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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

Preexisting immunity against seasonal coronaviruses (CoVs) represents an important variable in predicting antibody responses and disease severity to severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infections. We used electron microscopy–based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against β-CoV spike proteins in prepandemic (PP) sera or SARS-CoV-2 convalescent (SC) sera. We observed that most PP sera had antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the SC sera showed reactivity across all human β-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by preexisting antibodies and SC serum antibodies. Our studies provide an antigenic landscape to β-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-CoV-2–infected individuals.

Objectives Understanding the nature of Mycobacterium leprae transmission is vital to implement better control strategies for leprosy elimination. The present study expands the knowledge of county-level strain diversity, distribution, and transmission patterns of leprosy in endemic provinces of China. Methods We genetically characterized 290 clinical isolates of M. leprae from four endemic provinces using variable number tandem repeats (VNTR) and single nucleotide polymorphisms (SNPs). Attained genetic profiles and cluster consequences were contrasted with geographical and migration features of leprosy at county levels. Results Considering the allelic variability of 17 VNTR loci by the discriminatory index, (GTA)9, (AT)17, (AT)15, (TA)18, (TTC)21, and (TA)10 are reported to be more highly polymorphic than other loci. The VNTR profile generated the low-density clustering pattern in the counties of Sichuan and Yunnan, whereas clusters have been observed from the isolates from Huayuan (N = 6), Yongding (N = 3), Zixing (N = 3), Chenxi (N = 2) and Zhongfang (N = 2) counties of Hunan, and Zhijin (N = 3), Anlong (N = 2), Zhenning (N = 2), and Xixiu (N = 2) counties of Guizhou. In some clusters, people’s social relations have been observed between villages. From the 290 clinical isolates, the most predominantly reported SNP was 3K (278, 95.8%), followed by SNP 1D (10, 3.4%), which are typically observed to be predominant in China. We also detected the novel SNP 3J (2, 0.8%), which has not yet been reported in China. Conclusion The clustering pattern of M. leprae indicates the transmission of leprosy still persists at county levels, suggesting that there is a need to implement better approaches for tracing the close contacts of leprosy patients.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8+ T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8+ T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8+ T cells were not recognized by VACV-reactive CD8+ T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R70–78 variant of the dominant VACV B8R70–78 epitope. C4R70–78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

Cancer vaccines targeting patient-specific neoantigens have emerged as a promising strategy for improving responses to immune checkpoint blockade. However, neoantigenic peptides are poorly immunogenic and inept at stimulating CD8(+) T cell responses, motivating a need for new vaccine technologies that enhance their immunogenicity. The stimulator of interferon genes (STING) pathway is an endogenous mechanism by which the innate immune system generates an immunological context for priming and mobilizing neoantigen-specific T cells. Owing to this critical role in tumor immune surveillance, a synthetic cancer nanovaccine platform (nanoSTING-vax) was developed that mimics immunogenic cancer cells in its capacity to efficiently promote co-delivery of peptide antigens and the STING agonist, cGAMP. The co-loading of cGAMP and peptides into pH-responsive, endosomolytic polymersomes promoted the coordinated delivery of both cGAMP and peptide antigens to the cytosol, thereby eliciting inflammatory cytokine production, costimulatory marker expression, and antigen cross-presentation. Consequently, nanoSTING-vax significantly enhanced CD8(+) T cell responses to a range of peptide antigens. Therapeutic immunization with nanoSTING-vax, in combination with immune checkpoint blockade, inhibited tumor growth in multiple murine tumor models, even leading to complete tumor rejection and generation of durable antitumor immune memory. Collectively, this work establishes nanoSTING-vax as a versatile platform for enhancing immune responses to neoantigen-targeted cancer vaccines.

