Immune Responses Of Recombinant Adenovirus Containing MOMP Gene Of Chlamydia Trachomatis Serovar E And Dendritic Cells Transfected With Recombinant Adenovirus

Chlamydia trachomatis (Ct) is the most prevalent sexually transmitted bacterial pathogen in the world, with an estimated 100 million clinically diagnosed cases occurring annually. The incidence rate of genital Ct infection has increased significantly in china with 2.5% of people of 20-64 years of age infected. Sexually transmitted C. trachomatis infection is an important public-health concern because of its adverse effects on reproduction. Ct infection causes urethritis and epididymis. Although antibiotic therapy appears to effectively eliminate chlamydial infection, asymptomatic infections are common (>50%) and, when left untreated, can progress to chronic disease, including pelvic inflammatory disease, ectopic pregnancy, and infertility. More recently, genital chlamydial infection has been found to be highly associated with the increased risk for acquiring human immunodeficiency virus infection and the persistence of high-risk types of human papillomavirus infection. It is likely that the most effective form of intervention for diseases caused by C. trachomatis will be a vaccine.Early vaccine studies showed that whole inactivated bacterial cells administered intramuscularly were partially protective in human and primate trials. The protection was of short duration and was serovar specific and, in some of the vaccinated individuals that were reexposed to Chlamydia, a severe disease developed. This hypersensitivity reaction was thought to be due to a component present in the organism. Based on these observations, recent efforts have focused on developing a subunit vaccine using the major outer membrane protein (MOMP) of C. trachomatis as the antigen. Mouse models of genital infection with C. muridarum, which has most of the same genes as the human strains of C. trachomatis, have been widely used for Ct vaccine research. However, there are some important differences between C muridarum and C. trachomatis that might affect the immunobiology of infection. C trachomatis shows substantial allelic variation of its dominant surface protein MOMP, whereas C. muridarum has a single allele.Based mainly on the differences in the amino acid sequence of the variable domains (VD) of the major outer membrane protein, C. trachomatis is grouped to 19 human serovars, among which serovar E is most prevalent. There may be some biological properties in serovar E in favor of its transmission. Human C. trachomatis may be less virulent in mice than C. muridarum. The low degree of pathology has been observed in the oviducts of mice infected with Human C. trachomatis serovar E. Ito JI Jr et al. has established genital tract infection in the CF-1 mouse by intravaginal inoculation of a human oculogenital isolate of C. trachomati serovar E. Using this mice model, Lyons JM et al. prove that C. trachomati serovar E isolates from patients with different clinical manifestations have similar courses of infection. It is reported that prior genital tract infection in C3H/HeN mice with a human biovar of Chlamydia trachomatis E protects mice against heterotypic challenge infection. In 2006 Wang et al. have successfully established a BALB/c murine model of genital tract infection with Ct serovar E through vaginal inoculation. Previous studies confirm the availability of murine model genitally infected with C.trachomatis serovar E. Therefore, we have carried out some research on vaccines of C.trachomatis serovar E MOMP.Construction of recombinant adenovirus and DNA vaccine carrying Chlamydia trachomatis serovar E MOMP geneAdenovirus (Ad) vectors have several advantages over many conventional vaccines. They are relatively stable and easy to manipulate, can be readily obtained in high titers (>10⁹ plaque-forming units per ml) by propagation on 293 or PER.C6 cells, and can be easily purified. Importantly, the Ad genome has been shown to persist episomally rather than to integrate into its host DNA. Ad vectors can be administered by various routes such as intranasal, oral, intratracheal, intraperitoneal, intravenous, intramuscular, or subcutaneous ones. Consequently, Ad-based vaccines are able to induce not only a systemic immune response, but also, upon application to the respiratory or intestinal tract, a good mucosal immune response. Mucosal immune responses may be particularly useful to prevent infections by C.trachomatis that infect the host via genital mucosal membranes. DNA vaccines represent a new and promising technology that uses DNA to encode the antigen(s) of interest. Antigen is produced within the transfected cells minaicking a real life viral infection. Therefore, we constructed recombinant adenovirus and DNA vaccine carrying Chlamydia trachomatis serovar E MOMP gene which provide the basis of further research on the immune responses in murine model and on the biological functions of MOMP protein.The MOMP gene fragments of Chlamydia trachomatis serovar E were amplified from genome DNA by PCR method. They were cloned into pcDNAII vector and then were subcloned into the pshuttle vector pDC316. Recombinant pshuttle vectors pDC316-MOMP were confirmed by enzyme digestion and PCR. The pshuttle vector pDC316-MOMP and the adenovirus genomic plasmids PBHGloxAEl,3Cre were cotransfected into 293 cells for packaging and amplification of recombinant