Three Specific Potential Epitopes That Could Be Recognized by T Cells of Convalescent COVID-19 Patients Were Identified From Spike Protein.

The current coronavirus disease 2019 (COVID-19) vaccines are used to prevent viral infection by inducing neutralizing antibody in the body, but according to the existing experience of severe acute respiratory syndrome coronavirus (SARS) infection, T-cell immunity could provide a longer durable protection period than antibody. The research on SARS-CoV-2-specific T-cell epitope can provide target antigen for the development and evaluation of COVID-19 vaccines, which is conducive to obtain COVID-19 vaccine that can provide long-term protection. For screening specific T-cell epitopes, a SARS-CoV-2 S protein peptide library with a peptide length of 15 amino acids was synthesized. Through flow cytometry to detect percentage of IFN-γ+ T cells after mixed COVID-19 convalescent patients' peripheral blood mononuclear cell with peptide library, seven peptides (P77, P14, P24, P38, P48, P74, and P84) that can be recognized by the T cells of COVID-19 convalescent patients were found. After excluding the nonspecific cross-reactions with unexposed population, three SARS-CoV-2-specific T-cell potential epitopes (P38, P48, and P84) were finally screened with the positive reaction rates between 15.4% and 48.0% in COVID-19 convalescent patients. This study also provided the HLA allele information of peptide-positive-response COVID-19 convalescent patients, thus predicting the population coverage of these three potential epitopes. Some HLA alleles showed higher frequency of occurrence in COVID-19 patients than in total Chinese population but no HLA alleles related to the T-cell peptide response and the severity of COVID-19. This research provides three potential T-cell epitopes that are helpful for the design and efficacy evaluation of COVID-19 vaccines. The HLA information provided by this research supplies reference significance for subsequent research such as finding the relation of HLA genotype with disease susceptibility.

Seropositive Reaction Rates of 9 B-Cell Epitopes of the SARS-CoV-2 Spike Protein and the Relationship between the Epitopes and Neutralizing Antibody.

OBJECTIVE: The aim of the study was to analyze the relationship between serum antibody and neutralizing antibody titers in convalescent coronavirus disease 2019 (COVID-19) patients with different disease severities, and the seropositive reaction rates of 9 reported B-cell epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Serum IgG and total antibody titers of 165 convalescent COVID-19 patients were determined by chemiluminescence, the serum neutralization antibody titers were determined by microneutralization assay, and the S/CO values of 9 peptides were detected by indirect enzyme-linked immunosorbent assay. Correlations between the aforementioned indexes were statistically analyzed, and differences in patients with different diseases severities were evaluated. RESULTS: IgG, total antibody, and neutralizing antibody titers increased with disease severity. The positive rate of the receptor-binding region (RBD) was 100%, and the average positive rate for all the 9 peptides was above 50% in 165 patients. IDf showed the highest rate of positivity (86.06%), with a rate of 95% for the (IDf + IDa) pattern. Moreover, S/CO values of RBD and mix (IDh) were significantly correlated with IgG, total antibody titers, and neutralizing antibody titers (p < 0.001), whereas the S/CO values for other 8 peptides showed no obvious correlation. CONCLUSION: In this study, a large sample was used to confirm that the peptide IDf had a high positive reaction rate for all patients (86.06%) and also had the highest detection rate in asymptomatic patients (86.67%). Only long peptide and mixed peptide showed correlation with neutralizing antibody titers, suggesting that the ability of SARS-CoV-2 antibody to neutralize virus infectivity may require the interaction of multiple sites.

A newly identified linear epitope on non-RBD region of SARS-CoV-2 spike protein improves the serological detection rate of COVID-19 patients.

BACKGROUND: Serological test is helpful in confirming and tracking infectious diseases in large population with the advantage of fast and convenience. Using the specific epitope peptides identified from the whole antigen as the detection antigen is sensitive and relatively economical. The development of epitope peptide-based detection kits for COVID-19 patients requires comprehensive information about epitope peptides. But the data on B cell epitope of SARS-CoV-2 spike protein is still limited. More importantly, there is a lack of serological data on the peptides in the population. In this study, we aimed to identify the B cell epitope peptides of spike protein and detect the reactivity in serum samples, for further providing data support for their subsequent serological applications. RESULTS: Two B cell linear epitopes, P104 and P82, located in non-RBD region of SARS-CoV-2 S protein were identified by indirect ELISA screening of an overlapping peptide library of the S protein with COVID-19 patients' convalescent serum. And the peptides were verified by testing with 165 serum samples. P104 has not been reported previously; P82 is contained in peptide S21P2 reported before. The positive reaction rates of epitope peptides S14P5 and S21P2, the two non-RBD region epitopes identified by Poh et al., and P82 and P104 were 77.0%, 73.9%, 61.2% and 30.3%, respectively, for 165 convalescent sera, including 30 asymptomatic patients. Although P104 had the lowest positive rate for total patients (30.3%), it exhibited slight advantage for detection of asymptomatic infections (36.7%). Combination of epitopes significantly improved the positive reaction rate. Among all combination patterns, (S14P5 + S21P2 + P104) pattern exhibited the highest positive reaction rate for all patients (92.7%), as well as for asymptomatic infections (86.7%), confirming the feasibility of P104 as supplementary antigen for serological detection. In addition, we analyzed the correlation between epitopes with neutralizing antibody, but only S14P5 had a medium positive correlation with neutralizing antibody titre (rs = 0.510, P < 0.01). CONCLUSION: Our research proved that epitopes on non-RBD region are of value in serological detection especially when combination more than one epitope, thus providing serological reaction information about the four epitopes, which has valuable references for their usage.