Human immunodeficiency virus-tuberculosis (HIV-TB) co-infection poses a challenge to the immunologists in developing new diagnostic and therapeutic tools. Mechanisms behind the breakdown of the immune defense of the co-infected individual are poorly known. Numerous studies in HIV alone have revealed the role of PD1, TAP, and IL-10, but not in co-infection. The interaction of the 2 distinct bugs, which is resulting in domination over the host immune system, is still a lacuna. Hence, we aimed to portray functions of IL-10, TAP, and PD1 molecules in HIV-TB co-infection. Co-culture cells challenged with γ-irradiated M.Tb under various conditions resulted in high interleukin (IL)-10 secretion and high percentage of PD1 expression on CD8 T cells, which might be due to defective antigen presentation of TAP on dendritic cells and macrophages. Herein our observations provide an insight into the escape mechanisms by M.Tb in HIV-infected individuals from the host immune responses leading to TB co-infection.

Preexisting immunity against seasonal coronaviruses (CoV) represents an important variable in predicting antibody responses and disease severity to Severe Acute Respiratory Syndrome CoV-2 (SARS-2) infections. We used electron microscopy based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against β-CoV spike proteins in sera from healthy donors (HDs) or SARS-2 convalescent donors (CDs). We observed that most HDs possessed antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the CDs showed reactivity across all human β-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by antibodies in preexisting and convalescent serum. Our studies provide an antigenic landscape to β-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-2 -infected individuals.One-Sentence summaryWe present the epitope mapping of polyclonal antibodies against beta-coronavirus spike proteins in human sera.

Broadly reactive antibodies targeting the influenza A virus hemagglutinin (HA) head domain are thought to be rare and to require extensive somatic mutations or unusual structural features to achieve breadth against divergent HA subtypes. Here we describe common genetic and structural features of protective human antibodies from several individuals recognizing the trimer interface (TI) of the influenza A HA head, a recently identified site of vulnerability. We examined the sequence of TI-reactive antibodies, determined crystal structures for TI antibody-antigen complexes, and analyzed the contact residues of the antibodies on HA to discover common genetic and structural features of TI antibodies. Our data reveal that many TI antibodies are encoded by a light chain variable gene segment incorporating a shared somatic mutation. In addition, these antibodies have a shared acidic residue in the heavy chain despite originating from diverse heavy chain variable gene segments. These studies show that the TI region of influenza A HA is a major antigenic site with conserved structural features that are recognized by a common human B cell public clonotype. The canonical nature of this antibody-antigen interaction suggests that the TI epitope might serve as an important new target for structure-based vaccine design.

SUMMARYRNA-based vaccines against SARS-CoV-2 are critical to limiting COVID-19 severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. We used single-cell technologies to identify and characterized antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 in longitudinal samples from a cohort of healthy donors. Mass cytometry and machine learning pinpointed a novel expanding, population of antigen-specific non-canonical memory CD4+ and CD8+ T cells. B cell sequencing suggested progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlated with eventual SARS-CoV-2 IgG and a donor lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms reveal an antigen-specific cellular basis of RNA vaccine-based immunity.ONE SENTENCE SUMMARYSingle-cell profiling reveals the cellular basis of the antigen-specific response to the BNT162b2 SARS-CoV-2 RNA vaccine.

Tissue-resident memory T cells (TRM) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective TRM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting TRM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8+ TRM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8+ TRM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting TRM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8+ T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8+ TRM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of TRM-related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8+ TRM cells in the lungs.

Reports on antimicrobial resistance (AMR) of Mycobacterium leprae, relationship with bacteriological index (BI), and transmission in China are limited. We investigated the emergence of AMR ...

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody–ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen–ligand interactions.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis—and in some cases trogocytosis—but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies., Graphical abstract, Shiakolas et al. demonstrate that cross-reactive coronavirus antibodies induced by natural infection display a spectrum of epitope specificities across the spike protein and exhibit in vitro and in vivo antiviral functions.

