Antibody effector functions are associated with protection from respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory tract infection and death in young infants and the elderly. With no effective prophylactic treatment available, current vaccine candidates aim to elicit neutralizing antibodies. However, binding and neutralization have poorly predicted protection in the past, and accumulating data across epidemiologic cohorts and animal models collectively point to a role for additional antibody Fc-effector functions. To begin to define the humoral correlates of immunity against RSV, here we profiled an adenovirus 26 RSV-preF vaccine-induced humoral immune response in a group of healthy adults that were ultimately challenged with RSV. Protection from infection was linked to opsonophagocytic functions, driven by IgA and differentially glycosylated RSV-specific IgG profiles, marking a functional humoral immune signature of protection against RSV. Furthermore, Fc-modified monoclonal antibodies able to selectively recruit effector functions demonstrated significant antiviral control in a murine model of RSV.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy.

Maternal immunity impacts the infant, but how is unclear. To understand the implications of the immune exposures of vaccination and infection in pregnancy for neonatal immunity, we evaluated antibody functions in paired peripheral maternal and cord blood. We compared those who in pregnancy received mRNA coronavirus disease 2019 (COVID-19) vaccine, were infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the combination. We found that vaccination enriched a subset of neutralizing activities and Fc effector functions that was driven by IgG1 and was minimally impacted by antibody glycosylation in maternal blood. In paired cord blood, maternal vaccination also enhanced IgG1. However, Fc effector functions compared to neutralizing activities were preferentially transferred. Moreover, changes in IgG posttranslational glycosylation contributed more to cord than peripheral maternal blood antibody functional potency. These differences were enhanced with the combination of vaccination and infection as compared to either alone. Thus, Fc effector functions and antibody glycosylation highlight underexplored maternal opportunities to safeguard newborns.

Hemagglutinin stalk-specific Fc-mediated functions are associated with protection against influenza-illness after seasonal influenza vaccination in pregnant women.

BACKGROUND: Several influenza vaccine candidates aim to elicit antibodies against the conserved hemagglutinin stalk domain. Understanding the protective mechanism of these antibodies, which mediate broad neutralization and Fc-mediated functions, following seasonal vaccination is critical. METHODS: Plasma samples were obtained from a subset of pregnant women living with or without HIV-1 enrolled in a randomised trial (138 trivalent inactivated vaccine [TIV] and 145 placebo recipients). Twenty-three influenza-illness cases were confirmed within 6 months postpartum. We measured H1 stalk-specific antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD) and cellular cytotoxicity (ADCC) at enrolment and 1-month post-vaccination. The association between these Fc-mediated functions and protection against influenza-illness following vaccination was examined using multiple logistic regression analysis and risk reduction thresholds were defined by the score associated with the lowest odds of influenza-illness. RESULTS: Amongst TIV and placebo recipients, lower H1 stalk-specific ADCP and ADCD activity was detected for participants with confirmed influenza compared with individuals without confirmed influenza-illness 1-month post-vaccination. Pre-existing ADCP scores ≥250 reduced the odds of A/H1N1 infection (odds ratio 0.11; p=0.01) with an 83% likelihood of risk reduction. Following TIV, ADCD scores of ≥25 and ≥15 significantly reduced the odds against A/H1N1 (0.10; p=0.01) and non-group 1 (0.06; p=0.0004) influenza virus infections, respectively. These ADCD scores were associated with >84% likelihood of risk reduction. H1 stalk-specific ADCC potential was not associated with protection against influenza-illness. CONCLUSION: H1 stalk-specific ADCD correlates with protection against influenza-illness following influenza vaccination during pregnancy. These findings provide insight into the protective mechanisms of HA stalk antibodies.

Vaccine-induced antibody Fc-effector functions in humans immunized with a combination Ad26.RSV.preF/RSV preF protein vaccine.

IMPORTANCE: Respiratory syncytial virus (RSV) can cause serious illness in older adults (i.e., those aged ≥60 years). Because options for RSV prophylaxis and treatment are limited, the prevention of RSV-mediated illness in older adults remains an important unmet medical need. Data from prior studies suggest that Fc-effector functions are important for protection against RSV infection. In this work, we show that the investigational Ad26.RSV.preF/RSV preF protein vaccine induced Fc-effector functional immune responses in adults aged ≥60 years who were enrolled in a phase 1/2a regimen selection study of Ad26.RSV.preF/RSV preF protein. These results demonstrate the breadth of the immune responses induced by the Ad26.RSV.preF/RSV preF protein vaccine.

