Dots represent individual animals and the horizontal bars correspond to the medians. a P<0.05 by the Mann-Whitney Rank Sum Test compared to the OVA/Z3-14 immunized group. b P<0.05 by the Mann-Whitney Rank Sum Test compared to the IFUs/mouse recovered over the course of the experiment was observed in the groups vaccinated with nMOMP/Z3-14 (median: 147,031; range: <2C1,921,070), or nMOMP/A8-35 (14,125; <2C2,817,998), when compared to their respective negative controls immunized with OVA/Z3-14, (2,719,510; 272,555C9,862,992), or OVA/A8-35 (1,886,340; 1,159,330C4,800,672), respectively (P<0.05) (Table III). fertility rates equivalent to the positive control group immunized with live EB and the fertility controls. In conclusion, increased accessibility of epitopes in the nMOMP/A8-35 preparation may account for the very robust protection against infection and disease elicited by this vaccine. Keywords: Amphipols, detergents, Chlamydia, thermal stability, major outer membrane IOX1 protein, vaccine Mouse monoclonal to CD147.TBM6 monoclonal reacts with basigin or neurothelin, a 50-60 kDa transmembrane glycoprotein, broadly expressed on cells of hematopoietic and non-hematopoietic origin. Neutrothelin is a blood-brain barrier-specific molecule. CD147 play a role in embryonal blood barrier development and a role in integrin-mediated adhesion in brain endothelia protection INTRODUCTION is worldwide the leading cause of bacterial sexually transmitted diseases and can also produce ocular, gastrointestinal and respiratory infections (1, 2). Annually, up to 4C5 million new genital infections are reported in the United States (1, 3). Although effective antimicrobial therapy is available, over 50% of the chlamydial infections are asymptomatic and even in symptomatic cases treatment failures can occur (4). Furthermore, countries that have established screening programs, followed by antibiotic therapy, have observed an increase in the prevalence of the infection (5). This increase is thought to be due IOX1 to a block in the development of natural immunity as a result of the antibiotic therapy (5, 6). Therefore, the implementation of a vaccine appears to be the best approach to control and eradicate these diseases (7C10). In the 1960s various investigators tested live and inactivated whole-organism vaccines in humans and non-human primates to protect against trachoma (2, 11C13). Several vaccine formulations were reported to be effective. However, the protection was found to be short-lived and serovar or serogroup specific. Furthermore, some groups of immunized individuals suffered a hypersensitivity reaction upon reexposure to (2, 13C15). Although the cause of this hypersensitivity reaction is still under investigation, it is thought to be secondary to exposure to one of the antigenic components present in the whole bacterium (16). Thus, there is a need to formulate a subunit vaccine. When the sequence of the major outer membrane protein (MOMP) was analyzed, it was found to have regions of DNA unique to each serovar (17, 18). Therefore, the likelihood that the protection elicited by the trachoma vaccine was due to MOMP was considered (7, 8, 19). Unfortunately, attempts to elicit protection in several animal models using recombinant MOMP, MOMP peptides and DNA MOMP-based vaccines, yielded disappointing results (20C22). The possibility that the native conformation of MOMP, or post-translational modification of the protein, were necessary for protection instigated the search for preparations of the native MOMP. Using detergents a trimeric form of MOMP, considered to correspond to its native structure, was isolated. Mice vaccinated with this preparation mounted a strong immune response that was protective against genital and respiratory challenges (23C27). Subsequently, the same type of preparation was found to elicit protection in non-human primates against an ocular infection (28). Detergents however, can have toxic effects at high concentrations and tend to destabilize membrane proteins accelerating their denaturation (29C33). Thus, detergents are not considered ideal vehicles to formulate vaccines. In the 1990s, Tribet et al. developed amphipathic polymers, termed amphipols (APols), designed to keep membrane proteins soluble in water in the absence of detergent (34). This group of investigators showed that several integral membrane proteins, including OmpF, a protein similar in structure to MOMP, were kept soluble IOX1 in their native conformation by APols (35). Based on these findings, the nMOMP was extracted with detergents and transferred to APols. Immunization of mice with this preparation was found to elicit a more robust protection against an intranasal chlamydial challenge than the detergent-based formulation (31). Here, to assess the feasibility of eliciting protection against a vaginal challenge with and to characterize the immune mechanisms involved in protection, we tested in parallel nMOMP preparations formulated in detergents and in APols. Our results show that nMOMP formulated with APols induces very robust protection against a vaginal infection and preserves fertility. The protection is dependent on CD4+ T-cells and may result from the increased accessibility to the immune system of epitopes in the nMOMP/A8-35 formulation when compared to the nMOMP/A8-35 preparation. IOX1 MATERIALS AND METHODS Stocks of C. muridarum The (mouse pneumonitis (MoPn) biovar, strain Nig II; obtained from the American Type Culture Collection, ATCC; Manassas, VA) was grown in McCoy cells (36). Elementary bodies (nMOMP The nMOMP was extracted and purified as described (25, 26). IOX1 Briefly, was grown in McCoy cells, harvested and washed with PBS. Following digestion of the pellet with DNAse the nMOMP was extracted by incubating twice with [3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate] (CHAPS; Anatrace; Maumee, OH) and once with n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Anzergent? Z3-14; Anatrace). The supernatant was transfer to a hydroxyapatite column and the MOMP trimer was eluted using a phosphate buffer gradient (37). The purity of the nMOMP preparation was assessed by several methods. Using the limulus amoebocyte assay (BioWhittaker, Inc., Walkersville, MD) the nMOMP was found to have less than 0.05 EU of LPS/mg of protein (24). Mass spectrometry analyses.