Immunologically relevant strain polymorphism in the Amastigote Surface Protein 2 of Trypanosoma cruzi

Microbes and Infection 9 (2007) 1011e1019 www.elsevier.com/locate/micinf

Original article

Immunologically relevant strain polymorphism in the Amastigote Surface Protein 2 of Trypanosoma cruzi Carla Claser a,b, Noeli Maria Espı´ndola c, Gisela Sasso d, Adelaide Jose´ Vaz c, Silvia B. Boscardin a,b, Mauricio M. Rodrigues a,b,* a

Centro Interdisciplinar de Terapia Geˆnica (CINTERGEN), Universidade Federal de S~ao Paulo-Escola Paulista de Medicina, Rua Mirassol, 207, S~ao Paulo-SP 04044-010, Brazil b Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de S~ao Paulo-Escola Paulista de Medicina, Rua Mirassol, 207, S~ao Paulo-SP 04044-010, Brazil c Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de S~ao Paulo, Av. Prof. Lineu Prestes, 580, Cidade Universita´ria, S~ao Paulo, SP 05508-900, Brazil d Departamento de Morfologia, Universidade Federal de S~ao Paulo-Escola Paulista de Medicina, Rua Botucatu, 740, S~ao Paulo-SP 04023-062, Brazil Received 19 October 2006; accepted 11 April 2007 Available online 19 April 2007

Abstract Several evidences suggest that the Amastigote Surface Protein-2 (ASP-2) of Trypanosoma cruzi is an important target for immunity during infection. Based on this, we considered it important to evaluate its strain polymorphism. Initially, we observed the presence of conserved crossreactive epitopes in amastigotes of all parasite strains tested. In addition, the predicted amino acid sequences of the genes isolated from the cDNA of amastigotes of CL-Brener, Tulahuen, Colombian and G strains displayed a high degree of identity (>80%) to the previously described genes of ASP-2. Unexpectedly, Sylvio X10/4 and G strains expressed a new isoform of ASP-2 with limited identity to the previously described genes, but with a high degree of identity when compared to each other. Immunological studies confirmed the presence of cross-reactive epitopes between recombinant proteins representing the different isoforms of ASP-2. However, the genetic vaccination of mice with the new isoform of asp-2 gene expressed by the G strain failed to provide the same degree of protective immunity to a challenge by parasites of the Y strain as did asp-2 genes of Y or CL-Brener strains. In summary, we found that few strains can express different isoforms of ASP-2 which may not share cross-protective epitopes. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Trypanosoma cruzi; Amastigotes; Trans-sialidase

1. Introduction The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas’ disease, which chronically affects more than 15 million individuals in the Americas, causing an annual death toll of approximately 45,000 people [1]. T. cruzi invades and multiplies inside a variety of different host cells. After * Corresponding author. CINTERGEN, UNIFESP e Escola Paulista de Medicina, Rua Mirassol, 207, S~ao Paulo-SP 04044-010, Brazil. Tel./fax: þ55 11 5571 1095. E-mail address: [email protected] (M.M. Rodrigues). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2007.04.006

trypomastigotes invade, they transform into amastigotes, the intracellular replicative form of the parasite. Host cells containing amastigote nests are critically involved in chronic phase Chagas’ disease pathology. Not only are these cells destroyed by multiplying parasites, but they also stimulate inflammatory responses considered the main cause of chronic chagasic pathology [2e4]. Because amastigotes are important for the observed pathology, they can be considered primary targets for therapeutic drugs and host protective immune responses. Amastigotes express an 83 kDa molecule denominated Amastigote Surface Protein-2 (ASP-2, [5,6]) on their surface. This protein was first described in the Brazil strain

