SARS-CoV-2 variants Omicron BA.4/5 and XBB.1.5 significantly escape T cell recognition in solid organ transplant recipients vaccinated against the ancestral strain (preprint)

Background: Immune-suppressed solid organ transplant recipients (SOTRs) display impaired humoral responses to COVID-19 vaccination, but T cell responses are incompletely understood. The highly infectious SARS-CoV-2 variants Omicron BA.4/5 and XBB.1.5 escape neutralization by antibodies induced by vaccination or infection with earlier strains, but T cell recognition of these lineages in SOTRs is unclear. Methods: We characterized Spike-specific T cell responses to ancestral SARS-CoV-2, Omicron BA.4/5 and XBB.1.5 peptides in a prospective study of kidney, lung and liver transplant recipients (n = 42) throughout a three- or four-dose ancestral Spike mRNA vaccination schedule. Using an optimized activation-induced marker assay, we quantified circulating Spike-specific CD4+ and CD8+ T cells based on antigen-stimulated expression of CD134, CD69, CD25, CD137 and/or CD107a. Results: Vaccination strongly induced SARS-CoV-2-specific T cells, including BA.4/5- and XBB.1.5-reactive T cells, which remained detectable over time and further increased following a fourth dose. However, responses to Omicron BA.4/5 and XBB.1.5 were significantly lower in magnitude compared to ancestral strain responses. Antigen-specific CD4+ T cell frequencies correlated with anti-receptor-binding domain (RBD) antibody titres, with post-second dose T cell responses predicting subsequent antibody responses. Patients receiving prednisone, lung transplant recipients and older adults displayed weaker responses. Conclusions: Ancestral strain vaccination stimulates BA.4/5 and XBB.1.5-cross-reactive T cells in SOTRs, but responses to these variants are diminished. Antigen-specific T cells can predict future antibody responses and identify vaccine responses in seronegative individuals. Our data support monitoring both humoral and cellular immunity in SOTRs to track effectiveness of COVID-19 vaccines against emerging variants.

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains (preprint)

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

SARS-CoV-2 Omicron Variant: ACE2 Binding, Cryo-EM Structure of Spike Protein-ACE2 Complex and Antibody Evasion (preprint)

The newly reported Omicron variant is poised to replace Delta as the most rapidly spread SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, explaining our finding of similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion, with greater evasion observed in sera obtained from unvaccinated convalescent patients as compared to doubly vaccinated individuals (8- vs 3-fold). The retention of strong interactions at the ACE2 interface and the increase in antibody evasion are molecular factors that likely contribute to the increased transmissibility of the Omicron variant.

Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization (preprint)

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we report the discovery of a novel antibody fragment (V H ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants (preprint)

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding (preprint)

The recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-CoV-2 activation (preprint)

Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-coronavirus-2 (SARS-CoV-2). Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) Protein, enabling virus-host membrane fusion and infection of the lungs. We present here an efficient recombinant production strategy for enzymatically active TMPRSS2 ectodomain enabling enzymatic characterization, and the 1.95 A X-ray crystal structure. To stabilize the enzyme for co-crystallization, we pre-treated TMPRSS2 with the synthetic protease inhibitor nafamosat to form a stable but slowly reversible (15 hour half-life) phenylguanidino acyl-enzyme complex. Our study provides a structural basis for the potent but non-specific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that will guide future generations of inhibitors to improve selectivity. TMPRSS2 cleaved recombinant SARS-CoV-2 S protein ectodomain at the canonical S1/S2 cleavage site and at least two additional minor sites previously uncharacterized. We established enzymatic activity and inhibition assays that enabled ranking of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.7 nM to 120 uM and determination of inhibitor mechanisms of action. These results provide a body of data and reagents to support future drug development efforts to selectively inhibit TMPRSS2 and other type 2 transmembrane serine proteases involved in viral glycoprotein processing, in order to combat current and future viral threats.

Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody (preprint)

The recently reported ''UK variant'' of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. Here, we report cryo-EM structures of SARS-CoV-2 spike protein ectodomains with and without the N501Y mutation, in complex with the VH fragment of the potent neutralizing antibody, VH -Fc ab8. The mutation results in localized structural perturbations near Y501, but VH -Fc ab8 retains the ability to bind and neutralize pseudotyped viruses expressing the N501Y mutant with efficiencies comparable to that of unmutated viruses. Our results show that despite the higher affinity of ACE2 for the N501Y mutant, it can still be neutralized efficiently by an antibody that binds epitopes in the receptor binding domain of the SARS-CoV-2 spike protein.

Snake venom phospholipases A2 possess a strong virucidal activity against SARS-CoV-2 in vitro and block the cell fusion mediated by spike glycoprotein interaction with the ACE2 receptor (preprint)

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the absence of specific therapeutic and prophylactic agents, the virus has infected almost hundred million people, of whom nearly two million have died from the viral disease COVID-19. The ongoing COVID-19 pandemic is a global threat requiring new therapeutic strategies. Among them, antiviral studies based on natural molecules are a promising approach. The superfamily of phospholipases A2 (PLA2s) consists of a large number of members that catalyze the hydrolysis of phospholipids at a specific position. Here we show that secreted PLA2s from the venom of various snakes protect to varying degrees the Vero E6 cells widely used for the replication of viruses with evident cytopathic action, from SARS-CoV-2 infection PLA2s showed low cytotoxicity to Vero E6 cells and the high antiviral activity against SARS-CoV-2 with IC50 values ranged from 0.06 to 7.71 ug/ml. Dimeric PLA2 HDP-2 from the viper Vipera nikolskii, as well as its catalytic and inhibitory subunits, had potent virucidal (neutralizing) activity against SARS-CoV-2. Inactivation of the enzymatic activity of the catalytic subunit of dimeric PLA2 led to a significant decrease in antiviral activity. In addition, dimeric PLA2 inhibited cell-cell fusion mediated by SARS-CoV-2 spike glycoprotein. These results suggest that snake PLA2s, in particular dimeric ones, are promising candidates for the development of antiviral drugs that target lipid bilayers of the viral envelope and may be good tools to study the interaction of viruses with host cell membranes.

Novel RT-ddPCR assays for determining the transcriptional profile of SARS-CoV-2 (preprint)

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.

Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease (preprint)

The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

Glycan reactive anti-HIV-1 antibodies bind the SARS-CoV-2 spike protein but do not block viral entry (preprint)

The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via Fc{gamma}RII receptor engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate the ability for cross-reactive antibodies to interfere in immunoassays.