Adaptive advantage of deletion repair in the N terminal domain of the SARS-CoV-2 spike protein in variants of concern (preprint)

Mutations within the N-terminal domain (NTD) of the spike (S) protein play a pivotal role in the emergence of successful SARS-CoV-2 viral lineages. This study investigates the influence of novel combinations of NTD lineage-defining mutations found in the Alpha, Delta, and Omicron variants on viral success. We performed comparative genomics of more than 10 million public SARS-CoV-2 samples to decipher the transmission success of different NTD markers. Additionally, we characterized the viral phenotype of such markers in a surrogate in vitro system. We found that viruses bearing repaired deletions SDeltaH69/V70 and SDeltaY144 in Alpha background were associated with increased transmission rates. After the emergence of the Omicron BA.1 lineage, Alpha viruses harbouring both repaired deletions still showed increased transmission compared to their BA.1 counterparts. Remarkably, Alpha viruses with the SDeltaH69/V70 repair displayed the highest emergence rate, while those in BA.1 exhibited the lowest. Moreover, repaired deletions were more frequently observed among older individuals infected with Alpha, but not with BA.1. In vitro biological characterization of Omicron BA.1 spike deletion repair patterns revealed substantial differences with Alpha. In BA.1, SDeltaV143/Y145 repair enhanced fusogenicity and susceptibility to neutralization by vaccinated individuals' sera. In contrast, the SDeltaH69/V70 repair did not significantly alter these traits but reduced viral infectivity. Simultaneous repair of both deletions led to lower fusogenicity. These findings highlight the intricate genotype-phenotype landscape of the spike NTD in SARS-CoV-2, which impacts viral biology, transmission efficiency, and susceptibility to neutralization. Overall, this study advances our comprehension of SARS-CoV-2 evolution, carrying implications for public health and future research.

Cell type-specific adaptation of the SARS-CoV-2 spike (preprint)

SARS-CoV-2 can infect various human tissues and cell types, principally via interaction with its cognate receptor ACE2. However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After 20 passages, cell type-specific phenotypic changes were observed. Selected spike mutations were identified and functionally characterized in terms of entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, entry pathway and syncytia formation. We found that the effects of these mutations varied across cell types. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins (preprint)

Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety levels containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses and highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native as well as recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

Cumulative incidence of SARS-CoV-2 infection in the general population of the Valencian Community (Spain) after the surge of the Omicron BA.1 variant (preprint)

Background Studies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision- making. Methods The current cross-sectional, population-based study, conducted in April 2022 in the Valencian Community (VC), recruited 935 participants of all ages. Anti-SARS-CoV-2-Receptor Binding Domain-RBD- total antibodies and anti-Nucleocapsid (N)- IgGs were measured by electrochemiluminescence assays. To account for past SARS-CoV-2 infection the VC microbiology registry (RedMiVa) was interrogated. Quantitation of neutralizing antibodies (NtAb) against the ancestral and Omicron BA.1 and BA.2 (sub)variants by an S-pseudotyped neutralization assay and for enumeration of SARS-CoV-2-S specific-IFNgamma;-producing CD4+ and CD8+ T cells by Intracellular Cytokine Staining assay was performed in a subset of participants (n=100 and 137, respectively). Findings The weighted cumulative incidence was 51.9% (95% CI, 48.7-55.1), and was inversely related to age. Anti-RBD total antibodies were detected in 906/931 (97.3%) participants, those vaccinated and SARS-CoV-2-experienced (VAC-ex;=442) displaying higher levels (P<0.001) than vaccinated/naive (VAC-n;(n=472) and non-vaccinated/experienced (UNVAC-ex; n(n=63). Antibody levels correlated inversely with the time elapsed since receipt of last vaccine dose in VAC-n (Rho, -0.52; 95% CI, -0.59 to -0.45; P<0.001) but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75% and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively, while in 97%, 84% and 40%, against Omicron BA.2. SARS-CoV-2-S-reactive IFNgamma; T cells were detected in 73%, 75%, and 64% for VAC-ex, VAC-n, UNVAC-ex, respectively. Interpretation By April 2022 around half of the VC population had been infected with SARS-CoV-2 and due to extensive vaccination display hybrid immunity. The large percentage of participants with detectable functional antibody and T-cell responses against SARS-CoV-2, which may be cross-reactive to some extent, points towards lower expected severity than in previous waves.

