Construction Of Yersinia Pestis Candidate DNA Vaccines And Identification Of Their Immunoefficiency

Yersinia pestis is the etiological agent of bubonic, speticemic and pneumonic plague diseases which had caused over 200 milllion human deaths in the past. Plague still occurs throughout the world today and it is one of the agents of greatest concern with respect to illegitimate use in bioterrorism or biological warfare. A killed whole cell plague vaccine had been used in the past, but recent studies in animals had shown that this vaccine offers poor protection against pneumonic disease. A live attenuated vaccine is also available. Though this vaccine is effective, it retains some virulence and in most countries it is not considered to be suitable for use in humans. Thus it is very important to develop new plague vaccines. Recently, DNA vaccines become appreciated because they are able to induce cellular and humoral responses to the encoded antigens and can be used repeatedly due to lack of immue responses to the vector itself. The secretory signal sequence derived from gene encoding tPA, which was shown to promote efficient secretion of heterologous proteins and has been associated with enhanced immunogenicity. Yersinia Pestis F1 ,V gene were amplified from EV76 line by PCR and the products were 513bp and 981bp respectively. They were inserted into the eukaryotic expression vector pVAX1 and construct recombinant eukaryotic expression vectors pVAX1/F1, pVAX1/V and pVAX1/F1-V. tPA signal encoding sequence was amplified by PCR method and then was inserted to the 5'terminus of those recombinant plasmids respectively to construct secretion eukaryotic expression vector tPA-pVAX1/F1, tPA-pVAX1/V and tPA-pVAX1/F1-V. Immuno-cytochymistry method and western blot analysis indicated that Yersinia pestis F1, V and F1-V fusion proteins were expressed in COS-7 cells after transfected 48h by those recombinant plasmids respectively. When BALB/c mice were immunized with 100μg recombinant plasmids combined with plasmid DNA carrying GM-CSF gene via an intramuscular route, specific antibodies were detected in sera of mice by ELISA. The tPA-F1-V and tPA-V candidate DNA vaccines were able to induce a sharp increase in antibody response following a single immunization while other candidate DNA vaccines required at least two immunizations to produce a response of the similar level. Furthermore immunoglobulin G isotype analysis indicated that the tPA-F1-V and tPA-V groups displayed a large increase in IgG2a relative to their wild type groups. In mice, increased IgG2a level indicated a Th1 type immune response was induced which might play critical roles for the better protection provided by the tPA-F1-V and tPA-V candidate DNA vaccines. The mice immunized with those candidate DNA vaccines could induce IFN-γsecreting in spleen cells as determined by ELISPOT assay. As compared with their wild type groups, the tPA-F1-V and tPA-V groups secreted significant amounts (P<0.01). The result indicated that all those immunized animals could induce Yersinia pestis specific CTL response that could lysis Yersinia pestis target cells and produce IFN-γ. Furthermore, tPA-F1-V and tPA-V groups induced more spots than other groups indicated that those two tPA groups induced an enhanced Th1 type response. tPA-F1-V and tPA-V were able to induce effective immuno-protection against subcutaneous challenge of virulent Yersinia pestis with 400 LD50, the survival rate was 90%. In summary, We have Successfully constructed six recombinant eukaryotic expression plasmids pVAX1/F, pVAX1/V, pVAX1/F1-V, tPA-pVAX1/F1, tPA-pVAX1/V and tPA-pVAX1/ F1-V and all those candidate DNA vaccines could induce both humoral and cellular immune responses. Mice immunized with tPA-pVAX1/V or tPA-pVAX1/F1-V could provide a 90% protection against 400 LD50 subcutaneous challenge dose. Those two candidate DNA vaccines could be used as potential candidate vaccines for the control of Yersinia Pestis.