title: International network for comparison of HIV neutralization assays: the NeutNet report II.
abstract: BACKGROUND
  Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study).
  
  
  METHODS
  In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression.
  
  
  RESULTS
  Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma.
  
  
  CONCLUSIONS
  Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation.
authors.affiliation: Virology Unit, Biomedical Department, Institute of Tropical Medicine, Antwerp, Belgium. lheyndrickx@itg.be
authors.forename: Leo
authors.initials: L
authors.lastname: Heyndrickx
authors.forename: Alan
authors.initials: A
authors.lastname: Heath
authors.forename: Enas
authors.initials: E
authors.lastname: Sheik-Khalil
authors.forename: Jose
authors.initials: J
authors.lastname: Alcami
authors.forename: Vera
authors.initials: V
authors.lastname: Bongertz
authors.forename: Marianne
authors.initials: M
authors.lastname: Jansson
authors.forename: Mauro
authors.initials: M
authors.lastname: Malnati
authors.forename: David
authors.initials: D
authors.lastname: Montefiori
authors.forename: Christiane
authors.initials: C
authors.lastname: Moog
authors.forename: Lynn
authors.initials: L
authors.lastname: Morris
authors.forename: Saladin
authors.initials: S
authors.lastname: Osmanov
authors.forename: Victoria
authors.initials: V
authors.lastname: Polonis
authors.forename: Meghna
authors.initials: M
authors.lastname: Ramaswamy
authors.forename: Quentin
authors.initials: Q
authors.lastname: Sattentau
authors.forename: Monica
authors.initials: M
authors.lastname: Tolazzi
authors.forename: Hanneke
authors.initials: H
authors.lastname: Schuitemaker
authors.forename: Betty
authors.initials: B
authors.lastname: Willems
authors.forename: Terri
authors.initials: T
authors.lastname: Wrin
authors.forename: Eva Maria
authors.initials: EM
authors.lastname: Fenyö
authors.forename: Gabriella
authors.initials: G
authors.lastname: Scarlatti
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology / Q000627:therapeutic use; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000276:immunology; D001681:Biological Assay / Q000592:standards*; D005260:Female; D015483:HIV Antibodies / Q000737:chemistry* / Q000276:immunology; D015658:HIV Infections / Q000276:immunology* / Q000628:therapy; D015497:HIV-1; D006367:HeLa Cells; D006801:Humans; D008297:Male; D012680:Sensitivity and Specificity
chemical_list: D016915:AIDS Vaccines; D057134:Antibodies, Neutralizing; D015483:HIV Antibodies