Molecular Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Isolates From Central Inner Sardinia

BackgroundThe SARS-CoV-2 pandemic stimulated an outstanding global sequencing effort, which allowed to monitor viral circulation and evolution. Nuoro province (Sardinia, Italy), characterized by a relatively isolated geographical location and a low population density, was severely hit and displayed a high incidence of infection.MethodsAmplicon approach Next Generation Sequencing and subsequent variant calling in 92 respiratory samples from SARS-CoV-2 infected patients involved in infection clusters from March 2020 to May 2021.ResultsPhylogenetic analysis displayed a coherent distribution of sequences in terms of lineage and temporal evolution of pandemic. Circulating lineage/clade characterization highlighted a growing diversity over time, with an increasingly growing number of mutations and variability of spike and nucleocapsid proteins, while viral RdRp appeared to be more conserved. A total of 384 different mutations were detected, of which 196 were missense and 147 synonymous ones. Mapping mutations along the viral genome showed an irregular distribution in key genes. S gene was the most mutated gene with missense and synonymous variants frequencies of 58.8 and 23.5%, respectively. Mutation rates were similar for the S and N genes with one mutation every ∼788 nucleotides and every ∼712 nucleotides, respectively. Nsp12 gene appeared to be more conserved, with one mutation every ∼1,270 nucleotides. The frequency of variant Y144F in the spike protein deviated from global values with higher prevalence of this mutation in the island.ConclusionThe analysis of the 92 viral genome highlighted evolution over time and identified which mutations are more widespread than others. The high number of sequences also permits the identification of subclusters that are characterized by subtle differences, not only in terms of lineage, which may be used to reconstruct transmission clusters. The disclosure of viral genetic diversity and timely identification of new variants is a useful tool to guide public health intervention measures.

Seroprevalence, waning, and correlates of anti-SARS-CoV-2 IgG antibodies in Tyrol, Austria: Large-scale study of 35,193 blood donors conducted between June 2020 and September 2021

Background: There is uncertainty about the seroprevalence of anti-SARS-CoV-2 antibodies in the general population of Austria, and about the extent to which antibodies elicited by vaccination or infection wane over time. Aim: To estimate seroprevalence, waning, and correlates of anti-SARS-CoV-2 IgG antibodies in the Federal State of Tyrol, Austria. Methods: We conducted a seroepidemiological study between June 2020 and September 2021, enrolling blood donors aged 18-70 years across Tyrol, Austria (participation rate 84.0%). We analysed serum samples for antibodies against spike or nucleocapsid proteins of SARS-CoV-2 with Abbott SARS-CoV-2 IgG assays. Results: We performed 47,363 serological tests among 35,193 individuals (median age 43.1 years [IQR: 29.3-53.7], 45.3% women, 10.0% with prior SARS-CoV-2 infection). Seroprevalence increased from 3.4% (95% CI: 2.8-4.2%) in June 2020 to 82.7% (95% CI: 81.4-83.8%) in September 2021, largely due to vaccination. Anti-spike IgG seroprevalence was 99.6% (99.4-99.7%) among fully vaccinated individuals, 90.4% (88.8-91.7%) among unvaccinated with prior infection, and 11.5% (10.8-12.3%) among unvaccinated without known prior infection. Anti-spike IgG levels were reduced by 44.0% (34.9-51.7%) at 5-6 months compared to 0-3 months after infection. In fully vaccinated individuals, they decreased by 31.7% (29.4-33.9%) per month. In multivariable adjusted analyses, both seropositivity among unvaccinated and antibody levels among fully vaccinated individuals were higher at young age (<25 years), higher with a known prior infection, and lower in current smokers. Conclusion: Seroprevalence in Tyrol increased to 82.7% in September 2021, with the bulk of seropositivity stemming from vaccination. Antibody levels substantially and gradually declined after vaccination or infection.

