Tuberculosis is an infectious disease that is treatable using the available chemotherapeutic agents. However, the drugs are sometimes not effective due to patient noncompliance with the recommended treatment regimen or because disease is caused by antibiotic-resistant strains of M. tuberculosis
. Use of the M. bovis
BCG vaccine to prevent TB is controversial since its protective efficacy is variable, immunity is not long lasting (effective only in childhood), and the vaccine is not routinely used in some countries (for example, the United States). Several strategies have been developed to improve the efficacy of the BCG vaccine or to generate new vaccines that induce protective humoral and cell-mediated immunity against M. tuberculosis
infection, including the use of live attenuated bacteria such as Salmonella
for delivery of protective M. tuberculosis
antigens. Delivery of recombinant Salmonella
vaccines by oral, intranasal, intravenous, and intraperitoneal routes stimulates mucosal immunity with production of secretory IgA in all mucosal tissues and all secretory glands, primarily due to the cross-communication within the mucosal immune system (37
). The antibody responses induced in mice immunized with Salmonella
vaccines may contribute to the control of M. tuberculosis
infection since there are IgG and IgA antibodies present in the mucosal secretions of the lower respiratory tract (10
). Although M. tuberculosis
is primarily an intracellular pathogen, there is also an extracellular phase in its infectious cycle. There are studies that show the importance of antibody responses against M. tuberculosis
in controlling the infection (33
). Therefore, oral immunization with Salmonella
vaccine vectors may be effective in protecting mucosal surfaces such as those of the lungs. In addition, live recombinant Salmonella
vaccines induce Th-1 cytokines IFN-γ and TNF-α, which are important in controlling infections and preventing diseases caused by intracellular pathogens such as M. tuberculosis
Kong et al. have developed an Asd+
balanced-lethal host-vector lysis system for complementation of the lethal chromosomal deletion of both the asd
genes in RASV strains (46
). This system ensures the stability of plasmid vectors producing protective antigens in vivo
after immunization of animal hosts and eliminates the use of drug resistance markers. Furthermore, RASV strains have been constructed that are phenotypically similar to wild-type Salmonella
at the time of oral vaccination but display (i) regulated delayed attenuation (22
), (ii) regulated delayed synthesis of recombinant antigens (71
), and (iii) regulated delayed lysis to release protective antigens and confer complete biological containment, after colonizing the host (46
In this study, we evaluated the protective efficacy of two RASV strains that, in vivo
, exhibit regulated delayed attenuation (χ9879) and regulated delayed lysis (χ11021). Each of these strains produces secreted M. tuberculosis
proteins such as Ag85A294
alone or Ag85A294
produced with either SopENt80
-E2C or OmpCSS
-E2C (in χ11021) and delivers these protective antigens to the host immune system to elicit protective immunity against M. tuberculosis
infection. Antigens Ag85A, ESAT-6, and CFP-10 are effective as subunit vaccines (3
), and vaccines expressing these antigens alone or together afford protection against M. tuberculosis
Secreted antigenic proteins are generally more effective in inducing protective immunity against intracellular pathogens than proteins that remain in the cell cytosol. Therefore, we constructed RASVs producing chimeric proteins such as Bla-Ag85A294
-E2C that are efficiently exported to the periplasm and subsequently to the outside of the bacterial cell via the T2SS, while the chimeric protein SopENt80
-E2C is efficiently secreted to the supernatant and translocated to eukaryotic cell cytoplasm via the T3SS (42
; this study).
Moreover, the level of recombinant antigen synthesis directly influences the quality of the immune response induced (14
) since high expression of heterologous genes on plasmids can generate a metabolic burden that overattenuates the Salmonella
vaccine strains, resulting in impaired colonization and either a lack of or decreased immunogenicity. Therefore, we controlled the amount of heterologous antigen synthesis in RASV by two ways: first, using balanced-lethal Asd+
plasmids that carry the mycobacterial genes fused to T2SS or T3SS effector sequences, with their transcription under the control of the Ptrc
promoter, whose activity is in turn controlled by the chromosomal arabinose-regulated lacI
gene in the Salmonella
vaccine strain χ11021. The second control is through the use of isogenic Asd+
expression plasmids with different replication origins, such as pBR, p15A, and pSC101 for high-, low-, and very-low-copy-number plasmids, respectively, to carry the nucleotide sequences encoding the mycobacterial chimeric proteins. The Asd+
pYA3941 plasmid and all of the Asd+
lysis plasmid derivatives constructed in this study are stable (100%) in RASV strains for over 50 generations of growth in the presence of DAP, independent of their copy number.
