Vaccination Using Recombinants Influenza and Adenoviruses Encoding Amastigote Surface Protein-2 Are Highly Effective on Protection against Trypanosoma cruzi Infection

In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzis amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). the CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-alpha and IFN-gamma and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease.FIOCRUZ/PDTIS-VacinasConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Microbiol, Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Morfol, Belo Horizonte, MG, BrazilFiocruz MS, Ctr Pesquisas Rene Rachou, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, São Paulo, BrazilUniv Massachusetts, Sch Med, Dept Med, Div Infect Dis & Immunol, Worcester, MA USAUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, São Paulo, BrazilCNPq: 015/2008CNPq: 064/2008Web of Scienc

Altered cardiomyocyte function and trypanosoma cruzi persistence in chagas disease

Chagas disease, caused by the triatominae Trypanosoma cruzi, is one of the leading causes of heart malfunctioning in Latin America. The cardiac phenotype is observed in 20-30% of infected people 10-40 years after their primary infection. The cardiac complications during Chagas disease range from cardiac arrhythmias to heart failure, with important involvement of the right ventricle. Interestingly, no studies have evaluated the electrical properties of right ventricle myocytes during Chagas disease and correlated them to parasite persistence. Taking advantage of a murine model of Chagas disease, we studied the histological and electrical properties of right ventricle in acute (30 days postinfection [dpi]) and chronic phases (90 dpi) of infected mice with the Colombian strain of T. cruzi and their correlation to parasite persistence. We observed an increase in collagen deposition and inflammatory infiltrate at both 30 and 90 dpi. Furthermore, using reverse transcriptase polymerase chain reaction, we detected parasites at 90 dpi in right and left ventricles. In addition, we observed action potential prolongation and reduced transient outward K+ current and L-type Ca2+ current at 30 and 90 dpi. Taking together, our results demonstrate that T. cruzi infection leads to important modifications in electrical properties associated with inflammatory infiltrate and parasite persistence in mice right ventricle, suggesting a causal role between inflammation, parasite persistence, and altered cardiomyocyte function in Chagas disease. Thus, arrhythmias observed in Chagas disease may be partially related to altered electrical function in right ventricle.CNPq [404353/2012-6]FAPESP [2014/09861-1]Universidade Federal de Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, Minas Gerais, BrazilCentro de Pesquisas René Rachou, Fundação Oswaldo Cruz (FIOCRUZ), Lab Parasitol Celular & Mol, Belo Horizonte, Minas Gerais, BrazilEscola Paulista de Medicina, Universidade Federal de São Paulo, Departamento de Biofisica, São Paulo, BrazilUniversidade Federal de Minas Gerais, Inst Ciencias Biol, Dept Patol Geral, Belo Horizonte, Minas Gerais, BrazilDepartamento de Biofisica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 862, Vila Clementino, São Paulo, São Paulo, Brazil, CEP 04023062CNPq: 404353/2012-6FAPESP: 2014/09861-1Web of Scienc

Vaccination Using Recombinants Influenza and Adenoviruses Encoding Amastigote Surface Protein-2 Are Highly Effective on Protection against Trypanosoma cruzi Infection

In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzi ´s amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). The CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-α and IFN-γ and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease.status: publishe

Percentage of effector CD8+ T cells in splenocytes of immunized mice.

<p>Percentage of effector CD8+ T cells in splenocytes of immunized mice.</p

Cellular responses to immunodominant epitopes from ASP2 in mice immunized with recombinant viruses.

<p>C57BL/6 and C3H/He mice were immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061795#s2" target="_blank">Material and Methods</a>. Three weeks after the boost immunization, the presence of ASP-2 specific IFN-γ producing T cells in spleen cells of C57BL/6 (A) or C3H/He (B) mice were assessed by ELISPOT and culture supernatant ELISA (n = 8). To this aim, the spleen cells of individual mice were stimulated 18 hours (ELISPOT) or 72 hours (ELISA) <i>ex vivo</i> with VNHRFTLV (aa 553–560; for C57BL/6) or TEWETGQI (aa 320–327; for C3H/He) specific ASP2 peptides. Optical Density (OD) was measured at 450 nm.</p

Immunization Schedule and Induction of specific anti-ASP2 humoral immune response in mice vaccinated with recombinant viruses.

<p>Timeline representation of immunization schedule and experimental procedures (A). C57BL/6 mice were immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061795#s2" target="_blank">Material and Methods</a>. Two weeks after the boost immunization, the animals were bled and the presence of specific anti-ASP2 total IgG antibodies in mice sera was evaluated by western by incubating individual (lanes 1–9) or pooled (lanes 10 and 11) sera of C57BL/6 mice with nitrocellulose membranes loaded with recombinant ASP2 protein (His65KDa) as capture antigen blot (B). Alternatively, the antibodies levels were measured by ELISA using individual sera of C5BL/6 (C) or C3H/He (D) mice sera diluted 1∶100 and recombinant ASP2 protein as capture antigen. Optical Density (OD) was measured at 450 nm.</p

Phenotype of anti-ASP2 specific CD8+ T cells elicited by vaccination with recombinant viruses.

<p>C3H/He mice were immunized with recombinant viruses as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061795#s2" target="_blank">Material and Methods</a>. Two weeks after the last immunization, the spleen cells were harvested and cultivated <i>ex vivo</i> with specific TEWETGQI CD8+ T peptide and incubated with anti-CD3, anti-CD8, permeabilized and fixed and stained with anti-CD107a, anti-IFN-γ and anti-TNF-α antibodies and assessed by flow cytometry. Percentage of effector CD8+ T cells reacting to the presence of TEWETGQI peptide obtained from spleen cells of mice immunized with recombinant viruses (A). Percentage of CD8 T cells which produces IFN-γ or/and TNF-α or/and mobilizes the degranulation marker CD107a after stimulation with TEWETGQI (B), the statistics depicted are compared to groups of mice immunized with control recombinant viruses. The number and frequency of dextramer positive CD8+ T found in 3×10⁴ CD8+ T (C). Functional profile of CD8+ T cells subpopulations obtained from mice immunized with recombinant viruses (D). Response were depicted with different color patterns according to the number of assessed functions (IFN-γ, TNF-α and CD107a) displayed by each dextramer negative or dextramer positive CD8+ T cells subpopulations.</p

Construction and characterization of recombinant influenza viruses.

<p>Schematic representation of primary sequence of Amastigote Surface Protein 2 and its corresponding moieties, highlighting the mapped CD8 T cells epitopes (A). Schematic representation of the neuraminidase dicistronic segment. NA38 segment contains an A/WSN/33 (WSN) derived recombinant neuraminidase (NA) segment followed by a duplicated 3′non coding (NC) sequence, <i>Xho</i>I and <i>Nhe</i>I cloning sites, a duplication of the last 42 nucleotides of NA (dark box) and the original 5′NC sequence (28 nucleotides). The foreign sequences (open boxes) were cloned between <i>Xho</i>I and <i>Nhe</i>I cloning sites (B).The plaque phenotype of the wild type WSN and recombinant influenza viruses were assessed by standard agarose plaque assay in MDCK cells after 3 days of incubation at 35°C and 5% CO₂ (C).The NA segments of recombinant influenza viruses were analyzed by RT-PCR, using a set of primers that allows the amplification of the region containing the inserted foreign sequence. Corresponding plasmids DNAs were amplified in parallel as positive control. The amplified products were analyzes on a 1% agarose and visualized by ethidium bromide staining. The values depicted at the weight marker lane ar (D). W.M: weight marker; M: medial moiety of ASP2, C: carboxi-terminal moiety of ASP2; b.p.: base pairs.</p