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo), and others recognizing non-RBD epitopes that bound the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants., Graphical Abstract, Chen et al. describe neutralizing and non-neutralizing public antibodies elicited to SARS-CoV-2 spike in vaccinated and naturally infected individuals. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting common germline-encoded properties for recognition.

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies rEBOV-515 and rEBOV-442 that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity and resisted viral escape, and they were optimized for their Fc-mediated effector function activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes., Suryadevara et al. find neutralizing antibodies to the spike protein N-terminal domain that arise from natural infection with SARS-CoV-2. These antibodies inhibit post-attachment steps of the viral cycle and initiate protective immune responses via the antibody Fc domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 106 million people and causing 2.3 million deaths. The rapid deployment of antibody-based countermeasures has provided hope for curtailing disease and ending the pandemic1. However, the emergence of rapidly-spreading SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), and elsewhere with mutations in the spike protein has raised concern for escape from neutralizing antibody responses and loss of vaccine efficacy based on preliminary data with pseudoviruses2–4. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera, and human sera from recipients of the Pfizer-BioNTech (BNT162b2) mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, a chimeric Washington strain with a South African spike gene (Wash SA-B.1.351), and isogenic recombinant variants with designed mutations or deletions at positions 69–70, 417, 484, 501, and/or 614 of the spike protein. Several highly neutralizing mAbs engaging the receptor binding domain (RBD) or N-terminal domain (NTD) lost inhibitory activity against Wash SA-B.1.351 or recombinant variants with an E484K spike mutation. Most convalescent sera and virtually all mRNA vaccine-induced immune sera tested showed markedly diminished neutralizing activity against the Wash SA-B.1.351 strain or recombinant viruses containing mutations at position 484 and 501. We also noted that cell line selection used for growth of virus stocks or neutralization assays can impact the potency of antibodies against different SARS-CoV-2 variants, which has implications for assay standardization and congruence of results across laboratories. As several antibodies binding specific regions of the RBD and NTD show loss-of-neutralization potency in vitro against emerging variants, updated mAb cocktails, targeting of highly conserved regions, enhancement of mAb potency, or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 106 million people and causing 2.3 million deaths. The rapid deployment of antibody-based countermeasures has provided hope for curtailing disease and ending the pandemic1. However, the emergence of rapidly-spreading SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), and elsewhere with mutations in the spike protein has raised concern for escape from neutralizing antibody responses and loss of vaccine efficacy based on preliminary data with pseudoviruses2-4. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera, and human sera from recipients of the Pfizer-BioNTech (BNT162b2) mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, a chimeric Washington strain with a South African spike gene (Wash SA-B.1.351), and isogenic recombinant variants with designed mutations or deletions at positions 69-70, 417, 484, 501, and/or 614 of the spike protein. Several highly neutralizing mAbs engaging the receptor binding domain (RBD) or N-terminal domain (NTD) lost inhibitory activity against Wash SA-B.1.351 or recombinant variants with an E484K spike mutation. Most convalescent sera and virtually all mRNA vaccine-induced immune sera tested showed markedly diminished neutralizing activity against the Wash SA-B.1.351 strain or recombinant viruses containing mutations at position 484 and 501. We also noted that cell line selection used for growth of virus stocks or neutralization assays can impact the potency of antibodies against different SARS-CoV-2 variants, which has implications for assay standardization and congruence of results across laboratories. As several antibodies binding specific regions of the RBD and NTD show loss-of-neutralization potency in vitro against emerging variants, updated mAb cocktails, targeting of highly conserved regions, enhancement of mAb potency, or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

