Currently available human immunodeficiency virus-1 (HIV-1) reporter cell lines that are highly susceptible to HIV-1 infection with primary patient isolates are not based on T cell lines and gain their HIV-1 susceptibility mostly from the presence of surface molecules that nonspecifically trap HIV-1 (1,2,3,4). As primary T cells fail to express such molecules, data interpretation from these reporter assays is sometimes complicated. Lacking molecules that can nonspecifically capture HIV-1 particles, T cell-based reporter cell lines generally exhibit a relatively low susceptibility to infection with primary HIV-1 patient isolates, thereby limiting their application. To overcome these limitations, we developed a T cell-based reporter assay that derives its high susceptibility to infection with primary HIV-1 isolates from optimized HIV-1 receptor expression and from the utilization of a serum-free medium (SFM) composition optimized for this assay.

While most reporter cell lines use luciferase or alkaline phosphatase activity as quantitative read-outs for HIV-1 infection, which produce high signal-over-background ratios but require additional manipulations (5,6,7,8,9,10), we recently introduced a reporter T cell line in which HIV-1 infection is directly correlated to enhanced green fluorescence protein (EGFP) expression (JLTRG-R5 cells) (11). These cells hold a stably integrated copy of an HIV-1 long terminal repeat (LTR)-EGFP reporter construct, which is induced through the presence of HIV-1 Tat protein following infection. Infection levels can thus be directly quantified using flow cytometric analysis or plate-based fluorometry. However, JLTRG-R5 cells were designed for high-throughput drug screening (HTS) purposes, and while they can be readily infected with laboratory-adapted HIV-1 clones (e.g., NL43, SG3, BaL), susceptibility to infection with primary HIV-1 patient isolates varies, in particular, as CCR5 expression is low.

To maximize the susceptibility of JLTRG-R5 cells to infection with primary viruses, we initially optimized CD4 and CCR5 expression using retroviral vectors. For this purpose, we cloned the cDNA sequences coding for human CD4 and human CCR5 into a retroviral vector derived from the murine stem cell virus (pMSCVpuro; Clontech Laboratories, Mountain View, CA, USA) using standard PCR techniques. We find that in contrast to other retroviral vectors, gene transfer into human T cell lines using these murine stem cell virus (MSCV)-based vectors is mostly resistant to DNA methylation and the resulting inactivation of expression during the initial transduction. MSCV promoter-driven gene expression is high and very stable in long-term culture, which is a major prerequisite for cell line optimization. JLTRG-R5 cells ((Figure 1)A) were transduced with the CD4-and CCR5-expression vectors and the resulting cell population enriched for high transgene expression using fluorescence-activated cell sorting and finally cloned. The ensuing CD4^highCCR5^high reporter T cell line was termed NOMI cells ((Figure 1)A) and expressed significantly higher levels of CD4 and CCR5 than JLTRG-R5 cells. CD4/CCR5 expression levels are also superior to CD4/CCR5 expression on TZM-bl (JC53) cells (3,4). TZM-bl (JC53) cells, a frequently used HIV-1 reporter cell line developed from HeLa cells (a human cervical epithelial carcinoma cell line), derives its high HIV-1 susceptibility primarily from the expression of heparan sulfatebearing adhesion molecules, which are mostly absent on primary T cells (Reference 2, and data not shown). CD4 and CCR5 expression on NOMI cells were found to be stable for longer than 6 months in continuous culture. As predicted, dependent on the virus clone/isolate, NOMI cells, in comparison to JLTRG-R5 cells, were found to be up to two logs more susceptible to HIV-1 infection, in particular with CCR5-tropic primary HIV-1 isolates ((Figure 1)B).

Figure 1.

To further improve the assay, we investigated the possibility of utilizing serum-free media (SFM) to enhance HIV-1 infectivity. This strategy is based on previous reports that describe the nonspecific HIV-1 neutralizing capacity of serum proteins, which led to attempts to develop HIV-1 inhibitors based on modified negatively charged serum albumin derivates (12). For this purpose, we sought to adapt NOMI cells to growth in SFM; however, initial attempts to achieve this goal using commercially available SFMs from a panel of vendors failed. We thus embarked on the development of a SFM composition optimized for this assay using plant seed and animal tissue hydrolysates as replacement of some previously serum-supplied nutrients and cofactors. Such formulations can produce excellent performance in the absence of serum and can provide a robust support of many cell lines and clones in continuous passage. The proprietary medium composition that resulted from these efforts, coined TVR-23 (Thermo Scientific HyClone, Logan, UT, USA) is a cytokine- and mitogen-free SFM with an unusually complex composition of hydrolysates derived from several plant and animal sources. The TVR-23 formula was developed by optimizing the ratio of soluble fractions from hydrolysates of various plant endosperm and of autolysed Saccharomyces cerevisiae. NOMI cells, as well as several other tested T cell lines, exhibited growth performance similar to that observed in RPMI supplemented with 10% fetal bovine serum (FBS) ((Figure 2)A).