Research progress in Fc-effector functions against SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused more than 676 million cases in the global human population with approximately 7 million deaths and vaccination has been proved as the most effective countermeasure in reducing clinical complications and mortality rate of SARS-CoV-2 infection in people. However, the protective elements and correlation of protection induced by vaccination are still not completely understood. Various antibodies with multiple protective mechanisms can be induced simultaneously by vaccination in vivo, thereby complicating the identification and characterization of individual correlate of protection. Recently, an increasing body of observations suggests that antibody-induced Fc-effector functions play a crucial role in combating SARS-CoV-2 infections, including neutralizing antibodies-escaping variants. Here, we review the recent progress in understanding the impact of Fc-effector functions in broadly disarming SARS-CoV-2 infectivity and discuss various efforts in harnessing this conserved antibody function to develop an effective SARS-CoV-2 vaccine that can protect humans against infections by SARS-CoV-2 virus and its variants of concern.

Repeated COVID-19 mRNA vaccination results in IgG4 class switching and decreased NK cell activation by S1-specific antibodies in older adults.

BACKGROUND: Previous research has shown that repeated COVID-19 mRNA vaccination leads to a marked increase of SARS-CoV-2 spike-specific serum antibodies of the IgG4 subclass, indicating far-reaching immunoglobulin class switching after booster immunization. Considering that repeated vaccination has been recommended especially for older adults, the aim of this study was to investigate IgG subclass responses in the ageing population and assess their relation with Fc-mediated antibody effector functionality. RESULTS: Spike S1-specific IgG subclass concentrations (expressed in arbitrary units per mL), antibody-dependent NK cell activation, complement deposition and monocyte phagocytosis were quantified in serum from older adults (n = 38-50, 65-83 years) at one month post-second, -third and -fifth vaccination. Subclass distribution in serum was compared to that in younger adults (n = 64, 18-47 years) at one month post-second and -third vaccination. Compared to younger individuals, older adults showed increased levels of IgG2 and IgG4 at one month post-third vaccination (possibly related to factors other than age) and a further increase following a fifth dose. The capacity of specific serum antibodies to mediate NK cell activation and complement deposition relative to S1-specific total IgG concentrations decreased upon repeated vaccination. This decrease associated with an increased IgG4/IgG1 ratio. CONCLUSIONS: In conclusion, these findings show that, like younger individuals, older adults produce antibodies with reduced functional capacity upon repeated COVID-19 mRNA vaccination. Additional research is needed to better understand the mechanisms underlying these responses and their potential implications for vaccine effectiveness. Such knowledge is vital for the future design of optimal vaccination strategies in the ageing population.

Measles Vaccination Elicits a Polyfunctional Antibody Response, Which Decays More Rapidly in Early Vaccinated Children.

BACKGROUND: Measles outbreaks are reported worldwide and pose a serious threat, especially to young unvaccinated infants. Early measles vaccination given to infants under 12 months of age can induce protective antibody levels, but the long-term antibody functionalities are unknown. METHODS: Measles-specific antibody functionality was tested using a systems serology approach for children who received an early measles vaccination at 6-8 or 9-12 months, followed by a regular dose at 14 months of age, and children who only received the vaccination at 14 months. Antibody functionalities comprised complement deposition, cellular cytotoxicity, and neutrophil and cellular phagocytosis. We used Pearson's r correlations between all effector functions to investigate the coordination of the response. RESULTS: Children receiving early measles vaccination at 6-8 or 9-12 months of age show polyfunctional antibody responses. Despite significant lower levels of antibodies in these early-vaccinated children, Fc effector functions were comparable with regular-timed vaccinees at 14 months. However, 3-year follow-up revealed significant decreased polyfunctionality in children who received a first vaccination at 6-8 months of age, but not in children who received the early vaccination at 9-12 months. CONCLUSIONS: Antibodies elicited in early-vaccinated children are equally polyfunctional to those elicited from children who received vaccination at 14 months. However, these antibody functionalities decay more rapidly than those induced later in life, which may lead to suboptimal, long-term protection.

