Francis O. Eko

Suppression of Endogenous IL-10 Gene Expression in Dendritic Cells Enhances Antigen Presentation for Specific Th1 Induction: Potential for Cellular Vaccine Development

A new paradigm for designing vaccines against certain microbial pathogens, including Chlamydia trachomatis, is based on the induction of local mucosal Th1 response. IL-10 is an anti-inflammatory cytokine that exerts negative immunoregulatory influence on Th1 response. This study investigated whether biochemical modulation of endogenous IL-10 expression at the level of APCs is a practical strategy for enhancing the specific Th1 response against pathogens controlled by Th1 immunity. The results revealed that the high resistance of genetically engineered IL-10−/− (IL-10KO) mice to genital chlamydial infection is a function of the predilection of their APCs to rapidly and preferentially activate a high Th1 response. Thus, in microbiological analysis, IL-10KO mice suffered a shorter duration of infection, less microbial burden, and limited ascending infection than immunocompetent wild-type mice. Also, IL-10KO were resistant to reinfection after 8 wk of the primary infection. Cellular and molecular immunologic evaluation indicated that IL-10KO mice induced greater frequency of chlamydial-specific Th1 response following C. trachomatis infection. Moreover, IL-10KO APCs or antisense IL-10 oligonucleotide-treated wild-type APCs were potent activators of Th1 response from naive or immune T cells. Furthermore, both Ag-pulsed dendritic cells from IL-10KO mice and IL-10 antisense-treated dendritic cells from wild-type mice were efficient cellular vaccines in adoptive immunotherapeutic vaccination against genital chlamydial infection. These findings may furnish a novel immunotherapeutic strategy for boosting the Th1 response against T cell-controlled pathogens and tumors, using IL-10-deficient APCs as vaccine delivery agents.

Fc Receptor–Mediated Antibody Regulation of T Cell Immunity against Intracellular Pathogens

Immunity to intracellular microbial pathogens, including Chlamydia species, is controlled primarily by cell-mediated effector mechanisms, yet, the absence of antibodies results in inefficient microbial clearance. We investigated the hypothesis that certain Fc receptor functions promote the rapid induction of elevated T helper type 1 (Th1) response, which effectively clears chlamydiae. FcR(-/-) mice exhibited a delayed and reduced frequency of Chlamydia-specific Th1 cells, compared to FcR(+/+) mice. In vitro, antichlamydial antibodies increased the rate of Th1 activation by FcR(+/+) but not FcR(-/-) antigen-presenting cells. FcR(-/-) dendritic cells and the T cell-associated IgG2A and IgA mediate enhanced Th1 activation by antibodies. Immunization with chlamydia-antibody complexes induced elevated and protective Th1 response. These results provide a mechanistic basis for requiring both T cell and humoral immune responses in protective immunity and vaccine evaluation. Findings offer a paradigm in host defense wherein different effector components function indirectly to maximize the principal effector mechanism.

Fc receptor regulation of protective immunity against Chlamydia trachomatis

The prevailing paradigm for designing potentially efficacious vaccines against the obligate intracellular bacterium, Chlamydia trachomatis, advocates regimens capable of inducing a mucosal antigen‐specific T helper type 1 (Th1) response. However, recent reports indicate that rapid and efficient clearance of a secondary infection also requires certain B‐cell functions. We investigated the hypothesis that Fc receptor (FcR)‐mediated antibody effector mechanisms are important B‐cell‐related functions involved in controlling a chlamydial genital reinfection. Microbiological analysis of genital chlamydial infection in FcR knockout (FcRKO) mice lacking the activatory FcγRI (CD64) and FcRγIII (CD16), as well as the inhibitory FcγRIIB1 (CD32), revealed a greater intensity of secondary infection (i.e. bacterial shedding) in FcR−/− as compared to FcR+/+ mice; however, the course of the primary infection was indistinguishable in both animals. Pathologically, FcRKO mice suffered greater ascending infection than immunocompetent wild‐type (WT) mice after a secondary infection. Immunological evaluation indicated that the presence of specific anti‐chlamydial antibodies enhanced chlamydial antigen presentation for induction of a Th1 response by FcR+/+, but not FcR−/−, antigen‐presenting cells. In addition, specific anti‐chlamydial antibodies augmented both macrophage killing of infected epithelial cells by antibody‐dependent cellular cytotoxicity (ADCC) and macrophage inhibition of productive growth of chlamydiae in co‐cultures. These results indicate that B cells participate in anti‐chlamydial immunity via FcR‐mediated effector functions of antibodies, which are operative during reinfections. Such effector functions include ADCC, and possibly enhanced uptake, processing and presentation of chlamydial antigens for rapid induction of a Th1 response, all facilitating the early clearance of an infection. These findings suggest that a future anti‐chlamydial vaccine should elicit both humoral and T‐cell‐mediated immune responses for optimal memory response and vaccine efficacy.