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of T. cruzi being encoded by a gene member of the subfamily II of the trans-sialidase gene family. This gene family was the largest found in the recently sequenced T. cruzi genome with 1430 genes [7]. Although the importance of this molecule for amastigote survival inside host cells remains unknown, various evidences from different groups strongly suggest that this antigen can be a target for important host immune responses. Chronically infected humans display antibodies, as well as cytotoxic CD8þT cells specific for ASP-2 [6,8]. In mice, upon experimental infection, intense cellular immune responses mediated by cytotoxic and IFN-g producing CD8þT-cells have also been noted [9,10]. The most compelling evidence showing the importance of immune response to ASP-2 is based on vaccination studies. Immunization with genes or recombinant proteins of ASP-2 induced CD4þ Th1 and CD8þ Tc1 immune cells which elicited strong protective immunity in mice of three different genetic backgrounds challenged with parasites of two distinct strains [11e15]. Vaccination with ASP-2 genes/proteins not only diminished acute phase parasitemia/mortality, but also reduced the chronic phase pathology in at least two distinct experimental models [11,14]. These interesting results obtained with immunization using ASP-2 suggest that this protein could be part of a subunit prophylactic or therapeutic vaccine against Chagas’ disease. Nevertheless, the fact that T. cruzi displays a strain polymorphism indicates that these results should be interpreted with prudence. Based on this, the first objective of the present study was to evaluate what type of strain polymorphism could be present in ASP-2. The subsequent objective would be the evaluation of the presence of strain-variant or cross-reactive epitopes and their possible relevance for immunity.

2. Materials and methods 2.1. Mice and parasites Female 8-week-old BALB/c, A/Sn and C57BL6 Interferon (IFN)-g knock out (KO) female and male mice used in this study were purchased from University of S~ao Paulo. Parasites of the Y, Sylvio X10/4, Dm28c, CL-Brener, Tulahuen, G or Colombian strains of T. cruzi were used. Epimastigotes and trypomastigotes were obtained as described [12]. Extracellular amastigotes were generated by incubation of trypomastigotes derived from infected LLC-MK2 in LIT medium during 24e 48 h at 37  C. Infection with bloodstream trypomastigotes of the Y strain and parasitemia were performed as described [12]. Each mouse was inoculated intraperitoneally (i.p.) with 250 trypomastigotes. The values of peak parasitemia of each individual mouse were log transformed before being compared by OneWay Anova followed by Tukey HSD tests available at the website http://faculty.vassar.edu/lowry/VassarStats.html. LogRank test was used to compare mouse survival rate after challenge with T. cruzi. The differences were considered significant when the P value was <0.05.

2.2. ASP-2 bacterial recombinant proteins and eukaryotic expression vectors Several bacterial recombinant proteins representing different domains of the ASP-2 expressed by amastigotes of the Y strain of T. cruzi were used in the present study [12,15]. A new His-65 kDa recombinant protein containing the gene isolated from the G strain was generated as follows. The asp-2 gene isolated from the G strain was amplified using the forward oligonucleotide (50 -GGG CCA TGG GG CTG CCG CAG GAG GTT GAT-30 ) and the reverse oligonucleotide (50 -GG GGT ACC TCA GAC CAT TTT TAG TTC AC-30 ) designed to anneal with nucleotides 226e243 and 2072e2091 of gene clone 62, respectively. Underlined nucleotides represent NcoI and KpnI sites, respectively. The PCR product was cloned in the pHis vector [12]. The resulting recombinant protein encoded AA 76e696 of ASP2 (clone 62, G strain). The plasmids pIgSPclone9 (Y strain), pIgSPclone50 (CLBrener strain) and pIgSPclone62 (G strain) containing the asp-2 gene were generated and purified as described [12]. A/ Sn mice were immunized 3 times (100 mg of plasmid DNA per mouse per dose) as described [12,14]. Two weeks after the last dose, mice were challenged with bloodstream trypomastigotes of the Y strain. 2.3. ASP-2 specific polyclonal and monoclonal antibodies BALB/c mice were immunized with 50 mg of plasmid DNA pIgSPclone9 (Y strain) or pIgSPclone62 (G strain) intramuscularly (i.m.) in each of the Tibialis anterioris muscles at 0 and 3 weeks. A third dose was provided 2 weeks later and consisted of an i.p. injection of 25 mg of recombinant protein His-65 kDa/Y strain or His-65 kDa/G strain (clone 62) adsorbed to alum (Alhydrogel ‘‘85’’ Superfos Biosector, Vedbaek, Denmark). Two weeks later, mice were bled and their sera used for immunological assays. BALB/c mice immunized with plasmid DNA pIgSPclone9 and boosted with recombinant protein HIS-65 kDa/Y strain had their spleen removed and fused with myeloma cells (SP2/O) using polyethylene glycol 4000 (Merck, Darmstadt, Germany). Hybridomas were grown for two weeks and samples of medium from these cultures were screened by indirect immunofluorescence assay (IIA) for antibodies reacting with amastigotes of the Y strain. The positive hybridomas were cloned and recloned by limiting dilution. Hybridoma supernatants were used for the immunological assays. 2.4. Immunoblot The recombinant proteins (0.5 mg/lane) or parasite extracts (equivalent to 107 amastigotes) were loaded onto SDS-PAGE and after electrophoresis, the samples were transferred to nitrocellulose membranes (Millipore Corp., Bedford) and the immunoblot performed as described [12]. Pool of sera (diluted 1:500) from mice immunized with the different plasmids

