Neutralizing antibodies against Mayaro virus require Fc effector functions for protective activity

Despite causing outbreaks of fever and arthritis in multiple countries, no countermeasures exist against Mayaro virus (MAYV), an emerging mosquito-transmitted alphavirus. We generated 18 neutralizing mAbs against MAYV, 11 of which had “elite” activity that inhibited infection with EC50 values of <10 ng/ml. Antibodies with the greatest inhibitory capacity in cell culture mapped to epitopes near the fusion peptide of E1 and in domain B of the E2 glycoproteins. Unexpectedly, many of the elite neutralizing mAbs failed to prevent MAYV infection and disease in vivo. Instead, the most protective mAbs bound viral antigen on the cell surface with high avidity and promoted specific Fc effector functions, including phagocytosis by neutrophils and monocytes. In subclass switching studies, murine IgG2a and humanized IgG1 mAb variants controlled infection better than murine IgG1 and humanized IgG1-N297Q variants. An optimally protective antibody response to MAYV and possibly other alphaviruses may require tandem virus neutralization by the Fab moiety and effector functions of the Fc region.

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Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

mBio ◽

10.1128/mbio.01624-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 64

Author(s):

Caitlin E. Mullarkey ◽

Mark J. Bailey ◽

Diana A. Golubeva ◽

Gene S. Tan ◽

Raffael Nachbagauer ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Fc Receptor ◽

Igg Antibodies ◽

Specific Antibodies ◽

Effector Functions ◽

Iga Antibodies ◽

Specific Igg ◽

Optimal Protection ◽

Specific Igg Antibodies

ABSTRACTBroadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protectionin vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using anin vitroassay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.IMPORTANCEThe present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to neutralize, this class of antibodies has been shown to rely on Fc-Fc receptor interactions for optimal protectionin vivo. Curiously, neutralizing antibodies that bind the HA head domain do not require such interactions. Our findings build on these previous observations and provide a more complete picture of the relationship between stalk-specific antibodies and cells of the innate immune compartment. Furthermore, our data suggest that the ability of HA stalk-specific antibodies to mediate Fc-Fc receptor engagement is epitope dependent. Overall, this work will inform the rational design of improved influenza virus vaccines and therapeutics.

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Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609316113 ◽

2016 ◽

Vol 113 (42) ◽

pp. 11931-11936 ◽

Cited By ~ 88

Author(s):

Wenqian He ◽

Gene S. Tan ◽

Caitlin E. Mullarkey ◽

Amanda J. Lee ◽

Mannie Man Wai Lam ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Neutralizing Antibodies ◽

Influenza A ◽

Critical Role ◽

Host Cells ◽

Viral Glycoprotein ◽

Effector Functions ◽

Dependent Cell

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

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Mayaro Virus Induction of Oxidative Stress is Associated With Liver Pathology in a Non-Lethal Mouse Model

Scientific Reports ◽

10.1038/s41598-019-51713-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Camila Carla da Silva Caetano ◽

Fernanda Caetano Camini ◽

Letícia Trindade Almeida ◽

Ariane Coelho Ferraz ◽

Tales Fernando da Silva ◽

...

Keyword(s):

Oxidative Stress ◽

Neutralizing Antibodies ◽

Redox Homeostasis ◽

Antioxidant Compounds ◽

Viral Particles ◽

Lower Weight Gain ◽

Mayaro Virus ◽

High Virus ◽

A Site

Abstract Mayaro virus (MAYV) causes Mayaro fever in humans, a self-limiting acute disease, with persistent arthralgia and arthritis. Although MAYV has a remerging potential, its pathogenic mechanisms remain unclear. Here, we characterized a model of MAYV infection in 3–4-week BALB/c mice. We investigated whether the liver acts as a site of viral replication and if the infection could cause histopathological alterations and an imbalance in redox homeostasis, culminating with oxidative stress. MAYV-infected mice revealed lower weight gain; however, the disease was self-resolving. High virus titre, neutralizing antibodies, and increased levels of aspartate and alanine aminotransferases were detected in the serum. Infectious viral particles were recovered in the liver of infected animals and the histological examination of liver tissues revealed significant increase in the inflammatory infiltrate. MAYV induced significant oxidative stress in the liver of infected animals, as well as a deregulation of enzymatic antioxidant components. Collectively, this is the first study to report that oxidative stress occurs in MAYV infection in vivo, and that it may be crucial in virus pathogenesis. Future studies are warranted to address the alternative therapeutic strategies for Mayaro fever, such as those based on antioxidant compounds.

