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New Inhibitor of HIV gp120: N-Butyldeoxynojirimycin is a broadly effective anti-HIV therapy significantly enhanced by targeted liposome delivery
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AIDS:Volume 22(15)1 October 2008p 1961-1969
Pollock, Stephaniea; Dwek, Raymond Aa; Burton, Dennis Rb; Zitzmann, Nicolea
aOxford Antiviral Drug Discovery Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, UK
bDepartments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
"In this report, we have re-introduced NB-DNJ as a potential addition to the anti-HIV arsenal. With modern-day advances in drug delivery systems and an obvious need for new molecular targets, the usefulness of NB-DNJ as a broadly active antiviral is now revisited and further optimized. By targeting the host pathway of calnexin-mediated glycoprotein folding, we hope to have exposed a major weakness within HIV, minimizing the possibility for viral escape. Using a diverse panel of HIV-1 isolates from different genetic subtypes and geographic regions, NB-DNJ inhibited viral infectivity in all isolates assayed by an average of 80%....In summary, we demonstrate the ability of targeted NB-DNJ liposomes to treat HIV infections by efficiently reducing viral infectivity to below detectable levels. Because of their broad range of activity, targeted NB-DNJ liposomes could be used in both salvage therapy and during the early stages of infection. With different molecular targets, the combination of NB-DNJ and current antiretrovirals should have an even greater effect, allowing for overall lower drug doses and less toxicity. Similar strategies are currently being developed for the treatment of other viral infections sensitive to glucosidase inhibition.....Therapies targeting virus-dependent host pathways, rather than the virus, provide an attractive strategy for the development of broad-spectrum antivirals, as the development of escape mutations is less likely. An example of this type of antiviral activity is seen with the endoplasmic reticulum α-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), which has been shown to be effective against a range of human viruses including HIV, hepatitis C virus (HCV), hepatitis B virus (HBV), parainfluenza, West Nile and Dengue viruses....NB-DNJ CD4 liposomes demonstrated additional antiviral effects, reducing viral secretion by 81% and effectively neutralizing free viral particles to prevent further infections."
Abstract
Objective: N-Butyldeoxynojirimycin (NB-DNJ), an inhibitor of HIV gp120 folding, was assessed as a broadly active therapy for the treatment of HIV/AIDS. Furthermore, to reduce the effective dose necessary for antiviral activity, NB-DNJ was encapsulated inside liposomes and targeted to HIV-infected cells.
Methods: Thirty-one primary isolates of HIV (including drug-resistant isolates) were cultured in peripheral blood mononuclear cells to quantify the effect of NB-DNJ on viral infectivity. pH-sensitive liposomes capable of mediating the intracellular delivery of NB-DNJ inside peripheral blood mononuclear cells were used to increase drug efficacy.
Results: NB-DNJ decreased viral infectivity with a single round of treatment by an average of 80% in HIV-1-infected and 95% in HIV-2-infected cultures. Two rounds of treatment reduced viral infectivity to below detectable levels for all isolates tested, with a calculated IC50 of 282 and 211 νmol/l for HIV-1 and HIV-2, respectively. When encapsulated inside liposomes, NB-DNJ inhibited HIV-1 with final concentrations in the nmol/l range (IC50 = 4 nmol/l), a 100 000-fold enhancement in IC50 relative to free NB-DNJ. Targeting liposomes to the gp120/gp41 complex with a CD4 molecule conjugated to the outer bilayer increased drug/liposome uptake five-fold in HIV-infected cells compared with uninfected cells. NB-DNJ CD4 liposomes demonstrated additional antiviral effects, reducing viral secretion by 81% and effectively neutralizing free viral particles to prevent further infections.
Conclusion: The use of targeted liposomes encapsulating NB-DNJ provides an attractive therapeutic option against all clades of HIV, including drug-resistant isolates, in an attempt to prevent disease progression to AIDS.
Introduction
Numerous therapies against HIV/AIDS have been developed that have greatly improved both the quantity and quality of life of those infected. Unfortunately, drug-resistant isolates exist for every antiviral currently in use, and approximately 20% of all new HIV-1 infections involve drug-resistant variants [1]. Efforts toward the discovery of novel drug targets must persist if continued viral repression and, ultimately, virus eradication are to be achieved. Therapies targeting virus-dependent host pathways, rather than the virus, provide an attractive strategy for the development of broad-spectrum antivirals, as the development of escape mutations is less likely. An example of this type of antiviral activity is seen with the endoplasmic reticulum α-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), which has been shown to be effective against a range of human viruses including HIV, hepatitis C virus (HCV), hepatitis B virus (HBV), parainfluenza, West Nile and Dengue viruses [2-7]. In this case, the protective glycan shield used by the virus to evade host immune responses may, in fact, present a vulnerable target for therapy.
