**3. Use and prospects of recombinant DNA technology**

Since the emergence of recombinant DNA technology, many protein molecules have been designed and prepared to eventually be assessed for serological diagnosis. The proteins obtained through this technology may be used as antigens to capture antibodies, to evaluate exclusively defined molecules, avoiding potential interferences from other components that usually occur when the antigens have been obtained by purifying native source proteins. (da Silveira *et al.*, 2001) It follows that the usage of recombinant proteins as antigens to detect or quantify specific antibodies markers of a disease permits enhancing the specificity of the immunological reaction involved, therefore leading to more accurate diagnosis.(Aguirre *et al.*, 2006;Camussone *et al.*, 2009)

In this methodology the proteins are usually prepared by heterologous expression, mainly in *Escherichia coli* cells.(da Silveira *et al.*, 2001) Sequences of *T. cruzi*-protein codifying-DNA are inserted in a bacterial plasmid, which is transformed in competent bacteria. The proteins encoded by the plasmid are expressed in the bacterial culture, and are afterward purified into a highly pure product. The advantage of the proteins thus obtained is that they are an entirely characterized antigen, which may be evaluated individually for antibody determination in different clinical conditions. The prepared antigens can therefore be characterized considering the clinical information they provide, and may then be used to prepare specific diagnostic reagents. These proteins count with one desired feature of diagnostic reagents, as it is that their production and evaluation can be highly standardized. From another point of view, recombinant antigens do not require manipulation of the infective agent as do the antigens obtained by purification procedures from rough cultures. This has been a significant progress when considering the characteristics of viral infective agents, for which reagents production has substantially switched to that derived from recombinant DNA technology. Not less important is the major saving financial benefit of these reagents. Indeed, once bacteria are transformed into competent, protein producing strain, they can be used to prepare substantial amounts of antigen with low cost of production.

Using this technology, many gene expression clones have been create, a fact that has made available the obtainment of massive amounts of highly pure, standardized *T. cruzi* proteins.(da Silveira *et al.*, 2001)

recommendations states that *T. cruzi* infection must be diagnosed when the sample produces positive results by two different serological methods, whereas the undetermined condition is

Traditionally, whole parasites, or extracts from laboratory strains of *T. cruzi* epimastigotes cultures, have been the source of antigens used for the serological infection diagnosis. However, this yields to complex protein mixtures of unknown composition, which display

The diagnostic problems arising from serology deficient specificity, as well as the deprived reagents standardization, can be resolved through the use of defined antigens, such as the proteins obtained by molecular biology technology procedures.(Saez-Alquezar *et al.*,

The following sections will be focused in this issue and the most important contributions

Since the emergence of recombinant DNA technology, many protein molecules have been designed and prepared to eventually be assessed for serological diagnosis. The proteins obtained through this technology may be used as antigens to capture antibodies, to evaluate exclusively defined molecules, avoiding potential interferences from other components that usually occur when the antigens have been obtained by purifying native source proteins. (da Silveira *et al.*, 2001) It follows that the usage of recombinant proteins as antigens to detect or quantify specific antibodies markers of a disease permits enhancing the specificity of the immunological reaction involved, therefore leading to more accurate

In this methodology the proteins are usually prepared by heterologous expression, mainly in *Escherichia coli* cells.(da Silveira *et al.*, 2001) Sequences of *T. cruzi*-protein codifying-DNA are inserted in a bacterial plasmid, which is transformed in competent bacteria. The proteins encoded by the plasmid are expressed in the bacterial culture, and are afterward purified into a highly pure product. The advantage of the proteins thus obtained is that they are an entirely characterized antigen, which may be evaluated individually for antibody determination in different clinical conditions. The prepared antigens can therefore be characterized considering the clinical information they provide, and may then be used to prepare specific diagnostic reagents. These proteins count with one desired feature of diagnostic reagents, as it is that their production and evaluation can be highly standardized. From another point of view, recombinant antigens do not require manipulation of the infective agent as do the antigens obtained by purification procedures from rough cultures. This has been a significant progress when considering the characteristics of viral infective agents, for which reagents production has substantially switched to that derived from recombinant DNA technology. Not less important is the major saving financial benefit of these reagents. Indeed, once bacteria are transformed into competent, protein producing strain, they can be used to prepare substantial

Using this technology, many gene expression clones have been create, a fact that has made available the obtainment of massive amounts of highly pure, standardized *T. cruzi*

severe difficulties to be standardized, and additionally lead to false-positive results.

