title: Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP).
abstract: BACKGROUND
  Plasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen.
  
  
  METHODS
  Limited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence.
  
  
  RESULTS
  Each of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A24/A24) and one 9mer was restricted by three HLA supertypes (A01A24/A24/B27) indicating that some CSP class I-restricted epitopes, like DR epitopes, may be HLA-promiscuous.
  
  
  CONCLUSIONS
  This study identified nine and confirmed five novel class I epitopes restricted by six HLA supertypes, suggesting that an adenovirus-vectored CSP vaccine would be immunogenic and potentially protective in genetically diverse populations.
authors.affiliation: US Military Malaria Vaccine Program, Naval Medical Research Center, Walter Reed Army Institute of Research, Silver Spring, MD, USA. martha.sedegah@med.navy.mil
authors.forename: Martha
authors.initials: M
authors.lastname: Sedegah
authors.forename: Yohan
authors.initials: Y
authors.lastname: Kim
authors.forename: Harini
authors.initials: H
authors.lastname: Ganeshan
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.forename: Maria
authors.initials: M
authors.lastname: Belmonte
authors.forename: Esteban
authors.initials: E
authors.lastname: Abot
authors.forename: Jo Glenna
authors.initials: JG
authors.lastname: Banania
authors.forename: Fouzia
authors.initials: F
authors.lastname: Farooq
authors.forename: Shannon
authors.initials: S
authors.lastname: McGrath
authors.forename: Bjoern
authors.initials: B
authors.lastname: Peters
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sette
authors.forename: Lorraine
authors.initials: L
authors.lastname: Soisson
authors.forename: Carter
authors.initials: C
authors.lastname: Diggs
authors.forename: Denise L
authors.initials: DL
authors.lastname: Doolan
authors.forename: Cindy
authors.initials: C
authors.lastname: Tamminga
authors.forename: Eileen
authors.initials: E
authors.lastname: Villasante
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hollingdale
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Richie
mesh_terms: D002986:Clinical Trials as Topic; D019295:Computational Biology; D018604:Epitope Mapping; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006805:Human Experimentation; D006801:Humans; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D017780:Malaria Vaccines; D015800:Protozoan Proteins; C041595:circumsporozoite protein, Protozoan