key: cord-0893250-caqhfjd7
authors: Prakash, Swayam; Srivastava, Ruchi; Coulon, Pierre-Gregoire; Dhanushkodi, Nisha R.; Chentoufi, Aziz A.; Tifrea, Delia F.; Edwards, Robert A.; Figueroa, Cesar J.; Schubl, Sebastian D.; Hsieh, Lanny; Buchmeier, Michael J.; Bouziane, Mohammed; Nesburn, Anthony B.; Kuppermann, Baruch D.; BenMohamed, Lbachir
title: Genome-Wide Asymptomatic B-Cell, CD4+ and CD8+ T-Cell Epitopes, that are Highly Conserved Between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Immune Targets for Pre-Emptive Pan-Coronavirus Vaccines
date: 2020-09-28
journal: bioRxiv
DOI: 10.1101/2020.09.27.316018
sha: a730cb1ad5fed9ad7cc4b1a928ddd4785d3d4c81
doc_id: 893250
cord_uid: caqhfjd7

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal “flu-like” global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+ and CD8+ T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines 1. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+ and CD8+ T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the “Common Cold”; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+ and CD8+ T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in “humanized” Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.

Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine

As deforestation continues to expand and humans progressively conquer wildlife habitats 59 around the globe, the wildlife "fights back" by spilling over many zoonotic viruses into human 60 populations 2, 3 . Among these, is the large family of RNA viruses called Coronaviruses (a name 61 alluding to the small pointy spikes that give them the appearance of a corona). Since the first human 62 Coronavirus was identified in 1965, many additional strains of Coronaviruses have continued to 63 emerge 4, 5, 6 . These set the stage for several major human disease outbreaks within the last two 64 decades (i.e., from 2002 to 2019): SARS-CoV 7 ; CoV-NL63 8 ; CoV-HKU1 9 ; CoV-229E 9 ; CoV-OC43 epitopes in both asymptomatic SARS-CoV-2 patients and unexposed healthy individuals; and their 108 immunogenicity in "humanized" Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine Altogether, the phylogenetic analysis and genetic distance suggest that the highly contagious 142 and deadly human-SARS-CoV-2 strain originated from bats, likely from either the Bat-CoV-19-ZXC21 143 or Bat-CoV-RaTG13 strains, that spilled over into humans after further mutations and/or recombination 144 with a yet-to-be determined CoV strain from the pangolin, as the likely intermediate animal reservoir. 

GeneBank accession number MN908947.3) 42, 43, 44, 45, 46, 47, 48 . For this, we used multiple databases 

CD4 + T cell epitopes are 100% conserved and common among 81,963 SARS-CoV-2 strains currently 205 circulating in 6 continents ( Fig. 3) . High degree of sequence similarities were also identified in the 206 sequences of most 16 CD4 + T cell epitopes among the SARS-CoV-2 strains and the six strains of 207 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, high degree of sequence 209 similarities were also identified among the SARS-CoV-2 strains and the SL-CoV strains isolated from 210 bats and pangolins. In contrast, a low sequence similarity, of around 20%-40%, was identified among Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine nucleoprotein (N) protein, and 1 epitope from the spike protein (S). The remaining CD4 + T cell 220 epitopes are distributed among the ORF6, ORF7a, ORF7b and ORF8 proteins (Figs. 3 and 12) .

Altogether, these results identified 16 potential CD4 + T cell epitopes from the whole sequence 222 of SARS-CoV-2 that cross-react and have high sequence similarity among 81,963 SARS-CoV-2 223 strains, the main 4 major ''common cold'' Coronaviruses and the SL-CoV strains isolated from bats 224 and pangolins. Similar to CD8 + T cell epitopes, the replicase polyprotein ORF1ab appeared to be the 225 most immunodominant antigen with high number of conserved epitopes that maybe targeted by 226 human CD4 + T cells.

230 immunogenic in "humanized" HLA transgenic mice: Next, we assessed whether the potential 231 SARS-CoV-2 CD4 + and CD8 + T cell epitopes, that are highly conserved between human and animal 232 Coronaviruses, would recall memory CD8 + T cells from COVID-19 patients as well as from healthy 233 individuals, who have never been exposed to SARS-CoV-2 or to COVID-19 patients (i.e. from healthy highly conserved cross-reactive SARS-CoV-2 CD8 + T cell epitopes, significant T cell responses were 

ORF6 4-11 , ORF10 3-11 and ORF10 5-13 ) (Figs. 2D, 4B and 4D) . Moreover, among the 24 SARS-CoV-2 248 epitopes, 11 epitopes recalled memory CD8 + T cells from unexposed healthy individuals (i.e.

ORF1ab 1675-1683 , ORF1ab 3732-3740 , ORF1ab 4283-4290 , ORF1ab 6419-6427 , ORF1ab 6749-6757 , S 2-10 , S 958-966 , S 976-250 984 , S 1220-1228 , ORF10 3-11 , and ORF10 5-13 ) (Figs. 4C and Fig. 4D) . However, the unexposed healthy 251 individuals exhibited a different pattern of CD8 + T cell immunodominance as compared to 252 patients. We then compared the epitopes-specificity and function of memory CD8 + T cells in HLA-253 *A0201-positive COVID-19 patients and healthy individuals (Fig. 4E ) using flow cytometry (Fig. 4F ).

