Multi-antigen Vaccination With Simultaneous Engagement of the OX40 Receptor Delays Malignant Mesothelioma Growth and Increases Survival in Animal Models

Malignant Mesothelioma (MM) is a rare and highly aggressive cancer that develops from mesothelial cells lining the pleura and other internal cavities, and is often associated with asbestos exposure. To date, no effective treatments have been made available for this pathology. Herein, we propose a novel immunotherapeutic approach based on a unique vaccine targeting a series of antigens that we found expressed in different MM tumors, but largely undetectable in normal tissues. This vaccine, that we term p-Tvax, is comprised of a series of immunogenic peptides presented by both MHC-I and -II to generate robust immune responses. The peptides were designed using in silico algorithms that discriminate between highly immunogenic T cell epitopes and other harmful epitopes, such as suppressive regulatory T cell epitopes and autoimmune epitopes. Vaccination of mice with p-Tvax led to antigen-specific immune responses that involved both CD8+ and CD4+ T cells, which exhibited cytolytic activity against MM cells in vitro. In mice carrying MM tumors, p-Tvax increased tumor infiltration of CD4+ T cells. Moreover, combining p-Tvax with an OX40 agonist led to decreased tumor growth and increased survival. Mice treated with this combination immunotherapy displayed higher numbers of tumor-infiltrating CD8+ and CD4+ T cells and reduced T regulatory cells in tumors. Collectively, these data suggest that the combination of p-Tvax with an OX40 agonist could be an effective strategy for MM treatment

Galectin-9 Mediates HIV Transcription by Inducing TCR-Dependent ERK Signaling

Endogenous plasma levels of the immunomodulatory carbohydrate-binding protein galectin-9 (Gal-9) are elevated during HIV infection and remain elevated after antiretroviral therapy (ART) suppression. We recently reported that Gal-9 regulates HIV transcription and potently reactivates latent HIV. However, the signaling mechanisms underlying Gal-9-mediated viral transcription remain unclear. Given that galectins are known to modulate T cell receptor (TCR)-signaling, we hypothesized that Gal-9 modulates HIV transcriptional activity, at least in part, through inducing TCR signaling pathways. Gal-9 induced T cell receptor ζ chain (CD3ζ) phosphorylation (11.2 to 32.1%; P = 0.008) in the J-Lat HIV latency model. Lck inhibition reduced Gal-9-mediated viral reactivation in the J-Lat HIV latency model (16.8–0.9%; P < 0.0001) and reduced both Gal-9-mediated CD4+ T cell activation (10.3 to 1.65% CD69 and CD25 co-expression; P = 0.0006), and IL-2/TNFα secretion (P < 0.004) in primary CD4+ T cells from HIV-infected individuals on suppressive ART. Using phospho-kinase antibody arrays, we found that Gal-9 increased the phosphorylation of the TCR-downstream signaling molecules ERK1/2 (26.7-fold) and CREB (6.6-fold). ERK and CREB inhibitors significantly reduced Gal-9-mediated viral reactivation (16.8 to 2.6 or 12.6%, respectively; P < 0.0007). Given that the immunosuppressive rapamycin uncouples HIV latency reversal from cytokine-associated toxicity, we also investigated whether rapamycin could uncouple Gal-9-mediated latency reactivation from its concurrent pro-inflammatory cytokine production. Rapamycin reduced Gal-9-mediated secretion of IL-2 (4.4-fold, P = 0.001) and TNF (4-fold, P = 0.02) without impacting viral reactivation (16.8% compared to 16.1%; P = 0.2). In conclusion, Gal-9 modulates HIV transcription by activating the TCR-downstream ERK and CREB signaling pathways in an Lck-dependent manner. Our findings could have implications for understanding the role of endogenous galectin interactions in modulating TCR signaling and maintaining chronic immune activation during ART-suppressed HIV infection. In addition, uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of recombinant Gal-9 within the reversal of HIV latency eradication framework