A Novel Recombinant Multisubunit Vaccine against Chlamydia

The administration of an efficacious vaccine is the most effective long-term measure to control the oculogenital infections caused by Chlamydia trachomatis in humans. Chlamydia genome sequencing has identified a number of potential vaccine candidates, and the current challenge is to develop an effective delivery vehicle for induction of a high level of mucosal T and complementary B cell responses. Vibrio cholerae ghosts (VCG) are nontoxic, effective delivery vehicles with potent adjuvant properties, and are capable of inducing both T cell and Ab responses in mucosal tissues. We investigated the hypothesis that rVCG could serve as effective delivery vehicles for single or multiple subunit chlamydial vaccines to induce a high level of protective immunity. rVCG-expressing chlamydial outer membrane proteins were produced by a two-step genetic process, involving cloning of Omp genes in V. cholerae, followed by gene E-mediated lysis of the cells. The immunogenicity and vaccine efficacy of rVCG-expressing single and multiple subunits were compared. Immunologic analysis indicated that i.m. immunization of mice with either vaccine construct induced a strong mucosal and systemic specific Th1 response against the whole chlamydial organism. However, there was an immunogenic advantage associated with the multiple subunit vaccine that induced a higher frequency of Th1 cells and a relatively greater ability to confer protective immunity, compared with the single subunit construct. These results support the operational theory that the ability of a vaccine to confer protective immunity against Chlamydia is a function of the level of Th1 response elicited.

Recombinant Vibrio cholerae ghosts as a delivery vehicle for vaccinating against Chlamydia trachomatis.

An efficacious vaccine is needed to control the morbidity and burden of rising healthcare costs associated with genital Chlamydia trachomatis infection. Despite considerable efforts, the development of reliable chlamydial vaccines using conventional strategies has proven to be elusive. The 40kDa major outer membrane protein (MOMP) of C. trachomatis is so far the most promising candidate for a subunit vaccine. The lack of satisfactory protective immunity with MOMP-based vaccine regimens to date would suggest that either MOMP alone is inadequate as a vaccine candidate or better delivery systems are needed to optimize the effect of MOMP. Recombinant Vibrio cholerae ghosts (rVCG) are attractive for use as non-living vaccines because they possess strong adjuvant properties and are excellent vehicles for delivery of antigens of vaccine relevance to mucosal sites. The suitability of the ghost technology for designing an anti-chlamydial vaccine was evaluated by constructing a rVCG vector-based candidate vaccine expressing MOMP (rVCG-MOMP) and assessing vaccine efficacy in a murine model of C. trachomatis genital infection. Intramuscular delivery of the rVCG-MOMP vaccine induced elevated local genital mucosal as well as systemic Th1 responses. In addition, immune T cells from immunized mice could transfer partial protection against a C. trachomatis genital challenge to nai;ve mice. These results suggest that rVCG expressing chlamydial proteins may constitute a suitable subunit vaccine for inducing an efficient mucosal T cell response that protects against C. trachomatis infection. Altogether, the potency and relatively low production cost of rVCG offer a significant technical advantage as a chlamydial vaccine.

Bacterial ghosts as carrier and targeting systems for mucosal antigen delivery.

The application of new strategies to develop effective vaccines is essential in modern medicine. The bacterial ghost system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures including bioadhesive properties. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria. The intrinsic adjuvant properties of bacterial ghost preparations enhance immune responses against envelope-bound antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens can be expressed in the envelope complex of ghosts before E-mediated lysis, multiple antigens of various origin can be presented to the immune system simultaneously. In addition, the extended bacterial ghost system represents a platform technology for specific targeting of DNA-encoded antigens to primary antigen-presenting cells. The potency, safety and relatively low production cost of bacterial ghosts offer a significant technical advantage, especially when used as combination vaccines.

Contemporary approaches to designing and evaluating vaccines against Chlamydia

The clinically relevant pathologic consequences of primary ocular, genital, or respiratory human infection by members of the genus Chlamydia are conjunctivitis, cervicitis, urethritis and sinusitis. The major complications and sometimes debilitating evolutionary outcomes of these infections include: trichiasis and cicatrizing trachoma, endometritis or pelvic inflammatory disease and involuntary tubal factor infertility and bronchopulmonary pneumonia. These diseases, in addition to other chlamydia-associated chronic syndromes (e.g., artherosclerosis, multiple sclerosis and Alzheimer’s disease), pose serious public healthcare and huge budgetary concerns. The current medical opinion is that an efficacious prophylactic vaccine is a sine qua non – to control the morbidity of chlamydial infection in the human population. The research goal for an efficacious human chlamydial vaccine has faced key challenges to define the elements of protective immunity to facilitate vaccine evaluation, the judicious selection of appropriate vaccine candidates that possess stable antigenic and immunologic properties and the development of effective delivery vehicles and adjuvants to boost immune effectors to achieve long-term protective immunity. Progress in the functional immunobiology of Chlamydia has established the essential immunologic paradigms for vaccine selection and evaluation, including the obligatory requirement for a vaccine to induce T-helper Type 1 immune response that controls chlamydiae. Recent advances in chlamydial genomics and proteomics should enhance the identification of likely chlamydial gene products that fulfill the antigenic requirements of putative vaccine candidates. Major inroads are however needed in the construction and development of novel and effective delivery systems, such as vectors and adjuvants. This review summarizes the status of contemporary chlamydial vaccine research and promising trends fueling the growing optimism for an efficacious vaccine. The unified approach to vaccines for the genus Chlamydia is validated by the several conserved genes and common immunogenic proteins among member species and the similarity of immune effectors controlling Chlamydia species in animals and humans.