C. Claser et al. / Microbes and Infection 9 (2007) 1011e1019

followed by the recombinant proteins or the monoclonal antibody (MAb, diluted 1:200) were used as antibody source. 2.5. ELISA and IIA Antibodies to His-65 kDa of Y or G strains were detected by ELISA as described [12]. ELISA plates were coated with 250 ng/well of each recombinant protein. For IIA, parasites or HeLa cells infected 6 or 48 h before with trypomastigotes of the Y strain (50 parasites per cell) were fixed for 30 min in PBS containing the non-permeabilizing reagent formaldehyde. After fixation and washes in PBS, their concentration was adjusted to 107 parasites per ml. Parasite suspension was layered onto coated round coverslips and let stand overnight at rt. Coverslips were stored at 20  C until use. Infected cells were permeabilized by treatment with PBS containing 0.2% (vol/vol) Triton X-100 for 15 min at rt. IIA reaction was performed as described [12]. Nomarski differential interference contrast or fluorescence images were acquired with a Zeiss Axiovert microscope (100 M), 63  1.3 oil objective using the software LSM 510 Expert Mode SP2. 2.6. Immunohistochemistry The slices containing the heart or liver tissue from IFN-g KO mice were used due the higher density of parasite nests. After blockage of endogenous peroxidase activity, the samples were incubated in 5% bovine serum for 30 min and then with the MAb K22 (undiluted supernatant) for 1 h each. After washed in PBS-Tween and incubated with goat anti-mouse IgG coupled to peroxidase (1:200, Life Technologies), the peroxidase activity was developed with H2O2-DAB. The samples were counterstained with Harris’ hematoxilin, then dehydrated and covered with synthetic resin. 2.7. RNA purification, RT-PCR, gene cloning and sequencing Parasite RNA was purified using TRIzolÒ (Invitrogen). Subsequently, the RNA was treated with RNAse-free DNAse I (Invitrogen). The cDNA reaction was performed with total RNA using the ThermoScript RT-PCR System (Invitrogen) in the presence of oligo (dT) primer supplied in the kit. Initial PCR reaction was performed with spliced-leader (50 -AAC CGGATC CAACGC TATTATTGAT-30 ) and reverse (50 GGGTCTAGATCAGACCATTTTTAGTTCACC-30 ) primers. Subsequently, we performed a second PCR reaction using the forward primer (50 -GGGGGTACCATGCTCTCACGT GTTGCT-30 ) and the reverse primer. We also used for some strains (Y, Sylvio X10/4 and G) a reverse primer representing the VTV box domain (50 -GGGTCTAGAAGAAAGACGTT CGTCACTGTCAC-30 ). PCR were performed using the enzyme PLATINUM Pfx DNA Polymerase (Invitrogen) according to the protocol supplied by the manufacturer. The annealing temperature used was 48  C and the number of cycles was 35. PCR were performed in a Perkin-Elmer 2400 GeneAmp PCR System.

