Identification of HLA-A*24:02-Restricted CTL Candidate Epitopes Derived from the Nonstructural Polyprotein 1a of SARS-CoV-2 and Analysis of Their Conservation Using the Mutation Database of SARS-CoV-2 Variants

ABSTRACT COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.

1. The work presented by Takagi, A. is mostly based on a computational algorithm-based identification of conserved epitope in evolving SARS-CoV-2 variants, and their studies indicate that a few of those epitopes have the ability to induce significantly higher CTL immune response. However, it would be important to show corresponding S protein neutralizing antibody production in the test mice. 2. Please refer and discuss relevant recent work directed to identify the epitopes for subunit peptide vaccine using a multiepitopic and cross-reactive humoral neutralizing and cellular CD4 and CD8 response strategy against COVID19 (Aparicio, et al., Emerg Microbes Infect. 2021 Dec;10(1):1931-1946 3. Please simplify figure 3 by adding unified statistical data (as in Table 4) in graph format and showing representative flow cytometry histograms only.
Reviewer #2 (Comments for the Author): One major concern this reviewer has is about relevance of model to humans. The analysis of SARS-CoV-2 peptides that authors developed and tested in mice may not be relevant to humans. SARS-CoV-2 does not affect murine epithelial cells, so the in vivo CTL responses may not be relevant. Authors must explain or clear this point. Authors can also choose to test these peptides using relevant model system Staff Comments:

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Thank you for submitting your paper to Microbiology Spectrum.
Thank you for inviting me to review the manuscript entitled, "Identification of HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 and analysis of their conservation using the mutation database of SARS-CoV-2 variants" by Takagi, A., and Matsui, M.
This work focused on understating and solving an important aspect of enhanced transmissibility and/or immunoevasion by the novel variants to escape from vaccine-elicited immunity. In addition to tradition antibody response, the work is dedicated to explore potential precision anti SARS-CoV-2 spike protein immune responses of CD8 + cytotoxic T lymphocytes (CTLs). The authors identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptideencapsulated liposomes. This study revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants and predicted that these conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. This work highlights aspects of designing the next-generation COVID-19 vaccine in order to compensate for the viral evolution that might be universally effective against the new SARS-CoV-2 variants.

Concerns and Suggestions:
1. The work presented by Takagi, A. is mostly based on computational algorithm based identification of conserved epitope in evolving SARS-CoV-2 variants, and their studies indicate that a few of those epitopes have the ability to induce significantly higher CTL immune response. However, it would be important to show corresponding S protein neutralizing antibody production in the test mice.
2. Please refer and discuss relevant recent work directed to identify the epitopes for subunit peptide vaccine using a multiepitopic and cross-reactive humoral neutralizing and cellular CD4 and CD8 response strategy against COVID19 (Aparicio, et al., Emerg Microbes Infect. 2021 Dec;10(1):1931-1946

Response to Reviewers
Manuscript number: Spectrum01659-21 1) Reviewer #1 Thank you very much for the very helpful comments and suggestions.
1. We feel that this comment of Reviewer #1 results from a misinterpretation of the data.
In this paper, mice were immunized with liposomal 9-mer short peptides derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2 for the induction of pp1a-specific CTLs. Because mice were not immunized with S protein or S protein-derived peptides, anti-S protein neutralizing antibodies were not produced in the test mice. We just suggested in the discussion (Pages 15-16, lines 288-294) that if the nucleotide sequences encoding pp1a-derived CTL epitopes are inserted into the current mRNA vaccine or vectored vaccine, the new vaccine would be very effective for any variants because of the induction of both anti-S protein neutralizing antibodies and pp1a-specific CTLs.
2. We are very grateful to Reviewer #1 for suggesting the addition of a good reference. We have referred (reference #43, Page 34, lines 627-632) and discussed (Page 16, lines 294-303) about this paper (Emerg Microbes Infect 10:1931-1946. Page 16, lines 294-303: "Recently, Aparicio et al. (43) showed the polyepitope at region 446-480 in the receptor binding domain of S protein that elicited neutralizing antibodies cross-recognizing SARS-CoV-2 variants of concern. The peptide 446-480 contained murine CD4 + and CD8 + T cell epitopes as well. Hence, they suggested this polyepitope could be the basis for a peptide vaccine or other vaccine platforms such as mRNA vaccine and vectored vaccine against COVID-19. This nice idea seems to be similar to ours that induction of both neutralizing antibodies and T cells will generate the desired next-generation vaccine. However, they could not find any dominant human T cell epitopes in the short region 446-480 (43). Finding human T cell epitopes restricted by a variety of HLA class I & class II alleles would require searching from large proteins such as pp1a." 3. We agree with the referee in this regard. We have simplified Fig. 3  Thirty-six peptides tested were divided into 4 groups with ICS values of 1% or higher, 0.5-1%, 0.1-0.5%, and ND (not detected). (B) Representative flow cytometry histograms are shown. Numbers shown indicate the percentages of intracellular IFN- + cells within CD8 + T cells stimulated with (+) or without (-) a relevant peptide." 2) Reviewer #2 Thank you very much for the very helpful comments.
We absolutely agree with Reviewer #2 in this point. As Reviewer #2 mentioned, the analysis of SARS-CoV-2 peptides that we developed and tested in mice may not be relevant to humans. However, we already described several sentences about this issue in the discussion as follows. Pages16-17, lines311-315: "However, we have to take into account that the immunogenic variation in HLA class I transgenic mice may not be identical to that in humans because the antigen processing and presentation differ between them. In addition, we did not present data showing that viral infection in a mouse model induces T cells targeting these epitopes because liposomal peptides were used as an immunogen. Page 17, lines316-318: "Hence, there is no guarantee that the candidate epitopes identified here are real pp1a-derived epitopes that are presented by human cells during live infection with SARS-CoV-2." Therefore, we do not think we need to write more about this issue. However, according to the comment of Reviewer #2, we have added the following new sentence. Page 17, lines 315-316: "Since SARS-CoV-2 does not affect murine epithelial cells in the first place, the in vivo CTL responses may not be relevant." As Reviewer #2 suggested in the last sentence of this comment, we will check these peptides with human cells in the near future.

3) A minor point:
We have added the following sentence in the acknowledgements. Page 24, lines 433-434: "The authors also thank Dr. T. Nakatsura (National Cancer center, Japan) for his help of preparing HLA-A*24:02 transgenic mice." Dr. Masanori Matsui Saitama Medical University Microbiology 38 Morohongo Moroyama-cho Iruma-gun, Saitama 350-0495 Japan Re: Spectrum01659-21R1 (Identification of HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 and analysis of their conservation using the mutation database of SARS-CoV-2 variants) Dear Dr. Masanori Matsui: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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