PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response

Abstract

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein.

FIG. 1.

In silico analysis of PPE ORFs. Only the ORFs belonging to subgroup 3 and encoding products ≤200 amino acids in length were subjected to analysis. The presence of stretches of high antigenic index are shown. The x axis indicates the ORF product length in amino acids, while the y axis represents the antigenic index.

FIG. 2.

(A) Transcription of hypothetical PPE ORF Rv2430c. RNA extracted from the virulent H37Rv laboratory strain of M. tuberculosis was used in an RT-PCR. A 597-bp RT-PCR product was observed after electrophoresis in a 1% agarose gel (lane 1). Lane M is the 100-bp DNA molecular size marker run alongside. The arrow on the right indicates the position of the 597-bp PCR product. (B) Expression and purification of the recombinant PPE protein. The recombinant protein was expressed in strain M15pREP4 of E. coli and was purified to homogeneity using the NiNTA protein purification kit. Lane 1, marker; lane 2, purified protein. The arrow on the right indicates the position of the 23-kDa protein.

FIG. 3.

The recombinant Rv2430c PPE protein elicits strong antibody responses in M. tuberculosis-infected patients as opposed to healthy controls. ELISA reactivities of IgG anti-Rv2430c antibodies were assayed in sera of either M. tuberculosis-infected patients or healthy controls (P < 0.0001). Symbols, patient population.

FIG. 4.

PPE Rv2430c protein shows strong reactivities to sera from all three patient categories. Reactivities to both recombinant Rv2430c and Hsp10 of M. tuberculosis in the three categories of patients were estimated by ELISA. The patients belonging to categories 2 (B) and 3 (C) displayed similar antibody responses to both antigens. However, the antibody responses of category 1 patients (A) to Rv2430c were higher than those to Hsp10 (P < 0.003) or PPD (P < 0.0001).

FIG. 5.

Serological sensitivities of PPE Rv2430c and Hsp10 as a function of percentages of individuals. (A) The results shown in Fig. 4 were recalculated as percentages of individuals showing A₄₉₂ values of >0.65. The anti-IgG responses against Rv2430c or Hsp10 were compared for all three categories of patients studied. A higher percentage of individuals belonging to category 1 showed stronger reactivity to Rv2430c than to Hsp10, but for the other two categories, the values were comparable. (B) Percentage of individuals in category 1 showing anti-IgM antibody A₄₉₂ of >0.5. A higher percentage of individuals showed anti-IgM antibody A₄₉₂ of >0.5 against Rv2430c than against Hsp10.