Molecular understanding of neutralizing antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could accelerate vaccine design and drug discovery. We analyzed 294 ...anti-SARS-CoV-2 antibodies and found that immunoglobulin G heavy-chain variable region 3-53 (IGHV3-53) is the most frequently used IGHV gene for targeting the receptor-binding domain (RBD) of the spike protein. Co-crystal structures of two IGHV3-53-neutralizing antibodies with RBD, with or without Fab CR3022, at 2.33- to 3.20-angstrom resolution revealed that the germline-encoded residues dominate recognition of the angiotensin I converting enzyme 2 (ACE2)-binding site. This binding mode limits the IGHV3-53 antibodies to short complementarity-determining region H3 loops but accommodates light-chain diversity. These IGHV3-53 antibodies show minimal affinity maturation and high potency, which is promising for vaccine design. Knowledge of these structural motifs and binding mode should facilitate the design of antigens that elicit this type of neutralizing response.
Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered ...participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.
Rational immunogen design aims to focus antibody responses to vulnerable sites on primary antigens. Given the size of these antigens, there is, however, potential for eliciting unwanted, off-target ...responses. Here, we use our electron microscopy polyclonal epitope mapping approach to describe the antibody specificities elicited by immunization of non-human primates with soluble HIV envelope trimers and subsequent repeated viral challenge. An increased diversity of epitopes recognized and the approach angle by which these antibodies bind constitute a hallmark of the humoral response in most protected animals. We also show that fusion peptide-specific antibodies are likely responsible for some neutralization breadth. Moreover, cryoelectron microscopy (cryo-EM) analysis of a fully protected animal reveals a high degree of clonality within a subset of putatively neutralizing antibodies, enabling a detailed molecular description of the antibody paratope. Our results provide important insights into the immune response against a vaccine candidate that entered into clinical trials in 2019.
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•Electron microscopy polyclonal epitope mapping of immunized and challenged macaques•Diversity of epitopes and specific angles of approach are hallmarks of protection•Neutralization breadth is due in part to fusion peptide-specific antibodies•Cryo-EM analysis of a protected animal details a putatively neutralizing paratope
Nogal et al. use electron microscopy polyclonal epitope mapping of BG505 Env-immunized and matched SHIVBG505-challenged non-human primates to identify hallmarks of protection. Additionally, cryo-EM polyclonal analysis of a fully protected animal reveals a high degree of clonality, allowing detailed characterization of a putative neutralizing paratope.
Thymocyte-expressed, positive selection-associated 1 (Tespa1) is important in T cell receptor (TCR)-driven thymocyte development. Downstream of the TCR, Tespa1 is a crucial component of the linker ...for activation of T cells (LAT) signalosome, facilitating calcium signalling and subsequent MAPK activation. However, it is unknown how Tespa1 elicits calcium signalling. Here, we show that inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is crucial for Tespa1-optimized, TCR-induced Ca
flux and thymocyte development. Upon TCR stimulation, Tespa1 directly interacts with IP3R1 and recruits it to the TCR complex, where IP3R1 is phosphorylated at Y353 by Fyn. This Tespa1-IP3R1 interaction is mediated by the F187 and F188 residues of Tespa1 and the amino-terminus of IP3R1. Tespa1-F187A/F188A mutant mice phenocopy Tespa1-deficient mice with impaired late thymocyte development due to reduced IP3R1 translocation to the TCR-proximal region. Our work elucidates the function of Tespa1 in T cell development and the regulation of TCR-induced Ca
signalling through IP3R1.
Nature Communications 8: Article number: 15732 (2017); Published: 9 June 2017; Updated: 12 March 2018 In the original version of this Article, the affiliation details for Yan Fang were incorrectly ...given as “ZJU-UoE Institute, Zhejiang University School of Medicine, Hangzhou 310058, China”. This has now been corrected in both the PDF and HTML versions of the Article.
The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein ...have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.
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•Cryo-EM analysis of Class 2 and Class 3 SARS-CoV-2 neutralizing monoclonal antibodies•Engineered antibodies with higher affinity neutralize emerging SARS-CoV-2 variants•The higher affinity antibodies reduce the pathways for viral escape•Engineered antibodies improve protection in hamster model
Immunology; Virology; Structural biology.
Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells and is partly caused by deficiencies in the Foxp3+ regulatory T‐cell (Treg) compartment. Conversely, therapies ...that increase Treg function can prevent autoimmune diabetes in animal models. The majority of Tregs develop in the thymus (tTregs), but a proportion of Foxp3+ Tregs is generated in the periphery (pTregs) from Foxp3−CD4+ T‐cell precursors. Whether pTregs play a distinct role in T1D has not yet been explored. We report here that pTregs are a key modifier of disease in the nonobese diabetic (NOD) mouse model for T1D. We generated NOD mice deficient for the Foxp3 enhancer CNS1 involved in pTreg induction. We show that CNS1 knockout decreased the frequency of pTregs and increased the risk of diabetes. Our results show that pTregs fulfill an important non‐redundant function in the prevention of beta cell autoimmunity that causes T1D.
NOD mice, susceptible to autoimmune diabetes, harbor regulatory T cells both of thymic origin (tTregs) and induced in the periphery (pTregs). We deleted the Foxp3 enhancer CNS1 in NOD mice to decrease the frequency of pTregs but not tTregs, and found that this increased the risk of diabetes.
