Protective Immunity Against<I> Trypanosoma cruzi</I> Infection in a Highly Susceptible Mouse Strain After Vaccination with Genes Encoding the Amastigote Surface Protein-2 and<I> Trans</I>-Sialidase

ABSTRACT We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethal Trypanosoma cruzi infection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathione S-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8+ T cells, but not CD4+ T cells, and was associated with the presence of CD8+ T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8+-T-cell-dependent protective immunity against T. cruzi infection.

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Immunization with cDNA Expressed by Amastigotes of Trypanosoma cruzi Elicits Protective Immune Response against Experimental Infection

Infection and Immunity ◽

10.1128/iai.71.5.2744-2757.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2744-2757 ◽

Cited By ~ 48

Author(s):

Silvia B. Boscardin ◽

Sheila S. Kinoshita ◽

Adriana E. Fujimura ◽

Mauricio M. Rodrigues

Keyword(s):

Trypanosoma Cruzi ◽

Vaccine Development ◽

Surface Protein ◽

Dna Vaccination ◽

Eukaryotic Expression ◽

Host Cells ◽

Specific Antibodies ◽

C Terminus ◽

Lethal Infection ◽

Genes Encoding

ABSTRACT Immunization of mice with plasmids containing Trypanosoma cruzi genes induced specific antibodies, CD4+ Th1 and CD8+ Tc1 cells, and protective immunity against infection. In most cases, plasmids used for DNA vaccination contained genes encoding antigens expressed by trypomastigotes, the nonreplicative forms of the parasite. In this study, we explored the possibility of using genes expressed by amastigotes, the form of the parasite which replicates inside host cells, for experimental DNA vaccination. For that purpose, we selected a gene related to the amastigote surface protein 2 (ASP-2), an antigen recognized by antibodies and T cells from infected mice and humans, for our study. Using primers specific for the asp-2 gene, four distinct groups of genes were amplified from cDNA from amastigotes of the Y strain of T. cruzi. At the nucleotide level, they shared 82.3 to 89.9% identity with the previously described asp-2 gene. A gene named clone 9 presented the highest degree of identity with the asp-2 gene and was selected for immunological studies. Polyclonal antisera raised against the C terminus of the recombinant protein expressed by the clone 9 gene reacted with an antigen of approximately 83 kDa expressed in amastigotes of T. cruzi. Immunization of BALB/c mice with eukaryotic expression plasmids containing the clone 9 gene elicited specific antibodies and CD4+ T-cell-dependent gamma interferon secretion. Upon challenge with trypomastigotes, mice immunized with plasmids harboring the clone 9 gene displayed reduced parasitemia and survived lethal infection. We concluded that amastigote cDNA is an interesting source of antigens that can be used for immunological studies, as well as for vaccine development.

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Novel Protective Antigens Expressed by Trypanosoma cruzi Amastigotes Provide Immunity to Mice Highly Susceptible to Chagas' Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00142-08 ◽

2008 ◽

Vol 15 (8) ◽

pp. 1292-1300 ◽

Cited By ~ 17

Author(s):

Eduardo L. V. Silveira ◽

Carla Claser ◽

Filipe A. B. Haolla ◽

Luiz G. Zanella ◽

Mauricio M. Rodrigues

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Protective Immunity ◽

Protective Antigen ◽

Surface Protein ◽

Cross Reactivity ◽

Eukaryotic Expression ◽

Lethal Challenge ◽

Reading Frame ◽

Antibody Recognition

ABSTRACT Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease.

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Efficient protective immunity against Trypanosoma cruzi infection after nasal vaccination with recombinant Sendai virus vector expressing amastigote surface protein-2

Vaccine ◽

10.1016/j.vaccine.2009.08.026 ◽

2009 ◽

Vol 27 (44) ◽

pp. 6154-6159 ◽

Cited By ~ 16

Author(s):

Xuefeng Duan ◽

Yoshikazu Yonemitsu ◽

Bin Chou ◽

Kumi Yoshida ◽

Sakura Tanaka ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Protective Immunity ◽

Sendai Virus ◽

Surface Protein ◽

Virus Vector ◽

Nasal Vaccination ◽

Cruzi Infection

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Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

