High-resolution map of the Fc functions mediated by COVID-19-neutralizing antibodies.

A growing body of evidence shows that fragment crystallizable (Fc)-dependent antibody effector functions play an important role in protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To unravel the mechanisms that drive these responses, we analyzed the phagocytosis and complement deposition mediated by a panel of 482 human monoclonal antibodies (nAbs) neutralizing the original Wuhan virus, expressed as recombinant IgG1. Our study confirmed that nAbs no longer neutralizing SARS-CoV-2 Omicron variants can retain their Fc functions. Surprisingly, we found that nAbs with the most potent Fc function recognize the N-terminal domain, followed by those targeting class 3 epitopes in the receptor binding domain. Interestingly, nAbs direct against the class 1/2 epitopes in the receptor binding motif, which are the most potent in neutralizing the virus, were the weakest in Fc functions. The divergent properties of the neutralizing and Fc function-mediating antibodies were confirmed by the use of different B cell germlines and by the observation that Fc functions of polyclonal sera differ from the profile observed with nAbs, suggesting that non-neutralizing antibodies also contribute to Fc functions. These data provide a high-resolution picture of the Fc-antibody response to SARS-CoV-2 and suggest that the Fc contribution should be considered for the design of improved vaccines, the selection of therapeutic antibodies, and the evaluation of correlates of protection.

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism.

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.

Polyfunctional antibodies: a path towards precision vaccines for vulnerable populations.

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.

Heterologous SARS-CoV-2 IgA neutralising antibody responses in convalescent plasma.

Objectives: Following infection with SARS-CoV-2, virus-specific antibodies are generated, which can both neutralise virions and clear infection via Fc effector functions. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. By comparison, other antibody isotypes including IgA have been poorly characterised. Methods: Here, we characterised plasma IgA from 41 early convalescent COVID-19 subjects for neutralisation and Fc effector functions. Results: Convalescent plasma IgA from > 60% of the cohort had the capacity to inhibit the interaction between wild-type RBD and ACE2. Furthermore, a third of the cohort induced stronger IgA-mediated ACE2 inhibition than matched IgG when tested at equivalent concentrations. Plasma IgA and IgG from this cohort broadly recognised similar RBD epitopes and had similar capacities to inhibit ACE2 from binding to 22 of the 23 prevalent RBD mutations assessed. However, plasma IgA was largely incapable of mediating antibody-dependent phagocytosis in comparison with plasma IgG. Conclusion: Overall, convalescent plasma IgA contributed to the neutralising antibody response of wild-type SARS-CoV-2 RBD and various RBD mutations. However, this response displayed large heterogeneity and was less potent than IgG.

Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays.

Antibody Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies. Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding the Fc effector function during monoclonal antibody development. This article covers two cell-based ADCC bioassays which can quantitatively measure the antibody potency in ADCC. Basic Protocol 1 describes the ADCC reporter bioassay using engineered ADCC effector cells which measures the FcγRIIIa-mediated luciferase reporter activation upon the binding of antibody-coated target cells. Basic Protocol 2 describes the PBMC ADCC bioassay using primary peripheral blood mononuclear cells (PBMC) as effector cells and engineered HiBiT target cells in an assay that measures the release of HiBiT from target cells upon antibody-mediated target lysis. Optimization of several key assay parameters including cell handling, effector:target (E:T) ratios, assay plate, and plate reader requirement, and how these parameters impact assay performance are discussed. © 2021 Promega Corporation. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ADCC reporter bioassay using engineered ADCC bioassay effector cells Basic Protocol 2: PBMC ADCC bioassay using primary PBMC and engineered HiBiT target cells.

Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination.

Recently approved vaccines have shown remarkable efficacy in limiting SARS-CoV-2-associated disease. However, with the variety of vaccines, immunization strategies, and waning antibody titers, defining the correlates of immunity across a spectrum of antibody titers is urgently required. Thus, we profiled the humoral immune response in a cohort of non-human primates immunized with a recombinant SARS-CoV-2 spike glycoprotein (NVX-CoV2373) at two doses, administered as a single- or two-dose regimen. Both antigen dose and boosting significantly altered neutralization titers and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were associated with distinct levels of protection in the upper and lower respiratory tract. Moreover, NVX-CoV2373 elicited antibodies that functionally targeted emerging SARS-CoV-2 variants. Collectively, the data presented here suggest that a single dose may prevent disease via combined Fc/Fab functions but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.

Antibodies for Human Immunodeficiency Virus-1 Cure Strategies.

Human immunodeficiency virus (HIV) infection leads to the establishment of a long-lived latent cellular reservoir. One strategy to eliminate quiescent reservoir cells is to reactivate virus replication to induce HIV envelope glycoprotein (Env) expression on the cell surface exposing them to subsequent antibody targeting. Via the interactions between the antibody Fc domain and Fc-γ receptors (FcγRs) that are expressed on innate effector cells, such as natural killer cells, monocytes, and neutrophils, antibodies can mediate the elimination of infected cells. Over the last decade, a multitude of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes have been identified and are currently being explored for HIV eradication strategies. Antibody development also includes novel Fc engineering approaches to increase engagement of effector cells and optimize antireservoir efficacy. In this review, we discuss the usefulness of antibodies for HIV eradication approaches specifically focusing on antibody-mediated strategies to target latently infected cells and options to increase antibody efficacy.

Delayed fractional dosing with RTS,S/AS01 improves humoral immunity to malaria via a balance of polyfunctional NANP6- and Pf16-specific antibodies.

BACKGROUND: Malaria remains a key cause of mortality in low-income countries. RTS,S/AS01 is currently the most advanced malaria vaccine, demonstrating ∼50% efficacy in controlled human malaria infection (CHMI) studies in malaria-naive adults and ∼30%-40% efficacy in field trials in African infants and children. However, a higher vaccine efficacy is desirable. METHODS: Modification of the vaccine regimen in a CHMI trial in malaria-naive individuals resulted in significant increase in protection. While three equal monthly RTS,S/AS01 doses (RRR) were used originally, the administration of a delayed third dose with 20% of the original antigen dose (RRr) resulted in ∼87% protection, linked to enhanced antibody affinity maturation. Here, we sought to identify a novel molecular basis for this higher protective efficacy using Systems Serology. FINDINGS: We demonstrate that the delayed fractional dose maintains monocyte phagocytosis and NK activation mediated by NANP6-specific antibodies, key correlates of protection for the RRR regimen. However, it is also marked by a higher breadth of C-term Fc effector functions, including enhanced phagocytosis. The RRr regimen breaches immunodominance of the humoral immune response, inducing a balanced response across the C-terminal (Pf16) and NANP region of CSP, both of which were linked to protection. CONCLUSIONS: Collectively, these data point to an unexpectedly concordant evolution in Fab avidity and expanded C-term Fc effector functions, providing novel insights into the basis for higher protection conferred by the delayed fractional dose in malaria-naive individuals. FUNDING: This research was supported by PATH's Malaria Vaccine Initiative and the MGH Research Scholars program.

Collaborative study for the establishment of human immunoglobulin BRP replacement batches.

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of these BRPs were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Seventeen laboratories, including manufacturers and Official Medicines Control Laboratories, took part in the study. Several batches of candidate BRPs were calibrated against human immunoglobulin (ACA and molecular size) BRP batch 1 and human immunoglobulin (Fc function and molecular size) BRP batch 1 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. human immunoglobulin for anticomplementary activity BRP batch 1 and batch 2, Ph. Eur. human immunoglobulin for Fc function BRP batch 1 and batch 2 and Ph. Eur. human immunoglobulin (molecular size) BRP batch 2 and batch 3.