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Novel low-avidity glypican-3 specific CARTs resist exhaustion and mediate durable antitumor effects against hepatocellular carcinoma

10.1101/2021.07.09.451642 ◽

2021 ◽

Author(s):

Leidy Caraballo-Galva ◽

Mohamed Hussein ◽

Xiaotao Jiang ◽

Huajun Zhang ◽

Rui Mao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Solid Tumors ◽

High Avidity ◽

Antitumor Effects ◽

Tumor Mass ◽

Effector Functions ◽

Engineered T Cells ◽

Effector Cytokines

Chimeric antigen receptor engineered T cells (CARTs) are being developed to treat solid tumors including hepatocellular carcinoma (HCC). However, thus far, CARTs have not been as effective against solid tumors as compared to blood cancers. A main reason is that, once infiltrating into a solid tumor mass, CARTs are surrounded and chronically stimulated by persistent target antigens, which may drive them to exhaustion. We hypothesize that, due to weak engagement, low-avidity CARTs will resist the antigen-driven exhaustion and apoptosis and maintain effector functions in solid tumors, generating durable antitumor effects. To test this idea, we developed a novel human glypican-3 (hGPC3) specific antibody (8F8) that binds an epitope close to that of GC33 (the frequently used high-affinity antibody), but with ~17 folds lower affinity. In vitro, the low-avidity 8F8 CART killed tumor cells and produced effector cytokines to the same extent as high-avidity GC33 CART. Remarkably, however, 8F8 CART expanded and persisted to a greater extent than GC33 CART in vivo, resulting in durable responses against HCC xenografts. Compared to GC33 CARTs, there were significantly more (5 times) 8F8-BBz CART detected in the tumor mass. Importantly, the tumor infiltrating 8F8 CARTs were less apoptotic and more resistant to exhaustion, revealed by their enhanced and durable effector functions overtime. We predict that this novel low-avidity 8F8-BBz CART has a greater potential than mainstream high-avidity CARTs in effectively treating patients with HCC or other hGPC3+ solid tumors.

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An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice

10.1101/2021.09.08.459408 ◽

2021 ◽

Author(s):

Guillaume Beaudoin-Bussières ◽

Yaozong Chen ◽

Irfan Ullah ◽

Jérémie Prévost ◽

William D. Tolbert ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Animal Models ◽

Mouse Model ◽

Transgenic Mouse ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Transgenic Mouse Model ◽

Effector Function ◽

Effector Functions

SummaryEmerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

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Systematic assessment of antiviral potency, breadth, and synergy of triple broadly neutralizing antibody combinations against Simian-Human Immunodeficiency Viruses

Journal of Virology ◽

10.1128/jvi.01667-20 ◽

2020 ◽

Author(s):

Stella J Berendam ◽

Tiffany M Styles ◽

Papa K Morgan-Asiedu ◽

DeAnna Tenney ◽

Amit Kumar ◽

...

Keyword(s):

Antiviral Activity ◽

Neutralizing Antibodies ◽

Passive Immunization ◽

Neutralizing Antibody ◽

Systematic Assessment ◽

Effector Functions ◽

Cd4 Binding Site ◽

Human Immunodeficiency Viruses ◽

Antiviral Activities

Daily burden and clinical toxicities associated with antiretroviral therapy (ART) emphasize the need for alternative strategies to induce long-term HIV remission upon ART cessation. Broadly neutralizing antibodies (bNAbs) can both neutralize free virions and mediate effector functions against infected cells and therefore represent a leading immunotherapeutic approach. To increase potency and breadth as well as to limit the development of resistant virus strains, it is likely that bNAbs will need to be administered in combination. It is therefore critical to identify bNAb combinations that can achieve robust polyfunctional antiviral activity against a high number of HIV strains. In this study, we systematically assessed the ability of single bNAbs and triple bNAb combinations to mediate robust polyfunctional antiviral activity against a large panel of cross-clade simian-human immunodeficiency viruses (SHIVs), commonly used as tools for validation of therapeutic strategies targeting the HIV envelope in nonhuman primate models. We demonstrate that most bNAbs are capable of mediating both neutralizing and non-neutralizing effector functions against cross-clade SHIVs, although the susceptibility to V3 glycan-specific bNAbs is highly strain-dependent. Moreover, we observe a strong correlation between the neutralization potencies and non-neutralizing effector functions of bNAbs against the transmitted/founder SHIV CH505. Finally, we identify several triple bNAb combinations comprised of CD4 binding site, V2-glycan, and gp120-gp41 interface targeting bNAbs that are capable of mediating, synergistic polyfunctional antiviral activities against multiple clade A, B, C, and D SHIVs. IMPORTANCE Optimal bNAb immunotherapeutics will need to mediate multiple antiviral functions against a broad range of HIV strains. Our systematic assessment of triple bNAb combinations against SHIVs will identify bNAbs with synergistic, polyfunctional antiviral activity that will inform the selection of candidate bNAbs for optimal combination designs. The identified combinations can be validated in vivo in future passive immunization studies using the SHIV challenge model.

