Antibodies targeting the glycan cap of Ebola virus glycoprotein are potent inducers of the complement system

Antibodies to Ebola virus glycoprotein (EBOV GP) represent an important correlate of the vaccine efficiency and infection survival. Both neutralization and some of the Fc-mediated effects are known to contribute the protection conferred by antibodies of various epitope specificities. At the same time, the role of the complement system remains unclear. Here, we compare complement activation by two groups of representative monoclonal antibodies (mAbs) interacting with the glycan cap (GC) or the membrane-proximal external region (MPER) of GP. Binding of GC-specific mAbs to GP induces complement-dependent cytotoxicity (CDC) in the GP-expressing cell line via C3 deposition on GP in contrast to MPER-specific mAbs. In the mouse model of EBOV infection, depletion of the complement system leads to an impairment of protection exerted by one of the GC-specific, but not MPER-specific mAbs. Our data suggest that activation of the complement system represents an important mechanism of antiviral protection by GC antibodies.

Antigen specificity shapes antibody functions in tuberculosis.

Tuberculosis (TB) is the number one infectious disease cause of death worldwide due to an incomplete understanding of immunity. Emerging data highlight antibody functions mediated by the Fc domain as immune correlates. However, the mechanisms by which antibody functions impact the causative agent Mycobacterium tuberculosis (Mtb) are unclear. Here, we examine how antigen specificity determined by the Fab domain shapes Fc effector functions against Mtb. Using the critical structural and secreted virulence proteins Mtb cell wall and ESAT-6 & CFP-10, we observe that antigen specificity alters subclass, antibody post-translational glycosylation, and Fc effector functions in TB patients. Moreover, Mtb cell wall IgG3 enhances disease through opsonophagocytosis of extracellular Mtb . In contrast, polyclonal and a human monoclonal IgG1 we generated targeting ESAT-6 & CFP-10 inhibit intracellular Mtb . These data show that antibodies have multiple roles in TB and antigen specificity is a critical determinant of the protective and pathogenic capacity.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.

Altered COVID-19 immunity in children with asthma by atopic status.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a spectrum of clinical outcomes that may be complicated by severe asthma. Antiviral immunity is often compromised in patients with asthma; however, whether this is true for SARS-CoV-2 immunity and children is unknown. Objective: We aimed to evaluate SARS-CoV-2 immunity in children with asthma on the basis of infection or vaccination history and compared to respiratory syncytial viral or allergen (eg, cockroach, dust mite)-specific immunity. Methods: Fifty-three children from an urban asthma study were evaluated for medical history, lung function, and virus- or allergen-specific immunity using antibody or T-cell assays. Results: Polyclonal antibody responses to spike were observed in most children from infection and/or vaccination history. Children with atopic asthma or high allergen-specific IgE, particularly to dust mites, exhibited reduced seroconversion, antibody magnitude, and SARS-CoV-2 virus neutralization after SARS-CoV-2 infection or vaccination. TH1 responses to SARS-CoV-2 and respiratory syncytial virus correlated with antigen-respective IgG. Cockroach-specific T-cell activation as well as IL-17A and IL-21 cytokines negatively correlated with SARS-CoV-2 antibodies and effector functions, distinct from total and dust mite IgE. Allergen-specific IgE and lack of vaccination were associated with recent health care utilization. Reduced lung function (forced expiratory volume in 1 second ≤ 80%) was independently associated with (SARS-CoV-2) peptide-induced cytokines, including IL-31, whereas poor asthma control was associated with cockroach-specific cytokine responses. Conclusion: Mechanisms underpinning atopic and nonatopic asthma may complicate the development of memory to SARS-CoV-2 infection or vaccination and lead to a higher risk of repeated infection in these children.

Divergent antibody recognition profiles are generated by protective mRNA vaccines against Marburg and Ravn viruses.