adenovirus Ad-MOMP, which was identified by PCR, RT-PCR and Western blotting.MOMP were released by double-enzyme digestion of pcDNAII-MOMP and were subcloned into the vector pVAX1 to construct the recombinant eukaryotic expression plasmid pVAX1-MOMP. The recombinant expression plasmids were transfected into COS-1 cells. The expression of MOMP protein in COS-1 cells was confirmed by Western blotting.Combined immunization of DNA vaccine and recombinant adenovirusIt has been well recognized that heterologous prime-boost immunization confers a stronger response to immunogens and greater protection in several infectious disease models than immunization with either vector alone. It has been reported that priming with MoPn MOMP DNA followed by MOMP ISCOM boosting enhanced protection and was associated with increased immunoglobulin A and Th1 cellular immune responses. Here, we investigated the specific humoral and cellular immune response induced by prime-boost immunization of MOMP DNA vaccine and recombinant adenovirus.Six to eight week old BALB/c mice were inoculated intramuscularly with 100 ug of plasmids pVAX1-MOMP and were boosted twice, two weeks apart, by the same route and with the same dose. DNA vaccination was able to elicit partial cellular immune responses (spleen T cells producing minor IFN-γ) and serum IgG and IgA antibodies, but did not generate mucosal immune responses.Mice immunized intranasally with 10⁸ pfu (final volume of 30μl) of recombinant adenovirus Ad-MOMP could generate higher IFN-γsecretion from spleen cells, higher serum antibodies level than DNA vaccination. More importantly, mucosal IgA antibodies were induced. In addition, IgG2a levels were higher than IgG1, suggesting a Chlamydia-specific Th1-biased immune response.A predominant Th1-type response, serum antibody response and genital mucosal response were significantly enhanced by both DNA prime-adenovirus boost and adenovirus prime-DNA boost strategy. While humoral immune response including mucosal IgA production was stronger in DNA/Ad immunization, and Th1-type cell response was more visible in Ad/DNA immunization, indicating that the order of combined immunization is relevant to the type of immune response.It is necessary for a desirable antichlamydial vaccine to induce both CD4+ Th1 and mucosal IgA immunity. The present study is the first demonstration that Chlamydia-specific immune responses can be enhanced significantly by using prime-boost regimen based on DNA vaccine and recombinant adenovirus. In general, DNA appears to be most effective at priming immune responses and is somewhat less effective as a boosting agent. Khanam(2006) examined the relative efficacies of plasmid prime/Ad boost (P/A) and Ad prime/plasmid boost (A/P) regimens in eliciting DEN-2 virus-specific immune responses, and found the immune response induced by the A/P regimen appeared to be relatively more potent. Our result that DNA prime-adenovirus boost enhance the immune responses is consistent with most studies. But why Ad/DNA regimen is more effective in Th1 response than DNA/Ad immunization, however weaker in humoral immune responses. One reason the Ad vector is better than the plasmid vector in priming is likely to be the result of superior antigen gene transduction into antigen presenting cells by the former, compared to the latter. Another reason is that plasmids inoculated intramuscularly induce primarily Th1 responses.Above study indicate that the order of combined immunization is very important and this shows promise for the enhancement and development of vaccines against Chlamydia trachomatis.Immune function of dendritic cells transfected with recombinant adenovirusDendritic cells (DC) are the most potent antigen-presenting cells (APC) and appear optimally capable of initiating and modulating the T-cell immune response. Presently, the proven means of eliciting potent protective immunity against C. trachomatis infection in murine genital mucosae is infection itself. Interestingly, adoptive immunization with dendritic cells (DC) pulsed ex vivo with killed chlamydial EBs elicits levels of protective immunity equivalent to those generated by infection. But a pathological component present in the chlamydial organism limited the use of this kind of vaccine. Shaw et al. (2002) have reported dendritic cells pulsed with a recombinant MoPn MOMP antigen (rMOMP) elicited a CD4+ Type 2 rather than type 1 immune response that was not protective, though rMOMP-pulsed DC produced IL-12 and stimulated infection-sensitized CD4+ T cells to proliferate and secreted IFN-γ.Modification of gene expression in dendritic cells could provide important advantages over protein antigen pulsing. Expression of a transgene could extend the duration of antigen presentation. Replication-deficient recombinant adenovirus (rAd) vectors have been shown to be particularly effective at infecting DC. Furthermore, rAd infection causes DC maturation, making them more effective stimulators of T cell responses. Here, we used recombinant adenovirus delivering Ct E serovar MOMP to modify dendritic cells, and examined the immune function effects of modified dendritic cells in vivo and ex vivo. Bone marrow-derived DC were generated from female BALB/c mice and induced with recombinant cytokines GM-CSF and IL-4. At the 8^th day, DC were harvested for DC typical phenotypes by fluorescence-activated cell sorter (FACS) analysis, and DC expressed high levels of CD11c and MHC-II surface markers, moderate levels of CD80.Then DC were infected with adenovirus encoding fluorescin EGFP at various values of multiplicity of infection (MOI). Ad vector transduction effciency was consistently >90% at 1000 MOI, as determined by fluorescence expression following transduction with Ad-EGFP. Therefore DC were transfected with recombinant adenovirus Ad-MOMP (rAd) and MOMP expression was detected by Western blotting.rAd transfection caused increased expression of CD80 and MHC II in DC; the expression of CD80 was significantly higher in Ad-MOMP-DC than Ad-DC and untransfected DC; the expression of MHC II was slightly higher in Ad-DC than untransfected DC; the expression of CD80 was slightly lower in Ad-DC than untransfected DC; however, either rAd or Ad control transfection did not result in significant increases or decreases of CD11c.Ad-MOMP transfection enhanced the production of proinflammatory cytokines IL-12 but not IL-10, increased the ability to induce allogeneic T-cell proliferation, and enhanced IFN-γsecretion by stimulated T cells. In addition, IFN-γsecretion stimulated by Ad-DC was slightly higher than negative control. These observations indicate that rAd induces a mature DC phenotype, which imply that rAd-transfected DC would be potent inducers of MOMP-specific CD4+ Th1 immunity in vivo.Mice adoptively immunized with rAd-transfected DC generated a CD4+ Th1 response, as shown by the predominant serum IgG2a antibody response and the higher secretion of IFN-γbut not IL-10 by cultured T cells, which was not consistent with the immune response elicited by MoPn rMOMP-pulsed DC. rAd-transfected DC elicited potent protective Th1 response, and secretion of IFN-γby spleen cells is 1.2 or 0.6 times higher than DNA/Ad or Ad/DNA immunization. An interesting finding in this study was the induction of IgA antibody response against chlamydial EBs in the group immunized with rAd-transfected DC. But serum IgG and IgA, muscol IgA of mice immunized with rAd-transfected DC were just similar to Ad/DNA group.In conclusion, our results demonstrate that DC transfected with Ad-MOMP secrete IL-12 and stimulate naive T cells to proliferate and secrete gamma interferon ex vivo, and elicit a Chlamydia-specific Th1-biased immune response and immunoglobulin A response in vivo which is opposite to the immune responses resulting from mice immunized with MoPn rMOMP-pulsed DC. We speculate that as an efficient adjuvant adenovirus vector may help to enhance immunological properties of MOMP, or the variation of MOMP leads to this kind of discrepancy. These findings might prove useful for chlamydial vaccine design.Analysis of synonymous codon usage bias in ChlamydiaIt has been demonstrated that codon optimization can enhance the expression and immunogenicity of the target gene. Meanwhile, from above study we found that recombinant plasmid pVAX1-MOMP and recombinant adenovirus Ad-MOMP both had a poor expression of MOMP in eukaryotic cells. These findings suggest that codon optimization may enhance the expression and immunogenicity of recombinant adenovirus Ad-MOMP. But whether human strains of C. trachomatis and other species of Chlamydia such as C. pneumoniae show similar codon usage pattern to MoPn are still need to be resolved.To explore the availability of codon optimization of MOMP gene, we analyzed the codon usage data of MoPn, Ct (here indicating biovar trachoma and LGV), C. pneumoniae and C. psittaci and compared also the codon preferences of Ct with those of Escherichia coli, Saccharomyces cerevisiae, adenovirus and homo sapiens using bioinformatics softwares such as DNAStar, codon usage database, EMBOSS and so on.The results have revealed that 1) MoPn, Ct, C. pneumoniae and C. psittaci adopt similar codon usage patterns though C. psittaci shows the greatest bias towards optimal codons; 2) the chlamydial species prefer to use the codons with A and T at the third codon position. Meantime, the biased trend towards A and T is coincided with high A+T content at silent sites in Chlamydia (mean value is 70%); 3) the gene codon usage pattern is significantly different between Chlamydia (MoPn, Ct, C. pneumoniae and C psittaci) and human genomes. Compared with human, the four chlamydial genomes do not have the same preferred codons; 4) Codon usage of Ct genes more closely resembles that of yeast genes than that of E.coli, adenovirus and human genes, which may provide some explanation for the poor expression of Ct E servoar MOMP in eukaryotic cells.These findings suggest that optimized codons of human strains of C. trachomatis gene may enhance the immunogenicity of chlamydial vaccine, and it is still necessary to differentiate various chlamydial species even biovars when designing specific strategies for optimizing chlamydial codons even though there are significant similarities in codon usage pattern among all tested Chlamydia species. We also analyzed the codon usage pattern between adenovirus and human genes, and found their codon bias is similar which support the efficacy of MOMP gene optimized according to human optimal codon. On further studies we are planning to test our hypothesis. At the same time, we can speculate that the Chlamydia genes may be more efficiently expressed in the yeast system.Knowledge of codon usage pattern in Chlamydia may therefore assist the development of Chlamydia vaccines and improve understanding of the evolution and pathogenesis of Chlamydia.