Recombinant Vibrio cholerae ghosts as a delivery vehicle for vaccinating against Chlamydia trachomatis
Introduction
Infection of the genitourinary tract of humans with the obligate intracellular bacterium, Chlamydia trachomatis, is a major cause of sexually transmitted diseases [1]. Genital infection of women poses a significant risk often leading to pelvic inflammatory disease, ectopic pregnancy and infertility [2], [3]. Most genital tract infections in women are asymptomatic with severe complications often being the first symptoms of an infection. Although antibiotic therapy effectively eliminates chlamydial infection, it does not always affect established pathology [4], and the rampant asymptomatic infections make treatment of symptomatic individuals alone unlikely to be a successful control strategy. A vaccine against C. trachomatis is an attractive approach and the most promising and effective strategy for controlling these diseases.
Early human trials of candidate vaccines composed of whole chlamydial cells revealed that vaccinated individuals suffered exacerbated disease during subsequent infection episodes [5], [6], [7], [8]. Thus, the use of whole chlamydial agents as vaccine candidates appears to be unattractive due to the potential existence of immunopathogenic components [9]. Besides, progress made in molecular immunology and biotechnology in the last two decades has led to a gradual shift from the classical whole vaccines, consisting of inactivated and live-attenuated intact pathogens or their inactivated toxins, to peptide or subunit vaccines. Thus, current research has focused on the development of vaccines based on chlamydial subunit components. Although additional immunogenic proteins have recently been predicted [10], [11], [12], [13], there are eight major serologically defined chlamydial antigens recognized during human infection by immunoblotting analysis of sera from women with C. trachomatis cervical infections [9], [14], [15], [16]. Among potential protective antigens, the immunodominant 40 kDa MOMP protein is the best characterized, and the antigen that has shown most promise as a candidate vaccine. Purified MOMP and a number of other immunogenic subunits have been evaluated for their prophylactic and therapeutic potential in several animal models [17], [18], [19], [20]. To date these efforts have only been partially effective. This may suggest that MOMP alone is inadequate, calling for a multi-subunit approach, or that more effective delivery systems and adjuvants are needed to boost immune responses. As an alternative to purified antigens, cloned recombinant fragments of chlamydial proteins [18], [21], [22], [23] as non-replicating antigens, naked DNA or live recombinant vectors [24], [25] are other promising vaccine candidates.
Genetic inactivation of pathogenic bacteria by the controlled expression of cloned bacteriophage PhiX174 lysis gene E offers a promising new approach in non-living vaccine technology [26], [27]. Expression of plasmid-encoded gene E leads to the formation of a transmembrane tunnel structure through the cell envelope of Gram-negative bacteria and the loss of cytoplasmic contents. The resulting bacterial ghosts share the functional and antigenic determinants of the envelope with their living counterparts [28]. Recombinant Vibrio cholerae ghosts (rVCG) are attractive for use as non-living vaccines because they possess intrinsic adjuvant properties, maintain the structural and functional integrity of expressed antigens, and are excellent vehicles for delivery of foreign or heterologous proteins and other antigens of vaccine relevance to the primary antigen-presenting cells (APCs) [27]. It has been demonstrated that the cell targeting and adjuvant properties of ghosts are superior to Alum and complete Freund’s adjuvant [27]. Furthermore, the ghost technology can accommodate multiple proteins that can lead to a multi-subunit vaccine with greater potential for immunogenicity and protection.
To test the adjuvant properties of rVCG in enhancing the efficacy of an anti-chlamydial subunit vaccine, we have constructed a rVCG vector-based candidate vaccine expressing MOMP (rVCG-MOMP) and evaluated its ability to induce a local genital Thl response and confer protection against a C. trachomatis genital infection in mice. The results showed that rVCG-MOMP administered intramuscularly (IM) induced a genital mucosal chlamydial-specific Thl response. Also, the adoptive transfer of T cells from immunized mice into naı̈ve recipients conferred protection from significant genital chiamydial challenge. These results suggest that appropriately attenuated or inactivated bacterial vectors may be effective alternative methods of delivering subunit chlamydial antigens that will elicit protective cellular and humoral immune responses.
Section snippets
DNA preparation and PCR amplification
Stock preparations of C. trachomatis serovar D strain were generated by propagating elementary bodies (EBs) in HeLa cells as previously described [29]. All stocks were titrated on HeLa cell monolayers followed by purification of EBs over renografin gradients [29] and stored at −70 °C. Genomic DNA was purified from 1×108 chlamydial EBs using the QIAGEN DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The MOMP gene, omp1, was obtained by polyrmerase chain
Cloning and expression of chlamydial MOMP in V. cholerae 01
V. cholerae 01 clone HM12 harbors plasmid pCOM12, which was obtained by cloning the MOMP gene, omp1 into the membrane-targeting vector pKSEL5-2 (Fig. 1). Restriction enzyme and sequencing analyses confirmed the plasmid to contain omp1 sequences downstream from the lac promoter. In pCOM12, rMOMP protein is expressed under the transcriptional control of the lacpo promoter. The expression and identity of rMOMP protein was confirmed by Western blotting using a mouse monoclonal antibody to MOMP (
Discussion
A vaccine represents the best approach to protect the greatest number of people from the widespread and often devastating consequences of genital chlamydial infections. In the continued search for an efficacious vaccine, it appears that the use of whole chlamydial agents is unattractive due to the potential existence of pathologic reactions to certain chlamydial antigens [6], [9]. With attention focused on designing vaccines based on subunit components, the challenge to overcome poor
Acknowledgements
This work was supported by PHS grants (AI4123 1, GM 08248 and RRO3034) from the National Institutes of Health, and the Centers for Disease Control and Prevention (CDC). We thank Dr. Harlan D. Caldwell for kindly providing the monoclonal antibody L2 1–1 0.
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