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71 Both Cross-Clade Neutralization and Cross-Clade Enhancement of HIV-1 Primary Isolates Are Observed Using an Assay Based on CD4+CCR5+CXCR4+ HeLa Cells
F. Barin*, S. Brunet, D. Brand, and R. Peyre
Univ. F Rabelais, Tours, France
Background: Simple and rapid assays for evaluation of neutralization of HIV-1 primary isolates (PIs) are necessary. Due to the high HIV-1 diversity PIs belonging to different clades must be used.
Methods: CD4+CXCR4+CCR5+ HeLa cells that carry the LacZ gene under the control of the HIV-1 LTR (P4P cells, P. Charneau, Inst. Pasteur, Paris) were used. Diluted sera (1:10(1:320) were incubated with 100 TCID50 of each strain, and each mixture was incubated in 4 wells of 96-well microplates containing P4P cells. After 1 h of incubation at 37°C and washings, the cells were incubated for 48 h. Neutralization titers (90%) were determined after immunostaining using a monoclonal antibody to beta galactosidase, and numeration of positive cells. Eight PIs (genotypes: 1A, 2B, 2D, 1E, 1F, and 1G; phenotypes: 4R4, 3R5, and 1X4R5) and 18 sera (5A, 5B, 1C, 2D, 2E, 1F, and 2G) were first included. A second series of 17 sera from long-term infected patients (>15 years; clade B viruses) was also studied with 4 of the most resistant PIs (1A, 1B, 1E, and 1F).
Results: Cross-clade neutralization was observed for 9 sera of the first series, 1 being able to neutralize 7 strains of 6 genotypes. Five sera of the second series were able to neutralize 4 strains of 4 different genotypes. Cross-clade enhancement (nb of positive cells > 1.5 negative control) was also observed for 3 sera. One serum was able to neutralize the homologous strain (subtype G) but enhanced all the heterologous strains (first series). The 2 other sera enhanced 2 of 4 and 3 of 4 PIs, respectively (second series).
Conclusions: We confirm the cross-clade neutralization already reported using classical assays based on PBMCs, but also that cross-clade enhancement can be detected using CD4+CXCR4+CCR5+ HeLa cells. Our results suggest that this new, simple, and rapid assay is suitable for analysis of HIV-1 neutralization and/or enhancement by antibodies.
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