Introduction
Current HIV therapies employ combinations of small molecule inhibitors that target viral proteins at different steps in the HIV replication cycle in order to prevent the emergence of HIV resistance to therapy [1,2,3,4]. Despite this strategy, resistance to one or more drug classes can emerge, resulting in a population of patients requiring salvage therapy [5]. The development of new anti-HIV therapeutics that target host proteins important for the virus life cycle could circumvent the problem of viral resistance. Host cell proteins that influence viral replication are less mutable than viral proteins, possibly offering an increased genetic barrier to the development of drug resistance. An analogous therapeutic concept has already proven efficacious in the treatment of HCV: stimulation of the host innate immune response using interferonbased therapy effectively blocks viral replication without induction of viral resistance [6]. Endogenous serine protease inhibitors (serpins) are part of the early innate immune response to viral infection that includes mannose binding lectins, soluble CD14, defensins and antimicrobial peptides [7]. The main biologic function of serpins is the blockage of protease activity involved in blood clotting and complement activation. Serpins belong to a superfamily of proteins that also regulate other inflammatory processes [8].
Serine protease inhibitors have a broad spectrum of anti-viral activity against HIV, HCV, HSV and the influenza virus [9,10]. A number of clinical observations suggest a role for the serpins in controlling HIV infection and disease progression in the mucosa and the peripheral blood. For example, (1) there is a barrier to HIV transmission via the oral mucosa; this may be due to the antiviral activity of Secretory Leukocyte Inhibitor (SLPI) in saliva [11]. (2) a1-anti-trypsin, the most abundant serpin in blood, prevents HIV replication in vitro at physiological concentrations; in addition, HIV replicates at a much higher rate in the blood of a1-antitrypsin-deficient individuals, suggesting a1-anti-trypsin might reduce viral replication in vivo [12]. (3) The anti-HIV activity of a1anti-trypsin is believed to be responsible for the relatively low transmission rates of HIV through contaminated needles, compared to that of HCV and HBV. (4) Furthermore, presence of the a1-anti-trypsin allelic variants M2 and A332A is associated with enhanced HIV-1 acquisition [13]. Antithrombin III (ATIII), a serpin with a role in the coagulation cascade, exhibits potent anti-HIV activity. ATIII exists in three different forms under physiological conditions. In its inactive latent (L) form, ATIII circulates with its reactive COOH-terminal loop not fully exposed, thereby preventing its binding to thrombin. Upon binding to heparin, ATIII undergoes a conformational change to an activated, or stressed (S) form (here also termed hep1 ATIII) allowing the exposure of the reactive COOH-terminal loop thus increasing the binding of thrombin by 100-fold. The resultant ATIII-thrombin complex eventually dissociates with the release of thrombin and an ATIII with a cleaved reactive loop, inducing a conformational change of ATIII to a relaxed (R) form. A proteolytically cleaved form of ATIII was originally discovered to be a CD8+ T cell anti-HIV factor (CAF) – a noncytolytic innate immune response in HIV-1 long-term nonprogressors [14,15]. The S form of ATIII has greater antiviral activity against HIV and the simian immunodeficiency virus (SIV) than the R form; the L form has no anti-viral activity [14]. HepATIII is up to 10-fold more potent at inhibiting HIV than the nonactivated form of ATIII [16]. When compared to other serpins with anti-HIV activity, a1-antitrypsin and SLPI, heparin-activated antithrombin III (hep-ATIII) displays up to 106 fold higher antiHIV activity in vitro [11,12,14,16,17,18]. The anti-viral activity of hep-ATIII and ATIII is mediated at least in part by host cell factors prostaglandin synthetase 2 (PTGS2) and transcription factor NFkB [9]. Two hundred-fold less hep-ATIII was required as compared to non-activated ATIII to elicit equivalent changes in gene transcription of these host cell factors [9]. In the present study, we sought to validate hep-ATIII as an HIV therapeutic using in vitro, humanized mouse and preclinical primate models of HIV infection. In order to evaluate the potential utility of ATIII as a salvage agent in patients with multidrug resistant HIV, we assessed the ability of hep-ATIII to inhibit a range of drug-resistant HIV-1 isolates in vitro, and in humanized mice infected with highly drug resistant HIV-1. In addition, we studied the effects of ATIII treatment in rhesus macaques chronically infected with SIV. In a novel therapeutic approach, we used anti-HLA-DR antibodies engrafted into immunoliposomes to encapsulate hep-ATIII (termed ET-ATIII): it has been shown that these immunoliposomes specifically target HLA-DR positive cells in lymph nodes including monocytes, macrophages and activated CD4+ T lymphocytes, allowing concentration of therapeutic ATIII in the main cellular reservoirs of HIV and SIV [19].
Finally, we sought to understand the mechanism by which hep-ATIII exerts its antiviral activity. We studied the gene expression profiles of peripheral blood mononuclear cells (PBMC) from SIV-infected macaques treated with hep-ATIII, and identified the transcriptional networks activated or repressed by hep-ATIII treatment. By elaborating the biologic networks associated with HIV inhibition by the innate immune system, we hoped to identify potential biomarkers of drug efficacy, as well as potential future drug targets.the Assessment and Accreditation of Laboratory Animal Care International. Research was conducted in compliance with the Animal Welfare Act and other US federal statutes and regulations relating to animals and experiments involving animals, and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. All steps were taken to ameliorate the welfare and to avoid the suffering of the animals in accordance with the “Weatherall report for the use of non-human primates” recommendations. Animals were housed either socially or in adjoining individual primate cages allowing social interactions, under controlled conditions of humidity, temperature and light (12-hour light/12-hour dark cycles). Food and water were available ad libitum. Animals were fed commercial monkey chow and treats by trained personnel. Environmental enrichment consisted of commercial toys. Blood draws were conducted under sedation by trained personnel under the supervision of veterinarians.MiceNOD/scid/scid-beta-2 microglobulin (b2m) knockout mice (Nod/Scid/b2mnull mice) (6? weeks) and C57BL/6 mice were from the Jackson Laboratory (Bar Harbor, ME).
