Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease

Abstract

Background: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease.

Methods and results: Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st)-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd)-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcG(mix) were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcG(mix)- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects.

Conclusions: Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

Figure 1. Antigenicity of TcG1, TcG2 and TcG4 in inhabitants of Salta Argentina.

Sera (A–C) and plasma (D–I) samples obtained in year 2010 (A–F) and year 2009 (G–I) from volunteers in Salta Argentina were identified as seropositive (+ve) or seronegative (−ve) in 1^st-phase screening by using conventional approaches. The 2^nd-phase screening for antigen-specific antibody response was conducted by an ELISA using the recombinant TcG1, TcG2 and TcG4 proteins. Data (mean of four observations from each sample) are presented as box plot. The horizontal lines of the box (bottom to top) depict the lower quartile (Q1, cuts off lowest 25% of the data), median (Q2, middle value), and upper quartile (Q3, cuts off the highest 25% of the data). The lower and upper whiskers depict the smallest and largest non-outlier observations, respectively, and solid dots represent the outliers. The spacing between the different parts of the box indicates the degree of dispersion (spread). Standard deviation for all samples was <12%.

Figure 2. TcG1, TcG2 and TcG4 are recognized by antibody response in human subjects from Mexico.

Sera samples obtained from volunteers living in the endemic areas of Chiapas Mexico were characterized as seropositive (+ve) and seronegative (−ve) by whole-parasite antigen based serology tests in the 1^st phase. The TcG1 (A), TcG2 (B) and TcG4 (C) specific antibody response was measured by ELISA, and data are presented as box plot (details in Fig. 1).

Figure 3. TcG_mix-based ELISA provides superior efficacy in identifying exposure to T. cruzi infection among inhabitants of Salta Argentina.

Sera (A&B) and plasma (C–F) samples, characterized as seropositive (+ve) and seronegative (−ve) by conventional approaches, were submitted to multiplex ELISA using the mixture of recombinant TcG1, TcG2 and TcG4 proteins for capturing the antigen-specific antibody response (A,C,E). Shown are the antibody response captured using the T. cruzi trypomastigote lysate (TcTL) as a source of crude antigen (B,D,F) for comparison purpose.

Figure 4. TcG_mix-based ELISA was effective in diagnosing exposure to T. cruzi infection among inhabitants of Chiapas Mexico.

Sera samples obtained from volunteers living in the endemic areas of Chiapas Mexico were characterized as seropositive (+ve) and seronegative (−ve) by whole-parasite antigen based serology tests in the 1^st phase. Shown are the antibody response captured by an ELISA using mixture (TcG_mix) of recombinant TcG1, TcG2 and TcG4 proteins (A) or T. cruzi trypomastigote lysate (TcTL) (B) as a source of antigen.

Figure 5. Pair-wise correlation analysis.

Shown is pair-wise correlation analysis of antibody response to TcG_mix (A) and TcTL (B) with clinical disease category in patients enrolled in the study from Argentina-Bolivia border area. For this, seronegative/healthy subjects were labeled as 0, and patients classified as 0, I, II and III (Materials and Methods) were labeled as 1, 2, 3, and 4, respectively. Dots, individual subjects.