Structural insights into protection against a SARS-CoV-2 spike variant by T cell receptor diversity.

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell immunity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2 T cell epitopes elicit clonally restricted CD8+ T cell responses characterized by T cell receptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2-restricted spike protein epitope (RLQ) that elicits CD8+ T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recognizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ-HLA-A2 and T1006I-HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants.

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.

An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking.

ß-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors.

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.

Structural and energetic profiling of SARS-CoV-2 receptor binding domain antibody recognition and the impact of circulating variants.

The SARS-CoV-2 pandemic highlights the need for a detailed molecular understanding of protective antibody responses. This is underscored by the emergence and spread of SARS-CoV-2 variants, including Alpha (B.1.1.7) and Delta (B.1.617.2), some of which appear to be less effectively targeted by current monoclonal antibodies and vaccines. Here we report a high resolution and comprehensive map of antibody recognition of the SARS-CoV-2 spike receptor binding domain (RBD), which is the target of most neutralizing antibodies, using computational structural analysis. With a dataset of nonredundant experimentally determined antibody-RBD structures, we classified antibodies by RBD residue binding determinants using unsupervised clustering. We also identified the energetic and conservation features of epitope residues and assessed the capacity of viral variant mutations to disrupt antibody recognition, revealing sets of antibodies predicted to effectively target recently described viral variants. This detailed structure-based reference of antibody RBD recognition signatures can inform therapeutic and vaccine design strategies.

Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development.

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

CoV3D: a database of high resolution coronavirus protein structures.

SARS-CoV-2, the etiologic agent of COVID-19, exemplifies the general threat to global health posed by coronaviruses. The urgent need for effective vaccines and therapies is leading to a rapid rise in the number of high resolution structures of SARS-CoV-2 proteins that collectively reveal a map of virus vulnerabilities. To assist structure-based design of vaccines and therapeutics against SARS-CoV-2 and other coronaviruses, we have developed CoV3D, a database and resource for coronavirus protein structures, which is updated on a weekly basis. CoV3D provides users with comprehensive sets of structures of coronavirus proteins and their complexes with antibodies, receptors, and small molecules. Integrated molecular viewers allow users to visualize structures of the spike glycoprotein, which is the major target of neutralizing antibodies and vaccine design efforts, as well as sets of spike-antibody complexes, spike sequence variability, and known polymorphisms. In order to aid structure-based design and analysis of the spike glycoprotein, CoV3D permits visualization and download of spike structures with modeled N-glycosylation at known glycan sites, and contains structure-based classification of spike conformations, generated by unsupervised clustering. CoV3D can serve the research community as a centralized reference and resource for spike and other coronavirus protein structures, and is available at: https://cov3d.ibbr.umd.edu.