**6. Hsps as antigens in** *M. tuberculosis*

Hsps may be released extracellularly upon necrotic cell death or independent of cell death. The mechanism of the release of Hsps is not clear (Tsan & Gao, 2004). The human as well bacterial Hsps stimulate immune response. The bacterial Hsps might modulate immunity by rapidly and directly increasing cytokine production in macrophages. T cells reacting to Hsp65 appear to play an important role in the control of *M. leprae* infection (de la Barrera et al., 1995). Hsp65 directly activates monocytes during mycobacterial infection. It leads to the production of TNF (tumor necrosis factor), IL-6 and IL-8. These cytokines are important in developing antigen specific T-cell mediated host immunity (Friedland et al., 1993). The murine intraepithelial lymphocytes (IEL), when exposed to soluble extract from *M. tuberculosis* showed elevated expression of IL-3, interferon-γ and IL-6 (Mendez-Samperio et al., 1995). Peripheral blood mononuclear cells (PBMC) from TB patients showed proliferative response to the Hsp65 (Mendez-Samperio et al., 1995). The *in vitro* immune responses to *M. tuberculosis* Hsp65 were checked in TB patients, and their PBMC showed high IFN-γ levels (Antas et al., 2005). When guinea pigs were vaccinated or infected with *M. bovis* (BCG) and virulent *M. tuberculosis*, cellular and humoral immune responses to mycobacterial stress proteins Hsp65 and Hsp70 were detected (Bartow & McMurray, 1997).

The C-terminal portion of heat shock protein Hsp70 was shown to be responsible for stimulating Th1-polarizing cytokines in human monocytes to produce IL-12, TNF-α, NO, and C-C chemokines (Wang et al., 2002). Hsp70 induces the expression of IL-10 and inhibits T-cell proliferation *in vitro*. Hsp70 appears to have immunosuppressive properties rather than inflammatory potential (Motta et al., 2007).

Hsp71 and Hsp65 are the major active components of the soluble extract of *M. tuberculosis.*  Murine IELs were induced to divide and to secrete cytokines by Hsp71 and Hsp65 (Beagley et al., 1993). *M. tuberculosis* Hsp70, *M. leprae* Hsp65, and *M. bovis* BCG Hsp65 increased the levels of cytokines IL-1α, IL-1β, IL-6, TNFα, and GMCSF in macrophages (Retzlaff et al., 1994). *M. tuberculosis* contains multiple genes encoding Cpn 60 proteins, and these chaperonins have been involved in directly activating human monocytes and vascular

Heat Shock Proteins in *Mycobacterium tuberculosis*:

**7. Hsps in vaccine development against TB** 

1996).

(Skinner et al., 2003).

et al., 1993).

Involvement in Survival and Virulence of the Pathogen 265

multiple sclerosis and tuberculosis were assessed, it was seen that Hsp70 is an antigen in TB as well as an autoantigen in multiple sclerosis (Salvetti et al., 1996). Immunizations with recombinant Hsp65, and Hsp65 rich *M. tuberculosis* in C57BL/6J mice induced atherogenesis

Hsps are among the proteins that are expressed at high level during the TB infection and are highly conserved. They could mediate the T cell sensitization required for the production of antibodies and can be used in the development of vaccine(s) against TB (Lussow et al., 1991). Inside the host, T cells are involved in activating macrophages and controlling the mycobacterial infection. Both the macrophage and mycobacterium synthesize heat shock proteins in order to facilitate their survival, and these Hsps possess potent immunogenicity (Munk & Kaufmann, 1991). The Hsp70 of *M. tuberculosis* has been shown to have antiinflammatory properties and immunosuppressive role in a graft rejection system (Borges et al 2010). Hsp70 is recognized by human CD4+ T-cells and it leads to the secretion of TNF, IL-6 and IL-1β (Asea et al., 2000). Mycobacterial Hsp70 can be used in subunit vaccine design since it contains a variety of T-cell epitopes (Oftung et al., 1994). Studies have been done to map the epitopes of Hsp70, so as to eliminate the autoimmune response in humans (Adams et al., 1993). A synthetic peptide, non-covalently bound to *M. tuberculosis* Hsp70 generated a very strong specific proliferative T-cell response in the spleen of mice (Roman & Moreno,

Hsp16 also induces T-cells to proliferate and secret cytokines, and therefore can be used as a potential subunit vaccine candidate (Agrewala & Wilkinson, 1999). A DNA vaccine combination expressing mycobacterial Hsp65 and IL-12 provided high degree of protection against TB (Okada et al., 2007). The vaccine was delivered by the hemagglutinating virus of Japan (HVJ)-envelope and liposome. This vaccine provided remarkable protection in mice and monkeys compared to the BCG vaccine, demonstrating the potential of Hsps to be used in vaccine development (Okada, 2006; Okada & Kita; Okada et al., 2007; Okada et al., 2009). A prime-boost strategy was investigated in cattle, using a combination of three DNA vaccines coding for Hsp65, Hsp70, and another mycobacterial protein Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent *M. bovis*

