title: Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.
abstract: BACKGROUND
  Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans.
  
  
  METHODS
  Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated.
  
  
  RESULTS
  Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity.
  
  
  CONCLUSIONS
  The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.
authors.forename: Robert
authors.initials: R
authors.lastname: Schwenk
authors.forename: Glenna
authors.initials: G
authors.lastname: Banania
authors.forename: Judy
authors.initials: J
authors.lastname: Epstein
authors.forename: Yohan
authors.initials: Y
authors.lastname: Kim
authors.forename: Bjoern
authors.initials: B
authors.lastname: Peters
authors.forename: Maria
authors.initials: M
authors.lastname: Belmonte
authors.forename: Harini
authors.initials: H
authors.lastname: Ganeshan
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.forename: Sharina
authors.initials: S
authors.lastname: Reyes
authors.forename: Anette
authors.initials: A
authors.lastname: Stryhn
authors.forename: Christian F
authors.initials: CF
authors.lastname: Ockenhouse
authors.forename: Soren
authors.initials: S
authors.lastname: Buus
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Richie
authors.affiliation: Malaria Department, Infectious Disease Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA. Martha.Sedegah@med.navy.mil.
authors.forename: Martha
authors.initials: M
authors.lastname: Sedegah
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis*; D000953:Antigens, Protozoan / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology*; D006684:HLA-DR Antigens / Q000032:analysis*; D064368:Healthy Volunteers; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D017780:Malaria Vaccines / Q000008:administration & dosage / Q000276:immunology*; D016778:Malaria, Falciparum / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000032:analysis*; D008565:Membrane Proteins / Q000276:immunology; D015800:Protozoan Proteins / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods; D016176:T-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology
chemical_list: D000953:Antigens, Protozoan; D006684:HLA-DR Antigens; D017780:Malaria Vaccines; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic; C090077:apical membrane antigen I, Plasmodium; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1