Most human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2 isolated to date recognize the spike (S) protein receptor-binding domain (RBD) and block virus interaction with the cellular receptor angiotensin converting enzyme 2 (ACE2). We isolated a panel of human mAbs binding to diverse epitopes on the spike protein N-terminal domain (NTD) from SARS-CoV2 convalescent donors and found that a minority of these possessed neutralizing activity. Two mAbs (designated COV2-2489 and COV2-2676) inhibited infection of both authentic SARS-CoV-2 and recombinant chimeric VSV/SARS-CoV-2 reporter viruses. We mapped their binding epitopes by alanine scanning mutagenesis and selection of functional SARS-CoV-2 S protein variants with mutations in the NTD that conferred resistance to recombinant chimeric VSV/SARS-CoV-2 reporter virus. Mechanistic studies showed that the antibodies neutralize in part by inhibiting at a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 prevented infection and disease in mice when administered either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, a subset of human NTD-specific mAbs protects against infection using multiple functional attributes including neutralizing activity and Fc-mediated activities.

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels ofNr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearrangedVα14-Jα18TCR-α chain gene into the Nur77tg(Nur77tg;Vα14tg) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4–producing NKT2 cell subset but not IFN-γ–producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions.

SUMMARYBroadly reactive antibodies targeting the influenza A hemagglutinin (HA) head domain are thought to be rare and to require extensive somatic mutations or unusual structural features to achieve breadth against divergent HA subtypes. Here we describe common genetic and structural features of diverse human antibodies from several individuals recognizing the trimer interface (TI) of the influenza HA head, a recently identified site of vulnerability1–3. We examined the sequence of TI-reactive antibodies, determined crystal structures for TI antibody-antigen complexes, and analyzed the contact residues of the antibodies on HA to discover common genetic and structural features of TI antibodies. Our data reveal that many TI antibodies are encoded by a light chain variable gene segment incorporating a shared somatic mutation. In addition, these antibodies have a shared acidic residue in the heavy chain despite originating from diverse heavy chain variable gene segments. These studies show that the TI region of influenza HA is a major antigenic site with conserved structural features that are recognized by a common human B cell public clonotype. The canonical nature of this antibody-antigen interaction suggests that the TI epitope might serve as an important new target for structure-based vaccine design.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails—including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution., Graphical Abstract, Highlights • Develop system to map all SARS-CoV-2 RBD mutations that escape antibody binding • Escape maps predict which mutations emerge when virus grown in presence of antibody • Escape maps inform surveillance for possible antigenic evolution, Greaney et al. develop a method to map all mutations to the SARS-CoV-2 RBD that escape antibody binding and apply this method to 10 antibodies. The resulting escape maps predict which mutations arise when virus is grown in the presence of antibody and can inform the design of antibody therapeutics.

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.

Wide heterogeneity of disease course ranging from asymptomatic spread to respiratory failure and death has become a hallmark of the SARS-CoV-2 pandemic. While this clinical spectrum is well documented, its immunologic underpinnings are less clear. We have therefore, initiated studies of the B cell responses as they would participate in both early effector responses and in the initiation of memory formation. In terms of effector responses, we were particularly interested in the engagement and clinical correlates of the extra-follicular pathway (EF), we recently described in flaring SLE. In this systemic autoimmune disease, the EF pathway is initiated by newly activated naive B cell (aN) leading to large expansion of autoantibody-producing antibody-secreting cells through the generation of an epigenetically primed B cell precursor which are double negative (DN) for naive (IgD) and memory markers (CD27) and lacking expression of CXCR5 and CD21 (DN2). These highly activated D2 cells are also distinguished by high expression of CD11c and T-bet and are TLR7-driven. Both, TLR7-stimulation which is triggered by ssRNA and the central role played by their murine counterparts (typically characterized as Age-Associated B cells), in viral clearance, strongly supported the hypothesis that DN2 cells and the global EF pathway could be prominently engaged in COVID-19 patients. Also of note, EF B cell activation is particularly prominent in SLE patients of African-American ancestry, a population disproportionately represented in severe COVID-19. In this study we find that critically-ill patients with COVID-19 robustly upregulate constituents of the extrafollicular pathway, produce enormous numbers of antibody secreting cells, and lose unique transitional B cell populations that correlate with positive prognosis. This patient cluster associates tightly with biomarkers of poor outcomes and exhibits high rates of mortality. Thus, this B cell phenotype might serve as an immunological marker of severe COVID infection at early stages and could therefore identify a patient subset likely to benefit from targeted immunomodulatory therapy aimed at alleviating disease burden.