The latent HIV reservoir: current advances in genetic sequencing approaches

ABSTRACT Multiple cellular HIV reservoirs in diverse anatomical sites can undergo clonal expansion and persist for years despite suppressive antiretroviral therapy, posing a major barrier toward an HIV cure. Commonly adopted assays to assess HIV reservoir size mainly consist of PCR-based measures of cell-associated total proviral DNA, intact proviruses and transcriptionally competent provirus (viral RNA), flow cytometry and microscopy-based methods to measure translationally competent provirus (viral protein), and quantitative viral outgrowth assay, the gold standard to measure replication-competent provirus; yet no assay alone can provide a comprehensive view of the total HIV reservoir or its dynamics. Furthermore, the detection of extant provirus by these measures does not preclude defects affecting replication competence. An accurate measure of the latent reservoir is essential for evaluating the efficacy of HIV cure strategies. Recent approaches have been developed, which generate proviral sequence data to create a more detailed profile of the latent reservoir. These sequencing approaches are valuable tools to understand the complex multicellular processes in a diverse range of tissues and cell types and have provided insights into the mechanisms of HIV establishment and persistence. These advancements over previous sequencing methods have allowed multiplexing and new assays have emerged, which can document transcriptional activity, chromosome accessibility, and in-depth cellular phenotypes harboring latent HIV, enabling the characterization of rare infected cells across restrictive sites such as the brain. In this manuscript, we provide a review of HIV sequencing-based assays adopted to address challenges in quantifying and characterizing the latent HIV reservoir

Geriatric Syndromes in Older Adults Living with HIV and Cognitive Impairment

ObjectivesNearly half of the population living with human immunodeficiency virus (HIV) in the United States is now older than 50 years with at least 6% over age 65. Between 35% and 50% live with mild to moderate cognitive impairment. Older persons living with HIV (PLWH) also have a substantial burden of HIV-associated non-acquired immunodeficiency syndrome medical conditions and are at risk for frailty, geriatric syndromes, and early mortality compared with HIV-uninfected peers. We sought to define the magnitude of geriatric conditions and multimorbidity in PLWH older than 60 years who are living with symptomatic cognitive impairment. In a subset of participants, we examined associations between these geriatric conditions.DesignRetrospective cohort study.SettingHIV Elders Study at the University of California, San Francisco, Memory and Aging Center.ParticipantsParticipants were HIV infected, virally suppressed, 60 years or older, and clinically diagnosed with mild neurocognitive disorder (MND).MeasurementsWe conducted standardized assessment of geriatric conditions and everyday function and investigated multimorbidity burden using the Veterans Aging Cohort Study (VACS) index.ResultsAmong 141 older PLWH with MND, 58% report incontinence, 55% meet criteria for pre-frailty, and a substantial proportion report dependence with instrumental activities of daily living (52%) or activities of daily living (41%). The mean VACS index score is 33 (standard deviation = 14), suggesting a 13.8% 5-year all-cause mortality risk.ConclusionsOlder PLWH with symptomatic cognitive impairment carry a substantial burden of other geriatric conditions. Our work supports the need for comprehensive geriatric systems of care for cognitively impaired individuals aging with HIV. J Am Geriatr Soc 67:1913-1916, 2019

Associations Between Plasma Immunomodulatory and Inflammatory Mediators With VACS Index Scores Among Older HIV-Infected Adults on Antiretroviral Therapy.

The prevalence of age-related comorbidities is increased in people living with HIV, even in those well-controlled on combination antiretroviral therapy (ART). Persistent immune activation and inflammation may play pivotal roles in the pathogenesis; however, the burden of morbidities in the older HIV infected population may be exacerbated and driven by distinct mechanisms. In a cross sectional study of 45 HIV-infected participants 60 years or older, we examined the relationships between 14 immunomodulatory and inflammatory factors and the Veterans Aging Cohort Study (VACS) Index, a metric of multimorbidity and mortality comprised of age, CD4 count, hemoglobin, Fibrosis-4 [FIB-4], and estimated glomerular filtration rate [eGFR], by linear regression analysis. All participants were virally suppressed (<50 HIV RNA copies/mL), on ART, and primarily Caucasian (86.7%), and male (91.1%). Plasma levels of monocyte/macrophage-associated (neopterin, IP-10, sCD163, sCD14, and MCP-1) and glycan-binding immunomodulatory factors (galectin (Gal)-1, Gal-3, and Gal-9) were assessed, as well as inflammatory biomarkers previously linked to the VACS Index (i.e., CRP, cystatin C, TNF-α, TNFRI, IL-6, and D-dimer) for comparison. In regression analysis, higher VACS index scores were associated with higher levels of neopterin, cystatin C, TNFRI, and Gal-9 (all p < 0.05), potentially driven by correlations found with individual VACS components, including age, CD4 count, FIB-4, and eGFR. Gal-9, cystatin C, and TNFRI directly correlated with the extent of multimorbidity. Multiple correlations among markers were observed, suggesting an interplay of overlapping, but distinct, pathways. Collectively, in addition to cystatin C and TNFRI, both galectin-9 and neopterin, independently emerged as novel fluid markers of the VACS Index and burden of comorbidity and may further guide in understanding pathogenic mechanisms of age-related disorders in older HIV-infected individuals on suppressive ART

Plasma inflammatory biomarkers link to diffusion tensor imaging metrics in virally suppressed HIV-infected individuals.

