Role of BCG in Reduction of the Spread and Severity of COVID-19

Bacille Calmette Guerin (BCG) vaccine has existence of more than 80 years with its widest use among all currently available vaccines [1]. BCG vaccine developed from a virulent attenuated M. bovis strain against tuberculosis (TB) by deleting more than 14 different regions in the genome of Mycobacterium [2]. Several countries are having high prevalence rate of TB, BCG vaccine is used as preventive measure for tubercle meningitis in children. The countries with low risk usually do not include BCG vaccine in their vaccination schedule. BCG is administered immediately to new-borns within a few hours or days of birth. BCG vaccine has 70% potency against meningitis and miliary tuberculosis, whereas the efficacy against pulmonary TB, 52% was reported [3-6]. The intracellular surviving ability of Mycobacteria in phagocytic cells has been tackled with the development of a vaccine with front focus on cell-mediated immunity [7]. The innate immunity being the barrier for every infection has proved to reduce the severity with early recovery. Phagocytosis is the wellestablished system for the recognition and killing of microorganisms [7]. Various studies have shown the role of BCG vaccine in multiple signalling pathways that have the potent to stimulate the innate myeloid and lymphoid cellular population. Dendritic cells, macrophages, monocytes and neutrophils are activated by BCG vaccine and eventually induce T cell mediated immunity [8,9]. Several studies have shown the reduction in hospitalization due to respiratory infections in case of BCG vaccinated individuals. BCG vaccination has been reduced child mortality all over the world, including Haiti, India, Sweden, South or Southeast Asia, and the United Kingdom [10-13]. A high childhood mortality rate in Guinea-Bissau associated with unclassified fever or malaria associated to be diminished with BCG-vaccination. Abstract


Introduction
Bacille Calmette Guerin (BCG) vaccine has existence of more than 80 years with its widest use among all currently available vaccines [1]. BCG vaccine developed from a virulent attenuated M. bovis strain against tuberculosis (TB) by deleting more than 14 different regions in the genome of Mycobacterium [2]. Several countries are having high prevalence rate of TB, BCG vaccine is used as preventive measure for tubercle meningitis in children. The countries with low risk usually do not include BCG vaccine in their vaccination schedule. BCG is administered immediately to new-borns within a few hours or days of birth. BCG vaccine has 70% potency against meningitis and miliary tuberculosis, whereas the efficacy against pulmonary TB, 52% was reported [3][4][5][6]. The intracellular surviving ability of Mycobacteria in phagocytic cells has been tackled with the development of a vaccine with front focus on cell-mediated immunity [7]. The innate immunity being the barrier for every infection has proved to reduce the severity with early recovery. Phagocytosis is the wellestablished system for the recognition and killing of microorganisms [7]. Various studies have shown the role of BCG vaccine in multiple signalling pathways that have the potent to stimulate the innate myeloid and lymphoid cellular population. Dendritic cells, macrophages, monocytes and neutrophils are activated by BCG vaccine and eventually induce T cell mediated immunity [8,9]. Several studies have shown the reduction in hospitalization due to respiratory infections in case of BCG vaccinated individuals. BCG vaccination has been reduced child mortality all over the world, including Haiti, India, Sweden, South or Southeast Asia, and the United Kingdom [10][11][12][13]. A high childhood mortality rate in Guinea-Bissau associated with unclassified fever or malaria associated to be diminished with BCG-vaccination. BCG vaccine stimulates the innate immune response or cell-mediated immune response against similar types of pathogens by developing the immune memory. Several studies proved that the BCG vaccine protects from various types of infections like parasitic, bacterial, and viral by its non-specific activity for the pathogens causing pneumonialike pathology. Maternal vaccination against the measles might be associated with child mortality reduction by maternal antibodies. This might be sharing the same mechanism, which acts in case of BCG to enhance the nonspecific efficacy [14]. Intravenous injection of BCG in mice has reported to protect against Babesia and Plasmodium species and proved that it is not associated with specific antibody [15,16]. BCG may have role in the protection beyond the target pathogens that could elevate through the memory of innate immune response or heterologous lymphocyte activation. BCG vaccine has also shown a role in the reduction of acute lower respiratory tract infections (ALRI) risk among vaccinated children as compared to the non-vaccinated child population [17,18]. Apart from TB, the BCG vaccine also showed a specific role in children's fatality rate reduction by its non-specific potential against unrelated pathogens [10,19]. Effect of the BCG vaccine in other respiratory tract infections reported in the older Indonesian population [20]. A clinical trial conducted in Japan on the BCG vaccination in aged tuberculinnegative population with pneumonia that shows adequate protection against pneumonia [21]. Several studies showed a 40% reduction in mortality by malaria, sepsis, respiratory infections, and leprosy in BCG vaccinated people, conducted in West Africa [22][23][24][25][26]. A study conducted in the South African adolescent population also showed that the BCG vaccine reduces upto 70% respiratory tract infections [27].
A study reported that BCG vaccinated infants' exhibit upregulated IgG production against the antigens of the hepatitis B vaccine [28]. BCG vaccination showed the association with IgG's high titer against H-influenzae type b polysaccharide, pneumococcal capsular polysaccharide antigens, and tetanus toxoid in vaccinated and non-BCG vaccinated Australian's infants' population [29]. Clinical trials also showed that BCG decreases unrelated respiratory tract infections [21,30]. Moreover, viruses are specific for their hosts with intracellular obligate parasitic nature. Viruses become more pathogenic when these change the host or undergo antigenic mutations. Innate immune response might eliminate the viruses and virusinfected cells. Various clinical trials launched intending to generate data from all around the world to find out the most efficient treatment for COVID-19. However, it is not possible to prevent the spread of any infection through the vaccination. Vaccination might be able to increase the immune response by reducing the severity of infections. SARS viruses also reported in the respiratory tract related medical conditions like inflammation, pneumonia, etc., and BCG already reported in the reduction of respiratory tract related infection after vaccination. A study reported that BCG has the potential to protect individuals from infection up to 60 years of age. A high mortality rate has found among the aged and co-morbid population infected with SARS-CoV-2 [31]. The mechanism of innate immune response plays an essential role for the elimination of pathogens. The versatile existence and diversity at every level of life in evolutionary tree has set up its well-marked role.

