We have shown that nMOMP is highly immunogenic in non-human primates. Systemic immunization elicited high levels of serovar-specific serum IgG and IgA antibodies, but very low levels of tear IgG and undetectable levels of tear IgA antibodies, as measured by ELISA. Notably, serum from vaccinated monkeys was shown to contain high strain-specific neutralizing antibodies. Conversely, immunization induced a broad trachoma strain cross-reactive IFN-γ response. This immune response resulted in highly significant protection against homotypic ocular challenge, reducing the infectious burden greater than 70-fold over the first two weeks post-challenge. However, protection was restricted to early time periods post-challenge, with minimal differences observed between vaccinated and control monkeys in either infectious burden or duration at later time points. Surprisingly, these marked early differences in organism burden in the conjunctival epithelia did not reduce the severity of ocular disease. Nevertheless, this is the first time a subunit vaccine has shown a significant reduction in ocular shedding in non-human primates. Although two previous studies described partial protection in non-human primates after subunit vaccination (33
), this protection was limited to a transient decrease in clinical response with no significant reduction observed in shedding.
A major and perhaps important finding of this work was the high strain-specific neutralizing titers generated following immunization with nMOMP. We believe the native trimeric structure of MOMP could be the reason for achieving such high strain-specific neutralizing titers. Interestingly, neither this high titer nor strain specificity was found by ELISA when using purified formalin-fixed EBs as antigen. Serum ELISA antibody titers showed virtually identical titers against the two serovar A strains (A2497 and AHAR-13), with lower but measurable titers against the heterologous Ba and C trachoma serovars. The significance of this finding is unclear, but it is consistent with previous findings that protective immunity against C. trachomatis
ocular infection is serovar-specific, with little to no cross protection against different serovars (39
). Indirectly, these findings implicate serovar-specific neutralizing antibodies in ocular immunity. The exquisite degree of strain specificity found in serum neutralizing antibodies of nMOMP immunized monkeys was unexpected and unpredicted, as the MOMPs of strains A2497 and AHAR-13 differ by only four amino acids (42
). Two of these differences are located in MOMP variable domains (amino acid #80 and #153 in VDs I and II, respectively), and the other two in constant regions. According to the 2-D model of MOMP (43
), VD I is the latching loop for the trimers and that loop should be critical for trimer formation. Also, recombinant phage clones expressing MOMP antigenic determinants revealed that protective serotype-specific mAbs recognized epitopes in VD I and II (26
). Although speculative, these findings argue the four amino acid substitutions, either independently or collectively, may change the structural properties of trimeric nMOMP. These substitutions could thus generate immunodominant determinants recognized by highly efficient infection blocking neutralizing antibodies.
Immunization with nMOMP resulted in an antigen-specific production of IFN-γ by PBMC. The response was broadly cross-reactive, as different trachoma strains were equally effective in its induction. IFN-γ is thought to play an important role in resolving C. trachomatis
infection; however our findings indicate it is not sufficient to significantly alter the course of ocular infection. Possible explanations for this finding are that systemic cellular immunity was ineffective mucosally or perhaps the levels of IFN-γ generated by systemic immunization were simply insufficient. A more accurate role for IFN-γ in ocular immunity against chlamydial infection may require, like neutralizing antibodies, strategies capable of targeting local ocular immune responses. Typically, C. trachomatis
infections generate primarily homotypic immunity, providing less protection against heterotypic challenges (39
). Our findings suggest protective immunity is not solely reliant on the cytokine-mediated immune response — homotypic neutralizing antibodies could also be important factors. Due to the limited number of primates available for experiments, we were unable to challenge nMOMP vaccinated animals with strains other than A2497. Heterotypic challenge using other serovars could better define the role of IFN-γ in achieving the protection observed in our experiments. Similarly, heterotypic challenge with A/HAR-13 would help characterize the in vivo
function of the strain specificity of the neutralizing serum antibodies.
The immunity induced by systemic immunization with nMOMP was equivalent or even better than the immunity induced by experimental infection in non-human primates. In a previous experiment, monkeys were infected with A2497 twice and immunity was evaluated 3 weeks later (unpublished data). The serum ELISA IgG and IgA titers and the IFN-γ response of PBMCs were comparable to that induced by nMOMP immunization. However, serum neutralizing titers were about 10 fold lower (4000-8000 versus 32000-76800) than those measured after nMOMP immunization. Because the nMOMP immunizations induced chlamydial-specific IFN-γ and serum neutralizing antibody responses that correlated with a significant reduction in the level of early bacterial shedding, the lack of impact on gross pathology post-challenge was puzzling. In mice, systemic immunization with nMOMP induced protective immunity comparable to that induced by infection with live bacteria. Discrepancies between the results of the murine and non-human primate experiments underscore the importance of using the non-human primate trachoma model in pre-clinical studies. Aside from the inherent differences between host and chlamydial species, these discrepancies likely result from differences in infection sites. Both the ocular conjunctiva and the upper female genital tract are mucosal sites; however, the ocular mucosa is probably more regionally isolated. Lacking a practical genital model in non-human primates, we were unable to investigate this further. Another interesting aspect of the partial protection induced by nMOMP immunization in non-human primates is that it does not differ much from the protection induced by experimental infection. A single experimental infection in primates also induces only partial protection, with a 1-2 log decrease in shedding and limited reduction in pathology (41
). This suggests an ideal vaccine of non-human primates and humans will need to produce immunity that actually exceeds that of natural infection. At present the underlying mechanisms for this disparity remain unclear, but it is certain that a better understanding of human immunity and C. trachomatis
virulence factors capable of altering natural and vaccine-mediated immunity are needed. Vaccines that can provide effective protection of the eye will probably require immunization strategies that target regional ocular immune induction sites.
Computer modeling studies have predicted even a partially efficacious vaccine would have a significant effect on decreasing chlamydial transmission (11
). Considerably reducing chlamydial shedding throughout communities could successfully interrupt the trachoma re-infection transmission cycle. This approach would in fact be similar to that of mass antibiotic treatment, which reduces transmission by temporarily reducing the total infectious burden in a community. However, the effect of mass antibiotic treatments is unsustainable and treatment must be repeated as infections return over time (45
). In contrast, a vaccine providing long-lasting partial protection need be administered only once, or at least less frequently, to achieve a sustainable effect on transmission. In our study, systemic immunization with nMOMP reduced the total infectious burden by 94% (18-fold). Thus, a community-wide nMOMP immunization could significantly impact the fight against blinding trachoma by interrupting the re-infection cycle. Admittedly, this vaccination campaign could face significant logistical difficulties, such as the high production cost of the immunogen and the delivery and storage of the vaccine in rural African villages. This might be overcome by expressing nMOMP in a surrogate system and combining it with other childhood vaccines. Nevertheless, it is foreseeable that nMOMP vaccination in combination with antibiotic treatment could be the most effective way to eliminate trachoma.