Immunization with a Combination of Integral Chlamydial Antigens and a Defined Secreted Protein Induces Robust Immunity against Genital Chlamydial Challenge

We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF; a secreted chlamydial protein) in inducing antigen-specific CD4⁺ T cell/gamma interferon (IFN-γ)-mediated but not antibody-mediated chlamydial clearance and reduction of upper genital tract (UGT) pathological sequelae. Since chlamydial integral antigens may induce neutralizing antibody protection, we further evaluated induction of protective immunity using a combination of rCPAF and UV-inactivated chlamydial elementary bodies (UV-EB) against vaginal chlamydial challenge in comparison to immunization with the individual components or live EB. The rCPAF-UV-EB immunization induced a significantly enhanced anti-UV-EB cellular and antibody response and a reduced anti-CPAF cellular and antibody response, compared to immunization with the respective individual components. Moreover, vaccination with UV-EB and rCPAF-UV-EB induced serum antibodies that neutralized chlamydial infectivity. The rCPAF-UV-EB immunization resulted in a significant reduction of vaginal chlamydial shedding and induced earlier bacterial clearance than vaccination of mice with the individual components. Importantly, the UGT sequelae were significantly reduced in mice immunized with rCPAF or rCPAF-UV-EB, but not in those immunized with UV-EB alone, and approached the levels of protection induced by live EB. These results collectively suggest that a combination of neutralizing antibodies induced by integral chlamydial antigens and cell-mediated responses induced by secreted proteins such as CPAF induces optimal protective immunity against genital chlamydial infections.There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of bacterial sexually transmitted disease worldwide (2, 16). We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF) in inducing protective immunity against genital chlamydial challenge (23). Immunization using rCPAF with a T helper 1 (Th1)-type adjuvant induces significantly enhanced bacterial clearance and robust protection against upper genital tract (UGT) pathology following vaginal challenge with homologous or heterologous serovars/species of Chlamydia (5, 6, 23). The high degree of cross-serovar/species protection against UGT sequelae highlights the importance of further characterizing the potential of rCPAF as a component of an antichlamydial vaccine for humans (25). rCPAF-vaccinated mice display significant protection against UGT chlamydial sequelae and clear the bacteria with significantly accelerated kinetics, achieving complete clearance by day 18 (day 30 in mock-vaccinated mice) after challenge. However, vaginal bacterial shedding in rCPAF-vaccinated mice is comparable to the level for mock-vaccinated controls during the initial week after challenge (6, 23). Such enhanced clearance kinetics, in the absence of resistance to infection, may be attributed to the dependence of the protective response on gamma interferon (IFN-γ)-producing CPAF-specific CD4⁺ T cells (15), a limited role for anti-CPAF antibody (22), and the restriction of CPAF to replicating reticulate bodies.Chlamydia muridarum infection in mice induces a high level of protective immune responses, including a certain degree of resistance to reinfection, mediated by robust IFN-γ-producing CD4⁺ T cell responses (4, 11-13, 16, 17, 20, 28-31, 34) and antibodies (16,18-20). A single immunogenic subunit that induces protective immunity comparable to that induced by live, replicating chlamydial organisms has yet to be identified (2, 16, 25). The immunogenic proteins that serve as targets for antibody and T cell responses may be broadly categorized, albeit with some overlap, as proteins that are integral to the chlamydial organism and those that are secreted from the organism, respectively. Specifically, proteins integral to the chlamydial organism would likely serve as targets for neutralizing infectivity extracellularly but may not be candidates of choice for eliciting T cell-mediated killing, due to the sturdy inclusion membrane barrier between the organisms and antigen-presentation pathways during the intracellular developmental cycle (25). On the other hand, secreted proteins such as CPAF are not present on the infectious chlamydial elementary body (EB) and therefore would not be expected to serve as targets for neutralizing chlamydial infectivity (25). However, proteins secreted into the host cytosol, and thereafter into extracellular compartments, may serve as exogenous antigens and a suitable target for CD4⁺ T cell-mediated effector responses (25, 37). Thus, it would appear that both integral and secreted proteins of Chlamydia may serve as targets for complementary immune responses and that the greatest potential for successful vaccination could be derived by combining them in a multisubunit vaccine.In this study, we compared the protective immunities induced by intranasal (i.n.) immunization with rCPAF, UV-inactivated EBs (UV-EB), rCPAF-UV-EB, or live EB against genital C. muridarum challenge in female BALB/c mice. The combination of integral and secreted proteins enhanced protective immunity compared to the individual components and approached the high level of protection induced by live, replicating chlamydial organisms.