Previous studies have shown that Pmps of Chlamydia spp. are immunogenic; however, relatively little is known regarding the specificity of the immune response against these antigens in the case of C. trachomatis. The availability of a complete panel of rPmps and serum samples from C. trachomatis-infected patients allowed for the examination of Pmp-specific antibody responses at the Pmp subtype level in four different patient groups. Immunoblot analysis revealed that 156 out of 159 patients exhibited serum antibody reactivity to at least one rPmp. Reactivity to rPmp also correlated with the reactivity to high-molecular-mass protein bands ranging from 75 to 150 kDa in purified EBs of C. trachomatis (Fig. ). This strongly suggests that the observed antibody reactivity to rPmps is representative of the serum immune response to native chlamydial Pmps. Conversely, this result suggested that immunoblot analysis using denatured rPmp antigen would be a suitable assay of antibody reactivity against native Pmps in infected patients. Notwithstanding, it is likely that patient antibodies also recognize Pmp conformational epitopes that are not represented in denatured rPmps and thus would not be detected by our method. Hence, although the immunoblot method developed in this study provides a semiquantitative assay representative of the patient antibody response to each Pmp, which is suitable for comparative analysis, it falls short of providing a true antibody titer to each specific Pmp subtype.
We quantified the antibody reactivity to each rPmp subtype for each patient. Since it was not experimentally feasible to distinguish reactivity to degraded Pmp-specific immunoreactive peptides from that to E. coli
contaminating proteins, only the reactivity against the intact full-size protein was used for quantification. Our results reveal that Pmps are capable of eliciting quantitatively and qualitatively different antibody responses in patients with C. trachomatis
genital infection and that individual patients mount antibody responses to different subsets of Pmp proteins. A possible cause for the observed differential Pmp immunoreactivity resides in the variable in vivo expression of the pmp
genes, which may be highly regulated in the context of a genital infection. Regulated pmp
) may allow Chlamydia
to alter its antigenic coat to evade host immunity, thus facilitating the development of chronic infection. A graphic summary of the anti-Pmp response in all patient serum samples is shown in Fig. .
Although the sequence and content of the pmp
loci are highly conserved between different strains and serovars of C. trachomatis
), differential transcriptional regulation or posttranslational modification in different strains cannot be ruled out. Moreover, intrinsic geographical, behavioral (e.g., increased frequency of repeat infection with age), or gender-specific factors are also not accounted for in our study. For instance, our comparison of C. trachomatis
-infected males and females from different locations does not account for potential distinct pathogenic or serovar-specific properties of the infecting strain(s) or possible genetic differences or differences in antigen presentation in the infected human populations. A logical extension of our study will be a similar comparison of Pmp-specific antibody profiles in C. trachomatis
-infected males and females from the same geographical location.
Table summarizes the relative frequencies of the antibody response to each rPmp subtype in the three different patient populations. PmpB, -C, -D, and -I were more frequently recognized than were the other Pmps in all groups. This indicates that some Pmps, including PmpB, -C, -D, and -I, may be more abundantly expressed or specifically exposed at the chlamydial surface to elicit a relatively stronger antibody response. Alternatively, the antibody response to each Pmp subtype may be differentially regulated. For instance, the host immune system may preferentially recognize protein structures that are present in some but not all Pmps. Swanson et al. have recently shown that the native form of PmpD of C. trachomatis
exists as an oligomer with a flower-like structure (43
). They also found out that PmpD is a target of broadly cross-reactive neutralizing antibodies (10
). The relatively high prevalence of PmpD-specific antibodies (73% of patients) supports the concept that PmpD may be important in the development of a multicomponent Chlamydia
vaccine in the future. Gomes et al. (15
) and Nunes et al. (29
) have investigated the serum antibody response to recombinant forms of PmpC, -D, and -F in a group of female adolescent patients from Oakland, CA, by dot blot analysis. Results from their studies indicate that patients infected with specific strains of C. trachomatis
make antibodies reactive to PmpC and PmpD, but not to PmpF, which is consistent with our observations.
Cross-sectional comparisons of anti-Pmp antibody reactivities in different patient populations are shown in Fig. . The two adolescent female populations were pooled for the purpose of statistical analysis since both groups include patients of similar age and from similar urban areas. Although differences in ethnicity between the two urban sites may impact disease development, the focus of our analysis is the Pmp-specific antibody profile in a heterogeneous population, irrespective of the infecting strain or the infection status. Indeed, several infecting strains are usually found in urban sites with potential additional variation within strains (25
). When comparing the frequencies of the anti-Pmp response between populations, the least frequently recognized rPmps (rPmpA, -E, -F, and -H) tend to also be similarly less immunoreactive in all populations. In contrast, rPmps that are recognized more strongly and more frequently (rPmpB, -C, -D, and -I) reveal some differences in antibody response between populations. The major differences in relative reactivity of Pmp-specific antibody occur between the male population and either of the two adolescent female populations. Male patients tend to display overall stronger anti-Pmp reactivity, suggesting a possible gender specificity of the antibody response. The PID and adolescent female patients displayed similar antibody responses, except for PmpB and PmpI, for which the PID group showed stronger reactivity than did the adolescent females. This suggests that during chronic or repeated infection, infecting chlamydiae stimulate the host immune response against Pmps, further suggesting that the Pmp-specific response contributes to the development of chronic inflammation leading to PID and infertility. Furthermore, the development of strong antibody reactivity to specific Pmp subtypes (PmpB and PmpI) in PID patients implies that these patients may be more susceptible to develop inflammatory outcomes. The possibility that PmpB and PmpI antibody titers could be used as predictable markers of the onset of PID and other long-term sequelae should be investigated.
Figure shows that the majority of the patient serum samples recognize multiple rPmps. Pmps are similar in sequence; hence, the possibility that some of the observed reactivity to multiple Pmps may be due to cross-reactivity cannot be excluded. Although serum samples from all four C. trachomatis-negative control samples were nonreactive against any rPmp, respiratory infection was not assessed in these patients, so the possibility that potentially cross-reactive Pmps of C. pneumoniae contributed to the observed antibody responses cannot be excluded. Antigenic cross-reactivity between PmpB and PmpC of C. trachomatis is nevertheless possible since these Pmps belong to the same subfamily and share 44% sequence identity. However, our experimental design using a complete panel of rPmps allowed heterologous rPmps to serve as internal negative controls for one another. Moreover, the human immune system most likely generates polyclonal antibodies that recognize mutually exclusive epitopes on individual Pmps. In an independent part of this project, guinea pig polyclonal antibodies to each Pmp subtype were obtained. All antibodies recognized rPmps by immunoblot analysis and specifically labeled C. trachomatis inclusions by immunofluorescence, strongly supporting the antigenic and immunogenic specificity of individual anti-Pmp antibody responses in vivo (Tan and Bavoil, unpublished).
Our study reveals varied anti-Pmp antibody profiles in patients from four geographically distinct C. trachomatis-infected populations. In addition, Pmp subtype-specific and gender-specific antibody responses are demonstrated. These observations imply variable expression of the pmp gene family during infection, suggesting that the C. trachomatis pmp gene family is the basis of a mechanism of antigenic variation for the purpose of immune evasion. Ongoing efforts include in vitro investigations of the prediction that pmp expression varies in a highly regulated manner.