We assessed the viral inhibitory activity of unencapsulated hepATIII, hep-ATIII encapsulated into conventional liposomes lacking anti-HLA-DR, and ET-ATIII against the dual-tropic (X4R5) HIV 89.6, and the macrophage-tropic (R5) SF162 in cell culture. These two HIV-1 isolates are derived from two different viral reservoirs: 89.6 was isolated from PBMC, and SF162 from the cerebrospinal fluid. 89.6 is a prototype for syncytium-forming, highly cytopathic HIV-1 viruses that replicate to high titers in cells bearing either CXCR4 or CCR5 receptors, reflective of isolates that may be found in individuals with AIDS [41]. In contrast, SF162 has more restricted co-receptor usage, is less cytopathic and more typical of primary, transmitted HIV isolates [42]. We infected hPBMC with SF162 and 89.6, and treated cells with hep-ATIII either unencapsulated or encapsulated into eitherconventional liposomes or sterically-stabilized anti-HLA-DR immunoliposomes (ET-ATIII) (Table 2). We found that encapsulation into immunoliposomes increased the anti-viral potency of hep-ATIII 107?50old, resulting in sub-nanomolar IC50 values of 0.4?.7 nM. In contrast the use of conventional liposomes had only a limited effect on hep-ATIII potency. The IC90 for immunoliposomes was 3.7 nM for the 89.6 strain and 4.1 nM for the SF162 strain. We also compared the anti-HIV activity of ETATIII to that of the integrase inhibitor, 118-D-24. We observed an IC50 for 118-D-24 of 1750 nM, similar to what has been previously reported [28], suggesting a 1000-fold greater activity for ET-ATIII on a molar basis (Table 2). A vehicle liposome control was used for both liposome constructs to confirm that the liposomal vehicle did not have antiviral properties itself, consistent with prior studies [43]. Conventional liposomes and sterically-stabilized immunoliposomes have been used to encapsulate imaging reagents, small molecule drugs and proteins in both pre-clinical experiments and clinical settings, including in HIV patients, to minimize drug sideeffects by increasing target tissue specificity [19,40,43,44,45,46,47]. There has been no cell toxicity reported after administration of proteins encapsulated in conventional or sterically stabilized liposomes [43,48,49].
ET-ATIII up to 30 nmol/kg, 100-fold the effective dose for in vivo SIV inhibition. We measured the complete blood count (CBC) to assess for the hematologic effects of the ET-ATIII formulation. In particular, the hematocrit (HCT) was used as a surrogate marker for internal bleeding, a potential side effect of the use of hepATIII. We found no significant change in animal weight, white blood cell count or HCT. All other blood parameters as well as liver function tests were within the expected normal range (data not shown). Thus, no in vivo cytotoxicity could be detected in mice at concentrations significantly in excess of the therapeutically effective anti-viral dose.
Gene Expression in PBMC from Hep-ATIII Treated Rhesus Macaques
The exact molecular mechanisms by which hep-ATIII exerts its anti-HIV effect in vivo is unknown. We used transcriptional profiling of PBMC taken from SIV-infected, hep-ATIII-treated rhesus macaques to identify host molecular pathways that might contribute to viral suppression in an extension of our prior in vitro investigations [9]. We compared the transcriptional profiles of 47,000 mRNAs at day 7 post-hep-ATIII therapy to the pretreatment controls, when SIV inhibition was maximal. We also compared transcriptional profiles from day 15 post-therapy, when SIV inhibition had dissipated, to pre-treatment controls. We found that the expression of only a limited number of genes were significantly affected (P,0.01, DDCt method) by hep-ATIII therapy when compared to pretreatment (Fig. 6). We grouped genes into three groups dependent on the gene-expression pattern at the treatment time point compared to pre-treatment (Fig. 6A/ B/C; abbreviations of gene names are specified in Table S1). (1) We identified 18 genes and 15 gene loci that were over-expressed at both day 7 and day 15 in relation to pretreatment (Fig. 6A). (2) We found 20 genes and 12 loci that were significantly downregulated at the time of maximal inhibition (day 7) compared to pre-treatment controls, but were up-regulated by day 15 compared to pre-treatment controls, when inhibition had diminished (Fig. 6B). (3) One gene and 2 loci were up-regulated after 7 days but down-regulated at day 15 (Fig. 6C). We also found that six genes and 7 loci were down regulated at both time points after therapy (Fig. 6C).
index (TI) of our liposomes we tested for cytotoxicity over a wide dose range. We calculated TI as the ratio of the 50% cytotoxic dose (CD50) to the IC50. To measure the CD50 we tested the effect of ET-ATIII on cell viability using the Guava ViaCountH Assay. For these assays we used hPBMC from an HIV-1 infected patient as the indicator of cytotoxicity, as these best reflect cell populations that would be targeted by the liposome treatment in vivo. Additionally, we also assessed the toxicity of the liposomes against endothelial cells as ATIII is known to affect this cell type, inducing the release of anti-inflammatory prostacyclin and prostaglandins [7,8]. We found no significant decrease in viability of either cell population in response to an escalating dose of encapsulated hep-ATIII (3?0 nM). We found that encapsulated hep-ATIII had a very favorable TI of .100. A TI of .10 is considered feasible for an antiviral drug.
In vivo Anti-viral Activity of hep-ATIII after Encapsulation into Sterically Stabilized Anti-HLA-DR Immunoliposomes
Under normal physiologic conditions, ATIII is not detectable in the lymphatic system [50]. As the lymph nodes are a major compartment for HIV-1 replication, we hypothesized that targeting hep-ATIII directly to the lymphatic system may increase the in vivo activity of hep-ATIII in our chronically SIVinfected macaque model. It has been previously shown that sterically stabilized anti-HLA-DR immunoliposomes accumulate in cervical and brachial lymph nodes, suggesting that they may effectively target HLA-DR positive cells, i.e. monocytes/macrophages and activated CD4+ T lymphocytes that are the primary cellular targets of HIV-1 [19]. Hep-ATIII was encapsulated in immunoliposomes (0.05 mg/mL) of 100 nm diameter with 46104 mol/ml anti-HLA-DR antibody incorporated. We subcutaneously injected chronically two SIV-infected animals with 0.3 nmol/kg of ET-ATIII. The overall quantity of hep-ATIII inoculated was 2000-fold less than the dose of unencapsulated hep-ATIII used in our previous in vivo experiments. We determined this dose based on two prior findings: firstly, our in vitro inhibition data suggested that ET-ATIII was at least 23fold more effective than hep-ATIII compared to the lowest IC50 found for the resistant strains (Table 1); secondly, prior reports have suggested a 100-fold increase of efficacy in antiHIV-1 drugs activity when these are delivered directly to lymph tissue [40]. We observed that ET-ATIII decreased plasma SIV viral RNA by an average of 2 log10 (range 0.97?.5 log10) at day 7 (Fig. 5A/B) – 5 days after the 2nd treatment, demonstrating the potent anti-viral activity of ET-ATIII. Control immunoliposomes did not alter viral load (Fig. 5B).