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation (preprint)

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

Neutralizing antibodies against SARS-CoV-2 variants of concern elicited by the Comirnaty COVID-19 vaccine in nursing home residents (preprint)

Immunosenescence may impact the functionality and breadth of vaccine-elicited humoral immune responses. The ability of sera to neutralize the SARS-CoV-2 spike protein (S) from Beta, Gamma, Delta, and Epsilon variants of concern (VOCs) relative to the ancestral Wuhan-Hu-1 strain was compared in Comirnaty COVID-19-vaccinated elderly nursing home residents (n=30) or younger individuals (n=18) and non-vaccinated individuals who recovered from severe COVID-19 (n=19). In all groups, some participants lacked NtAb against one or more VOCs, mainly the Beta variant (15-20%). Serum NtAb titers were lowest against the Beta variant followed by Gamma, Epsilon, and Delta variants. Fold change reduction in NtAb titers relative to the ancestral strain was greatest for the Beta variant (6.7-18.8) followed by Gamma (3.6-6.2), Epsilon (2.9-5.8), and Delta (3.5-4.3) variants, regardless of the study group considered. In summary, older age, frailty, and concurrence of co-morbidities had no impact on the serum NtAb activity profile against SARS-CoV-2 VOCs.

Evolutionary and phenotypic characterization of spike mutations in a new SARS-CoV-2 Lineage reveals two Variants of Interest (preprint)

Molecular epidemiology of SARS-CoV-2 aims to monitor the appearance of new variants with the potential to change the virulence or transmissibility of the virus. During the first year of SARS-CoV-2 evolution, numerous variants with possible public health impact have emerged. We have detected two mutations in the Spike protein at amino acid positions 1163 and 1167 that have appeared independently multiple times in different genetic backgrounds, indicating they may increase viral fitness. Interestingly, the majority of these sequences appear in transmission clusters, with the genotype encoding mutations at both positions increasing in frequency more than single-site mutants. This genetic outcome that we denote as Lineage B.1.177.637, belongs to clade 20E and includes 12 additional single nucleotide polymorphisms but no deletions with respect to the reference genome (first sequence in Wuhan). B.1.177.637 appeared after the first wave of the epidemic in Spain, and subsequently spread to eight additional countries, increasing in frequency among sequences in public databases. Positions 1163 and 1167 of the Spike protein are situated in the HR2 domain, which is implicated in the fusion of the host and viral membranes. To better understand the effect of these mutations on the virus, we examined whether B.1.177.637 altered infectivity, thermal stability, or antibody sensitivity. Unexpectedly, we observed reduced infectivity of this variant relative to the ancestral 20E variant in vitro while the levels of viral RNA in nasopharyngeal swabs did not vary significantly. In addition, we found the mutations do not impact thermal stability or antibody susceptibility in vaccinated individuals but display a moderate reduction in sensitivity to neutralization by convalescent sera from early stages of the pandemic. Altogether, this lineage could be considered a Variant of Interest (VOI), we denote VOI1163.7. Finally, we detected a sub-cluster of sequences within VOI1163.7 that have acquired two additional changes previously associated with antibody escape and it could be identified as VOI1163.7.V2. Overall, we have detected the spread of a new Spike variant that may be advantageous to the virus and whose continuous transmission poses risks by the acquisition of additional mutations that could affect pre-existing immunity.

Suitability of Two Rapid Lateral Flow Immunochromatographic Assays for Predicting SARS-CoV-2 Neutralizing Activity of Sera (preprint)

PurposeAssessment of commercial SARS-CoV-2 immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially-available lateral flow immunochromatographic assays (LFIC) (Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for COVID-19 diagnosis in hospitalized patients, and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Patients and MethodsNinety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). ResultsOverall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%,93.9% and 93.9%, respectively). Reactivities equal to or more intense than the positive control line ([≥]2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb50 titers ([≥]1/160). ConclusionsOur findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb50 titers.

Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays (preprint)

BackgroundWhether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. ObjectivesTo evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and MethodsNinety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. ResultsOverall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay ({kappa}, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho = 0.73) and moderate for the remaining assays (Rho = 0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. ConclusionsThe suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

SARS-CoV-2 antibodies, serum inflammatory biomarkers and clinical severity of hospitalized COVID-19 Patients (preprint)

Background: The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. Patients and Methods: This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers were measured in a GFP reporter-based pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts. Results: The overall correlation between levels of both antibody measurements was good (Rho=0.79; P=0<0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). The percentage of patients who exhibited high NtAb50 titers ([≥]160) was similar (P=0.20) in ICU (79%) and non-ICU (60%) patients. Four ICU patients died; two of these achieved NtAb50 titers [≥]1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0-<0.2) or weak (Rho=>0.2-<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers. Conclusions: The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity