Mycobacterium tuberculosis serine/threonine protein kinases (STPKs) are key regulators of growth and metabolism; however, evidence for their roles in virulence is limited. In a preliminary screen based on comparative expression between strains H37Rv and H37Ra, six STPK genes, pknD, pknG, pknH, pknJ, pknK and pknL, showed higher expression in H37Rv. In the second screen, STPK expression was analysed in H37Rv-infected human macrophages. Interestingly, significant expression of pknK was detected only at 18 h post-infection, suggesting its involvement in early infection events. We have investigated the roles of PknK in vitro and in vivo. PknK levels were induced under stationary phase and deletion of pknK resulted in increased resistance of the mutant to acidic pH, hypoxia, oxidative and stationary-phase stresses in vitro. These results, together with the increased survival of the ΔpknK strain during persistent infection in mice, reveal a role for PknK in adaptive mechanisms that slow the growth of mycobacteria. A novel finding of this study was the inhibition of growth of ΔpknK strain during acute infection in mice that correlated with the significant upregulation of tumour necrosis factor as well as the simultaneous downregulation of interleukin-12p40, interferon-γ and induced nitric oxide synthase transcripts. Finally, we provide evidence for the localization of PknK during infection and discuss its implications in pathogenesis.
Tuberculosis remains a global health threat, and there is dire need to develop a vaccine that is safe and efficacious and confers long-lasting protection. In this study, we constructed recombinant attenuated Salmonella vaccine (RASV) strains with plasmids expressing fusion proteins consisting of the 80 amino-terminal amino acids of the type 3 secretion system effector SopE of Salmonella and the Mycobacterium tuberculosis antigens early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10). We demonstrated that the SopE-mycobacterial antigen fusion proteins were translocated into the cytoplasm of INT-407 cells in cell culture assays. Oral immunization of mice with RASV strains synthesizing SopE-ESAT-6 -CFP-10 fusion proteins resulted in significant protection of the mice against aerosol challenge with M. tuberculosis H37Rv that was similar to the protection afforded by immunization with Mycobacterium bovis bacillus Calmette-Guérin (BCG) administered subcutaneously. In addition, oral immunization with the RASV strains specifying these mycobacterial antigens elicited production of significant antibody titers to ESAT-6 and production of ESAT-6-or CFP-10-specific gamma interferon (IFN-␥)-secreting and tumor necrosis factor alpha (TNF-␣)-secreting splenocytes.
Vaccines are frequently less efficacious in the elderly and correlates of protection in older adults often differ from those seen in younger individuals. However, clinical testing of new vaccine formulations is typically limited and does not include elderly subjects. In order to identify a nonhuman primate (NHP) model that would recapitulate the effects of immunosenescence in humans, we assessed immune responses in three different NHPs: baboons (Papio), rhesus macaques (Macaca), and African green monkeys (Chlorocebus). The F1-antigen, a capsular protein encoded on the plague bacterium Y. pestis pFra plasmid, was chosen because it should elicit naive responses in all subjects. The animals were bled following primary and booster immunizations; B cell (antibody titer) and T cell parameters were assessed. As in humans, significant heterogeneity between individuals was seen. In rhesus and vervets, T cell responses to F1 were generally lower with increasing age. However, less of an age effect was observed in the baboons; this is consistent with the finding that antibody titers to F1 were not significantly diminished in older baboons and with our previous work showing immunization of old baboons with Y. pestis LcrV elicited high titer protective antibodies [JI 181:109]. Flow cytometric analyses of the T cell populations in old and young NHPs reveal differences across the species and provide additional insight into the immune parameters that correlate with healthy aging.
The ability of humans and rodents to mount effective immune responses declines with age. Yet, when old baboons (19-24 years) were immunized with LcrV, a protective antigen from Y. pestis, antibody titers were significantly higher than in immunized young animals (2½ years) [JI 181:109]. It may be that baboons are unusual, aging without losing immune competence or alternatively, that LcrV is enhancing humoral immunity in older animals. To address this, a second NHP species was tested; young (4 years), middle-age (11 years), and older (18-24 years) rhesus macaques were immunized with LcrV. Significant humoral responses were observed in all age groups, so like baboons, there was no loss of immune reactivity with aging. However, the greatly enhanced response in older baboons was not recapitulated in rhesus. To test the possibility that LcrV is unique, young (6 years), middle-age (12-13), and older (21-23) baboons were immunized with a second plague antigen, F1; a more typical response was seen whereby F1 antibody titers elicited in younger baboons were higher than those seen in the older cohorts. T cell proliferative and cytokine responses to these antigens have been assessed and no definitive age-associated changes were noted. On the other hand, circulating Tregs and some serum cytokines were higher in older NHPs. These experiments provide novel insights into aging effects on primate immunity and suggest a unique function for plague LcrV in enhancing responses in old animals.
Vaccine efficacy declines significantly with age, yet old baboons immunized with Yersinia pestis LcrV antigen generated vigorous humoral responses [JI 181:109 (2008)] while aged rhesus macaques did not. To assess whether these non-human primate (NHP) species are differentially impacted by immunosenescence, animals were immunized with Y. pestis F1, a plasmid-encoded protein which is known to elicit naïve B and T cell responses. Interestingly, age-associated decreases in antibody titers to F1 were observed in both species. However, defects in T cell proliferative responses were seen only in the macaques; T cell reactivity to both mitogen and antigenic stimulation was similar in the old and young baboons. We therefore asked whether other aspects of immune regulation are affected by age. The loss of naive, relative to memory, T cells was seen, as expected. Moreover, there was a decrease, albeit relatively small, in the production of naive T cells (TRECs). However, several parameters known to change with aging were not affected. For example, AID induction was maintained in most, but not all, old baboons. Moreover, the T cell fine specificity was similar in old and young animals. Lastly, TCR-Vβ spectratype analysis revealed that the age-associated repertoire skewing reported in old rhesus and humans is not seen in baboons, even in the CD8+ T cell subset. Thus, baboons do appear to be less affected by aging and this NHP species may provide novel insights into healthy immune aging.
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