key: cord-0812202-qeo5dfxg
authors: Feng, Ye; Qiu, Min; Liu, Liang; Zou, Shengmei; Li, Yun; Luo, Kai; Guo, Qianpeng; Han, Ning; Sun, Yingqiang; Wang, Kui; Zhuang, Xinlei; Zhang, Shanshan; Chen, Shuqing; Mo, Fan
title: Multi-epitope vaccine design using an immunoinformatics approach for 2019 novel coronavirus (SARS-CoV-2)
date: 2020-06-30
journal: bioRxiv
DOI: 10.1101/2020.03.03.962332
sha: 202383e9c79c6dac9c6de7622681a2b2fdd699b6
doc_id: 812202
cord_uid: qeo5dfxg

A new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. An in silico approach based on the available virus genome was applied to identify 19 high immunogenic B-cell epitopes and 499 human-leukocyte-antigen (HLA) restricted T-cell epitopes. Thirty multi-epitope peptide vaccines were designed by iNeo Suite, and manufactured by solid-phase synthesis. Docking analysis showed stable hydrogen bonds of epitopes with their corresponding HLA alleles. When four vaccine peptide candidates from the spike protein of SARS-CoV-2 were selected to immunize mice, a significantly larger amount of IgG in serum as well as an increase of CD19+ cells in ILNs was observed in peptide-immunized mice compared to the control mice. The ratio of IFN-γ-secreting lymphocytes in CD4+ or CD8+ cells in the peptides-immunized mice were higher than that in the control mice. There were also a larger number of IFN-γ-secreting T cells in spleen in the peptides-immunized mice. This study screened antigenic B-cell and T-cell epitopes in all encoded proteins of SARS-CoV-2, and further designed multi-epitope based peptide vaccine against viral structural proteins. The obtained vaccine peptides successfully elicited specific humoral and cellular immune responses in mice. Primate experiments and clinical trial are urgently required to validate the efficacy and safety of these vaccine peptides. Importance So far, a new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. Different from the development approaches for traditional vaccines, the development of our peptide vaccine is faster and simpler. In this study, we performed an in silico approach to identify the antigenic B-cell epitopes and human-leukocyte-antigen (HLA) restricted T-cell epitopes, and designed a panel of multi-epitope peptide vaccines. The resulting SARS-CoV-2 multi-epitope peptide vaccine could elicit specific humoral and cellular immune responses in mice efficiently, displaying its great potential in our fight of COVID-19.

(29). All predicted structures or models were decorated and displayed by the open source 136 version of pymol program (https://github.com/schrodinger/pymol-open-source).

Immuno-stimulation of B lymphocytes 138 The selected 4 peptides were synthesized with the solid phase synthesis method by 139 GenScript Biotech Company (Nanjing, China), and were mixed at an equal concentration 140 of 1mg/ml. The immunization experiment constituted three groups, each consisting of 12 On the 28 th day after the 1 st immunization, 6 randomly selected mice were euthanized.

The inguinal lymph nodes (ILNs) were harvested and processed into single cell 

The design of immunization experiment was similar to that for the B cells, but the suspensions. Splenocytes (1×10 6 /well) and ILN lymphocyte (1×10 6 /well) were cultured 178 overnight with the peptide mixture (5μg/ml) or in RPMI-1640 alone (negative control).

PerCP/Cyanine5.5-conjugated anti-mouse CD8a antibody (Biolegend, San Diego, US), cells were resuspended in 500l PBS for flow cytometry analysis.

The IFN-γ-secreting T lymphocytes were also quantified on 6 randomly selected containing T-cell epitopes only. Based on both the epitope counts and HLA score, we 250 eventually selected 13 T-cell epitopes-only vaccine peptides.

Taken together, a total of 30 peptide vaccine candidates were designed (Table 3) . 26 252 of them were from the spike protein, two from the membrane protein and two from the 253 envelope protein. Five peptides were located in the RBD region, indicating they were 254 likely to induce the production of neutralizing antibody. The 30 vaccine peptides covered 255 all structural proteins that may induce immune response against SARS-CoV-2 in theory; 256 and the multi-peptide strategy we applied would better fit the genetic variability of the 257 human immune system and reduce the risk of pathogen's escape through mutation (33).

Interaction of predicted peptides with HLA alleles 259 To further inspect the binding stability of T-cell epitopes against HLA alleles, the T-cell 260 epitopes involved in the above designed vaccine peptides were selected to conduct an 261 interaction analysis. Figure 3 illustrated the docking results against the most popular HLA 262 types for the two epitopes from vaccines peptide 25 and 27 (Table 3; Table 4 ), which 263 showed relatively higher HLA score. The MDockPep scores were between -148 ~ -136, 264 indicating that the predicted crystal structures were stable. All epitopes were docked (Table 4 ). Taken together, the epitopes included in our vaccine 269 peptides can interact with the given HLA alleles by in silico prediction.

P12, P14 and P15, were chosen as the candidates for the downstream validation 273 experiments because of their relatively higher counts of B-cell and T-cell epitopes and the 274 higher frequencies of their epitopes' corresponding HLA alleles (Table 3) . We immunized 275 mice by subcutaneous injection of the mixture of these synthesized peptides plus 

To evaluate whether these peptides induce B cells to produce specific antibody 280 against the S protein, an ELISA assay was conducted to detect IgG in the sera of mice. Fig. 3 . Interaction between the predicted peptides (by yellow sticks) and different HLA 495 alleles (by green cartoons). Amino acids were labeled adjacent to the contact sites. Table   496 3 displays the detailed docking information. 

Surveillance case definitions for human infection with novel 376 coronavirus (nCoV): interim guidance v1

The authors declare that they have no competing interests.