A new cell line-based neutralization assay for primary HIV type 1 isolates

Simple and standardized assays for detection and quantification of neutralizing antibodies to primary HIV-1 isolates are needed in research on HIV-1 vaccines and pathogenesis. Here we describe a new HIV-1 neutralization assay that is based on plaque formation in U87.CD4-CCR5 and U87.CD4-CXCR4 cells, which is an attractive alternative to peripheral blood mononuclear cell-based assays. Infected cells form syncytia, that is, plaques, that can be stained with hematoxylin and enumerated by light microscopy. Neutralization is determined by the ability of a serum to reduce the number of plaque-forming units (PFU) relative to controls exposed to medium or negative serum. The intraassay variation of the plaque-forming unit determinations was tested with 15 serum-virus combinations and showed good reproducibility. The differences ranged from -19 to +27% and had a standard deviation of +/- 9.1%. On the basis of these data the cutoff for neutralization (i.e., plaque reduction) was set to 30% (3.3 standard deviations). Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10-100 PFU/well. The reproducibility of the new neutralization assay was tested with 4 primary viruses and 9 sera for a total of 20 virus-serum combinations. The mean difference in neutralization (i.e. plaque reduction) determinations performed on different days was as small as 11%. None of 10 Swedish sera and 1 Ugandan plasma pool from HIV-1-uninfected subjects were positive for neutralization, indicating that the assay has high specificity. In summary, the new U87.CD4 cell line-based neutralization assay for primary HIV-1 isolates is a highly reproducible, sensitive, and high-throughput assay that is well suited for large-scale HIV-1 neutralization studies.