BCG Induced Immune Response
BCG involves in the stimulation of Pathogen Recognition Receptors (PRRs) like Toll-like receptors (TLR) receptors and nucleotide-binding oligomer domain-like receptors (NOD) [8,32] and activates the innate immune system. BCG antigens mainly act on TLR2, TLR4, TLR7/8 and TLR9 receptors and initiate the activation of macrophages and DCs later on maturation. TLRs further start the cascade through the activation of the MYD88 adapter molecule, which helps in the TRAF6 (TNF receptor-associated factor 6), IRAK (IL1 receptor-associated kinase), and MAPK (Mitogen-activated protein kinase) activations. TRAF6, IRAK, and MAPK consequently activate the nuclear transcription factor (NF-kB), which activates the genes associated with the secretion of the pro-inflammatory cytokine [33].
The mechanism of BCG vaccine-induced immune response and stimulates the c chemokine and cytokines secretion in the microenvironment that besides spurs other immune cells in a non-specific manner. BCG vaccine stimulates the secretion of different cytokines like IL-2, TNF-α, and IFN-γ by the activation of macrophages and T cells. IFN-γ secreting T cells showed the CCR7+ CD62L phenotype expressing the central memory T cells in an invitro experiment [34] and also found in neonates till 27 weeks of BCG vaccination [35]. However, BCG vaccination also stimulates the smaller subset of T effector cells. IFN-γ secretion regulates by the IL-10 secreted through T regulatory cells (Tregs). BCG might activate the Tregs with CD4+CD25+ phenotype that acts non-specifically against non-TB related health problems [36]. Tregs cells stimulated by BCG secrete cytokines such as TGF-β and IL-10 [37]. Sebina et. al., have shown that the PPD-specific IFN-γ levels are higher in BCG vaccinated individuals whereas lower in unvaccinated individuals. The study has proved the robust activation of long-lived T cells in association with BCG vaccination [38]. Professional Antigen Presenting Cells (APCs) represent BCG antigens to the helper T-cells by using the MHC molecules. These interactions need to expressions of both types of MHCs molecules identified through TCR of CD4 + T-cells and CD8 + T-cells [39]. T-cells differentiation into Th-17 and Th-22 depends on the levels of cytokines and their downstream signaling. Th-17 cells are differentiated from T-cells by the IL-6, IL-23, and TGFβ cytokines actions, which produce the cytokine IL-17. Whereas, Th-22 cells differentiate by the action of IL-6 and TNF-α on Th-cells that secrets IL-22 [40]. BCG vaccine also stimulates the differentiation of mononuclear cells into a specific group of cells that are BCG Activated Killer cells (BAK) [41]. Targeted killing cells are lymphocyte-activated killer cells (LAK), CD8+, and CD56+ that help in the clearance of pathogens. Th-1 cytokines such as IL-2 and IFN-γ activate the BAK cells for cytotoxicity [41][42][43] (Figure 1 [44]).
BCG vaccine helps in the reduction of inflammation and symptoms of infectious diseases by inhibiting the inflammasome activation via Zinc-metalloproteases [45]. Zinc signaling exhibits an essential role in the immune response against viral infections by maintaining the proper immune functions [46]. A study showed that the BCG mutant zmp1 deletion enhanced immunogenicity by increased IFN-γ production, antigen presentation, and DTH [47]. Currently, physicians prescribing the Zinc consumption in coronavirus infection that might help to reduce the disease pathology and severity of the infection. Moreover, the BCG vaccine already activated the similar immunogenicity in the vaccinated population.

