key: cord-1024721-cjy2cmf0 authors: Querne, Lana Bazan Peters; Bastos, Fernanda Zettel; Beirão, Breno Castello Branco title: Potential cross-protection against SARS-CoV-2 from previous exposure to bovine coronavirus date: 2021-12-14 journal: bioRxiv DOI: 10.1101/2021.12.13.472476 sha: 6c1d08d63a31f635e1d5073235cbd8b18a50c748 doc_id: 1024721 cord_uid: cjy2cmf0 Humans have long shared infectious agents with cattle, and the common cold OC-43 CoV is a not-so-distant example of cross-species viral spillover. Human exposure to BCoV is certainly common, as the virus is endemic in cattle-raising regions. This article shows an in silico investigation of shared viral epitopes between BCoV and SARS-CoV-2. HLA recognition and lymphocyte reactivity were assessed using freely-available resources. Several epitopes were shared between BCoV and SARS-CoV-2, both for B and T lymphocytes. These data demonstrate that possible cross-protection is being induced by human exposure to cattle. Coronaviruses are single-strand RNA viruses belonging to the Coronaviridae family, capable of infecting several animals causing respiratory, gastrointestinal and neurological diseases. The four genera that compose this family are: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus [5] . Among the beta-coronaviruses are the aforementioned SARS-CoV-2 and the bovine coronavirus (BCoV), being the latter responsible for livestock losses causing diarrhea in newborn calves and respiratory infections in calves and confined cattle [6, 7] . Both viruses encode four structural proteins in the genome: envelope protein (E), membrane protein (M), nucleocapsid protein (N) and spike protein (S); in addition to non-structural proteins (NSP) and open reading frame polyproteins (ORF) [8, 9] . Cross-reactivity between coronavirus strains may be able to induce an adaptive immunity that would help reduce the severity and spread of disease. This pre-existing immunity from contact with other coronaviruses, such as BCoV, possibly can provide protection against the SARS-CoV-2 [10] . Nevertheless, previous works predicting cross-immunity have focused on coronaviruses from different genera [11] . As adaptive immunity is generated by the recognition of epitopes by T and B cells [12] , the present study aimed to search for peptides originated from BCoV proteins M, N, S and ORF that presented T and B cell responses in humans and high identity with SARS-CoV-2. The proteome sequences of bovine coronavirus were obtained from the NCBI database and focused on four proteins (Table 1) : spike protein, membrane protein, nucleocapsid protein and replicase polyprotein (Orf1ab). The entire protein sequences were organized in 15-mer peptides that overlapped by 10 amino acids. T cell reactivity of bovine coronavirus peptides was assessed by predicting their binding to human leukocyte antigen class II (HLA II) molecules using IEDB MHC II binding predictions tool (http://tools.iedb.org/mhcii/). Peptide binding was predicted to all HLA class II molecules. A 20% percentile rank cutoff was chosen as a universal prediction threshold [13] . B cell reactivity of bovine coronavirus peptides was assessed using IEDB Bepipred Linear Epitope Prediction 2.0 (http://tools.iedb.org/bcell/). The residues with scores above the threshold (0.5) and with 5 amino acids or more were predicted to be part of an epitope. All bovine peptides that were above the threshold for T cells and B cells were analyzed for identity to the corresponding proteins of human SARS-CoV-2 (Table 1) using the Multiple Sequence Alignment (Clustal Omega, https://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences with an identity greater than or equal to 80% were selected as peptide matches [14] . COVID-19 epidemiology was assessed from publicly available data [15] . The slope of increase of cases/100,000 people for each city in the Brazilian State of Mato Grosso do Sul (MS) was used (between January, 2020 and September, 2021) [16] . The slope of COVID-19 cases was compared to the number of cattle/100,000 people for each municipality in the state [17] . As a control, the distance from each municipality to the major city in the subregion of the state was compared to the slope of COVID-19 cases [18] . General efficiency of public spending (not directly correlated with COVID-19) was also used as a control in a correlation analysis with COVID-19 prevalence. Data from the literature on public investment were used. Spending rigour was scored from 1-4, with four being the best-quality public use of resources [19] . The correlation of the data with COVID-19 prevalence was assessed with run's test in a linear correlation. GraphPad Prism 8 (GraphPad Software, Inc., USA) was used for graphing and for statistical analysis. All the data used for this analysis is available as supplementary