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Cloning and sequencing were performed exactly as described [12]. DNA and predicted AA sequences were analyzed using the DNASTAR package version 5.00 (DNASTAR Inc.). Alignments were produced through the Clustal W program. 3. Results In order to study the expression of ASP-2 in amastigotes of different strains of T. cruzi, we produced polyclonal and monoclonal antibodies specific to the recombinant protein derived from the Y strain of T. cruzi (clone 9). Immunoblot studies showed that both antibodies recognized a band of w83 kDa in amastigote extracts of the Y strain (Fig. 1A). In Fig. 1B, using recombinant proteins representing different portions of ASP-2, we observed that the polyclonal sera recognized the recombinant proteins His-65 kDa (lane 1), GSTP1P3 (N-terminal, lane 2), GST-P4P7 (Central domain, lane 3), and His-25 kDa (C-terminal, lane 4). In contrast, MAb K22 recognized His-65 kDa (lane 5) and His-25 kDa (C-terminal, lane 8). Several other MAb tested also recognized the Cterminal portion of ASP-2 (data not shown). Next, we determined by IIA that the recognition of both antibodies was restricted to the amastigote forms of the parasite (Fig. 1D). A study of the expression of ASP-2 in T. cruzi infected cultured cells showed a continued expression, as detected by MAb K22, from 6 to 48 h after invasion (Fig. 1E). The expression of ASP-2 by amastigotes in vivo was confirmed by using the MAb K22 to stain cardiac or liver tissues from IFN-g KO mice infected with T. cruzi (Fig. 1F,G). IIA performed using polyclonal and monoclonal antibodies showed that both antibodies recognized amastigotes of all strains tested (Fig. 2A). Immunoblot analysis confirmed that a band of w83 kDa was recognized in amastigote extracts of the different strains by MAb K22 (Fig. 2B) In an attempt to characterize genes expressed by the different strains of T. cruzi related to the previously described asp-2 gene, RT-PCR reactions were performed as described in Methods section. A single band of w2.1 Kb was obtained in RT-PCR. PCR products were not obtained in the absence of reverse transcriptase. DNA products obtained from PCR using amastigotes cDNA were cloned, and 13e35 clones from each strain were subjected to restriction and sequence analysis. One to four different restriction patterns were observed as we previously found for the Y strain of T. cruzi [12]. One to two ORFs were found per strain with the exception of strain Dm28c. All 35 clones sequenced from Dm28c strain had genes with premature stop codons. We selected clones 9 (Y), 25 (Sylvio X10/4), 50 (CL-Brener), 45 (Tulahuen), 62 (G1), 31 (G2), 22 (Colombian), and 19 (Dm28c) for further comparison. These clones were representatives of the most abundant clones or the only clone found in the cDNA of each strain. Nucleotide and predicted AA identities among clones 9, 25, 50, 45, 62, 22, 31 and asp-2 genes are shown in Table 1. The predicted AA sequences of the genes isolated from CL-Brener, Tulahuen, Colombian or G (G2) presented a high degree of identity (>81%) to the previously described asp-2 genes from Y and Brazil strains. In contrast, the predicted AA

C. Claser et al. / Microbes and Infection 9 (2007) 1011e1019

1014

A

1

2

3

B kDa

4

kDa

1

2

3

4

5

6

7

8

66-

100-

43-

7029554033Polyclonal anti-His 65kDa/Y strain

C

MAb K22

1

694

NH2-

- COOH

ASP-2 (clone 9) His-65kDa (78-694)

GST

(67-260)

P1-P3 GST

P4-P7

(261-500) His-25kDa

(492-694)

D Epi

Ama

Trypo

Polyclonal anti-His 65kDa/Y strain

MAb K22

E

F

6h

H

48h MAb K22

Fig. 1. Characterization of mouse-derived polyclonal and monoclonal antibodies specific to ASP-2. (A) Extracellular amastigote extracts of the Y strain (equivalent to 107 parasites) were added to each lane of an SDS-PAGE performed under reducing conditions. Immunoblot strips were incubated with the following antibodies: polyclonal anti-His 65 kDa/Y strain (lane 1), MAb K22 (lane 2), control sera from mice immunized with pcDNA3 and Alum (lane 3) or unrelated MAb anti-gp82 (lane 4, [26]). (B) Recombinant proteins (0.5 mg) were added to each lane of an SDS-PAGE performed under reducing conditions. Lanes contained the recombinant protein His-65 kDa/Y strain (1 and 5), GST-P1P3 (N-terminal, 2 and 6), GST-P4P7 (Central domain, 3 and 7) and His-25 kDa (C-terminal, 4 and 8). After transfer to nitrocellulose, immunoblot strips were incubated with the following antibodies: polyclonal anti-His 65 kDa/Y strain (lanes 1e4) or MAb K22 (lanes 5e8). (C) Schematic representation of the recombinant proteins used for immunoblot analysis. (D) Analysis by IIA of epimastigotes, amastigotes and trypomastigotes of the