2000;Umezawa *et al.*, 2003;Umezawa *et al.*, 2004;Aguirre *et al.*, 2006)

**3. Use and prospects of recombinant DNA technology** 

established for samples rendering dissimilar outcomes.

that several research groups have recently made.

diagnosis.(Aguirre *et al.*, 2006;Camussone *et al.*, 2009)

amounts of antigen with low cost of production.

proteins.(da Silveira *et al.*, 2001)

During the latest three decades, many parasite antigens have been cloned and characterized. The cloned antigens correspond to different parasite stages namely, the trypomastigote sanguineous, the amastigote intracellular and the epimastigote, which is the form found inside the vector bowel and that can be cultured. Several of these antigens were obtained by immunological tracing through expression of cDNA libraries from chagasic patient sera, as well as from immunized animals.(Lafaille *et al.*, 1989;Affranchino *et al.*, 1989;Levin *et al.*, 1989;Cotrim *et al.*, 1990;Gruber & Zingales, 1993) The antigen codifying genes have been identified from cDNA present in the libraries accomplished from epimastigote or trypomastigote forms.(Affranchino *et al.*, 1989;Levin *et al.*, 1989;Gruber & Zingales, 1993;Godsel *et al.*, 1995) Lately, Da Rocha et al. have proposed using amastigote proteins since this is the intracellular parasite form, being these antigens more significant for serodiagnosis.(DaRocha *et al.*, 2002)

The usage of DNA technology brought into light the existence of many parasite antigens with repetitive sequences, a fact that had been previously described when cloning proteins of other parasites.(Hoft *et al.*, 1989) Generally, these are the most immunogenic antigens, and are the mainly selected when performing immunological tracing in cDNA libraries cloned in phages. Therefore, it was initially stated that these were the most valuable antigens for diagnosis.(Frasch & Reyes, 1990) However, it was afterward proved that some nonrepetitive antigens have equivalent diagnostic value than repetitive ones. Certainly, a multicenter study evaluated in parallel 4 repetitive recombinants antigens (H49, JL7, B13, JL8) together with 2 non-repetitive ones (A13 y 1F8).(Umezawa *et al.*, 1999) The results demonstrated that both type of antigens were similarly useful for *T. cruzi* infection diagnosis, and the authors suggested that if they were to be used together in a mixture, they could supplemented each other enhancing the sensitivity of the assay. This was afterwards proved by the same group, see Tables 1 A,B and C.(Umezawa *et al.*, 2003)

Once the complete genome sequence of *Trypanosoma cruzi* was annotated, (El Sayed *et al.*, 2005) alternative antigenic candidates have been searched in the parasite genome. The studies have been supported by bioinformatic prediction of putative proteins and antigenicity predictors.(Goto *et al.*, 2008;Cooley *et al.*, 2008;Hernandez *et al.*, 2010) Using these tools, it has been possible to choose antigens which display the lowest homology level with proteins of organisms related to *T. cruzi*.(Hernandez *et al.*, 2010) Moreover, the bioinformatic analysis has allowed describing for the first time a specific antigen to type discrete typing units (DTUs). (Di Noia *et al.*, 2002)

The results published by many different laboratories point towards considering recombinant proteins as the chosen molecules to be used in immunoassays to diagnose *T. cruzi* infection. Moreover, the lack of specificity leading to false-positive results can be overcome by deleting sequence regions encoding for proteins which cross-react when analyzing negative sera,(Aguirre *et al.*, 2006), or using recombinant proteins that are specific for anti-*T. cruzi* antibodies, yet keeping a high sensitivity.(Belluzo *et al.*, 2011;Camussone *et al.*, 2009) Indeed, the largest studies on the diagnosis reveal the convenience of using these antigens, regarding not only specificity but also the possibility of standardizing both, the methodology and the protein production.(Umezawa *et al.*, 1999;Saez-Alquezar *et al.*, 2000;Umezawa *et al.*, 2003)

The following table display the key recombinant antigens discussed in the present chapter, which were evaluated by several authors for *T. cruzi* infection diagnosis. Notice,

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 281

Antigen name Characteristics Diagnostic use Described by

infections.