We further extended our study to determine the ability of our RASV strains to induce both humoral and cellular immune responses. We detected anti-Ag85A IgG titers in mice orally vaccinated with RASV strain χ9879(pYA3941) or χ11021 independently harboring each constructed Asd+/MurA+ lysis vector producing and delivering Ag85A294. Higher anti-Ag85A IgG titers were obtained in mice immunized with the χ11021 strains harboring the Asd+/MurA+ lysis vectors producing Ag85A294 than in mice immunized with χ9879(pYA3941). These data suggest that Salmonella vaccines displaying regulated delayed lysis were superior in inducing IgG antibody production in vaccinated mice compared to RASVs that had only attenuating mutations. Interestingly, among the vaccine strains harboring isogenic plasmids with different replication origins, we observed that the anti-Ag58A IgG titers were influenced by the copy number of the plasmids, obtaining the highest titers with plasmids containing the p15A ori (χ11021 harboring pYA4894 or pYA4891).
Anti-ESAT-6 IgG titers were detected in all mice vaccinated with χ11021 harboring the Asd+
lysis vector derivatives expressing and delivering either the SopENt80
-E2C or OmpCSS
-E2C chimeric proteins. Similar to the pattern of humoral responses to Ag85A, the antibody response against ESAT-6 was inversely related to the copy number of the plasmids, suggesting that a metabolic burden was imposed on the RASV by the synthesis of the protective antigens. A similar observation has been described previously, where the immune response to antigens delivered by Salmonella
Typhi vaccines was improved when the ClyA-PA83 antigen was expressed from low-copy-number plasmids (pSC101 ori
), which induced the highest antibody responses (32
). In this study, the highest anti-ESAT-6 IgG titers were observed in the mice vaccinated with χ11021(pYA4894 [p15A ori
]) alone or in mice vaccinated simultaneously with both χ11021(pYA4894) and χ11021(pYA4892 [pSC101 ori
]). In addition to the copy number of the plasmid, the OmpC signal peptide also positively influenced the humoral response, since the OmpCSS
-E2C chimeric proteins were more effective in eliciting higher antibody titers than the SopENt80
-E2C chimeric proteins.
The IgG responses to CFP-10 in immunized mice were similar to the patterns observed with ESAT-6. Induction of anti-CFP-10 IgG was also inversely related to the copy number of the plasmid, although the highest titers were seen in mice vaccinated with χ11021(pYA4892 [pSC101 ori]). Mice immunized with χ11021(pYA4894 [p15A ori]) had the second highest titer of anti-CFP IgG, suggesting that the OmpC signal sequence in the OmpCSS-E2C chimeric protein produced by χ11021(pYA4894) did not increase the humoral response to CFP-10 as it appeared to do for both ESAT-6 and Ag85A (B and C). It is unclear why this difference occurred.
Analysis of the IgG subclasses showed higher levels of anti-Ag85A IgG2b antibodies than IgG1 titers, which is typical for a Th1 response. The switch between secretion of IgG2b or IgG1 is determined by the differential production of cytokines; thus, the stimulation of IgG2b production is dependent on IFN-γ production. Th1-type immune responses were observed in all mice immunized with either strain χ9879 or χ11021 delivering M. tuberculosis chimeric proteins. These results are in agreement with the T-cell response that was characterized by Ag85A-specific secretion of IFN-γ, TNF-α, and IL-2, which are more favorable for a protective immune response against M. tuberculosis. The secretion of Ag85A294-specific cytokines was higher in the mice vaccinated with Salmonella vaccine strain χ11021 harboring Asd+/MurA+ lysis plasmids delivering Ag85A294 and displaying regulated delayed lysis than in the mice vaccinated with Salmonella strain χ9879 harboring the Asd+ pYA3941 plasmid. Interestingly, the induction of Ag85A-specific secretion of IFN-γ, TNF-α, and IL-2 in vaccinated mice was directly related to the copy number of the plasmids producing SopENt80-E2C/Ag85A294 chimeric proteins (pYA4890, pYA4891, and pYA4892) but not those pro-ducing OmpCSS-E2C/Ag85A294 chimeric proteins (pYA4893 and pYA4894), where a slightly greater cytokine production was observed in mice vaccinated with χ11021(pYA4894) than mice vaccinated with χ11021(pYA4893). These results suggest that, in mice orally vaccinated with Salmonella vaccines secreting the combined chimeric proteins Ag85A294 and SopENt80-E2C, there is better stimulation of the Th1-associated cytokines. The highest induction of IFN-γ production was observed in mice vaccinated with both RASV strains χ11021(pYA4892 [p15A ori]) synthesizing SopENt80-E2C/Ag85A294 and χ11021(pYA4894 [p15A ori]) synthesizing OmpCSS-E2C/Ag85A294, indicating a synergistic effect of both vaccines in the enhancement of the T-cell immune response.
