Jacqueline Kaye Flynn

Oncolytic Virus and Anti–4-1BB Combination Therapy Elicits Strong Antitumor Immunity against Established Cancer

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b(+) and CD11c(+) myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8(+) effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer.

Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy.

The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously. A tissue-specific response to Tri-mAb was confirmed by ex vivo separation of subcutaneous (SC) or orthotopic tumor cells from stromal cells, followed by reinjection of tumor cells into the opposite site. Compared with SC tumors, orthotopic tumors had a microenvironment associated with a type 2 immune response, related to immunosuppression, and an involvement of alternatively activated macrophages in the kidney model. Orthotopic kidney tumors were more highly vascularized than SC tumors. Neutralizing the macrophage- and Th2-associated molecules chemokine (C-C motif) ligand 2 or interleukin-13 led to a significantly improved therapeutic effect. This study highlights the importance of the tissue of implantation in sculpting the tumor microenvironment. These are important fundamental issues in tumor biology and crucial factors to consider in the design of experimental models and treatment strategies.

Early IL-10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users

Summary.  The critical events in clearance or persistence of hepatitis C virus (HCV) infection are unknown but likely to be determined early in acute infection. Type 1 and type 2 cytokine production was assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays in longitudinally collected samples from 20 untreated participants enrolled in the Australian Trial in Acute Hepatitis C (ATAHC); a prospective cohort of acute HCV infection (77% injecting drug users, IDU). Significantly higher interleukin‐10 (IL‐10) production (P = 0.048), in the relative absence of interferon‐gamma (IFN‐γ) and IL‐2 production, was present early in HCV infection in those who progressed to chronic infection. In contrast, viral clearance was associated with a greater magnitude and broader specificity of IFN‐γ (magnitude P < 0.001, breadth P = 0.004) and IL‐2 responses, in the relative absence of IL‐10. Early IL‐10 production was correlated with higher HCV RNA level at baseline (P = 0.046) and week 12 (P = 0.018), while IFN‐γ and IL‐2 production was inversely correlated with HCV RNA level at baseline (IFN‐γP = 0.020, IL‐2 P = 0.050) and week 48 (IFN‐γP = 0.045, IL‐2 P = 0.026). Intracellular staining (ICS) indicated the HCV‐specific IFN‐γ response was primarily from CD8+ T cells and NK cells, whereas IL‐10 production was predominantly from monocytes, with a subset of IL‐10 producing CD8+ T cells present only in those who progressed to chronic infection. IL‐10, an immunoregulatory cytokine, appears to play a key role in progression to chronic HCV infection.

Is the central nervous system a reservoir of HIV-1?

Purpose of reviewTo summarize the evidence in the literature that supports the central nervous system (CNS) as a viral reservoir for HIV-1 and to prioritize future research efforts. Recent findingsHIV-1 DNA has been detected in brain tissue of patients with undetectable viral load or neurocognitive disorders, and is associated with long-lived cells such as astrocytes and microglia. In neurocognitively normal patients, HIV-1 can be found at high frequency in these cells (4% of astrocytes and 20% of macrophages). CNS cells have unique molecular mechanisms to suppress viral replication and induce latency, which include increased expression of dominant negative transcription factors and suppressive epigenetic factors. There is also evidence of continued inflammation in patients lacking a CNS viral load, suggesting the production and activity of viral neurotoxins (for example, Tat). SummaryTogether, these findings provide evidence that the CNS can potentially act as a viral reservoir of HIV-1. However, the majority of these studies were performed in historical cohorts (absence of combination antiretroviral therapy or presence of viral load), which do not reflect modern day patients (combination antiretroviral therapy-treated and undetectable viral load). Future studies will need to examine patient samples with these characteristics to conclusively determine whether the CNS represents a relevant and important viral reservoir.

A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations

BackgroundThe CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC.ResultsEnvs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively “weak” and “strong” resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus.ConclusionsClinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Naïve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.

Stem memory T cells (TSCM)—their role in cancer and HIV immunotherapies

Stem memory T cells (TSCM) have been described in mice, non‐human primates and in humans, constituting approximately 2–4% of the total CD4+ and CD8+ T‐cell population in the periphery. TSCM represent the earliest and long‐lasting developmental stage of memory T cells, displaying stem cell‐like properties, and exhibiting a gene profile between naïve and central memory T cells. Their self‐renewal capacity and long‐term survival has sparked interest in the cancer and human immunodeficiency virus (HIV) fields. How and when the formation of TSCM occurs during the immune response to pathogens and the therapeutic potential of these cells are currently being investigated. This review will explore the potential role of TSCM to be used as, or targeted by, immunotherapies and vaccines for treatment of cancer and HIV.

