title: A flow cytometric method for measuring neutralization of HIV-1 subtype B and E primary isolates.
abstract: BACKGROUND
  Clinical trials testing candidate human immunodeficiency virus type 1 (HIV-1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV-1. The variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV-1 primary isolates.
  
  
  METHODS
  A modified neutralization assay was performed using CD8-depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV-1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV-1 subtype B' and E primary isolates were tested using pooled sera or plasma from subtype B' or E infected patients.
  
  
  RESULTS
  Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24(+) cells, depending on the virus. Less than 0.2% p24(+) cells were detected in uninfected cultures. Subtype-specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells.
  
  
  CONCLUSIONS
  Flow cytometric detection of intracellular HIV-1 p24 can be used as an endpoint assay to assess neutralization of HIV-1 subtypes B' and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It also provides an alternative quantitative endpoint for HIV neutralization assays.
authors.affiliation: Office for Research and Development, Center of Excellence for Flow Cytometry, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: J M
authors.initials: JM
authors.lastname: Darden
authors.forename: V R
authors.initials: VR
authors.lastname: Polonis
authors.forename: M S
authors.initials: MS
authors.lastname: deSouza
authors.forename: S
authors.initials: S
authors.lastname: Chantakulkij
authors.forename: A E
authors.initials: AE
authors.lastname: Brown
authors.forename: D L
authors.initials: DL
authors.lastname: Birx
authors.forename: K
authors.initials: K
authors.lastname: Pattanapanyasat
mesh_terms: D016915:AIDS Vaccines; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000032:analysis / Q000276:immunology; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D016655:HIV Core Protein p24 / Q000032:analysis / Q000276:immunology; D015658:HIV Infections / Q000517:prevention & control*; D015497:HIV-1 / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D006801:Humans; D009500:Neutralization Tests / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D016915:AIDS Vaccines; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D005456:Fluorescent Dyes; D016655:HIV Core Protein p24; D016650:Fluorescein-5-isothiocyanate