| Yersinia pestis, the causative agent of plague, causes rapidly progressing disease in humans with a high mortality, especially in the pneumonic form of the disease. Due to the severe nature of plague, its ability for aerosol transmission, and the potential for human to human transmission, plague is considered to be a disease of high concern as an agent of biological warfare or biological terrorism. For this reason, an improved vaccine for plague is desirable. Current efforts for vaccine development have focused on two proteins: LcrV (also known as the V antigen) and the capsular F1 antigen. The best results to date have been obtained by using a combination of recombinant LcrV and F1 subunits, respectively, or as a fusion protein. These subunit vaccines demonstrate very good protection against both pneumonic and systemic forms of plague in mouse models. One of the potential limitations of these subunit vaccines is that F1 is not required for full virulence of Y. pestis, as F1-negative strains have the same LD50 value as F1-positive strains. The second limitation that could result in undesired side-effects in immunized individuals is the demonstrated immunosuppressive effect of LcrV. Additionally, serologic diversity of LcrV in Yersinia species other than Y. pestis could theoretically limit the use of an LcrV based vaccine. While the recombinant subunit vaccines are very effective in experimental animals and offer protection against F1 minus strains of Y. pestis, the inclusion of other antigens with the LcrV/F1 subunit vaccine candidates could improve the ability of the resulting vaccine to offer protection against multiple Y. pesti strains, or the new antigens could be developed as separate vaccine candidates.Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen. In the recent, pneumonic plague which is transmitted person-to-person by the respiratory route or as the result of the spread of bubonic or septicemic plague by an untreated person, is the most fatal form of plague. Administration of adjuvant vaccines by mucosal routes is important and imperative way of inducing long term immune responses to protect pneumonic plague. In this study, the outer membrane protein proteosome adjuvant was extracted from group B type 2 Neisseria meningitides; recombinant PorA, PorB and Class 4 protein were derived from gene recombinance. The studies reported here were conducted to determine which mucosa adjuvant vaccine was efficient and which adjuvant could be used as a mucosal adjuvant.1. YscF gene fragment of Y. pestis EV76 was amplified by PCR and cloned, inserted into pET32a vector. The recombinant YscF antigen of Y. pestis expressed in Escherichia coli BL21 (DE3) cells was purified by way of Ni2+ affinity chromatography. The purity was more than 80%. An proteosome-adsorbed vaccine of this YscF antigen has been intranarally immunized with there doses into experimental mice. Results demonstrated that intranasal immunization of mice with proteosome combined plague recombinant protein YscF mucosal vaccine induced higher systemic immune response and mucosal immune response. It was able to induce an effective immunoprotection against intranarally challenge of virulent Y. pestis. The survival rate was 20%. The recombinant YscF is a possible alternative or adjunct protective vaccine antigen against plague. YscF is an important protective antigen of Y. pestis.2. High purity proteosome was extracted from group B type 2 Neisseria meningitides. The endotoxin, nucleic acid and capsule polysaccharide contents of proteosome were lower than 1% of it. Plague YscF antigen formulated with proteosome adjuvant in a ratio of 3:1, and the plague adjuvant vaccines were used to immunize mice intranasally. The results demonstrated that the proteosome adjuvant vaccine induced better mucosal and systemic immune responses compared with the aluminium adjuvant vaccine.3. Proteosome was comprosed of three major proteins, PorA, PorB and Class 4. The gene of PorA, PorB and Class 4 were obtained by using PCR technology, cloned into pET-28a(+) vector, and expressed in E. coli. Western-blotting results confirmed that PorA, PorB and Class 4 proteins had immunogenicity. H5N1 splitted vaccine formulated with PorA, PorB and Class 4 proteins, and used to immunize mice intranasally. PorA-H5N1 vaccine demonstrated an ability to induce potent mucosal and systemic immune responses like proteosome adjuvant. So, PorA protein was shown to possess strong adjuvant activity. This is a new viewpoint in the vaccine adjuvant research.
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