ObjectiveInflammation may contribute to brain white matter health in people living with HIV who report cognitive symptoms despite adherence to combination antiretroviral therapy and viral suppression. We explored relationships between diffusion tensor imaging (DTI) metrics of white matter, plasma biomarkers of immune activation, and cognitive function in the HIV-infected population.DesignRetrospective study of older adults living with HIV who are combination antiretroviral therapy adherent, virally suppressed, and self-report cognitive symptoms.MethodsMRI, blood draws, and standardized neuropsychological test scores were collected from HIV-infected individuals. DTI metrics (fractional anisotropy, mean diffusivity, radial diffusivity, axial diffusivity) and plasma biomarkers (soluble CD163, soluble CD14, neopterin, IFN γ-induced protein 10, monocyte chemoattractant protein 1) were quantified. Statistical analysis explored associations between biomarker levels or neuropsychological test scores and DTI metrics using region of interest analyses and a voxelwise approach.ResultsA total of 43 participants with median (interquartile range) age of 64 (62-66 years), CD4 cell count of 600 (400-760 cell/μl) who were all virally suppressed (<100 copies/ml) were selected. Higher levels of monocyte chemoattractant protein 1 associated with lower fractional anisotropy and higher mean diffusivity (P < 0.05) across white matter tracts including corpus callosum, corona radiata, and superior longitudinal fasciculus. Higher neopterin associated with higher mean diffusivity in the genu of corpus callosum, and higher soluble CD14 associated with lower fractional anisotropy in the bilateral superior corona radiata (P < 0.05). Worse global performance and speed domain scores associated with higher mean diffusivity and lower fractional anisotropy, and worse executive domain scores associated with lower fractional anisotropy (P < 0.05).ConclusionElevated inflammatory plasma biomarkers link to white matter abnormalities among virally suppressed individuals. DTI abnormalities associate to cognitive performance. We conclude that inflammatory processes impact clinically relevant brain health indices despite viral suppression

Retroviral b‐Zip protein (HBZ) contributes to the release of soluble and exosomal immune checkpoint molecules in the context of neuroinflammation

Abstract HTLV‐I‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neuroinflammatory demyelinating condition of the spinal cord. We have previously shown that aberrant expression and activity of immune checkpoint (ICP) molecules such as PD‐1 and PD‐L1/PD‐L2, negatively associates with the cytolytic potential of T cells in individuals with HAM/TSP. Interestingly, ICPs can exist in a soluble cell‐free form and can be carried on extracellular vesicles (EVs) and exosomes (small EVs, <300 nm) while maintaining their immunomodulatory activity. Therefore, we investigated the role of soluble and exosomal ICPs in HTLV‐1 associated neuroinflammation. For the very first time, we demonstrate a unique elevated presence of several stimulatory (CD27, CD28, 4‐1BB) and inhibitory (BTLA, CTLA‐4, LAG‐3, PD‐1, PD‐L2) ICP receptors in HAM/TSP sera, and in purified exosomes from a HAM/TSP‐derived HTLV‐1‐producing (OSP2) cells. These ICPs were found to be co‐localized with the endosomal sorting complex required for transport (ESCRT) pathway proteins and exhibited functional binding with their respective ligands. Viral proteins and cytokines (primarily IFNγ) were found to be present in purified exosomes. IFNγ exposure enhanced the release of ICP molecules while antiretroviral drugs (Azidothymidine and Lopinavir) significantly inhibited this process. HTLV‐1 b‐Zip protein (HBZ) has been linked to factors that enhance EV release and concurrent knockdown here led to the reduced expression of ESCRT associated genes (e.g., Hrs, Vsp4, Alix, Tsg101) as well as abrogated the release of ICP molecules, suggesting HBZ involvement in this process. Moreso, exosomes from OSP2 cells adversely affected CD8 T‐cell functions by diminishing levels of cytokines and cytotoxic factors. Collectively, these findings highlight exosome‐mediated immunomodulation of T‐cell functions with HBZ and ESCRT pathways as an underlying mechanism in the context of HTLV‐1‐induced neuroinflammation