| Gonorrhoeae is one of the most prevalent sexually transmitted diseases, caused by Neisseria gonorrhoeae. Gonococcal diseases mainly include acute or chronic purulent infection on the mucous membrane of the urogenital system, such as urethritis, prostatitis, epididymitis in men. Asymptomatic infection is more often seen in female, so the infected women will not seek medical treatment in time. This asymptomatic condition is significant as it not only contributes to the persistence of the gonococcus within the general population, but it can also lead to more serious conditions, e.g. pelvic inflammatory disease (PID), infertility, ectopic pregnancy and disseminated gonococcal infection (DGI). In addition, antibiotic-resistance in N. gonorrhoeae is an important problem. The prevention of gonococcal infection has been the goal of many investigators in ongoing attempts to develop an effective anti-gonococcal vaccine.Many efforts have been made to develop an effective vaccine. In 1970's, crude killed whole cell vaccines have been tried in humans and chimpanzees, but showed no efficacy. Gonococcal pilus vaccines have been developed but a clinical trial of a pilus vaccine failed, probably because of the extensive antigenic variation of Pili. Opa protein vaccines have encounted the same problem of antigenic variation. This indicates that the conservation of the vaccine candidates is important.Plante recently cloned and sequenced the gene of Neisseria surface protein A (NspA) and proved that this protein is highly conserved and constantly expresses at the surface of intact bacterial cells of all N. gonorrhoeae isolates tested. Our previous work showed that NspA could elicit protective antibodies, which emphasized its potential feasibility as a vaccine candidate. Porin B (PorB) is also an important conserved out membrane protein. But its immune protection was inhibited by the small amount of contaminated Rmp when purified PorB from gonococcal cells was used as a subunit vaccine.In this study, we further explored the potential feasibility of NspA and PorB as effective veacines against gonorrhea. To improve the antibodies and protection level of gene immunization, gene immunized BALB/c mice were boosted with recombinant NspA or/and PorB. A prokaryotic expression vector pQE31NspA was constructed and proved to express rNspA in E. coli by SDS-PAGE and Western-blotting. The purified and renatured rNspA was used as immunogen. Then mice were divided into 3 groups and vaccinated with different antigens. The 1st group of mice were immunized with eukaryotic expression vector pCNspA for 4 times and then boosted with rNspA; the 2nd group was vaccinated solely with rNspA; the rest mice inoculated with blank vector and adjuvant were negative control. After immunization, high level of gonococcus-specific serum IgG was detected by ELISA in both of the 2 experimental groups.Antibody-complement-mediated bactericidal activity assay and antibody-mediated opsonophagocytosis assay indicated that the immune sera from the 2 groups of mice both had gonococcus-specific antibiosis activity. Serum from the gene immunization and rNspA boosting mice showed higher antibiosis activity.In order to avoid the contamination of PorB by Rmp, we used gene engineering products of recombinant PorB as immnogen; and to perform gene immunization, a PorB eukaryotic expression vector pcDNA3.1PorB was contructed with the PorB gene gifted by Dr. Christopher. The expression of PorB by pcDNA3.1PorB in COS-1 cells was revealed by Western-blotting, demonstrating that recombinant vector could express PorB gene in eukaryotic cells. Then we immunized mice with rPorB solely or with pcDNA3.1PorB first and boosting rPorB later. The 2 groups of mice both produced high level of gonococcus-specific serum IgG. Serum from the later group showed higher bactericidal activity and stronger opsonophagocytosis.The cooperation of the immune protection induced by NspA and PorB was explored. Mice were immunized with combination of rPorB and rNspA, or with pCNspA and pcDNA3.1PorB followed by the boosting of rPorB and rNspA. The antibody titer induced by combination immunization was higher than that induced by each of the immunogens. Serum from groups vaccinated with combinational immunogens showed stronger immune protection.These experimental data indicated that the two gonococcal proteins, NspA and PorB, possessed the potential value in developing effective vaccines against gonococcal diseases; and that the prime-boost protocol was a better vaccination procedure.
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