vaccine include the induction of a broadly neutralizing antibody response and the recruitment of potent effector functions of immune cells via the constant antibody Fc region. In this study, we show that nonprogressing HIV-infected children mounted antibody responses against HIV that were able to mediate potent Fc effector functions, which may contribute to the control of HIV replication. Children who had specific glycan structures on the Fc portion of antibodies against HIV were able to neutralize a broader range of HIV variants, providing evidence of a potential role of Fc glycovariation in the development of bnAbs against HIV. These findings complement our knowledge of the distinct immune landscape in early life that could be exploited in the development of vaccine strategies.

Antibody Fc effector functions contribute to HIV control and have been implicated in the partial efficacy seen in the RV144 vaccine trial. Fc-mediated trogocytosis has been previously described for anti-cancer antibodies and results in the removal of membrane fragments from target cells. Here we developed a flow cytometry-based assay which measures the transfer of membrane fragments from a gp120-coated CD4+ lymphocytic cell line (CEM.NKR-CCR5 cells stained with a membrane dye PKH26) to monocytic cells (THP-1 cells stained with CFSE). We showed that this transfer occurred rapidly, within 1 h, and was mediated through engagement of the FcγRIIa/b receptors on the THP-1 cells. HIV-specific IgG as well as gp120 and CD4 could be detected on the surface of THP-1 cells in a process that we demonstrated was distinct from phagocytosis. Furthermore, while the THP-1 effector cells remained intact following the receipt of new membrane proteins, the viability of the target CEM.NKR-CCR5 cells decreased over time. Analysis of HIV-specific plasma revealed that antibodies with trogocytic activity were common in acute and chronic HIV infection but were higher in individuals with broadly neutralizing antibody responses We also examined trogocytosis mediated by broadly neutralizing antibodies (bNAbs) targeting multiple epitopes on the BG505.SOSIP.664 trimer and show that levels of binding correlated with the trogocytosis score. Overall, our data describe a new antiviral Fc effector function mediated by HIV-specific antibodies that could be harnessed for vaccination and cure strategies.

SUMMARY Antibodies that bind residue K169 in the V2 region of the HIV-1 envelope correlated with reduced risk of infection in the RV144 vaccine trial but were restricted to two ED-motif-encoding light chain genes. Here, we identify an HIV-infected donor with high-titer V2 peptide-binding antibodies and isolate two antibody lineages (CAP228-16H/19F and CAP228–3D) that mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC). Both lineages use the IGHV5–51 heavy chain germline gene, similar to the RV144 antibody CH58, but one lineage (CAP228-16H/19F) uses a light chain without the ED motif. A cocrystal structure of CAP228-16H bound to a V2 peptide identified a IGLV3–21 gene-encoded DDxD motif that is used to bind K169, with a mechanism that allows CAP228-16H to recognize more globally relevant V2 immunotypes. Overall, these data further our understanding of the development of cross-reactive, V2-binding, antiviral antibodies and effectively expand the human light chain repertoire able to respond to RV144-like immunogens., In Brief V2-directed antibodies from the RV144 vaccine trial correlated with reduced HIV-1 infection risk but exhibited restricted light chain gene usage. Here, van Eeden et al. isolate similar antibodies from an HIV-1-infected individual and identify a third V2-reactive light chain gene, increasing the antibody repertoire potentially elicited by vaccination., Graphical Abstract

The α4β7 integrin is a non-essential HIV-1 adhesion receptor, bound by the gp120 V1V2 domain, facilitating rapid viral dissemination into gut-associated lymphoid tissues. Antibodies blocking this interaction early in infection can improve disease outcome, and V1V2-targeted antibodies were correlated with moderate efficacy reported from the RV144 HIV-1 vaccine trial. Monoclonal α4β7-blocking antibodies recognise two slightly different helical V2 conformations, and current structural data suggests their binding sites are occluded in prefusion envelope trimers. Here, we report cocrystal structures of two α4β7-blocking antibodies from an infected donor complexed with scaffolded V1V2 or V2 peptides. Both antibodies recognised the same helix-coil V2 conformation as RV144 antibody CH58, identifying a frequently sampled alternative conformation of full-length V1V2. In the context of Envelope, this α-helical form of V1V2 displays highly exposed α4β7-binding sites, potentially providing a functional role for non-native Envelope on virion or infected cell surfaces in HIV-1 dissemination, pathogenesis, and vaccine design., Antibodies blocking the V1V2 domain of HIV Envelope from binding integrin are associated with positive disease outcomes. Here, Wibmer et al. determine the structure of full length V1V2 bound to these antibodies, revealing an alternative fold of V1V2 with exposed integrin-binding sites that functions on non-native Envelope.

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.

Background The integrin α4β7 mediates the trafficking of immune cells to the gut associated lymphoid tissue (GALT) and is an attachment factor for the HIV gp120 envelope glycoprotein. We developed a viral replication inhibition assay to more clearly evaluate the role of α4β7 in HIV infection and the contribution of viral and host factors. Results Replication of 60 HIV-1 subtype C viruses collected over time from 11 individuals in the CAPRISA cohort were partially inhibited by antibodies targeting α4β7. However, dependence on α4β7 for replication varied substantially among viral isolates from different individuals as well as over time in some individuals. Among 8 transmitted/founder (T/F) viruses, α4β7 reactivity was highest for viruses having P/SDI/V tri-peptide binding motifs. Mutation of T/F viruses that had LDI/L motifs to P/SDI/V resulted in greater α4β7 reactivity, whereas mutating P/SDI/V to LDI/L motifs was associated with reduced α4β7 binding. P/SDI/V motifs were more common among South African HIV subtype C viruses (35%) compared to subtype C viruses from other regions of Africa (