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The Janssen (Johnson & Johnson) Ad26.COV2.S non-replicating viral vector vaccine has been widely deployed for COVID-19 vaccination programs in resource-limited settings. Here we confirm that neutralizing and binding responses to Ad26.COV2.S vaccination are stable for 6 months post-vaccination, when tested against multiple SARS-CoV-2 variants. Secondly, using longitudinal samples from individuals who experienced clinically mild breakthrough infections 4 to 5 months after vaccination, we show dramatically boosted binding antibodies, Fc effector function and neutralization. These high titer responses are of similar magnitude to humoral immune responses measured in severely ill, hospitalized donors, and are cross-reactive against diverse SARS-CoV-2 variants, including the extremely neutralization resistant Omicron (B.1.1.529) variant that currently dominates global infections, as well as SARS-CoV-1. These data have implications for population immunity in areas where the Ad26.COV2.S vaccine has been widely deployed, but where ongoing infections continue to occur at high levels.

SummarySARS-CoV-2 variants of concern (VOCs) exhibit escape from neutralizing antibodies, causing concern about vaccine effectiveness. However, while non-neutralizing cytotoxic functions of antibodies are associated with decreased disease severity and vaccine protection, Fc effector function escape from VOCs is poorly defined. Furthermore, whether VOCs trigger Fc functions with altered specificity, as has been reported for neutralization, is unknown. Here, we demonstrate that the Beta VOC partially evades Fc effector activity in individuals infected with the original (D614G) variant. However, not all functions are equivalently affected, suggesting differential targeting by antibodies mediating distinct Fc functions. Furthermore, Beta infection triggered responses with significantly improved Fc cross-reactivity against global VOCs compared to either D614G infected or Ad26.COV2.S vaccinated individuals. This suggests that, as for neutralization, the infecting spike sequence impacts Fc effector function. These data have important implications for vaccine strategies that incorporate VOCs, suggesting these may induce broader Fc effector responses.

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.

Summary The Johnson and Johnson Ad26.COV2.S single dose vaccine, designed as an emergency response to the pandemic, represents an attractive option for the scale-up of COVID-19 vaccination in resource-limited countries. We examined the effect of prior infection with ancestral (D614G) or Beta variants on Ad26.COV2.S immunogenicity approximately 28 days post-vaccination. We compared healthcare workers who were SARS-CoV-2 naive (n=20), to those infected during the first wave prior to the emergence of Beta (n=20), and those infected in the second wave (n=20), when Beta was the dominant variant. We demonstrate that a priming exposure from infection significantly increased the magnitude of spike binding antibodies, neutralizing antibodies and antibody-dependent cellular cytotoxicity activity (ADCC) against D614G, Beta and Delta variants. The magnitude of antibody boosting was similar in both waves, despite the longer time interval between wave 1 infection and vaccination (7 months), compared to wave 2 (2 months). ADCC and binding cross-reactivity was similar in both waves. However, neutralization cross-reactivity varied by wave, showing that the antibody repertoire was shaped by the spike sequence of the infecting variant. Robust CD4 and CD8 T cell responses to spike of similar or higher magnitude as those elicited by infection were induced after vaccination. In contrast to antibody responses, prior infection was not required for the generation of high magnitude T cell responses, and T cell recognition of the Beta variant was fully preserved. Therefore, Ad26.COV2.S vaccination following prior infection, even >6 months previously, may result in substantially enhanced protection against COVID-19, of particular relevance in settings of high SARS-CoV-2 seroprevalence. Furthermore, the dominant impact of the infecting variant on neutralization breadth after vaccination has important implications for the design of second-generation vaccines based on variants of concern.

The Johnson and Johnson Ad26.COV2.S single dose vaccine represents an attractive option for COVID-19 vaccination in resource limited countries. We examined the effect of prior infection with different SARS-CoV-2 variants on Ad26.COV2.S immunogenicity. We compared participants who were SARS-CoV-2 naïve with those either infected with the ancestral D614G virus or infected in the second wave when Beta predominated. Prior infection significantly boosts spike binding antibodies, antibody-dependent cellular cytotoxicity and neutralizing antibodies against D614G, Beta and Delta, however neutralization cross-reactivity varied by wave. Robust CD4 and CD8 T cell responses are induced after vaccination, regardless of prior infection. T cell recognition of variants is largely preserved, apart from some reduction in CD8 recognition of Delta. Thus, Ad26.COV2.S vaccination following infection may result in enhanced protection against COVID-19. The impact of the infecting variant on neutralization breadth after vaccination has implications for the design of second-generation vaccines based on variants of concern., Graphical Abstract, Keeton, Richardson, Moyo-Gwete et al show that SARS-CoV-2 infection prior to Ad26CoV2.S vaccination significantly boosts cross-reactive ADCC, binding and neutralizing antibodies, and moderately boosts T cell responses against variants of concern. Furthermore, the infecting virus spike sequence determines the cross-reactivity of neutralizing responses, with implications for second generation vaccine design.

