Genome-Wide B Cell, CD4⁺, and CD8⁺ T Cell Epitopes That Are Highly Conserved between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Targets for Preemptive Pan-Coronavirus Vaccines

Evolutionary convergence of human SARS-CoV-2 into bat- and pangolin-derived SL-CoVs

Understanding the animal origins of SARS-CoV-2 is critical for the development of a preemptive pan-coronavirus vaccine to protect from future human outbreaks and deter future zoonosis.

We first screened for the evolutionary relationship among human SARS-CoV-2 and SARS-CoV/MERS-CoV strains from previous outbreaks (i.e., Urbani, MERS-CoV, OC43, NL63, 229E, and HKU1–genotype B) along with 25 SL-CoV genome sequences obtained from different animal species: bats (R. affinis and R. malayanus), civet cats (Paguma larvata), and pangolins (M. javanica) and MERS-CoVs from camels (Camelus dromedarius and C. bactrianus) (Fig. 1). These sequence alignments revealed similarity of the original human-SARS-CoV-2 strain found in Wuhan, China, to four bat SL-CoV strains: hCoV-19-bat-Yunnan-RmYN02, bat-CoV-19-ZXC21, and hCoV-19-bat-Yunnan-RaTG13 obtained from the Yunnan and Zhejiang provinces of China (Fig. 1A). With further genetic distance analysis, we discovered the least evolutionary divergence between SARS-CoV-2 isolate Wuhan-Hu-1 and the above-mentioned three SL-CoV isolates from bats, namely 1) Bat-CoV-RaTG13 (0.1), 2), bat-CoV-19-ZXC21 (0.1), and 3) Bat-CoV-YN02 (0.2) (Fig. 1B, 1C). Moreover, the phylogenetic analysis performed among the whole genome sequences of a total of 81,963 SARS-CoV-2 strains for which sequences have been reported in circulation in 190 countries suggest an evolutionary convergence of bat and pangolin SL-CoVs into the human SARS-CoV-2 strains (Fig. 1D, 1E). Furthermore, through a complete genome tree derived from the 81,963 SARS-CoV-2 genome sequences submitted from Asian, African, North American, South American, European, and Oceanian regions, we confirmed that the least evolutionary divergence for SARS-CoV-2 strains is in SL-CoVs isolated from bats and pangolins (Fig. 1D–F).

Altogether, the phylogenetic analysis and genetic distance suggest that the highly contagious and deadly human-SARS-CoV-2 strain originated from bats, most likely from either the Bat-CoV-19-ZXC21 (MG772934.1) or Bat-CoV-RaTG13 (MN996532.2) strains that spilled over into humans after further mutations and/or recombination. These mutations and/or recombination(s) possibly contributed to the rapid global expansion of the highly contagious and deadly SARS-CoV-2 (51, 52).

Genome-wide identification of SARS-CoV-2 CD8⁺ T cell epitopes that are highly conserved between human and bat/pangolin coronaviruses

We first predicted potential CD8⁺ T cell epitopes from the entire genome sequence of the first SARS-CoV-2-Wuhan-Hu-1 strain (NCBI GenBank accession number MN908947.3) (53–59). For this, we used multiple databases and algorithms, including the SYFPEITHI, MHC-I processing predictions, MHC-I binding predictions, MHC-I immunogenicity, and IEDB (56, 60). We focused on epitopes restricted to the five most frequent HLA class I alleles with large coverage in worldwide human populations, regardless of race and ethnicity (i.e., HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-A*23:01) (61–63) (Supplemental Fig. 1A, 1C).

Using the aforementioned criteria, we originally identified a total of 9,660 potential CD8⁺ T cell epitopes derived from 12 structural proteins (S, membrane glycoprotein, and nucleocapsid phosphoprotein) and ORFs of SARS-CoV-2-Wuhan-Hu-1 strain (MN908947.3) (Supplemental Table I). Subsequently, this large pool of epitopes was narrowed down to 91 epitopes that are highly conserved among 1) over 81,000 SARS-CoV-2 strains (that currently circulate in 190 countries on six continents); 2) the four major common cold coronaviruses that caused previous outbreaks (i.e., hCoV-OC43 [KF923903], hCoV-229E [KY983587], hCoV-HKU1 genotype B [AY884001], and hCoV-NL63 [NC_005831]); and 3) the SL-CoVs that are isolated from bats, civet cats, pangolins, and camels (Fig. 2A). Although the highest degree of similarity (expressed as percentage of resemblance) was identified among 81,963 SARS-CoV-2 strains, 6 strains of previous human SARS-CoVs and 18 animal SL-CoVs strains isolated from bats and pangolins, only a small percentage of similarity was found between the SARS-CoV-2 and MERS-CoV strains (Supplemental Fig. 2). However, a significantly lower degree of similarity was recorded among the SARS-CoV-2 and the SL-CoVs strains isolated from civet cats’ and camels’ CoVs (Supplemental Fig. 2).

