Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease

    Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease Malcolm S. Duthie, Jeffrey A. Guderian, Aarthy C. Vallur, Ayesha Misquith, Hong Liang, Raodoh Mohamath, Alejandro O. Luquetti, Darrick Carter, Suelene N.B. Tavares, Steven G. Reed PII: DOI: Reference:

S0732-8893(15)00405-8 doi: 10.1016/j.diagmicrobio.2015.11.006 DMB 13954

To appear in:

Diagnostic Microbiology and Infectious Disease

Received date: Revised date: Accepted date:

7 August 2015 2 November 2015 7 November 2015

Please cite this article as: Duthie Malcolm S., Guderian Jeffrey A., Vallur Aarthy C., Misquith Ayesha, Liang Hong, Mohamath Raodoh, Luquetti Alejandro O., Carter Darrick, Tavares Suelene N.B., Reed Steven G., Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease, Diagnostic Microbiology and Infectious Disease (2015), doi: 10.1016/j.diagmicrobio.2015.11.006

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ACCEPTED MANUSCRIPT Multi-epitope proteins for improved serological detection

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of Trypanosoma cruzi infection and Chagas Disease

By Malcolm S. Duthie1, Jeffrey A. Guderian1, Aarthy C. Vallur1, Ayesha Misquith1, Hong Liang1, Raodoh Mohamath1, Alejandro O. Luquetti2, Darrick Carter1,3, Suelene N. B. Tavares2

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and Steven G. Reed1

From the 1Infectious Disease Research Institute, Suite 400, 1616 Eastlake Ave E, Seattle, WA

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98102, 2Laboratorio de Chagas, Hospital das Clinicas, Universidade Federal de Goiás, Goiânia,

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Goias, Brazil and 3PAI Life Sciences Inc., Suite 550, 1616 Eastlake Ave E, Seattle, WA 98102.

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This work was supported by IDRI internal funds. Address correspondence and reprint requests:

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Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102

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Tel: 206-858-6012; Fax: 206-381-3678; Email address: [email protected]

Abbreviations used in this paper: EC, endemic control ELISA, enzyme-linked immunosorbent assay IIF, indirect immunofluorescence NEC, non-endemic control RDT, rapid diagnostic tests TR, tandem repeat 1

ACCEPTED MANUSCRIPT Running title: Diagnostic antigens for Chagas disease.

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Keywords for submission: Chagas disease; serological diagnosis; T. cruzi; antigen; antibodies

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ACCEPTED MANUSCRIPT Abstract We previously reported that tandem repeat (TR) proteins from Trypanosoma cruzi could

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serve as targets of the antibody response and be useful as diagnostic indicators. To optimize

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reagents for detecting T. cruzi infection we evaluated individual TR proteins and identified several that were recognized by the majority of Chagas patient’s sera collected from individuals form Brazil. We then produced novel, recombinant fusion proteins to combine the reactive TR

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proteins into a single diagnostic product. Direct comparison of the antibody response of serum samples that were readily detected by the established fusion antigen used in commercial

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detection of Chagas disease, TcF, revealed strong responses to TcF43 and TcF26 proteins. While the TcF43 and TcF26 antigens enhanced detection and strength of signal, they did not

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compromise the specificity of detection compared to that obtained with TcF. Finally, it was apparent by testing against a panel of 84 serum samples assembled on the basis of moderate or weak reactivity against TcF (mostly signal:noise <5) that TcF43 and TcF26 could more strongly

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detected by many of the sera that had low TcF antibody levels. Taken together, these data indicate that TcF43 and TcF26 could be used to enhance the detection of T. cruzi infection as

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well as supporting a diagnosis of Chagas disease.