Biochemical Journal ◽

10.1042/bj20061049 ◽

2006 ◽

Vol 401 (1) ◽

pp. 279-285 ◽

Cited By ~ 30

Author(s):

Ana L. Stern ◽

Emmanuel Burgos ◽

Laurent Salmon ◽

Juan J. Cazzulo

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Pentose Phosphate ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Kinetic Properties ◽

Type B ◽

Nucleotide Synthesis ◽

Gene Coding ◽

Genes Encoding

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

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Intranasal immunization with yeast‐expressed 19 kD carboxyl‐terminal fragment ofPlasmodium yoeliimerozoite surface protein‐1 (yMSP119) induces protective immunity to blood stage malaria infection in mice

Parasite Immunology ◽

10.1046/j.1365-3024.1998.00161.x ◽

1998 ◽

Vol 20 (9) ◽

pp. 413-420 ◽

Cited By ~ 37

Author(s):

CHAKRIT HIRUNPETCHARAT ◽

DANIELLE STANISIC ◽

XUE QIN LIU ◽

JIM VADOLAS ◽

RICHARD A. STRUGNELL ◽

...

Keyword(s):

Malaria Infection ◽

Protective Immunity ◽

Surface Protein ◽

Blood Stage ◽

Intranasal Immunization ◽

Terminal Fragment ◽

Carboxyl Terminal ◽

Blood Stage Malaria

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CD4+ T Lymphocytes from Calves Immunized withAnaplasma marginale Major Surface Protein 1 (MSP1), a Heteromeric Complex of MSP1a and MSP1b, Preferentially Recognize the MSP1a Carboxyl Terminus That Is Conserved among Strains

Infection and Immunity ◽

10.1128/iai.69.11.6853-6862.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6853-6862 ◽

Cited By ~ 50

Author(s):

Wendy C. Brown ◽

Guy H. Palmer ◽

Harris A. Lewin ◽

Travis C. McGuire

Keyword(s):

T Lymphocytes ◽

T Lymphocyte ◽

Protective Immunity ◽

B Lymphocyte ◽

Surface Protein ◽

Specific Response ◽

Major Surface Protein ◽

Ifn Γ ◽

Major Surface ◽

Lymphocyte Responses

ABSTRACT Native major surface protein 1 (MSP1) of the ehrlichial pathogenAnaplasma marginale induces protective immunity in calves challenged with homologous and heterologous strains. MSP1 is a heteromeric complex of a single MSP1a protein covalently associated with MSP1b polypeptides, of which at least two (designated MSP1F1 and MSP1F3) in the Florida strain are expressed. Immunization with recombinant MSP1a and MSP1b alone or in combination fails to provide protection. The protective immunity in calves immunized with native MSP1 is associated with the development of opsonizing and neutralizing antibodies, but CD4+ T-lymphocyte responses have not been evaluated. CD4+ T lymphocytes participate in protective immunity to ehrlichial pathogens through production of gamma interferon (IFN-γ), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine for A. marginaleand related organisms should contain both T- and B-lymphocyte epitopes that induce a strong memory response that can be recalled upon challenge with homologous and heterologous strains. This study was designed to determine the relative contributions of MSP1a and MSP1b proteins, which contain both variant and conserved amino acid sequences, in stimulating memory CD4+ T-lymphocyte responses in calves immunized with native MSP1. Peripheral blood mononuclear cells and CD4+ T-cell lines from MSP1-immunized calves proliferated vigorously in response to the immunizing strain (Florida) and heterologous strains of A. marginale. The conserved MSP1-specific response was preferentially directed to the carboxyl-terminal region of MSP1a, which stimulated high levels of IFN-γ production by CD4+ T cells. In contrast, there was either weak or no recognition of MSP1b proteins. Paradoxically, all calves developed high titers of IgG antibodies to both MSP1a and MSP1b polypeptides. These findings suggest that in calves immunized with MSP1 heteromeric complex, MSP1a-specific T lymphocytes may provide help to MSP1b-specific B lymphocytes. The data provide a basis for determining whether selected MSP1a CD4+ T-lymphocyte epitopes and selected MSP1a and MSP1b B-lymphocyte epitopes presented on the same molecule can stimulate a protective immune response.

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