Hsp65 as an antigen can confer protection equal to that from live BCG vaccine (Silva, 1999). The mycobacterial Hsp65 and Hsp70 acted as carrier molecules in mice previously primed with *M. tuberculosis* and showed high and long-lasting titers of IgG (Barrios et al., 1992; Perraut et al., 1993). The mycobacterial Hsp65 conjugated to peptides or oligosaccharides in the absence of adjuvants, induced antibodies which cross-reacted well with Hsp homologues from other prokaryotes, but weakly with the human Hsp homologue (Barrios et al., 1994). The PBMCs and T-cell lines from *M. leprae* and *M. bovis* BCG vaccinated subjects showed proliferation in response to Hsp18 and Hsp65 of *M. leprae*, Hsp65 of *M. bovis* BCG, and the Hsp70 of *M. tuberculosis* (Mustafa et al., 1993). The response of T cells to these Hsps makes them eligible for their application in the next generation of subunit vaccines (Mustafa

indicating the involvement of Hsp65 in atherogenesis (George et al., 1999).

endothelial cells. Among them the Cpn 60.2 protein activates human PBMCs by a CD14 independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and Cpn 60.1 is a more potent cytokine stimulator than Cpn 60.2 (Lewthwaite et al., 2001). Cpn 60.1 is said to play more important role in *M. tuberculosis* virulence than Cpn 60.2 (Lewthwaite et al., 2001).

*M. tuberculosis* can manipulate and inhibit the host response to ensure survival within macrophage. The anti-inflammatory cytokine IL-10 is shown to inhibit phagosome maturation in macrophages infected with *M. tuberculosis* (O'Leary et al., 2010). HspX/Acr is among the dormancy regulon whose expression is increased in hypoxia and on nitric oxide exposure essential for the survival of bacteria during persistence *in vivo*. Acr is also an immunodominant antigen during infection (Roupie et al., 2007). The T cells primed with Hsp65 of *M. tuberculosis*/*M.bovis* showed a response to the epitpoes shared by human Hsp65 and mycobacterial Hsp65, demonstrating that if activated these T cells can develop autoimmunity (Munk et al., 1989). The immune system, once primed for one Hsp might recognize other Hsps as well. Sarcoidosis (SA) is a multisystem granulomatous autoimmune disorder. The clinical and histopathological pictures of SA and TB are similar. *M. tuberculosis* heat shock proteins have been considered as proteins involved in the genesis of SA (Chen & Moller, 2008; Rajaiah & Moudgil, 2009). *M. tuberculosis* Hsps are also proposed to have a role in apoptosis which might be important in the pathogenesis of SA and TB granuloma formation (Dubaniewicz et al., 2006a; Dubaniewicz et al., 2006b). There is high cross reactivity between human and mycobacterial Hsp65. This could be the reason for the development of SA, however another hypothesis is that the BCG vaccination can develop autoimmunty in a pre-disposed host (Dubaniewicz, 2010). T-cells from SA patients produced a CD4+ response to multiple mycobacterial antigens including Hsps. These T cells were present at the site of active SA inside the human body (Oswald-Richter et al., 2010). The sera of the patients of rheumatoid arthritis (RA) showed increased levels of IgG and IgA antibody to the mycobacterial Hsp65 (Tsoulfa et al., 1989). The synovial membrane from rats and humans with arthritis appeared positive for mycobacterial Hsp65 showing the possible role of mycobacterial antigens in autoimmune diseases like arthritis (de Graeff-Meeder et al., 1990; Karopoulos et al., 1995). A survey of antigen-specific antibody isotypes from rheumatoid patients showed that antimycobacterial Hsp65 antibodies clearly do not appear to be disease specific markers for RA; however this does not exclude the possibility of mycobacterial Hsp65 in the pathogenesis of RA (Lai et al., 1995; Minota, 1997). The mycobacterial 71kDa Hsp antigen in lower concentration inhibits arthritis and at higher concentrations completely protects rats from arthritis (Kingston et al., 1996). Mycobacterial Hsp65 has also shown crossreactive epitopes of epidermal cytokeratins which is a protein from epidermal keratinocytes of the normal human skin (Rambukkana et al., 1992). *Mycobacterium paratuberculosis* Hsp65 has been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease (el-Zaatari et al., 1995).