The HIV envelope glycoprotein, gp160 (gp120/gp41 precursor), is glycosylated and folded by the concerted actions of molecular chaperones and a system for processing oligosaccharides within the lumen of the endoplasmic reticulum. In HIV infections, blocking gp160 entry into the calnexin-mediated glycoprotein folding pathway (or calnexin cycle) by inhibiting the process of glucose trimming results in a localized conformational defect within the V1/V2 hypervariable loops of gp120 so that newly formed viral particles are less infectious [8,9]. NB-DNJ treatment does not affect the interaction of gp120 with CD4; however, upon binding, exposure of gp41 fusion peptides is reduced and there is no fusion of viral and host membranes [3,10]. To date, there are no known escape mutants against NB-DNJ treatment.
NB-DNJ has been tested in combination with several common antiretroviral therapies both in cell culture and in the clinic, and, although antiviral effects were evident, the concentrations necessary to achieve significant activity in vivo were excessive [11-14]. Observed side-effects at increased concentrations were due to inhibition of intestinal disaccharides in vivo, which led to carbohydrate malabsorbtion and gastrointestinal disturbances (diarrhea, gastric irritation, nausea, and emesis). In these trials, NB-DNJ was administered orally; therefore, correct localization for antiviral activity was dependent on passive diffusion of this water-soluble molecule across both plasma and endoplasmic reticulum membranes. Drug delivery systems in which water-soluble molecules are directly delivered into the intracellular space should bypass these membrane barriers, significantly increasing the effective potency. For this problem, pH-sensitive liposomes provide an attractive solution as their ability to efficiently mediate the intracellular delivery of a wide variety of molecules has been reported [15,16].
In this report, NB-DNJ was tested for the first time in monotherapy against a genetically diverse panel of 29 HIV-1 isolates and two HIV-2 isolates. Liposomes encapsulating NB-DNJ were used to enhance drug activity and further optimized by targeting to HIV-infected cells. Finally, the emergence of NB-DNJ-resistant variants of HIV-1 was monitored over a 15-week period.
Discussion
In this report, we have re-introduced NB-DNJ as a potential addition to the anti-HIV arsenal. With modern-day advances in drug delivery systems and an obvious need for new molecular targets, the usefulness of NB-DNJ as a broadly active antiviral is now revisited and further optimized. By targeting the host pathway of calnexin-mediated glycoprotein folding, we hope to have exposed a major weakness within HIV, minimizing the possibility for viral escape.
Using a diverse panel of HIV-1 isolates from different genetic subtypes and geographic regions, NB-DNJ inhibited viral infectivity in all isolates assayed by an average of 80%. Two HIV-2 isolates were included in our assays and were shown to be particularly sensitive to NB-DNJ, reducing viral infectivity by an average of 95%. Our most important finding, however, is that we were able to render all isolates completely noninfectious with just two rounds of NB-DNJ treatment. Overall, NB-DNJ treatment was shown to be similarly effective against all clades of HIV-1 assayed. Mutations conferring resistance to current antiretrovirals had no effect on NB-DNJ activity; therefore, NB-DNJ could be used in both salvage therapy and during early stages of infection.
In addition to its antiviral activity, NB-DNJ treatment will also affect the folding of host glycoproteins; however, the continued use of NB-DNJ (Zavesca) for treatment of Gaucher's disease suggests this drug is well tolerated in low doses [22]. The side effects observed during HIV clinical trials were due to a separate activity against the sucrase isomaltose enzyme of the intestinal lumen [14], and, although this side-effect is easily managed, the excessive concentrations needed to decrease viral titers and increase CD4+ cell counts highlight the need for an alternative delivery method. To address this issue, we have successfully utilized a liposome-based drug delivery system in an attempt to lower the effective dose of NB-DNJ necessary for antiviral activity. When encapsulated inside pH-sensitive liposomes, the mean IC50 value for NB-DNJ treatment of HIV-1 dropped to 4.0 nmol/l, signifying a 100 000-fold enhancement compared to free delivery. This concentration of drug therapy is now well below the limit approved by the FDA for safe NB-DNJ treatment in patients.
