title: Application of rapid in vitro co-culture system of macrophages and T-cell subsets to assess the immunogenicity of dogs vaccinated with live attenuated Leishmania donovani centrin deleted parasites (LdCen-/-).
abstract: BACKGROUND
  Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge.
  
  
  METHODS
  Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines.
  
  
  CONCLUSIONS
  Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells.
  
  
  CONCLUSIONS
  The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.
authors.affiliation: Laboratory of Cell-Cell Interactions, Morphology Department, Institute of Biological Science, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais, 31270-901, Brazil.|Laboratory of Biomolecules and Vaccines, Agrarian Sciences and Technologic Department, Federal University of Tocantins, Gurupi, Tocantins, Brazil.
authors.forename: Kelvinson Fernandes
authors.initials: KF
authors.lastname: Viana
authors.affiliation: Laboratory of Cellular and Molecular Immunology, René Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte, MG, Brazil.
authors.forename: Jacqueline Araújo
authors.initials: JA
authors.lastname: Fiuza
authors.affiliation: Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD, 20993, USA.
authors.forename: Sreenivas
authors.initials: S
authors.lastname: Gannavaram
authors.affiliation: Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD, 20993, USA.
authors.forename: Ranadhir
authors.initials: R
authors.lastname: Dey
authors.affiliation: Institute of Molecular Medicine, 254 Okhla Industrial Estate, Phase III, New Delhi, 110020, India.
authors.forename: Angamuthu
authors.initials: A
authors.lastname: Selvapandiyan
authors.affiliation: Laboratory of Immunology and Genome of Parasites, Parasitology Department, Institute of Biological Science, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Daniella Castanheira
authors.initials: DC
authors.lastname: Bartholomeu
authors.affiliation: Laboratory of Diagnosis Biomarkers and Monitoring, René Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Denise
authors.initials: D
authors.lastname: da Silveira-Lemos
authors.affiliation: Laboratory of Immunology and Genome of Parasites, Parasitology Department, Institute of Biological Science, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Lilian Lacerda
authors.initials: LL
authors.lastname: Bueno
authors.affiliation: Laboratory of Cell-Cell Interactions, Morphology Department, Institute of Biological Science, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais, 31270-901, Brazil.
authors.forename: Walderez Ornelas
authors.initials: WO
authors.lastname: Dutra
authors.affiliation: Laboratory of Immunology and Genome of Parasites, Parasitology Department, Institute of Biological Science, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Ricardo Toshio
authors.initials: RT
authors.lastname: Fujiwara
authors.affiliation: Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD, 20993, USA.
authors.forename: Hira L
authors.initials: HL
authors.lastname: Nakhasi
authors.affiliation: Laboratory of Cell-Cell Interactions, Morphology Department, Institute of Biological Science, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, Minas Gerais, 31270-901, Brazil. giunchetti@gmail.com.
authors.forename: Rodolfo Cordeiro
authors.initials: RC
authors.lastname: Giunchetti
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology*; D018920:Coculture Techniques; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004283:Dog Diseases / Q000469:parasitology / Q000517:prevention & control*; D004285:Dogs; D005260:Female; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000276:immunology; D007893:Leishmania donovani / Q000276:immunology*; D054332:Leishmaniasis Vaccines / Q000276:immunology*; D008264:Macrophages / Q000502:physiology*; D008297:Male; D015662:Trimethoprim, Sulfamethoxazole Drug Combination / Q000378:metabolism*
chemical_list: D016207:Cytokines; D054332:Leishmaniasis Vaccines; D015662:Trimethoprim, Sulfamethoxazole Drug Combination
keywords: CD4+ and CD8+ T-cells; Canine visceral leishmaniasis LdCen−/−; In vitro co-culture; Leishmune®; Vaccine