The world is facing the rising emergency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since December 2019 and the outbreak of coronavirus disease 2019 (COVID-19) has caused a global public health and economic crisis.
Many countries are being forced to introduce strict limitations in order to reduce risk of contagion and COVID-19 is considered pandemic since 11th March 2020 [1] [2] [3] . Given the severity of the disease, vaccines and therapeutics for SARS-CoV-2 are urgently needed [1] . Accumulating evidence suggests that the human CD4+ and CD8+ T cells can response to SARS-CoV-2 [4-7], however, the knowledge about immune reactions against SARS-CoV2 in human body is still partial [1] .
Mycobacterium bovis that protects against tuberculosis. A preclinical study has shown that BCG vaccination induces genome-wide epigenetic reprogramming of human monocytes that correlates with protection against experimental viral infection [8] .
Although conflicting results exist [9] [10] [11] , recent epidemiological studies have shown that a possible association of BCG vaccination program with decreased COVID-19-related risks, suggesting that BCG vaccination may provide protection against COVID- 19 [2, 3, 10, 12, 13] .
Clinical trials to determine whether BCG vaccine can protect healthcare workers from the COVID-19 are currently underway (NCT04327206, NCT04328441, NCT04348370) [14] . These clinical studies have been initiated based on the idea that BCG vaccine may have non-specific immune protective effects against viral infections including COVID-19, highlighting the importance of innate immunity [2, 3]. Studies have shown that BCG vaccination induces histone modifications and epigenetic reprogramming of innate immune cells, resulting in a more active innate immune response upon re-stimulation and a decreased susceptibility to respiratory tract infections, which has been termed trained immunity [8, 15, 16] .
We thought that understanding the mechanism of protective effect of BCG vaccine against SARS-CoV2 may guide the development of COVID-19 vaccine. To defend the body against viral infections, the cooperation between innate (non-specific) and adaptive (specific) immune system plays a crucial role [1, 17] . Adaptive immunity, which is characterized by three unique features: specificity, diversity, and memory, is essential for regulating viral infections [17] . Therefore, we hypothesized that BCG may carry similar T cell epitopes with SARS-CoV2. If similar T cell epitopes with SARS-CoV2 are included in the amino-acid sequences of Mycobacterium bovis, BCG vaccination may activate not only non-specific innate immune system but also adaptive immune system and induce memory T cells specific to SARS-CoV2, which leading to a lower COVID-19 growth rate in countries with known BCG policy.
Cross-reactivity is a term that was used to indicate unexpected reactivity to targets that differed from those used to initially define the T cell clone [17, 18] . Studies revealed that T cells can recognize dissimilar epitopes [17] . Importantly, T cell crossreactivity have been reported for cytotoxic T lymphocyte (CTL) responses to viruses including coronaviruses [5, 6, 17] . It has been shown that CTL elicited during a primary viral infection also can respond when the same host is re-infected with unrelated viruses in preclinical murine model [18] . By mapping the different viral epitopes to which a particular T cell clone responds, it was demonstrated that T cells can recognize similar but not identical peptides, suggesting that T cells specific to SARS-CoV2 can be generated by BCG vaccination if Mycobacterium bovis contains similar T cell epitopes with SARS-CoV2 [17, 19] . Intriguingly, we found that Mycobacterium bovis has total seven similar 9amino acids sequences, which include only one amino acid substitution, with SARS-CoV2. Six out of 7 sets of different but closely related peptides between SARS-CoV2
and Mycobacterium bovis are shown in Table 1 . Unexpectedly, we also found that Mycobacterium bovis contains many 7-amino acid sequences completely identical to SARS-CoV2. More than 40 identical heptamer amino acid sequences with SARS-CoV2 were found in Mycobacterium bovis BCG str. Tokyo 172 (data not shown).
To evaluate whether the seven similar 9-amino acids sequences can bind to common HLA class I (HLA-A, -B, or -C alleles) and can be recognized by CTL, the amino acid sequences of both SARS-CoV2 and Mycobacterium bovis were analyzed by two computer algorithms; IEDB analysis resource (T cell epitope prediction tool, http://tools.iedb.org/main/tcell/) and NetMHCpan 4.1 (http://www.cbs.dtu.dk/services/NetMHCpan/).
One out of 7 sets of these similar peptides (SARS-CoV2, ELAKMVSLD;
Mycobacterium bovis, ELAKLVSLD) was not predicted to bind to any HLA class I molecules tested. However, analyses using IEDB analysis resource demonstrated that six of 7 sets of different but closely related peptides between SARS-CoV2 and Mycobacterium bovis had moderate to high binding affinity for multiple common HLA class I molecules ( Table 1) . Analyses using NetMHCpan 4.1 demonstrated that four sets of these similar peptides had weak to high binding affinity for common HLA class I molecules ( Table 2) . These results suggest that the similar peptides between SARS-CoV2 and Mycobacterium bovis can be presented by common HLA class I and have the potential to induce cross-reactive T cells.