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ACCEPTED MANUSCRIPT Introduction Chagas disease is the illness caused by infection with the parasite Trypanosoma cruzi,

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characterized by serious morbidity and mortality. Despite attempts to eliminate the transmitting

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triatomine bugs in many Latin American countries, Chagas disease remains a major health problem. It is estimated that 8-10 million people living in endemic countries are infected with T. cruzi and another 100 million are at risk for infection. While transmission is traditionally thought

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of as being vector-based, T. cruzi can also be transmitted through contaminated blood products. Additional concerns arise due to the potential for congenital transmission, with 5% of infected

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pregnant women giving birth to infected babies, and via the ingestion of parasite-contaminated food, which has caused outbreaks in endemic countries [1, 2 , 3, 4]. Given the risks of

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inadvertent transmission, screening of donated blood, blood components, and solid organ donors, as well as donors of cells, tissues, and cell and tissue products for T. cruzi is now mandated in all Chagas-endemic countries. Migration of infected individuals and the expansion of the vector

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range into other countries, including the United States, represent additional and alternative mechanisms of transmission into non-endemic countries. Testing is therefore now also conducted

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on a selective basis in some non-endemic countries, including some blood centers in the United

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States.

While the symptoms of Chagas disease can be managed, control largely relies on the

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early and accurate detection of T. cruzi-infected people. Proactive screening programs that could lead to early detection through intervention measures to prevent development of chronic disease are not, however, routinely conducted. Over the last decades, the need for new and simplified diagnostic tools to increase the diagnosis of T. cruzi infection/Chagas disease has grown. Due to low or absent parasitemia after the acute phase, direct parasitological diagnosis of T. cruzi infection from blood is difficult and unreliable. Many antibody-detection tests, using different antigenic targets and various principles such as enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, indirect hemagglutination assay and chemiluminescent assays have become available and are now routinely used for laboratory diagnosis of Chagas disease [5-8]. However, a meta-analysis of the currently available tests indicated that significant heterogeneity exists in results from all targeted subgroups [9]. Overall sensitivity across 18 studies and 61 assays was 90% with an overall specificity of 98%, indicating that the 4

ACCEPTED MANUSCRIPT performance specifications of existing serological assays for the diagnosis of Chagas disease are lower than previously thought. Improvements in the available diagnostics, in terms of sensitivity

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and specificity, are required.

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One of the antigens used for the diagnosis of Chagas disease is TcF, a multiepitope, recombinant protein containing four immunodominant and repeating peptide epitopes. The reactivity of TcF, and that of related peptides, has been described extensively and this protein

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demonstrates a high level of sensitivity and specificity when used in ELISA with T. cruzipositive sera [10-16]. This is most clearly observed with Chagasic sera from South America,

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which were also used in the initial selection of the epitopes to include within this fusion construct. Further evaluations indicate that TcF has varying sensitivity or intensity of signal

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when tested against T. cruzi-positive blood donors or patient sera from the United States and Central America. This indicates that enhancements can be made to TcF to render improved or

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expanded diagnostic capabilities.

Targets of antibody responses can now be predicted with some accuracy using

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bioinformatic searches. We recently predicted, and following recombinant expression identified, a number of novel tandem repeat (TR) domains in the T. cruzi proteome that are recognized by

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antibodies circulating in serum from T. cruzi-infected individuals (Chagasic patient serum). With a goal of maximizing reactivity, we generated and evaluated the diagnostic potential of novel

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chimeric fusion proteins/polyproteins that included multiple antibody-binding epitopes from a variety of individual diagnostic candidate antigens.

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ACCEPTED MANUSCRIPT Materials and Methods Patient and control samples. Several panels of sera were assessed. The preliminary panel

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consisted of sera from 9 healthy US controls and 40 confirmed Chagas disease patients collected

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in the state of Bahia, Brazil in 1993 and 1994. A second panel consisted of 36 healthy US controls and 162 confirmed Chagas disease patients. Finally, a third panel from endemic areas of Brazil was selected to prioritize samples with moderate to low TcF reactivity from Chagas

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disease patients with clinical and electrocardiographic alterations and/or mega disease.

