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. 2023 Nov 8;8(21):e172488.
doi: 10.1172/jci.insight.172488.

Multiantigen pan-sarbecovirus DNA vaccines generate protective T cell immune responses

Jeroen van Bergen  1 , Marcel Gm Camps  2 , Iris N Pardieck  2 , Dominique Veerkamp  2 , Wing Yan Leung  1   3 , Anouk A Leijs  4 , Sebenzile K Myeni  4 , Marjolein Kikkert  4 , Ramon Arens  2 , Gerben C Zondag  1   3 , Ferry Ossendorp  2
Affiliations

Multiantigen pan-sarbecovirus DNA vaccines generate protective T cell immune responses

Jeroen van Bergen et al. JCI Insight. .

Abstract

SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell-based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2-transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.

Keywords: COVID-19; MHC class 1; MHC class 2; T cells; Vaccines.

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Figures

Figure 1
Figure 1. Antigen selection for T cell–based pan-sarbecovirus vaccine CoVAX_MNS.
(A) Using the SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2) as the reference sequence, M, N, and S aa sequences from 41 sarbecoviruses (Supplemental Table 2) were aligned using Clustal Omega. For every Wuhan aa position in these proteins, the fraction of sarbecovirus sequences with a different aa at that position is plotted (difference to Wuhan). Full alignment results are presented in Supplemental Figures 1–3. (B) The selected conserved antigenic regions were incorporated into a multiantigen DNA vaccine in which these regions were separated by AAA spacers and reordered to minimize artificial junctional epitopes containing spacer-derived alanines. (C and D) Subsequently, the number of predicted HLA class I binders and experimentally validated CD8+ T cell epitopes present in the resulting vaccines were calculated using online tools. (C) First, peptides predicted to bind any of the most prominent HLA-A (A*01:01, A*02:01, A*03:01, A*11:01, A*23:01, A*24:02) or HLA-B (B*07:02, B*08:01, B*35:01, B*40:01, B*44:02, B*44:03) alleles (83) with affinities below 50 nM (black) or 500 nM (gray) were identified using MHCflurry (54). At every aa position, the number of predicted HLA-binding peptides to which this aa residue contributes is indicated (HLA binders). (D) Next, known human SARS-CoV-2 CD8+ T cell epitopes presented via the abovementioned HLA alleles were obtained from the IEDB database (55). For every aa position, the number of confirmed CD8+ T cell epitopes this aa residue contributes to is plotted (T cell epitopes).
Figure 2
Figure 2. Antigen selection for T cell–based pan-sarbecovirus vaccine CoVAX_ORF1ab.
(A) Using the SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2) as the reference sequence, ORF1ab aa sequences from 41 sarbecoviruses (Supplemental Table 2) were aligned using Clustal Omega. For every Wuhan aa position in these proteins, the fraction of sarbecovirus sequences with a different aa at that position is plotted (difference to Wuhan). For the full alignment, see Supplemental Figure 4. (B) The selected conserved antigenic regions were incorporated into a multiantigen DNA vaccine in which these regions were separated by AAA spacers and reordered to minimize artificial junctional epitopes containing spacer-derived alanines. (C and D) Subsequently, the number of predicted HLA class I binders and experimentally validated CD8+ T cell epitopes present in the resulting vaccines were calculated using online tools. (C) First, peptides predicted to bind any of the most prominent HLA-A (A*01:01, A*02:01, A*03:01, A*11:01, A*23:01, A*24:02) or HLA-B (B*07:02, B*08:01, B*35:01, B*40:01, B*44:02, B*44:03) alleles (83) with affinities below 50 nM (black) or 500 nM (gray) were identified using MHCflurry (54). At every aa position, the number of predicted HLA-binding peptides to which this aa residue contributes is indicated (HLA binders). (D) Next, known human SARS-CoV-2 CD8+ T cell epitopes presented via the abovementioned HLA alleles were obtained from the IEDB database (55). For every aa position, the number of confirmed CD8+ T cell epitopes this aa residue contributes to is plotted (T cell epitopes).
