Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4+ T memory stem cells (TSCM) harbor high per-cell levels of HIV-1 DNA, and make increasing contributions to the total viral CD4+ T cell reservoir over time. Moreover, phylogenetic studies suggested long-term persistence of viral quasispecies in CD4+ TSCM cells. Thus, HIV-1 may exploit stem cell characteristics of cellular immune memory to promote long-term viral persistence.

Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCEAntiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

Combined treatment with interferon alpha (IFN-α) and ribavirin (RBV) can effectively cure HCV infection in a significant proportion of patients, but effects of this regimen on cellular reservoirs for human immunodeficiency virus type 1 (HIV-1) are unknown. Here, we show that treatment with IFN-α/RBV led to a moderate but significant and sustained decline of HIV-1 DNA in CD4 T cells from HIV-1/hepatitis C virus-coinfected patients receiving highly active antiretroviral therapy (n = 12). However, in vitro experiments failed to demonstrate an effect of pharmacological doses of IFN-α on HIV-1 reactivation. Together, these data suggest that treatment with IFN-α/RBV can moderately reduce the reservoir of HIV-1-infected CD4 T cells that persists despite suppressive antiretroviral therapy.

Bovine herpesvirus 1 (BHV-1), an alphaherpesvirus, is a major pathogen that causes respiratory and reproductive infections. We observed tyrosine phosphorylation of a 95-kDa viral protein and dephosphorylation of 55- and 103-kDa cellular proteins during the course of BHV-1 infection. We demonstrated BHV-1 glycoprotein E (gE) to be the tyrosine phosphorylated viral protein by immunoprecipitation. Inhibition of phosphorylation of BHV-1 gE by tyrosine kinase inhibitors genistein and tyrphostin AG1478 substantially lowered the viral titer in Madin-Darby bovine kidney cells. The decrease in viral titer was directly proportional to the decrease in phosphorylation of the BHV-1 gE. Interestingly, these kinase inhibitors did not inhibit the replication of the BHV-1 gE deletion mutant virion (BHV-1gEDelta3.1). Our findings suggest that the wild-type BHV-1, with a functional gE protein, uses a different pathway of signaling events than the BHV-1 gE deletion mutant in replication. Our results indicate that the tyrosine phosphorylation of the cytoplasmic tail of BHV-1 gE is an important post-translational modification of the functional protein. An application of this study may be the use of tyrosine kinase inhibitors in controlling the BHV-1 infection.

Cell therapies have demonstrated clinical benefit for some patients; however, in more resistant tumors, efficacy, persistence, and cell expansion may be improved with co-administration of an inflammatory cytokine such as interleukin 15 (IL-15). Clinical use of IL-15 has been limited due to systemic toxicity resulting in a narrow therapeutic index. Torque has advanced a “backpack” technique to attach IL-15 to the surface of T cells for adoptive transfer. IL-15 backpacks are ~120 nanometer particles consisting of multiple chemically crosslinked human IL-15/IL-15Rα/Fc heterodimers. Following crosslinker cleavage the IL-15 fusion proteins are released and drive T cell division in an autocrine fashion, providing a targeted, controllable and time-dependent immune stimulus which we term Deep Priming. Torque's lead program, TRQ15-01, consists of antigen-directed, autologous T cells carrying IL-15 backpacks. To explore the effects of IL-15 backpacks on T cells, we characterized backpack-carrying capacity of T cells, IL-15 heterodimer release kinetics, and T cell expansion by incubating IL-15 backpacks with human T cells activated by CD3/CD28 beads. Across 4 donors, T-cells were loaded in a reproducible manner proportional to the backpack labeling solution concentration. Backpacks promote the exponential expansion of activated T-cells for over one week through sustained release of IL-15 into the extracellular space. To explore the effect of IL-15 backpacks on CAR-T cell activity, IL-15 backpacks were loaded onto anti-EGFR CAR-expressing human CD3 cells. NSG mice bearing human H1299 tumor xenografts received CAR-T therapy with and without backpacks 7 days after tumor implantation. IL-15 backpacks significantly improved in vivo expansion of CAR-T cells (p < 0.008, at Day 10) and improved tumor growth inhibition (p < 0.003, 2-way ANOVA). To enable exploration of the clinical utility of backpacking using antigen-directed T-cells, we developed a novel, fully-closed, semi-automated cell manufacturing process with a yield of up to several billion antigen-directed cells. This process utilizes monocyte-derived dendritic cells pulsed with peptides from multiple tumor-associated antigens to expand cytotoxic T lymphocytes (CTLs). In a final step the antigen-directed CTLs are loaded with IL-15 backpacks to generate the TRQ15-01 cell product. Characterization of TRQ15-01 reveals controllable, dose-dependent loading of IL-15 backpacks on the antigen-directed T cells. Compared to non-backpacked antigen-directed cells, TRQ15-01 products displayed significantly higher expansion levels both in vitro (10-fold increase) and in NSG mice (20-fold increase). IL-15 backpack loading on human T cells is tunable and produces slow IL-15 release with sustained cell expansion and enhanced anti-tumor activity. Clinical trials with TRQ15-01 will be initiated in 2018. Citation Format: Pengpeng Cao, De-Kuan Chang, Andy Rakestraw, Amy Shaw, Ferdinando Pucci, Fabio Fachin, Christine McInnis, Shawn Carey, Austin Boesch, David Chirgwin, Carlos Tassa, Aaron Handler, Khristianna Jones, Philip D. Bardwell, Elena Geretti, Jon Nardozzi, Doug Jones, Jesse Lyons, Jonathan B. Fitzgerald, Becker Hewes, Ulrik B. Nielsen, Thomas Andresen. Application of deep IL-15 backpacks to human T cells demonstrates tunable loading with enhanced cell proliferation and antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3577.