CT043, a Protective Antigen That Induces a CD4+ Th1 Response during Chlamydia trachomatis Infection in Mice and Humans

Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4⁺ Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4⁺ Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.Chlamydia trachomatis is an obligate intracellular human pathogen which exists in two highly specialized morphological forms: the infectious elementary body (EB) and the replicative reticulate body (RB). The pathogen has been classified into 19 different serotypes (serovars), on the basis of which variant of the major outer membrane protein (MOMP) is expressed on its surface. Worldwide, C. trachomatis is responsible for more than 92 million sexually transmitted infections and 85 million ocular infections per year (45). These infections cause several types of diseases (35, 44), including trachoma-induced blindness (serovars A to C), pelvic inflammatory disease, ectopic pregnancy and infertility (serovars D to K), and lymphogranuloma venereum (serovars L1 to L3). Furthermore, recent studies indicate that chlamydia infections facilitate the transmission of both human immunodeficiency virus (28) and human papillomavirus (16).Although effective antibiotic treatments are available, they are often unsuccessful in halting the spread of the infection or inadequate to prevent the chlamydia-mediated long-term sequelae for a number of reasons. First, urogenital tract infections are frequently asymptomatic and therefore not properly treated in due time. Second, although C. trachomatis is generally sensitive to a panel of antibiotics, multiple-antibiotic-resistant strains of chlamydia have been reported (34). Third, there are indications from in vitro studies that antibiotic treatment could lead to the formation of aberrant chlamydia forms that remain dormant within inclusions and may eventually turn into EBs under favorable environmental conditions (9, 47). Finally, epidemiological data in industrialized countries indicate that the rate of chlamydia reinfection is rising, and this has been attributed to the interference of early antibiotic treatment with the acquisition of immunity against chlamydia (2).For these reasons, the development of an effective vaccine against C. trachomatis infection is urgently needed. Early trials with heat-killed preparations of whole EBs have been shown to elicit short-term protection, but a few cases of immunopathological reactions upon reexposure to chlamydia have also been reported (2). Based on these early findings, further efforts have been focused on the development of subunit vaccines (2, 13, 14). In particular, several studies have described the use of MOMP, the immunodominant chlamydial antigen accounting for 60% of the total mass of the chlamydia outer membrane (6). Immunization with MOMP purified from C. trachomatis elicited an immune response that fully protected mice against an intra-ovarian bursa chlamydia challenge (27). However, only correctly folded MOMP appears to provide protection, and no adequate, scalable processes for the production of soluble and properly folded recombinant MOMP have been developed yet. This has so far hampered the use of MOMP for vaccine development. Moreover, due to the sequence variability of MOMP, a broadly protective chlamydial vaccine will probably require the use of other immunogenic antigens in addition to, or in place of, MOMP.The search for other chlamydial antigens has been driven by the elucidation of the mechanisms of immune responses to chlamydia infection and the demonstration of the importance of CD4⁺ T cells in natural immunity. Animal models have shown that protective immunity to C. trachomatis depends on the elicitation of a Th1-polarized cell-mediated immune response, in particular, gamma interferon (IFN-γ)-secreting CD4⁺ lymphocytes. In mice, the depletion of CD4⁺ T cells results in the loss of protective immunity and adoptive transfer of chlamydia-specific CD4⁺ T cells confers protection against C. trachomatis challenge (36). Studies in primates have also emphasized the role of Th1 cells in immunity to chlamydia (42, 43).Here we report experimental evidence that CT043, a protein annotated as hypothetical, is a novel protective chlamydial antigen. This antigen appears to be associated with the bacterial cell surface and is expressed within the chlamydia inclusion in infected HeLa cells throughout the infection cycle. We show that CT043 is the target of CD4⁺ Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. Moreover, by using a DNA priming/protein boost immunization protocol, we show that the antigen significantly reduces the bacterial load in a mouse model of intranasal (i.n.) infection. Finally, sequence analysis of the CT043 gene in 22 C. trachomatis strains belonging to the most representative genital serovars revealed a high degree of conservation (99.4% amino acid identity), suggesting that the antigen could provide cross-serotype protection.Altogether, our study demonstrates that this antigen is a promising candidate to be included in a subunit-based vaccine against C. trachomatis.