The magnitude of HIV-1 resistance to the CCR5 antagonist maraviroc may impart a differential alteration in HIV-1 tropism for macrophages and T-cell subsets

Human immunodeficiency virus type 1 (HIV-1) resistance to CCR5 antagonists, including maraviroc (MVC), results from alterations in the HIV-1 envelope glycoproteins (Env) enabling recognition of antagonist-bound CCR5. Here, we characterized tropism alterations for CD4+ T-cell subsets and macrophages by Envs from two subjects who developed MVC resistance in vivo, which displayed either relatively efficient or inefficient recognition of MVC-bound CCR5. We show that MVC-resistant Env with efficient recognition of drug-bound CCR5 displays a tropism shift for CD4+ T-cell subsets associated with increased infection of central memory T-cells and reduced infection of effector memory and transitional memory T-cells, and no change in macrophage infectivity. In contrast, MVC-resistant Env with inefficient recognition of drug-bound CCR5 displays no change in tropism for CD4+ T-cell subsets, but exhibits a significant reduction in macrophage infectivity. The pattern of HIV-1 tropism alterations for susceptible cells may therefore be variable in subjects with MVC resistance.

Differences in coreceptor specificity contribute to alternative tropism of HIV-1 subtype C for CD4(+) T-cell subsets, including stem cell memory T-cells.

BackgroundCD4+ memory T-cells are a major target for infection by HIV-1, whereby latent provirus can establish and endure suppressive antiretroviral therapies. Although HIV-1 subtype C strains (C-HIV) account for the majority of HIV-1 infections worldwide, the susceptibility of CD4+ memory T-cells to infection by CCR5- (R5) and CXCR4-using (X4) C-HIV is unknown. Here, we quantified the susceptibility of naïve and memory CD4+ T-cell subsets, including stem cell memory T-cells (TSCM), to infection by HIV-1 subtype C (C-HIV) strains from treatment-naïve subjects who progressed from chronic to advanced stages of disease whilst either maintaining CCR5-using (R5) viruses (subjects 1503 and 1854), or who experienced emergence of dominant CXCR4-using (X4) strains (subject 1109).FindingsWe show that R5 and X4 C-HIV viruses preferentially target memory and naïve CD4+ T-cell subsets, respectively. While TSCM were susceptible to infection by both R5 and X4 C-HIV viruses, the proportion of infected CD4+ T-cells that were TSCM was higher for R5 strains. Mutagenesis studies of subject 1109 viruses established the V3 region of env as the determinant underlying the preferential targeting of naïve CD4+ T-cells by emergent X4 C-HIV variants in this subject. In contrast, the tropism of R5 C-HIV viruses for CD4+ T-cell subsets was maintained from chronic to advanced stages of disease in subjects 1503 and 1854.ConclusionsThis study provides new insights into the natural history of tropism alterations for CD4+ T-cell subsets by C-HIV strains during progression from chronic to advanced stages of infection. Although not preferentially targeted, our data suggest that TSCM and other memory CD4+ T-cells are likely to be viral reservoirs in subjects with X4 C-HIV infection.

Quantifying Susceptibility of CD4+ Stem Memory T-Cells to Infection by Laboratory Adapted and Clinical HIV-1 Strains

CD4+ T cells are principal targets for human immunodeficiency virus type 1 (HIV-1) infection. CD4+ T cell subsets are heterogeneous cell populations, divided by functional and phenotypic differences into naïve and memory T cells. The memory CD4+ T cells are further segregated into central, effector and transitional memory cell subsets by functional, phenotypic and homeostatic characteristics. Defining the distribution of HIV-1 infection in different T cell subsets is important, as this can play a role in determining the size and composition of the viral reservoir. Both central memory and transitional memory CD4+ T cells have been described as long-lived viral reservoirs for HIV. Recently, the newly described stem memory T cell subset has also been implicated as a long-lived HIV reservoir. Using green fluorescent protein (GFP) reporter strains of HIV-1 and multi parameter flow cytometry, we developed an assay to simultaneously quantify the susceptibility of stem memory (TSCM), central memory, effector memory, transitional memory and naïve CD4+ T cell subsets, to HIV-1 infection in vitro. We show that TSCM are susceptible to infection with laboratory adapted and clinical HIV-1 strains. Our system facilitates the quantitation of HIV-1 infection in alternative T cell subsets by CCR5- and CXCR4-using viruses across different HIV-1 subtypes, and will be useful for studies of HIV-1 pathogenesis and viral reservoirs.