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individuals were specifically treated for Chagas disease before sample collection. This third

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panel consisted of 84 Chagas sera that were confirmed by positive results additional diagnostic tests and 51 samples that were negative were used as controls (Table 1). These samples were

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repeatedly tested by the following conventional serological tests: Indirect immunofluorescence (IIF) assays; Chagatest-HAI (Wiener, Rosario, Argentina), which detects anti-T. cruzi antibodies by observation of a specific agglutination when a patient's serum is mixed with red blood cells

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sensitized with T. cruzi cytoplasmic and membrane antigens and; Test ELISA Chagas III (BiosChile, Santiago, Chile), which detects IgG antibodies against whole extracts, including

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highly immunogenic membrane antigens, of the T. cruzi Tulahuén and Mn strains.

Protein production and purification. Nucleotide sequences encoding combinations of reactive

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TR domains were synthesized at Blue Heron Biotechnology (Bothell, WA, USA) and were designed to incorporate specific restriction enzyme sites 5’ and 3’ of the gene of interest and excluded in the target gene for directional cloning into the expression vector pET28a (Novagen, Madison, WI). The fusion polypeptide referred to as TcF26 was generated by the tandem linkage of an open reading frame of polynucleotides encoding a methionine initiation codon (ATG) added to the 5’ end of a fragment of the carboxy-terminus of the Tc11r3 polynucleotide, the open reading frame of polynucleotides encoding the TcFr2 polypeptide, and the open reading frame of polynucleotides encoding the Tc13r2 polypeptide. The fusion polypeptide referred to as TcF43 was generated by the tandem linkage of an open reading frame of polynucleotides encoding a methionine initiation codon (ATG) added to the 5’ end of a fragment of the carboxy-terminus of the Tc11r9 polynucleotide, the open reading frame of polynucleotides encoding the TcFr2 polypeptide, and the open reading frame of polynucleotides encoding the Tc13r5 polypeptide. 6

ACCEPTED MANUSCRIPT After cloning, the expression vectors were transformed into the E. coli. E. coli cultures were grown overnight at 37 °C as seeds and saturated cultures diluted fifty fold in 4 liter shaker flasks filled with 1 liter 2 × YS medium. Protein expression was induced with 1 mM IPTG once the

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optical density at 600 nm reached 0.3-0.5 OD units. Cultured bacteria were harvested 4 hours

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later by centrifugation and cell pellets frozen at -80 °C. Protein was extracted by lysing the cells with 3 rounds of sonication on ice. Debris was removed by centrifugation and the supernatant

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and pellet evaluated for the presence of recombinant protein. Depending on the location of the expressed recombinant protein, the supernatant was either used directly for purification or the

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pellet was dissolved in 8 M urea. Target proteins were then purified using Nickel-NTA columns (Qiagen, Germantown, MD) for standard nickel affinity chromatography, followed by ion

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exchange chromatography. Purity was monitored using SDS-PAGE and Western Blot analysis using anti-E. coli antibodies to determine residual host strain contamination.

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Antigen-specific antibody ELISA. ELISA plates were coated with 1 µg/ml antigen in 0.1 M bicarbonate buffer and blocked with 0.1% BSA-PBS. Following washes in PBS/Tween, diluted

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serum samples were added and incubated. After washes, anti-human IgG-HRP conjugate antibodies (Zymed, Grand Island, NY) were added and incubated. After further washing, TMB

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Sureblue Peroxidase (Kirkegaard Perry laboratories, Gaithersburg, MD) was added to reveal any reactions, which were subsequently stopped by the addition of 0.1N H2SO4. Plates were read at

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405nm using an ELX808 plate reader and OD values determined.

Statistics. The threshold for positive responses was calculated as OD of sera from healthy US controls plus 2 x standard deviation (SD). Per cent sensitivity was determined by number of positive samples among disease samples divided by the total number of disease samples, while specificity was determined as 100 – (number of positive samples among negative control samples divided by the total number of negative control samples). Statistical significance was assessed using unpaired t-test for comparison between two groups. Results were considered statistically significant when p-values < 0.05 were obtained.

