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Fc mediated effector functions of antibodies play important roles in immunotherapies and vaccine efficacy but assessing those functions in animal models can be challenging due to species differences. Rhesus macaques, Macaca mulatta (Mm) share approximately 93% sequence identity with humans but display important differences in their adaptive immune system that complicates their use in validating therapeutics and vaccines that rely on Fc effector functions. In contrast to humans, macaques only have one low affinity FcγRIII receptor, CD16, which shares a polymorphism at position 158 with human FcγRIIIa with Ile 158 and Val 158 variants. Here we describe structure-function relationships of the Ile/Val 158 polymorphism in Mm FcγRIII. Our data indicate that the affinity of the allelic variants of Mm FcγRIII for the macaque IgG subclasses vary greatly with changes in glycan composition both on the Fc and the receptor. However, unlike the human Phe/Val 158 polymorphism in FcγRIIIa, the higher affinity variant corresponds to the larger, more hydrophobic side chain, Ile, even though it is not directly involved in the binding interface. Instead, this side chain appears to modulate glycan-glycan interactions at the Fc/FcγRIII interface. Furthermore, changes in glycan composition on the receptor have a greater effect for the Val 158 variant such that with oligomannose type glycans and with glycans only on Asn 45 and Asn 162 , Val 158 becomes the variant with higher affinity to Fc. These results have implications not only for the better interpretation of nonhuman primate studies but also for studies performed with human effector cells carrying different FcγRIIIa alleles.

Vaccine efficacy determined within the controlled environment of a clinical trial is usually substantially greater than real-world vaccine effectiveness. Typically, this results from reduced protection of immunologically vulnerable populations, such as children, elderly individuals and people with chronic comorbidities. Consequently, these high-risk groups are frequently recommended tailored immunisation schedules to boost responses. In addition, diverse groups of healthy adults may also be variably protected by the same vaccine regimen. Current population-based vaccination strategies that consider basic clinical parameters offer a glimpse into what may be achievable if more nuanced aspects of the immune response are considered in vaccine design. To date, vaccine development has been largely empirical. However, next-generation approaches require more rational strategies. We foresee a generation of precision vaccines that consider the mechanistic basis of vaccine response variations associated with both immunogenetic and baseline health differences. Recent efforts have highlighted the importance of balanced and diverse extra-neutralising antibody functions for vaccine-induced protection. However, in immunologically vulnerable populations, significant modulation of polyfunctional antibody responses that mediate both neutralisation and effector functions has been observed. Here, we review the current understanding of key genetic and inflammatory modulators of antibody polyfunctionality that affect vaccination outcomes and consider how this knowledge may be harnessed to tailor vaccine design for improved public health.

Substantial population-level variation in vaccine-specific antibody responses has been observed following global coronavirus disease 2019 (COVID-19) vaccination efforts. Beyond the influence of clinical and demographic features, immunogenetic variation is suggested to underlie divergent serological responses following COVID-19 vaccination of distinct populations. Immunoglobulin G1 (IgG1) allotypic markers (G1m) for 121 COVID-19 vaccinated healthy adults were genotyped via Sanger sequencing. Vaccine-specific IgG and Fc gamma receptor (FcγR) engagement were characterised via bead-based multiplex array. Following two COVID-19 vaccine doses, G1m1,17+/+ compared to G1m-1,3+/+ vaccinees had increased IgG and FcγR engagement specific for the antigenically conserved SARS-CoV-2 Spike 2 (S2) domain. IgG targeting antigenically novel SARS-CoV-2 receptor binding domain (RBD) trended higher in G1m1,17+/+ vaccinees, facilitating increased RBD-specific FcγR2a-R131 and FcγR2b binding. Primary COVID-19 vaccination induced increased S2-specific IgG in G1m1,17+/+ vaccinees, facilitating enhanced anti-viral FcγR engagement and suggesting immunogenetics may be a valuble consideration for next-generation vaccine design. •G1m1,17+/+IGHG1 haplotype linked to increased COVID-19 vaccine-specific IgG and IgG1.•Elevated vaccine-specific IgG promoted enhanced SARS-CoV-2-directed FcγR2 binding.•Elevated G1m1,17 IgG predominantly targeted antigenically conserved Spike 2 domain.