material (Supplementary Table 1 ). A total of 136, 23, 45 and 709 15-mer peptides that overlapped by 10 amino acids were obtained for proteins S, M, N and ORF1ab respectively. From the results obtained by the IEDB MHC II binding prediction tool, 106 peptides from protein S, 20 peptides from protein M, 24 peptides from protein N and 566 peptides from ORF1ab protein had a percentile rank equal to or less than 20%. From the results obtained by the IEDB Bepipred Linear Epitope Prediction 2.0, 70 peptides from protein S, 9 peptides from protein M, 38 peptides from protein N and 386 peptides from ORF1ab protein had scores above the threshold. Among the peptides that showed good results for T or B cells, only 2 peptides from protein S, 1 peptide from protein M, and 2 peptides from protein N showed at least 80% similarity with SARS-CoV-2 (Table 2) . For these three proteins, no sequence was found to be within the cutoff values for both T cells and B cells. Regarding the ORF1ab protein,107 peptides showed good results for T or B cells (Table 3 ). In this case, 28 peptides were found to be within the cutoff values for both T cells and B cells. We analysed the correlation of COVID-19 prevalence to the density of cattle in the Brazilian state of MS as an example of a possible epidemiological association between human exposure to the Bovine Coronavirus (BCoV) and altered pandemic spread. Cattle density (cattle/100,000 people) negatively correlated with the slope of COVID-19 case increase in MS. In opposition, confounding factors in this epidemiological analysis showed no association with the slope of COVID-19 cases in the state (assessed factors were distance of each municipality to the main regional hub city and quality of public spending) (Fig. 1) . The premise of this article is the hypothesis that if BCoV influenced human immune responses to COVID-19, the epidemiology of the pandemic would have been altered by human exposure to cattle, since BCoV naturally occurs in bovine herds [20] , in addition to other factors that can influence COVID-19 geographic spread, such as income rates and social vulnerability levels [21] . The Brazilian state of MS was chosen as an hypothesis-raising case study, as it is a large beef productor with no megacities, which can "distort" the local epidemiological status due to their large influence on the statistics and their worldwide connections [22, 23] . Within-state infrastructure, scholarity, income and animal production conditions are more homogeneous than in inter-state comparisons, and a whole-country analysis would be biased by these and therefore was not performed [24, 25] . Further, general efficiency of public spending was an important factor in the spread and control of the pandemic in Brazil [26] , being another discrepant factor in the analysis of the whole-country. Municipalities with more cattle are expected to be further away from regional hubs, since large land areas are needed for farming. Therefore, any association between COVID-19 cases with cattle density could only indicate lower connectivity of the municipality, which is a major cause of spatial proliferation of the disease [27] . Bronchitis is being clinically tested for COVID-19 vaccination, for instance [11, 29] . Nevertheless, such considerations have not yet been directed to BCoV. It was not the goal of this study to confirm the association of BCoV with COVID-19 using epidemiological data. Our analysis is exceedingly restricted for this purpose. We used epidemiological data of COVID-19 for hypothesis-raising, and they showed a possible association of human exposure to cattle with the development of the pandemic. This work evaluated in silico if BCoV epitopes could be recognized by human B and T lymphocytes. Here, we report several epitopes which are likely to be important in the response for COVID-19 and which are shared with BCoV. This analysis is valuable in understanding the impact that exposure to the bovine coronavirus may have had on COVID-19. It is possible that COVID-19 epidemiology was shaped by human exposure to BCoV, much as smallpox was naturally curtailed by the exposure to cowpox [34] , and this paper offers evidence for viral crossprotection from the analysis of common epitopes. SARS-CoV-2 and BCoV share several common epitopes, which may confer cross-immunity. The relevance of this connection for the development of the pandemic is of yet not known, and should be proven with controlled trials of human responses to the bovine virus. One important outcome of this analysis is that if veterinary vaccines are to be considered for human use against COVID-19, as is being tested in Brazil, BCoV is a likely candidate. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein World Health Organization. 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