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ACCEPTED MANUSCRIPT Results Antigenicity of individual T. cruzi TR proteins. We previously reported that T. cruzi TR proteins

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could serve as targets of the antibody response and therefore hold diagnostic potential [17]. To

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prioritize TR proteins, we extended upon our prior report by assessing the antibody binding of an alternate panel of sera from Chagas disease patients. Although each TR protein could be bound by antibodies from Chagas disease patients, the responses were heterogeneous and only a few

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TR proteins were recognized by more than 50% of the Chagas patient sera (Figure 1 and Table 2). To identify TR proteins that might enhance the diagnostic sensitivity over that provided by

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TcF, one of the recombinant antigens currently used within tests for Chagas disease, we contrasted the seroreactivity of each TR protein against that of TcF. As expected, TcF was

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strongly recognized by many of these sera. Of note, however, 12 of the 40 samples within this particular evaluation panel had TcF ELISA OD less than 1.5 and some of the TR proteins provided a stronger signal (Figure 1 and Table 2). The single sample that tested negative by TcF

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was detected by Tc11 (data not shown). Taken together, these data identify several TR proteins that could supplement TcF for the enhanced and clearer detection of T. cruzi infection/ Chagas

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disease.

Evaluation of fusion proteins. To combine selected TR proteins within a single product we

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produced recombinant multiepitope fusion proteins which were then evaluated against sera from Chagas disease patients from coastal Brazil. By directly contrasting the response of each individual sample, we observed that these novel fusion proteins strongly identified the majority of sera with only a subset of samples providing moderate responses (Table 3). Both TcF43 and TcF26 yielded strong responses against sera that were also strongly detected by TcF (Figure 2). More importantly, TcF43 and TcF26 typically gave stronger responses and could supplement the recognition of sera that were moderately or weakly reactive with TcF. Taken together these data suggest that TcF26 and TcF43 could be used for the confirmation of Chagas disease.

Supplementation of diagnosis by TcF46 and TcF26. Low antibody responders account for around 20% of T. cruzi-infected individuals, a third serum panel was selected to evaluate the fusion 8

ACCEPTED MANUSCRIPT proteins under the most stringent diagnostic conditions . Results from a test panel assembled on the basis of moderate or weak recognition by TcF (mostly signal:noise <5) (Table 1) indicated that TcF43 and TcF26 typically yielded stronger responses than TcF and it was again apparent

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that TcF43 and TcF26 could more strongly recognize many of the sera that had low antibody

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levels against TcF (Figure 3 and Table 4). Examination of negative control sera from a T. cruziendemic region (samples designated as NEG) indicated that TcF43 and TcF26 did not

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compromise the specificity of detection but actually, despite enhancing signal in positive samples, slightly improved specificity over TcF (Table 4). Taken together, these data indicate

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that TcF26 and TcF43 react with many Chagasic sera that are only weakly detected by TcF. Given that all of these sera were also submitted to at least three serological tests that are

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conventionally used to confirm Chagas disease, and were selected because of their low antibody concentration (defined in IIF or IHA as titers among 1/80 and 1/640 or as an ELISA index (OD of the sample/OD of cut-off) below 2.5), we contrasted the magnitude and clarity of responses

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against TcF43 and TcF26 versus those obtained in these other tests. By arranging the data from each of those tests in rank order it was apparent that there was widespread disagreement with the

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antibody tests against recombinant antigens TcF, TcF26 and TcF43 (Figure 4). Of note, however, several samples that were assessed as low responders in each test (plotted on the right side of

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each panel) were readily detected by the fusion proteins. These data further support the use TcF26 and TcF43 in antibody-based tests for the detection of T. cruzi infection and the diagnosis

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of Chagas disease.

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ACCEPTED MANUSCRIPT Discussion The detection of T. cruzi infection and diagnosis of Chagas disease is difficult due to the frequently symptomless acute and typically asymptomatic chronic phases of the disease.