The first-ever recent Marburg virus (MARV) outbreak in Ghana, West Africa and Equatorial Guinea has refocused efforts towards the development of therapeutics since no vaccine or treatment has been approved. mRNA vaccines were proven successful in a pandemic-response to severe acute respiratory syndrome coronavirus-2, making it an appealing vaccine platform to target highly pathogenic emerging viruses. Here, 1-methyl-pseudouridine-modified mRNA vaccines formulated in lipid nanoparticles (LNP) were developed against MARV and the closely-related Ravn virus (RAVV), which were based on sequences of the glycoproteins (GP) of the two viruses. Vaccination of guinea pigs with both vaccines elicited robust binding and neutralizing antibodies and conferred complete protection against virus replication, disease and death. The study characterized antibody responses to identify disparities in the binding and functional profiles between the two viruses and regions in GP that are broadly reactive. For the first time, the glycan cap is highlighted as an immunoreactive site for marburgviruses, inducing both binding and neutralizing antibody responses that are dependent on the virus. Profiling the antibody responses against the two viruses provided an insight into how antigenic differences may affect the response towards conserved GP regions which would otherwise be predicted to be cross-reactive and has implications for the future design of broadly protective vaccines. The results support the use of mRNA-LNPs against pathogens of high consequence.

Passive antibody transfer from pregnant women to their fetus are maximized after SARS-CoV-2 vaccination irrespective of prior infection.

Background: Pregnancy is associated with a higher risk of adverse symptoms and outcomes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection for both mother and neonate. Antibodies can provide protection against SARS-CoV-2 infection and are induced in pregnant women after vaccination or infection. Passive transfer of these antibodies from mother to fetus in utero may provide protection to the neonate against infection. However, it is unclear whether the magnitude or quality and kinetics of maternally derived fetal antibodies differs in the context of maternal infection or vaccination. Objective: We aimed to determine whether antibodies transferred from maternal to fetus differed in quality or quantity between infection- or vaccination-induced humoral immune responses. Methods: We evaluated 93 paired maternal and neonatal umbilical cord blood plasma samples collected between October 2020 and February 2022 from a birth cohort of pregnant women from New Orleans, Louisiana, with histories of SARS-CoV-2 infection and/or vaccination. Plasma was profiled for the levels of spike-specific antibodies and induction of antiviral humoral immune functions, including neutralization and Fc-mediated innate immune effector functions. Responses were compared between 4 groups according to maternal infection and vaccination. Results: We found that SARS-CoV-2 vaccination or infection during pregnancy increased the levels of antiviral antibodies compared to naive subjects. Vaccinated mothers and cord samples had the highest anti-spike antibody levels and antiviral function independent of the time of vaccination during pregnancy. Conclusions: These results show that the most effective passive transfer of functional antibodies against SARS-CoV-2 in utero is achieved through vaccination, highlighting the importance of vaccination in pregnant women.

Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

Nearly one-half of patients with cystic fibrosis (CF) carry the homozygous F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene but exhibit variable lung function phenotypes. How adaptive immunity influences their lung function remains unclear, particularly the serological antibody responses to antigens from mucoid Pseudomonas in sera from patients with CF with varying lung function. Sera from patients with CF with reduced lung function show higher anti-outer membrane protein I (OprI) immunoglobulin G1 (IgG1) titers and greater antibody-mediated complement deposition. Induction of anti-OprI antibody isotypes with complement activity enhances lung inflammation in preclinical mouse models. This enhanced inflammation is absent in immunized Rag2-/- mice and is transferrable to unimmunized mice through sera. In a CF cohort undergoing treatment with elexacaftor-tezacaftor-ivacaftor, the declination in anti-OprI IgG1 titers is associated with lung function improvement and reduced hospitalizations. These findings suggest that antibody responses to specific Pseudomonas aeruginosa (PA) antigens worsen lung function in patients with CF.

Antiviral protection by antibodies targeting the glycan cap of Ebola virus glycoprotein requires activation of the complement system.