For a better comparison, a similar FACS gating strategy was applied to PBMCs-derived T cells from 255 both COVID-19 and healthy donors (not shown). The COVID-19 patients appeared to have a higher 256 frequency of CD8 + T cells compared to healthy donors (Fig. 4F) . Tetramer staining showed that many 257 of SARS-CoV-2 epitope-specific CD8 + T cells are multifunctional producing IFN-γ, TNF-α and 258 expressing CD69 and CD107 a/b markers of activation and cytotoxicity in COVID-19 patients (Fig. 4F) .

Similar to SARS-CoV-2 memory CD8 + T cells, memory CD4 + T cells specific to several highly 260 conserved SARS-CoV-2 epitopes were detected in both COVID-19-recovered patients and unexposed 261 healthy individuals (Figs. 5B-D). Significant responses were detected in COVID-19 patients against 10 262 epitopes derived from both structural proteins (i.e. S 1-13 , E 26-40 , M 176-190 and N 388-403 ) and non-structural 263 proteins (i.e. ORF1a 1350-1365 , ORF1a 1801-1815 , ORF6 12-26 , ORF7a 3-17 , ORF7a 98-112 , ORF7b 8-22 and Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine proportions of SARS-CoV-2 epitopes-specific CD4 + T cells, expressing CD69, CD107 a/b and TNF-α, 271 were detected using specific tetramers in PBMCs of HLA-DR positive COVID-19 patients, as 272 compared to healthy patients (Figs. 5E and 5F).

The immunogenicity of the identified SARS-CoV-2 human CD4 + and CD8 + T cell epitopes was 

The gating strategy employed in mice is shown in Figs 6B and 7B. Two weeks after the second 281 immunization with the mixture of CD8 + T-cell peptides, 11 out of 24 highly conserved SARS-CoV-2 282 human CD8 + T cell epitope peptides were immunogenic in "humanized" HLA-DR/HLA-A*02:01 double 283 transgenic mice (Figs. 6C and 6D) . The immunogenic epitopes were derived from both structural 284 Spike protein (S 2-10 , S 958-966 , S 1000-1008 and S 1220-1228 ) and Envelope protein (E 20-28 ) and from non-285 structural proteins (i.e. ORF1ab 2363-2371 , ORF1ab 3732-3740 , ORF1ab 5470-5478 , ORF8 [73] [74] [75] [76] [77] [78] [79] [80] [81] 

Altogether, these results indicate that pre-existing memory CD4 + T and CD8 + T cells specific to 292 both structural and non-structural protein antigens and epitopes are present in COVID-19 patients and 293 unexposed healthy individuals. Most T cell epitopes are concentrated in the non-structural proteins 294 which appeared to be more targets for human CD4 + T and CD8 + T cells. These memory T cells Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine recognized highly conserved SARS-CoV-2 epitopes that cross-react with the human and animal 296 Coronaviruses. It is likely that infection with "common cold" Coronavirus induced long-lasting memory 297 CD4 + and CD8 + T cells specific to the structural and non-structural SARS-CoV-2 epitopes in healthy 298 unexposed individuals. Heterologous immunity and heterologous immunopathology orchestrated by 299 these cross-reactive epitope-specific memory CD4 + and CD8 + T cells, following previous multiple 300 exposures to "common cold" Coronaviruses, may have shaped protection vs. susceptibility to SARS-

CoV-2 infection and disease, with a yet-to-be determined mechanism. 

GeneBank accession number MN908947.3) using BepiPred 2.0, with a cutoff of 0.55 (corresponding 307 to a specificity greater than 0.81 and sensitivity below 0.30) and considering sequences having more 308 than 5 amino acid residues 54 . This stringent screening process initially resulted in the identification of 309 28 linear B-cell epitopes (Supplemental Table S3 ). From this pool of 28 potential epitopes, we later 310 selected 10 B-cell epitopes, (19 to 62 amino acids in length), based on: (i) their sequences being 311 highly conserved between SARS-CoV-2, the main 4 major ''common cold'' Coronaviruses (CoV-312 OC43, CoV-229E, CoV-HKU1, and CoV-NL63 55 ), and the SARS-like SL-CoVs that are isolated from 313 bats, civet cats, pangolins and camels; and (ii) the probability of exposure each linear epitope to the Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine interaction similarity scores were observed for RBD-derived epitopes S 369-393 and S 471-501 when 321 molecular docking was performed against the ACE2 receptor (Supplemental Fig. S4B ).

We later determined the ability of each of the 10 B-cell epitopes selected from the Spike 323 protein, that showed a high conservancy between human and animal Coronaviruses, to induce in B6 324 mice SARS-CoV-2 epitope-specific antibody-producing plasma B cells and IgG antibodies (Fig. 9) .

Synthetic peptides corresponding to each linear B cell epitope were produced. Since 4 epitopes were 326 too long to synthetize (e.g. 62 amino acids), they were divided into 2 or 3 short fragments resulting in 327 a total of 17 B-cell epitope peptides (Supplemental Table S3 ). As illustrated in Fig. 9A Ags. The frequency of antibody-producing plasma B cells and the level of IgG antibodies specific to 331 each SARS-CoV-2 B cell epitope were determined in the spleen and in the serum using FACS 332 staining of CD138 and B220 surface markers and IgG-ELISpot and ELISA assays, respectively. The 333 gating strategy used to determine the frequencies of plasma B-cells in the spleen is shown in Fig. 9B .