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Parasitological diagnosis in the acute phase can be achieved by microscopy of blood films or by

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haematocrit centrifugation and examination of the buffy coat, with the latter being particularly recommended for congenital cases. Parasitological diagnosis in the chronic phase may be achieved by amplification of parasite DNA, by growth of parasites from blood cultures or by

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xenodiagnoses, but these usually require multiple blood samplings and even then still have limited sensitivity. Diagnosis during the chronic phase is typically achieved with the support of

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indirect antibody-detection tests that are reported to have high sensitivities and specificities, although manufacturer-listed performance parameters are generally better than those achieved in

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subsequent validation studies [9]. As a strategy to improve upon the performance of TcF, one of the antigens used in current diagnostic tests, we identified TR proteins that were reactive with

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Chagasic sera and then generated fusion proteins that were predicted to improve the sensitive detection of Chagas patients. Our data indicate that TcF26 and TcF43 provide incremental, but

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important, enhancements for the diagnosis of Chagas disease. A wide variety of factors influence the performance of diagnostic tests for Chagas

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disease. It is now recognized that T. cruzi can be segregated into six distinct lineages based upon genetic diversity (TcI-TcVI) [18]. These lineages have partially overlapping geographical

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distributions and circumstantial evidence suggests that they may present in distinct patterns epidemiologically [19, 20]. TcII, TcV and TcVI are the principal agents of Chagas disease in the Southern Cone region of South America, where Chagasic cardiomyopathy, megaoesophagus and megacolon are found. TcV and TcVI are known to be relatively recent hybrids of TcII and TcIII [20, 21]. TcIII is seldomly isolated from humans and TcIV is a sporadic secondary agent of Chagas disease in Venezuela [22]. TcI is the principal agent north of the Amazon River, where infection is associated with Chagasic heart disease but rarely with megaesophagus. Although a previous study correctly diagnosed Chagasic sera from Mexico with reagents made with parasites of TcII or TcV-VI lineages [23], it remains possible that the infecting T. cruzi lineage and associated antigenic variability could impact antibody-detection tests. This would be particularly detrimental when tests are used in regions where genetic lineages of T. cruzi different from those used to develop the tests are prevalent and indicates the need for extensive 10

ACCEPTED MANUSCRIPT evaluations across all T. cruzi-endemic regions. Further consideration is also required to ensure the specificity of testing, as cross-reactivity against other pathogens that are distributed throughout T. cruzi-endemic regions, particularly the Leishmania species, could potentially

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confound diagnosis. Expansion of TcF43 and TcF26 testing to serum panels from alternate

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regions and from patients infected with defined T. cruzi lineages appears warranted. The intended purpose of tests is also a critical consideration in their development.

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Laboratory tests are best suited to screening of large numbers of samples, such as blood donations etc., and require qualified staff, specific equipment, and infrastructure. Given the risks

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of inadvertent transmission, screening of donated blood, blood components, and solid organ donors, as well as donors of cells, tissues, and cell and tissue products for T. cruzi is now

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mandated in all endemic countries. T. cruzi infection and Chagas disease is also, however, highly pertinent to health services in non-endemic countries. It is commonly predicted that approximately 300,000 symptomatic cases exist in the United States, although an alternative

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estimate suggests that more than one million more cases may be present: conservative estimates of over $118 million in terms of direct financial impact each year Chagas disease-related

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healthcare costs in the US are second globally to only Brazil [24-27]. The use of antigens that provide incremental improvements over current levels, such as a 1% increase in sensitivity,

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could therefore have a large impact and would potentially allow recognition of another 3,000 to 10,000 infected individuals in the United States alone. Although screening of the United States

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blood supply was instituted in 2007, it was observed that only 4 of the 58 organ procurement organizations required testing of every donor and it is estimated that over 300 transplant patients may have contracted Chagas disease. The expansion of reference laboratory testing is readily achievable with standardized ELISA, and large-scale manufacturing is typically more consistent and sustainable through the use of recombinant proteins over antigens purified from crude parasites. TcF26 and TcF43 would therefore appear well placed for use in reference and screening laboratories. Reference laboratory testing is either unaffordable or unavailable in many regions where Chagas disease is endemic, however, and this is one of the main obstacles to initiating appropriate treatment [28]. The lack of access in remote areas also results in a disconcerting number of missed diagnoses and rapid diagnostic tests (RDT) would therefore be preferable to permit point-of-care use in these areas. The need for such RDT for T. cruzi infection/ Chagas 11

ACCEPTED MANUSCRIPT disease was reiterated in a 2010 World Health Assembly resolution (WHA63.20) that outlined the need “to promote and encourage operational research on control of Chagas disease in order to establish systems of early detection” and “to integrate, at the primary health care level, diagnosis

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and treatment of Chagas disease in patients in both acute and chronic phases of the disease”.

