Development of salmonella typhi ty21a as a potential oral vaccine against tuberculosis: surface display and DNA vaccine carrier of a synthetic multi-epitope mycobacterial gene

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Date
2004-06
Authors
Ahmad Sarhan, Mohammed Abdel Aziz
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Abstract
Despite the discovery of the causative agent of tuberculosis (TB), Mycobacterium tuberculosis, more than 120 years ago TB remains a major worldwide health problem. Currently, the attenuated strain of M. bovis, Bacille Calmette-Giierin (BCG) is the only vaccine available against TB. Although BCG is the world's most widely used vaccine, its protective value as an anti-TB vaccine for adults in certain areas of the world, has been shown to be low or even non-existent. Thus there is general agreement that new novel vaccines are required for TB control and prevention especially in developing countries. In this study, the use of the live attenuated typhoid vaccine, S. typhi Ty21a, for development as candidate vaccines against TB was explored in which the organism was utilized in a surface display system as well as a carrier of a DNA vaccine. In the surface display approach, a surface display expression system was developed by the construction of a synthetic gene coding for the N-terminal of the ice nucleation protein (Inak-n) from Pseudomonas syringae using a method called assembly polymerase chain reaction (PCR). In this method, the Inak-n gene was assembled from 34 overlapping chemically synthesized oligonucleotides in a single step and amplified by PCR using specific cloning primers. The gene was cloned into the pCR®2.1-TOPO® vector to create a recombinant plasmid designated as pMSinak. Cloning of a previously constructed 0.82kb synthetic gene known as Vacll [which contained selected T cell epitopes of several M. tuberculosis genes namely ESAT6, MTP40, 38 kDa and MPT64 and further modified to include six consecutive histidine (6xH) residues at the C-terminal end for affinity purification purposes] ir..~o pMSinak resulted in the fusion of the lnak-n and Vacii genes and the resultant recombir..~nt plasmid was designated as pMSinak-nVacii. The fused Inak-n::Vacii (Inak-nVacii) gene from pMSinak-nVacii was then cloned into an expression vector pKK223-3 resulting in the final construct designated as pKMSinak-nVacii which when transformed into S. typhi Ty21a (creating the recombinant strain, r-STVII) and expressed allowed the fusion protein, Inak-nVacii, to be displayed on the surface of the host bacterial cells. In the :.econd approach, S. typhi Ty21a was utilized as a ca!Tier of DNA vaccine. In this study, S. typhi Ty21a was transformed with a previously constructed DNA vaccine cailed pJWVacii to create a strain called STVII-c. Both newly constructed vaccine candidates, r-STVII and STVII-c, were shown to be safe when tested in C57BL/6 mice. The immunogenicity of the two vaccine candidates in C57BL/6 mice were compared with each other and with the appropriate controls. Each mouse was immunized orally with a dose of 2Xl09 CFU of r-STVII or STVII-c (or controls) on Day 0 and Day 14 respectively and analyses were performed two weeks after the second immunization. The spleen cells of vaccinated mice were harvested and tested with the following assays: (i) Proliferation of T cells by thymidine uptake (ii) IFN-y in spleen cell culture supernatant by ELISA and (iii) intracellular expression of IFN-y by flow cytometry. In these studies, the purified recombinant protein (lnaknVacll) and the synthetic peptides corresponding to single epitopes in the Vacii protein were used as antigen specific stimulants. The stimulation index of splenocytes from vaccinated mice with r-STVII was found~to be about 2 fold higher than that of mice vaccinated with STV!I-c. Conversely how.: 1er, the concentration of IFN-y secreted in the culture medium of splenocytes from mice vaccinated with STVII-c was 2 fold higher than that of r-STVII. Intracellular cytokines analysis showed that both CD4+ and CDS+ T cells produced IFN-y when splenocytes were stimulated in vitro with purified Inak-nVacii orthe single epitope peptides. The data also showed that IFN-y produced by CD4+ T-cells from mice vaccinated with STVII-c was 1.3 fold higher than mice vaccinated with r-STVII when the cells were stimulated with purified Inak-nVacli. However, the data also showed that COS+ T-cells from mice vaccinated with STVII-c secreted 1.5 fcld higher IFN-y than mice vaccinated with r-STVII when stimulated with the same protein. The importance of targeting both CD4+ and COS+ T cells to stimulate effective protection against M. tuberculosis have been noted by many workers. In conclusion, the results obtained suggest that oral vaccination with the two new vaccine candidates produced in this study might be an efficient method for generating a broad and protective immune response against TB in the mouse model. The data generated by this study therefore may have an important impact in the strategy for developing newer vaccines against TB in humans.
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Salmonella typhi ty21A , Vaccine against tuberculosis , DNA vaccine carrier , Synthetic multi-epitope
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