Evaluation of the protective efficacy of Vibrio cholerae ghost (VCG) candidate vaccines in rabbits.

An effective Vibrio cholerae vaccine is needed to reduce the morbidity and mortality caused by this pathogen. Despite the availability of current oral vaccines with measurable efficacy, there is need for more effective vaccines with broad-spectrum efficacy in target populations. Recent studies have shown that bacterial ghosts, produced by the expression of cloned lysis gene E, possess adjuvant properties and are immunogenic. In this study, ghosts were prepared from V. cholerae O1 or O139 and evaluated as vaccines in the reversible intestinal tie adult rabbit diarrhea (RITARD) model. Rabbits were orally immunized with different doses of V. cholerae ghost (VCG) formulations. The vaccine formulations elicited high levels of serum vibriocidal titers against indicator strains. The magnitude of the response was measured as the geometric mean titer (GMT) increase for all rabbits in relation to prevaccination titers. The induction of cross protection was evidenced by the ability of serum from VCG-immunized rabbits to mediate complement-dependent killing of both the homologous and the heterologous strains. Immunized rabbits were protected against intraduodenal challenge 30 days after primary immunization. Protective immunity against challenge appeared to be dose dependent and was associated with marked inhibition of colonization. These results indicate that VCGs represent a novel approach to cholera vaccine development and constitute an effective vaccine delivery vehicle.

Chemokine and Chemokine Receptor Dynamics during Genital Chlamydial Infection

ABSTRACT Current design strategies for vaccines against certain microbial pathogens, including Chlamydia trachomatis, require the induction and targeting of specific immune effectors to the local sites of infection known as the mucosal effector sites. Chemokines and their receptors are important mediators of leukocyte trafficking and of the controlled recruitment of specific leukocyte clonotypes during host defense against infections and during inflammation. We analyzed the dynamics of chemokine and chemokine receptor expression in genital mucosae during genital chlamydial infection in a murine model to determine how these molecular entities influence the development of immunity and the clearance of infection. A time course study revealed an increase of up to threefold in the levels of expression of RANTES, monocyte chemotactic protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), macrophage inflammatory protein 1α (MIP-1α), and intercellular adhesion molecule type 1 (ICAM-1) after genital infection with the C. trachomatis agent of mouse pneumonitis. Peak levels of expression of RANTES, MCP-1, and MIP-1α occurred by day 7 after primary infection, while those of IP-10 and ICAM-1 peaked by day 21. Expression levels of these molecules decreased by day 42 after primary infection, by which time all animals had resolved the infection, suggesting an infection-driven regulation of expression. A rapid upregulation of expression of these molecules was observed after secondary infection. The presence of cells bearing the chemokine receptors CCR5 and CXCR3, known to be preferentially expressed on Th1 and dendritic cells, was also synchronous with the kinetics of immune induction in the genital tract and clearance of infection. Results demonstrated that genital chlamydial infection is associated with a significant induction of chemokines and chemokine receptors that are involved in the recruitment of Th1 cells into the site of infection. Future studies will focus on how selective modulation of chemokines and their receptors can be used to optimize long-term immunity against Chlamydia.

Role of T Lymphocytes in the Pathogenesis of Chlamydia Disease

Vaccines are needed to prevent the oculogenital diseases of Chlamydia trachomatis. Infected hosts develop immunity, although temporary, and experimental vaccines have yielded significant protective immunity in animal models, fueling the impetus for a vaccine. Because infections cause sequelae, the functional relationship between infection- and vaccine-induced immunity is unclear. We hypothesized that infection- and vaccine-induced immunity are functionally distinct, particularly in the ability to prevent sequelae. Chlamydia-immune mice, with immunity generated by either a previous infection or vaccination, exhibited a significant degree of protective immunity, marked by a lower-intensity, abbreviated course of infection. However, vaccinated mice were protected from infertility, whereas preinfected mice were not. Thus, infection-induced immunity does not prevent the pathologic process leading to infertility. Furthermore, T cell subsets, especially CD8 T cells, play a major role in Chlamydia-induced infertility. The results have important implications for the immunopathogenesis of chlamydial disease and new vaccine strategies.