Comparative analyses of the Mycobacterium tuberculosis genome with the genomes of other mycobacteria have led to the identification of several genomic regions of difference (RDs) between M. tuberculosis and M. bovis BCG. The identification of immunodominant and HLA-promiscuous antigens and peptides encoded by these RDs could be useful for diagnosis and the development of new vaccines against tuberculosis. The analysis of RD proteins and peptides by in silico methods (using computational programs to predict major and HLA-promiscuous antigenic proteins and peptides) and experimental validations (using peripheral blood mononuclear cells and sera from tuberculosis patients and BCG-vaccinated healthy subjects to assess antigen-specific cellular and humoral immune responses in vitro) identified several major antigens and peptides. To evaluate the in vivo potentials, the genes of immunodominant antigens were cloned and expressed in DNA vaccine vectors. Immunizations of experimental animals with the recombinant constructs induced antigen-specific cellular responses. Further experiments showed that each of these proteins had several T and B cell epitopes scattered throughout their sequence, which confirmed their strong immunogenicity. In conclusion, the bioinformatics-based in silico identification of promiscuous antigens and peptides of M. tuberculosis is a useful approach to identify new candidates important for diagnosis and vaccine applications.

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In silico Analysis and Experimental Validation of Mycobacterium tuberculosis-Specific Proteins and Peptides of Mycobacterium tuberculosis for Immunological Diagnosis and Vaccine Development

The cytokines produced by T helper (Th)1 cells (IFN-γ, IL-2 and TNF-β) correlate with protection, whereas the cytokines released by Th2 cells (IL-4, IL-5) and the anti-inflammatory cytokine IL-10 correlate with pathogenesis of tuberculosis (TB). However, the pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α and IL-12p70) are responsible for both protection and pathogenesis of TB. The aim of this work was to carry out a comparative analysis of cytokines present in early (day 2) and late (day 6) cultures of peripheral blood mononuclear cells (PBMCs) obtained from pulmonary tuberculosis patients. PBMCs were cultured in vitro in the absence and presence of exogenously added complex mycobacterial antigens and RD1 peptide pool. The supernatants were collected on day 2 and day 6 of culture and assayed for secreted cytokines using the flow cytomix assay. All of the cytokines, except for IL-12p70, were spontaneously secreted by PBMCs of 27–100% TB patients, but only TNF-α concentration was significantly higher on day 2 than day 6 (P < 0.05). Two days following antigenic stimulation, only IL-1β, IL-6, TNF-α and IL-10 were secreted in response to some mycobacterial antigens. However, 6 days later, all of the cytokines, except for IL-2, IL-4, IL-5 and IL-8, were secreted significantly in response to all complex antigens and RD1 peptides, compared with the non-stimulated cultures (P < 0.05). In conclusion, the study shows that the longer in vitro stimulation time (6 days) was necessary for the optimal induction of IFN-γ and TNF-β, and practically convenient for the detection of IL-10, IL-1 β, TNF-α and IL-6.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-3083.2012.02692.x

Comparative analysis of spontaneous and mycobacterial antigen-induced secretion of Th1, Th2 and pro-inflammatory cytokines by peripheral blood mononuclear cells of tuberculosis patients.

Comparative genomic studies have identified several Mycobacterium tuberculosis-specific genomic regions of difference (RDs) which are absent in the vaccine strains of Mycobacterium bovis BCG and which may be useful in the specific diagnosis of tuberculosis (TB). In this study, a total of 775 synthetic peptides covering the sequences of 39 open reading frame (ORF) proteins encoded by genes predicted in five RDs of M. tuberculosis, i.e., RD1, RD4, RD5, RD6, and RD7, were tested by enzyme-linked immunosorbent assays for antibody reactivity with sera from HIV-negative pulmonary TB patients (n = 100) and M. bovis BCG-vaccinated healthy subjects (n = 100). The results identified three immunodominant peptides reactive with TB sera, i.e., amino acids (aa) 346 to 370 of RD1ORF Rv3876, aa 241 to 265 of RD6ORF Rv1508c, and aa 325 to 336 of RD6ORF Rv1516c. These peptides had significantly stronger antibody reactivity with sera from TB patients than with sera from healthy subjects (P < 0.05) and significantly higher rates of positivity with TB sera (positives = 66 to 93%) than sera from healthy subjects (positives = 10 to 28%). Antipeptide antibodies were raised in rabbits after immunization with pools of 11 peptides corresponding to each protein. Probing of culture filtrates and whole-cell lysates of M. tuberculosis with antipeptide antibodies suggested the natural expression of Rv1516c in whole-cell lysates of M. tuberculosis. The results suggest the potential of the identified immunodominant RD peptides in the serodiagnosis of TB.

https://cvi.asm.org/content/cdli/18/3/477.full.pdf

Identification, diagnostic potential, and natural expression of immunodominant seroreactive peptides encoded by five Mycobacterium tuberculosis-specific genomic regions.