Out of the 17 Spike B-cell epitopes, 7 epitopes (S 13-37 , S 287-317 , S 524-558 , S 544-578 , S 565-598 , S 601-628 , and 335 S 614-640 ) induced high frequencies of CD138 + B220 + plasma B cells in the spleen of B6 mice (Fig. 9C) .

The IgG ELISpot assay confirmed that 7 out of the 17 Spike B-cell epitopes induced significant 337 numbers of IgG-producing B cells in the spleen (Fig. 9D) . Moreover, significant amount of IgG were 338 detected in the serum of the immunized B6 mice. These IgG antibodies were specific to 5 out of the 17

CD8 + T cell epitopes that are associated with asymptomatic SARS-CoV-2 infection: We next 348 investigated whether antibodies, CD4 + and CD8 + T cells from symptomatic vs. asymptomatic COVID-349 19 patients and unexposed healthy individuals had different profile of epitopes specificities.

The COVID-19 patients were divided into 4 groups depending on the severity of the 351 symptoms: Group 1 that comprised SARS-CoV-2 infected patients that never develop any symptoms 352 (i.e. asymptomatic patients, n = 11); Group 2 with mild symptoms (i.e. Inpatient only, n = 32); Group 3 353 with moderate symptoms (i.e. ICU admission, n = 11) and Group 4 with severe symptoms (i.e. ICU 354 admission +/-Intubation or death, n = 9). As expected, compared to the asymptomatic group, all of 355 the 3 symptomatic groups (i.e. mild, moderate and severe) had higher percentages of comorbidities,

including diabetes (22% to 64%), hypertension (64% to 78%), cardiovascular disease (11% to 18%) 357 and obesity (9% to 50%) ( Table 1 ). The final Group 5 comprised of unexposed healthy individuals 358 (controls), with no history of COVID-19 or contact with COVID-19 patients (n = 10).

The COVID-19 patients that present severe symptoms had significantly higher frequencies of 360 ORF1ab 1675-1683 and ORF1ab 2210-2218 epitopes-specific CD8 + T cells compared to asymptomatic 361 patients (Fig. 10) . In contrast, a significantly higher frequencies of S 691-699 , ORF6 4-11 , and ORF8a 31-39 362 epitope-specific CD8 + T cells were detected in asymptomatic patients and unexposed healthy 363 individuals as compared to COVID-19 patients with moderate to severe symptoms (Fig. 10A ).

However, there were no significant correlations between the overall frequencies of SARS-CoV-2-365 specific CD8 + T cells and the severity of the COVID-19 disease (Fig. 10B , P = 0.6088).

In contrast to CD8 + T cells, we detected a significantly higher frequencies of memory CD4 + T 367 cells specific to 8 highly conserved epitopes in asymptomatic COVID-19 patients compared to 368 symptomatic COVID-19 patients, regardless of level of severity of symptoms ( Fig. 10C , P < 0.005).

Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine COVID-19 symptoms (Fig. 10D , P = 0.0157). Finally, we found the symptomatic patients with the 373 most severe symptoms had significantly higher IgG antibodies specific to some linear B-cell epitopes 374 (e.g. S 544-578 and S 565-598 ) compared to asymptomatic patients, suggesting some Spike-specific 375 antibodies may not be protective, but instead maybe immune enhancing (Fig. 10E , P < 0.05). No 376 significant correlations was found between the overall Spike-specific antibody responses and the low 377 severity of symptoms in COVID-19 patients (Fig. 10F , P > 0.05).

Circulating memory CD4 + and CD8 + T CIRC cell populations are categorized into two major 379 phenotypically distinct sub-populations: effector memory T cells (CD62L low CD44 high T EM ) and central 380 memory T cells (CD62L high CD44 high T CM ) 56, 57 . Next, we investigated whether symptomatic vs.

asymptomatic COVID-19 patients had different frequencies of SARS-CoV-2-specific memory CD4 + 382 and CD8 + T EM and T CM sub-populations (Fig. 11A) . We gated on CD3 + CD4 + T cells and CD3 + CD8 + T 383 cells specific to the immunodominant SARS-CoV-2 epitopes (Fig. 11B) 

Altogether, these results suggest a dichotomy in the phenotype of memory CD4 + and CD8 + T 388 cell sub-populations in COVID-19 patients. More of central memory CD4 + T CM cells and more of 389 effector memory CD8 + T cells appeared present in the blood of COVID-19 patients, regardless of the 390 severity of symptoms. Moreover, we discovered that, in contrast to SARS-CoV-2 epitopes-specific 391 CD8 + T cells and IgG antibodies, high frequencies of IFN-γ-producing CD4 + T cells specific to 8 highly 

have focused primarily on COVID-19 and mainly used the Spike protein as target antigen, our 410 strategy has been to develop a pre-emptive pan-Coronavirus vaccine, that incorporates several 411 SARS-CoV-2 epitopes, and designed to target past, present and future Coronavirus outbreaks.

