Surprisingly, the plasmid encoded pgp3 was as immunodominant as CPAF, a chlamydial secreted protease factor known to be most immunodominant when evaluated together with many other C. trachomatis selleck inhibitor genome encoded proteins and to induce protective immunity against chlamydial diseases when tested in a murine urogenital infection model. The immunodom inance was not only reflected by the high frequency of human Inhibitors,Modulators,Libraries antiserum recognition but also the high antibody binding titers. Both pgp3 and CPAF displayed the highest antibody binding titers when the titers of each individual antiserum binding to the fusion proteins were averaged or when the 15 antiserum samples were pooled at equal ratio and assayed against the fusion proteins. A pooled negative antiserum failed to react with any of the fusion proteins significantly.
Inhibitors,Modulators,Libraries To further confirm the antibody binding specificity, we used the lysates made from either C. trachomatis infected HeLa cells or HeLa cells alone to pre absorb the human antisera prior to reacting with the fusion proteins in ELISA. We found that the reac tivity of the pooled positive antiserum with the fusion proteins was completely removed by the absorp tion with the C. trachomatis Inhibitors,Modulators,Libraries infected cell lysates but not HeLa alone lysates, demonstrating that the human antibodies reactive with the fusion proteins were also able to recognize the chlamydial endogenous antigens produced during infection in HeLa cells. The above observations together have suggested that all 8 plas mid proteins are expressed by C. trachomatis during human infection and pgp3 is most immunogenic.
Since both pgp3 and CPAF were dominantly recognized by the human antibodies and the antibody reactivity with Inhibitors,Modulators,Libraries both was blocked by the C. trachomatis infected cell lysates, we further evaluated whether there was a cross reactivity between pgp3 and CPAF by human antibodies. We found that preabsorption of the pooled human posi tive antiserum with pgp3 fusion proteins only blocked the antibody reactivity with pgp3 but not CPAF, MOMP or HSP60 while preabsorption with CPAF fusion protein blocked the antibody binding to CPAF but not pgp3, MOMP or HSP60, demonstrating that the pgp3 reactive human antibodies did not cross react with CPAF or vice versa. Interestingly, the human antibodies purified with pgp3 or CPAF fusion proteins both detected the endogenous proteins in the cytosol of the C. trachom atis infected cells in addition to their intra inclusion Inhibitors,Modulators,Libraries local ization while the MOMP or HSP60 fusion protein purified human antibodies detected sig nals only inside the inclusions. The locali zation sellekchem of the corresponding endogenous chlamydial proteins was confirmed with antigen specific mouse anti bodies. 2.