Evaluating the biophysical and functional nature of IgG is key to defining correlates of protection in infectious disease, and autoimmunity research cohorts, as well as vaccine efficacy trials. These studies often require small quantities of IgG to be purified from plasma for downstream analysis with high throughput immunoaffinity formats which elute IgG at low-pH, such as Protein G and Protein A. Herein we sought to compare Protein G purification of IgG with an immunoaffinity method which elutes at physiological pH (Melon Gel). Critical factors impacting Fc functionality with the potential to significantly influence FcγR binding, such as IgG subclass distribution, -glycosylation, aggregation, and IgG conformational changes were investigated and compared. We observed that transient exposure of IgG to the low-pH elution buffer, used during the Protein G purification process, artificially enhanced recognition of Fcγ Receptors (FcγRs) as demonstrated by Surface Plasmon Resonance (SPR), FcγR dimer ELISA, and a functional cell-based assay. Furthermore, low-pH exposed IgG caused conformational changes resulting in increased aggregation and hydrophobicity; factors likely to contribute to the observed enhanced interaction with FcγRs. These results highlight that methods employed to purify IgG can significantly alter FcγR-binding behavior and biological activity and suggest that the IgG purification approach selected may be a previously overlooked factor contributing to the poor reproducibility across current assays employed to evaluate Fc-mediated antibody effector functions.

Antibodies against the SARS-CoV-2 spike protein are a critical immune determinant for protection against the virus. While virus neutralization is a key function of spike-specific antibodies, antibodies also mediate Fc-dependent activities that can play a role in protection or pathogenesis. This study characterized serum antibody responses elicited after two doses of heterologous adenovirus-vectored (Ad26/ Ad5) vaccines. Vaccine-induced antibody binding titers and Fc-mediated functions decreased over six months, while neutralization titers remained stable. Comparison of antibody isotypes elicited after Ad26/Ad5 vs. LNP-mRNA vaccination and after infection showed that anti-spike IgG1 were dominant and produced to high levels in all groups. The Ad26/Ad5 vaccines also induced IgG4 but not IgG2 and IgG3, whereas the LNP-mRNA vaccines elicited a full Ig spectrum (IgM, IgG1-4, IgA1-2). Convalescent COVID-19 patients had mainly IgM and IgA1 alongside IgG1. Despite these differences, the neutralization potencies against early variants were similar. However, both vaccine groups had antibodies with greater Fc potencies of binding complement and Fcg receptors than the COVID-19 group. The Ad26/Ad5 group also displayed a greater potency of RBD-specific antibody-mediated cellular phagocytosis. Antibodies with distinctive quality were induced by different vaccines and infection. The data imply the utility of different vaccine platforms to elicit antibody responses with fine-tuned Fc activities.

Antibodies play a critical role in immunity in part by mediating clearance of pathogens and infected cells by antibody-dependent cellular phagocytosis (ADCP) through engagement of Fc gamma receptors (FcγRs) on innate immune cells. Among these, FcγRIIa (CD32a) is a key activating receptor expressed on macrophages, dendritic cells, and other antigen-presenting cells. Its affinity for IgG and ability to mediate ADCP is influenced by allelic polymorphisms. In humans, a single amino acid polymorphism at position 131, where histidine (H) is substituted with arginine (R), leads to decreased IgG1 and IgG2 subclass binding affinity and, consequently, lower efficiency of phagocytic responses. Rhesus macaques ( ), which are widely used as nonhuman primate models, exhibit a similar polymorphism at position 131 of FcγRIIa, but with arginine replaced by proline (P). Here, we investigated structure-function relationships associated with the FcγRIIa polymorphism at position 131 in both species, specifically with respect to IgG1 and IgG2. We determined the structures of complexes formed by each variant with IgG1 Fc and those formed by the higher affinity variant with IgG2 Fc for both species by x-ray crystallography and linked these structures to affinity and activity using SPR and an ADCP assay. We also determined the structure of human inhibitory FcγRIIb (CD32b) in complex with IgG1 Fc by x-ray crystallography. Through analysis of these structures, our studies reveal that FcγRIIa engagement is minimally influenced by Fc glycan composition, distinguishing it from FcγRIIIa whose affinity is strongly influenced by glycan-composition. Comparative structures of human and macaque FcγRIIa variants demonstrate species- and allele-specific differences in Fc binding, but our functional assays showed only minimal allele-specific effects in humans. In contrast, allele-specific effects in macaques were highly significant; the macaque P variant showing uniformly reduced IgG affinity. These insights highlight fundamental interspecies and allelic distinctions that are critical for interpreting FcγRIIa-mediated effector functions in macaque models and for optimizing translational antibody and vaccine design.

