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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., (© 2024. The Author(s).)

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.

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. T H 1 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., (© 2024 The Authors.)

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., Competing Interests: Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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., (© 2023 The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Author MD is a consultant for Inbios, Vir Biotechnology, Moderna, and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Emergent BioSolutions, and Moderna. Authors GB and AB are employees and shareholders of Zepteon. The Glycap-3A resin and method are subject to issued and pending patents US20130084648, WO20123013193. Author GA is an employee and shareholder in Moderna. GA is a founder and equity holder in Seroymx Systems and Leyden Labs. GA has collaborative agreements with Sanaria, Merck, Abbvie, Medicago, Sanofi, BMS, Moderna, BioNtech, Gilead, SK Biosciences, GlaxoSmithKline, and Novavax and has served as an advisor for Sanofi Vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fox, Roy, Gunn, Bolton, Fremont, Alter, Diamond and Boesch.)

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., Competing Interests: Declaration of interests S.D., M.E., B.-A.C., and J.K.S. are employees of Merck & Co., T.J.S. is an employee of SeromYx, Inc., and G.A. is the founder of SeromYx, Inc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022. Published by Elsevier Ltd.)

Competing Interests: PP is an employee of Lava Therapeutics, a publicly traded biotechnology company that develops therapeutic antibodies including bispecific antibodies and bispecific antibody technology. He obtains stock options as part of his employment.

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.

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., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: SB and NC are employees of Abterra Biosciences. JL, ArG, ED, and BD are employees of Integral Molecular. GA is a founder of Seromyx Systems Inc. LZ is employer and co-owner of Mapp Biopharmaceutical. BD is a shareholder of Integral Molecular. AdG was an employee of Mapp Biopharmaceutical. JC has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gilchuk, Guthals, Bonissone, Shaw, Ilinykh, Huang, Bombardi, Liang, Grinyo, Davidson, Chen, Gunn, Alter, Saphire, Doranz, Bukreyev, Zeitlin, Castellana and Crowe.)

Although substantial progress has been made with Ebola virus (EBOV) vaccine measures, the immune correlates of vaccine-mediated protection remain uncertain. Here, five mucosal vaccine vectors based on human and avian paramyxoviruses provided nonhuman primates with varying degrees of protection, despite expressing the same EBOV glycoprotein (GP) immunogen. Each vaccine produced antibody responses that differed in Fc-mediated functions and isotype composition, as well as in magnitude and coverage toward GP and its conformational and linear epitopes. Differences in the degree of protection and comprehensive characterization of the response afforded the opportunity to identify which features and functions were elevated in survivors and could therefore serve as vaccine correlates of protection. Pairwise network correlation analysis of 139 immune- and vaccine-related parameters was performed to demonstrate relationships with survival. Total GP-specific antibodies, as measured by biolayer interferometry, but not neutralizing IgG or IgA titers, correlated with survival. Fc-mediated functions and the amount of receptor binding domain antibodies were associated with improved survival outcomes, alluding to the protective mechanisms of these vaccines. Therefore, functional qualities of the antibody response, particularly Fc-mediated effects and GP specificity, rather than simply magnitude of the response, appear central to vaccine-induced protection against EBOV. The heterogeneity of the response profile between the vaccines indicates that each vaccine likely exhibits its own protective signature and the requirements for an efficacious EBOV vaccine are complex., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Neutrophil activation and the formation of neutrophil extracellular traps (NETs) are hallmarks of innate immune activation in systemic lupus erythematosus (SLE). Here we report that the expression of an endogenous retrovirus (ERV) locus ERV-K102, encoding an envelope protein, was significantly elevated in SLE patient blood and correlated with autoantibody levels and higher interferon status. Induction of ERV-K102 in SLE negatively correlated with the expression of epigenetic silencing factors. Anti-ERV-K102 IgG levels in SLE plasma correlated with higher interferon stimulated gene expression, and further promoted enhanced neutrophil phagocytosis of ERV-K102 envelope protein through immune complex formation. Finally, phagocytosis of ERV-K102 immune complexes resulted in the formation of NETs consisting of DNA, neutrophil elastase, and citrullinated histone H3. Together, we identified an immunostimulatory ERV-K envelope protein that in an immune complex with SLE IgG is capable of activating neutrophils., Competing Interests: Disclosures: M.J. Townsend reported "other" from Genentech, a member of the Roche Group, outside the submitted work. G. Alter reported "other" from Seromyx Systems Inc. and grants from Pfizer, GSK, BMS, Merck, Gilead, Novavax, Janssen, Sanofi outside the submitted work; in addition, G. Alter had a patent to Systems Serology Platform pending. A. Iwasaki reported grants from AbbVie during the conduct of the study; "other" from RIGImmune; and personal fees from InProTher and Boehringer Ingelheim outside the submitted work. No other disclosures were reported., (© 2021 Tokuyama et al.)

