Contents lists available at ScienceDirect Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis João A. Oliveira-da-Silva a , Amanda S. Machado a , Fernanda F. Ramos a , Grasiele S.V. Tavares a , Daniela P. Lage a , Débora V.C. Mendonça a , Isabela A.G. Pereira a , Thaís T.O. Santos a , Vívian T. Martins a , Lívia M. Carvalho b , Camila S. Freitas a , Fernanda Ludolf a , Thiago A.R. Reis a , Raquel S. Bandeira a , Alessandra M. Silva a , Lourena E. Costa a , Jamil S. Oliveira c , Mariana C. Duarte a,d , Bruno M. Roatt b , Antônio L. Teixeira a,e , Eduardo A.F. Coelho a,d, ⁎ a 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 b Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas/NUPEB, Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil c Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil d Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil e Neuropsychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA ARTICLE INFO Keywords: Visceral leishmaniasis Hypothetical proteins Vaccine Immune response Amastigote antigens ABSTRACT Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote- expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and para- sitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8 + T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4 + T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be con- sidered in future studies as a vaccine candidate to protect against VL. 1. Introduction Leishmaniasis is a vector-borne tropical disease caused by ap- proximately 20 distinct species of protozoan parasites of the Leishmania genus. This neglected disease complex is a major health problem in several countries around the world, with approximately 380 million people at risk of contracting infection and 2.0 million cases registered annually [41]. This vector-borne disease complex is caused by parasites https://doi.org/10.1016/j.cellimm.2020.104194 Received 24 April 2020; Received in revised form 13 July 2020; Accepted 7 August 2020 ⁎ Corresponding author at: Laboratório de Pesquisa do Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Avenida Prof. Alfredo Balena, 190, 30.130-100, Belo Horizonte, Minas Gerais, Brazil. E-mail address: eduardoferrazcoelho@yahoo.com.br (E.A.F. Coelho). Cellular Immunology 356 (2020) 104194 Available online 13 August 2020 0008-8749/ © 2020 Elsevier Inc. All rights reserved. T