Fc-independent functions of anti-CTLA-4 antibodies contribute to anti-tumor efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T-lymphocyte associated protein 4 (CTLA-4), was the first immune checkpoint inhibitor approved by the FDA to treat metastatic melanoma patients. Multiple preclinical studies have proposed that Fc effector functions of anti-CTLA-4 therapy are required for anti-tumor efficacy, in part, through the depletion of intratumoral regulatory T cells (Tregs). However, the contribution of the Fc-independent functions of anti-CTLA-4 antibodies to the observed efficacy is not fully understood. H11, a non-Fc-containing single-domain antibody (VHH) against CTLA-4, has previously been demonstrated to block CTLA-4-ligand interaction. However, in vivo studies demonstrated lack of anti-tumor efficacy with H11 treatment. Here, we show that a half-life extended H11 (H11-HLE), despite the lack of Fc effector functions, induced potent anti-tumor efficacy in mouse syngeneic tumor models. In addition, a non-Fc receptor binding version of ipilimumab (Ipi-LALAPG) also demonstrated anti-tumor activity in the absence of Treg depletion. Thus, we demonstrate that Fc-independent functions of anti-CTLA-4 antibodies contributed to anti-tumor efficacy, which may indicate that non-Treg depleting activity of anti-CTLA-4 therapy could benefit cancer patients in the clinic.

Use of broadly neutralizing antibodies for HIV-1 prevention.

Antibodies have a long history in antiviral therapy, but until recently, they have not been actively pursued for HIV-1 due to modest potency and breadth of early human monoclonal antibodies (MAbs) and perceived insurmountable technical, financial, and logistical hurdles. Recent advances in the identification and characterization of MAbs with the ability to potently neutralize diverse HIV-1 isolates have reinvigorated discussion and testing of these products in humans, since new broadly neutralizing MAbs (bnMAbs) are more likely to be effective against worldwide strains of HIV-1. In animal models, there is abundant evidence that bnMAbs can block infection in a dose-dependent manner, and the more potent bnMAbs will allow clinical testing at infusion doses that are practically achievable. Moreover, recent advances in antibody engineering are providing further improvements in MAb potency, breadth, and half-life. This review summarizes the current state of the field of bnMAb protection in animal models as well as a review of variables that are critical for antiviral activity. Several bnMAbs are currently in clinical testing, and we offer perspectives on their use as pre-exposure prophylaxis (PrEP), potential benefits beyond sterilizing immunity, and a discussion of future approaches to engineer novel molecules.

Deciphering Fc-effector functions against SARS-CoV-2.

Major efforts were deployed to study the antibody response against SARS-CoV-2. Antibodies neutralizing SARS-CoV-2 have been extensively studied in the context of infections, vaccinations, and breakthrough infections. Antibodies, however, are pleiotropic proteins that have many functions in addition to neutralization. These include Fc-effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Although important to combat viral infections, these Fc-effector functions were less studied in the context of SARS-CoV-2 compared with binding and neutralization. This is partly due to the difficulty in developing reliable assays to measure Fc-effector functions compared to antibody binding and neutralization. Multiple assays have now been developed and can be used to measure different Fc-effector functions. Here, we review these assays and what is known regarding anti-SARS-CoV-2 Fc-effector functions. Overall, this review summarizes and updates our current state of knowledge regarding anti-SARS-CoV-2 Fc-effector functions.

Multivalent Rhamnose-Modified EGFR-Targeting Nanobody Gains Enhanced Innate Fc Effector Immunity and Overcomes Cetuximab Resistance via Recruitment of Endogenous Antibodies.