Towards the goal of developing a multi-epitopes pre-emptive pan-Coronavirus human vaccine; 413 in the present study; we identified the asymptomatic and cross-reactive human B and T cell epitopes 414 of SARS-CoV-2 that are highly conserved among the human SARS-CoVs and animal SL-CoVs. While 415 antiviral SARS-CoV-2-specific antibodies and CD4 + and CD8 + T cell responses appear crucial in 416 protecting asymptomatic COVID-19 patients and convalescent patients, very little information exists 417 with regards to the repertoire of targeted SARS-CoV-2 B and T cell epitopes that are common within a 418 substantial group of human and animal Coronaviruses 45, 60 . The highly conserved human B and T cell 419 epitopes reported in this study have huge implications for the development of an universal pre-420 emptive pan-Coronavirus vaccine to induce (or to boost) neutralizing antibodies (Abs), CD4 + T helpers 421 (Th1), and antiviral CD8 + cytotoxic T-cells (CTL) 21, 61 . Some of our identified epitopes are similar to 422 those recently reported by Grifoni, et al, 45, 60 while other epitopes have never been reported. 

With no universal pan-Coronavirus vaccine is yet available, five short-and long-term COVID-427 19 scenarios may occur: (1) the COVID-19 pandemic will be controlled and disappear, with through 428 implementation of physical distancing/barriers or yet-to-be determined mechanism(s) and factor(s).

Nevertheless, because small outbreaks SARS-CoV-2 virus will still likely occur around the world, 430 maintaining these small outbreaks in check will still require the implementation of preventive 431 Coronavirus vaccination; (2) COVID-19 pandemic will be controlled by current measures, but there 432 will be localized clusters that may occur in some regions and/or countries of the world, which may 433 lead to another pandemic.; (3) A gradual spread of asymptomatic SARS-CoV-2 infections, that are 434 undetectable, hence may later lead to a resumption of another COVID pandemic; (4) This COVID-19 435 outbreak returns and transforms into a seasonal "flu-like" global pandemic; and (5) Other global 436 COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an 437 unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human 438 population. In all five scenarios; a pre-emptive pan-Coronavirus vaccine that incorporates highly 439 conserved and common epitopes could target not only past and current COVID outbreaks, but may 440 also target future Coronavirus outbreaks that may be caused by yet another spillover of bat SL-CoVs 

S5). incorporates multiple human asymptomatic B and CD4 + /CD8 + T cell epitopes that are selected 446 carefully from the whole genome of SARS-CoV-2 for being recognized by antibodies and CD4 + /CD8 + 447 T cells from asymptomatic and convalescent patients that are "naturally protected" from COVID-19.

increase viral load or exacerbate COVID-19 disease 50, 51, 52 . The present study employed a 451 combinatorial approach for designing an all-in-one multi-epitope pan-Coronavirus vaccine candidate 452 (Supplemental Fig. S5 ) by applying highly conserved genome-wide human B-and T-cell epitopes 453 from 12 genome derived antigenic proteins of SARS-CoV-2. While this study focused on HLA-454 A*02:01-restricted epitopes, an HLA class I haplotype that is represented similar to 50% of the human 

inflammatory cytokine storm appears to lead to acute respiratory distress syndrome in many 471 symptomatic individuals 33, 34, 35, 36, 37, 38, 39 . This cytokine storm contributes to the immunopathology 472 seen in severe COVID-19 patients, mandating caution in selecting a potentially protective 473 "asymptomatic" CD4 + and CD8 + T cell epitopes to be incorporated in future pan-Coronavirus vaccines 474 33, 34, 35, 36, 70, 71, 72 . The "asymptomatic" epitopes recognized by CD4 + and CD8 + T cells associated with Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine of a safe and efficient pan-Coronavirus vaccine. Besides, these epitopes can also be used in 477 diagnostics of human and animal Coronaviruses.

While SARS-CoV-2 appeared to originate from the natural reservoirs horseshoe bats 479 (Rhinolophus spp.) there was a lack of clarity around how the virus was passed onto humans 73 .

Questions remain as to whether the highly contagious and deadly SARS-CoV-2 was: (1) transmitted 481 naturally to humans through a yet-to-be identified intermediate animal reservoir; or (2) accidentally 482 transmitted to humans as a "natural" but recombinant bat virus, or even as a man-made "artificial" 483 recombinant bat virus 18, 19, 20, 74 Fig. S5 ) includes several highly conserved human B-, CD4 + and CD8 + T cell epitopes 504 identified from the entire genome sequences of human SARS-CoVs that cross-react and shared with 505 bat and pangolin 76, 77, 78, 79 . Unfortunately, for the past two decades, on one hand the 506 governments decided that developing pre-emptive pan-Coronavirus vaccines is costly. On the other 507 hand, private pharmaceutical companies, which mainly operate for profit, won't invest in potentially 508 unprofitable pre-emptive pan-Coronavirus vaccines for a "Disease X" that doesn't exist yet, or that has 509 yet to emerge. Thus, development of pre-emptive pan-vaccines against potential zoonotic viruses with 510 a higher probability to emerge and spillover into humans should be funded by WHO and/or non-511 governmental nonprofit health organizations. The current ongoing collaborative research efforts 512 should not only focus on developing a vaccine for COVID-19, but should also be oriented towards 513 developing pre-emptive pan-Coronavirus vaccines. Such a proactive vaccine strategy would help fight 514 and contain future local outbreaks and epicenters of highly contagious and deadly zoonotic 515 Coronaviruses, globally, before they become a next deadly pandemic worldwide 21, 61 . Moreover, 516 because it is impossible to predict the time and location of next deadly global pandemic, it is essential 517 to have ready, at least pre-clinically, pan-Coronavirus vaccine candidates that would be quickly 518 implemented in a clinical trial against a substantial group of Coronaviruses before an outbreak 519 spreads into global pandemics.