Significant increases in production of the IL-4 cytokine and low or moderate levels of IFN-γ were detected in mice vaccinated with the χ11021(pYA3681) vector control and with χ11021(pYA4892). It has been reported that significant production of the Th2-associated IL-4 cytokine is related to failure to control M. tuberculosis
). Consistent with these reports, mice immunized with the RASV χ11021(pYA4892) strain showed no protection against M. tuberculosis
In mice orally vaccinated with the RASV strain χ9879(pYA4257) synthesizing SopENt80
-E2C and displaying regulated delayed attenuation, we previously detected induction of a significant increase in the number of ESAT-6-specific IFN-γ- and TNF-α-secreting T cells in mouse spleens 1 week after the last immunization (42
). However, here, neither ESAT-6-specific nor CFP-10-specific T-lymphocyte activity was observed in mice orally vaccinated with χ11021 delivering either SopENt80
chimeric proteins. In this study, the ESAT-6-specific and CFP-10-specific stimulation of T-cell responses was analyzed at 3 weeks after the last immunization. If the stimulation of ESAT-6- and CFP-10-specific T-lymphocyte activity occurs at 1 week postboost and rapidly declines, as has been reported (4
), this needs to be further investigated. Other studies have determined that vaccination with the ESAT-6 protein results in low immunogenicity and requires a strong adjuvant to prime specific immune responses (11
). Moreover, secretion of ESAT-6 alone or as a fusion protein with Ag85B delivered by recombinant BCG induces weakened T-cell responses or completely diminishes T-cell responses, respectively (59
). If the absence of ESAT-6-specific and CFP-10-specific stimulation of T-cell responses (or dominant Ag85A immune responses) is the result of competition between both Ag85A294
-E2C or OmpCSS
-E2C, resulting in a dominant Ag85A immune response over the response to ESAT-6 and/or CFP-10, remains to be determined.
In spite of the lack of ESAT-6- and CFP-10-specific T-cell activity observed in mice vaccinated with the Salmonella
vaccines synthesizing SopENt80
at the specific times assessed, we cannot rule out the possibility that the antibody responses against ESAT-6, CFP-10, and Ag85A may also play a role in the induction of T-cell activity. In addition to the role of serum antibodies in classical opsonization, phagocytosis, and killing of pathogens, there exist an interdependence and synergy between humoral and cell-mediated immunity (1
). B cells are necessary for rapid T-cell activation via Fc receptors (FcR) by an FcR-dependent antibody-enhanced uptake, processing, and presentation of pathogen-derived antigens by FcR-bearing antigen-presenting cells (36
). The enhanced uptake of BCG coated with antimycobacterial antibodies by dendritic cells and the subsequent processing of these bacteria, resulting in the proliferation of mycobacterium-specific CD4+
T cells secreting IFN-γ, support the role of specific antimycobacterial antibodies in the uptake of bacteria via Fc receptors by professional antigen-presenting cells for the activation of T cells (27
The mycobacterial loads were significantly reduced in both the lungs and spleens of mice vaccinated with the monovalent Salmonella strain χ9879(pYA3941) and with the trivalent Salmonella vaccine χ11021 harboring the Asd+/MurA+ lysis plasmids and displaying the regulated delayed lysis phenotype. Among the trivalent vaccines, those harboring pYA4891, pYA4893, and pYA4894 were the most effective in conferring protection in the lungs against aerosol challenge with M. tuberculosis, and this level of protection was slightly better than that afforded by BCG. On the other hand, mice immunized with strain χ11021(pYA4890) or with both χ11021(pYA4892) and χ11021(pYA4894) achieved a protection in the lungs comparable to that generated with BCG. In contrast, in the spleens of vaccinated mice, the reduction in number of CFU was similar to that observed with BCG, with the exception of the reduction caused by strains χ11021(pYA4893) and χ11021(pYA4892), which did not afford significant protection.
All the observations made in the present study indicate that there is a requirement for an optimum level of synthesis of the protective antigens to induce a robust immune response and protection against M. tuberculosis, without impairing the capability of the live Salmonella vaccine to colonize the host lymphoid tissues. In this work, this was achieved by employing low-copy-number Asd+/MurA+ lysis vectors containing the p15A ori to produce secreted recombinant antigens. Thus, the trivalent Salmonella vaccine harboring either of the Asd+/MurA+ lysis plasmids with the p15A ori and expressing SopENt80-E2C/Ag85A294 or OmpCSS-E2C/Ag85A294, pYA4891 and pYA4894, respectively, afforded the most effective protection against M. tuberculosis challenge in orally immunized mice.
Our results show the efficacy of the Salmonella/Asd+/MurA+ lysis system for delivery of mycobacterial antigens to confer protection against M. tuberculosis infection and encourage further development of improved vaccines based on the Salmonella/Asd+/MurA+ lysis system to prevent M. tuberculosis infection. Since BCG is used in many countries to immunize infants, it is likely that an RASV-M. tuberculosis vaccine would be used as a boosting immunization in BCG-vaccinated individuals. Thus, in future experiments, we will explore the use of improved RASV-M. tuberculosis vaccines in prime-boost immunization regimens with BCG as the priming vaccine. In such experiments, antigens other than ESAT-6 and CFP-10 will be included, since the genes encoding these antigens are absent from the BCG genome. Alternatively, prime-boost vaccination strategies using RASV-M. tuberculosis vaccines with subunit vaccines with the same antigens will also be assessed.