Vaccine Strain-Specificity of Protective HLA-Restricted Class 1 P. falciparum Epitopes

RESEARCH ARTICLE Vaccine Strain-Specificity of Protective HLARestricted Class 1 P. falciparum Epitopes Martha Sedegah1, Bjoern Peters2, Michael R. Hollingdale1,3*, Harini D. Ganeshan1,3, Jun Huang1,3, Fouzia Farooq1,3, Maria N. Belmonte1,3, Arnel D. Belmonte1,3, Keith J. Limbach1,3, Carter Diggs4, Lorraine Soisson4, Ilin Chuang1, Eileen D. Villasante1 1 Malaria Department, Naval Medical Research Center, Silver Spring, MD, 20910, United States of America, 2 La Jolla Institute for Allergy and Immunology, La Jolla, CA, 92037, United States of America, 3 Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, MD, 20817, United States of America, 4 USAID, Washington, DC, 20523, United States of America a11111 * Abstract OPEN ACCESS Citation: Sedegah M, Peters B, Hollingdale MR, Ganeshan HD, Huang J, Farooq F, et al. (2016) Vaccine Strain-Specificity of Protective HLARestricted Class 1 P. falciparum Epitopes. PLoS ONE 11(10): e0163026. doi:10.1371/journal. pone.0163026 Editor: Adrian J.F. Luty, Institut de recherche pour le developpement, FRANCE Received: May 13, 2016 Accepted: September 1, 2016 Published: October 3, 2016 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. A DNA prime/adenovirus boost malaria vaccine encoding Plasmodium falciparum strain 3D7 CSP and AMA1 elicited sterile clinical protection associated with CD8+ T cell interferon-gamma (IFN-γ) cells responses directed to HLA class 1-restricted AMA1 epitopes of the vaccine strain 3D7. Since a highly effective malaria vaccine must be broadly protective against multiple P. falciparum strains, we compared these AMA1 epitopes of two P. falciparum strains (7G8 and 3D7), which differ by single amino acid substitutions, in their ability to recall CD8+ T cell activities using ELISpot and flow cytometry/intracellular staining assays. The 7G8 variant peptides did not recall 3D7 vaccine-induced CD8+ T IFN-γ cell responses in these assays, suggesting that protection may be limited to the vaccine strain. The predicted MHC binding affinities of the 7G8 variant epitopes were similar to the 3D7 epitopes, suggesting that the amino acid substitutions of the 7G8 variants may have interfered with TCR recognition of the MHC:peptide complex or that the 7G8 variant may have acted as an altered peptide ligand. These results stress the importance of functional assays in defining protective epitopes. Clinical Trials Registrations: NCT00870987, NCT00392015 Data Availability Statement: All relevant data are within the paper. Introduction Funding: This study was supported by the United States Agency for International Development "Development of Adenovirus-Vectored Malaria Vaccines" interagency agreement # GHA-P-00-0300006-01, project number 936–3118, www.usaid. gov, recipient EV; Congressionally Directed Medical Research Program (https://cdmrp.org) "Development of Recombinant Adenoviral-based Vaccines against Malaria", grant # W81XWH-05-20041, recipient EV; Military Infectious Research Recently we demonstrated that a heterologous DNA-prime/human adenovirus 5 (HuAd5) boost vaccine encoding two Plasmodium falciparum 3D7 strain antigens, circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), induced sterile protection against controlled human malaria infection (CHMI) in four of 15 immunized subjects [1]. Protection was associated with ELISpot and CD8+ T cell interferon-gamma (IFN-γ) responses to AMA1 using peripheral blood mononuclear cells (PBMC) taken just prior to CHMI [1]. Without DNApriming the HuAd5 vaccine alone did not elicit sterile protection, but often elicited similar or higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1 than the protected subjects [2]. PLOS ONE | DOI:10.1371/journal.pone.0163026 October 3, 2016 1 / 14 Malaria Vaccine T-Epitope Polymorphism Program "Phase 1/2a clinical trials assessing the safety, tolerability, immunogenicity & protective efficacy of Ad5-CA, a two-antigen, adenovirusvectored Plasmodium falciparum malaria vaccine, in healthy, malaria-naive adults", work unit number 62787A 870 F 1432, https://midrp.amedd.army. mil, recipient EV. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. We sought to further investigate these differences and found that quality rather than quantity of CD8+ T cell responses were crucial. We used 12 peptide pools containing 15mer peptides spanning the entire sequence of AMA1, and found that responses of three of the four protected subjects were narrowly focused on discrete regions of AMA1 represented by single peptide pools, designated Ap8 and Ap10, whereas responses of non-protected subjects from the DNA/HuAd5 and HuAd5 alone trials were more broadly reactive to multiple regions of AMA1 [3]. Activities of protected subjects to Ap8 or Ap10 represented a higher percent of the total response to all peptide pools than non-protected subjects to Ap8 or Ap10 [3]. We suggested that these focused responses were genetically-restricted as the protected subjects recognized single 15mer peptides within Ap8 and Ap10, and these 15mers also recalled ELISpot and CD8+ T cell IFN-γ responses from these subjects. The NetMHC [4] algorithm which predicts peptide binding to MHC class I molecules in terms of 50% inhibitory concentration (IC50) values was used to predict putative 3D7 AMA1 class I epitopes within these 15mers. Experimental testing of peptides representing these predicted epitopes indeed recalled T cell responses from protected subjects. Ap10 contained the predicted HLA A 11:01-restricted epitope NSTCRFFVCK that recalled responses from one DNA/Ad-protected subject (v11) expressing HLA A 11:01 (HLA supertype A 03), and Ap8 contained the predicted HLA B 57:01/B 58:01 epitope KSHGKGYNW that recalled responses from the two DNA/Ad-protected subjects (v10 and v18) expressing HLA B 57:01 or B 58:01 (HLA B 58 supertype) [3]. Among the non-protected subjects in the HuAd5 trial, one subject (v194) who expressed HLA B 58:01 and recognized the B 58 epitope showed a significant delay to patency (suggesting reduction in numbers of liver stage parasites), suggesting partial protection [3]. The fine specificity of the HLA expressed by protected and non-protected subjects was crucial as two non-protected subjects (v135, and v179) from the HuAd5 alone trial also strongly recognized the same protection associated epitope within Ap8 but expressed A 32:01 (HLA supertype A 01), and two non-protected subjects (v126, and v172) from the HuAd5 trial recognized the same protection associated epitope within Ap10 but expressed HLA A 30:01 or A 03:01 (HLA A (...truncated)