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.
While the current COVID-19 pandemic will likely disappear through implementation of physical distancing, barriers and mass immunization with a COVID-19 vaccine, it is indispensable that a safe and effective pre-emptive vaccine be developed, and in place ready to protect, against another inevitable COVID pandemic that will emerge in the years to come. More than 169 vaccine candidates against COVID-19 are currently being pre-clinically developed globally Many of these vaccines are targeting exclusively the SARS-CoV-2 that causes COVID-19 but no other human and animal Coronaviruses. Among these, eighteen COVID-19 vaccine candidates, using a variety of different approaches - live attenuated virus, viral vectors and sub-unit mRNA or protein/adjuvant – have already entered Phase I/II/III clinical trials. While the current vaccine efforts, have focused primarily on COVID-19 and mainly used the Spike protein as target antigen, our strategy has been to develop a pre-emptive pan-Coronavirus vaccine, that incorporates several 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; in the present study; we identified the asymptomatic and cross-reactive human B and T cell epitopes of SARS-CoV-2 that are highly conserved among the human SARS-CoVs and animal SL-CoVs. While antiviral SARS-CoV-2-specific antibodies and CD4+ and CD8+ T cell responses appear crucial in protecting asymptomatic COVID-19 patients and convalescent patients, very little information exists with regards to the repertoire of targeted SARS-CoV-2 B and T cell epitopes that are common within a substantial group of human and animal Coronaviruses. The highly conserved human B and T cell epitopes reported in this study have huge implications for the development of an universal pre-emptive pan-Coronavirus vaccine to induce (or to boost) neutralizing antibodies (Abs), CD4+ T helpers (Th1), and antiviral CD8+ cytotoxic T-cells (CTL). Some of our identified epitopes are similar to those recently reported by Grifoni, et al, while other epitopes have never been reported. Moreover, in agreement with recent reports, our study revealed a high degree of similarity among SARS-CoV-2, SARS-CoV and bat-SL-CoVs epitope sequences, but not the MERS-CoV epitope sequences
With no universal pan-Coronavirus vaccine is yet available, five short- and long-term COVID-19 scenarios may occur: (1) the COVID-19 pandemic will be controlled and disappear, with through 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, maintaining these small outbreaks in check will still require the implementation of preventive Coronavirus vaccination; (2) COVID-19 pandemic will be controlled by current measures, but there will be localized clusters that may occur in some regions and/or countries of the world, which may lead to another pandemic.; (3) A gradual spread of asymptomatic SARS-CoV-2 infections, that are undetectable, hence may later lead to a resumption of another COVID pandemic; (4) This COVID-19 outbreak returns and transforms into a seasonal “flu-like” global pandemic; and (5) 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. In all five scenarios; a pre-emptive pan-Coronavirus vaccine that incorporates highly conserved and common epitopes could target not only past and current COVID outbreaks, but may also target future Coronavirus outbreaks that may be caused by yet another spillover of bat SL-CoVs into humans.
Unlike most protein/adjuvant or vector-based Coronavirus subunit vaccines, that use either whole proteins or whole DNA/mRNA, and whole attenuated or weakened viruses our multi-epitope Coronavirus vaccine (e.g. the pan-Coronavirus candidate #1 illustrated in Supplemental Fig. S5). incorporates multiple human asymptomatic B and CD4+/CD8+ T cell epitopes that are selected carefully from the whole genome of SARS-CoV-2 for being recognized by antibodies and CD4+/CD8+ T cells from asymptomatic and convalescent patients that are “naturally protected” from COVID-19. This “asymptomatic” vaccine excludes potentially harmful “symptomatic” epitopes that are highly recognized by antibodies and CD4+/CD8+ T cells from symptomatic and hence would otherwise increase viral load or exacerbate COVID-19 disease 50–52. The present study employed a combinatorial approach for designing an all-in-one multi-epitope pan-Coronavirus vaccine candidate (Supplemental Fig. S5) by applying highly conserved genome-wide human B- and T-cell epitopes from 12 genome derived antigenic proteins of SARS-CoV-2. While this study focused on HLA-A*02:01-restricted epitopes, an HLA class I haplotype that is represented similar to 50% of the human population, when epitopes restricted to other HLA-A, HLA-B and HLA-C haplotype including the forecasted population coverage of chosen T cell epitope ensemble (combined HLA class I) is expected to cover 99.8% of the worldwide human population regardless of race and ethnicity. In addition, for a wider vaccine coverage (i.e., close to 99%), our multi-epitope pan-Coronavirus vaccine platform would be easily adapted to include CD8+ T cell epitopes for other HLA supertypes that are distributed in the various human populations. The polymorphic HLA molecules can be clustered into a handful of HLA-A supertypes that bind largely overlapping peptide repertoires . Moreover, such a multi-epitope vaccine would be easily adapted to exclude undesirable epitopes that are restricted to HLA-B*44 and HLA-C*01 alleles which appear to correlate with SARS-CoV-2 virus spreading across certain countries and HLA-B*35 allele which appear to be associated with severe pneumonia developed by SARS-CoV-2 in young patients.
