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. 2021 Oct 13;26(20):6182.
doi: 10.3390/molecules26206182.

In Silico and In Vivo Evaluation of SARS-CoV-2 Predicted Epitopes-Based Candidate Vaccine

Mahmoud M Shehata  1   2 , Sara H Mahmoud  1 , Mohammad Tarek  3 , Ahmed A Al-Karmalawy  4 , Amal Mahmoud  5 , Ahmed Mostafa  1 , Mahmoud M Elhefnawi  6 , Mohamed A Ali  1
Affiliations

In Silico and In Vivo Evaluation of SARS-CoV-2 Predicted Epitopes-Based Candidate Vaccine

Mahmoud M Shehata et al. Molecules. .

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of coronavirus disease (COVID-19)) has caused relatively high mortality rates in humans throughout the world since its first detection in late December 2019, leading to the most devastating pandemic of the current century. Consequently, SARS-CoV-2 therapeutic interventions have received high priority from public health authorities. Despite increased COVID-19 infections, a vaccine or therapy to cover all the population is not yet available. Herein, immunoinformatics and custommune tools were used to identify B and T-cells epitopes from the available SARS-CoV-2 sequences spike (S) protein. In the in silico predictions, six B cell epitopes QTGKIADYNYK, TEIYQASTPCNGVEG, LQSYGFQPT, IRGDEVRQIAPGQTGKIADYNYKLPD, FSQILPDPSKPSKRS and PFAMQMAYRFNG were cross-reacted with MHC-I and MHC-II T-cells binding epitopes and selected for vaccination in experimental animals for evaluation as candidate vaccine(s) due to their high antigenic matching and conserved score. The selected six peptides were used individually or in combinations to immunize female Balb/c mice. The immunized mice raised reactive antibodies against SARS-CoV-2 in two different short peptides located in receptor binding domain and S2 region. In combination groups, an additive effect was demonstrated in-comparison with single peptide immunized mice. This study provides novel epitope-based peptide vaccine candidates against SARS-CoV-2.

Keywords: SARS-CoV-2; epitopes; prediction; vaccine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Shows the structural configuration of the spike glycoprotein (green) binding with receptor ACE2 (white) (PDB: 7DF4). The receptor-binding domain of the spike glycoprotein shows two distinctive regions essential for binding; RBDg (cyan) and RBDp (magenta). (B) Shows the cleavage sites of TMPRSS2 (yellow), furin (blue) in addition to N-acetylglucosamine (NAG) moieties (white). (C) shows the selected epitopes within RBDg (peptides A, D) and RBDp (peptides B, C) colored differently including: peptide A (red), peptide B (yellow), peptide C (black), peptide D (orange).
Figure 2
Figure 2
(A) Shows the amino-acid sequence alignment of RBDg epitopes among SARS-CoV-2 variants of concern (VOCs). Peptide A (bold red) is conserved in five variants out of seven, however it harbors K417N mutation within variants B.1.351 and B.1.617.2.1, In-addition to mutation K417T within P.1 variant. Peptide D (bold black) also includes the aforementioned mutations. (B) Shows the amino-acid sequence alignment of RBDp epitopes among SARS-CoV-2 VOCs. While peptide C (bold orange) is conserved among all studied variants, peptide B (bold yellow) is only conserved among three variants including; Wuhan reference, B.1.1.7 and A.23.1. In addition, peptide B harbors mutation E484K within B.1.351, B.1.525 and P.1 variants, peptide B also includes a delta variant B.1.617.2 T478K mutation. (C) Shows peptide E targeting TMPRSS2 cleavage site (yellow highlight) is completely conserved within the studied variants. (D) Shows peptide F targeting S2 fragment (cyan highlight) is completely conserved within the studied variants.
Figure 3
Figure 3
Shows the Bepipred 2.0 predictions for neutralizing antibody epitopes within RBD and cleavage regions of interest. The figure also shows the effect of evolved mutations of different SARS-CoV-2 variants on epitope prediction. (A) Shows that mutation K417N of variants B.1.351 and AY.1 does not affect the prediction score for RBDg epitopes A and D. However, mutation K417T of variant P.1 causes mild decrease in prediction scores which still does not affect the positivity of the selected epitopes. (B) Shows that neither E484K nor T478K negatively affect the positivity of RBDp peptides B and C. (C) Shows the positivity of peptide E Bepipred 2.0 prediction within the TMPRSS2 cleavage site region. (D) Shows the positivity of peptide F within S2 fragment (897–908).
Figure 4
Figure 4
Optical density (O.D) obtained from ELISA for sera of BALB/C mice immunized with short-peptides from A to E and combination (AB, CD, EF) groups in comparison with SARS-CoV-2 inactivated vaccine. Cut-off value for assay validation = 0.1 O.D reading at 490 nM. Statistical changes marked by * p value < 0.05, ** p value < 0.01 and *** p value < 0.001.
Figure 5
Figure 5
Timeline of the immunization protocol of predicted short peptides in BALB/C mice. W; week.
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