CD4 liposomes and antigp120/gp41 immunoliposomes are used to target HIV-infected cells in an attempt to further optimize NB-DNJ delivery. Only CD4 was able to target all infected cells with an average increase of 460% in drug/liposome uptake compared with uninfected cells. The usefulness of immunoliposomes was more isolate or clade specific or both and corresponds with neutralization data for each antibody [23]. Targeting drug-loaded liposomes directly to virus-infected cells increased the specificity of NB-DNJ delivery to infected cells and simultaneously neutralized free viral particles significantly reducing infectivity through a secondary mechanism. CD4 liposomes were also shown to decrease the secretion of HIV, which may be due to interactions between the CD4-conjugated lipids and gp160/gp120 inside the cell.
Based on a strong correlation between NB-DNJ IC50 and N-glycans spanning the C2/V3 regions of gp120, we speculate that these sites may be important for interactions with calnexin and associated disulfide isomerases within the endoplasmic reticulum. gp160 molecules inherently predisposed to misfolding may have evolved to accumulate a greater number of N-glycan sites within this cluster, favoring calnexin recognition and prolonging associations with crucial folding enzymes. Glycans within this site are predominantly oligomannose and their positions within the amino acid sequence are more strongly conserved than those associated with complex-type carbohydrates [24]. A detailed analysis of glycan diversity within divergent HIV quasi-species has demonstrated a remarkable degree of convergent evolution at the C2 domain [25], highlighting the importance of these glycans for viral fitness and suggesting a constraint on the potential diversity of HIV.
In an attempt to force the development of HIV mutants resistant to the treatment of NB-DNJ, assays with extended viral passages of HIV-infected PBMCs in the presence of free or encapsulated drug were conducted over a 15-week period. Throughout the treatment period, no NB-DNJ-resistant HIV-1 variants were detected within the population of four individual isolates, suggesting the virus is incapable of escaping from this type of inhibition.
In-vivo treatment regimes based on liposome-mediated delivery of NB-DNJ will likely consist of subcutaneous injections resulting in liposome uptake by draining lymphatic capillaries and active capture by macrophages in regional lymph nodes [26]. The use of long-circulating, slow-release liposome formulations would aid in minimizing the burden of constant injections, promoting patient compliance. Because of its mode of action, NB-DNJ treatment in vivo would not clear HIV from cells already infected. Instead, production of noninfectious viral particles would prevent further spread within the body, allowing for the regeneration of CD4+ cells, and preventing disease progression to AIDS. Moreover, widespread treatment of HIV infections with NB-DNJ could conceivably reduce the spread of the virus, as those receiving treatment would be transmitting viral particles less capable of establishing new infections.
In summary, we demonstrate the ability of targeted NB-DNJ liposomes to treat HIV infections by efficiently reducing viral infectivity to below detectable levels. Because of their broad range of activity, targeted NB-DNJ liposomes could be used in both salvage therapy and during the early stages of infection. With different molecular targets, the combination of NB-DNJ and current antiretrovirals should have an even greater effect, allowing for overall lower drug doses and less toxicity. Similar strategies are currently being developed for the treatment of other viral infections sensitive to glucosidase inhibition.
Results
We sought to quantify the effect of NB-DNJ on the infectivity of 25 genetically and geographically diverse isolates of HIV-1, four drug-resistant isolates of HIV-1, and two isolates of HIV-2 using a single round infectivity assay. One millimolar per liter NB-DNJ was shown to effectively inhibit glucosidase activity with no cytotoxicity (data not shown), and was therefore chosen as the maximum drug concentration for all assays. On average, the incubation of infected PBMCs with 1 mmol/l NB-DNJ reduced HIV secretion by 44% (data not shown). The infectivity of HIV particles secreted from NB-DNJ-treated cells was determined by evaluating their ability to infect naive PBMCs. Secretion of HIV from cells infected with treated and untreated virus is shown in Fig. 1a. On average, NB-DNJ reduced the infectivity of all 31 isolates by 80% (SEM = 2.3%). HIV-2-MIR was the most sensitive to treatment (99% reduction) and 92UG021 the most resistant (56% reduction).