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre‐symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti‐lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11‐dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell‐dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ‐binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization‐independent T cell‐mediated effects, even against an established CNS infection by a lethal neurotropic virus. Synopsis Rabies is a fatal viral disease of humans, with uniform mortality once central nervous system (CNS) invasion occurs and symptoms appear. This study demonstrates that a single‐dose monoclonal (mAb) therapy can yield a functional cure for rabies, even after robust CNS replication. Peripheral administration of mAb F11 reduces CNS viral replication and prevents mortality, following infection of mice with a lethal dose of either Australian bat lyssavirus (ABLV) or rabies virus (RABV). Therapeutic efficacy of F11, a human IgG1, requires a functional antibody Fc region, implicating the mechanistic involvement of immune cells bearing FcRγ. F11 efficacy requires an intact host adaptive immune response, particularly CD4 T cells. Administration of F11 alters both the proportions and phenotypes of immune cells in the brains of ABLV‐infected animals. Virus persists chronically at a low level in the brains of F11‐treated animals, but animals remain free of disease signs. Graphical Abstract Rabies is a fatal viral disease of humans, with uniform mortality once central nervous system (CNS) invasion occurs and symptoms appear. This study demonstrates that a single‐dose monoclonal (mAb) therapy can yield a functional cure for rabies, even after robust CNS replication.

Despite decades of research, the development of an effective HIV vaccine remains a significant challenge. Recent findings from three large vaccine efficacy trials have identified antibodies against the V1V2 domain of the HIV envelope glycoprotein as a potential correlate of reduced infection risk, offering a promising avenue for improving vaccine efficacy. Vaccine-elicited anti-V1V2 antibodies do not mediate potent virus-neutralizing activities, but they mediate Fc-dependent effector functions. This study evaluated the capacity of V1V2-scaffold vaccines in different formulations to generate antibody responses with Fc-mediated functions. BALB/c mice were immunized with V1V2-scaffold proteins formulated with one of the following adjuvants: MF59-like squalene-based oil-in-water emulsion (Addavax), a combination of TLR7/8 and RIG-I agonists (IMDQ-PC/IVT), nanoemulsion and RIG-I agonist (NE/IVT), or empty lipid nanoparticles (eLNP). All formulations were administered intramuscularly except NE/IVT, which was given intranasally. For comparison, we also tested a V1V2-scaffold-expressing mRNA-LNP vaccine delivered intramuscularly and an Env gp140 protein with liposomal MPLA/DDA adjuvant administered subcutaneously. Among the six vaccine formulations tested, V1V2-scaffold immunogens adjuvanted with LNP (eLNP and mRNA-LNP) elicited the most robust and cross-reactive serum IgG responses that recognized native Env on cell surfaces or virions. The eLNP and mRNA-LNP groups, along with IMDQ-PC/IVT, also elicited functional IgG2a, and correspondingly displayed Fc-mediated activities, as measured by antibody-dependent cellular phagocytosis and FcγRIV binding. Notably, IMDQ-PC/IVT elicited predominantly IgG2a with minimal IgG1, eLNP stimulated IgG1 and IgG2a with IgG1 dominance, whereas mRNA-LNP yielded more balanced IgG2a/IgG1 responses. Data from this study provide new insights into the utility of novel formulations for V1V2-scaffold immunogens as a strategy for optimizing the induction of functional V1V2-specific antibodies to improve HIV vaccine efficacy.