Vaccine 27 (2009) 2870–2876 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Immunogenicity and protection-inducing ability of recombinant Plasmodium vivax rhoptry-associated protein 2 in Aotus monkeys: A potential vaccine candidate Jose Rojas-Caraballo a , Alvaro Mongui a,b , Manuel A. Giraldo a,b , Gabriela Delgado c , Diana Granados c , Diana Millan-Cortes a,b , Paola Martinez a,b , Raul Rodriguez a,b , Manuel A. Patarroyo a,b,∗ a Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá, Colombia b Universidad del Rosario, Bogotá, Colombia c Universidad Nacional de Colombia, Bogotá, Colombia article info Article history: Received 10 October 2008 Received in revised form 19 February 2009 Accepted 24 February 2009 Available online 9 March 2009 Keywords: Plasmodium vivax Rhoptry-associated protein Immune response Vaccine candidate abstract Rhoptry proteins have been extensively shown to be important in invasion and parasitophorous vacuole (PV) formation. This work evaluates the immunogenicity and protective efficacy of Plasmodium vivax RAP2 in the non-human Aotus primate model, when expressed as a recombinant molecule in E. coli and formulated in Freund and Alum hydroxide adjuvants. Our results show that rPvRAP2 is immunogenic in both formulations, finding a trend of higher cytokine levels in immunized monkeys, specially in IL- 4 levels (using Freund’s adjuvant) and IL-5 (using Alum hydroxide). RAP2 is suggested as a P. vivax- vaccine candidate since immunized monkeys exhibited lower parasitemias than control groups after being experimentally challenged with the P. vivax VCG-I strain. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Malaria is one of the most prevalent parasitic diseases among tropical countries. Every year nearly 500 million new cases are reported worldwide, of which an estimated 1–2 million result in death [1]. Epidemiologically speaking, Plasmodium vivax is consid- ered as the second most important malarial parasite species, as it accounts for more than 75 million annual cases of malaria occurring mainly in Asia, Central and South America [2,3]. Despite the great efforts in developing an effective strategy for the control of this scourging disease, malaria remains a public health threat worldwide [4]. One of the most appealing strategies for eradicating this disease is the development of a vaccine. Nev- ertheless, obtaining a fully effective vaccine has been hindered by Abbreviations: RAP2, rhoptry-associated protein 2; rPvRAP2, recombinant rhoptry-associated protein 2; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; VCG-I, Vivax Colombia Guaviare I; PHA, phytohemagglutinin; RBCs, red blood cells; iRBCs, infected red blood cells. ∗ Corresponding author at: Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50 # 26-20, Bogotá, Colombia. Tel.: +57 1 3244672x143; fax: +57 1 4815269. E-mail address: mapatarr@fidic.org.co (M.A. Patarroyo). the great diversity of surface proteins expressed throughout the parasite’s life cycle, its polymorphism and the fact that immunity induced by a natural infection is gradually acquired and species- , stage- and strain-specific [5–7]. Aiming at developing a vaccine against P. falciparum, several research groups have characterized new antigens in this species, among which the rhoptry-associated protein 2 (RAP2) has long been considered a major vaccine candi- date due to its ability to induce antibodies capable of inhibiting in vitro invasion of red blood cells (RBCs) [8], its low degree of genetic polymorphism [9], its binding ability to RBCs [10], its recog- nition by sera from patients living in endemic areas [11] and, more importantly, its capacity to confer protection against experimen- tal challenge with P. falciparum in Saimiri boliviensis monkeys [12]. All this evidence has encouraged its recent characterization in P. vivax [13], even though its role in RBCs invasion is still not clearly elucidated in this parasite species. Diverse components of the immune system are involved in the response against Plasmodium, depending on the parasite’s life stage. In the liver stage, specific parasite antigens are recog- nized by Interferon- (IFN-)-secreting CD8+ T lymphocytes [14]. IFN--activated macrophages display a direct antiparasitic effect by secreting nitric oxide (NO) through an inducible nitric oxide synthase (iNOS) [15]. Regarding the erythrocytic stage, antibod- ies directed against merozoite surface antigens are able to block RBC invasion and/or facilitate phagocytosis [16,17]. Both Th1 and 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.02.083