Our pre-emptive multi-epitope pan-Coronavirus vaccine is highly adaptable to new 521 Coronavirus strains that may appear in the future. If an epitope from a human or an animal 522 Coronavirus mutates, then that epitope can be easily adjusted and replaced in the multi-epitope 523 vaccine with the new mutated epitope 80 . Thus, it is not excluded that the highly conserved B-cell,

CD4 + and CD8 + T cell epitopes identified in this study from bat's Coronavirus variants will mutate, 525 following recombination that often occurs for zoonotic events before an animal SL-CoV spills over into Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine humans. Thus, our pre-emptive multi-epitope pan-Coronavirus vaccine strategy could be easily 527 adapted, not only to any zoonotic bat SL-CoVs that may spill over into humans, but also to any 528 mutations and shifts of SARS-CoV-2 variants that may emerge in the future. This high adaptability is 529 expected to speed up the implementation of a future pre-emptive multi-epitope vaccine before an 530 outbreak spreads into global pandemics. Besides, the multi-epitope vaccine can also be adapted to 531 any antigen delivery system that will deliver the identified highly conserved SARS-CoV-2 epitopes 81 .

In the present study, CD4 + and CD8 + T cells specific to highly conserved SARS-CoV2 epitopes 533 were detected in healthy adults, recruited between 2014 and 2018, who have never been exposed to 534 the SARS-CoV-2 virus. These findings suggest cross-reactive T cell responses between SARS-CoV-2 535 and circulating ''common cold'' Coronaviruses as confirmed by recent reports 60, 82, 83 . However, since 536 we do not have available history of whether the healthy adults used in our study were exposed to any 537 ''common cold'' Coronaviruses, such an assertion may not be conclusive. Among the many circulating 538 ''common cold'' Coronaviruses known to infect humans, four serotypes that cause severe respiratory 539 infections are highly seasonal: CoV-OC43, CoV-229E, CoV-HKU1, and CoV-NL63 55 and appear to 540 have a similar transmission potential to influenza A (H3N2) but their seasonality was more predictable 541 as their outbreaks often emerged in December, peaked in January/February, and began to decrease 542 in March of each year 55 . The human SARS-CoV-2 CD4 + and CD8 + T cell epitopes identified in this 543 study are highly conserved between 81,963 strains of SARS-CoV-2 and CoV-OC43, CoV-229E, CoV-544 HKU1, and CoV-NL63 (Figs. 2D and 3) . Whether these apparent cross-reactive CD4 + and CD8 + T 545 cells play a protective or a harmful role or an entirely negligible role SARS-CoV-2 infection and 546 disease remains to be determined 82, 83 . Nevertheless, since ''common cold'' Coronaviruses are usual 547 in children, it will be interesting to determine whether children who appeared more resistant to COVID-548 19 compared to adults will have robust antiviral memory T cell responses to some asymptomatic 549 epitopes. A stronger CD4 + and CD8 + T cell responses to common Coronavirus epitopes in children 550 would shed some light on the unique situation currently seen in COVID-19 where immune children result would also imply that a pan-Coronavirus vaccine incorporating cross-reactive highly conserved 553 SARS-CoV-2 human CD4 + and CD8 + T cell epitopes in the next pan-Coronavirus vaccine would boost 554 protective T cell immunity that is previously induced by a ''common cold'' Coronavirus, thus protecting 555 not only from seasonal circulating ''common cold'' Coronaviruses, but also from SARS-CoV-2 infection 556 and disease. Currently, we are in the process of determining whether the individuals that were 557 exposed to ''common cold'' Coronaviruses will develop frequent cross-reactive tissue-resident memory 558 CD4 + and CD8 + T cells (T RM cells) that would better protect them from SARS-CoV-2, compared to 559 individuals who have never been exposed to ''common cold'' Coronaviruses. The results from those 560 studies will be the subject of a future report. It is also likely that these cross-reactive SARS-CoV-2 561 human CD4 + and CD8 + T cells might be the result of a heterologous immunity with yet-to-be 562 determined pathogen(s), within or outside the Coronavirus family, initiated by the development of 563 these cross-reactive CD4 + and CD8 + T cells.

