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.

CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice.

In this preclinical study, we evaluated the efficacy and feasibility of creating broad human immunodeficiency virus (HIV) resistance by simultaneously disrupting the human CCR5 and CXCR4 genes, which encode cellular co-receptors required for HIV-1 infection. Using a clinically scalable system for transient ex vivo delivery of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes, we demonstrated that CRISPR-mediated disruption of CCR5 and CXCR4 in T lymphocyte cells significantly reduced surface expression of the co-receptors, thereby establishing resistance to HIV-1 infection by CCR5 (R5)-tropic, CXCR4 (X4)-tropic, and dual (R5/X4)-tropic strains. Similarly, disruption of CCR5 alleles in human CD34+ hematopoietic stem and progenitor cells (HSPCs) successfully led to the differentiation of HIV-resistant macrophages. In a humanized mouse model under HIV-1 challenge, CXCR4-disrupted CD4+ T cells were enriched in the peripheral blood and spleen, indicating survival advantage because of resistance to viral infection. However, in human CD4+ T cells with both CCR5 and CXCR4 disruption, we observed poor engraftment in bone marrow, although significant changes were not observed in the lung, spleen, or peripheral blood. This study establishes a clinically scalable strategy for the dual knockout of HIV-1 co-receptors as a therapeutic strategy, while also raising caution of disrupting CXCR4, which may abate engraftment of CD4+ T cells in bone marrow.

Novel Humanized Peripheral Blood Mononuclear Cell Mouse Model with Delayed Onset of Graft-versus-Host Disease for Preclinical HIV Research.

Humanized mouse models are based on the engraftment of human cells in immunodeficient mouse strains, most notably the NSG strain. Most used models have a major limitation in common, the development of graft-versus-host disease (GVHD). GVHD not only introduces variabilities into the research data but also leads to animal welfare concerns. A new mouse strain, B6.129S-Rag2tm1Fwa CD47tm1Fpl Il2rgtm1Wjl/J, which lacks Rag1, IL2rg, and CD47 (triple knockout [TKO]), is resistant to GVHD development. We transplanted TKO mice with human peripheral blood mononuclear cells (PBMCs) to establish a new humanized PBMC (hu-PBMC) mouse model. A cohort of these mice was infected with HIV-1 and monitored for plasma HIV viremia and CD4+ T cell depletion. The onset and progression of GVHD were monitored by clinical signs. This study demonstrates that TKO mice transplanted with human PBMCs support engraftment of human immune cells in primary and secondary lymphoid tissues, rectum, and brain. Moreover, the TKO hu-PBMC model supports HIV-1 infection via the intraperitoneal, rectal, or vaginal route, as confirmed by robust plasma HIV viremia and CD4+ T cell depletion. Lastly, TKO mice showed a delayed onset of GVHD clinical signs (∼24 days) and exhibited significant decreases in plasma levels of tumor necrosis factor beta (TNF-ß). Based on these results, the TKO hu-PBMC mouse model not only supports humanization and HIV-1 infection but also has a delayed onset of GVHD development, making this model a valuable tool in HIV research. IMPORTANCE Currently, there is no cure or vaccine for HIV infection; thus, continued research is needed to end the HIV pandemic. While many animal models are used in HIV research, none is used more than the humanized mouse model. A major limitation with current humanized mouse models is the development of graft-versus-host disease (GVHD). Here, we describe a novel humanized-PBMC mouse model that has a delayed onset GVHD development and supports and models HIV infection comparably to well-established humanized mouse models.

Identification of a therapeutic interfering particle-A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance.

Viral-deletion mutants that conditionally replicate and inhibit the wild-type virus (i.e., defective interfering particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., therapeutic interfering particles, TIPs) must conditionally spread between cells with R0 >1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0 >1, and inhibit viral replication 10- to 100-fold. Inhibition occurs via competition for viral replication machinery, and a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100-fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof of concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.

Exosome-mediated stable epigenetic repression of HIV-1.

Human Immunodeficiency Virus (HIV-1) produces a persistent latent infection. Control of HIV-1 using combination antiretroviral therapy (cART) comes at the cost of life-shortening side effects and development of drug-resistant HIV-1. An ideal and safer therapy should be deliverable in vivo and target the stable epigenetic repression of the virus, inducing a stable "block and lock" of virus expression. Towards this goal, we developed an HIV-1 promoter-targeting Zinc Finger Protein (ZFP-362) fused to active domains of DNA methyltransferase 3 A to induce long-term stable epigenetic repression of HIV-1. Cells were engineered to produce exosomes packaged with RNAs encoding this HIV-1 repressor protein. We find here that the repressor loaded anti-HIV-1 exosomes suppress virus expression and that this suppression is mechanistically driven by DNA methylation of HIV-1 in humanized NSG mouse models. The observations presented here pave the way for an exosome-mediated systemic delivery platform of therapeutic cargo to epigenetically repress HIV-1 infection.