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FIGURE 2.

Identification of highly conserved potential SARS-CoV-2–derived human CD8⁺ T cell epitopes that bind with high affinity to HLA-A*02:01 molecules. (A) Ninety-one genome-wide, in silico predicted, and highly conserved SARS-CoV-2–derived CD8⁺ T cell epitope peptides were synthetized and were tested for their binding affinity in vitro to HLA-A*02:01 molecules expressed on the surface of T₂ cells. (B) Out of the 91 CD8⁺ T cell epitopes, four epitopes were selected as high binders to HLA-A*02:01 molecules, even at the lowest molarity of 3 μM. Further, 20 epitopes with high and three epitopes with moderate binding affinity were found to stabilize the expression of HLA-A*02:01 molecules on the surface of the T₂ cells. The levels of HLA-A*02:01 surface expression was determined by MFI, measured by flow cytometry on T₂ cells following an overnight incubation of T₂ cells at 26°C with decreasing peptide epitopes molarity (30, 15, and 5 μM) as shown in graphs. Percentage of MFI increase was calculated as follows: percentage MFI increase = (MFI with the given peptide − MFI without peptide)/(MFI without peptide) × 100.

We further identified 27 SARS-CoV-2 human CD8⁺ T cell epitopes out of the 91 epitopes that bound with high affinity with HLA-A*02:01 molecules using in vitro peptide–HLA binding assay (Fig. 2A). Four epitopes were found to be very high affinity binders (Fig. 2B). The 27 epitopes with high binding affinity were later confirmed in silico using molecular docking models across five major HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-A*23:01 haplotypes (Supplemental Fig. 3) (64). The highest binding affinity to HLA-A*02:01 molecules with the highest S_inter scores (blue squares) were recorded for ORF1ab_6749–6757, S_2–10, S_958–966, S_1220–1228, E_26–34, ORF8_83–91, ORF10_3–11, and ORF10_5–13, whereas minimum S_inter score was observed for ORF1ab_3732–3740, S_691–699, and membrane protein (M)_89–97. Other CD8⁺ T cell epitopes like ORF1ab_1675–1683, ORF1ab_2363–2371, ORF1ab_3013–3021, and ORF7b_26–34 were also found with intermediate S_inter scores (Supplemental Fig. 3A, 3B). Although the identified highly conserved CD8⁺ T cell epitopes were distributed within 8 of the 12 structural and nonstructural ORFs (i.e., ORF1ab, S, E, M, ORF6, ORF7b, ORF8, and ORF10), the highest numbers of epitopes were localized in the replicase polyprotein 1ab/1a (ORF1ab) (nine epitopes) followed by the spike glycoprotein (five epitopes) (Supplemental Figs. 2, 8).

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FIGURE 3.

CD8⁺ T cells specific to highly conserved SARS-CoV-2 epitopes detected in COVID-19 patients and unexposed healthy individuals. (A) Experimental design: PBMCs from HLA-A*02:01 positive COVID-19 patients (n = 30) (B) and controls unexposed healthy individuals (n = 10) (C) were isolated and stimulated overnight with 10 μM of each of the 27 SARS-CoV-2–derived CD8⁺ T cell epitopes. The number of IFN-γ–producing cells were quantified using ELISpot assay (B, C, and D). Dotted lines represent threshold to evaluate the relative magnitude of the response: mean SFCs between 25 and 50 correspond to a medium/intermediate response, whereas a strong response is defined for mean SFCs >50. PBMCs from HLA-A*02:01–positive COVID-19 patients (E) were further stimulated for an additional 5 h in the presence of mAbs specific to CD107^a and CD107^b and GolgiPlug and GolgiStop. Tetramers specific to Spike epitopes, CD107^a/b and CD69, and TNF-α expression were then measured by FACS. Representative FACS plot showing the frequencies of tetramer⁺CD8⁺ T cells, CD107^a/b+CD8⁺ T cells, CD69⁺CD8⁺ T cells, and TNF-α⁺CD8⁺ T cells following priming with a group of four Spike CD8⁺ T cell epitope peptides. Average frequencies of tetramer⁺CD8⁺ T cells, CD107^a/b+CD8⁺ T cells, CD69⁺CD8⁺ T cells, and TNF-α⁺CD8⁺ T cells. *p < 0.05.

