Studies On The Immunological Effect Of Codon Optimized Chlamydia Trachomatis MOMP DNA Vaccine

Chlamydia trachomatis (Ct) is an obligate intracellular bacterial pathogen that infects the epithelium of the genital and ocular mucosa. It is a common cause of several sexually transmitted diseases and is the causative agent of trachoma, the leading cause of preventable blindness worldwide. In addition, infection with Ct facilitates the transmission of human immunodeficiency virus (HIV) and might be a co-factor in human papilloma virus (HPV)-induced cervical neoplasia. Female genital tract infection can lead to serious complications which include cervicitis, endometritis, salpingitis, and therefore result in infertility and ectopic pregnancy. Although effective antimicrobial therapy is available, this has been largely unsuccessful in halting the spread of infection, most likely due to the high rate of asymptomatic infections, which may persist for months to years. Thus, improved means of prevention and control of Ct genital tract infections are urgently required, particularly the development of a vaccine to prevent infection.Chlamydiae are distinguished from all other intracelluar pathogens by their characteristic developmental cycle and two morphologically and metabolically distict cellular forms: the infectious form of the organism, termed the elementary body (EB) and the replictive form, termed the reticulate body (RB). Outer membrane compoments of the EB are determinants of chlamydia pathogenicity and primary targets of the host immune system. Among them the major outer membrane protein (MOMP) is regarded as a major protective antigen and a primary target antigen for current chlamyidal vaccine development. It has B cell and T cell antigenic determinants (especially in its variable domains) that could elicit T-cell responses and neutralizing antibodies, and is regarded as the most promising vaccine candidate. But so far studies in MOMP vaccination are not very satisfactary: the immunogenicity is poor, MOMP vaccination only elicit low serological response and produce partial protective immunity. Recently, scholars did lots of researches on codon optimization, which uses mammal preferable codon replace corresponding codon of low-expression pathogeny organism gene and furthermore amino acid coded by rebuilt codon is invariable, as a result the level of protein expression is significantly improved. The immunogenecity of the codon optimized DNA vaccine may be improved. With this method great progress has made in the research of the virus vaccine and also in some prokaryotic organisms. But there is no relevant research work done on codon optimized Ct vaccine throughout the world.Previous studies of our research group had analyzed the difference of preferable codon usage between Ct and mammal gene, constructed a codon optimized gene (HuMOMP, GeneBank accession NO. AY732495) through substitute human preferred codons for rarely used codons, and constructed the HuMOMP and wild type MOMP DNA vaccine. In this study we aimed at evaluating the immunological effect of codon optimized C. muridarum MOMP DNA Vaccine, finding a better immune route to induce stronger protection against Chlamydia infection. Our study conducted for the first time on the study of human-optimzed Chlamydial DNA vaccine.First we cultured C. muridarum in Hela229 cells, abstracted and purified it; constructed a mouse model of chlamydial genital tract infection. Then we evaluated the immunological effect of human-optimized vaccine on mice through intramuscular immunization. Result demonstrated that codon optimization enhanced the cellular and humoral immune responses caused by MOMP DNA vaccine. In order to found out the mechanism of it, we compared the in vivo protein expression of the humanized and wild type MOMP DNA, and the mRNA expression levels of them. On the bases of the data got from intramuscular immunized mice, we optimized the immune conditions by changing inoculate route and using adjuvant, and got better immunological effect and stronger protective immunity against C. muridarum genital infection. Our study conducted a primary exploration for the design of Ct relevant vaccine and even effective vaccine of other pathogen.1. Culture, purification of C. muridarum and construction of a chlamydial genital tract infection model with mouseC. muridarum was cultrured in Hela-229 cells. First we use DEAE-dextran treat the Hela-229 cells, make it susceptible to C. muridarum infection, then add the infectious sample(containing living C. muridarum), cultured for 2-3h; discard the infectious sample add cell culture liquid and cultured for 42-48h, the chlamydial inclusion will be found in the cells. Collect the infected cells and sonicate to set the Ct elementary bodies free. Centrifuge, purify the Ct elementary bodies using discontinuous renograffin gradient.Next we constructed a mouse chlamydial genital tract infection model by vaginal inoculation of C. muridarum, found out the suitable number of IFU to infect mice, and studied the characteristic of chlamydial genital tract infection of this mouse model.Our results show that, the vaginal inoculation of C. muridarum results in an infection that naturally ascends from the lower genital tract (vagina and cervix) to upper genital tract tissues (uterine horns and oviducts). In our study, we found that postinfection sequelae were noted in groups inoculated with 10~7,10~6 and 10~5 IFU C. muridarum. 10~5 IFU is the suitable number to the mouse model of chlamydial upper genital tract infection. The pathological characteristic of genital tract tissues can vary with diferent time of postinoculation. Pathologic findings include: The initial inflammatory response elicited by infection is characterized by a marked mucosal edema, focal erosion and necrosis, infiltration of neutrophils. In later stage, fibrosis is noted; the mucosa and the surrounding soft tissue were infiltrated with lymphocytes.2. Comparation of the immunological effects induced by intramuscular immunization with HuMOMP and WtMOMP DNA vaccine1). To examine in vivo immune responses induced by the wild-type and codon modified MOMP expression plasmids, female BALB/c mice were immunized intramuscularly with the pcDNA3-huM0MP and pcDNA3-wtM0MP respectively. The results showed that the mice immunized with HuMOMP produced higher levels of antigen-specific IgG antibody, showed stronger DTH reaction and proliferative T cell responses than those with WtMOMP immunization. Antigen-specific stimulation of spleen cells obtained from HuMOMP DNA immunized mice produced higher IFN-γ than those with WtMOMP DNA.These data confirm that codon optimization enhances the immunogenicity of DNA vaccine encoding MOMP gene. Codon optimized MOMP DNA vaccine can induce higher specific CMI and serum IgG antibody immune response than the wild type DNA vaccine. However, it did not induce detectable mucosal secretory IgA, just as the wild type DNA vaccine. The protection against C. muridarum vaginal infection induced by the HuMOMP DNA vaccine has no significant differences compared with the wild type one.2). the mechanism for the increase in imunogenicity of codon modified MOMP DNA vaccine was analyzed as follows:We first compared the in vivo protein expression levels of wild-type and codon modified MOMP gene, plasmids DNA pcDNA3-HuMOMP and pcDNA3-WtMOMP were respectively injected into tibial muscles of mice. At 72 h after DNA injection, muscle samples from the DNA inoculation sites were collected, lysed, and homogenized with protein extraction buffer. The expressed MOMP protein was detected by Western blot. Results showed that HuMOMP gene produced significantly higher levels of MOMP protein than WtMOMP.Codon optimization enhances the expression of MOMP protein in mammalian cells and as a result the immunogenicity of DNA vaccine encoding the MOMP gene, but very little is known about the moclecular mechanisms.To study the mechanism of the enhanced protein expression of codon optimized MOMP gene in mammalian cells, we compared the mRNA expression levels of codon optimized MOMP gene and wild type MOMP gene.The mammalian cells, COS-1 cell, were transfected with plasmid pcDNA3-HuM0MP or pcDNA3-WtMOMP. The MOMP mRNA expression levels of the two MOMP genes were analysed by real time RT-PCR. Results showed that the cpm for HuMOMP mRNA was only 1.36 fold that of WtMOMP mRNA, representing little increase of mRNA expression of codon-optimized MOMP gene. Statistical analysis indicated that no obvious difference existed between modified and native MOMP in the level of mRNA expression. These results suggested that it is likely that posttranscriptional mechanisms account for the low expression of C. muridarum protein in mammalian cells.3. Studies on the immunological effect induced by intranasal immunization with codon optimized Chlamydia trachomatis MOMP DNA vaccineSince an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In order to get this goal we decided to use B subunit of cholera toxin and CpG as adjuvant, study the immunological effect of codon modified MOMP DNA vaccine by intranasal immune route.HuMOMP plasmids or vector plasmid were mixed with B subunit of cholera toxin and CpG adjuvant, administered into each of the external nares of mice using a micropipette. The other group of mice received HuMOMP plsmids only, Control mice were inoculated with PBS or C. muridarum EBs. The immune efects of Ct DNA vaccine and the adjuvant were evaluated by detecting of antibodies, lymphocytes, and observing of DTH response, proliferation assay of spleen lymphocytes, clearance ability for Ct challenge from genital tract, and local histomorphology.Results showed that intranasal immunization with HuMOMP DNA vaccine in combination with both CTB and CpG oligodeoxynucleotides elicits MOMP-specific slgA in vaginal lavage fluid, MOMP-specific IgG in serum. Annlysis of serum IgGland IgG2a levels and cytokines producted by the spleen cells of immunized mice proved that the HuMOMP DNA vaccine in combination with CTB and CpG elicits Th1 predominat CMI. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice. The protection effect induced by intranasally immunization with HuMOMP DNA vaccine combined with CTB and CpG was similar to the C. muridarum EB immunized group.Taken together, this study suggests a novel strategy in the enhancement of the immunogenicity of Chlamydia MOMP DNA vaccine, and lays an experimental foundation for developing Ct MOMP prophylactic and therapeutic vaccine.