regions

TcD Trans-sialidase family Chronic and acute infection Burns, Jr. *et al.*, 1992

infection

monitoring

monitoring Cure monitoring

*T. cruzi* typing

Table 1B. Relevant recombinant antigens which belong to tran-sialidase (TS) and TS-like family, proposed for diagnostic uses. Abbreviations used: CEA, chronic exoantigen (160 KDa); CRP, complement regulatory protein; FL-160, surface flagellar protein (160 KDa); RP2,

Confirmation of chronic

Chronic infection and cure

Chronic infection and cure

(named lineage Tc I, in the previous nomenclature)

*T. cruzi* typing (named lineage Tc II, in the previous nomenclature) DTUII, V and VI in the current nomenclature Confirmatory diagnostic in Chagas and leishmaniasis co-endemic regions

Acute and congenital

Frasch & Reyes, 1990 Russomando *et al*.,

Breniere et al., 1997 Gil *et al*., 2011 Camussone *et al*.,

Buchovsky *et al.*, 2001

Cetron *et al.*, 1992

Jazin *et al.*, 1995

Meira *et al.*, 2004

Di Noia *et al*., 2002

Di Noia *et al*., 2002

Bhattacharyya *et al.*,

Cimino *et al.*, 2011

2010

2010

2009

Chronic infection Houghton *et al*., 1999

Chronic infection in leishmaniasis endemic

Trans-sialidase

family

family

family

TSSAI Trypomastigote

family

family 

Trans-sialidase

Complement regulatory protein from TS-like family

muscin of TS-like

Trypomastigote muscin of TS-like

repetitive peptide 2; SAPA, shed-acute phase antigen.

TcLo1.2 Trans-sialidase

SAPA

RP2

Trans-sialidase catalytic region

FL-160

CEA

CRP160

TSSAII 

that many of these antigens particularly named by one author have amino acid sequences, which may be very similar to those obtained by other authors who have named them differently (e.g. FRA, Ag1, JL7, H49). Identical or highly similar antigens were grouped in the same row.


Table 1A. Relevant repetitive recombinant antigens proposed for diagnostic uses. Abbreviations used: CRA, cytoplasmic repetitive antigen; FRA, flagellar repetitive antigen; MAP, microtubule associated protein. RP1, RP3, RP4 and RP5, repetitive peptide 1, 3, 4 and 5, respectively.

that many of these antigens particularly named by one author have amino acid sequences, which may be very similar to those obtained by other authors who have named them differently (e.g. FRA, Ag1, JL7, H49). Identical or highly similar antigens were grouped in

Characteristics Diagnostic use Described by

Chronic infection Lafaille *et al*., 1989

Chronic infection Lafaille *et al*., 1989

Chronic infection Gruber *et al*., 1993

Chronic and acute

Antibodies against this protein render crossreactions with mammal cell cytoskeleton.

infection.

Table 1A. Relevant repetitive recombinant antigens proposed for diagnostic uses.

Abbreviations used: CRA, cytoplasmic repetitive antigen; FRA, flagellar repetitive antigen; MAP, microtubule associated protein. RP1, RP3, RP4 and RP5, repetitive peptide 1, 3, 4 and

Ibáñez *et al.*, 1988 Levin et al., 1989 Hoft *et al*., 1989 Camussone *et al*., 2009

Ibañez *et al*., 1988 Levin *et al*., 1989 Cotrim *et al.*, 1995 Camussone *et al*., 2009

Ibañez *et al*., 1988 Hoft *et al*., 1989 Peralta *et al*., 1994 Camussone *et al*., 2009

Ibañez *et al*., 1988

Levin *et al*., 1989 Kerner *et al.*, 1991 Camussone *et al*., 2009

the same row.

Antigen (grouped by high identity)

> Cytoplasmic antigen

Cytoskeleton associated protein

Trypomastigote surface protein

Microtubule associated protein

CRA Ag30 JL8 TCR27 RP4

FRA Ag1 JL7 H49 RP1

B13 Ag2 TCR39 PEP-2 RP5

Ag36

JL9 MAP-like RP3

5, respectively.


Table 1B. Relevant recombinant antigens which belong to tran-sialidase (TS) and TS-like family, proposed for diagnostic uses. Abbreviations used: CEA, chronic exoantigen (160 KDa); CRP, complement regulatory protein; FL-160, surface flagellar protein (160 KDa); RP2, repetitive peptide 2; SAPA, shed-acute phase antigen.

