title: Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis.
abstract: In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Vivian Tamietti
authors.initials: VT
authors.lastname: Martins
authors.affiliation: Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Chávez-Fumagalli
authors.affiliation: Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Daniela Pagliara
authors.initials: DP
authors.lastname: Lage
authors.affiliation: Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Mariana Costa
authors.initials: MC
authors.lastname: Duarte
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Garde
authors.affiliation: Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Lourena Emanuele
authors.initials: LE
authors.lastname: Costa
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Viviane Grazielle
authors.initials: VG
authors.lastname: da Silva
authors.forename: Viviane Gomes da
authors.initials: VG
authors.lastname: Silva
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Jamil Silvano
authors.initials: JS
authors.lastname: Oliveira
authors.affiliation: Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Danielle Ferreira de
authors.initials: DF
authors.lastname: Magalhães-Soares
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Santuza Maria Ribeiro
authors.initials: SM
authors.lastname: Teixeira
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Fernandes
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Soto
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Carlos Alberto Pereira
authors.initials: CA
authors.lastname: Tavares
authors.affiliation: Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Eduardo Antonio Ferraz
authors.initials: EA
authors.lastname: Coelho
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000627:therapeutic use*; D016207:Cytokines / Q000378:metabolism; D004285:Dogs; D005260:Female; D056724:Immunity, Humoral; D018314:Leishmania infantum / Q000254:growth & development / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000517:prevention & control*; D008807:Mice, Inbred BALB C; D009573:Nitrites / Q000378:metabolism; D059208:Parasite Load; D015800:Protozoan Proteins / Q000276:immunology*; D016052:Protozoan Vaccines / Q000627:therapeutic use*
chemical_list: D000953:Antigens, Protozoan; D016207:Cytokines; D009573:Nitrites; D015800:Protozoan Proteins; D016052:Protozoan Vaccines