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

10.1101/2021.03.22.436337 ◽

2021 ◽

Author(s):

Irfan Ullah ◽

Jeremie Prevost ◽

Mark S. Ladinsky ◽

Helen Stone ◽

Maolin Lu ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Bioluminescence Imaging ◽

Inflammatory Responses ◽

Immune Protection ◽

In Vivo Efficacy ◽

Effector Functions ◽

Systemic Spread ◽

The Brain ◽

Established Infection

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 in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread 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 of infection. In addition to direct neutralization, in vivo efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal in vivo efficacy afforded by NAbs against SARS-CoV-2.

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A Novel Double Mosaic Virus-like Particle-Based Vaccine against SARS-CoV-2 Incorporates Both Receptor Binding Motif (RBM) and Fusion Domain

Vaccines ◽

10.3390/vaccines9111287 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1287

Author(s):

Xinyue Chang ◽

Andris Zeltins ◽

Mona O. Mohsen ◽

Zahra Gharailoo ◽

Lisha Zha ◽

...

Keyword(s):

Mosaic Virus ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Scale Up ◽

Fusion Peptide ◽

Binding Motif ◽

High Avidity ◽

Major Health Problem ◽

Bacterial Fermentation ◽

Virus Like Particle

COVID-19 has emerged, and has rapidly become a major health problem worldwide, causing millions of mortalities. Vaccination against COVID-19 is the most efficient way to stop the pandemic. The goal of vaccines is to induce neutralizing antibodies against SARS-CoV-2 virus. Here, we present a novel double mosaic virus-like particle (VLP) displaying two independent neutralizing epitopes, namely the receptor binding motif (RBM) located in S1 and the fusion peptide (AA 817–855) located in S2. CuMVTT virus-like particles were used as VLP scaffold and both domains were genetically fused in the middle of CuMVTT subunits, which co-assembled into double mosaic particles (CuMVTT-DF). A single fusion mosaic particle (CuMVTT-FP) containing the fusion peptide only was used for comparison. The vaccines were produced in E. coli, and electron microscopy and dynamic light scattering confirmed their integrity and homogeneity. In addition, the CuMVTT-DF vaccine was well recognized by ACE2 receptor, indicating that the RBM was in native conformation. Both CuMVTT-FP and CuMVTT-DF vaccines induced high levels of high avidity IgG antibodies as well as IgA recognizing spike and RBD in the case of CuMVTT-DF. Both vaccine candidates induced virus-neutralizing antibodies indicating that the fusion peptide can independently induce virus-neutralizing antibodies. In contrast, CuMVTT-DF containing both RBM and fusion peptide induced a higher level of neutralizing antibodies suggesting that the new double mosaic vaccine candidate CuMVTT-DF consisting of two antigens in one VLP maybe an attractive candidate for scale-up in a bacterial fermentation process for clinical development.

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Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection

10.1101/2020.12.28.424554 ◽

2020 ◽

Author(s):

Emma S. Winkler ◽

Pavlo Gilchuk ◽

Jinsheng Yu ◽

Adam L. Bailey ◽

Rita E. Chen ◽

...

Keyword(s):

Lung Disease ◽

Neutralizing Antibodies ◽

Immune Cell ◽

Transcriptional Profiling ◽

Therapeutic Benefit ◽

Loss Of Function ◽

Effector Functions ◽

Innate Immune Signaling ◽

Optimal Protection

SUMMARYSARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease.

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The Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing antibody, SC31, isolated from an early convalescent COVID-19 patient, are essential for the optimal therapeutic efficacy of the antibody

10.1101/2020.10.26.355107 ◽

2020 ◽

Author(s):

Conrad E.Z. Chan ◽

Shirley G.K. Seah ◽

De Hoe Chye ◽

Shane Massey ◽

Maricela Torres ◽

...

Keyword(s):

Viral Load ◽

Therapeutic Efficacy ◽

Neutralizing Antibodies ◽

Inflammatory Responses ◽

Systemic Disease ◽

Severe Disease ◽

Neutralizing Antibody ◽

Effector Functions ◽

Igg1 Antibody

AbstractSARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFNγ-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite FcγR binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development.One Sentence SummaryAnti-SARS-CoV-2 IgG1 antibody SC31 controls infection in vivo by blocking SP:ACE2 binding and triggering a Fc-mediated anti-viral response.

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