Recent studies have reported that BCG vaccination has the potential to provide protective effects against viral infections [8, 16] . In addition, epidemiological studies suggested a possible association of BCG vaccination program with decreased COVID-19-related risks [2, 8, 10, [12] [13] [14] . Clinical studies have been initiated based on the idea that BCG vaccine has non-specific immune protective effects against COVID-19 [10, 14] .
However, the essential role of vaccination is to generate immunological memory, which is an exclusive property of "adaptive" or acquired" immune system, to viral infections [17] . Antigen-specific T cell receptor expressing T cells proliferate and differentiate in response to a primary infection and remain in the host after resolution of the infection. When host immune system encounter reinfection, memory T cell clones equipped with antiviral effector functions can rapidly expand and confer resistance to reinfection. Effector CD8+ T cells (called cytotoxic T lymphocytes, or CTLs) play a crucial role in destroying virus-infected cells. CTLs recognize viral-derived peptide-HLA complexes and kill specifically viral-infected cells. Importantly, recent studies suggested that cross-reactive T cell recognition between "common cold" coronaviruses and SARS-CoV-2 can be exist [5, 6]. Cross-reactive CTL epitopes have been shown to exist between human immunodeficiency virus and Mycobacterium tuberculosis [17, 19] . Therefore, we hypothesized that BCG may carry similar T cell epitopes with SARS-CoV2, which leading to the potential to generate cross-reactive T cells response to SARS-CoV2.
We investigated the similarity of amino acid sequences between SARS-CoV2 and Mycobacterium bovis and found that BCG contains closely similar 9-amino acids sequences with SARS-CoV2. HLA binding affinity analyses in silico demonstrated that these closely similar 9-mer peptides can be T cell epitopes. It has been known that HLA-class I molecules can bind peptides 8 amino acids in length and induce antigenspecific T cells [20, 21] . In addition to seven similar 9 amino acids sequences, we found more than 40 identical heptamer amino acids sequences with SARS-CoV2 in Mycobacterium bovis BCG str. Tokyo 172. Although we did not assess the binding capacity of the 8-amino acids sequences of BCG including the identical heptamer amino acids sequences with SARS-CoV2, our study implies that BCG may contains amino acids sequences which have the potential to induce cross-reactive T cells against SARS-CoV-2 other than the similar 9-mer peptides.
In a recent study, NetMHCpan 4 has been used to predict potential T cell epitopes derived from SARS-CoV2 [21, 22] . Thus, we have used two computer algorithms, NetMHCpan 4.1 and IEDB analysis resource, to predict HLA class I-binding peptides. We have analyzed the closely related 9-mer amino acids sequences between SARS-CoV2 and Mycobacterium bovis by using these two computer algorithms and the analyses showed different results ( Table 1 and Table 2 ). Among these similar peptides, both computer algorithms demonstrated that the sequence 1, 2, 3, and 4 have good binding affinity for common HLA class I molecules, suggesting that these four closely related 9-mer peptides are candidates for further investigation. CTLs. In current study, we evaluate whether the similar 9-amino acids sequences can bind to common HLA class I molecules by using publicly available computer algorithms predicting HLA class I-binding peptides. However, we did not evaluate natural processing of the closely similar 9-mer peptides or binding to specific HLA class I molecules on human cells, which need be validated by future research. against viral infection requires CD4+ T cell activation [17, 26] . BCG vaccine has been shown to induce human memory CD4+ T cells [27] , suggesting BCG vaccine may have the potential to induce cross-reactive memory T cells efficiently.
Data shown in this study are entirely originated form in silico analyses. Thus, our hypothesis needs to be further investigated. Although we are unable to prove our hypothesis at this time, our hypothesis proposed here could be validated by in vitro experiments. First, we need to induce HLA-class I-restricted CTL clones that specifically recognize Mycobacterium bovis-derived peptides by using candidate peptides ( Table 1 and Table 2 ) and healthy donor-derived peripheral blood mononuclear cells. Second, SARS-CoV2 infected target cells need to be prepared from the same donor-derived and SARS-CoV-2 entry receptor ACE2 (angiotensin converting enzyme 2)-expressing cells. Third, cytotoxicity and cytokine-release assays need to be done to evaluate the specific SARS-CoV2-infected cell recognition and cytotoxic function of Mycobacterium bovis-specific CTL clones by using these effector and target cells.
In conclusion, although the mechanisms of protective effect of BCG vaccine against SARS-CoV2 need further investigation, our results and accumulating evidence suggest that BCG vaccine has the potential to generate cross-reactive T cells against SARS-CoV-2. The present study may provide new insights into the immune response to 