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 283

The first works dealing with a single recombinant protein for diagnostic purposes reported lack of sensitivity when using only one of those antigens.(Levin *et al.*, 1991;Moncayo & Luquetti, 1990;Peralta *et al.*, 1994) Consequently, most of these proteins have been evaluated not only alone and independently from others, but also together as part of mixtures or as fusion proteins, carrying several recombinant epitopes.(Umezawa *et al.*, 1999;Umezawa *et al.*, 2004;Camussone *et al.*, 2009;Foti *et al.*, 2009) Accordingly, a multicenter study evaluating 6 recombinant proteins separately with a serum panel composed by sera from patients of several countries, described that using the set of results of the 6 proteins together had yield a sensitivity and specificity compatible with the reference assays.(Umezawa *et al.*, 1999) Later, the same group evaluated the mixture of the 6 proteins, supporting the use of the mixture to reach the same sensitivity and specificity.(Umezawa *et al.*, 2003) Soon after, the reactivity of individual antigens vs. antigen mixtures was systematically assessed by ELISA.(Umezawa *et al.*, 2004) This study confirmed that the results obtained with recombinant protein mixtures led to higher media values of optical densities, ODs, than the results produced when using the individual recombinant proteins. Moreover, sera rendering low ODs when examined with individual recombinant proteins produced higher ODs outcomes when using the protein mixtures. Along with this, several commercial ELISA kits with recombinant protein mixtures display equivalent or even higher sensitivities and specificities than those produced by kits with total parasite homogenate.(Gadelha *et al.*, 2003;Pirard *et al.*, 2005;Remesar *et al.*, 2009;Caballero *et al.*, 2007) These works have studied kits using Ag1, Ag2, Ag30, Ag13 together with Ag36 recombinant antigens (Chagatest Rec from Wiener lab, Argentina), and FRA and CRA recombinant antigens (Biomanguinhos, Friocruz, Brazil). However, another study reported that Chagatest Rec v3.0 (Wiener) displayed a rather low

One of the strategies proposed to enhance reagents production standardization is to obtain multiepitope molecules, designed as a unique construction by fusing several relevant diagnostic antigens.(Houghton *et al.*, 1999;Aguirre *et al.*, 2006;Camussone *et al.*, 2009) It has recently been proved that when using these constructions, the ODs of sera with low reactivity increases, as well as it had been reported for mixtures.(Camussone *et al.*, 2009) Moreover, by this approach the attachment of the antigen turned out to be homogenous and reproducible when using different surfaces such as ELISA plaques, latex particles or bioelectrodes.(Camussone *et al.*, 2009;Gonzalez *et al.*, 2010;Belluzo *et al.*, 2011) It has been proposed that when there is only one molecule exposed to the surface, competition for the active sites is prevented, therefore resulting in a uniform attachment. Furthermore, sensitivity may be increased because a higher number of freely accessible epitopes are available to capture the antibodies present in samples, as depicted in Fig. 1.(Camussone *et* 

A few articles report on the use of this strategy to produce commercial ELISA kits which have demonstrated to be highly satisfying. One of these works, analyzes the performance of the TcF antigen, previously described by Houghton et al in 1999, with which the Biolab Merieux reagent was prepared.(Ferreira *et al.*, 2001) In this case, the recombinant protein used bears the PEP2, TcD, TcE and TcLo1.2 peptides. Recently, Abbot Laboratories have presented a new kit which uses a 4-antigen multiepitope protein containing TcF, FP3 -built up with TcR27 and FcaBP-, FP6 –with TcR39 and FRA- and FP10 -with SAPA and MAP-.(Praast *et al.*, 2011)

According to the authors, this kit performed even better than the Biolab Merieux one.

**4. Recombinant proteins use: Mixtures vs. fusion proteins** 

95% sensitivity.(Ramirez *et al.*, 2009)

*al.*, 2009)


Table 1C. Other relevant recombinant antigens proposed for diagnostic uses. Abbreviations used: cy-hsp70, cytoplasmic thermal-shock protein; FCaBP, flagellar calcium-binding protein; grp.hsp 78, endoplasmic reticule thermal-shock protein (78 KDa); mt-hsp 70, thermal-shock mitochondrial protein (70 KDA).