The effect of NB-DNJ (concentrations ranging from 0 to 1 mmol/l) was assessed over three rounds of treatment in all isolates to monitor viral infectivity over time. In these assays, HIV secretion reached below detectable limits by the third round of treatment for all isolates, signifying the inability of treated cells to produce infectious virions during the second round of NB-DNJ treatment at concentrations of more than 750 νmol/l. IC50 values for each isolate are listed in Fig. 1a, and profiles of HIV secretion throughout NB-DNJ treatment are shown for the most sensitive (93TH051) and the most tolerant (93RW024) isolates as references (Fig. 1b). The mean IC50 values for NB-DNJ activity against HIV-1 and HIV-2 were calculated to be 282 νmol/l (SEM = 2.1 νmol/l) and 211 νmol/l (SEM = 5.3 νmol/l), respectively. Neither clade nor co-receptor specificity appears to have an influence on IC50 values. NB-DNJ activity is not affected by the resistance of HIV-1 to common antiretroviral therapies.
To examine the effects of gp120 N-glycosylation on NB-DNJ sensitivity, gp120 protein sequences were obtained for 18 HIV-1 isolates, and occupied N-glycan sites were predicted using the NetNGlyc 1.0 server. IC50 values of individual HIV-1 isolates were plotted against the number of occupied sites in the entire gp120 molecule as well as individual gp120 domains. Results demonstrate a strong negative correlation between IC50 and a cluster of sites covering the C2/V3 domains (r2 = 0.66; Table 1). In other words, 66% of the variation in NB-DNJ sensitivity between isolates can be explained by differences in the number of occupied glycan sites on the C2/V3 region, so that isolates with more glycans are more sensitive to NB-DNJ treatment. None of the other gp120 domains demonstrated a significant correlation with the IC50 data set (Table 1).
The antiviral activity of NB-DNJ when encapsulated inside pH-sensitive liposomes was compared with free drug delivery. Assays to measure HIV infectivity over three rounds of treatment were carried out as previously described, except treatments included liposomes encapsulating NB-DNJ at final concentrations ranging from 0 νmol/l (encapsulated PBS) to 3.75 νmol/l. Figure 2 shows average viral secretion following the third round of treatment with both free and liposome-encapsulated NB-DNJ for 12 diverse HIV-1 isolates. Overall, calculated IC50 values for NB-DNJ liposome treatment averaged 4 nmol/l (SEM = 0.1 nmol/l), representing a 100 000-fold enhancement compared with free NB-DNJ treatment. Table 2 lists IC50 values for isolates treated with free NB-DNJ and NB-DNJ liposomes, as well as the corresponding enhancement as a result of intracellular delivery. Liposomes encapsulating PBS had no effect on viral infectivity.
Increased cellular uptake by targeting liposomes to the gp120/gp41 complex on the surface of HIV-1-infected cells was measured following conjugation of a soluble CD4 molecule, or monoclonal antibodies known to bind this complex (IgGs 2F5, 2G12, 4E10, and b12) [18-20], onto the surface of NB-DNJ liposomes. The CD4 and IgG molecules were modified to form free thiols for cross-linking to 3% maleimide-PE/1% rhodamine-PE-containing liposomes via a thioester bond, with an average yield of 30 νg protein/νmol lipid. The 4E10-liposome conjugate was excluded as this antibody caused liposome precipitation.
Figure 3a represents the uptake of CD4 liposomes and immunoliposomes encapsulating NB-DNJ (final concentration = 375 nmol/l) in HIV-infected PBMCs, which is expressed as the percentage of liposome uptake per cell in relation to the 'non-targeted liposome, no infection' control. The relative uptake data were tested separately for each infection using a series of one-way analyses of variance followed by post-hoc Tukey tests to test for differences between the effectiveness of different targeting molecules. 2G12-liposomes did not target any of the nine isolates tested, most likely a result of the unique structure of this antibody necessary for epitope recognition [21], and was excluded from the data set. For each HIV infection, there are significant differences in uptake between CD4 liposomes and the nontargeted liposome control (P < 0.0001), with an average increase of 460% (SEM = 9.4%; range 331-534%). b12-liposomes were able to target six of nine infections with an average increase of 394% (SEM = 10.2%; range 307-452%). 2F5-liposomes targeted five of nine infections with an average increase of 363% (SEM = 6.6%; range 337-399%). None of the targeting molecules caused a significant increase in liposome uptake by nonspecific interactions.