Control of the SARS-CoV-2 viral infection and disease in asymptomatic individuals requires collaborations among helper CD4+ T cells, neutralizing antibodies, IFN-γ-producing effector CD4+ and cytotoxic CD8+ T cells. While SARS-CoV-2-induced antibody and CD4+ and CD8+ T cell responses are critical to reducing viral infection, in a majority of asymptomatic individuals, an excessive pro-inflammatory cytokine storm appears to lead to acute respiratory distress syndrome in many symptomatic individuals. This cytokine storm contributes to the immunopathology seen in severe COVID-19 patients, mandating caution in selecting a potentially protective “asymptomatic” CD4+ and CD8+ T cell epitopes to be incorporated in future pan-Coronavirus vaccines. The “asymptomatic” epitopes recognized by CD4+ and CD8+ T cells associated with “natural protection” seen in asymptomatic individuals would be highly informative in the development of a safe and efficient pan-Coronavirus vaccine. Besides, these epitopes can also be used in diagnostics of human and animal Coronaviruses.
While SARS-CoV-2 appeared to originate from the natural reservoirs horseshoe bats (Rhinolophus spp.) there was a lack of clarity around how the virus was passed onto humans. Questions remain as to whether the highly contagious and deadly SARS-CoV-2 was: (1) transmitted naturally to humans through a yet-to-be identified intermediate animal reservoir; or (2) accidentally transmitted to humans as a “natural” but recombinant bat virus, or even as a man-made “artificial” recombinant bat virus. Our study revealed that two SL-CoVs strains, called RATG13 and RmYN02, found in horseshoe bats Rhinolophus affinis that live in caves of Yunnan province, shared 96% of their genetic sequence with the sequences of over 81,000 SARS-CoV-2 strains and were phylogenetically the closest to the first human SARS-CoV-2 strain reported from Wuhan. Evidence suggests that the previous SARS-CoV passed on to humans from bats through civets, as intermediate animal reservoirs, while the previous MERS-CoV passed to humans from bats through camels as intermediate animal reservoir. Sequence alignments in study identified the pangolin as the likely intermediate animal reservoir of SARS-CoV-2. Regardless of the intermediate animal reservoir of SARS-CoV-2, it appears that all the three deadly Coronavirus outbreaks in the 21st century have originated from bats as natural hosts. The present study highlights the utility to identify highly conserved immune target epitopes that are common among the pre-pandemic SL-CoVs strains currently harbored in Rhinolophus spp., horseshoes bats and the post-pandemic human SARS-CoVs. The conserved epitopes between human and animal Coronaviruses would be excellent immune targets to be incorporated in pre-emptive pan-Coronavirus vaccines.
It is inevitable that other COVID-like outbreaks, caused by yet another spillover of a bat SL-CoV, could lead to another COVID-like pandemic with global health, social and economic disasters in the years to come. However, because it is almost impossible to predict which viral strain might cause the next Coronavirus pandemic, it is urgent to develop a pan-Coronavirus vaccine that targets a wide range of human and animal Coronavirus strains. Unlike conventional monovalent vaccines made from epitopes selected from a single virus strain, our pre-emptive multi-epitope pan-Coronavirus vaccine (Supplemental Fig. S5) includes several highly conserved human B-, CD4+ and CD8+ T cell epitopes identified from the entire genome sequences of human SARS-CoVs that cross-react and shared with bat and pangolin SL-CoVs.
Unfortunately, for the past two decades, on one hand the governments decided that developing pre-emptive pan-Coronavirus vaccines is costly. On the other hand, private pharmaceutical companies, which mainly operate for profit, won’t invest in potentially unprofitable pre-emptive pan-Coronavirus vaccines for a “Disease X” that doesn’t exist yet, or that has yet to emerge. Thus, development of pre-emptive pan-vaccines against potential zoonotic viruses with a higher probability to emerge and spillover into humans should be funded by WHO and/or non-governmental nonprofit health organizations. The current ongoing collaborative research efforts should not only focus on developing a vaccine for COVID-19, but should also be oriented towards developing pre-emptive pan-Coronavirus vaccines. Such a proactive vaccine strategy would help fight and contain future local outbreaks and epicenters of highly contagious and deadly zoonotic Coronaviruses, globally, before they become a next deadly pandemic worldwide. Moreover, because it is impossible to predict the time and location of next deadly global pandemic, it is essential to have ready, at least pre-clinically, pan-Coronavirus vaccine candidates that would be quickly implemented in a clinical trial against a substantial group of Coronaviruses before an outbreak spreads into global pandemics.
Our pre-emptive multi-epitope pan-Coronavirus vaccine is highly adaptable to new Coronavirus strains that may appear in the future. If an epitope from a human or an animal Coronavirus mutates, then that epitope can be easily adjusted and replaced in the multi-epitope vaccine with the new mutated epitope. 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, following recombination that often occurs for zoonotic events before an animal SL-CoV spills over into humans. Thus, our pre-emptive multi-epitope pan-Coronavirus vaccine strategy could be easily adapted, not only to any zoonotic bat SL-CoVs that may spill over into humans, but also to any mutations and shifts of SARS-CoV-2 variants that may emerge in the future. This high adaptability is expected to speed up the implementation of a future pre-emptive multi-epitope vaccine before an outbreak spreads into global pandemics. Besides, the multi-epitope vaccine can also be adapted to any antigen delivery system that will deliver the identified highly conserved SARS-CoV-2 epitopes.
In the present study, CD4+ and CD8+ T cells specific to highly conserved SARS-CoV2 epitopes were detected in healthy adults, recruited between 2014 and 2018, who have never been exposed to the SARS-CoV-2 virus. These findings suggest cross-reactive T cell responses between SARS-CoV-2 and circulating ‘‘common cold’’ Coronaviruses as confirmed by recent reports. However, since we do not have available history of whether the healthy adults used in our study were exposed to any ‘‘common cold’’ Coronaviruses, such an assertion may not be conclusive. Among the many circulating ‘‘common cold’’ Coronaviruses known to infect humans, four serotypes that cause severe respiratory infections are highly seasonal: CoV-OC43, CoV-229E, CoV-HKU1, and CoV-NL63 and appear to have a similar transmission potential to influenza A (H3N2) but their seasonality was more predictable as their outbreaks often emerged in December, peaked in January/February, and began to decrease in March of each year. The human SARS-CoV-2 CD4+ and CD8+ T cell epitopes identified in this study are highly conserved between 81,963 strains of SARS-CoV-2 and CoV-OC43, CoV-229E, CoV-HKU1, and CoV-NL63. Whether these apparent cross-reactive CD4+ and CD8+ T cells play a protective or a harmful role or an entirely negligible role SARS-CoV-2 infection and disease remains to be determined. Nevertheless, since ‘‘common cold’’ Coronaviruses are usual in children, it will be interesting to determine whether children who appeared more resistant to COVID-19 compared to adults will have robust antiviral memory T cell responses to some asymptomatic epitopes. A stronger CD4+ and CD8+ T cell responses to common Coronavirus epitopes in children would shed some light on the unique situation currently seen in COVID-19 where immune children tend to be more resistant to SARS-CoV-2 infection and disease, as compared to adults . Such a result would also imply that a pan-Coronavirus vaccine incorporating cross-reactive highly conserved SARS-CoV-2 human CD4+ and CD8+ T cell epitopes in the next pan-Coronavirus vaccine would boost protective T cell immunity that is previously induced by a ‘‘common cold’’ Coronavirus, thus protecting not only from seasonal circulating ‘‘common cold’’ Coronaviruses, but also from SARS-CoV-2 infection and disease.
Currently, we are in the process of determining whether the individuals that were exposed to ‘‘common cold’’ Coronaviruses will develop frequent cross-reactive tissue-resident memory CD4+ and CD8+ T cells (TRM cells) that would better protect them from SARS-CoV-2, compared to individuals who have never been exposed to ‘‘common cold’’ Coronaviruses. The results from those studies will be the subject of a future report. It is also likely that these cross-reactive SARS-CoV-2 human CD4+ and CD8+ T cells might be the result of a heterologous immunity with yet-to-be determined pathogen(s), within or outside the Coronavirus family, initiated by the development of these cross-reactive CD4+ and CD8+ T cells.
It was recently suggested that a majority of recovered patients produce antibodies against SARS-CoV-2 that would protect against re-infections. This concept leads to a so called “immunity passport” or “risk-free certificate” that would enable immune recovered individuals to return to work during the COVID-19 pandemic. However, the quality, the quantity and the epitope specificities of protective antibodies developed by immune recovered individuals remains to be yet determined. In a disagreement with a recent reports in this study we observed differences in the levels of IgG specific to SARS-CoV-2-Spike epitopes, with some spike-specific humoral responses were enriched among COVID-19 patients with severe symptoms, whereas asymptomatic COVID-19 patients develop rather lower levels of these IgG antibodies. This suggest that high titer IgG antibodies specific to some Spike epitopes might be involved in enhancing immunity while IgG specific to different Spike epitopes may be involved in protection.