C. Claser et al. / Microbes and Infection 9 (2007) 1011e1019

A

CL-Brener

G

CL-Brener

G

Tulahuen

SylvioX10/4

Tulahuen

SylvioX10/4

Colombian

Dm28c

Colombian

Dm28c

Polyclonal anti-His 65kDa/Y strain

B

1015

kDa

MAb K22

1

2

3

4

5

6

120100705540-

33-

Fig. 2. Presence of ASP-2 cross-reactive epitopes on amastigotes of different T. cruzi strains. (A) Extracellular amastigotes from different strains of the parasite were analyzed by IIA. Parasites were incubated with polyclonal anti-His 65 kDa/Y strain or MAb K22 as indicated and imaged under fluorescence microscopy. Bars, 14 mM. (B) Extracellular amastigote extracts (equivalent to 107 parasites) of the different strains were added to each lane of an SDS-PAGE performed under reducing conditions. Immunoblot strips were incubated with MAb K22. Lane 1: Sylvio X10/4; Lane 2: G; Lane 3: Tulahuen; Lane 4: Colombian; Lane 5: Dm28c; Lane 6: CL-Brener.

sequences of asp-2 genes from Sylvio X10/4 and G (G1) strains displayed limited identity to the other asp-2 genes (<50%). Interestingly, they displayed a high degree of identity when compared to each other (78.7%). Fig. 3 shows a schematic representation of the predicted AA sequences of asp-2 genes. The predicted AA sequences of different clones contain a sequence encoding a highly

probable signal peptide sequence at the N-terminus, which contains a strongly hydrophobic non-charged core. Also, ASP-2 of Brazil, Y, CL-Brener, Tulahuen, Colombian and G (G2) contain 1 or 2 ASP box motifs (SxDxGxTW) and the highly conserved motif VTVxNVxLYNR characteristic of bacterial and viral sialidases. In contrast, ASP-2 from Sylvio X10/4 and G (G1) strains did not express either motifs (Fig. 3).

Y strain of T. cruzi. Parasites were incubated with polyclonal anti-His 65kDa/Y strain or MAb K22 as indicated and imaged under Nomarski differential interference contrast (left) or fluorescence microscopy (right). Bars, 14 mM. (E) HeLa cells were infected for 6 h or 48 h with parasites of the Y strain. After fixation, IIA was performed as described in Section 2 using polyclonal anti-His 65 kDa/Y strain or MAb K22 as indicated and imaged under fluorescence microscopy. Arrows indicate amastigotes. Bars, 14 mM. (F) Heart tissue of IFNg-KO mice infected 14 days earlier with bloodstream trypomastigotes were removed and use for immunohistochemistry by staining with MAb K22 (magnification 400). (G) Liver tissue of IFNg-KO mice infected 14 days earlier with bloodstream trypomastigotes were removed and use for immunohistochemistry by staining with MAb K22 (magnification 1000).

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Table 1 Comparison of nucleotide and AA identities among the distinct genes isolated from cDNA of amastigotes of different T. cruzi strains

Nucleotide sequences (% of identity a) Strains

Brazilb

Brazilb

Dm28cd

Yc 89.3

CLBrenerd

Colom Tulahuend Sylvio X10/4d biand

G1d

G2d

87.6

90.2

89.9

90.5

68.8

69.2

84.3

88.4

91.0

98.7

98.2

69.1

69.7

90.8

94.0

88.7

88.8

67.9

68.2

84.0

91.0

90.9

68.9

69.4

85.7

98.0

69.6

70.3

90.8

70.0

90.0

97.7

68.2

Yc

82.4

Dm28cd

NC e

NC

CL-Brenerd

85.4

83.5

NC

Colombiand 81.7

97.0

NC

82.5

Tulahuend

84.1

96.7

NC

83.4

95.4

Sylvio X10/4d G1d

48.3

48.7

NC

47.9

47.5

49.0

43.8

44.2

NC

43.6

43.4

45.0

78.7

G2d

73.5

84.4

NC

73.6

83.7

82.7

45.9

69.3

68.7 42.6

Predicted AA sequences (% of identity a) a

Identities were calculated by the Clustal W alignment. GenBank accession number: U77951 (17). c GenBank accession number: AY186572 (5). d We used genes clones 19 (Dm28c, EF583446), 50 (CL-Brener, EF579918), 22 (Colombian, EF579919), 45 (Tulahuen, EF579921), 25 (Sylvio X10/4, EF579920), 62 (G1, EF579922) and 31 (G2, EF579923) for comparisons. e NC ¼ not compared. Comparisons of predicted AA sequences were not performed due to the presence of stop codons. b

COOH

NH2 Brazil Strain Y Strain CL-Brener Strain Tulahuen Strain Colombian Strain

G Strain (G2)

Sylvio X 10/4 Strain

G Strain (G1) -Signal peptide

-ASP BOX (SxDxGxTW) -VTV BOX (VTVxNVxLYNR)

Fig. 3. Schematic representation of the predicted AA sequences of asp-2 genes isolated from cDNA of intracellular amastigotes of different strains of T. cruzi.

