title: Neutralizing antibodies against HIV determined by amplification of viral long terminal repeat sequences from cells infected in vitro by nonneutralized virions.
abstract: Based on the earliest intracellular synthesis of nascent HIV-1 long terminal repeat (LTR) fragments, we have established a heminested polymerase chain reaction (HNPCR) amplification of the 5' LTR sequences (LTR-HNPCR) for molecular assay of virus-neutralizing antibodies (VNAb). We incubated HIV antibodies with virus isolates for an hour, followed by addition of lymphoid cells (H9 or peripheral blood mononuclear cells [PBMC]) and further incubation for an hour. After washing the cells three times for thorough removal of free virions and antibodies, LTR-HNPCR consistently revealed HIV DNA in H9 cells after 15 minutes, in PBMC after 4 hours, and corresponding virion expression after 7 days in culture. Replication-competent HIV detected by LTR-HNPCR following overnight culture of infected PBMC for 16 to 18 hours was comparable with tissue culture infectivity measured by p24 antigen expression at 7 days. After establishing a molecular assay for in vitro HIV neutralization by HIV Ig, a panel of five HIV isolates tested with 6 monoclonal antibodies and HIV Ig revealed that LTR-HNPCR was comparable with other VNAb assays. These preliminary data indicate that the molecular assay for HIV neutralization has a clear-cut end point, is specific, reliable, and more rapid than other VNAb assays. Therefore, it offers potential utility in evaluating immune response to candidate vaccines.
authors.affiliation: Department of Laboratory Medicine, University of California, San Francisco 94143, USA.
authors.forename: G
authors.initials: G
authors.lastname: Yang
authors.forename: M P
authors.initials: MP
authors.lastname: D'Souza
authors.forename: G N
authors.initials: GN
authors.lastname: Vyas
mesh_terms: D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D015483:HIV Antibodies / Q000032:analysis*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000276:immunology*; D009500:Neutralization Tests / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral; D015483:HIV Antibodies