It was recently suggested that a majority of recovered patients produce antibodies against 565 SARS-CoV-2 that would protect against re-infections 86 . This concept leads to a so called "immunity 566 passport" or "risk-free certificate" that would enable immune recovered individuals to return to work 567 during the COVID-19 pandemic 87 . However, the quality, the quantity and the epitope specificities of 568 protective antibodies developed by immune recovered individuals remains to be yet determined 88 . In a 569 disagreement with a recent reports 89, 90 , in this study we observed differences in the levels of IgG 570 specific to SARS-CoV-2-Spike epitopes, with some spike-specific humoral responses were enriched 571 among COVID-19 patients with severe symptoms, whereas asymptomatic COVID-19 patients develop 572 rather lower levels of these IgG antibodies. This suggest that high titer IgG antibodies specific to some 573 Spike epitopes might be involved in enhancing immunity while IgG specific to different Spike epitopes 574 may be involved in protection. Accordingly, one cannot rule out that vaccination with the whole 575 attenuated virus or even with whole proteins (e.g. Spike protein) can induce both protective and 576 pathogenic immune responses. Such a vaccine may induce antibodies and T cells specific to Towards A Multi-Epitopes Pre-Emptive Pan-Coronavirus Human Vaccine specific to other "symptomatic" epitopes from the same protein that may actually accelerate the 579 infection exacerbate the disease. Therefore, a multi-epitope vaccine that selectively incorporates 580 selected "asymptomatic" B and T cell epitopes, while excluding "symptomatic" epitopes would be 581 expected to protect from SARS-CoV-2 infection, while avoiding exacerbation of the infection and/or 582 disease. Besides, unfortunately, the concept of "immunity passport" was mainly based on antibodies 583 while ignoring the quality, the quantity and the epitope specificities of T cells developed by immune 584 recovered individuals that may also be involved in protection from a second infection. The present 585 report found that, in contrast to CD8 + T cells, we detected a significantly higher frequencies of memory 586 CD4 + T cells specific to 8 highly conserved epitopes in asymptomatic COVID-19 patients compared to 587 symptomatic COVID-19 patients, regardless of level of severity of symptoms. This. it is likely that, 588 besides antibodies, the SARS-CoV-2 infection may simultaneously induce protective and pathogenic T 589 cells specific to asymptomatic and symptomatic epitopes, respectively.

The highly contagious and transmissibility characteristics of SARS-CoV-2, compared to 591 previous Coronavirus outbreaks, is likely due to its high ability to mutate 91 . Dynamic tracking of variant 592 frequencies revealed that the originally SARS-CoV-2 variant that appeared in January 2020 in Wuhan,

China has a D amino acid in position 614 of the spike protein (D614) mutated into a G amino acid in 

Even though the highly conserved Coronavirus human CD4 + and CD8 + T cell epitopes 610 identified in this report can be enlightening for a pan-Coronavirus vaccine, humans are not 611 immunologically naive, and they often have memory CD4 + and CD8 + T cell populations that can cross-612 react with, and respond to, other infectious agents, a phenomenon termed heterologous immunity.

Therefore, we cannot exclude that some SARS-CoV-2-specific CD4 + and CD8 + T cell epitopes 614 identified in this study are cross-reactive with other viral pathogens-derived epitopes, such as epitopes 615 from circulating seasonal influenza or ''common cold'' Coronaviruses 92, 93 . This may explain, in part, 616 the high proportion of asymptomatic infections with SARS-CoV-2 in the current pandemic. The latter is 617 supported by a recent elegant study that detected SARS-CoV-2-reactive CD8 + and CD4 + T cells in 618 healthy individuals that were never exposed to SARS-CoV-2 60 . SARS-CoV-2-specific, but cross-619 reactive, CD4 + and CD8 + T cells can become activated and modulate the immune responses and 620 clinical outcome of subsequent heterologous SARS-CoV-2 infections. Therefore, T cell cross-621 reactivity may be crucial in protective heterologous immunity instead of damaging heterologous 622 immunopathology, as has been reported in other systems 94 . To confirm SARS-CoV-2 heterologous 623 CD4 + and CD8 + T cell epitopes that may potentially cross-react with other pathogenic (non-

Coronaviruses) epitopes, we are currently comparing the CD4 + and CD8 + T-cell response to those 625 highly conserved SARS-CoV-2 epitopes identified using humans CD4 + and CD8 + T-cell responses to 626 those of "pathogen-free" SARS-CoV-2-infected transgenic mice.

In summary, we report several human "universal" B, and CD4 + and CD8 + T cell target epitopes 628 identified from the whole SARS-CoV-2 genome that are highly conserved and common between previous human SARS and MERS outbreaks 61 ; (2) 81,963 strains of human SARS-CoV-2 that now 631 circulate in six continents; (3) bat-derived SARS-like strains 14, 15 ; and (4) SL-CoV strains isolated from 632 pangolins 95 . While the current COVID-19 pandemic will likely disappear through implementation 633 physical distancing and mass vaccination, another COVID pandemic will likely emerge in coming 634 years (the question is not "if" the question is "when"). This work paves the way for the design and 635 evaluation of "pre-emptive" pan-Coronavirus vaccine candidates that will target not only the current 636 human SARS-CoV-2, but also possible future bat-derived SARS-like Coronavirus strains, that might 637 transition and spill over into humans, thus potentially causing future global outbreaks.

Human study population: Sixty-three COVID-19 patients and ten unexposed healthy 643 individuals, who had never been exposed to SARS-CoV-2 or COVID-19 patients, were enrolled in this 644 study ( Table 1) . Seventy-eight percent were non-White (African, Asian, Hispanic and others) and 22% 645 were white. Forty-four percent were females, and 56% were males with an age range of 26-95 infected patients that never developed any symptoms or any viral diseases (i.e. asymptomatic 654 patients) (n = 11); Group 2 with mild symptoms (i.e. Inpatient only, n = 32); Group 3 with moderate 655 symptoms (i.e. ICU admission, n = 11) and Group 4 with severe symptoms (i.e. ICU admission +/-656 Intubation or death, n = 9). As expected, compared to the asymptomatic group, all of the 3 657 symptomatic groups (i.e. mild, moderate and severe) had higher percentages of comorbidities, 658 including diabetes (22% to 64%), hypertension (64% to 78%), cardiovascular disease (11% to 18%) 659 and obesity (9% to 50%) ( Table 1) 

For each protein, the epitope probability score for each amino acid and the probability of exposure 707 was retrieved. Potential B cell epitopes were predicted using a cutoff of 0.55 (corresponding to a 708 specificity greater than 0.81 and sensitivity below 0.3) and considering sequences having more than 5 709 amino acid residues. This screening process resulted in 28 B-cell peptides (Supplemental Table S3 ).

