| Background Plague is a category A infectious disease which caused by Yersinia pestis. The appearance of multi-antibiotic resistant strains and the potential misuse for biological warfare or bioterrorism alert us the threat brought by Y. pestis. Vaccination is an effective countermeasure to prevent the spread of plague and eliminate the bacterium at last. Unfortunately, there has no any effective vaccine that can be used widely against Y. pestis until now. The live attenuated and killed whole cell vaccines were once used in the epidemic areas of Asia. The variation of immunogenicity and the short time of protection suggested the impossibly wide use of these vaccines. Currently, the subunit and DNA vaccines of Y. pestis were focused on the known protective proteins F1 and LcrV. Although the vaccines based on these antigens were proved to be efficient against bubonic plague, the efficiency of them against pneumonic one need to be improved. At the other end, the F- strain found in natural plague foci was able to cause animal and human infection, and immunogenicity of LcrV antigen varied by different strains isolated from different foci. These facts made it an urgent demand that identification of novel protective antigens and use of them in combination with F1 and V antigens as multi-component subunit vaccines have been widely considered as one of the leading strategies for plague vaccine development in the future. Beside humoral immunity, cellular immunity also plays an important part in the defense against Y. pestis. The vaccine that can bring both humoral and cellular immunity will be the ideal target for the development of Y. pestis vaccine. There is few research about the cellular immunity of plague. According to the studies of Smily, there have other T cell antigens in the defense against Y. pestis except for F1 and LcrV. Thus, to effectively incorporate cellular immunity into subunit plague vaccines, it is now imperative to define the specific Y. pestis proteins that elicit protective cellular immune responses. The aim of this research is using the protein microarray and Elispot technology to find the proteins that can induce humoral and/or cellular immune response.Methods All putative open reading fragments (ORFs) of Y. pestis CO92 strain were submitted to computer analysis to identify genes potentially encoding surface-exposed, membrane-associated or exported proteins. The ORFs that have above three trans-membrane domains were discarded. Among the left ORFs, the genes that were likely acquired during the evolution of Y. pestis, the genes which encode homologous proteins that provide protection in other bacterial infection and the genes which may contribute to the virulence of Y. pestis according to the current studies were selected out to be expressed in E. coli. Then, the fusion recombinant proteins were purified by affinity chromatography on Ni2+-conjugated chelateing sepharose. The splenocytes from immunized or non-immunized mice were stimulated with purified recombinant proteins (10μg/ml), concanvalin A (ConA) (5μg/ml) or complete medium (RPMI1640) in triplicate to find the IFN-γsecreting cells to define the T cell antigen. Meanwhile, the protein microarray was fabricated by spotting the purified proteins on glass slides and the serum antibodies of Y. pestis infected in domestic dogs, cats and humans infected by Y. pestis were profiled by the microarray. At last, the protection efficacy of some selected proteins that can induced humoral or/and cellular immunity was evaluated using BAL B/c mouse as model. The proteins that can provide protection will be the targets for the next vaccine development.Results By in silico analysis, 430 ORFs that potentially encoded surface-exposed, member-associated or exported proteins and had no more than 4 trans-membrane domains were found. Among them, 261 proved or putative pathogenesis associated factors disclosed by STM(signature tagged mutation), proteomics, comparative genomics or gene expression profile and the proteins that have homologous to the protection antigens previously characterized in other microorganisms were selected out. All selected ORFs were cloned into plasmids pDEST17 or pET32a and expressed in E.coli BL21 (DE3). According to the SDS-PAGE analysis, 174 ORFs (67%) were successfully expressed. As most of the proteins were membrane-associated, 124 (71.3%) of them were expressed as inclusion bodies. At last, only 101 proteins (38.7%) were successfully purified to evaluate the ability to stimulate T cell response in vitro assay. Through the Elispot technology, the IFN-γproducing T-cell profiles from splenocytes of EV76 immunized or non-immunized control mice were examined with in vitro stimulation by each recombinant protein of Y. pestis. Using the spot number >100/106 splenocytes as positive criterion, 34 proteins stimulated stronger IFN-γresponses than other proteins. Meanwhile, a protein microarray including 218 recombinant proteins of Y. pestis was fabricated and used to profile the sera of Y. pestis infected cats, dogs and humans infected by Y. pestis. Twenty proteins that maybe contribute to the virulence of Y. pestis were discovered. At last, the protection efficacies of 28 proteins that can induce humoral and/or cellular were evaluated. Nine proteins can provide partial protection against the subcutaneous challenge of 20×LD50 Y. pestis 201 strain. |
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