HIV and SARS-CoV-2 infections have become major global pandemics, and the envelope protein on both viruses is the sole target of neutralizing antibody. Identification of (broadly) neutralizing ...antibodies have revealed vulnerable sites on HIV Env and SARS-CoV-2 spike proteins that help to guide rational immunogen design strategies to elicit (broadly) neutralizing antibody responses. This thesis firstly describes the isolation of HIV autologous monoclonal neutralizing antibodies from recombinant HIV Env trimer-immunized and naturally infected macaques. The epitope specificities are shared between immunized and infected animals, supporting the notion that the recombinant Env trimer is a reasonable mimic of the native trimer on virions. Autologous neutralizing antibodies might sterically hinder the elicitation of broadly neutralizing antibody responses, as their neutralization epitopes are close. The results highlight important considerations for HIV vaccine design and help to anticipate the outcomes of the Env trimer immunogen in clinical trials.The antibody-mediated protection trials indicate that broadly neutralizing antibodies can protect humans against HIV exposure, albeit at relatively high concentrations. This observation encourages interest in investigating whether lower antibody levels may be beneficial in the presence of cellular immunity. However, key cellular immune protection studies have been carried out in macaques using the virus SIVmac239 for which very few neutralizing antibodies are available. This thesis reports the isolation of neutralizing monoclonal antibodies from SIVmac239-infected macaques that facilitate high-resolution structural analysis of the SIVmac239 Env trimer. Passively administered neutralizing antibodies protect macaques against repeated SIVmac239 challenges, which provides the opportunity to investigate protective synergy with a combination of T cell vaccine and antibody-mediated protection.Finally, this thesis discovers and engineers neutralizing antibodies against SARS-CoV-2. Most of the neutralizing antibodies isolated from convalescent COVID-19 patients directly interact with receptor binding site on SARS-CoV-2 spike with low levels of somatic mutation. The antibodies are further affinity matured to increase neutralization potency and reduce viral escape pathways. Moreover, a SARS-CoV-1 neutralizing antibody is engineered to broaden neutralization to the closely related SARS-CoV-2 virus via directed evolution. Overall, discovery and engineering of neutralizing antibody against different viruses help to characterize anti-viral antibody features, which consequently guide for specific vaccine strategies and provide better candidates for antibody-mediated protection trial.
Coronaviruses have caused several human epidemics and pandemics including the ongoing coronavirus disease 2019 (COVID-19). Prophylactic vaccines and therapeutic antibodies have already shown striking ...effectiveness against COVID-19. Nevertheless, concerns remain about antigenic drift in SARS-CoV-2 as well as threats from other sarbecoviruses. Cross-neutralizing antibodies to SARS-related viruses provide opportunities to address such concerns. Here, we report on crystal structures of a cross-neutralizing antibody, CV38-142, in complex with the receptor-binding domains from SARS-CoV-2 and SARS-CoV. Recognition of the N343 glycosylation site and water-mediated interactions facilitate cross-reactivity of CV38-142 to SARS-related viruses, allowing the antibody to accommodate antigenic variation in these viruses. CV38-142 synergizes with other cross-neutralizing antibodies, notably COVA1-16, to enhance neutralization of SARS-CoV and SARS-CoV-2, including circulating variants of concern B.1.1.7 and B.1.351. Overall, this study provides valuable information for vaccine and therapeutic design to address current and future antigenic drift in SARS-CoV-2 and to protect against zoonotic SARS-related coronaviruses.
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•X-ray and EM structures of CV38-142 with SARS-CoV-2 and SARS-CoV RBDs and spikes•N343 glycan and bound waters facilitate cross-reactivity to SARS-related viruses•CV38-142 synergizes with CR3022 conserved site antibodies, in particular COVA1-16•These two cross-neutralizing antibodies neutralize SARS-CoV-2 variants of concern
Antibody CV38-142 from a COVID-19 patient cross-reacts with SARS-related viruses. Liu et al. reveal that CV38-142 interacts with the S309 N343 glycan site and synergizes with COVA1-16 that binds the conserved CR3022 site. Combination of the two cross-neutralizing antibodies shows enhanced neutralization to circulating variants of concern B.1.1.7 and B.1.351.
The potential emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) escape mutants is a threat to the efficacy of existing vaccines and neutralizing antibody (nAb) ...therapies. An understanding of the antibody/S escape mutation landscape is urgently needed to preemptively address this threat. Here we describe a rapid method to identify escape mutants for nAbs targeting the S receptor binding site. We identified escape mutants for five nAbs, including three from the public germline class VH3-53 elicited by natural coronavirus disease 2019 (COVID-19) infection. Escape mutations predominantly mapped to the periphery of the angiotensin-converting enzyme 2 (ACE2) recognition site on the RBD with K417, D420, Y421, F486, and Q493 as notable hotspots. We provide libraries, methods, and software as an openly available community resource to accelerate new therapeutic strategies against SARS-CoV-2.
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•Developed a method to identify antibody escape mutants on SARS-CoV-2 S RBD•Identified mutations that have been seen on emerging variants of concern•Identified vulnerabilities for antibody therapy escape, including D420 and Y421
Francino-Urdaniz et al. describe a deep mutational scanning method to identify escape mutants on SARS-CoV-2 S RBD. Escape mutants were identified for five neutralizing antibodies elicited from natural infection, revealing hotspots at positions K417, D420, Y421, F486, and Q493 at the periphery of the ACE2 recognition site.
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