CD4 liposomes were used in infectivity assays to compare their activity to that seen with nontargeted NB-DNJ liposomes. Assays were performed as described and included a group of eight primary isolates treated with final NB-DNJ concentrations ranging from 0 to 375 nmol/l encapsulated inside liposomes. The increase in drug/liposome uptake as a result of targeted delivery should correspond to a lower effective dose for NB-DNJ treatments; however, drug activity could not be properly quantified as CD4 liposomes, including those encapsulating PBS, neutralized secreted virions during the initial treatment preventing a second round of infection (Fig. 3b). In addition to neutralizing free viral particles, 375 nmol/l NB-DNJ CD4 liposomes led to a decrease in HIV secretion when compared with the results seen with 375 nmol/l NB-DNJ non-targeted liposomes. As shown in Fig. 3c, the mean decrease in p24 secretion as a direct result of the CD4-lipid conjugate was 65% (SE = 4.6%; range 53-76%; paired-sample t7 = 7.54, P < 0.0001). On average, 375 nmol/l NB-DNJ CD4 liposomes reduced the secretion of HIV-1 by 81% (SEM = 2.5%; range 69-91%; t7 = 32.9, P < 0.0001).
In an attempt to induce the formation of escape mutants of HIV-1 with increased resistance to NB-DNJ, PBMCs infected with isolates JR-FL, 92UG046, 93TH051, or TDC-r were cultured in the presence of either 250 νmol/l NB-DNJ free in the medium or 4 nmol/l NB-DNJ encapsulated inside liposomes for 15 weeks. Every week, a sample of culture supernatant from treated and mock-treated cultures was used to calculate the IC50 of the viral population at that stage of the incubation (Fig. 4). At the end of the 15 weeks of treatment, there was no significant difference in the IC50 of viral populations grown in the presence of NB-DNJ compared with the untreated control as determined by t-tests (n = 4 isolates; mean free NB-DNJ = 95%, SEM = 2%, t3 = 2.8, P = 0.97; mean NB-DNJ liposomes = 95%, SEM = 3%, t3 = 1.9, P = 0.92). Isolates tested included a clinical isolate of HIV which is already resistant to triple drug combination therapy.
Materials and methods
HIV primary isolates
All HIV isolates were obtained from The National Institute for Biological Standards and Control-Center for AIDS Reagents and the World Health Organization-UNAIDS Network for HIV Isolation and Characterization.
HIV infectivity assays
Peripheral blood mononuclear cells (PBMCs) from four uninfected donors were isolated using Histopaque density gradient centrifugation (Sigma-Aldrich, Gillingham, UK), pooled and stimulated with phytohemagglutinin (PHA; 5 νg/ml) for 48 h followed by interleukin-2 (IL2; 40 U/ml) for 72 h in complete RPMI (RPMI and 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 νg/ml streptomycin, and 2 mmol/l L-glutamine). All incubations were at 37C/5% CO2. To infect cells, 4 X 105 PBMCs were incubated with 100 TCID50 (tissue culture infectious dose 50%) of virus for 16 h, washed three times, and resuspended in media containing the appropriate free drug or liposome treatment (final lipid concentration of 50 νmol/l). After 4 days, the presence of HIV particles in supernatant was quantified by HIV core protein (p24) capture ELISA. All supernatants were diluted to a final p24 concentration of 5 ng/ml in complete RPMI/IL2, added to 4 X 105 PHA-activated PBMCs to infect as described, and then incubated for 4 days (untreated) before supernatant was assayed for p24 content. For multiple round treatments, supernatants were diluted to 5 ng p24/ml of the untreated sample and added to 4 X 105 PHA-activated PBMCs for two more rounds of infection and treatment. IC50 values were calculated by plotting NB-DNJ concentration versus p24 secretion detected following the third round of treatment.
Cultures were monitored for cellular viability using an MTS-based cell proliferation assay (CellTiter 96; Promega, San Luis Obispo, California, USA) following the manufacturers' protocol. All reported p24 concentrations were normalized to cellular viability data.
Quantitative p24 enzyme-linked immunosorbent assay
HIV samples were inactivated with 1% Empigen (vol/vol) prior to p24 quantification. Enzyme-linked immunosorbent assays (ELISAs) were carried out using the anti-p24 D7324 antibody (Aalto Bioreagents, Dublin, Eire) to capture p24 from treated supernatant and detected using the anti-p24 secondary antibody, BC1071-AP (Aalto Bioreagents), following the manufacturers' protocol. Alkaline phosphatase activity was measured using the AMPAK ELISA kit (Dako, Ely, UK) following the manufacturers' protocol. Samples were standardized using recombinant p24 protein (Aalto Bioreagents).