To confirm whether these strains indeed expressed distinct ASP-2 proteins, we used an antiserum against the epitope TEWETGQI. The predicted AA sequence representing this epitope can be found in all ASP-2 except in the new isoforms detected on the G and Sylvio X10/4. By immunofluorescence, we could detect this epitope on amastigotes of Y and G strains, but not in Sylvio X10/4 (data not shown). Therefore, we concluded that this epitope is absent from parasites of the Sylvio X10/4 strain. In order to study the immunological impact of the polymorphism found among the ASP-2 proteins expressed in the different strains of the parasite, a recombinant protein containing the extracellular domain of the ASP-2 expressed by amastigotes of the G strain (G1, clone 62) was produced and used for development of specific antibodies. Subsequently, the recognition of recombinant proteins His65 kDa/Y strain and His-65 kDa/G strain (G1) by polyclonal and monoclonal antibodies generated against these proteins was evaluated by ELISA. As shown in Fig. 4A, polyclonal antibodies raised against the His-65 kDa/Y strain recognized the heterologous protein His-65 kDa/G strain with significantly less intensity when compared to the homologous recombinant protein. Conversely, the polyclonal antibody raised against His-65 kDa/G strain recognized better the homologous recombinant protein (His-65 kDa/G strain, Fig. 4B). The MAb K22 also recognized the heterologous protein His-65 kDa/G strain with significantly less intensity

C. Claser et al. / Microbes and Infection 9 (2007) 1011e1019

2

B

1

3

0.8

3.2

12.8

1

51.2

3.2

D

2

OD492

OD492

MAb K22

1

12.8

51.2

204.8

Antibody dilution, X103

Antibody dilution, X103 3

0.26

1.02

4.10

16.38

1.5

1.0

0.5

0.0

2

8

10

12

14

Days after infection

1

B 0.5

Antibody dilution, X103

2.0

Anti-Histidine tag

3

0

0

A 2

0

0

C

Anti-His-65kDa/G Strain

Parasitemia, X10 6

Anti-His-65kDa/Y Strain

2.0

8.0

32.0

Antibody dilution, X103

His-65kDa/Y strain His-65kDa/G Strain Fig. 4. Analysis of the presence of strain-variant and cross-reactive epitopes between recombinant proteins representing ASP-2 expressed by different strains of T. cruzi. ELISA was performed with recombinant proteins His65 kDa/Y strain or His-65 kDa/G strain as substrates. The antibodies used were: (A) polyclonal antibodies anti-His-65 kDa/Y strain; (B) polyclonal antibodies anti-His-65 kDa/G strain (G1 gene); (C) MAb K22; and (D) AntiHistidine tag antibodies.

100 80

Survival (%)

3

OD492

OD492

A

1017

60 40 20 0 20

40

60

80

Days after infection

when compared to the homologous recombinant protein (Fig. 4C). Control polyclonal antibodies to the Histidine tag recognized both recombinant proteins almost identically well (Fig. 4D). Although these results clearly indicated the presence of cross-reactive, as well as strain-specific epitopes, it failed to demonstrate that this polymorphism would have a significant impact on the immunity induced against ASP-2. To determine the importance of the cross-reactive or the strain-variant epitopes for anti-parasitic immunity, we vaccinated highly susceptible A/Sn mice with eukaryotic expression vectors containing asp-2 genes of the Y strain (pIgSP-clone9), the CL-Brener strain (pIgSP-clone50), or the G strain (pIgSP-clone62, gene G1). Control mice received the vector pcDNA3. After immunization, mice were challenged with bloodstream trypomastigotes of Y strain. In mice immunized with plasmids pIgSP-clone9 (Y strain) or pIgSP-clone50 (CL-Brener strain), we observed a significant reduction in the peak parasitemia when compared with animals immunized with pcDNA3 (P < 0.01). In contrast, mice immunized with pIgSP-clone62 (G strain, G1 gene) did not show any reduction in the peak parasitemia when compared to control mice (Fig. 5A). Control mice injected with pcDNA3 died within 16e 25 days of infection (Fig. 5B). Animals immunized with pIgSP-clone62 (G strain, G1 gene) survived significantly longer than control mice (P < 0.0001). Nevertheless, 93.34% of these mice died. Mice immunized with plasmids pIgSP-clone9

pcDNA3 pIgSP-clone9 (Y strain) pIgSP-clone50 (CL-Brener strain) pIgSP-clone62 (G strain)