Cetuximab resistance is a significant challenge in cancer treatment, requiring the development of novel therapeutic strategies. In this study, a series of multivalent rhamnose (Rha)-modified nanobody conjugates are synthesized and their antitumor activities and their potential to overcome cetuximab resistance are investigated. Structure-activity relationship studies reveal that the multivalent conjugate D5, bearing sixteen Rha haptens, elicits the most potent innate fragment crystallizable (Fc) effector immunity in vitro and exhibits an excellent in vivo pharmacokinetics by recruiting endogenous antibodies. Notably, it is found that the optimal conjugate D5 represents a novel entity capable of reversing cetuximab-resistance induced by serine protease (PRSS). Moreover, in a xenograft mouse model, conjugate D5 exhibits significantly improved antitumor efficacy compared to unmodified nanobodies and cetuximab. The findings suggest that Rha-Nanobody (Nb) conjugates hold promise as a novel therapeutic strategy for the treatment of cetuximab-resistant tumors by enhancing the innate Fc effector immunity and enhancing the recruitment of endogenous antibodies to promote cancer cell clearance by innate immune cells.

Effector functions are required for broad and potent protection of neonatal mice with antibodies targeting HSV glycoprotein D.

Multiple failed herpes simplex virus (HSV) vaccine candidates induce robust neutralizing antibody (Ab) responses in clinical trials, raising the hypothesis that Fc-domain-dependent effector functions may be critical for protection. While neonatal HSV (nHSV) infection results in mortality and lifelong neurological morbidity in humans, it is uncommon among neonates with a seropositive birthing parent, supporting the hypothesis that Ab-based therapeutics could protect neonates from HSV. We therefore investigated the mechanisms of monoclonal Ab (mAb)-mediated protection in a mouse model of nHSV infection. For a panel of glycoprotein D (gD)-specific mAbs, neutralization and effector functions contributed to nHSV-1 protection. In contrast, effector functions alone were sufficient to protect against nHSV-2, exposing a functional dichotomy between virus types consistent with vaccine trial results. Effector functions are therefore crucial for protection by these gD-specific mAbs, informing effective Ab and vaccine design and demonstrating the potential of polyfunctional Abs as therapeutics for nHSV infections.

Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions.

Background: Fc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity. Methods: To explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions. Results: Immunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants. Conclusion: Depending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.

Antibody effector functions are required for broad and potent protection of neonates from herpes simplex virus infection.

The failure of multiple herpes simplex virus (HSV) vaccine candidates that induce neutralizing antibody responses raises the hypothesis that other activities, such as Fc domain-dependent effector functions, may be critical for protection. While neonatal HSV (nHSV) infection result in mortality and lifelong neurological morbidity in humans, it is uncommon among neonates with a seropositive birthing parent, suggesting the potential efficacy of antibody-based therapeutics to protect neonates. We therefore investigated the mechanisms of monoclonal antibody (mAb)-mediated protection in a mouse model of nHSV infection. Both neutralization and effector functions contributed to robust protection against nHSV-1. In contrast, effector functions alone were sufficient to protect against nHSV-2, exposing a functional dichotomy between virus types that is consistent with vaccine trial results. Together, these results emphasize that effector functions are crucial for optimal mAb-mediated protection, informing effective Ab and vaccine design, and demonstrating the potential of polyfunctional Abs as potent therapeutics for nHSV infections.

Despite delayed kinetics, people living with HIV achieve equivalent antibody function after SARS-CoV-2 infection or vaccination.

The kinetics of Fc-mediated functions following SARS-CoV-2 infection or vaccination in people living with HIV (PLWH) are not known. We compared SARS-CoV-2 spike-specific Fc functions, binding, and neutralization in PLWH and people without HIV (PWOH) during acute infection (without prior vaccination) with either the D614G or Beta variants of SARS-CoV-2, or vaccination with ChAdOx1 nCoV-19. Antiretroviral treatment (ART)-naïve PLWH had significantly lower levels of IgG binding, neutralization, and antibody-dependent cellular phagocytosis (ADCP) compared with PLWH on ART. The magnitude of antibody-dependent cellular cytotoxicity (ADCC), complement deposition (ADCD), and cellular trogocytosis (ADCT) was differentially triggered by D614G and Beta. The kinetics of spike IgG-binding antibodies, ADCC, and ADCD were similar, irrespective of the infecting variant between PWOH and PLWH overall. However, compared with PWOH, PLWH infected with D614G had delayed neutralization and ADCP. Furthermore, Beta infection resulted in delayed ADCT, regardless of HIV status. Despite these delays, we observed improved coordination between binding and neutralizing responses and Fc functions in PLWH. In contrast to D614G infection, binding responses in PLWH following ChAdOx-1 nCoV-19 vaccination were delayed, while neutralization and ADCP had similar timing of onset, but lower magnitude, and ADCC was significantly higher than in PWOH. Overall, despite delayed and differential kinetics, PLWH on ART develop comparable responses to PWOH, supporting the prioritization of ART rollout and SARS-CoV-2 vaccination in PLWH.