Immune Activation against SARS-CoV-2
The PRRs of the TLR family or DExD/H box RNA helicase family initiates the immune response inside the host upon virus entry. The cell receptors on activation give a signal to increase the levels of IFNs, especially IFN-α, and INF-β, and various other cytokines [48]. Different TLRs are inducible although, some are constitutively present on specialized immune cells like dendritic cells for the rapid production of IFNs and cytokines [49].
The TLRs molecules are too selective for their purpose, like to recognition of specific viral particles. TLR7/8 receptors bind with the single ssRNA and initiate the immune response with inflammatory cytokines secretion. TLR3 receptors are specific for double-stranded RNA (dsRNA) as a ligand and form an active signaling complex that initiates the immune response [50][51][52][53][54]. SARS viruses 14 Figure 1: Cell-mediated immune response. activate the immune system in the same way as the BCG vaccine induces. SARS-CoV-2 activates the immune system through the TLRs and PAMPs [55]. The innate and adaptive immune responses occur against the SARS infections through the phagocytic process of macrophages and antigen presentation to the naive T-cells. It starts T-cells activation and proliferation with differentiation by the secretion of various types of cytokines that help in the differentiation of T-cells sub-groups like Th-17, etc. The huge cytokines production increases the amplification of immune response. The constant production of these mediators due to the viral persistency might alter the CD8 + T cell Natural-Killer cell activation. Though, CD8 + T-cells are the most powerful mediators in cell-mediated immune actions to the clearance of SARS-CoV-2. Viral dsRNA might initiate the immune response during CoV replication via TLR3 signaling cascades like IRFs and NF-κB activation inside the infected cells or antigenpresenting cells. These cells produce a large amount of type I IFNs and pro-inflammatory cytokines [56,57]. For the protection of uninfected cells from the viral proteins, type-I IFNs plays a significant role in the clearance of the viral proteins. Essential proteins of SARS viruses are interfered with in TLRs signaling and try to escape the immune response via TLRs activation [54,58]. Viral spike proteins are recognized by the TLRs and get activated that stimulate pro-inflammatory cytokines secretion by using MyD88-dependent pathways. Virus and host-cell interactions enhance the immune mediators' secretions like chemokines and cytokines (IL-1, IL-2, etc.). Chemokines and cytokines help in activation of leukocytes and lymphocytes. Thus, activating the immune system against the SARS-CoV-2 infection. BCG vaccine already initiated that type of immune response in the BCG vaccinated population, which might be activated by the SARS viruses. Hence BCG vaccine decreases the time of immune response against the COVID-19 and reducing the severity of the infection with an increasing rate of recovery [59] (Figure 2  The Data of SARS-CoV-2 infections, severity, and recovery has shown a strong association among BCG vaccination. The universal BCG implemented countries are showing the very low severity and high rate of recovery as compared to the not universally BCG vaccination implemented countries. BCG trained immunity against the SARS-CoV-2 might have a role in the mechanism by which induces its beneficial effects. BCG vaccination has shown the reduction of viremia as compared to yellow fever vaccination (live attenuated) in healthy human volunteers. The immune response in healthy human volunteers might be associated with epigenetic changes in monocytes that could help to improve the antiviral responses [60]. Trained immunity of BCG might have a role in important protection against various viral infections. Thus, the BCG vaccine could play an important role in the reduction of SARS-CoV-2 infection severity and recovery in universally BCG implemented countries.

Status of SARS-CoV-2 in Vaccinated and Non-vaccinated Countries
By date 28 August 2020, the status of SARS-CoV-2 positive cases in countries vaccinated with BCG are 16,755,837, mortality in percentage is 3%, and recovery in percentage is 76.256%, whereas, in non-vaccinated countries, the SARS-CoV-2 positive cases are 7,751,688, mortality in percentage is 4.13%, and recovery rate is 52.45%. Thus, the data showed that BCG might play an essential role in the reduction of deaths by COVID-19 and increase recovery [61].

Conclusion
The BCG vaccine is the oldest vaccine that is being used against TB in all over the world. The efficacy of BCG to boost immunity against M. Tuberculosis is not 100 percent, but it also protects from other pathogens like bacteria, fungi, and viruses. It activates the innate immune response and cell-mediated immune response, which is related to the non-specific efficacy of BCG. The nonspecific immune response is significantly essential to clear viral-like pathogens. BCG vaccine dominantly activates the cell-mediated immunity via TLRs and IFNs, and it also develops the memory against similar types of pathogens, which cause TB like symptoms such as pneumonia. Several studies have reported the BCG vaccine's nonspecific role against various viral infections. Currently, the infection of SARS-CoV-2 is less symptomatic in the population vaccinated with BCG at all over the globe. The SARS-CoV-2 virus severity decreases, whereas recovery increases in vaccinated countries populations as compared to the non-vaccinated.