Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections

Abstract

Background: The OmcB protein is one of the most immunogenic proteins in C. trachomatis and C. pneumoniae infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of C. trachomatis infections.

Results: Using the ClustalW and Antigenic programs, we have selected two predicted specific and immunogenic regions in the OmcB protein: the N-terminal (Nt) region containing three epitopes and the C-terminal (Ct) region containing two epitopes with high scores. These regions were cloned into the PinPoint Xa-1 and pGEX-6P-1 expression vectors, incorporating a biotin purification tag and a glutathione-S-transferase tag, respectively. These regions were then expressed in E. coli. Only the pGEX-6P-1 has been found suitable for serological studies as its tag showed less cross reactivity with human sera and was retained for the evaluation of the selected antigens. Only the Ct region of the protein has been found to be well expressed in E. coli and was evaluated for its ability to be recognized by human sera. 384 sera were tested for the presence of IgG antibodies to C. trachomatis by our in house microimmunofluorescence (MIF) and the developed ELISA test. Using the MIF as the reference method, the developed OmcB Ct ELISA has a high specificity (94.3%) but a low sensitivity (23.9). Our results indicate that the use of the sequence alignment tool might be useful for identifying specific regions in an immunodominant antigen. However, the two epitopes, located in the selected Ct region, of the 24 predicted in the full length OmcB protein account for approximately 25% of the serological response detected by MIF, which limits the use of the developed ELISA test when screening C. trachomatis infections.

Conclusion: The developed ELISA test might be used as a confirmatory test to assess the specificity of serological results found by MIF.

Figure 1

Multiple sequence alignment of the OmcB proteins of C. trachomatis, C. pneumoniae and C. psittaci. The multiple sequence alignment was performed using the ClustalW program. The selected N-terminal and C-terminal regions, which represent a lesser degree of similarity, are boxed. Stars mark identical amino acid residues in all sequences in the alignment. Dashes represent gaps in the alignment introduced by the ClustalW program. Conserved amino acids are shaded dark grey and physico-chemical conserved amino acids are shaded in light grey.

Figure 2

Expression and purification of the recombinant proteins and the vector tags. All proteins of interest are indicated with arrows. (A): Immunoblot analysis of the BPT tagged proteins expressed using the PinPoint Xa-1 vector in E. coli BL21 (DE3). Lane 1: molecular weight marker (broad range protein molecular marker, PROMEGA), lane 2: BPT (13 kDa), lane 3: BPT-Nt (24.5 kDa), lane 4: BPT-Ct (18.5 kDa). The star marks an endogenous biotinylated protein of 22.5 kDa expressed in E. coli cells. (B): SDS-PAGE (15%) analysis of GST tagged proteins expressed using the pGEX-6P-1 vector in E. coli BL21 (DE3). Lane 1: molecular weight marker, lane 2: GST (24 kDa), lane 3: GST-Nt (35.5 kDa), lane 4: GST-Ct (29.5 kDa). (C): SDS-PAGE (15%) analysis of the semi purified BPT-Ct by heat treatment. Lane 1: molecular weight marker, lane 2: 40°C 5 min, lane 3: 55°C 5 min, lane 4: 70°C 5 min. (D): SDS-PAGE (15%) analysis of purified GST and GST-Ct. Lane 1: molecular weight marker, lane 2: purified GST, lane 3: purified GST-Ct.

Figure 3

Immunoreactivity of the recombinant proteins and the vector tags. (A): Immunoblot analysis of the BPT-Ct, the BPT, the GST-Ct and the GST using 6 human sera. Lane 1: purified BPT-Ct, lane 2: purified BPT, lane 3: purified GST-Ct, lane 4: purified GST. Each column represents the reactivity of one serum to the four antigens. Column 1 and 2: sera from healthy blood donors, column 3: serum from prostitute, column 4, 5 and 6: sera from MIF C. pneumoniae positive patients. (B): ELISA results using the GST and the GST-Ct as antigens obtained with the 356 sera. Each dot represents the GST-Ct reactivity according to that of the GST for the same serum.

Figure 4

Reproducibility of the OmcB Ct ELISA assay. The figure shows OD values, at 450 nm, of the 12 tested sera obtained in two ELISA tests conducted on two different days. Each line indicates the reactivity of one serum on the two days. The differences between the OD values for all sera were less than 20%.

Figure 5

Correlations between the MIF and OmcB Ct ELISA tests. *: Number of tested sera at the corresponding MIF titer range. The mean OD values significantly correlated with the MIF antibody titers (p < 0.01).

Figure 6

Performance of the OmcB Ct ELISA in selected populations. MIF CT-CP+: patients MIF C. trachomatis negative C. pneumoniae positive, HBD: healthy blood donors, MIF CT+CP-: patients MIF C. trachomatis positive C. pneumoniae negative, STI patients: patients referred to sexually transmitted infection clinics. *: Number of tested sera according to the population tested. Significant differences were found between the mean OD values of the OmcB Ct ELISA of children's sera as well as prostitutes' sera in comparison with those of the other populations tested (p < 0.01). No significant difference was found between the mean OD values of sera from patients MIF C. trachomatis positive, patients MIF C. pneumoniae positive, patients referred to STI clinics and from healthy blood donors.