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RDT can be developed on different principles but must be inexpensive and remove the need for skilled laboratory staff, external equipment, or refrigeration [29-32]. In general, the transition

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from ELISA to RDT is reasonably simple when the targets are single recombinant antigens. TcF43 and TcF26 are therefore likely very amenable to use in various test platforms.

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While a variety of tests are now available for the diagnosis of Chagas disease and detection of T. cruzi infection, field testing has revealed that there is still a need for

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improvements. Our data, generated across various serum panels, indicates that the TcF43 and TcF26 derived from the fusion of selected T. cruzi TR proteins can provide incremental improvement in overall sensitivity while also providing a greater distinction between negative

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and positive sera. Further evaluation against samples from other Chagas-endemic regions and in

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alternate test platforms appears warranted.

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ACCEPTED MANUSCRIPT Acknowledgements The authors are extremely grateful to the patients for generous participation and Rozangela

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Amaral de Oliveira and Liliane da Rocha Siriano for their excellent technical work.

Conflict of Interest

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The authors declare no conflicts of interest.

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. TR proteins detect antibodies in serum from Chagas disease patients. ELISA were

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conducted to capture antigen-specific IgG in sera from patients with Chagas disease (n = 40) or

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healthy US-based controls (NEC = 8). Each point represents the response of each individual

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sample.

Figure 2. The TcF43 and TcF26 fusion proteins are recognized by antibodies in sera from

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patients with Chagas disease. In A, fusion proteins were generated by recombinant methods and purity assessed by SDS-PAGE. In B, ELISA were conducted to capture antigen-specific IgG

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in sera from patients with Chagas disease (n = 162) or healthy US-based controls (n = 36). Data are arranged by magnitude of response to TcF, with each point representing the response of an

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individual sample.

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Figure 3. TcF26 and TcF43 enhance the detection of Chagas disease over that achieved with TcF. ELISA were conducted to capture antigen-specific IgG against recombinant fusion

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proteins A. TcF43 and B. TcF26. Chagas samples were selected on the basis of results from 3 alternative tests; NEG were negative in all 3 tests. Data are arranged by magnitude of response to

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TcF, with each point representing the response of an individual sample.

Figure 4. Comparison of antigen-specific responses versus alternative Chagas disease tests. ELISA were conducted to capture antigen-specific IgG against recombinant fusion proteins. Samples were selected on the basis of results from 3 alternative Chagas disease tests and each panel is arranged by magnitude of response in the indicated test.

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Hotez PJ: Neglected infections of poverty in the United States of America. PLoS Negl Trop Dis 2008, 2(6):e256. Lee BY, Bacon KM, Bottazzi ME, Hotez PJ: Global economic burden of Chagas disease: a computational simulation model. Lancet Infect Dis 2013, 13(4):342-348. Gomes YM, Lorena VM, Luquetti AO: Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies? Mem Inst Oswaldo Cruz 2009, 104 Suppl 1:115-121. Senior K: Chagas disease: moving towards global elimination. Lancet Infect Dis 2007, 7(9):572. Villa L, Morote S, Bernal O, Bulla D, Albajar-Vinas P: Access to diagnosis and treatment of Chagas disease/infection in endemic and non-endemic countries in the XXI century. Mem Inst Oswaldo Cruz 2007, 102 Suppl 1:87-94. Basile L, Jansa JM, Carlier Y, Salamanca DD, Angheben A, Bartoloni A, Seixas J, Van Gool T, Canavate C, Flores-Chavez M et al: Chagas disease in European countries: the challenge of a surveillance system. Euro Surveill 2011, 16(37). New global effort to eliminate Chagas disease. Wkly Epidemiol Rec 2007, 82(2829):259-260.

