Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 103per cell) compared to cell lines with high CCR5 concentration (about 104or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches. Keywords:HIV, antibody, X5, 2F5, 4E10, CCR5, envelope glycoprotein, neutralization Human immunodeficiency computer virus type I (HIV-1) enters cells by binding its envelope glycoprotein (Env, gp120gp41) gp120 to CD4 and coreceptor (typically CCR5 or CXCR4) leading to activation of the gp41 fusion machinery and membrane fusion [1;2]. Antibodies can bind to the Env and interfere with the fusion process mostly by competing with CD4, coreceptors or fusion intermediates. Thus one can expect that Env-specific antibody inhibitory activity may depend on the cell surface receptor concentration as has been also previously exhibited for other HIV-1 entry inhibitors [3]. Fab X5 was selected from a human antibody phage library by using gp120-CD4-CCR5 complexes as an antigen, and showed a potent and broad neutralizing activity comparable on average to that of IgG b12 as tested with a panel of primary isolates from different clades [4]. It was initially proposed that similarly to b12 the full antibody (in an IgG format) would exhibit even ABT333 greater potency [4]. Because X5 binds better to gp120 complexed with CD4 than to gp120 alone, i.e. it is a CD4i (CD4 induced) antibody, it was affordable to hypothesize that vaccine immunogens able to elicit X5-like antibodies, e.g. complexes of gp120 with CD4 or CD4 mimics, could have potential as AIDS vaccines. Mouse monoclonal to TRX However, later it was found that for most of the tested isolates IgG X5, which is bivalent but of relatively large size is usually less potent than Fab X5, which has smaller size. The most potent antibody format for most but not all isolates was the scFv X5 which is the smallest ([5], and unpublished data); this obtaining dashed hopes to use the X5 epitope as a template for design of vaccine immunogens but still offered the possibility for use of its epitope as a target for potent inhibitors of small size. Indeed, based on scFv X5, a very potent antibody in a scFv format, m9, was developed [6]; however, an IgG-like format of this antibody also showed on average lower inhibitory activity compared to the scFv format (Zhang et al., in preparation). Recently, it was reported that IgG X5 neutralizes significantly ABT333 higher number of primary isolates (7 vs 1) in ABT333 a PBMC-based neutralization assay compared to a cell line-based assay, and proposed that one of the factors that could contribute to these differences is related to differences in the surface concentrations of receptors molecules [7]. In the same report it was also described that 2F5 also exhibits higher neutralizing activity in a PBMC-based assay compared to a cell line-based assay although the difference between the two assays was significantly smaller than for X5; interestingly the other gp41-specific antibody tested in the same study exhibited much broader and higher neutralizing activity in the cell line/pseudovirus-based assay than in the PBMC-based assay. It was previously found that the coreceptor surface concentrations ABT333 can reach up to 105antibody binding sites (ABS) for some cell lines, while ABT333 they do not exceed 104ABS for CD4 T lymphocytes [812]. Although activation of T cells can lead to an increase in the receptor and coreceptor concentrations, the CCR5 surface concentration is relatively low in PBMCs unless specific activation aimed to induce high CCR5 concentration is used [13]. To elucidate the mechanism of CCR5 concentration on the inhibitory activity of CD4i antibodies and other antibodies, we used a panel of HeLa cell lines with well-defined differential surface concentrations of CCR5 and CD4 in a pseudovirus neutralization assay. Here we report significant effect of the CCR5 cell surface concentration on the inhibitory activity of X5 [4;1416] and several other control antibodies..