Antibodies to Ebola virus glycoprotein (EBOV GP) represent an important correlate of the vaccine efficiency and infection survival. Both neutralization and some of the Fc-mediated effects are known to contribute the protection conferred by antibodies of various epitope specificities. At the same time, the role of the complement system in antibody-mediated protection remains unclear. In this study, we compared complement activation by two groups of representative monoclonal antibodies (mAbs) interacting with the glycan cap (GC) or the membrane-proximal external region (MPER) of the viral sole glycoprotein GP. Binding of GC-specific mAbs to GP induced complement-dependent cytotoxicity (CDC) in the GP-expressing cell line via C3 deposition on GP in contrast to MPER-specific mAbs that did not. Moreover, treatment of cells with a glycosylation inhibitor increased the CDC activity, suggesting that N-linked glycans downregulate CDC. In the mouse model of EBOV infection, depletion of the complement system by cobra venom factor led to an impairment of protection exerted by GC-specific but not MPER-specific mAbs. Our data suggest that activation of the complement system is an essential component of antiviral protection by antibodies targeting GC of EBOV GP.

Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography.

Introduction: Chikungunya virus (CHIKV) is a re-emerging mosquito transmitted alphavirus of global concern. Neutralizing antibodies and antibody Fc-effector functions have been shown to reduce CHIKV disease and infection in animals. However, the ability to improve the therapeutic activity of CHIKV-specific polyclonal IgG by enhancing Fc-effector functions through modulation of IgG subclass and glycoforms remains unknown. Here, we evaluated the protective efficacy of CHIKV-immune IgG enriched for binding to Fc-gamma receptor IIIa (FcγRIIIa) to select for IgG with enhanced Fc effector functions. Methods: Total IgG was isolated from CHIKV-immune convalescent donors with and without additional purification by FcγRIIIa affinity chromatography. The enriched IgG was characterized in biophysical and biological assays and assessed for therapeutic efficacy during CHIKV infection in mice. Results: FcγRIIIa-column purification enriched for afucosylated IgG glycoforms. In vitro characterization showed the enriched CHIKV-immune IgG had enhanced human FcγRIIIa and mouse FcγRIV affinity and FcγR-mediated effector function without reducing virus neutralization in cellular assays. When administered as post-exposure therapy in mice, CHIKV-immune IgG enriched in afucosylated glycoforms promoted reduction in viral load. Discussion: Our study provides evidence that, in mice, increasing Fc engagement of FcγRs on effector cells, by leveraging FcγRIIIa-affinity chromatography, enhanced the antiviral activity of CHIKV-immune IgG and reveals a path to produce more effective therapeutics against these and potentially other emerging viruses.

Antibodies against the Ebola virus soluble glycoprotein are associated with long-term vaccine-mediated protection of non-human primates.

The 2013 Ebola epidemic in Central and West Africa heralded the emergence of wide-spread, highly pathogenic viruses. The successful recombinant vector vaccine against Ebola (rVSVΔG-ZEBOV-GP) will limit future outbreaks, but identifying mechanisms of protection is essential to protect the most vulnerable. Vaccine-induced antibodies are key determinants of vaccine efficacy, yet the mechanism by which vaccine-induced antibodies prevent Ebola infection remains elusive. Here, we exploit a break in long-term vaccine efficacy in non-human primates to identify predictors of protection. Using unbiased humoral profiling that captures neutralization and Fc-mediated functions, we find that antibodies specific for soluble glycoprotein (sGP) drive neutrophil-mediated phagocytosis and predict vaccine-mediated protection. Similarly, we show that protective sGP-specific monoclonal antibodies have elevated neutrophil-mediated phagocytic activity compared with non-protective antibodies, highlighting the importance of sGP in vaccine protection and monoclonal antibody therapeutics against Ebola virus.

Antibody-mediated protection against symptomatic COVID-19 can be achieved at low serum neutralizing titers.

Multiple studies of vaccinated and convalescent cohorts have demonstrated that serum neutralizing antibody (nAb) titers correlate with protection against coronavirus disease 2019 (COVID-19). However, the induction of multiple layers of immunity after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure has complicated the establishment of nAbs as a mechanistic correlate of protection (CoP) and hindered the definition of a protective nAb threshold. Here, we show that a half-life-extended monoclonal antibody (adintrevimab) provides about 50% protection against symptomatic COVID-19 in SARS-CoV-2-naïve adults at serum nAb titers on the order of 1:30. Vaccine modeling results support a similar 50% protective nAb threshold, suggesting that low titers of serum nAbs protect in both passive antibody prophylaxis and vaccination settings. Extrapolation of adintrevimab pharmacokinetic data suggests that protection against susceptible variants could be maintained for about 3 years. The results provide a benchmark for the selection of next-generation vaccine candidates and support the use of broad, long-acting monoclonal antibodies as alternatives or supplements to vaccination in high-risk populations.