From this pool, we selected 10 B-cell epitopes with 19 to 62 amino acid lengths in our current study.

Three B-cell epitopes were observed to possess receptor binding domain (RBD) region specific amino 712 acids. Structure-based antibody prediction was performed by using Discotope 2.0, and a positivity 713 cutoff greater than -2.5 was applied (corresponding to specificity greater than or equal to 0.80 and 714 sensitivity below 0.39), using the SARS-CoV-2 spike glycoprotein structure (PDB ID: 6M1D).

6M1D available on the Protein Data Bank. The 6M1D with a structural weight of 334.09 kDa, 719 possesses 2 unique protein chains, 2,706 residues, and 21,776 atoms. In this study, flexible target 720 docking based on an energy-optimization algorithm was carried out on the ligand-binding domain 721 containing ACE2 within the 4GBX structure. Similarity scores were calculated for protein-peptide 722 interaction pairs for each residue. The prediction accuracy is estimated from a linear model as the 723 relationship between the fraction of correctly predicted binding site residues and the template-target 724 similarity measured by the protein structure similarity score and interaction similarity score (S Inter ) 725 obtained by linear regression. S Inter shows the similarity of amino acids of the B-cell peptides aligned 726 to the contacting residues in the amino acids of the ACE2 template structure. Higher S Inter score 727 represents a more significant binding affinity among the ACE2 molecule and B-cell peptides.

Subsequently, molecular docking models were built based on distance restraints for protein-peptide 729 pairs using GalaxyPepDock. Based on optimized energy scores docking models were ranked.

While performing the protein-peptide docking analysis for CD8 + T cell epitope peptides, we 731 used the X-ray Crystal structure of HLA-A*02:01 in complex-4UQ3 available on the Protein Data Bank 732 and for CD4 peptides X-ray crystallographic structure HLA-DM-HLA-DRB1 Complex-4GBX.

Epitope conservancy analysis: The Epitope Conservancy Analysis tool was used to 734 compute the degree of conservancy of CD8 + T cell, CD4 + T cell, and B-cell epitopes within a given 735 protein sequence of SARS-CoV-2 set at 100% identity level. The fraction of protein sequences that 736 contain the epitope, and identity was defined as the degree of similarity or correspondence among 737 two sequences. The CD8 + T cell, and CD4 + T cell epitopes were screened against all the twelve 738 structural and non-structural proteins of SARS-CoV-2 namely YP_009724389. 

After incubation in humidified 5% CO 2 at 37°C for 72 hours, cells were removed by washing (using 838 PBS and PBS-Tween 0.02% solution) and 100 µl of biotinylated secondary anti-IFN-γ antibody (clone 839 7-B6-1, Mabtech) in blocking buffer (PBS 0.5% BSA) was added to each well. Following a 2 hour 840 incubation and washing, HRP-conjugated streptavidin was diluted 1:1000 and wells were incubated 841 with 100 µl for 1 hour at room temperature. Following washing, wells were incubated for 1 hour at 842 room temperature with 100 µl of TMB detection reagent and spots counted with an automated EliSpot

Reader System (ImmunoSpot reader, Cellular Technology).

first coated overnight at 4°C with 10μg/ml of each B cell peptide epitope. Subsequently plates were 847 washed five times with PBS-Tween 0.01% before to start the blocking by adding PBS 1% BSA for 3 h 848 at room temperature, followed by a second wash. Sera of C57BL/6 mice immunized either with pool B 849 cell peptides alum/CpG or adjuvant alone (control) were added into the wells at various dilutions (1/5, 850 1/25, 1/125 and 1/625 or PBS only, in triplicate). Plates were incubated at 4°C overnight with the 851 sera, then washed with PBS-Tween 0.01% before to add anti-mouse IgG antibody (Mabtech -1/500 852 dilution). After the last washing, Streptavidin-HRP (Mabtech -1/1000 dilution) was added for 30 853 minutes at room temperature. Finally, we added 100μl of filtered TMB substrate for 15 minutes and 854 blocked the reaction with H 2 S0 4 before the read-out (OD measurement was done at 450nm on the 855 Bio-Rad iMark microplate reader).

Constructing the Phylogenetic Tree: Phylogenetic analyses were conducted in MEGA X .

