In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

RESEARCH ARTICLE In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9 Rodrigo Nunes Rodrigues-da-Silva1, João Hermínio Martins da Silva2, Balwan Singh3, Jianlin Jiang3, Esmeralda V. S. Meyer4, Fátima Santos5, Dalma Maria Banic6, Alberto Moreno3,7, Mary R. Galinski3,7, Joseli Oliveira-Ferreira1*, Josué da Costa LimaJunior1* OPEN ACCESS Citation: Rodrigues-da-Silva RN, Martins da Silva JH, Singh B, Jiang J, Meyer EVS, Santos F, et al. (2016) In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9. PLoS ONE 11(1): e0146951. doi:10.1371/journal.pone.0146951 Editor: Érika Martins Braga, Universidade Federal de Minas Gerais, BRAZIL Received: August 13, 2015 Accepted: December 22, 2015 Published: January 20, 2016 Copyright: © 2016 Rodrigues-da-Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Brazilian National Research Council – CNPq/PAPES, (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Programa de Apoio Pesquisa Estratégica em Saúde) Fiocruz, the National Institute of Health (NIH Grant #RO1 1R01AI24710), and the Yerkes National Primate Research Center Base Grant (ORIP/OD P51OD011132) awarded by the National Center for Research Resources of the 1 Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ, Brazil, 2 Computational Modeling Group—FIOCRUZ-CE, Fortaleza, Brazil, 3 Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, GA, United States of America, 4 Environmental Health and Safety Office, Emory University, Atlanta, GA, United States of America, 5 National Health Foundation, Department of Entomology, Central Laboratory, Porto Velho, RO, Brazil, 6 Laboratory of Simulids and Onchocerciasis "Malaria and Onchocerciasis Research", Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 7 Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Emory University, Atlanta, GA, United States of America * (JCLJ); (JO-F) Abstract Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with PLOS ONE | DOI:10.1371/journal.pone.0146951 January 20, 2016 1 / 18 Identification and Validation of a Linear B-Cell Epitope in PvMSP-9 National Institutes of Health. JCLJ is recipient of a FAPERJ-APQ1 (E-26/111.248/2014) and CPNqUniversal research grants (445150/2014-9), JOF is recipient of CNPq Productivity Fellowship. Competing Interests: The authors have declared that no competing interests exist. the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines. Introduction Despite global investments in the control and elimination of malaria, the disease remains a major public health burden worldwide. According to the World Health Organization (WHO), more than 3 billion people are still at risk of infection, with an estimated 197 million of cases and 584 thousand deaths [1]. Among the species that infect humans Plasmodium falciparum and P. vivax are considered the two most important malaria parasites. Although P. falciparum is responsible for the major number of cases and deaths, especially in children, P. vivax is the most prevalent species outside the African continent [1]. Aside from the enormous socioeconomic impact caused by P. vivax prevalence [2], an increased number of publications reporting severe disease [3–8] and the emergence of strains resistant to chloroquine [9–11] and primaquine [12–14], make the development of a safe and affordable vaccine an important component in P. vivax control strategies. Although the epidemiological importance of P. vivax malaria worldwide is evident, the research on potential P. vivax vaccine candidates lags behind that on P. falciparum. Currently, there are only four P. vivax vaccine candidates or components in advanced preclinical studies and only one in clinical development, while 34 P. falciparum candidates are as listed in the WHO’s Malaria Vaccine Rainbow Tables [15]. These data show the continued global commitment to control and eliminate malaria with strategies that include vaccination, and highlight the specific need for identifying and testing additional vaccine candidates against P. vivax. Recent advances in adjuvant composition, delivery systems and the design of subunit vaccine constructs, support the use of synthetic peptides containing B and T-cell epitopes as a va (...truncated)