Anti-SARS-CoV-2 Immunoglobulin Isotypes, and Neutralization Activity Against Viral Variants, According to BNT162b2-Vaccination and Infection History

PurposeTo compare SARS-CoV-2 antigen-specific antibody production and plasma neutralizing capacity against B.1 wild-type-like strain, and Gamma/P.1 and Delta/B.1.617.2 variants-of-concern, in subjects with different Covid-19 disease and vaccination histories.MethodsAdult subjects were: 1) Unvaccinated/hospitalized for Covid-19; 2) Covid-19-recovered followed by one BNT162b2 vaccine dose; and 3) Covid-19-naïve/2-dose BNT162b2 vaccinated. Multiplex Luminex® immunoassays measured IgG, IgA, and IgM plasma levels against SARS-CoV-2 receptor-binding domain (RBD), spike-1 (S), and nucleocapsid proteins. Neutralizing activity was determined in Vero E6 cytopathic assays.ResultsMaximum anti-RBD IgG levels were similar in Covid-19‑recovered individuals 8‒10 days after single-dose vaccination and in Covid-19-naïve subjects 7 days after 2nd vaccine dosing; both groups had ≈2‑fold higher anti-RBD IgG levels than Unvaccinated/Covid-19 subjects tracked through 2 weeks post-symptom onset. Anti-S IgG expression patterns were similar to RBD within each group, but with lower signal strengths. Viral antigen-specific IgA and IgM levels were more variable than IgG patterns. Anti-nucleocapsid immunoglobulins were not detected in Covid-19-naïve subjects. Neutralizing activity against the B.1 strain, and Gamma/P.1 and Delta/B.1.617.2 variants, was highest in Covid‑19-recovered/single-dose vaccinated subjects; although neutralization against the Delta variant in this group was only 26% compared to B.1 neutralization, absolute anti-Delta titers suggested maintained protection. Neutralizing titers against the Gamma and Delta variants were 33‒77% and 26‒67%, respectively, versus neutralization against the B.1 strain (100%) in the three groups.ConclusionThese findings support SARS-CoV-2 mRNA vaccine usefulness regardless of Covid-19 history, and confirm remarkable protection provided by a single vaccine dose in people who have recovered from Covid-19.

Identification of Conserved Epitopes in SARS-CoV-2 Spike and Nucleocapsid Protein

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus that first occurred in Wuhan in December 2019. The spike glycoproteins and nucleocapsid proteins are the most common targets for the development of vaccines and antiviral drugs. Objective: We herein analyze the rate of evolution along with the sequences of spike and nucleocapsid proteins in relation to the spatial locations of their epitopes, previously suggested to contribute to the immune response caused by SARS-CoV-2 infections. Methods: We compare homologous proteins of seven human coronaviruses: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2. We then focus on the local, structural order-disorder propensity of the protein regions where the SARS-CoV-2 epitopes are located. Results : We show that most of nucleocapsid protein epitopes overlap the RNA-binding and dimerization domains, and some of them are characterized by a low rate of evolutions. Similarly, spike protein epitopes are preferentially located in regions that are predicted to be ordered and well-conserved, in correspondence of the heptad repeats 1 and 2. Interestingly, both the receptor-binding motif to ACE2 and the fusion peptide of spike protein are characterized by a high rate of evolution. Conclusion: Our results provide evidence for conserved epitopes that might help develop broad-spectrum SARS-CoV-2 vaccines.

Single immunization with recombinant ACAM2000 vaccinia viruses expressing the spike and the nucleocapsid proteins protect hamsters against SARS-CoV-2 caused clinical disease

SUMMARYIncreasing cases of SARS-CoV-2 breakthrough infections from immunization with predominantly spike protein based COVID-19 vaccines highlight the need for alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins in a novel vaccinia virus ACAM2000 platform (rACAM2000). Following a single intramuscular immunization, the rACAM2000 co-expressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and quicker recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titre and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that the rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate and further studies will investigate if the rACAM2000 vaccine candidate can induce a long lasting immunity against infection of SARS-CoV-2 variants of concern.