The evolutionary history was performed, and phylogenetic tree was constructed by using the 

The tables provide values of calculated scores for each residue. The larger score for the residues 1539 might be interpreted as that the residue might have a higher probability to be part of epitope. The 100%  67%  44%  44%  44%  11%  22%  78%  78%  67%  67%  100%  100%  100%  100%  44%  67%  67%  67%  100%  100%  100%  100%  100%  100%  100%  100%  100%  67%  67%  67%  89% C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y 100%  33%  56%  33%  22%  22%  22%  56%  56%  56%  56%  100%  100%  100%  100%  33%  56%  56%  56%  78%  78%  100%  100%  100%  100%  100%  100%  100%  33%  33%  33%  56%   100%  44%  56%  44%  11%  11%  33%  56%  56%  56%  56%  100%  100%  100%  100%  44%  56%  56%  56%  100%  100%  100%  100%  100%  100%  100%  100%  100%  44%  44%  44%  44%   100%  67%  22%  33%  11%  44%  33%  67%  67%  67%  67%  100%  100%  100%  100%  33%  67%  67%  67%  100%  100%  100%  100%  100%  100%  100%  100%  100%  44%  44%  44%  44%   100%  78%  67%  33%  -33%  -89%  89%  89%  89%  100%  100%  100%  100%  44%  78%  78%  78%  100%  100%  100%  100%  100%  100%  100%  100%  100%  78%  78%  78%  56%   100%  89%  33%  33%  11%  11%  11%  89%  89%  89%  89%  100%  100%  100%  100%  56%  89%  89%  89% 

6 4 2 0 -6 4 3 4 C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y C o n s e rv a n c y O R F 1 a Prakash et al. Fig. 3 

A HLA-A*0201 (+ ) Healthy Individuals

Analysis of SARS-CoV-2 specific CD8 + T cells (FACS)

In vitro stimulation of 0.5 x 10 6 PBMC with 10 μM of each of the 24 potential SARS-CoV-2-derived CD8 + T cell epitope peptides

In silico screening of 24 potential SARS-CoV-2-derived highly conserved CD8 + epitopes that showed high affinity to human HLA-A*0201 class I allele Phenotypic and functional CD8 + T cell responses 1 st Immunization with CD8 + T cell peptide epitopes (50 µM each) S.C.

2 nd Immunization with CD8 + T cell peptide epitopes (25 µM each) S.C. C o n s e r v a n c y S 2 8 7 -3 1 7 S 3 2 9 -3 6 3 C o n s e r v a n c y S 3 6 9 -3 9 3 S 4 4 0 -5 0 1 

Prakash et al. Fig. 9 o r f 1 a b 6 7 4 9 -6 7 5 7 S 2 -1 0 S 6 9 1 -6 9 9 S 9 5 8 -9 6 6 S 9 7 6 -9 8 4 S 1 2 2 0 -1 2 2 8 E 2 0 -2 8 E 2 

In vitro stimulation of 2 x 10 6 PBMC with 10 μM of SARS-CoV-2-derived CD4 + or CD8 + T cell epitope peptides

In silico screening of potential SARS-CoV-2derived CD4 + and CD8 + epitopes that showed high affinity to human HLA-A*0201 class I allele

Phenotype of SARS-CoV-2 specific CD4 + or CD8 + T cells Lymphocytes Singlets CD4 + or CD8 + T cells 

Prakash et al. Fig. 11 B TCM TEM CD44 CD62L COOH ORF1ab3183-3191 FLLNKEMYL 

HLA supertypes and supermotifs: a functional perspective on HLA 52

Identifying HLA supertypes by learning distance functions

HLA-A*0201-restricted CD8+ cytotoxic T lymphocyte epitopes identified 1066 from herpes simplex virus glycoprotein D

BepiPred-2.0: improving sequence-1069 based B-cell epitope prediction using conformational epitopes

Coronavirus Occurrence and Transmission Over 8 Years in the HIVE Cohort 1073 of Households in Michigan

Associations of HLA-A, HLA-B and HLA-C alleles frequency with 1076 prevalence of herpes simplex virus infections and diseases across global populations: 1077 implication for the development of an universal CD8+ T-cell epitope-based vaccine

Two subsets of memory T 1081 lymphocytes with distinct homing potentials and effector functions

Pandemic 1085 Preparedness: Developing Vaccines and Therapeutic Antibodies For COVID-19

Developing Covid-19 Vaccines at Pandemic 1088

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)

SARS-CoV-2-Wuhan (MN908947.3) SARS-CoV-Urbani (AY278741.1) hCoV-HKU1-Genotype-B (AY884001) hCoV-OC43 (KF923903) hCoV-NL63 (NC005831) hCoV-229E (KY983587)

MERS (NC019843) BAT-SL-CoV-WIV16 (KT444582) BAT-SL-CoV-WIV1 (KF367457.1) BAT-SL-CoV-YNLF31C (KP886808.1) BAT-SARS-CoV-RS672 (FJ588686.1) BAT-CoV-RATG13 (MN996532.1) BAT-CoV-YN01 (EPIISL412976) BAT-CoV-YN02 (EPIISL412977) BAT-CoV-19-ZXC21 (MG772934.1) BAT-SARS-RCoV (FJ211859.1)

SARS-CoV-Civet007 (AY572034.1) SARS-CoV-A022 (AY686863.1) SARS-CoV-B039 (AY686864.1) PCoV-GX-P2V(MT072864.1) PCoV-GX-P5E(MT040336.1) PCoV-GX-P5L (MT040335.1) PCoV-GX-P1E (MT040334.1) PCoV-GX-P4L (MT040333.1) PCoV-MP789 (MT084071.1) PCoV-GX-P3B (MT072865.1)

P2S (EPIISL410544) PCoV-Guangdong (EPIISL410721) Camel-CoV-HKU23 (KT368891.1) DcCoV-HKU23 (MN514967.1)