A Trypanosoma cruzi Trans-Sialidase Peptide Demonstrates High Serological Prevalence Among Infected Populations Across Endemic Regions of Latin America

Abstract

Infection by Trypanosoma cruzi, the agent of Chagas disease, can irreparably damage the cardiac and gastrointestinal systems during decades of parasite persistence and related inflammation in these tissues. Diagnosis of chronic disease requires confirmation by multiple serological assays due to the imperfect performance of existing clinical tests. Current serology tests utilize antigens discovered over three decades ago with small specimen sets predominantly from South America, and lower test performance has been observed in patients who acquired T. cruzi infection in Central America and Mexico. Here, we attempt to address this gap by evaluating antibody responses against the entire T. cruzi proteome with phage display immunoprecipitation sequencing comprised of 228,127 47-amino acid peptides. We utilized diverse specimen sets from Mexico, Central America and South America, as well as different stages of cardiac disease severity, from 185 cases and 143 controls. We identified over 1,300 antigenic T. cruzi peptides derived from 961 proteins between specimen sets. A total of 67 peptides were reactive in 70% of samples across all regions, and 3 peptide epitopes were enriched in ≥90% of seropositive samples. Of these three, only one antigen, belonging to the trans-sialidase family, has not previously been described as a diagnostic target. Orthogonal validation of this peptide demonstrated increased antibody reactivity for infections originating from Central America. Overall, this study provides proteome-wide identification of seroreactive T. cruzi peptides across a large cohort spanning multiple endemic areas and identified a novel trans-sialidase peptide antigen (TS-2.23) with significant potential for translation into diagnostic serological assays.

Fig. 1.. PhIP-seq library design and assay steps.

Phage library displays the proteome of T. cruzi in 47-aa peptides with a 19-aa step size on the capsid of T7 phage. The library includes all coding regions of the proteome and splice variants. We performed the PhIP-seq assay by incubating the phage library with human plasma, followed by immunoprecipitation of antibodies in the sample and enrichment of antibody-bound phage through lysis in E. coli. We performed two rounds of enrichment and then sequenced the enriched phage to obtain the identity of the immunoprecipitated peptides.

Fig. 2.. PhIP-seq captures known antigens across the T. cruzi life cycle stages.

(A,B) Heatmap of z-score enrichment over seronegative controls in the (A) blood donor (BD) specimens (n=64 seropositive, n=26 seronegative) and the (B) cardiac biomarker (CBM) specimens (n=114, seropositive; n=18, seronegative; n=95, healthy controls) for seroreactive peptides (rows) with >15% seropositivity within each cohort. Peptides are sorted by protein name and samples are sorted by patient region of origin (n=10, Central America [yellow]; n=13, Mexico [blue]; n=12, South America [light green]; n=1, USA [pink]; n=28, unknown [green]) (A), or cardiac disease stage (n=14 stage A seronegative [magenta]; n=6 stage B seronegative [orange]; n=38 stage A seropositive [magenta]; n=46 stage B seropositive [orange]; n=7 stage C seropositive [light green]; n=23 stage D seropositive [blue]) (B). Protein groups with well-characterized antigens are indicated by labels (Surface antigen, Surface antigen 2 (CA-2); NSP-like, Nucleoporin NSP1-like C-terminal domain-containing protein; MASP, Mucin-associated surface protein; Mucin, TcMUCII; MAP, Microtubule-associated protein; CCP, Calpain-like cysteine peptidase; 60S, 40S, ribosomal subunit proteins). (C) Breadth of antibody reactivity, shown as the number of seroreactive peptides in each person. The dotted red line and number signify the median number of seroreactive peptides in BD and CBM specimen sets. Samples are grouped by geographic region (BD specimens) and heart disease stage (CBM specimens). (D) Number of peptides identified as seroreactive in this study that are part of proteins expressed in specific stages of the T. cruzi life cycle (Tryp = trypomastigote; Ama = amastigote; Meta = metacyclic trypomastigote; Epi = epimastigote; Multiple = protein is expressed in trypomastigote, amastigote, and/or metacyclic trypomastigote stages). Stage expression analysis shows seroreactive peptides in every host-interfacing lifecycle stage. Stage-specific expression is based on the ‘Life cycle proteome (Brazil)’ data set from TriTrypDB. Gene IDs for stage-specific proteins were mapped onto the gene IDs that corresponded to seroreactive peptides. (E,F) Selected known seroreactive antigens are captured by T. cruzi PhIP-seq. Neg. is seronegative specimens from the respective specimen sets; Pos. is seropositive specimens from the respective cohorts; NYBC is NYBC US controls. Antibody reactivity to two known antigens (E) Ag2, a nucleoporin protein, and (F) TCE, a 60S ribosomal subunit protein are plotted as reads per 100,000 (RPK). The dotted red line signifies the RPK that corresponds to a z-score cutoff of 5 in the seronegative population of each cohort.

Fig. 3.. A novel trans-sialidase peptide sequence is a highly reactive serological antigen.

(A) Anti-trans-sialidase peptide antibody reactivity is plotted as RPK. The dotted red line signifies the RPK that corresponds to a z-score cutoff of 5 in the seronegative population of each cohort. (B) Trans-sialidase reactivity orthogonal validation using a split-luciferase binding assay (SLBA). Reactivity was tested against four seropositive blood donor specimens and five seronegative US healthy control specimens. (C) Alanine-scanning mutagenesis in 10-aa windows (highlighted in red) across the entire trans-sialidase antigenic fragment demonstrates the seroreactive epitope in Chagas disease. Values are normalized antibody indices and represent the averages of five seropositive blood donor specimens.

Fig. 4.. PhIP-seq antibody reactivity of current diagnostic antigens and TS-2.23 within individual Chagas disease seropositive specimens.

Recombinant antigens in current FDA-cleared serology tests include Ag 1, Ag 2, Ag 13, Ag 30, Ag 36 (33, 34), shed acute phase antigen (SAPA) (35), KMP-11 (36), TcD and TcE (30, 37). Note, TcD contains the same antigenic epitope as Ag 13. (A) Heatmap of z-score enrichment over seronegative controls in the seropositive blood donor (BD) specimens (n=64). Each antigen motif was derived using Multiple EM for Motif Elicitation (MEME) and then scored against the entire T. cruzi PhIP-seq proteome. The maximum z-score across all peptides with significant sequence matches to a given antigen motif was plotted for each sample and each antigen. (B) Percent of samples enriched (z-score ≥5) for each antigen in BD and cardiac biomarker (CBM) specimen sets.

Fig. 5.. Biolayer interferometry validates trans-sialidase antigen TS-2.23 seroreactivity in a large cohort.

(A) Schematic of biolayer interferometry (BLI) approach. BLI uses a fiberoptic probe to measure the wavelength of light reflected from the surface of a biosensor, which changes due to light interference when an analyte binds. First, an anti-His tag probe is incubated with His-tagged antigen. Then, the probe is incubated in diluted serum or plasma, and antibodies bind the immobilized antigen on the probe. To quantify IgG-specific reactivity, anti-IgG antibodies are added and bind the immobilized patient antibodies. (B) Seropositive blood donor specimens (n = 250) demonstrate a range of reactivity to the trans-sialidase antigen by quantitative BLI immunoassay, while seronegative blood donor specimens (n=86) do not (Wilcoxon rank-sum test). Reactivity, as denoted by wavelength shift, was higher in Central American (n=86) and South American (n=72) specimens than in Mexican specimens (n=92). Dashed line corresponds to the 25th percentile across all seropositive specimens.