Targets and cross-reactivity of human T cell recognition of Common Cold Coronaviruses (preprint)

The Coronavirus (CoV) family includes a variety of viruses able to infect humans. Endemic CoVs that can cause common cold belong to the alphaCoV and betaCoV genera, with the betaCoV genus also containing subgenera with zoonotic and pandemic concern, including sarbecoCoV (SARS-CoV and SARS-CoV-2) and merbecoCoV (MERS-CoV). It is therefore warranted to explore pan-CoV vaccine concepts, to provide adaptive immune protection against new potential CoV outbreaks, particularly in the context of betaCoV sub lineages. To explore the feasibility of eliciting CD4+ T cell responses widely cross-recognizing different CoVs, we utilized samples collected pre-pandemic to systematically analyze T cell reactivity against representative alpha (NL63) and beta (OC43) common cold CoVs (CCC). Similar to previous findings on SARS-CoV-2, the S, N, M, and nsp3 antigens were immunodominant for both viruses while nsp2 and nsp12 were immunodominant for NL63 and OC43, respectively. We next performed a comprehensive T cell epitope screen, identifying 78 OC43 and 87 NL63-specific epitopes. For a selected subset of 18 epitopes, we experimentally assessed the T cell capability to cross-recognize sequences from representative viruses belonging to alphaCoV, sarbecoCoV, and beta-non-sarbecoCoV groups. We found general conservation within the alpha and beta groups, with cross-reactivity experimentally detected in 89% of the instances associated with sequence conservation of >67%. However, despite sequence conservation, limited cross-reactivity was observed in the case of sarbecoCoV (50% of instances), indicating that previous CoV exposure to viruses phylogenetically closer to this subgenera is a contributing factor in determining cross-reactivity. Overall, these results provided critical insights in the development of future pan-CoV vaccines.

SARS-CoV-2 vaccination induces immunological memory able to cross-recognize variants from Alpha to Omicron (preprint)

We address whether T cell responses induced by different vaccine platforms (mRNA-1273, BNT162b2, Ad26.COV2.S, NVX-CoV2373) cross-recognize SARS-CoV-2 variants. Preservation of at least 83% and 85% for CD4+ and CD8+ T cell responses was found, respectively, regardless of vaccine platform or variants analyzed. By contrast, highly significant decreases were observed for memory B cell and neutralizing antibody recognition of variants. Bioinformatic analyses showed full conservation of 91% and 94% of class II and class I spike epitopes. For Omicron, 72% of class II and 86% of class I epitopes were fully conserved, and 84% and 85% of CD4+ and CD8+ T cell responses were preserved. In-depth epitope repertoire analysis showed a median of 11 and 10 spike epitopes recognized by CD4+ and CD8+ T cells from vaccinees. Functional preservation of the majority of the T cell responses may play an important role as a second-level defense against diverse variants.

Minimal in-school SARS-CoV-2 transmission with strict mitigation protocols at two independent schools in Nashville, TN (preprint)

ABSTRACT BACKGROUND The COVID-19 pandemic has greatly impacted school operations. To better understand the role of schools in COVID-19 transmission, we evaluated infections at two independent schools in Nashville, TN during the 2020-2021 school year. METHODS The cumulative incidence of COVID-19 within each school, age group, and exposure setting were estimated and compared to local incidence. Primary attack rates were estimated among students quarantined for in-school close contact. RESULTS Among 1401 students who attended school during the study period, 98 cases of COVID-19 were reported, corresponding to cumulative incidence of 7.0% (95% confidence interval (CI): 5.7-8.5). Most cases were linked to household (58%) or community (31%) transmission, with few linked to in-school transmission (11%). Overall, 619 students were quarantined, corresponding to >5000 person-days of missed school, among whom only 5 tested positive for SARS-CoV-2 during quarantine (primary attack rate: 0.8%, 95% CI: 0.3, 1.9). Weekly case rates at school were not correlated with community transmission. CONCLUSION These results suggest that transmission of COVID-19 in schools is minimal when strict mitigation measures are used, even during periods of extensive community transmission. Strict quarantine of contacts may lead to unnecessary missed school days with minimal benefit to in-school transmission.

Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections (preprint)

SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and day of illness when sampling occurred. A noncanonical subgenomic RNA which could represent ORF9b is significantly enriched in B.1.1.7 SARS-CoV-2 infections, potentially as a result of a triple nucleotide mutation leading to amino acid substitution D3L in nucleocapsid in this lineage which increases complementarity with the genomic leader sequence. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern.

Persistent Cellular Immunity to SARS-CoV-2 Infection (preprint)

SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4+ and CD8+ T cells, expression of activation/exhaustion markers, and cell division. SummaryWe show that SARS-CoV-2 infection elicits broadly reactive and highly functional memory T cell responses that persist 6 months after infection. In addition, recovered individuals show enduring immune alterations in CD4+ and CD8+ T cells compartments.

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases (preprint)

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4+ and CD8+ T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.

periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data (preprint)

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA in ARTIC network protocol generated Nanopore SARS-CoV-2 sequence data. We applied periscope to 1155 SARS-CoV-2 sequences from Sheffield, UK. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sub-genomic RNA. We were able to detect all canonical sub-genomic RNAs at expected abundances, with the exception of ORF10, suggesting that this is not a functional ORF. A number of recurrent non-canonical sub-genomic RNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sub-genomic RNA analysis. Finally variants found in genomic RNA are transmitted to sub-genomic RNAs with high fidelity in most cases. This tool can be applied to tens of thousands of sequences worldwide to provide the most comprehensive analysis of SARS-CoV-2 sub-genomic RNA to date.

Three adjacent nucleotide changes spanning two residues in SARS-CoV-2 nucleoprotein: possible homologous recombination from the transcription-regulating sequence (preprint)

The COVID-19 pandemic is caused by the single-stranded RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus of zoonotic origin that was first detected in Wuhan, China in December 2019. There is evidence that homologous recombination contributed to this cross-species transmission. Since that time the virus has demonstrated a high propensity for human-to-human transmission. Here we report two newly identified adjacent amino acid polymorphisms in the nucleocapsid at positions 203 and 204 (R203K/G204R) due to three adjacent nucleotide changes across the two codons (i.e. AGG GGA to AAA CGA). This new strain within the LGG clade may have arisen by a form of homologous recombination from the core sequence (CS-B) of the transcription-regulating sequences of SAS-CoV-2 itself and has rapidly increased to approximately one third of reported sequences from Europe during the month of March 2020. We note that these polymorphisms are predicted to reduce the binding of an overlying putative HLA-C*07-restricted epitope and that HLA-C*07 is prevalent in Caucasians being carried by >40% of the population. The findings suggest that homologous recombination may have occurred since its introduction into humans and be a mechanism for increased viral fitness and adaptation of SARS-CoV-2 to human populations.