Antibodies that mediate non-neutralizing functions play an important role in the immune response to Ebola virus (EBOV) and are thought to impact disease outcome. EBOV has also been associated with long term sequelae in survivors, however, the extent to which antibodies that mediate non-neutralizing functions are associated with the development of these sequelae is unknown. Here, the presence of antibodies mediating different effector functions and how they relate to long-term sequelae two years after the 2007 Bundibugyo Ebola virus (BDBV) outbreak was investigated. The majority of survivors demonstrated robust antibody effector functional activity and demonstrated persistent polyfunctional antibody profiles to the EBOV glycoprotein (GP) two years after infection. These functions were strongly associated with the levels of GP-specific IgG1. The odds of developing hearing loss, one of the more common sequelae to BDBV was reduced when antibodies mediating antibody dependent cellular phagocytosis (ADCP), antibody dependent complement deposition (ADCD), or activating NK cells (ADNKA) were observed. In addition, hearing loss was associated with increased levels of several pro-inflammatory cytokines and levels of these pro-inflammatory cytokines were associated with lower ADCP. These results are indicating that a skewed antibody profile and persistent inflammation may contribute to long term outcome in survivors of BDBV infection., Competing Interests: GA is a founder of SeroMyx Systems Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Paquin-Proulx, Gunn, Alrubayyi, Clark, Creegan, Kim, Kibuuka, Millard, Wakabi, Eller, Michael, Schoepp, Hepburn, Hensley, Robb, Alter and Eller.)

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design., Competing Interests: Declaration of interests G.A. is a founder of Seromyx Systems Inc., and T.J.S. is currently an employee of Seromyx Systems Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Typhoid Vi vaccines have been shown to be efficacious in children living in endemic regions; however, a widely accepted correlate of protection remains to be established. We applied a systems serology approach to identify Vi-specific serological correlates of protection using samples obtained from participants enrolled in an experimental controlled human infection study. Participants were vaccinated with Vi-tetanus toxoid conjugate (Vi-TT) or unconjugated Vi-polysaccharide (Vi-PS) vaccines and were subsequently challenged with Salmonella Typhi bacteria. Multivariate analyses identified distinct protective signatures for Vi-TT and Vi-PS vaccines in addition to shared features that predicted protection across both groups. Vi IgA quantity and avidity correlated with protection from S. Typhi infection, whereas higher fold increases in Vi IgG responses were associated with reduced disease severity. Targeted antibody-mediated functional responses, particularly neutrophil phagocytosis, were also identified as important components of the protective signature. These humoral markers could be used to evaluate and develop efficacious Vi-conjugate vaccines and assist with accelerating vaccine availability to typhoid-endemic regions., Competing Interests: Disclosures: C. Jin reported grants from the Bill and Melinda Gates Foundation and grants from European Commission FP7 Grant (Advanced Immunization Technologies) during the conduct of the study. K. Seaton reported grants from Duke University during the conduct of the study. G. Alter reported "other" from Seromyx Systems Inc outside the submitted work; in addition, G. Alter had a patent to Systems Serology Platform pending. A.J. Pollard reported grants from the Bill and Melinda Gates Foundation, the National Institute for Health Research, and the Wellcome Trust during the conduct of the study; is Chair of the UK Department of Health and Social Care's Joint Committee on Vaccination and Immunisation; and is a member of the World Health Organization's Strategic Advisory Group of Experts. A.J. Pollard is a National Institute for Health Research Senior Investigator. The views expressed in this article do not necessarily represent the views of the UK Department of Health and Social Care, Joint Committee on Vaccination and Immunisation, National Institute for Health Research, or World Health Organization. The University of Oxford has entered into a partnership with AstraZeneca on coronavirus vaccine development. No other disclosures were reported., (© 2020 Jin et al.)