Holding it together: Nanoparticle stabilization of LASV trimer.

The development of Lassa virus (LASV) vaccines has been challenging due to the instability of recombinant immunogens. In this issue of Cell Host & Microbe, Brouwer et al. use a two-component nanoparticle strategy that enables stable trimeric presentation of the LASV glycoprotein complex (GPC) and induction of broadly neutralizing antibodies.

Engineering the supernatural: monoclonal antibodies for challenging infectious diseases.

The COVID-19 pandemic demonstrated that monoclonal antibodies can be deployed faster than antimicrobials and vaccines. However, the majority of mAbs treat cancer and autoimmune diseases, whereas a minority treat infection. This is in part because targeting a single antigen by the antibody Fab domain is insufficient to stop the dynamic microbial life cycle. Thus, finding the 'right' antigens remains the focus of intense investigations. Equally important is the antibody-Fc domain that has the capacity to induce immune responses that enhance neutralization, and limit pathology and transmission. While Fc-effector functions have been less deeply studied, conceptual and technical advances reveal previously underappreciated antibody potential to combat diseases from microbes difficult to address with current diagnostics, therapeutics, and vaccines, including S. aureus, P. aeruginosa, P. falciparum, and M. tuberculosis. What is learned about engineering antibodies for these challenging organisms will enhance our approach to new and emerging infectious diseases.

Antibody-mediated protection against symptomatic COVID-19 can be achieved at low serum neutralizing titers.

Multiple studies of vaccinated and convalescent cohorts have demonstrated that serum neutralizing antibody (nAb) titers correlate with protection against COVID-19. However, the induction of multiple layers of immunity following SARS-CoV-2 exposure has complicated the establishment of nAbs as a mechanistic correlate of protection (CoP) and hindered the definition of a protective nAb threshold. Here, we show that a half-life extended monoclonal antibody (adintrevimab) provides approximately 50% protection against symptomatic COVID-19 in SARS-CoV-2-naive adults at low serum nAb titers on the order of 1:30. Vaccine modeling supports a similar 50% protective nAb threshold, suggesting low levels of serum nAb can protect in both monoclonal and polyclonal settings. Extrapolation of adintrevimab pharmacokinetic data suggests that protection against susceptible variants could be maintained for approximately 3 years. The results provide a benchmark for the selection of next-generation vaccine candidates and support the use of broad, long-acting monoclonal antibodies as an alternative or supplement to vaccination in high-risk populations.

An ACE2-dependent Sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines.

Spillover of sarbecoviruses from animals to humans has resulted in outbreaks of severe acute respiratory syndrome SARS-CoVs and the ongoing COVID-19 pandemic. Efforts to identify the origins of SARS-CoV-1 and -2 has resulted in the discovery of numerous animal sarbecoviruses-the majority of which are only distantly related to known human pathogens and do not infect human cells. The receptor binding domain (RBD) on sarbecoviruses engages receptor molecules on the host cell and mediates cell invasion. Here, we tested the receptor tropism and serological cross reactivity for RBDs from two sarbecoviruses found in Russian horseshoe bats. While these two viruses are in a viral lineage distinct from SARS-CoV-1 and -2, the RBD from one virus, Khosta 2, was capable of using human ACE2 to facilitate cell entry. Viral pseudotypes with a recombinant, SARS-CoV-2 spike encoding for the Khosta 2 RBD were resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Our findings further demonstrate that sarbecoviruses circulating in wildlife outside of Asia also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2.

Building a better antibody through the Fc: advances and challenges in harnessing antibody Fc effector functions for antiviral protection.

