First-in-human clinical trial of allogeneic, platelet-derived extracellular vesicles as a potential therapeutic for delayed wound healing.

The release of growth factors, cytokines and extracellular matrix modifiers by activated platelets is an important step in the process of healthy wound healing. Extracellular vesicles (EVs) released by activated platelets carry this bioactive cargo in an enriched form, and may therefore represent a potential therapeutic for the treatment of delayed wound healing, such as chronic wounds. While EVs show great promise in regenerative medicine, their production at clinical scale remains a critical challenge and their tolerability in humans is still to be fully established. In this work, we demonstrate that Ligand-based Exosome Affinity Purification (LEAP) chromatography can successfully isolate platelet EVs (pEVs) of clinical grade from activated platelets, which retain the regenerative properties of the parent cell. LEAP-isolated pEVs display the expected biophysical features of EV populations and transport essential proteins in wound healing processes, including insulin growth factor (IGF) and transforming growth factor beta (TGF-ß). In vitro studies show that pEVs induce proliferation and migration of dermal fibroblasts and increase dermal endothelial cells' angiogenic potential, demonstrating their wound healing potential. pEV treatment activates the ERK and Akt signalling pathways within recipient cells. In a first-in-human, double-blind, placebo-controlled, phase I clinical trial of healthy volunteer adults, designed primarily to assess safety in the context of wound healing, we demonstrate that injections of LEAP-purified pEVs in formulation buffer are safe and well tolerated (Plexoval II study, ACTRN12620000944932). As a secondary objective, biological activity in the context of wound healing rate was assessed. In this cohort of healthy participants, in which the wound bed would not be expected to be deficient in the bioactive cargo that pEVs carry, all wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed at the single dose tested. The outcomes of this study evidence that pEVs manufactured through the LEAP process can be injected safely in humans as a potential wound healing treatment, and warrant further study in clinical trials designed expressly to assess therapeutic efficacy in patients with delayed or disrupted wound healing.

Prospective Therapeutic Applications of Platelet Extracellular Vesicles.

There is much interest in the use of extracellular vesicles (EVs) as a subcellular therapy for regenerative medicine and drug delivery. Blood-borne platelets represent a source of therapeutic EVs that is so far largely unexplored. Advantages of platelets as a cellular source of EVs include their established clinical value, regulated collection procedures, availability in a concentrated form, propensity to generate EVs, and unique composition and tissue-targeting capacity. This review analyzes the unique potential of platelet-derived (p-) EVs as therapeutic modalities and presents their inherent translational advantages for hemostasis, for regenerative medicine, and as drug-delivery vehicles.

Viremic and Virologically Suppressed HIV Infection Increases Age-Related Changes to Monocyte Activation Equivalent to 12 and 4 Years of Aging, Respectively.

BACKGROUND: Chronic inflammation and immune activation occur in both HIV infection and normal aging and are associated with inflammatory disease. However, the degree to which HIV influences age-related innate immune changes, and the biomarkers which best reflect them, remains unclear. METHODS AND RESULTS: We measured established innate immune aging biomarkers in 309 individuals including 88 virologically suppressed (VS) and 52 viremic (viral load ≤ and >50 copies per milliliter, respectively) HIV-positive individuals. Levels of soluble (ie, CXCL10, soluble CD163, neopterin) and cellular (ie, proportions of inflammatory CD16 monocytes) biomarkers of monocyte activation were increased in HIV-positive individuals and were only partially ameliorated by viral suppression. Viremic and VS HIV-positive individuals show levels of age-related monocyte activation biomarkers that are similar to uninfected controls aged 12 and 4 years older, respectively. Viremic HIV infection was associated with an accelerated rate of change of some monocyte activation markers (eg, neopterin) with age, whereas in VS individuals, subsequent age-related changes occurred at a similar rate as in controls, albeit at a higher absolute level. We further identified CXCL10 as a robust soluble biomarker of monocyte activation, highlighting the potential utility of this chemokine as a prognostic marker. IMPLICATIONS: These findings may partially explain the increased prevalence of inflammatory age-related diseases in HIV-positive individuals and potentially indicate the pathological mechanisms underlying these diseases, which persist despite viral suppression.

Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG.

Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission.

Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation.

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART(-)), virologically suppressed patients receiving cART (ART(+)), and HIV-uninfected controls. CD8(+) T cells were activated, as assessed by CD38(+)HLA-DR(+) expression, in ART(-) patients (p < 0.0001), which was significantly reduced in ART(+) patients (p = 0.0005). In contrast, CD38(+)HLA-DR(+) NK cells were elevated in ART(-) patients (p = 0.0001) but did not decrease in ART(+) patients (p = 0.88). NK cells from both ART(-) and ART(+) patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART(+) patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.