Altogether, our findings identified 27 highly conserved potential human CD8⁺ T cell epitopes from the sequence of SARS-CoV-2 that are highly conserved among 81,963 SARS-CoV-2 strains, the four major common cold coronaviruses (i.e., hCoV-OC43, hCoV-229E, hCoV-HKU1 genotype B, and hCoV-NL63), newly found highly transmissible variants (Supplemental Fig. 9), and several SL-CoV strains that are isolated from bats and pangolins. These results suggest that both the structural and the nonstructural proteins are immunodominant Ags that are targeted by human CD8⁺ T cells from both COVID-19 patients and common cold coronavirus–infected healthy individuals.

In silico screening of potential promiscuous SARS-CoV-2 CD4⁺ T cell epitopes that are highly conserved between human and bat/pangolin coronaviruses

We subsequently identified a total of 9,594 potential HLA-DR–restricted CD4⁺ T cell epitopes from the whole genome sequence of SARS-CoV-2-Wuhan-Hu-1 strain (MN908947.3) using multiple databases and algorithms including the SYFPEITHI, MHC-II binding predictions, TepiTool, and TEPITOPEpan (Supplemental Table II). These potential promiscuous CD4⁺ T cell epitopes were screened in silico against the five most frequent HLA-DR alleles with large coverage in the human population, regardless of race or ethnicity: HLA-DRB1*01:01, HLA-DRB1*11:01, HLA-DRB1*15:01, HLA-DRB1*03:01, and HLA-DRB1*04:01 (Supplemental Fig. 1B, 1D). The number of potential CD4⁺ T cell epitopes was later narrowed down to 16 epitopes based on 1) the epitope sequences that are highly conserved among 81,963 SARS-CoV-2 strains, the four major common cold and 25 SL-CoV strains isolated from bats, civet cats, pangolins, and camels (Supplemental Fig. 4); and 2) their high binding affinity to HLA-DR molecules using in silico molecular docking models (Supplemental Fig. 5). The sequences of most of the 16 CD4⁺ T cell epitopes are 100% conserved and common among 81,963 SARS-CoV-2 strains currently circulating in six continents (Supplemental Fig. 4). A high degree of sequence similarities was also identified in the sequences of most 16 CD4⁺ T cell epitopes among the SARS-CoV-2 strains and the six strains of previous human SARS-CoVs (e.g., up to 100% sequence identity for epitopes ORF1ab_5019–5033, ORF1ab_6088–6102, ORF1ab _6420–6434, E_20–34, E_26–40, and M_176–190). Moreover, a high degree of sequence similarities was also identified among the SARS-CoV-2 and the SL-CoV strains isolated from bats and pangolins. In contrast, a lower sequence similarity was identified among CD4⁺ T cell epitopes from SARS-CoV-2 strains and the SL-CoV strains isolated from civet cats, followed by MERS-like CoV strains isolated from camels (Supplemental Figs. 4, 8).

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FIGURE 4.

CD4⁺ T cells specific to highly conserved SARS-CoV-2 epitopes detected in COVID-19 patients and unexposed healthy individuals. (A) Experimental design: PBMCs from HLA-DRB1–positive COVID-19 patients (n = 30) (B) and controls unexposed healthy individuals (n = 10) (C) were isolated and stimulated for 48 h. with 10 μM of each of the 16 SARS-CoV-2-derived CD4⁺ T cell epitopes. The number of IFN-γ–producing cells were quantified using ELISpot assay (B, C, and D). Dotted lines represent a threshold to evaluate the relative magnitude of the response: mean SFCs between 25 and 50 correspond to a medium/intermediate response, whereas a strong response is defined for mean SFCs >50. PBMCs from HLA-DRB1–positive COVID-19 patients (E) were further stimulated for an additional 5 h in the presence of mAbs specific to CD107^a and CD107^b, and GolgiPlug and GolgiStop. Tetramers specific to two Spike epitopes, CD107^a/b and CD69, and TNF-α expression were then measured by FACS. Representative FACS plot showing the frequencies of tetramer⁺CD4⁺ T cells, CD107^a/b+CD4⁺ T cells, CD69⁺CD4⁺ T cells, and TNF-α⁺CD4⁺ T cells following priming with a group of two Spike CD4⁺ T cell epitope peptides. Average frequencies are shown for tetramer⁺CD4⁺ T cells, CD107^a/b+CD4⁺ T cells, CD69⁺CD4⁺ T cells, and TNF-α⁺CD4⁺ T cells. *p < 0.05.

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FIGURE 5.