Anthroponotic cutaneous leishmaniasis (CL) caused by Leishmania tropica (L. tropica) represents a public health challenge in several resource poor settings. We herein employed a systems analysis approach to study molecular signatures of CL caused by L. tropica in the skin lesions of ulcerative CL (UCL) and non-ulcerative CL (NUCL) patients. Results from RNA-seq analysis determined shared and unique functional transcriptional pathways in the lesions of the UCL and NUCL patients. Several transcriptional pathways involved in inflammatory response were positively enriched in the CL lesions. A multiplexed inflammatory protein analysis showed differential profiles of inflammatory cytokines and chemokines in the UCL and NUCL lesions. Transcriptional pathways for Fcγ receptor dependent phagocytosis were among shared enriched pathways. Using L. tropica specific antibody (Ab)-mediated phagocytosis assays, we could substantiate Ab-dependent cellular phagocytosis (ADCP) and Ab-dependent neutrophil phagocytosis (ADNP) activities in the lesions of the UCL and NUCL patients, which correlated with L. tropica specific IgG Abs. Interestingly, a negative correlation was observed between parasite load and L. tropica specific IgG/ADCP/ADNP in the skin lesions of CL patients. These results enhance our understanding of human skin response to CL caused by L. tropica.

We introduce a new vaccine platform against Marburg virus (MARV) combining the advantages of the immunogenicity of a highly attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). Our vaccine, MVA-MARV-VLP, expresses the minimal components of MARV VLPs: the envelope glycoprotein GP and the matrix protein VP40. Electron microscopy confirmed self-assembly and budding of VLPs from infected cells. Prime/boost vaccination of guinea pigs with MVA-MARV-VLP-elicited MARV-specific binding and neutralizing antibody responses. Vaccination also induced Fc-mediated innate immune effector functions including activation of NK cells and antibody-dependent phagocytosis by neutrophils and monocytes. Inoculation of vaccinated animals with guinea pig-adapted MARV demonstrated 100% protection against death and disease with no viremia. Therefore, our vaccine platform, expressing two antigens resulting in assembly of VLPs in the native conformation in vaccinated hosts, can be used as a potent vaccine against MARV., Competing Interests: COMPETING INTERESTSA.D., M.J.C., and F.G. has competing interest as being a full time employee of GeoVax. D.C.M., M.M., B.M.G., G.A., and A.B. declare that there are no competing interests., (© The Author(s) 2020.)

Three months since the detection of the first COVID-19 case in Africa, almost all countries of the continent continued to report lower morbidity and mortality than the global trend, including Europe and North America. We reviewed the merits of various hypotheses advanced to explain this phenomenon, including low seeding rate, effective mitigation measures, population that is more youthful, favorable weather, and possible prior exposure to a cross-reactive virus. Having a youthful population and favorable weather appears compelling, particularly their combined effect; however, progression of the pandemic in the region and globally may dispel these in the coming months.

