A New Recombinant Multiepitope Chimeric Protein of Leptospira interrogans Is a Promising Marker for the Serodiagnosis of Leptospirosis

Abstract

The zoonotic disease leptospirosis is caused by pathogenic species of the genus Leptospira and was recently included in the list of Neglected Diseases by the World Health Organization. Leptospirosis burden is estimated to have over a million human cases and cause 60 thousand deaths annually, in addition to its economic impact and veterinary concern. The microscopic agglutination test (MAT), recommended by the World Health Organization, exhibits reduced sensitivity at the beginning of the disease, in addition to being technically difficult. New recombinant antigens are being pursued for rapid and specific serodiagnostic tests, especially in the initial phase of the disease, and chimeric multiepitope proteins are a strategy with a great potential to be implemented in serology. Based on previous subproteomic results, we designed a synthetic construct comprising 10 conserved leptospiral surface antigens, and the recombinant protein was purified and evaluated regarding its diagnostic potential. The protein termed rChi2 was recognized by antibodies in serum from patients both at the onset (MAT-) and in the convalescent (MAT+) phase in 75 and 82% of responders, respectively. In addition, rChi2 immunization in hamsters elicited a strong humoral response, and anti-rChi2 antibodies recognized several immobilized intact Leptospira species, validating its potential as an early, broad, and cross-reactive diagnostic test.

Keywords: Leptospira; MAT; chimeric protein; diagnosis; leptospirosis.

Figure 1

Chimeric protein design, expression, and characterization. Diverse regions of 10 conserved outer membrane proteins from L. interrogans were selected based on conserved domains and epitope prediction (A). The final amino acid sequence was used for protein modeling, indicating independent folding of each region that was interspaced by polypeptide linkers (B). Recombinant plasmid containing the chimeric protein coding sequence was used to transform E. coli BL21 (DE3) strain, and protein expression was induced by IPTG. After cell lysis, supernatant (SN) and pellet after 8M urea solubilization (IN) were evaluated via immunoblotting and probing with monoclonal anti-His antibodies (C). Recombinant protein was purified via metal-chelating chromatography from the insoluble fraction and reassessed via immunoblotting. Secondary structure content was evaluated via circular dichroism (D) and ELISA experiments with immobilized rChi2 demonstrated that the chimeric protein could be recognized by mouse hyperimmune serum against some of the proteins from which the portions were selected (E).

Figure 2

Pooled serum from patients at the onset (MAT−) and in the convalescent phase (MAT+) and normal human serum (NHS) were incubated at different dilutions (1:100 to 1:800) with the immobilized rChi2 onto ELISA plates. Reactivity was detected either with HRP-conjugated anti-human IgM (A) or IgG (B). For assessing the specificity of antigen–antibody recognition, immunoblots were performed, and transferred rChi2 or BSA (negative control) were incubated with the polled samples at 1:1000 dilution. Reactivity was detected either with HRP-conjugated anti-human IgM (C) or IgG (D).

Figure 3

Reactivity of rChi2 with confirmed leptospirosis cases and unrelated febrile illness. The reactivity of the chimeric protein experimentally with human leptospirosis paired serum samples (n = 36) at the onset of disease (MAT-negative) and in the convalescent phase of disease (MAT-positive) and additional MAT+ samples (n = 142) were determined via ELISA. The cutoff value is shown as a dashed line, and it was defined as the mean plus 3 standard deviations obtained with NHS (n = 50). Sera from patients with unrelated febrile illness, including dengue, Chagas’ disease, HIV, and malaria (n = 10 each), were also employed for assessing the specificity of the reactions.

Figure 4

Immune response elicited via rChi2 immunization and Leptospira spp. cross-reaction. Hamsters (n = 6) were immunized subcutaneously with 50 µg recombinant protein mixed with 12.5% Alhydrogel on day 0 and boosted after 2 weeks. Blood was collected 2 weeks after the last immunization and sera at 1:800 dilution were utilized for the determination of antibody titers regarding total IgG via ELISA (A). The hamsters in the negative control group (saline) were injected with Tris-NaCl buffer in 12.5% Alhydrogel. Pooled hyperimmune antiserum was used for antigen detection on the surface of intact immobilized Leptospira spp. via ELISA (B). Saprophyte L. biflexa was used as a control.