ABSTRACT
Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.
ABSTRACT
Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 [A]2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.
ABSTRACT
A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.
ABSTRACT
The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to [~]10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 [A]-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.
ABSTRACT
Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.
ABSTRACT
The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development we determined a 3.5 [A]-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.
