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Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.

In light of recent findings demonstrating that the rhesus TRIM-5a protein specifically binds to HIV-1 Gag and blocks the uncoating of viral particles, SIV-based lentiviral vectors have been proposed as a more appropriate vector than HIV-1 based vectors for the transduction of rhesus macaque hematopoietic stem cells. We evaluated the ability of a SIVmac239-based lentiviral vector (VRX859), which encodes 1275 bp antisense SIV envelope and eGFP transcriptionally regulated from the SIV LTR, to inhibit viral replication in vitro and compared transduction of rhesus CD34+ lymphoid progenitor cells with an HIV-1 based lentiviral vector. Initially, we evaluated inhibition by VRX859 of the homologous SIVmac239; the related SIVmac251; the heterologous SIVsmE660 (|[sim]|80% homologous in env); the chimeric SHIV 89.6P and HIV-1 NL4-3. CEMx174 cells, which support replication of both HIV-1 and SIV isolates, were transduced with VRX859 at MOI of 10 and the eGFP sorted population contained 1 proviral copy per cell. Following infection with SIVmac239 and SIVmac251, CEMx174-VRX859 cells exhibited over 1000- and 650-fold inhibition of viral replication compared to control cells. Inhibition of SIVsmE660 and SHIV 89.6P was weaker, ranging from 3 to 9-fold and 18 to 32-fold. HIV-1 NL4-3 replication in CEMx174-VRX859 cells was similar to control cells. To evaluate transduction of rhesus CD34+ cells and lymphoid progenitor cells, we prestimulated CD34+ cells with Tpo, Flt3L and SCF (50 ng/ml each) for 1 day and exposed cells to VRX859 vector (or the analogous HIV-1 based lentiviral vector VRX494) for 1, 2 or 3 days on Retronectin-coated plates. Transduction of rhesus CD34+ cells with the SIV vector VRX859 over a range of MOIs (3|[ndash]|50 IU per cell) resulted in similar percentages of eGFP expression as observed with the HIV vector VRX494 at similar MOIs. Transduced CD34+ cells were then cultured with rhesus fetal thymus stromal cells to induce T cell differentiation and analyzed by flow for expression of eGFP. eGFP expression in CD4+ T cells derived from CD34+ cells transduced with VRX859 at low MOIs (3|[ndash]|10 IU per cell) was increased from 13- to 2.2- fold compared cells transduced with VRX494. eGFP expression in CD4+ T cells derived from CD34+ cells transduced at higher MOIs (30 and 50 IU per cell) were basically equivalent between VRX859 and VRX494. Rhesus CD4+ T cells expressing eGFP were obtained by cell sorting and expanded with ConA, IL-2 and irradiated human feeders. GFP+ CD4+ T cells derived from VRX859-transduced cells strongly inhibited SIVmac239, SIVsmE660 and SHIV 89.6P replication (up to 1000-fold) as compared to control CD4+ T cells. These results demonstrate superior transduction of rhesus lymphoid progenitor cells with a SIV-based lentiviral vector, particularly at low MOI, and strong inhibition of viral replication, based partially on competition with genomic RNA, in CD4+ T cells derived from transduced CD34+ cells and in a CD4+ cell line. These results support using a SIV-based lentiviral vector in the rhesus macaque model as a valuable preclinical model for stem cell gene therapy for AIDS.