Figure 3
Figure 3. CoVAX_MNS and CoVAX_ORF1ab DNA vaccines generate CD8+ T cell responses to conserved antigens.
C57BL/6 mice (5 mice/group) were vaccinated intradermally 3 times at 3-week intervals with (A–D) CoVAX_MNS or (E–H) CoVAX_ORF1ab, both including a C-terminal H-2Kb–restricted S reporter epitope (VNFNFNGL). (A and E) Ten days after the third vaccination, CD8+ T cell responses to this H-2Kb/S reporter epitope were measured in blood, using tetramers. (B–D and F–H) Eleven days after the third vaccination, isolated splenocytes were exposed to DCs (D1), then either peptide loaded (+ peptide) or not (– peptide), and CD8+ T cell cytokine responses were evaluated by ICS for IFN-γ and TNF. (C and G) Summarized responses to the S reporter epitope, a known H-2Kb–restricted M epitope (RTLSYYKL), and a newly discovered H-2Kb–restricted ORF1ab epitope (TGYHFREL) are shown, as well as (D and H) representative FACS plots gated on CD8+ T cells. (A–C and E–G) Dots represent individual mice; bars and whiskers indicate means and SEM. Tetramer responses were evaluated using a 2-tailed Mann-Whitney test and ICS responses by 2-way ANOVA using Holm-Šídák multiple comparisons test. For multiplicity-adjusted P values: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.002.
Figure 4
Figure 4. CoVAX_MNS induces a CD4+ T cell response to a conserved S antigen.
C57BL/6 mice (5 mice/group) were vaccinated intradermally with CoVAX_MNS, from which the reporter antigens had been removed (“norep” in Supplemental Table 5), 3 times at 3-week intervals. Additional control mice (3 mice/group) were mock-vaccinated or vaccinated with DNA encoding SARS-CoV-2 Wuhan S (p393 or p422; see Supplemental Table 5). A week after the final vaccination, spleen cells were cultured for 7 days with peptide-loaded syngeneic DCs (D1) and another 2 days with IL-2, after which the cultured splenocytes were exposed for 6 hours to D1; preloaded (+ peptide), or not (– peptide), with shared sarbecovirus S peptides comprising (A–C) S residues 966–1020 (S991–1020) or (D and E) 893–921 (S893–921); and analyzed by ICS. Activated CD4+ T cells were identified by coexpression of CD40L and TNF after restimulation with these S peptides. (A) Representative dot plots for the S991–1020 peptide. (B–E) Summarized data.
Figure 5
Figure 5. CoVAX_MNS-induced CD4+ T cell responses improve CD8+ T and B cell responses to full-length S.
(A) Schematic representation of the S challenge experiment. C57BL/6 mice (5 mice/group) were vaccinated intradermally with mock (–), CoVAX_ORF1ab (ORF1ab), or CoVAX_MNS DNA vaccines, from which the reporter antigens had been removed (“norep” in Supplemental Table 5), on days 0, 3, 6, and 21. On day 46, CD4+ T cells were depleted, or not, by i.p. injection of CD4-specific Abs (αCD4), and 3 days later, mice were exposed to full-length WT SARS-CoV-2 (Omicron) S. To this end, mice were injected with DNA encoding S from SARS-CoV-2 Omicron VOC. (B) Eight days after exposure to these spikes, blood samples were analyzed for CD8+ T cell responses to the H-2Kb–restricted S reporter antigen VNFNFNGL (absent from the vaccines, present in full-length S) as well as (C) IgG and (D) IgG2c Ab responses to the SARS-CoV-2 Omicron spikes. As expected, in the absence of an S challenge, CoVAX_MNS-vaccinated mice did not generate detectable S-specific CD8+ T cell (VNFNFNGL) or Ab (IgG, IgG2c) responses (data not shown). Dots represent individual mice; bars and whiskers indicate means and SEM. Tetramer (B) and Ab (C and D) responses were evaluated by 2-way ANOVA using Holm-Šídák multiple comparisons test. For multiplicity-adjusted P values: *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0004.
Figure 6
Figure 6. CoVAX_MNS vaccination improves CD8+ T and B cell responses to multiple sarbecovirus S variants.