Fig. 5. Trypomastigote-induced parasitemia and mortality in A/Sn mice immunized with plasmids containing asp-2 genes expressed in different parasite strains. A/Sn mice were immunized with plasmids pIgSP-clone9 (Y strain), pIgSP-clone50 (CL-Brener strain), pIgSP-clone62 (G strain, G1 gene) or pcDNA3. Ten days after the last immunization, mice were challenged i.p. with 250 bloodstream trypomastigotes of the Y strain. (A) Course of infection, estimated by the number of trypomastigotes per mL of blood. Results represent the mean values  SD obtained from mice immunized with pIgSP-clone9 (n ¼ 7), pIgSP-clone50 (n ¼ 8), pIgSP-clone62 (n ¼ 8) or pcDNA3 (n ¼ 7). At the peak of infection (day 11), the parasitemia of mice immunized with each plasmid was compared by One-way Anova and Tukey HSD tests. The peak parasitemias of mice immunized with pIgSP-clone9 or pIgSP-clone50 were lower than mice immunized with pcDNA3 or pIgSP-clone62 (P < 0.05 in all cases). No significant differences were detected when we compared the peak parasitemias of mice immunized with pcDNA3 or pIgSP-clone62. Similarly, the peak parasitemia of mice immunized with pIgSP-clone9 or pIgSP-clone50 were not different. The results of the comparisons were as follows: (i) pcDNA3 X pIgSP-clone9 (P < 0.05); (ii) pcDNA3 X pIgSP-clone50 (P < 0.01); (iii) pcDNA3 X pIgSP-clone62 (Non-significant, NS); (iv) pIgSPclone9 X pIgSP-clone50 (NS); (vi) pIgSP-clone9 X pIgSP-clone62 (P < 0.01); and (vii) pIgSP-clone50 X pIgSP-clone62 (P < 0.01). (B) KaplaneMeier curves for survival of mice immunized with pIgSP-clone9 (n ¼ 14), pIgSPclone50 (n ¼ 15), pIgSP-clone62 (n ¼ 15) or pcDNA3 (n ¼ 13). Statistical analyses were performed using LogRank test. The results of the comparison were as follows: (i) pcDNA3 X pIgSP-clone9 (P < 0.0001); (ii) pcDNA3 X pIgSP-clone50 (P < 0.0001); (iii) pcDNA3 X pIgSP-clone62 (P < 0.001); (iv) pIgSP-clone9 X pIgSP-clone50 (NS); (v) pIgSP-clone9 X pIgSP-clone62 (P ¼ 0.0045); and (vi) pIgSP-clone50 X pIgSP-clone62 (P ¼ 0.020).