The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity1,2. From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type (wt) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD. Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBDand provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.

The COVID-19 pandemic revealed an urgent need for rapid profiling of neutralizing antibody responses and development of antibody therapeutics. The current Food and Drug Administration-approved serological tests do not measure antibody-mediated viral neutralization, and there is a need for standardized quantitative neutralization assays. We report a high-throughput two-step profiling approach for identifying neutralizing convalescent plasma. Screening and downselection for serum antibody binding to the receptor-binding domain are followed by quantitative neutralization testing using a chimeric vesicular stomatitis virus expressing spike protein of SARS-CoV-2 in a real-time cell analysis assay. This approach enables a predictive screening process for identifying plasma units that neutralize SARS-CoV-2. To calibrate antibody neutralizing activity in serum from convalescent plasma donors, we introduce a neutralizing antibody standard reagent composed of two human antibodies that neutralize SARS-CoV strains, including SARS-CoV-2 variants of concern. Our results provide a framework for establishing a standardized assessment of antibody-based interventions against COVID-19., Graphical abstract, Immunology; Virology

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

The emergence of SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs., Graphical Abstract, Kramer et al. demonstrate that antibody 54042-4 recognizes residues highly conserved across global SARS-CoV-2 isolates. Antibody 54042-4 potently neutralizes all known circulating variants of concern (VOCs), and could be developed as a clinical candidate to treat COVID-19 infection.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents. An analysis identifies human monoclonal antibodies that potently neutralize wild-type SARS-CoV-2 and protect animals from disease, including two that synergize in a cocktail, suggesting that these could be candidates for use as therapeutic agents for the treatment of COVID-19 in humans.

Type I natural killer T (NKT) cells are innate-like T lymphocytes that recognize glycolipid antigens presented by the MHC class I-like protein CD1d. Agonistic activation of NKT cells leads to rapid pro-inflammatory and immune modulatory cytokine and chemokine responses. This property of NKT cells, in conjunction with their interactions with antigen-presenting cells, controls downstream innate and adaptive immune responses against cancers and infectious diseases, as well as in several inflammatory disorders. NKT cell properties are acquired during development in the thymus and by interactions with the host microbial consortium in the gut, the nature of which can be influenced by NKT cells. This latter property, together with the role of the host microbiota in cancer therapy, necessitates a new perspective. Hence, this review provides an initial approach to understanding NKT cells from an ecological evolutionary developmental biology (eco-evo-devo) perspective.

Background Vitamin D Receptor ( VDR ) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. Objective To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. Methods The study group includes 404 participants of which 222 were leprosy patients (paucibacillary = 87, multibacillary = 135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. Results The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. Conclusion The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response.

CXCL-10 known as Interferon gamma-induced protein 10 (IP-10) or small-inducible cytokine 10 is a 8.7 kDa protein, which is secreted in response to IFN-γ by monocytes, endothelial cells and fi-broblasts. It has chemo-attraction for monocytes/macrophages, T cells, NK cells and dendritic cells in promotion of T cell adhesion to endothelial cells. In the present study, we investigated whether polymorphisms in CXCL-10 gene have any role in the manifestation of Tuberculous (TB) pleurisy. Two SNPs in CXCL-10 promoter region (﹣1447A > G and ﹣135G > A) were genotyped in patients with TB Pleurisy (n = 186), Pulmonary TB patients (n = 159) and healthy controls (n = 205) by PCR-RFLP. Disease associations were statistically analyzed by Fisher exact test. At the ﹣135G > A position, the frequencies of genotype GA and allele G were significantly high in TB pleurisy patients compared to healthy controls. While the frequencies of genotype AA and allele A were significantly low in TB pleurisy patients compared to healthy controls. The frequency of haplotype A-G with the combination of 1447A > G and ﹣135G > A was significantly high in TB pleurisy. Our results reveal that genotype GA and allele G at ﹣135G > A position were strongly associated with susceptibility to tuberculous pleurisy. The GA genotype may be a useful genetic marker for early detection of the disease in high risk individuals.

Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ 2 ) test and t -tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy.

Received 10 May 2013; revised 2 August 2013; accepted 5 August 2013Background: Polymorphisms in TLR4 may change the function of the protein and alter the efficiencyof immuneresponse of host to infection. The high relevance of host gene polymorphisms with outcome of Mycobacteriumleprae infection led us to study the genetic association of TLR4 G896A polymorphism in order to identify its riskamong contacts of affected leprosy patients.Methods and Results: For case-control study design a total of 628 individuals were recruited; 17 multicaseleprosy families which included 32 case-parent trios were considered for family-based study. Genotyping wasdone using PCR-RFLP method. In case-control study AA genotype was positively associated while GA genotypewas negatively associated with leprosy. In family based transmission disequilibrium test (TDT) analysis alleleG was found to be over transmitted to the affected individuals.Conclusion:Case-control studysuggeststhat homozygousAAgenotypemayconfersusceptibilityandheterozy-gousGAgenotypemayconferresistancetoleprosy,whilealleleAwasobservedtoincreaseriskandthatofalleleGmayconferresistancetoleprosy.Nostrongtransmissiondisequilibriumwasdetectedinfamily-basedTDTana-lysis, possibly due to lower numberof trios. In contrast to case-control data allele G was over transmitted to theaffected ones in TDTanalysis. To conclude, the frequencies of genotypes in household contacts were almost thesameasinleprosypatients,suggestingthatcontactswithAAgenotypemaybeathigherriskofleprosyandmaytherefore require prophylactic inputs.Keywords: TLR4, Polymorphism, Contacts, PCR-RFLP, Leprosy

Background Toll-like receptor 2 (TLR2) is critical in bringing up immune responses to mycobacterial infections. The mutations in TLR2 are known to confer susceptibility for severe infection with mycobacteria. TLR2 may diminish response to mycobacterial proteins and place individuals at risk of developing leprosy. We investigated the association of GT repeat polymorphism in intron2 of TLR2 gene with leprosy in south Indian patients.

Interleukin (IL-10), an anti-inflammatory cytokine, is known to have dual effect on the host immune system. One of these roles is that it provides an effective autoregulatory mechanism which protects the host from excessive inflammation and tissue damage which is in part initiated by the Th1 driven pro-inflammatory immune responses during infections (such as TB, HIV and malaria). However, though beneficial, this autoregulatory mechanism is at times exploited by pathogens which evade elimination by Th1 driven immune response leading to chronic infections. The main aim of this study therefore was to study the influence of IL-10 polymorphism in relation to its levels with respect to HIV-TB co-infection. A total of 452 participants were categorized into HIV (121), active tuberculosis (TB) (118), HIV-TB (HT) (106) groups and healthy control group (107). Polymorphism for IL-10 gene (positions -1082, -819, -592) was studied using ARMS-PCR, RFLP. IL-10 and IFN-γ levels in antigen stimulated cultures were measured using ELISA. Statistical analysis was performed using Chi-Square ( χ 2 ) test, One-way ANOVA and t -tests. IL-10 ( -1082 ) GG genotype was positively associated with HIV-TB, whereas AG with HIV and AA with TB. The cohort with GG genotype also had significantly high stimulated levels of IL-10 compared to AG and AA . AC genotype was significantly frequent in HIV-TB group at IL-10 (-592) position when compared with controls. HIV positive individuals with GG genotype at IL-10 (-1082) position and high IL-10 levels may have a high risk of developing TB co-infection.