Immunogenicity of genome-wide identified human SARS-CoV-2 CD8⁺ T epitopes in HLA-A*02:01/HLA-DRB1 double-transgenic mice. (A) Timeline of immunization and immunological analyses. Eight groups of age-matched HLA-A*02:01–transgenic mice (n = 3) were immunized s.c., on days 0 and 14, with a mixture of four SARS-CoV-2–derived human CD8⁺ T cell peptide epitopes mixed with pan DR epitope (PADRE) CD4⁺ Th epitope, delivered in alum and CpG₁₈₂₆ adjuvants. As a negative control, mice received adjuvants alone (mock immunized). (B) Gating strategy used to characterize spleen-derived CD8⁺ T cells. Lymphocytes were identified by a low forward scatter (FSC) and low side scatter (SSC) gate. Singlets were selected by plotting FSC area (FSC-A) versus FSC height (FSC-H). CD8-positive cells were then gated by the expression of CD8 and CD3 markers. (C) Representative ELISpot images (left panel) and average frequencies (right panel) of IFN-γ–producing cell spots from splenocytes (1 × 10⁶ cells per well) stimulated for 48 h with 10 µM of 10 immunodominant CD8⁺ T cell peptides and 1 subdominant CD8⁺ T cell peptide out of the total pool of 27 CD8⁺ T cell peptides derived from SARS-CoV-2 structural and nonstructural proteins. The number on the top of each ELISpot image represents the number of IFN-γ–producing, SFCs per one million splenocytes. (D) Representative FACS plot (left panel) and average frequencies (right panel) of IFN-γ and TNF-α production by CD107^a/b and CD69 expression on 10 immunodominant CD8⁺ T cell peptides and 1 subdominant CD8⁺ T cell peptide out of the total pool of 27 CD8⁺ T cell peptides derived from SARS-CoV-2 structural and nonstructural proteins determined by FACS. Numbers indicate frequencies of IFN-γ⁺CD8⁺ T cells, CD107⁺CD8⁺ T cells, CD69⁺CD8⁺ T cells, and TNF-α⁺CD8⁺ T cells detected in three immunized mice.

The 16 highly conserved CD4⁺ T cell epitopes are distributed within 9 out of the 12 structural and nonstructural ORFs (i.e., ORF1ab, S, E, M, ORF6, ORF7a, ORF7b, ORF8, and nucleoprotein [N]). The highest numbers of epitopes were localized in the replicase polyprotein ORF1ab/1a (five epitopes) followed by ORF7a (three epitopes) (Supplemental Figs. 4, 8). Unlike the human CD8⁺ T cell epitopes, the human CD4⁺ T cell epitopes are found to be expressed in each of the structural S, E, M, and N proteins. Two epitopes are from the E, one epitope from the M, one epitope from the N protein, and one epitope from the spike protein. The remaining CD4⁺ T cell epitopes are distributed among the ORF6, ORF7a, ORF7b, and ORF8 proteins (Supplemental Figs. 4, 8).

Altogether, these results identified 16 potential CD4⁺ T cell epitopes from the whole sequence of SARS-CoV-2 that cross-react and have high sequence similarity among 81,963 SARS-CoV-2 strains, the main four major common cold coronaviruses, and the SL-CoV strains isolated from bats and pangolins. Similar to CD8⁺ T cell epitopes, the replicase polyprotein ORF1ab appeared to be the most immunodominant Ag, with a high number of conserved epitopes that may possibly be targeted by human CD4⁺ T cells.

Cross-reactive human and animal coronavirus-derived epitopes, spanning the whole virus proteome, are targeted by memory CD4⁺ and CD8⁺ T cells from SARS-CoV-2 patients and unexposed healthy individuals

Next, we assessed whether the potential SARS-CoV-2 CD4⁺ and CD8⁺ T cell epitopes that are highly conserved between human and animal coronaviruses would recall memory CD8⁺ T cells from COVID-19 patients as well as from healthy individuals who have never been exposed to SARS-CoV-2 or to COVID-19 patients (i.e., from healthy individuals’ blood samples that were collected from 2014 to 2018; Figs. (3A, 4A). Detailed clinical and demographic characteristics of the COVID-19 patients and the unexposed healthy individuals enrolled in the current study, with respect to age, gender, HLA-A*02:01 and HLA-DRB1 distribution, COVID-19 disease severity, comorbidity, and biochemical parameters, are described in Table I and in Materials and Methods.

Blood-derived PBMCs from COVID-19 patients (black, (Fig. 3B) and healthy individuals (white, (Fig. 3C) were analyzed by ELISpot for frequencies in SARS-CoV-2 epitope–specific, IFN-γ–producing CD8⁺ T cells. As shown in (Fig. 3B and 3D, significant numbers of SARS-CoV-2 epitope–specific memory CD8⁺ T cells producing IFN-γ were detected in PBMCs of COVID-19 patients. Out of the 27 highly conserved cross-reactive SARS-CoV-2 CD8⁺ T cell epitopes (Supplemental Fig. 2) selected for their binding affinity with HLA-A*02:01 molecules (Fig. 2B), strong T cell responses (mean spot-forming T cells [SFCs] > 50 per 0.5 × 10⁶ PBMCs fixed as threshold) were detected in COVID-19 patients against 10 epitopes derived from 1) structural proteins like Spike (i.e., S_958–966, S_976–984, S_1000–1008, and S_1220–1228) or the Es (i.e., E_26–34); and 2) nonstructural proteins (i.e., ORF1ab_1675–1683, ORF1ab_2210–2218, ORF1ab_6749–6757, ORF6_3–11, and ORF10_3–11) (Fig. 3B, 3D). In addition, 12 other SARS-CoV-2 CD8⁺ T cell epitopes from structural of nonstructural SARS-CoV-2 proteins induced an intermediate response (with a mean SFCs between 25 and 50 per 0.5 × 10⁶ PBMCs) in COVID-19 patients: ORF1ab_84–92, ORF1ab_3013–3021, ORF1ab_3183–3191, ORF1ab_3732–3740, ORF1ab_4283–4291, ORF1ab_6419–6427, S_2–10, S_691–699, E_20–28, M_52–60, M_89–97, and ORF10_5–13.