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(Y strain) or pIgSP-clone50 (CL-Brener strain) survived longer than those immunized with pIgSP-clone62 (G strain, G1 gene) or pcDNA3 (P < 0.05 in all cases). Statistical comparison showed no significant difference between groups of mice vaccinated with pIgSP-clone9 (Y strain) or pIgSP-clone50 (CL-Brener strain). 4. Discussion Initially, we found that amastigotes of all strains that we tested expressed an 83 kDa protein containing epitopes cross-reactive with the previously characterized ASP-2 of the Y strain of T. cruzi (Fig. 2). Analysis of the predicted AA sequences of the genes isolated from the cDNA of intracellular amastigotes of CL-Brener, Tulahuen, Colombian and G strains indicated that these parasites expressed a similar isoform of ASP-2. The high degree of conservation between the ASP-2 of these different strains of T. cruzi is compatible with the hypothesis of its involvement in a relevant biological function for parasite survival. Because these strains belong to the phylogenetically distinct T. cruzi I (Tulahuen, Colombian and G), T. cruzi II (Y and Brazil), or a hybrid between the two (CL-Brener), our results failed to support any lineagerelated polymorphism [16]. When using polyclonal and monoclonal antibodies, we detected an 83 kDa molecule in amastigotes of the Dm28c strain (Fig. 2). However, all the 35 cDNA genes that we isolated from intra or extracellular amastigotes had premature stop codons. The exact reason for our failure to find an ORF is still unknown. Nevertheless, the nucleotide sequence of the genes that we sequenced had a high degree of identity to the genes of Tulahuen, Colombian, CL-Brener, Y, G, and Brazil strains (Table 1). In contrast to most strains, Sylvio X10/4 and G strains expressed an ASP-2 isoform with a low degree of identity at the predicted AA level with the previously described ASP-2. These strains differ widely in their biological behavior in experimental hosts and usually cause only sub-patent infection in immuno-competent hosts [17e20]. Because we found a new isoform of ASP-2, the second objective of our study was to determine the immunological significance of this strain polymorphism. For this purpose, we used a recombinant protein representing the ASP-2 of the G strain (His-65 kDa/G strain, G1 gene) and confirmed the presence of both cross-reactive and strain-variant epitopes recognized by murine polyclonal and monoclonal antibodies (Fig. 4). Finally, we evaluated the presence of cross-reactive and strain-variant protective epitopes. For this purpose, we vaccinated A/Sn mice with plasmids containing the asp-2 genes of the Y, CL-Brener or G (G1 gene) strains. In this experimental model, we had previously shown that protective immunity is mediated by CD4þ Th1 and CD8þ Tc1 cells [14]. Asp-2 genes from the Y and CL-Brener strains share 83.5% identity at the predicted AA level. The results of these vaccination studies showed that both genes elicited similar degrees of protective immunity after a lethal challenge with trypomastigotes of the Y strain. In

contrast, mice immunized with a plasmid containing the asp-2 gene of the G strain (G1 gene), which shares only 44.2% identity at the predicted AA sequences with the asp-2 gene of the Y strain, provided a significantly lower degree of protective immunity (Fig. 5). These results suggest that strain-variant epitopes are critical for mouse survival after the challenge of A/Sn mice by parasites of the Y strain of T. cruzi. Which are these strain-variant protective epitopes, precisely? Protective CD8þT cells from immunized or infected A/Sn (H-2a) mice recognize the epitope TEWETGQI [10,15]. This epitope is conserved between ASP-2 of Y and CL-Brener strains, a fact that may help to explain the similar degree of protective immunity provided by these two genes. In contrast, in the asp-2 gene of the G strain, the predicted AA sequence is AEWENREL. We speculate that due to this drastic change, there would be little or no cross-protective immunity. This hypothesis agrees with recent observations that CD8þT-cell responses to T. cruzi are focused on strain-variant epitopes encoded by members of the trans-sialidase gene family [21]. Protective CD4þT-cell epitopes of ASP-2 recognized by A/ Sn mice T cells are yet to be found. So far, only a single epitope recognized by protective CD4þT cells of BALB/c (H-2d) mice has been identified in T. cruzi (SHNFTLVASVIIEE APSGNT) and expressed in the protein SA85.11, a member of the trans-sialidase family of T. cruzi. This epitope contains a number of variant peptides in parasites of the same strain which may serve as antagonists modulating the specific immune response [22e24]. Based on this evidence, we can hypothesize that CD4þT-cell epitopes present in the ASP-2 of G strain are either different or perhaps act as antagonistic for epitopes of the ASP-2 of Y strain. Because very little is known about protective CD4þT-cell epitopes of T. cruzi, our experimental system may provide an interesting model in which to study them. Unfortunately, vaccination experiments followed by infection of mice with Sylvio X10/4 or G strains could not be performed because these parasites failed to cause parasitemia and mouse mortality after challenge. We are currently trying to find a more virulent strain of T. cruzi which expresses the isoform of ASP-2 similar to Sylvio X10/4 and G strains that would allow us to perform experiments for vaccination and challenge. Previous studies have shown the presence of strain-variant epitopes encoded by members of the glycosylphosphatidyl inositol-anchored mucin-like protein gene family of T. cruzi [25]. These strain-variant epitopes were restricted to the central region of the molecule, allowed discrimination between T. cruzi lineages I or II, and only one of the variants was recognized by chagasic human antibodies [25]. However, whether these strain-variant epitopes were in fact recognized by protective antibodies has not yet been determined. Very recently, strain-variant epitopes recognized by CD8þT cells have also been described in antigens encoded by members of the trans-sialidase gene family [21]. Considering the importance of CD8þT cells for mouse resistance to infection, it is very likely that these epitopes are also critical for host immune-protection.

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