Phylogenomics and population genomics of SARS-CoV-2 in Mexico during the pre-vaccination stage reveals variants of interest B.1.1.28.4 and B.1.1.222 or B.1.1.519 and the nucleocapsid mutation S194L associated with symptoms

Understanding the evolution of the SARS-CoV-2 virus in various regions of the world during the Covid-19 pandemic is essential to help mitigate the effects of this devastating disease. We describe the phylogenomic and population genetic patterns of the virus in Mexico during the pre-vaccination stage, including asymptomatic carriers. A real-time quantitative PCR screening and phylogenomic reconstructions directed at sequence/structure analysis of the spike glycoprotein revealed mutation of concern E484K in genomes from central Mexico, in addition to the nationwide prevalence of the imported variant 20C/S:452R (B.1.427/9). Overall, the detected variants in Mexico show spike protein mutations in the N-terminal domain (i.e. R190M), in the receptor-binding motif (i.e. T478K, E484K), within the S1–S2 subdomains (i.e. P681R/H, T732A), and at the basis of the protein, V1176F, raising concerns about the lack of phenotypic and clinical data available for the variants of interest we postulate: 20B/478K.V1 (B.1.1.222 or B.1.1.519) and 20B/P.4 (B.1.1.28.4). Moreover, the population patterns of single nucleotide variants from symptomatic and asymptomatic carriers obtained with a self-sampling scheme confirmed the presence of several fixed variants, and differences in allelic frequencies among localities. We identified the mutation N:S194L of the nucleocapsid protein associated with symptomatic patients. Phylogenetically, this mutation is frequent in Mexican sub-clades. Our results highlight the dual and complementary role of spike and nucleocapsid proteins in adaptive evolution of SARS-CoV-2 to their hosts and provide a baseline for specific follow-up of mutations of concern during the vaccination stage.

Conserved molecular signatures in the spike protein provide evidence indicating the origin of SARS-CoV-2 and a Pangolin-CoV (MP789) by recombination(s) between specific lineages of Sarbecoviruses

Both SARS-CoV-2 and SARS coronaviruses (CoVs) are members of the subgenus Sarbecovirus. To understand the origin of SARS-CoV-2, sequences for the spike and nucleocapsid proteins from sarbecoviruses were analyzed to identify molecular markers consisting of conserved inserts or deletions (termed CSIs) that are specific for either a particular clade of Sarbecovirus or are commonly shared by two or more clades of these viruses. Three novel CSIs in the N-terminal domain (NTD) of the spike protein S1-subunit (S1-NTD) are uniquely shared by SARS-CoV-2, Bat-CoV-RaTG13 and most pangolin CoVs (SARS-CoV-2r clade). Three other sarbecoviruses viz. bat-CoVZXC21, -CoVZC45 and -PrC31 (forming CoVZC/PrC31 clade), and a pangolin-CoV_MP789 also contain related CSIs in the same positions. In contrast to the S1-NTD, both SARS and SARS-CoV-2r viruses contain two large CSIs in the S1-C-terminal domain (S1-CTD) that are absent in the CoVZC/PrC31 clade. One of these CSIs, consisting of a 12 aa insert, is also present in the RShSTT clade (Cambodia-CoV strains). Sequence similarity studies show that the S1-NTD of SARS-CoV-2r viruses is most similar to the CoVZC/PrC31 clade, whereas their S1-CTD exhibits highest similarity to the RShSTT- (and the SARS-related) CoVs. Results from the shared presence of CSIs and sequence similarity studies on different CoV lineages support the inference that the SARS-CoV-2r cluster of viruses has originated by a genetic recombination between the S1-NTD of the CoVZC/PrC31 clade of CoVs and the S1-CTD of RShSTT/SARS viruses, respectively. We also present compelling evidence, based on the shared presence of CSIs and sequence similarity studies, that the pangolin-CoV_MP789, whose receptor-binding domain is most similar to the SARS-CoV-2 virus, has resulted from another independent recombination event involving the S1-NTD of the CoVZC/PrC31 CoVs and the S1-CTD of an unidentified SARS-CoV-2r related virus. The SARS-CoV-2 virus involved in this latter recombination event is postulated to be most similar to the SARS-CoV-2. Several other CSIs reported here are specific for other clusters of sarbecoviruses including a clade consisting of bat-SARS-CoVs (BM48-31/BGR/2008 and SARS_BtKY72). Structural mapping studies show that the identified CSIs form distinct loops/patches on the surface of the spike protein. It is hypothesized that these novel loops/patches on the spike protein, through their interactions with other host components, should play important roles in the biology/pathology of SARS-CoV-2 virus. Lastly, the CSIs specific for different clades of sarbecoviruses including SARS-CoV-2r clade provide novel means for the identification of these viruses and other potential applications.