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here, we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP, termed the wing region. One mAb targeting the GP2 wing, MR228, showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb, MR235, to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs, resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection., Competing Interests: Declaration of Interests P.A.I., J.E.C., and A.B. are listed as inventors on a submitted patent application, which covers antibodies described in the manuscript. J.E.C. has served as a consultant for Takeda Vaccines, Sanofi-Aventis U.S., Pfizer, and Novavax; is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines; and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Moderna, Sanofi-Aventis U.S., and IDBiologics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics., Competing Interests: Declaration of Interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Sanofi and is on the Scientific Advisory Boards of CompuVax and Meissa Vaccines, is a recipient of previous unrelated research grants from Moderna and Sanofi, and is founder of IDBiologics. Vanderbilt University has applied for a patent that is related to this work. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Monoclonal antibodies can mediate protection against Ebola virus (EBOV) infection through direct neutralization as well as through the recruitment of innate immune effector functions. However, the antibody functional response following survival of acute EBOV disease has not been well characterized. In this study, serum antibodies from Ebola virus disease (EVD) survivors from Sierra Leone were profiled to capture variation in overall subclass/isotype abundance, neutralizing activity, and innate immune effector functions. Antibodies from EVD survivors exhibited robust innate immune effector functions, mediated primarily by IgG1 and IgA1. In conclusion, development of functional antibodies follows survival of acute EVD., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Chikungunya virus (CHIKV) is an emerging mosquito-borne virus that has caused explosive outbreaks worldwide. Although neutralizing monoclonal antibodies (mAbs) against CHIKV inhibit infection in animals, the contribution of Fc effector functions to protection remains unknown. Here, we evaluated the activity of therapeutic mAbs that had or lacked the ability to engage complement and Fcγ receptors (FcγR). When administered as post-exposure therapy in mice, the Fc effector functions of mAbs promoted virus clearance from infected cells and reduced joint swelling-results that were corroborated in antibody-treated transgenic animals lacking activating FcγR. The control of CHIKV infection by antibody-FcγR engagement was associated with an accelerated influx of monocytes. A series of immune cell depletions revealed that therapeutic mAbs required monocytes for efficient clearance of CHIKV infection. Overall, our study suggests that in mice, FcγR expression on monocytes is required for optimal therapeutic activity of antibodies against CHIKV and likely other related viruses., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits, including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent vaccine immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV, and BDBV. As immunization with a cocktail of antigenically related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to sera from guinea pigs receiving the monovalent vaccines, sera from guinea pigs receiving the trivalent vaccine bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only a slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389 to 403, 397 to 415, and 477 to 493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus infection survivors demonstrated that the immune sera block the binding of antibodies specific for the GP glycan cap, the GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, administration of a cocktail of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus. IMPORTANCE The symptoms of the disease caused by the ebolaviruses Ebola, Bundibugyo, and Sudan are similar, and their areas of endemicity overlap. However, because of the limited antigenic relatedness of the ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous and not against heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, and this might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here, an in-depth analysis of the antibody responses to administration of a cocktail of human parainfluenza virus type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses compared to that induced by individual vaccines., (Copyright © 2019 American Society for Microbiology.)

Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134 AF , engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134 AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134 AF , a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134 AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134 AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent Ebola virus disease epidemics have highlighted the need for effective vaccines and therapeutics to prevent future outbreaks. Antibodies are clearly critical for control of this deadly disease; however, the specific mechanisms of action of protective antibodies have yet to be defined. In this Perspective we discuss the antibody features that correlate with in vivo protection during infection with Ebola virus, based on the results of a systematic and comprehensive study of antibodies directed against this virus. Although neutralization activity mediated by the Fab domains of the antibody is strongly correlated with protection, recruitment of immune effector functions by the Fc domain has also emerged as a complementary, and sometimes alternative, route to protection. For a subset of antibodies, Fc-mediated clearance and killing of infected cells seems to be the main driver of protection after exposure and mirrors observations in vaccination studies. Continued analysis of antibodies that achieve protection partially or wholly through Fc-mediated functions, the precise functions required, the intersection with specificity and the importance of these functions in different animal models is needed to identify and begin to capitalize on Fc-mediated protection in vaccines and therapeutics alike.

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interest: PAI, AIF, JECJ and AB hold a patent, which covers the antibodies described in the manuscript.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The recent Ebola virus (EBOV) epidemic highlighted the need for effective vaccines and therapeutics to limit and prevent outbreaks. Host antibodies against EBOV are critical for controlling disease, and recombinant monoclonal antibodies (mAbs) can protect from infection. However, antibodies mediate an array of antiviral functions including neutralization as well as engagement of Fc-domain receptors on immune cells, resulting in phagocytosis or NK cell-mediated killing of infected cells. Thus, to understand the antibody features mediating EBOV protection, we examined specific Fc features associated with protection using a library of EBOV-specific mAbs. Neutralization was strongly associated with therapeutic protection against EBOV. However, several neutralizing mAbs failed to protect, while several non-neutralizing or weakly neutralizing mAbs could protect. Antibody-mediated effector functions, including phagocytosis and NK cell activation, were associated with protection, particularly for antibodies with moderate neutralizing activity. This framework identifies functional correlates that can inform therapeutic and vaccine design strategies against EBOV and other pathogens., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses. IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses., (Copyright © 2018 Bricault et al.)

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis., (Copyright © 2017. Published by Elsevier Inc.)

Induction of pathogen-specific binding antibodies has long been considered a signature of protective immunity following vaccination and infection. The humoral immune response is a complex network of antibodies that target different specificities and drive different functions, collectively acting to limit and clear infection either directly, via pathogen neutralization, or indirectly, via pathogen clearance by the innate immune system. Emerging data suggest that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles. Here, we review the most recent advances in our understanding of protective functional antibody responses in natural infection, vaccination, and monoclonal antibody therapeutics. Moreover, we highlight opportunities to augment or modulate antibody-mediated protection through enhancement of antibody functionality., (Copyright © 2016. Published by Elsevier Ltd.)

Unlabelled: Ross River virus (RRV) is one of a group of mosquito-transmitted alphaviruses that cause debilitating, and often chronic, musculoskeletal disease in humans. Previously, we reported that replacement of the nonstructural protein 1 (nsP1) gene of the mouse-virulent RRV strain T48 with that from the mouse-avirulent strain DC5692 generated a virus that was attenuated in a mouse model of disease. Here we find that the six nsP1 nonsynonymous nucleotide differences between strains T48 and DC5692 are determinants of RRV virulence, and we identify two nonsynonymous nucleotide changes as sufficient for the attenuated phenotype. RRV T48 carrying the six nonsynonymous DC5692 nucleotide differences (RRV-T48-nsP1(6M)) was attenuated in both wild-type and Rag1(-/-) mice. Despite the attenuated phenotype, RRV T48 and RRV-T48-nsP1(6M) loads in tissues of wild-type and Rag1(-/-) mice were indistinguishable from 1 to 3 days postinoculation. RRV-T48-nsP1(6M) loads in skeletal muscle tissue, but not in other tissues, decreased dramatically by 5 days postinoculation in both wild-type and Rag1(-/-) mice, suggesting that the RRV-T48-nsP1(6M) mutant is more sensitive to innate antiviral effectors than RRV T48 in a tissue-specific manner. In vitro, we found that the attenuating mutations in nsP1 conferred enhanced sensitivity to type I interferon. In agreement with these findings, RRV T48 and RRV-T48-nsP1(6M) loads were similar in mice deficient in the type I interferon receptor. Our findings suggest that the type I IFN response controls RRV infection in a tissue-specific manner and that specific amino acid changes in nsP1 are determinants of RRV virulence by regulating the sensitivity of RRV to interferon., Importance: Arthritogenic alphaviruses, including Ross River virus (RRV), infect humans and cause debilitating pain and inflammation of the musculoskeletal system. In this study, we identified coding changes in the RRV nsP1 gene that control the virulence of RRV and its sensitivity to the antiviral type I interferon response, a major component of antiviral defense in mammals. Furthermore, our studies revealed that the effects of these attenuating mutations are tissue specific. These findings suggest that these mutations in nsP1 influence the sensitivity of RRV to type I interferon only in specific host tissues. The new knowledge gained from these studies contributes to our understanding of host responses that control alphavirus infection and viral determinants that counteract these responses.

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3(-/-) mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.

