Cells were washed of excess virus and 36106 hPBMC wereComplete Blood Count and Peripheral Blood CD4+ T Lymphocyte Count for Rhesus Macaques and C57BL/6 Mice
Complete Blood Count (CBC) testing white blood cell count (WBC), red blood cells (RBC), hemoglobolin (HGB), hematocrit (HCT), mean red cell volume (MCV), mean cell hemoglobin (MCH), platelet count (PLT) and mean platelet volume (MPV) was performed using the ADVIA Hematology System (Bayer, Leverkusen, Germany) according manufacturers protocol. Peripheral blood CD4+ T lymphocyte counts were calculated by multiplying the total lymphocyte count by the percentage of CD3+CD4+ T cells determined by mAb staining and flow cytometric analysis [32].
Data Analysis
The statistical significance of differences between groups was determined using the program GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA). A P value of ,0.05 was considered statistically significant. Statistical analysis was performed by use ofthe Mann-Whitney test, paired T-test and the DDCt method. Error bars represent standard error of the mean (S. E.).Anti-viral Activity of Activated and Non-activated ATIII in a Single-round HIV Pseudovirus Inhibition Assay
We used the single-round HIV inhibition assay to measure the anti-viral activity of activated ATIII, hep-ATIII, and compared that with unmodified ATIII, the 135 kDa ATIII complex and a heparin control. This system allowed the measurement of inhibition effects on all phases in a virus’ life-cycle during one round of replication. To investigate the susceptibility of different HIV-1 envelopes to hep-ATIII inhibition we used pseudoviruses with clade B (PVO.4, QH0692.42) and clade C (Du123.6, Du151.2) envelopes. We found that the 66?8 kDa fraction, containing heparin-activated ATIII, exhibited anti-viral activity. The IC50 of hep-ATIII was between 20?00 nM, and independent of envelope usage (Fig. 2). Unmodified ATIII had no inhibitory activity (Fig. 2) as well as the 135 kDa ATIII polymer (data not shown). Both demonstrated no inhibition as defined as inhibition below 25% in the pseudovirus inhibition assay. We also observed that heparin had an IC50 of 8 mM (Fig. 2), comparable to what has been previously reported.
Results Anti-viral Activation of ATIII
Anti-viral capacity of ATIII was activated by overnight incubation with heparin at 37uC. This resulted in a mixture containing three compounds, which we subsequently separated by Sephacyl S100 FPLC. We found 1?% w/w of a 135 kDa ATIII polymer with a retention volume of 7? ml. Ninety-five to ninetynine % (w/w) of the mixture was 66?8 kDa hep-ATIII, in which heparin was bound to ATIII, at a retention volume of 10?4 ml. At approximately 35 kDa a protein-free fraction comprised of unbound heparin polymer was contained (15?9 ml retention volume) (Fig. 1A). The amount of this free heparin was calculated to be routinely below 5% (w/w) of the total mixture. We used fractionated hep-ATIII for our experiments. We confirmed the purity of our fractionated hep-ATIII (66?68 kDa) preparations by SDS-PAGE and silver staining. We found that these hep-ATIII fractions were routinely more than 99% pure (Fig. 1B).Anti-viral Activity of Activated and Non-activated ATIII in PBMC Acutely Infected with Drug-resistant HIV-1
Anti-HIV-1 therapy that targets viral proteins must contend with the virus’ ability to rapidly evolve leading to the emergence of?Figure 1. Characterization of heparin-activated ATIII. (A) Sepharcyl S100 AKTA FPLC of purified hep-ATIII. A 9?4 ml fraction was separated, termed as hep-ATIII and used for our experiments. Detection at 260 nm for protein detection and 280 nm for heparin detection is shown. (B) HepATIII purity and molecular weight were also determined by SDS-PAGE and silver staining (Bio-Rad kit) of a 15% slab gel. For molecular weight determination a low-range protein molecular weight marker (Bio-Rad) was used. Lane 1:2 mg hep-ATIII; lane 2: phosphorylase B (97 kDa), serum albumin (69 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), aprotinin (6.5 kDa).Figure 2. Effect of heparin-activated ATIII in pseudovirus inhibition assay. Pseudoviruses with clade B envelopes (in A and C) and clade C envelopes (in B and D) were treated with three fold dilutions of heparin-activated ATIII (hep-ATIII), unmodified ATIII and heparin. Percentage of inhibition was calculated by comparing of residual luciferase activity and untreated control. Experiments were done in triplicates. Data are shown as mean 6 S.E. drug resistant isolates. Antiviral therapy that targets host proteins while stimulating host innate immunity may be able to circumvent antiviral resistance. We have previously shown that non-activated ATIII exerts limited anti-HIV-1 activity and has only limited effects on host cell gene expression. The anti-HIV-1 activity of ATIII can be augmented through overnight incubation with heparin [16], which results in non-covalent attachment of heparin to ATIII, creating activated hep-ATIII. We sought to determine if hep-ATIII could overcome current limitations in drug treatment options for multidrug resistant HIV. We tested the anti-HIV activity of hep-ATIII against an array of primary isolates from different clades, with T-cell tropism (X4), macrophage tropism (R5) or dual-tropism (X4R5), and differing in drug resistance profiles. After infection of hPBMC with the various HIV isolates, we then added hep-ATIII at the indicated concentrations (at day 1 and 4 of infection), and followed the infection by p24-antigen ELISA for 7 days. We then used the resulting dose-response curves for day 7 to calculate IC50 values. Our experiments demonstrated that hep-ATIII exhibited potent anti-HIV activity that was independent of prior drug exposure, clade or co-receptor usage (Table 1). Importantly, we found that the anti-viral activity of hep-ATIII was observed at therapeutically favorable levels with IC50 values ranging from 16?40 nM (Table 1), which was similar to those observed in the pseudovirus inhibition assays (Fig. 2).
Effect of Hep-ATIII on HIV-induced Cytotoxicity in Engrafted hPBMC in Nod/Scid/b2mnull Mice
We hypothesized that the immunomodulatory effects of hepATIII might not only inhibit HIV replication in target cells, but may also protect infected cells and uninfected bystander cells from HIV-related cytotoxic effects. We utilized the hPBMC engrafted, HIV-infected Nod/Scid/b2mnull mice to test this hypothesis.
enabling superior engraftment of hPBMC compared to other Nod/Scid mice [30]. To simulate therapy of the challenging multidrug resistant HIV patient, we engrafted mice with hPBMC that were infected in vitro with a highly resistant HIV-1 isolate. We assessed whether daily treatment with low doses of hep-ATIII (25 nmol/kg) might reduce virus-induced hPBMC cytotoxicity. We used 5 Nod/Scid/b2mnull mice per non-engrafted group, nonengrafted vehicle group, vehicle treated uninfected group, hepATIII treated uninfected group, vehicle treated infected group and hep-ATIII treated infected group (Fig. 3). We utilized a strain of HIV-1 that produced more than 90% virus-associated cell death of infected hPBMC in mice. We found that there was a 100% increase in splenocyte number after 14 days of treatment with hepATIII (P = 0.008, Mann-Whitney test) (Fig. 3) compared to untreated HIV-infected hPBMC engrafted control mice, suggesting protection of hPBMC from HIV cytotoxicity. This demonstration of in vivo efficacy of hep-ATIII against HIV in the humanized mouse model prompted us to further test hepATIII in a more sophisticated non-human primate model of chronic lentiviral infection.