Moreover, among the 27 SARS-CoV-2 epitopes, seven epitopes recalled a strong memory CD8⁺ T cells response (mean SFCs > 50) from unexposed healthy individuals (i.e., ORF1ab_1675–1683, ORF1ab_3732–3740, ORF1ab_4283–4290, ORF1ab_5470–5478, ORF1ab_6749–6757, S_976–984, S_1000–1008, and S_1220–1228), and five epitopes recalled a memory CD8⁺ T cells response that was intermediate (ORF1ab_6419–6427, S_2–10, E_26–34, ORF10_3–11, and ORF10_5–13) (Fig. 3C, 3D). However, the unexposed healthy individuals exhibited a different pattern of CD8⁺ T cell immunodominance as compared with COVID-19 patients. We then compared the epitopes specificity and function of memory CD8⁺ T cells in HLA-*A02:01–positive COVID-19 patients and healthy individuals using flow cytometry (Fig. 3E). For a better comparison, a similar FACS gating strategy was applied to PBMC-derived T cells from both COVID-19 and healthy donors (data not shown). Our COVID-19 patients appeared to have a higher frequency of CD8⁺ T cells compared with healthy donors (Fig. 3E). Tetramer staining showed that many of SARS-CoV-2 epitope–specific CD8⁺ T cells are multifunctional, producing IFN-γ and TNF-α and expressing CD69 and CD107^a/b markers of activation and cytotoxicity in COVID-19 patients (Fig. 3E).

Similar to SARS-CoV-2 memory CD8⁺ T cells, memory CD4⁺ T cells specific to several highly conserved SARS-CoV-2 epitopes were detected in both COVID-19–recovered patients and unexposed healthy individuals (Fig. 4B–D). Out of the 16 highly conserved cross-reactive SARS-CoV-2 CD4⁺ T cell epitopes (Supplemental Fig. 4), strong T cell responses (mean SFCs > 50 per 0.5 × 10⁶ PBMCs fixed as a threshold) were detected in COVID-19 patients against two epitopes, one derived from the structural protein M (M_176–190) and one from the nonstructural protein ORF1a (ORF1a_1350–1365) (Fig. 4B, 4D). Moreover, six additional SARS-CoV-2 CD8⁺ T cell epitopes from nonstructural SARS-CoV-2 proteins (i.e., ORF1a_1801–1815, ORF1a_6088–6102, ORF1a_6420–6434, ORF6_12–26, ORF7a_3–17, and ORF8b_1–15) and two more epitopes from structural proteins (i.e., S_1–13 and N_388–403) induced an intermediate CD4⁺ T cell response (mean SFCs between 25 and 50 per 0.5 × 10⁶ PBMCs) in COVID-19 patients (Fig. 4B, 4D).

Besides, among the 16 SARS-CoV-2 epitopes, two epitopes recalled a strong memory CD4⁺ T cells response (mean SFCs > 50) from unexposed healthy individuals with no history of COVID-19 (i.e., ORF1a_1350–1365 and ORF6_12–26) (Fig. 4C, 4D). Furthermore, five additional epitopes recalled an intermediate CD4⁺ T cells response in these unexposed healthy individuals (i.e., ORF1a_1801–1815, S_1–13, M_176–190, ORF8b_1–15, and N_388–403). Unlike for CD8⁺ T cell responses, the unexposed healthy individuals exhibited a similar pattern of CD4⁺ T cell immunodominance as compared with COVID-19 patients, with few differences in the magnitude of the responses only. Multifunctional SARS-CoV-2 epitope–specific CD4⁺ T cells expressing CD69, CD107^a/b, and TNF-α were detected using specific tetramers in PBMCs of HLA-DR1–positive COVID-19 patients and healthy individuals (Fig. 4E) with a trend showing higher percentage of these cells in COVID-19 patients, although not significantly higher.