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negatives 46.6 45.0 19 to 87* 21 F/30 M

22 11 6 12 51

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low positives 53.3 54.0 23 to 84 43 F/41 M 18** 46 9 11*** 44 19 11 10 84

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Main characteristics Age (years), mean Age (years), median Age (years), range Gender Cardiopathy Mega disease Associated (mega + cardiop.) Non-specific alterations Geographical origin: Goias State Bahia State Minas Gerais Other States TOTAL

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Table 1. Characteristics of a serum panel from T. cruzi-infected individuals with low antibody concentration and negative control individuals from an endemic area of central Brazil

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Tc6 r2 18

Tc7 r2 11

Tc9 r9 10

Tc11 r9 36

Tc12 r9 10

Tc13 r5 37

Tc14 r1 21

Tc15 r6 3

0

1

1

1

0

1

1

1

0

1

0

0

1

92. 5 10 0

52.5

77.5

27.5

50.0

45.0

27.5

25.0

90.0

25.0

92.5

52.5

7.5

88.9

88.9

88.9

100

88.9

88.9

88.9

100

88.9

100

100

88.9

1

3

0

0

2

0

0

8

0

5

3

0

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Tc5 r3 20

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positi ve, strong er than TcF

Tc4 r3 11

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Tc3 r2 31

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%

Tc1 r3 21

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positi ve

Tc F 37

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Table 2. Detection of Chagasic sera by recombinant tandem repeat (TR) proteins. The number and per cent of reactive sera within each category are indicated. Samples were considered positive if they generated an ELISA OD greater than mean plus 2 standard deviations of the NEC samples. For comparison, the reactivity of each TR protein with the 12 Chagasic sera that generated a signal :noise of < 1.5 versus TcF are indicated in the bottom row. NEC, nonendemic control.

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Table 3. Detection of Chagasic sera by fusion proteins. Per cent sera within each category are indicated. NEC, non-endemic control. TcF43 88.9 6.2

85.1 4.9 0 5.6

95.1 4.9 0 5.6

96.3 3.7 0 12.5

5.6 94.4

12.5 87.5

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TcF26 88.9 7.4

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TcF 77.2 17.9

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NEC (36)

signal:noise Strong (>5) Weak-Moderate (2-5) positive negative Strong (>5) Weak-Moderate (2-5) positive negative

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Tc6 r2 18

Tc7 r2 11

Tc9 r9 10

Tc1 1r9 36

0

1

1

1

0

1

1

1

0

92 .5 10 0

52. 5 88. 9

77. 5 88. 9

27. 5 88. 9

50. 0 100

45. 0 88. 9

27. 5 88. 9

25. 0 88. 9

1

3

0

0

2

0

0

Tc1 4r1 21

Tc1 5r6 3

1

0

0

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90.0

25.0

92.5

52.5

7.5

100

88.9

100

100

88.9

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Tc3 r2 31

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Tc1 r3 21

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Tc F 37

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Tc1 3r5 37

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Table 4. Detection of sera that were weakly reactive with TcF by alternate fusion antigens. Per cent sera within each category are indicated. EC, endemic control.

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Chagas

Figure 1 Tc15r6

Tc14r1

Tc13r5

Tc12r9

Tc11r9

Tc9r9

Tc7r2

Tc6r2

Tc5r3

Tc4r3

Tc3r2

Tc15r6

Tc14r1

Tc13r5

Tc12r9

Tc11r9

Tc9r9

Tc7r2

Tc6r2

Tc5r3

Tc4r3

1.5

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Tc1r3

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TcF

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Tc1r3

ELISA OD 450nm

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3.0

2.5

NEC

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Figure 3

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Designed novel recombinant fusion proteins combining several reactive tandem repeat proteins (TR)

Demonstrated that TcF43 and TcF26 antigens enhance detection and strength of signal without

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proteins into single diagnostic products for Chagas disease.

compromising the specificity of detection compared to that obtained with TcF, an established fusion antigen used in commercial test for detection of Chagas disease.

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TcF43 and TcF26 detect sera with weak reactivity towards TcF with a significantly stronger response.

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

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