Sustained Antibody-Dependent NK Cell Functions in Mild COVID-19 Outpatients During Convalescence.

The coronavirus disease 2019 (COVID19) pandemic has left researchers scrambling to identify the humoral immune correlates of protection from COVID-19. To date, the antibody mediated correlates of virus neutralization have been extensively studied. However, the extent that non-neutralizing functions contribute to anti-viral responses are ill defined. In this study, we profiled the anti-spike antibody subtype/subclass responses, along with neutralization and antibody-dependent natural killer cell functions in 83 blood samples collected between 4 and 201 days post-symptoms onset from a cohort of COVID-19 outpatients. We observed heterogeneous humoral responses against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Overall, anti-spike profiles were characterized by a rapid rise of IgA and sustained IgG titers. In addition, strong antibody-mediated natural killer effector responses correlated with milder disease and being female. While higher neutralization profiles were observed in males along with increased severity. These results give an insight into the underlying function of antibodies beyond neutralization and suggest that antibody-mediated natural killer cell activity is a key function of the humoral response against the SARS-CoV-2 spike protein.

HIV-1 Vpu restricts Fc-mediated effector functions in vivo.

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell-surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We find that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC. Moreover, administration of nnAbs in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wild-type virus. CD4-mimetics administration, known to "open" Env and expose nnAb epitopes, renders wild-type viruses sensitive to nnAbs Fc-effector functions. This work highlights the importance of Vpu-mediated evasion of humoral responses.

Nucleocapsid-specific antibody function is associated with therapeutic benefits from COVID-19 convalescent plasma therapy.

Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP), a passive polyclonal antibody therapeutic agent, has had mixed clinical results. Although antibody neutralization is the predominant approach to benchmarking CCP efficacy, CCP may also influence the evolution of the endogenous antibody response. Using systems serology to comprehensively profile severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) functional antibodies of hospitalized people with COVID-19 enrolled in a randomized controlled trial of CCP (ClinicalTrials.gov: NCT04397757), we find that the clinical benefits of CCP are associated with a shift toward reduced inflammatory Spike (S) responses and enhanced nucleocapsid (N) humoral responses. We find that CCP has the greatest clinical benefit in participants with low pre-existing anti-SARS-CoV-2 antibody function and that CCP-induced immunomodulatory Fc glycan profiles and N immunodominant profiles persist for at least 2 months. We highlight a potential mechanism of action of CCP associated with durable immunomodulation, outline optimal patient characteristics for CCP treatment, and provide guidance for development of a different class of COVID-19 hyperinflammation-targeting antibody therapeutic agents.

BNT162b2-induced neutralizing and non-neutralizing antibody functions against SARS-CoV-2 diminish with age.

Each severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant renews concerns about decreased vaccine neutralization weakening efficacy. However, while prevention of infection varies, protection from disease remains and implicates immunity beyond neutralization in vaccine efficacy. Polyclonal antibodies function through Fab domains that neutralize virus and Fc domains that induce non-neutralizing responses via engagement of Fc receptors on immune cells. To understand how vaccines promote protection, we leverage sera from 51 SARS-CoV-2 uninfected individuals after two doses of the BNT162b2 mRNA vaccine. We show that neutralizing activities against clinical isolates of wild-type and five SARS-CoV-2 variants, including Omicron BA.2, link to FcγRIIIa/CD16 non-neutralizing effector functions. This is associated with post-translational afucosylation and sialylation of vaccine-specific antibodies. Further, polyfunctional neutralizing and non-neutralizing breadth, magnitude, and coordination diminish with age. Thus, studying Fc functions in addition to Fab-mediated neutralization provides greater insight into vaccine efficacy for vulnerable populations, such as the elderly, against SARS-CoV-2 and novel variants.