The immunogenicity of the identified SARS-CoV-2 human CD4⁺ and CD8⁺ T cell epitopes was assessed in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice (Figs. 5A, 6A). A mixture of peptides incorporating CD4⁺ T cell or CD8⁺ T cell epitopes was delivered with CpG and alum, as shown in Figs. (5A and 6A and detailed in the Materials and Methods. As a negative control, mice received adjuvant alone. The induced SARS-CoV-2 epitope–specific CD4⁺ and CD8⁺ T cell responses were determined in the spleen using multiple immunological assays, including IFN-γ ELISpot, FACS surface markers of activation, markers of cytotoxic degranulation, and intracellular cytokine staining. The gating strategy used for mice is shown in Figs. (5B and 6B. Two weeks after the second immunization with the mixture of CD8⁺ T cell peptides, 10 out of 27 highly conserved SARS-CoV-2 human CD8⁺ T cell epitope peptides were immunogenic in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice (Fig. 5C, 5D). The remaining 17 CD8⁺ T cell epitopes presented moderate/low immunogenicity levels in HLA-DR1/HLA-A*02:01 double-transgenic mice. The immunogenic epitopes were derived from both structural Spike protein (S_2–10, S_958–966, S_1000–1008, and S_1220–1228) and E (E_20–28) and from nonstructural proteins (i.e., ORF1ab_2363–2371, ORF1ab_3732–3740, ORF1ab_5470–5478, ORF8_73–81, and ORF10_5–13). Moreover, 7 out of 16 SARS-CoV-2 peptides induced significant CD4⁺ T cell responses in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice (Fig. 6C, 6D). The immunogenic epitopes were derived from both structural Spike protein (S_1–13) and M (M_176–190) and from nonstructural proteins (ORF1a_1350–1365, ORF1a_5019–5033, ORF1a_6420–6434, ORF6_12–26, ORF7b_8–22, and ORF8b_1–15). The remaining 9 CD4⁺ T cell epitopes presented moderate/low level of immunogenicity in HLA-DR1/HLA-A*02:01 double-transgenic mice.

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FIGURE 6.

Immunogenicity of genome-wide identified human SARS-CoV-2 CD4⁺ T epitopes in HLA-A*02:01/HLA-DRB1 double-transgenic mice. (A) Timeline of immunization and immunological analyses. Four groups of age-matched HLA-DRB1–transgenic mice (n = 3) were immunized s.c. on days 0 and 14 with a mixture of four SARS-CoV-2–derived human CD4⁺ T cell peptide epitopes delivered in alum and CpG₁₈₂₆ adjuvants. As a negative control, mice received adjuvants alone (mock immunized). (B) Gating strategy used to characterize spleen-derived CD4⁺ T cells. CD4-positive cells were gated by the CD4 and CD3 expression markers. (C) Representative ELISpot images (left panel) and average frequencies (right panel) of IFN-γ–producing cell spots from splenocytes (1 × 10⁶ cells per well) stimulated for 48 h with 10 µM of seven immunodominant CD4⁺ T cell peptides and one subdominant CD4⁺ T cell peptide out of the total pool of 16 CD4⁺ T cell peptides derived from SARS-CoV-2 structural and nonstructural proteins. The number of IFN-γ–producing SFCs per one million of total cells is presented on the top of each ELISpot image. (D) Representative FACS plot (left panel) and average frequencies (right panel) show IFN-γ and TNF-α production by CD107^a/b and CD69 expression on seven immunodominant CD4⁺ T cell peptides and one subdominant CD4⁺ T cell peptide out of the total pool of 16 CD4⁺ T cell peptides derived from SARS-CoV-2 determined by FACS. The numbers indicate percentages of IFN-γ⁺CD4⁺ T cells, CD107⁺CD4⁺ T cells, CD69⁺CD4⁺ T cells, and TNF-α⁺CD4⁺ T cells detected in three immunized mice.

Altogether, these results indicate that preexisting memory CD4⁺ T and CD8⁺ T cells specific to both structural and nonstructural protein Ags and epitopes are present in COVID-19 patients and unexposed healthy individuals. Although SARS-CoV-2–specific CD4⁺ and CD8⁺ T cells in COVID-19 patients and healthy donors target epitopes from the whole virus proteome, most T cell epitopes are concentrated in the nonstructural proteins, with ORF1a/b being the most targeted Ags. These memory T cells recognized highly conserved SARS-CoV-2 epitopes that cross-react with the human and animal coronaviruses. It is likely that infection with a common cold coronavirus and/or human exposition with animal- and pet-related coronaviruses induced long-lasting memory CD4⁺ and CD8⁺ T cells specific to the structural and nonstructural SARS-CoV-2 epitopes in healthy unexposed individuals. Heterologous immunity and heterologous immunopathology orchestrated by these cross-reactive, epitope-specific memory CD4⁺ and CD8⁺ T cells following previous multiple exposures to common cold coronaviruses may have shaped protection versus susceptibility to SARS-CoV-2 infection and disease with a yet-to-be determined mechanism(s).

