extends these observations to include enhanced neutralization in the A3R5 cell line in the context of two disparate HIV-negative vaccine studies performed in Thailand[2]. luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera Diosmetin and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens. Introduction Modest protection against acquisition of HIV-1 was observed in a recent phase III clinical trial (RV144) of ALVAC-HIV and AIDSVAX B/E in Thailand [1]. The vaccine combination generated low levels of primarily tier 1, type-specific NAbs measured in the TZM-bl or T-cell line adapted assays[2,3]. These vaccine-induced antibodies were not identified as correlates of risk in RV144[4]. Nonetheless, broadly cross-reactive, potent neutralizing antibodies (bNAbs) may be an important consideration in future vaccine design [5,6]. Results of passive immunization studies in non-human primates [7C9] and the ability of NAbs to exert strong selection pressure on the virus in HIV-1-infected individuals support this proposition. However, NAbs induced by candidate HIV vaccines have typically proven weak, especially against circulating or transmitted strains of the virus[10C14]. The uncertainty surrounding the magnitude of neutralization necessary for protection in humans requires that vaccine induced NAb activity be accurately quantified by the most sensitive assays available[15,16] A variety of assay platforms have been used to assess NAb responses against HIV-1[14,17C20]. Among these, genetically Diosmetin engineered cells lines in combination with Tat-inducible luciferase (Luc) reporter genes have been extremely valuable for Diosmetin studies of HIV-1 neutralization and escape [21C23], the identification of HIV-1-infected subjects who possess broadly NAbs [24C27], the identification and characterization of broadly neutralizing mAbs [28C35] and the mapping of epitopes of autologous NAbs [36C43] and bNAb [32,44C50] in sera from HIV-1 infected subjects. The two most prevalent cell lines are TZM-bl[23,51] (HeLa derivative, human epithelial origin) and U87.CD4.CCR5 cells (human astroglioma cell line)[21,22]. However, evidence from several studies suggest that TZM-bl cells may not support the detection of neutralizing antibodies to certain epitopes, possibly owing to artificially high surface expression of NY-REN-37 CD4 and CCR5[52C54]. The observation that TZM-bl and U87.CD4.CCR5 cells exhibit similar levels of sensitivity[55] indicate limitations may exist for the latter assay as well. Here we describe a CD4+/CXCR4+/47+/CCR5+ T-cell line, A3R5.7 (designated A3R5), that supports the detection of HIV-1-specific neutralization by mAbs, sCD4 and polyclonal plasma from multiple subtypes encompassing a range of epitopes on the HIV-1 envelope with sensitivity similar to or greater than that observed in the TZM-bl line. Materials and Methods Cloning of pCMV-CCR5neo pCMV-CCR5neo consists of the ccr5 gene (nt positions 240 to 1298) amplified by PCR from PBMC DNA and inserted into the pCR3.1 expression vector (Invitrogen, Carlsbad, CA) downstream of the CMV immediate early (IE) promoter containing the neomycin phosphotransferase gene as a selectable marker. The PCR primers used to generate this fragment were: CCR5-1, and CCR5-2, gene sequencing (data not shown). Normal human serum (NHS) was purchased from Gemini Bio-Products (West Sacramento, CA) and used as a non-specific negative control for HIV-1 sera/plasma. All serum and plasma samples were stored at Diosmetin -80C and heat-inactivated at 56C for 1 hour prior to assay. Intravenous immunoglobulin (IVIG) is a pooled, polyvalent, IgG purchased from Bayer Healthcare, LLC (Clayton, NC) and used as a non-specific negative control for HIV-1 monoclonal antibodies. Virus stocks The uncloned R5-tropic HIV-1 subtype B isolate US1 was obtained from the NIH AIDS Reference and Reagent Program as contributed by Nelson Michael [69] and expanded in PHA/IL-2 stimulated PBMC as previously described[11,70]. PBMC derived US1 was grown in various A3R5 cell lines in the presence of 10 g/mL Polybrene (Sigma, St. Louis, MO) for up to fourteen days. Supernatants were harvested, lysed and assayed for HIV-1 core antigen p24 by sandwich ELISA according to the manufacturers protocol.