Antibodies can provide antiviral protection through neutralization and recruitment of innate effector functions through the Fc domain. While neutralization has long been appreciated for its role in antibody-mediated protection, a growing body of work indicates that the antibody Fc domain also significantly contributes to antiviral protection. Recruitment of innate immune cells such as natural killer cells, neutrophils, monocytes, macrophages, dendritic cells and the complement system by antibodies can lead to direct restriction of viral infection as well as promoting long-term antiviral immunity. Monoclonal antibody therapeutics against viruses are increasingly incorporating Fc-enhancing features to take advantage of the Fc domain, uncovering a surprising breadth of mechanisms through which antibodies can control viral infection. Here, we review the recent advances in our understanding of antibody-mediated innate immune effector functions in protection from viral infection and review the current approaches and challenges to effectively leverage innate immune cells via antibodies.

High seroprevalence of SARS-CoV-2 but low infection fatality ratio eight months after introduction in Nairobi, Kenya.

BACKGROUND: The lower than expected COVID-19 morbidity and mortality in Africa has been attributed to multiple factors, including weak surveillance. This study estimated the burden of SARS-CoV-2 infections eight months into the epidemic in Nairobi, Kenya. METHODS: A population-based, cross-sectional survey was conducted using multi-stage random sampling to select households within Nairobi in November 2020. Sera from consenting household members were tested for antibodies to SARS-CoV-2. Seroprevalence was estimated after adjusting for population structure and test performance. Infection fatality ratios (IFRs) were calculated by comparing study estimates with reported cases and deaths. RESULTS: Among 1,164 individuals, the adjusted seroprevalence was 34.7% (95% CI 31.8-37.6). Half of the enrolled households had at least one positive participant. Seropositivity increased in more densely populated areas (spearman's r=0.63; p=0.009). Individuals aged 20-59 years had at least two-fold higher seropositivity than those aged 0-9 years. The IFR was 40 per 100,000 infections, with individuals ≥60 years old having higher IFRs. CONCLUSION: Over one-third of Nairobi residents had been exposed to SARS-CoV-2 by November 2020, indicating extensive transmission. However, the IFR was >10-fold lower than that reported in Europe and the USA, supporting the perceived lower morbidity and mortality in sub-Saharan Africa.

Proteo-Genomic Analysis Identifies Two Major Sites of Vulnerability on Ebolavirus Glycoprotein for Neutralizing Antibodies in Convalescent Human Plasma.

Three clinically relevant ebolaviruses - Ebola (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) viruses, are responsible for severe disease and occasional deadly outbreaks in Africa. The largest Ebola virus disease (EVD) epidemic to date in 2013-2016 in West Africa highlighted the urgent need for countermeasures, leading to the development and FDA approval of the Ebola virus vaccine rVSV-ZEBOV (Ervebo®) in 2020 and two monoclonal antibody (mAb)-based therapeutics (Inmazeb® [atoltivimab, maftivimab, and odesivimab-ebgn] and Ebanga® (ansuvimab-zykl) in 2020. The humoral response plays an indispensable role in ebolavirus immunity, based on studies of mAbs isolated from the antibody genes in peripheral blood circulating ebolavirus-specific human memory B cells. However, antibodies in the body are not secreted by circulating memory B cells in the blood but rather principally by plasma cells in the bone marrow. Little is known about the protective polyclonal antibody responses in convalescent plasma. Here we exploited both single-cell antibody gene sequencing and proteomic sequencing approaches to assess the composition of the ebolavirus glycoprotein (GP)-reactive antibody repertoire in the plasma of an EVD survivor. We first identified 1,512 GP-specific mAb variable gene sequences from single cells in the memory B cell compartment. Using mass spectrometric analysis of the corresponding GP-specific plasma IgG, we found that only a portion of the large B cell antibody repertoire was represented in the plasma. Molecular and functional analysis of proteomics-identified mAbs revealed recognition of epitopes in three major antigenic sites - the GP head domain, the glycan cap, and the base region, with a high prevalence of neutralizing and protective mAb specificities that targeted the base and glycan cap regions on the GP. Polyclonal plasma antibodies from the survivor reacted broadly to EBOV, BDBV, and SUDV GP, while reactivity of the potently neutralizing mAbs we identified was limited mostly to the homologous EBOV GP. Together these results reveal a restricted diversity of neutralizing humoral response in which mAbs targeting two antigenic sites on GP - glycan cap and base - play a principal role in plasma-antibody-mediated protective immunity against EVD.