Identification of B cell epitopes from SARS-CoV-2 Spike protein that are highly conserved between human and animal coronaviruses that are antigenic in humans and immunogenic in humanized HLA-transgenic mice

We next predicted potential linear B cell (Ab) epitopes on Spike protein sequence of the first SARS-CoV-2-Wuhan-Hu-1 strain (NCBI GenBank accession number MN908947.3) using BepiPred 2.0, with a cutoff of 0.55 (corresponding to a specificity >0.81 and sensitivity <0.30) and considering sequences having more than 5-aa residues (65). This stringent screening process initially resulted in the identification of 28 linear B cell epitopes (Supplemental Table III). From this pool of 28 potential epitopes, we later selected 15 B cell epitopes (19–62 aa in length) based on 1) their sequences being highly conserved between SARS-CoV-2, the main four major common cold coronaviruses (CoV-OC43 [KF923903], CoV-229E [KY983587], CoV-HKU1 [AY884001], and CoV-NL63 [NC_005831]) (66), and the SL-CoVs that are isolated from bats, civet cats, pangolins, and camels; and 2) the probability of exposure each linear epitope to the surface of infected target cells (Supplemental Fig. 6). The Spike epitope sequences highlighted in blue indicate a high degree of homology among the currently circulating 81,963 SARS-CoV-2 strains and at least a 50% conservancy among two or more human SARS-CoV strains from previous outbreaks and the SL-CoV strains isolated from bats, civet cats, pangolins, and camels (Supplemental Fig. 6). Two of the fifteen B cell epitopes, namely S_369–393 and S_440–501, overlap with the Spike’s RBD regions that bind to the ACE2 receptor (designated as RBD-1 and RBD-2 in Supplemental Fig. 7A). Higher S_Inter scores were observed for RBD-derived epitopes S_369–393 and S_471–501 when molecular docking was performed against the ACE2 receptor (Supplemental Fig. 7B). Upon screening for the glycosylation regions, we observed B cell epitopes S_13–37, S_59–81, S_329–363, S_601–640, and S_1133–1172 with Asparagines predicted to be N-glycosylated. In contrast, B cell epitopes S_516–536, S_524–598, and S_802–819 were observed to be the O-glycosylated. The remaining B cell epitopes S_287–317, S_304–322, S_369–393, S_404–501, S_440–501, S_672–690, and S_888–909 were found to possess no glycosylation.

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FIGURE 7.

IgG Abs specific to SARS-CoV-2 Spike protein–derived B cell epitopes in immunized B6 mice and in convalescent COVID-19 patients. (A) Timeline of immunization and immunological analyses. A total of 22 SARS-CoV-2–derived B cell epitope peptides selected from SARS-CoV-2 Spike protein and tested in B6 mice were able to induce Ab responses. Four groups of age-matched B6 mice (n = 3) were immunized s.c. on days 0 and 14 with a mixture of four or five SARS-CoV-2–derived B cell peptide epitopes emulsified in alum and CpG₁₈₂₆ adjuvants. Alum/CpG₁₈₂₆ adjuvants alone were used as negative controls (mock immunized). (B and C) The frequencies of IgG-producing CD3^(–)CD138⁽⁺⁾B220⁽⁺⁾ plasma B cells were determined in the spleen of immunized mice by flow cytometry. (B) The gating strategy was as follows: lymphocytes were identified by a low forward scatter (FSC) and low side scatter (SSC) gate. Singlets were selected by plotting FSC area (FSC-A) versus FSC height (FSC-H). B cells were then gated by the expression of CD3^(–) and B220⁽⁺⁾ cells and CD138 expression on plasma B cells determined. (C) Representative FACS plot (left panels) and average frequencies (right panel) of plasma B cells detected in spleen of immunized mice. The percentages of plasma CD138^(–)B220⁽⁺⁾B cells is indicated on the top left of each dot plot. (D) SARS-CoV-2–derived B cell epitope–specific IgG responses were quantified in immune serum, 14 d after second immunization (i.e., day 28) by ELISpot (number of IgG⁽⁺⁾spots). Representative ELISpot images (left panels) and average frequencies (right panel) of anti-peptide specific IgG-producing B cell spots (1 × 10⁶ splenocytes per well) following 4 d in vitro B cell polyclonal stimulation with mouse Poly-S (ImmunoSpot). The top/left of each ELISpot image shows the number of IgG-producing B cells per half a million cells. ELISA plates were coated with each individual immunizing peptide. The B cell epitope–specific IgG concentrations (micrograms per milliliter) measured by ELISA in (E) levels of IgG detected in peptide-immunized B6 mice after subtraction of the background measured from mock-vaccinated mice. The dashed horizontal line indicates the limit of detection and in (F) level of IgG specific to each of the 22 Spike peptides detected SARS-CoV-2–infected patients (n = 40) after subtraction of the background measured from healthy nonexposed individuals, as shown in (G) (n = 10). Black bars and gray bars show high and medium immunogenic B cell peptides, respectively. The dashed line indicates the limit of detection.