Accordingly, one cannot rule out that vaccination with the whole attenuated virus or even with whole proteins (e.g. Spike protein) can induce both protective and pathogenic immune responses. Such a vaccine may induce antibodies and T cells specific to “asymptomatic” epitopes that are protective while, at the same time, may induce antibodies and T cells specific to other “symptomatic” epitopes from the same protein that may actually accelerate the infection exacerbate the disease. Therefore, a multi-epitope vaccine that selectively incorporates selected “asymptomatic” B and T cell epitopes, while excluding “symptomatic” epitopes would be expected to protect from SARS-CoV-2 infection, while avoiding exacerbation of the infection and/or disease. Besides, unfortunately, the concept of “immunity passport” was mainly based on antibodies while ignoring the quality, the quantity and the epitope specificities of T cells developed by immune recovered individuals that may also be involved in protection from a second infection. The present report found that, in contrast to CD8+ T cells, we detected a significantly higher frequencies of memory CD4+ T cells specific to 8 highly conserved epitopes in asymptomatic COVID-19 patients compared to symptomatic COVID-19 patients, regardless of level of severity of symptoms. This. it is likely that, besides antibodies, the SARS-CoV-2 infection may simultaneously induce protective and pathogenic T cells specific to asymptomatic and symptomatic epitopes, respectively.
The highly contagious and transmissibility characteristics of SARS-CoV-2, compared to previous Coronavirus outbreaks, is likely due to its high ability to mutate. Dynamic tracking of variant 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 the same position 614 of the spike protein (D614) while traveling to Europe and then to the Americas. The resulting new G614 SARS-CoV-2 variant may have a fitness advantage and appears to be the dominant pandemic variant in Europe and in the United States, leading to a higher magnitude of infection, a higher upper respiratory tract viral loads (although not with increased disease severity), compared to the original Wuhan SARS-CoV-2 variant. The SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. In this context, our anti-viral multi-epitope pan-Coronavirus vaccine candidate #1, which includes the Spike B8 epitope with D amino acid in position 14, could be adapted to target the new G614 SARS-CoV-2 variant by replacing the mutated B8 epitope with a G amino acid in the position 614. Thus, our multi-epitope pan-Coronavirus vaccine strategy could be easily adapted, not only to any zoonotic bat SL-CoVs that may spill over into humans in the future, but also to any mutations and shifts of future SARS-CoV-2 variants. Evidence of strong purifying selection around the receptor binding domains (RBD) in the Spike gene and in other genes among bat, pangolin and human Coronaviruses, indicating similarly strong evolutionary constraints in different host species has been reported.
Even though the highly conserved Coronavirus human CD4+ and CD8+ T cell epitopes identified in this report can be enlightening for a pan-Coronavirus vaccine, humans are not immunologically naive, and they often have memory CD4+ and CD8+ T cell populations that can cross-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 identified in this study are cross-reactive with other viral pathogens-derived epitopes, such as epitopes from circulating seasonal influenza or ‘‘common cold’’ Coronaviruses. This may explain, in part, the high proportion of asymptomatic infections with SARS-CoV-2 in the current pandemic. The latter is supported by a recent elegant study that detected SARS-CoV-2-reactive CD8+ and CD4+ T cells in healthy individuals that were never exposed to SARS-CoV-2. SARS-CoV-2-specific, but cross-reactive, CD4+ and CD8+ T cells can become activated and modulate the immune responses and clinical outcome of subsequent heterologous SARS-CoV-2 infections. Therefore, T cell cross-reactivity may be crucial in protective heterologous immunity instead of damaging heterologous immunopathology, as has been reported in other systems. To confirm SARS-CoV-2 heterologous 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 highly conserved SARS-CoV-2 epitopes identified using humans CD4+ and CD8+ T-cell responses to 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 identified from the whole SARS-CoV-2 genome that are highly conserved and common between SARS-CoV-2 Wuhan strain and: (1) circulating ‘‘common cold’’ human Coronaviruses that caused previous human SARS and MERS outbreaks; (2) 81,963 strains of human SARS-CoV-2 that now circulate in six continents; (3) bat-derived SARS-like strains; and (4) SL-CoV strains isolated from pangolins. While the current COVID-19 pandemic will likely disappear through implementation physical distancing and mass vaccination, another COVID pandemic will likely emerge in coming years (the question is not “if” the question is “when”). This work paves the way for the design and evaluation of “pre-emptive” pan-Coronavirus vaccine candidates that will target not only the current human SARS-CoV-2, but also possible future bat-derived SARS-like Coronavirus strains, that might transition and spill over into humans, thus potentially causing future global outbreaks.
Reference & source information: https://www.biorxiv.org/
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