The PE35 (Rv3872) gene of Mycobacterium tuberculosis is present in the region of difference (RD) one that is deleted in all vaccine strains of Mycobacterium bovis bacillus Calmette Guerin. The aim of this study was to clone PE35 DNA into a DNA vaccine plasmid with CMV promoter and interleukin-2 secretory signal and evaluate the recombinant plasmid for induction of antigen-specific cellular responses in mice. DNA corresponding to PE35 was PCR amplified from the genomic DNA of M. tuberculosis H37Rv, cloned into pGEMT-Easy vector and sub-cloned into the DNA vaccine vector pUMVC6. BALB/c mice were immunized with recombinant pUMVC6/PE35 and spleen cells were tested for T-helper (Th)1-type (antigen-induced proliferation and secretion of IFN-γ) and Th2-type (IL-5), and anti-inflammatory (IL-10) cytokine responses to pure recombinant PE35 protein and its synthetic peptides. Mice immunized with the recombinant plasmid DNA (pUMVC6/PE35) showed positive Th1-type cellular responses to pure PE35, but not to an irrelevant antigen, i.e. PPE68 (Rv3873). However, the vaccine construct did not induce antigen-specific Th2-type (IL-5) or anti-inflammatory (IL-10) reactivity to PE35. Testing with synthetic peptides showed that Th1-type cells recognizing various epitopes of PE35 were induced in mice immunized with pUMVC6/PE35 DNA. These results suggest that pUMVC6/PE35 may be useful as a safer vaccine candidate against TB.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-3083.2011.02604.x

Cellular Immune Responses in Mice Induced by M. tuberculosis PE35‐DNA Vaccine Construct

Besides being the most widely used vaccine directed against tuberculosis (TB) worldwide, Mycobacterium bovis BCG is also the most controversial vaccine in current use. Its protective efficacy varies widely in different parts of the world. One approach to improving the current BCG vaccine might be to produce recombinant BCG strains that express major antigens encoded by genes that are present in the M. tuberculosis-specific region of difference 1 (RD1), such as pe35, cfp10, and esat6. In this study, pe35, cfp10, and esat6 genes were cloned into shuttle plasmid pDE22 to generate the recombinant plasmids PDE22-PE35, PDE22-CFP10, and PDE22-ESAT6, which were electroporated into BCG to generate recombinant BCGs (rBCGs). The cellular immune responses (antigen-induced proliferation and secretion of selected T helper 1 [Th1], Th2, and anti-inflammatory cytokines, i.e., gamma interferon [IFN-γ], interleukin 5 [IL-5], and IL-10, respectively) that are specific to the proteins of cloned genes were studied by using spleen cells from mice immunized with native BCGs and rBCGs and synthetic peptides covering the protein sequence of the cloned genes. The results showed that the spleen cells did not secrete IL-5, whereas IL-10 was secreted in response to peptides of all three proteins from mice immunized with rBCGs only, suggesting expression of the cloned genes and in vivo priming of spleen cells to the expressed proteins. However, in Th1 cell assays that correlate with protective cellular immune responses, i.e., antigen-induced proliferation and IFN-γ secretion, only mice immunized with rBCG-pDE22-PE35 yielded positive responses to the peptides of PE35. These results suggest that rBCG-PDE22-PE35 is the only one of the three vaccines used in this work that is worthy of consideration as a new vaccine candidate against TB.

Cellular Immune Responses to Recombinant Mycobacterium bovis BCG Constructs Expressing Major Antigens of Region of Difference 1 of Mycobacterium tuberculosis

A. S. Mustafa

Papers

Cell mediated immunity assays identify proteins of diagnostic and vaccine potential from genomic regions of difference of Mycobacterium tuberculosis.