Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. http://www.sciencemag.org/about/science-licenses-journal-article-reuseThis is an article distributed under the terms of the Science Journals Default LicenseIn HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. A better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidence of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)-related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.This study was supported by the NIH Intramural Research Program, National Institute of Allergy and Infectious Diseases, and Bench-to-Bedside award R01HL117715-10S1 (to I.S. and I.P.). Part of this project has been also funded with federal funds from the National Cancer Institute, NIH, under contract no. HHSN261200800001E. The NHP study has also been funded in part with federal funds from the NIH (R01 HL123096 and RO1 HL117715 to I.P., R01 AI119346 to C.A., and R01AI104373 to R.M.R.).info:eu-repo/semantics/publishedVersio

Influence of Cytokines on HIV-Specific Antibody-Dependent Cellular Cytotoxicity Activation Profile of Natural Killer Cells

There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate “educated” KIR3DL1+ NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate “uneducated” KIR3DL1+ NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy

Inhibition of human immunodeficiency virus type 1 tat-trans-activation-responsive region interaction by an antiviral quinolone derivative

WM5, a 6-aminoquinolone derivative, binds with high affinity to the bulge of the trans-activation-responsive region (TAR), whereas it displays low binding affinity for the loop and stem regions of TAR and for random RNA and DNA sequences. Furthermore, WM5 disrupts the natural protein-nucleic acid complex with a 50% inhibitory concentration in the low micromolar range in both in vitro and in vivo assays

Characterization of immune responses elicited in mice by intranasal co immunization with HIV-1 Tat, gp140 \u394V2Env and/or SIV Gag proteins and the nontoxicogenic heat-labile Escherichia coli enterotoxin.

The development of a vaccine against HIV/AIDS capable of inducing broad humoral and cellular responses at both systemic and mucosal sites, able to stop or reduce viral infection at the portal of entry, represents the only realistic way to control the infection caused by HIV world-wide. The promising results obtained with the HIV-1 Tat-based vaccines in preclinical and clinical settings, the evidence that a broad immunity against HIV correlates with reduced viral load or virus control, as well as the availability of novel gp140 V2-loop deleted HIV-1 Env (DeltaV2Env) immunogens capable of inducing cross-reactive neutralizing antibodies, have led to the design of new vaccine strategies based on the combination of non-structural and structural proteins. In this study, we demonstrate that immunization with a biologically active HIV-1 Tat protein in combination with the oligomeric HIV-1 gp140 DeltaV2Env and/or SIV Gag proteins, delivered intranasally with the detoxified LTK63 mucosal adjuvant, whose safety has been recently shown in humans, elicits long-lasting local and systemic antibody and cellular immune responses against the co-administered antigens in a fashion similar to immune responses induced by vaccination with Tat, DeltaV2Env and Gag proteins alone. The results indicate lack of antigen interference implying that HIV-1 Tat is an optimal co-antigen for combined vaccine strategies employing DeltaV2Env and/or Gag proteins

The increase in intra-macrophage thiols induced by new pro-GSH molecules directs the Th1 skewing in ovalbumin immunized mice.

In the present work, the capacity of new pro-GSH molecules to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the immune response to Th1 in ovalbumin (Ova)-sensitized mice were examined. The molecules were the N-butanoyl GSH derivative, GSH-C4, and a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA), I-152. In vitro, 2h-incubation with both molecules was found to increase intra-macrophage thiol content; in vivo, Ova-sensitized mice pre-treated by intraperitoneal administration of the pro-GSH molecules showed an increase in plasma anti-Ova IgG2a and IgG2b, characterizing Th1 immune response, and a decrease in IgG1, typical of the Th2 response. Such findings were connected to a shift to a Th1 response also involving splenocyte IFN-\u3b3 production as revealed by ELISPOT assay and higher levels of IL-12 in circulation. Although immune responses are in vivo mediated both by dendritic cells and macrophages, the data reported in this paper corroborate the suggestion that the pro-GSH molecules, increasing the intra-cellular thiol pool, modulate the Th1/Th2 balance favouring Th1-type responses and may be employed as Th1-directing adjuvants in new vaccination protocols and as immunomodulators in those diseases where Th1 response patterns are compromised in favour of Th2

Preparation and Characterization of Innovative Protein-coated Poly(Methylmethacrylate) Core-shell Nanoparticles for Vaccine Purposes

PURPOSE: This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. MATERIALS AND METHODS: Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. RESULTS: The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. CONCLUSIONS: This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy

Induction of humoral and enhanced cellular immune responses by novel core-shell nanosphere- and microsphere-based vaccine formulations following systemic and mucosal administration

Anionic surfactant-free polymeric core-shell nanospheres and microspheres were previously described with an inner core constituted by poly(methylmethacrylate) (PMMA) and a highly hydrophilic outer shell composed of a hydiosoluble co-polymer (Eudragit L100-55). The outer shell is tightly linked to the core and bears carboxylic groups capable of adsorbing high amounts (antigen loading ability of up to 20%, w/w) of native basic proteins, mainly by electrostatic interactions, while preserving their activity. In the present study we have evaluated in mice the safety and immunogenicity of new vaccine formulations composed of these nano- and microspheres and the HIV-1 Tat protein. Vaccines were administered by different routes, including intramuscular, subcutaneous or intranasal and the results were compared to immunization with Tat alone or with Tat delivered with the alum adjuvant. The data demonstrate that the nano- and microspheres/Tat formulations are safe and induce robust and long-lasting cellular and humoral responses in mice after systemic and/or mucosal immunization. These delivery systems may have great potential for novel Tat protein-based vaccines against HIV-1 and hold promise for other protein-based vaccines

Preparation and characterization of innovative protein-coated poly(methylmethacrylate) core-shell nanoparticles for vaccine purposes.

PURPOSE: This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. MATERIALS AND METHODS: Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. RESULTS: The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. CONCLUSIONS: This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy