Pre-clinical data supporting immunotherapy for HIV using CMV-HIV-specific CAR T cells with CMV vaccine.

T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120. In this study, we use a GMP-compliant platform to engineer CMV-specific T cells to express a second-generation CAR derived from the N6 broadly neutralizing antibody, one of the broadest anti-gp120 neutralizing antibodies. These CMV-HIV CAR T cells exhibit dual effector functions upon in vitro stimulation through their endogenous CMV-specific T cell receptors or the introduced CARs. Using a humanized HIV mouse model, we show that CMV vaccination during ART accelerates CMV-HIV CAR T cell expansion in the peripheral blood and that higher numbers of CMV-HIV CAR T cells were associated with a better control of HIV viral load and fewer HIV antigen p24+ cells in the bone marrow upon ART interruption. Collectively, these data support the clinical development of CMV-HIV CAR T cells in combination with a CMV vaccine in HIV-infected individuals.

Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy.

Retroviral vector gene therapy is a promising approach to treating HIV-1. However, integrated vectors are mutagens with the potential to dysregulate nearby genes and cause severe adverse side effects. Leukemia has already been a documented severe adverse event in gene therapy clinical trials for the treatment of primary immunodeficiencies. These side effects will need to be reduced or avoided if retroviral vectors are to be used clinically for HIV-1 treatment. The addition of chromatin insulators to retroviral vectors is a potential strategy for reducing adverse side effects. Insulators have already been effectively used in retroviral vectors to reduce genotoxicity in pre-clinical studies. Here, we will review how insulators function, genotoxicity in gene therapy clinical trials, the design of insulated retroviral vectors, promising results from insulated retroviral vector studies, and considerations for the development of insulated retroviral treatment vectors for HIV-1 gene therapy.

Insulated Foamy Viral Vectors.

Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34(+) cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy.

Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites.

High-throughput mapping of retroviral vector integration sites (RIS) has become an invaluable tool to evaluate novel gene therapy vectors and to track clonal contribution in preclinical and clinical studies. Beard et al. (Methods Mol Biol 2014;1185:321-344) described an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. Here we describe adaptations to the previously published modified genomic sequencing PCR (MGS-PCR) protocol using the Illumina MiSeq paired-end sequencing platform. Lentiviral, gammaretroviral, and foamy virus vector integrations were analyzed. MGS-PCR using the MiSeq platform allows for the use of merged paired-end reads, which allows for efficient localization of RIS to published genomes.

Anergic CD4+ T cells form mature immunological synapses with enhanced accumulation of c-Cbl and Cbl-b.

CD4(+) T cell recognition of MHC:peptide complexes in the context of a costimulatory signal results in the large-scale redistribution of molecules at the T cell-APC interface to form the immunological synapse. The immunological synapse is the location of sustained TCR signaling and delivery of a subset of effector functions. T cells activated in the absence of costimulation are rendered anergic and are hyporesponsive when presented with Ag in the presence of optimal costimulation. Several previous studies have looked at aspects of immunological synapses formed by anergic T cells, but it remains unclear whether there are differences in the formation or composition of anergic immunological synapses. In this study, we energized primary murine CD4(+) T cells by incubation of costimulation-deficient, transfected fibroblast APCs. Using a combination of TCR, MHC:peptide, and ICAM-1 staining, we found that anergic T cells make mature immunological synapses with characteristic central and peripheral supramolecular activation cluster domains that were indistinguishable from control synapses. There were small increases in total phosphotyrosine at the anergic synapse along with significant decreases in phosphorylated ERK 1/2 accumulation. Most striking, there was specific accumulation of c-Cbl and Cbl-b to the anergic synapses. Cbl-b, previously shown to be essential in anergy induction, was found in both the central and the peripheral supramolecular activation clusters of the anergic synapse. This Cbl-b (and c-Cbl) accumulation at the anergic synapse may play an important role in anergy maintenance, induction, or both.