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis—and in some cases trogocytosis—but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies., Graphical abstract, Shiakolas et al. demonstrate that cross-reactive coronavirus antibodies induced by natural infection display a spectrum of epitope specificities across the spike protein and exhibit in vitro and in vivo antiviral functions.

Antibodies mediate pathogen neutralization in addition to several cytotoxic Fc functions through engaging cellular receptors and recruiting effector cells. Fc effector functions have been well described in disease control and protection against infectious diseases including HIV, Ebola, malaria, influenza and tuberculosis, making them attractive targets for vaccine design.We briefly summarize the role of Fc effector functions in disease control and protection in viral, bacterial and parasitic infectious diseases. We review Fc effector function in passive immunization and vaccination, and primarily focus on strategies to elicit and modulate these functions as part of a robust vaccine strategy.Despite their known correlation with vaccine efficacy for several diseases, only recently have seminal studies addressed how these Fc effector functions can be elicited and modulated in vaccination. However, gaps remain in assay standardization and the precise mechanisms of diverse functional assays. Furthermore, there are inherent difficulties in the translation of findings from animal models to humans, given the difference in sequence, expression and function of Fc receptors and Fc portions of antibodies. However, overall it is clear that vaccine development to elicit Fc effector function is an important goal for optimal prevention against infectious disease.

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.

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.

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

Antibody functions such as neutralization require recognition of antigen by the Fab region, while effector functions are additionally mediated by interactions of the Fc region with soluble factors and cellular receptors. The efficacy of individual antibodies varies based on Fab domain characteristics, such as affinity for antigen and epitope-specificity, and on Fc domain characteristics that include isotype, subclass, and glycosylation profile. Here, a series of HIV-specific antibody subclass and hinge variants were constructed and tested to define those properties associated with differential effector function. In the context of the broadly neutralizing CD4 binding site-specific antibody VRC01 and the variable loop (V3) binding antibody 447-52D, hinge truncation and extension had a considerable impact on the magnitude of phagocytic activity of both IgG1 and IgG3 subclasses. The improvement in phagocytic potency of antibodies with extended hinges could not be attributed to changes in either intrinsic antigen or antibody receptor affinity. This effect was specific to phagocytosis and was generalizable to different phagocytes, at different effector cell to target ratios, for target particles of different size and composition, and occurred across a range of antibody concentrations. Antibody dependent cellular cytotoxicity and neutralization were generally independent of hinge length, and complement deposition displayed variable local optima. In vivo stability testing showed that IgG molecules with altered hinges can exhibit similar biodistribution and pharmacokinetic profiles as IgG1. Overall, these results suggest that when high phagocytic activity is desirable, therapeutic antibodies may benefit from being formatted as human IgG3 or engineered IgG1 forms with elongated hinges., Author summary Target binding and receptor binding domains of antibodies are separated by a hinge region that differs among IgG subclasses. Among these, IgG3 has a uniquely elongated hinge, whose role in an immunoglobulin’s ability to facilitate different immune functions is incompletely understood. We evaluated the effect of varied hinge length and composition on diverse antibody activities and found that extended hinges enhance phagocytosis, a process through which the immune system can clear potentially harmful particles such as viruses. This enhancement was observed across diverse effector cell types, target particles, and for multiple HIV-specific antibodies. Antibodies with extended hinges showed in vivo persistence and localization in a mouse model similar to those with natural hinges. Consequently, immune responses or antibody therapies expected to benefit from a strong phagocytic response, such as viral sequestration, may be enhanced naturally by the IgG3 subclass or artificially by hinge extension. This work expands knowledge of the role that IgG3 plays in immunity and provides a framework for hinge engineering to improve antibodies.

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.

B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.

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 (