Fay E. Wong

SIV Infection Induces Accumulation of Plasmacytoid Dendritic Cells in the Gut Mucosa

Multiple studies suggest that plasmacytoid dendritic cells (pDCs) are depleted and dysfunctional during human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but little is known about pDCs in the gut-the primary site of virus replication. Here, we show that during SIV infection, pDCs were reduced 3--fold in the circulation and significantly upregulated the gut-homing marker α4β7, but were increased 4-fold in rectal biopsies of infected compared to naive macaques. These data revise the understanding of pDC immunobiology during SIV infection, indicating that pDCs are not necessarily depleted, but instead may traffic to and accumulate in the gut mucosa.

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

In Vivo Selection of CD4+ T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

We evaluated the potential of an anti-human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4(+) cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4(+) T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4(+) cell. Upon reinfusion of engineered autologous CD4(+) cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4(+) T cells progressively decreased, concurrent with loss of CD4(+) cells and elevated viral loads in both animals. However, CD4(+) T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4(+) cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo.

CD8 T Cell Response Maturation Defined by Anentropic Specificity and Repertoire Depth Correlates with SIVΔnef-induced Protection

The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.

Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T‐cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4+ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4+ T‐cell play roles in protection against wild‐type virus challenge.

SIVΔnef vaccination mobilizes systemic and mucosal natural killer cells in Mamu A*01+ macaques

Background Although vaccination with live attenuated SIV is the most effective means of inducing protection against lentiviruses, the immunologic mechanisms responsible remain unclear. Previous studies have yielded conflicting data regarding the role of adaptive immune responses in mediating protection, suggesting that innate immune responses, including natural killer (NK) cells, may play a role.

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay.

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, P<0.0001). 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.

Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter

BackgroundInternal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.ResultsA series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.ConclusionThe existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

546. Optimization of Expression of the SIV-Specific 9456 Ribozyme by the tRNAval PolIII Promoter within an MLV- or Lentiviral-Vector

The ribozyme 9456, a small RNA molecules having catalytic activity, has been designed to specifically cleave target sequences in the U3 region of SIV. Recent developments have indicated that lentiviral vectors may transduce quiescent cells and therefore provide more efficient gene transfer to important target populations for AIDS gene therapy. However, the optimal position within the lentiviral vector for expression of small RNA inhibitors, especially using internal polymerase III promoters, has not been determined. To compare expression of the tRNAval Pol III promoter/SIV-specific ribozyme 9456 inhibitor cassette, we generated a murine leukemia virus (MLV) vector with the inhibitor in the antisense orientation and a series of a self-inactivating lentiviral vectors (HRST) with the inhibitor in different positions in both the sense and antisense orientations (before RRE: P1/invP1, after CMV-GFP: P2/invP2, in the ΔU3 of the 3′ HIV LTR: P3/invP3). The CD4+ CEMx174 cell line was transduced with the MLV vector before selecting individual transduced clones or with the HRST vectors before sorting by green fluorescent protein (GFP) expression. Total RNA was isolated from transduced cells for analysis by quantitative, real-time RT-PCR for the expression of ribozyme 9456, MLV vector, HRST vector and β-actin sequences. To assess inhibition of viral replication, transduced cells were challenged with an MOI of 10−2 TCID50/cell of SIVmac239 and followed over fourteen days for viral replication. Expression of GFP from the internal CMV promoter with the HRST vectors was assessed by flow cytometry and transduced cells were >90% positive. Expression of the ribozyme 9456 inhibitor relative to expression of the β-actin gene in the MLV-transduced clones and the HRST-transduced populations ranged between 1.1 × 103 to 9.0 × 104 and 3.0x103 to 1.3 × 105 units, respectively. Expression of the ribozyme 9456 was highest in the sense orientation of P2 and P3 of the HRST vectors. The MLV vector backbone showed high levels of expression ranging from 2.2 × 106 to 2.9 × 107 units, while the HRST vector was lower but detectable ranging between 3.8 × 103 to 1.4 × 104 units, indicating that the ΔU3 promoter in the HIV LTR of the HRST vectors is not completely inactive. The MLV clones with the highest levels of 9456 expression also provided the strongest inhibition of viral replication. Many of the HRST vectors (P3, invP1, invP2, invP3) showed strong inhibition (>80%). Inhibition with P1 and P2 was modest even though P2 expressed the 9456 at the highest level. Inhibition with the HRST vectors was consistently stronger than in the MLV clones. These vectors demonstrate variable expression of the ribozyme inhibitor from a Pol III promoter in HRST and MLV vector backbones and may provide valuable insight into the expression of other small RNA molecules in transduced cells. Optimization of delivery of ribozymes by lentiviral vectors should facilitate analysis of the efficacy of ribozymes for stem cell gene therapy for AIDS in a nonhuman primate model.