HIV infection induces age-related changes to monocytes and innate immune activation in young men that persist despite combination antiretroviral therapy.

OBJECTIVES: To compare the impact of HIV infection and healthy ageing on monocyte phenotype and function and determine whether age-related changes induced by HIV are reversed in antiretroviral treated individuals. DESIGN: A cross sectional study of monocyte ageing markers in viremic and virologically suppressed HIV-positive males aged 45 years or less and age-matched and elderly (≥65 years) HIV-uninfected individuals. METHODS: Age-related changes to monocyte phenotype and function were measured in whole blood assays ex vivo on both CD14(++)CD16(-) (CD14(+)) and CD14(variable)CD16(+) (CD16(+)) subsets. Plasma markers relevant to innate immune activation were measured by ELISA. RESULTS: Monocytes from young viremic HIV-positive males resemble those from elderly controls, and show increased expression of CD11b (P < 0.0001 on CD14(+) and CD16(+)subsets) and decreased expression of CD62L and CD115 (P = 0.04 and 0.001, respectively, on CD14(+) monocytes) when compared with young uninfected controls. These changes were also present in young virologically suppressed HIV-positive males. Innate immune activation markers neopterin, soluble CD163 and CXCL10 were elevated in both young viremic (P < 0.0001 for all) and virologically suppressed (P = 0.0005, 0.003 and 0.002, respectively) HIV-positive males with levels in suppressed individuals resembling those observed in elderly controls. Like the elderly, CD14(+) monocytes from young HIV-positive males exhibited impaired phagocytic function (P = 0.007) and telomere-shortening (P = 0.03) as compared with young uninfected controls. CONCLUSION: HIV infection induces changes to monocyte phenotype and function in young HIV-positive males that mimic those observed in elderly uninfected individuals, suggesting HIV may accelerate age-related changes to monocytes. Importantly, these defects persist in virologically suppressed HIV-positive individuals.

Biomarkers of immune dysfunction following combination antiretroviral therapy for HIV infection.

Combination antiretroviral therapy (cART) has significantly reduced morbidity and mortality of HIV-infected patients, yet their life expectancy remains reduced compared with the general population. Most HIV-infected patients receiving cART have some persistent immune dysfunction characterized by chronic immune activation and premature aging of the immune system. Here we review biomarkers of T-cell activation (CD69, -25 and -38, HLA-DR, and soluble CD26 and -30); generalized immune activation (C-reactive protein, IL-6 and D-dimer); microbial translocation (lipopolysaccharide, 16S rDNA, lipopolysaccharide-binding protein and soluble CD14); and immune dysfunction of specific cellular subsets (T cells, natural killer cells and monocytes) in HIV-infected patients on cART and their relationship to adverse clinical outcomes including impaired CD4 T-cell recovery, as well as non-AIDS clinical events, such as cardiovascular disease.

Rapid detection of anti-Vaccinia virus neutralizing antibodies.

Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.

HIV inhibits early signal transduction events triggered by CD16 cross-linking on NK cells, which are important for antibody-dependent cellular cytotoxicity.

Measurement of NK cell cytolytic activity in the setting of chronic viral infection is important for determining viral pathogenicity. Mobilization of LAMP-1 (CD107a) to the NK cell surface is a surrogate marker for cytotoxic granule release and hence, NK cell cytotoxicity. We have developed a convenient, rapid, whole blood flow cytometric assay for measuring CD107a mobilization in response to CD16 cross-linking, a surrogate for NK cell ADCC activity ex vivo, which can be performed using small volumes of patient whole blood. Using this assay, we show that CD107a mobilization, in response to CD16 cross-linking, is triggered in CD56(dim) but not CD56(bright) NK cells, requiring Syk/Zap70 tyrosine kinase activity, and that there is a significant correlation between CD107a mobilization and pSyk/Zap70 in response to CD16 cross-linking. We compared whole blood from treatment-naïve, HIV-infected patients with age- and sex-matched HIV-uninfected control subjects and found a significant reduction in CD16-dependent pSyk/Zap70 (median=32.7% compared with 67.8%; P=0.0002) and CD107a mobilization (median=9.72% compared with 32.9%; P=0.046) in NK cells. Reduction of both correlated strongly with reduced CD16 surface expression on NK cells of HIV-infected individuals (P<0.01). These data suggest that ADCC is inhibited in NK cells from therapy-naïve, HIV-infected individuals at the level of early events in CD16 signal transduction, associated with low CD16R expression, and our method is a useful and reliable tool to detect pathological defects in NK cell degranulation.