(A) Schematic representation of the experiment. C57BL/6 mice (5 mice/group) were vaccinated intradermally with CoVAX_ORF1ab (1ab) or CoVAX_MNS (MNS) DNA vaccines, from which the reporter antigens had been removed (“norep” in Supplemental Table 5), 3 times at 3-week intervals (days 0, 21, and 42). Three weeks after the final vaccination (day 70), mice were challenged with full-length WT S from SARS-CoV-2 Omicron, SARS-CoV-2 Wuhan, or SARS-CoV-1, also by intradermal DNA injection. Eight days after exposure to these spikes (day 78), blood samples were analyzed for (B) CD8+ T cell responses to the H-2Kb–restricted S reporter antigen VNFNFNGL (absent from the vaccines, present in all 3 S DNAs) as well as (C) IgG1 and (D) IgG2c Ab responses to the different spikes. In the absence of an S challenge, CoVAX_MNS-vaccinated mice did not generate detectable S-specific CD8+ T cell (VNFNFNGL) or Ab (IgG, IgG2c) responses (data not shown). (E) Four weeks after exposure to full-length S (day 98), the specificity of these IgG responses was determined by testing the sera not only against the S to which the mice had been exposed (closed circles) but also against the other 2 spikes (open circles). Dots represent individual mice; bars and whiskers indicate means and SEM. Tetramer and Ab responses were evaluated by a 2-tailed Mann-Whitney (B–D) or Kruskal-Wallis test using Dunn’s multiple comparisons test (E). For multiplicity-adjusted P values: *P ≤ 0.05; **P ≤ 0.01.
Figure 7
Figure 7. Pan-sarbecovirus DNA vaccines induce CD8+ T cell responses and partial protection against SARS-CoV-2 in K18-hACE2tg mice.
(A) Schematic representation of the experiment. K18-hACE2tg mice (10 mice/group) were vaccinated intradermally with the indicated plasmid DNA vaccines thrice at 3-week intervals (days 0, 21, and 42). Mock-vaccinated animals served as negative controls. Nine days after the final vaccination (day 51), blood samples were collected to measure vaccine-specific CD8+ T cell responses. Three weeks after the final vaccination (day 63), the mice were challenged i.n. with a lethal dose of the Leiden-0008 SARS-CoV-2 isolate and BWs were measured daily as a parameter of disease. (B) CD8+ T cell responses in blood measured after 3 vaccinations (day 51) using H-2Kb tetramers containing ORF1ab nsp12-, M- (see Figure 3), or S-derived epitopes. In this experiment, CoVAX_MNS, but not CoVAX_ORF1ab, retained the C-terminal S reporter cassette; therefore, only CoVAX_MNS was able to induce responses to the S reporter epitope. Dots represent individual mice; bars and whiskers indicate means and SEM. These tetramer responses were evaluated by a Kruskal-Wallis test using Dunn’s multiple comparisons test. For multiplicity-adjusted P values: **P ≤ 0.01; ***P ≤ 0.002; ****P ≤ 0.0004. (C) BWs and survival of individual mice after SARS-CoV-2 challenge. Statistical analysis comparing the survival curves of vaccinated versus control mice by a log-rank (Mantel-Cox) test: for CoVAX_MNS, P = 0.06; for CoVAX_ORF1ab, P = 0.24.
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References

    1. Voysey M, et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet. 2021;397(10269):99–111. doi: 10.1016/S0140-6736(20)32661-1. - DOI - PMC - PubMed
    1. Polack FP, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020;383(27):2603–2615. doi: 10.1056/NEJMoa2034577. - DOI - PMC - PubMed
    1. Baden LR, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021;384(5):403–416. doi: 10.1056/NEJMoa2035389. - DOI - PMC - PubMed
    1. Sadoff J, et al. Safety and efficacy of single-dose Ad26.COV2.S vaccine against Covid-19. N Engl J Med. 2021;384(23):2187–2201. doi: 10.1056/NEJMoa2101544. - DOI - PMC - PubMed
    1. Muik A, et al. Neutralization of SARS-CoV-2 Omicron by BNT162b2 mRNA vaccine-elicited human sera. Science. 2022;375(6581):678–680. doi: 10.1126/science.abn7591. - DOI - PMC - PubMed
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