N-Glycan analysis
Analysis of conserved N-glycan sites on 976 gp120 sequences obtained from the Los Alamos HIV database (http://www.hiv.lanl.gov/content/hiv-db/mainpage.html ) was done using BioEdit Sequence Alignment Editor v7.0.5.3 (Ibis Therapeutics, Carlsbad, California, USA). Glycan occupancy for individual isolates was predicted by the NetNGlyc 1.0 server (Center for Biological Sequence Analysis, University of Denmark, Lyngby, Denmark). The number of occupied N-glycan sites was calculated by assigning a value of '1' for occupied, '0' for unoccupied, and '0.5' for low-confidence occupancies.
Lipids
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl) cyclohexane-carboxamide] (maleimide-PE) were all purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Cholesteryl hemisuccinate was purchased from Sigma-Aldrich.
Liposome preparation
PE:cholesteryl hemisuccinate liposomes (molar ratio 3: 2) were prepared fresh for each treatment. Chloroform solutions of lipids were evaporated under nitrogen gas. Lipid films were hydrated by vortexing in phosphate-buffered saline (PBS) buffer, with or without NB-DNJ (Sigma-Aldrich), to a final lipid concentration of 5 mmol/l. The resulting multilamellar vesicles were extruded 11 times through a polycarbonate filter of 100 nm pore diameter using a Mini-Extruder device (Avanti Polar Lipids). Drug-loaded liposomes were separated from unencapsulated NB-DNJ by size exclusion chromatography using sephadex-G50 (medium) resin (G.E. Healthcare, Giles, UK) and filter sterilized using a 0.22 νm filter unit. Drug encapsulation efficiency was calculated as previously described [17], and averaged 3.75% for all NB-DNJ concentrations.
CD4 liposome and immunoliposome conjugates
Liposomes were prepared as described incorporating 3% maleimide-PE. CD4 liposomes were prepared by first modifying a soluble recombinant CD4 molecule (amino acids 1-370; Promega) using N-succinimidyl-S-acetylthiopropionate (Perbio Science, Cramlington, UK) to produce a protected sulfhydryl at the C-terminus following the manufacturers' protocol. Addition of 10% 0.5 mol/l hydroxylamine/25 mmol/l EDTA (vol/vol) allowed for creation of a free sulfhydryl group. Antibodies were conjugated to liposomes by first reducing IgGs in 50 mmol/l 2-mercaptoethylamine HCl/10 mmol/l EDTA for selective reduction of the hinge disulfide bonds. Following buffer exchange of both modified CD4 and IgG molecules into PBS/10 mmol/l EDTA, each molecule was incubated with prepared liposomes overnight at room temperature. CD4-liposome and immunoliposome conjugates were separated from nonconjugated material using size exclusion chromatography and filter sterilized.
Liposome uptake assay
Uptake of liposomes into HIV-1-infected and uninfected PBMCs was monitored by the incorporation of 1% rhodamine-PE into liposomes prepared as previously described. Following a 24 h incubation of liposomes with infected or uninfected PBMCs (50 νmol/l final lipid concentration), cells were washed and counted using trypan blue staining. Fluorescence was measured at λex = 550 nm, λem = 590 nm, and normalized to the total number of cells in each incubation.
N-Butyldeoxynojirimycin resistance assay
In 24-well plates, 8 X 105 PHA-stimulated PBMCs were infected with HIV isolates at 200 TCID50 in 500 νl of RPMI/IL2 for 16 h before cells were washed and treatments were begun. On day 4, 250 νl of cellular supernatant was removed and replaced with 250 νl of fresh RPMI/IL2 with treatments. On day 7, cells were resuspended in their media and 250 νl of the cellular suspension was removed and kept aside. Two hundred and fifty microliter of fresh RPMI/IL2 containing 4 X 105 naive PBMCs was then added to each well. Cultures were maintained so that fresh media, treatments, and PBMCs were continuously replenished, and weekly harvests of supernatants were assayed to determine NB-DNJ IC50 as described.
All statistical analyses described throughout the text were carried out using JMP software v5.1.2 (SAS Institute Inc., Cary, North Carolina, USA).
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