We later determined the ability of each of the 15 B cell epitopes selected from the Spike protein, which showed a high conservancy between human and animal coronaviruses to induce SARS-CoV-2 epitope–specific, Ab-producing plasma B cells and IgG Abs in B6 mice (Fig. 7). Synthetic peptides corresponding to each linear B cell epitope were produced. Because four epitopes were too long to synthetize (e.g., 62 aa), they were divided into two or three short fragments, resulting in a total of 22 B cell epitope peptides (Supplemental Table III). As illustrated in (Fig. 7A, groups of five B6 mice each received two s.c. injections with mixtures of three to four B cell epitope peptides mixed with CpG and alum adjuvants. Negative control mice received adjuvant alone without Ags. The frequency of Ab-producing plasma B cells and the level of IgG Abs specific to each SARS-CoV-2 B cell epitope were determined in the spleen and in the serum using FACS staining of CD138 and B220 surface markers and IgG ELISpot and ELISA assays, respectively. The gating strategy used to determine the frequencies of plasma B cells in the spleen is shown in (Fig. 7B. Out of the 22 Spike B cell epitopes, seven epitopes (S_13–37, S_287–317, S_524–558, S_544–578, S_565–598, S_601–628, and S_614–640) induced high frequencies of CD138⁺B220⁺ plasma B cells in the spleen of B6 mice (Fig. 7C). The IgG ELISpot assay confirmed that 7 out of the 22 Spike B cell epitopes induced significant numbers of IgG-producing B cells in the spleen (Fig. 7D). Moreover, significant amounts of IgG were detected in the serum of the immunized B6 mice. These IgG Abs were specific to 6 out of the 22 Spike B cell peptide epitopes (S_13–37, S_59–81, S_287–317, S_565–598, S_601–628, and S_614–640) (Fig. 7E). As expected, nonimmunized animals or those that received adjuvant alone did not develop detectable IgG responses. Of these six highly immunogenic B cell peptides, five peptides (S_13–37, S_59–81, S_287–317, S_601–628, and S_614–640) were highly antigenic, as they were recognized by serum IgG from COVID-19 patients, confirming the presence of at least one native linear B cell epitope in each peptide (Fig. F). In summary, we identified five highly conserved immunogenic and antigenic human B cell target epitopes from the Spike SARS-CoV-2 virus that recall IgG Abs from COVID-19 patients (Fig. 8). This study further discovered five highly conserved B cell epitopes from SARS-CoV-2: S_13–37, S_287–317, S_338–363, S_614–640, and S_1133–1160, which are recognized by IgG Abs from healthy individuals who were never exposed to COVID-19, suggesting B cell epitopes cross-reactivity to other human coronaviruses (Fig. 7G).

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FIGURE 8.

Illustrations of the SARS-CoV/SARS-CoV-2 genome-wide location of the highly conserved, antigenic, and immunogenic CD4⁺ T cell, CD8⁺ T cell, and B cell epitopes. (A) Enveloped, spherical, ∼120 nm in diameter SARS-CoV/SARS-CoV-2 genome encodes four structural proteins: spike, E, M, and N, highlighted in blue, green, gray, and black, respectively. (B) The SARS-CoV/SARS-CoV-2 genome encodes two large nonstructural genes, ORF1a (green) and ORF1b (gray), encoding 16 nonstructural proteins (NSP1–NSP16). The genome encodes at least six accessory proteins (shades of light gray) that are unique to SARS-CoV/SARS-CoV-2 in terms of number, genomic organization, sequence, and function. The common SARS-CoV, SARS-CoV-2, and SL-CoVs–derived human B (blue), CD4⁺ (green), and CD8⁺ (black) T cell epitopes are shown. Structural and nonstructural ORFs used in this study were from SARS-CoV-2-Wuhan-Hu-1 strain (NCBI accession number MN908947.3). The amino acid sequence of the SARS-CoV-2-Wuhan-Hu-1 structural and nonstructural proteins was screened for human B and CD4⁺ and CD8⁺ T cell epitopes using different computational algorithms as previously described in Materials and Methods. Shown are genome-wide identified SARS-CoV-2 human B cell epitopes (in blue), CD4⁺ T cell epitopes (in green), and CD8⁺ T cell epitopes (in black) that are highly conserved between human and animal coronaviruses.