As seen in several studies, Hsp65 is involved in the development of autoimmune response because of its highly conserved sequence. Another important finding about Hsp65 is that the effector cells activated with Hsp65 strongly inhibited colony formation from live BCGinfected autologous macrophages (Ab et al., 1990). In the case of *M. lepare* infection, the Tcells from leprosy patients are exposed to a large variety of different antigens including *M. lepare* Hsp70, *M. tuberculosis* Hsp70 and Hsp65 (Janson et al., 1991). When patients with

endothelial cells. Among them the Cpn 60.2 protein activates human PBMCs by a CD14 independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and Cpn 60.1 is a more potent cytokine stimulator than Cpn 60.2 (Lewthwaite et al., 2001). Cpn 60.1 is said to play more important role in *M. tuberculosis* virulence than Cpn 60.2 (Lewthwaite et al., 2001). *M. tuberculosis* can manipulate and inhibit the host response to ensure survival within macrophage. The anti-inflammatory cytokine IL-10 is shown to inhibit phagosome maturation in macrophages infected with *M. tuberculosis* (O'Leary et al., 2010). HspX/Acr is among the dormancy regulon whose expression is increased in hypoxia and on nitric oxide exposure essential for the survival of bacteria during persistence *in vivo*. Acr is also an immunodominant antigen during infection (Roupie et al., 2007). The T cells primed with Hsp65 of *M. tuberculosis*/*M.bovis* showed a response to the epitpoes shared by human Hsp65 and mycobacterial Hsp65, demonstrating that if activated these T cells can develop autoimmunity (Munk et al., 1989). The immune system, once primed for one Hsp might recognize other Hsps as well. Sarcoidosis (SA) is a multisystem granulomatous autoimmune disorder. The clinical and histopathological pictures of SA and TB are similar. *M. tuberculosis* heat shock proteins have been considered as proteins involved in the genesis of SA (Chen & Moller, 2008; Rajaiah & Moudgil, 2009). *M. tuberculosis* Hsps are also proposed to have a role in apoptosis which might be important in the pathogenesis of SA and TB granuloma formation (Dubaniewicz et al., 2006a; Dubaniewicz et al., 2006b). There is high cross reactivity between human and mycobacterial Hsp65. This could be the reason for the development of SA, however another hypothesis is that the BCG vaccination can develop autoimmunty in a pre-disposed host (Dubaniewicz, 2010). T-cells from SA patients produced a CD4+ response to multiple mycobacterial antigens including Hsps. These T cells were present at the site of active SA inside the human body (Oswald-Richter et al., 2010). The sera of the patients of rheumatoid arthritis (RA) showed increased levels of IgG and IgA antibody to the mycobacterial Hsp65 (Tsoulfa et al., 1989). The synovial membrane from rats and humans with arthritis appeared positive for mycobacterial Hsp65 showing the possible role of mycobacterial antigens in autoimmune diseases like arthritis (de Graeff-Meeder et al., 1990; Karopoulos et al., 1995). A survey of antigen-specific antibody isotypes from rheumatoid patients showed that antimycobacterial Hsp65 antibodies clearly do not appear to be disease specific markers for RA; however this does not exclude the possibility of mycobacterial Hsp65 in the pathogenesis of RA (Lai et al., 1995; Minota, 1997). The mycobacterial 71kDa Hsp antigen in lower concentration inhibits arthritis and at higher concentrations completely protects rats from arthritis (Kingston et al., 1996). Mycobacterial Hsp65 has also shown crossreactive epitopes of epidermal cytokeratins which is a protein from epidermal keratinocytes of the normal human skin (Rambukkana et al., 1992). *Mycobacterium paratuberculosis* Hsp65 has been implicated as a possible cause of Crohn's disease, an

inflammatory bowel disease (el-Zaatari et al., 1995).

As seen in several studies, Hsp65 is involved in the development of autoimmune response because of its highly conserved sequence. Another important finding about Hsp65 is that the effector cells activated with Hsp65 strongly inhibited colony formation from live BCGinfected autologous macrophages (Ab et al., 1990). In the case of *M. lepare* infection, the Tcells from leprosy patients are exposed to a large variety of different antigens including *M. lepare* Hsp70, *M. tuberculosis* Hsp70 and Hsp65 (Janson et al., 1991). When patients with

multiple sclerosis and tuberculosis were assessed, it was seen that Hsp70 is an antigen in TB as well as an autoantigen in multiple sclerosis (Salvetti et al., 1996). Immunizations with recombinant Hsp65, and Hsp65 rich *M. tuberculosis* in C57BL/6J mice induced atherogenesis indicating the involvement of Hsp65 in atherogenesis (George et al., 1999).