The viral determinants of alphavirus-induced rheumatic disease have not been elucidated. We identified an RRV strain (DC5692) which, in contrast to the T48 strain, does not induce musculoskeletal inflammation in a mouse model of RRV disease. Substitution of the RRV T48 strain nonstructural protein 1 (nsP1) coding sequence with that from strain DC5692 generated a virus that was attenuated in vivo despite similar viral loads in tissues. In contrast, substitution of the T48 PE2 coding region with the PE2 coding region from DC5692 resulted in attenuation in vivo and reduced viral loads in tissues. In gain of virulence experiments, substitution of the DC5692 strain nsP1 and PE2 coding regions with those from the T48 strain was sufficient to restore full virulence to the DC5692 strain. These findings indicate that determinants in both nsP1 and PE2 have critical and distinct roles in the pathogenesis of RRV-induced musculoskeletal inflammatory disease in mice., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chikungunya virus (CHIKV), an emerging mosquito-borne Alphavirus, causes debilitating rheumatic disease in humans that can last for weeks to months. Starting in 2004, a CHIKV outbreak in the Indian Ocean region affected millions of people, and infected travelers introduced CHIKV to new regions. The pathogenesis of CHIKV is poorly understood, and no approved vaccines or specific therapies exist. A major challenge to the study of CHIKV disease is the lack of a small animal model that recapitulates the major outcomes of human infection. In this study, the pathogenesis of CHIKV in C57BL/6J mice was investigated using biological and molecular clones of CHIKV isolated from human serum (CHIKV SL15649). After 14-day-old mice were inoculated with CHIKV SL15649 in the footpad, they displayed reduced weight gain and swelling of the inoculated limb. Histologic analysis of hind limb sections revealed severe necrotizing myositis, mixed inflammatory cell arthritis, chronic active tenosynovitis, and multifocal vasculitis. Interestingly, these disease signs and viral RNA persisted in musculoskeletal tissues for at least 3 weeks after inoculation. This work demonstrates the development of a mouse model of CHIKV infection with clinical manifestations and histopathologic findings that are consistent with the disease signs of CHIKV-infected humans, providing a useful tool for studying viral and host factors that drive CHIKV pathogenesis and for evaluating potential therapeutics against this emerging viral disease., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

Recombinant bacterial vaccines must be safe, efficacious, and well tolerated, especially when administered to newborns and infants to prevent diseases of early childhood. Many means of attenuation have been shown to render vaccine strains susceptible to host defenses or unable to colonize lymphoid tissue effectively, thus decreasing their immunogenicity. We have constructed recombinant attenuated Salmonella vaccine strains that display high levels of attenuation while retaining the ability to induce high levels of immunogenicity and are well tolerated in high doses when administered to infant mice as young as 24 h old. The strains contain three means of regulated delayed attenuation, as well as a constellation of additional mutations that aid in enhancing safety, regulate antigen expression, and reduce disease symptoms commonly associated with Salmonella infection. The vaccine strains are well tolerated when orally administered to infant mice 24 h old at doses as high as 3.5 x 10(8) CFU.

We are developing a Salmonella vectored vaccine to prevent infant pneumonia and other diseases caused by Streptococcus pneumoniae. One prerequisite for achieving this goal is to construct and evaluate new recombinant attenuated Salmonella vaccine (RASV) strains suitable for use in neonates and infants. Salmonella enterica serovar Typhimurium strain chi9558(pYA4088) specifies delivery of the pneumococcal protective antigen PspA and can protect adult mice from challenge with S. pneumoniae. This strain is completely safe for oral delivery to day-old and infant mice. Here we assess the colonizing ability, immunogenicity, and protective efficacy of chi9558(pYA4088) in neonatal mice. Colonization was assessed in mice 0, 2, 4, or 7 days of age after oral inoculation. In the presence of maternal antibodies, the colonization of lymphoid tissues was delayed, but the immune responses were enhanced in mice born to immunized mothers. Both oral and intranasal routes were used to assess immunogenicity. All orally or intranasally immunized neonatal and infant mice born to either immunized or naïve mothers developed PspA-specific mucosal and systemic immune responses. Mice born to immunized mothers produced higher titers of PspA-specific antibodies in the blood and mucosa and greater numbers of PspA-specific interleukin-4 (IL-4)-secreting cells than mice born to naïve mothers. More importantly, mice born to immune mothers showed a significant increase in protection against S. pneumoniae challenge. These results suggest that strain chi9558(pYA4088) can circumvent some of the limitations of the immature immune system in neonatal and infant mice, generating enhanced protective immune responses in the presence of maternal antibodies.

Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host's ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1(-/-) mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1(-/-) mice displayed similar organ burdens to wild-type mice. CX3CR1(-/-) mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects immune-cell recruitment; however, loss of CX3CR1 alone does not render the host more susceptible to M. tuberculosis or F. tularensis.