Background

Cellulose acetate phthalate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was shown to inhibit infection by the human immunodeficiency virus type 1 (HIV-1) and several herpesviruses. CAP formulations inactivated HIV-1, herpesvirus types 1 (HSV-1) and 2 (HSV-2) and the major nonviral sexually transmitted disease (STD) pathogens and were effective in animal models for vaginal infection by HSV-2 and simian immunodeficiency virus.

Methods

Enzyme-linked immunoassays and flow cytometry were used to demonstrate CAP binding to HIV-1 and to define the binding site on the virus envelope.

Results

1) CAP binds to HIV-1 virus particles and to the envelope glycoprotein gp120; 2) this leads to blockade of the gp120 V3 loop and other gp120 sites resulting in diminished reactivity with HIV-1 coreceptors CXCR4 and CCR5; 3) CAP binding to HIV-1 virions impairs their infectivity; 4) these findings apply to both HIV-1 IIIB, an X4 virus, and HIV-1 BaL, an R5 virus.

Conclusions

These results provide support for consideration of CAP as a topical microbicide of choice for prevention of STDs, including HIV-1 infection.

Due to the current unavailability of anti-HIV vaccines, other preventive methods have to be developed to control the ongoing AIDS pandemic. This includes the design and application of safe and effective topical microbicides. Screening of pharmaceutical excipients revealed that cellulose acetate phthalate (CAP), commonly used for enteric coating of tablets and capsules [1], has anti-HIV-1 activity. CAP in micronized form and formulated into a cream, is a broad spectrum microbicide inactivating several sexually transmitted disease (STD) pathogens [2-4], including HIV-1 [2,5]. It was of interest to explore the mechanism(s) whereby CAP causes inactivation of HIV-1. Since CAP has a relatively high molecular weight (M_w ~ 60,000; [2]), its effect on HIV-1 virions would be expected to be confined to the virus surface, i.e. to the envelope glycoproteins gp120 and/or gp41. Thus, CAP would be expected to affect one or more steps required for HIV-1 entry into cells, i.e. binding to cellular CD4, to the major HIV-1 coreceptors CXCR4 or CCR5 for X4 and R5 viruses [6], respectively, and fusion with cell membranes [7-15]. Results presented here show that CAP pretreated HIV-1 has a reduced capacity to bind to the coreceptors leading to impaired virus infectivity.

Reagents

The following monoclonal antibodies (mAbs; the source is indicated in parentheses) were used: 2F5 and 588D (Drs. T. Muster and S. Zola-Pazner, respectively); 9305 and 9284 (NEN Research Products, Du Pont, Boston, MA); b12, 2G12 and 17b (AIDS Research and Reference Reagent Program, Rockville, MD; courtesy of Drs. D. Burton, H. Katinger and J.E. Robinson, respectively) and anti-p24 (ImmunoDiagnostics, Inc., Woburn, MA). Rabbit antibodies against peptides from HIV-1 IIIB gp120/gp41 and against the V3 loop of HIV-1 BaL (anti-V3 BaL) were prepared as described [16]. Antiserum to phthalate was prepared by immunization of rabbits with phthalic anhydride treated rabbit serum albumin [17]. Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA. Recombinant HIV-1 IIIB and MN gp120, biotinylated gp120 and biotinylated sCD4 were from ImmunoDiagnostics Inc. Protein A, the protease inhibitors phenylmethyl-sulfonyl fluoride, leupeptin and pepstatin, and 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) were all from Sigma, St. Louis, MO. Pelletted, 1000-fold concentrates of HIV-1 IIIB (6.8 × 10¹⁰ virus particles/ml) and BaL (1.8 × 10¹⁰ virus particles/ml) were from Advanced Biotechnologies Inc., Columbia, MD. Chicken serum was from OEM Concepts, Toms River, NJ. Horseradish peroxidase (HRP)- and phycoerythrin (PE)-labeled streptavidin were from Amersham, Arlington Heights, IL and R & D Systems, Minneapolis, MN, respectively. HRP was quantitated using a kit from Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD. Enzyme linked immunoassays (ELISA) kits for the HIV-1 p24 antigen and for the β-gal protein were from Coulter Immunology, Hialeah, FL and 5 Prime → 3 Prime Inc., Boulder, CO. CAP was a gift from Eastman, Kingsport, TN. H9 cells chronically infected with HIV-1 IIIB, HeLa-CD4-LTR-β-gal cells, GHOST CXCR4 and CCR5 cells and PM1 cells were obtained from the AIDS Research and Reference Reagent Program contributed by Drs. R. Gallo, M. Emerman, D. Littman, P. Lusso and M. Reitz, respectively. The Centricon centrifugal ultrafiltration devices were from Amicon/Millipore, Bedford, MA.

Measurement of HIV-1 infectivity

Serial two-fold dilutions of CAP treated and untreated HIV-1 IIIB (undiluted to 1/512) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) were mixed with MT-2 cells (10⁴ cells/well) and placed into 96-well polystyrene plates. The mixtures were incubated for 1 h at 37°C and the volume was adjusted with RPMI-1640 medium containing 10% FBS to 200 μl. On the 4^th and 6^th day after incubation at 37°C, 100 μl of culture supernatants were removed from each well and equal volumes of fresh medium were added. On the 6^th day, XTT dye (1 mg/ml) was added to the cells, Intracellular formazan was determined spectrophotometrically [18,19]. Similar experiments were done with HIV-1 BaL, except that PM1 cells were used instead of MT-2 cells, and virus production was measured by ELISA for p24 antigen one week after infection. The percentage of residual infectivity after CAP treatment was calculated from calibration curves relating absorbance (corresponding to formazan for HIV-1 IIIB and p24 antigen for HIV-1 BaL, respectively) to virus dilutions of untreated viruses. The results were plotted in Fig. 1.

Enzyme-linked immunosorbent assays (ELISA)

For virus capture assays, wells of 96-well polystyrene plates (Immulon II; Dynatech Laboratories Inc., Chantilly, VA) were coated either with sCD4 (1 μg/well) or with monoclonal or polyclonal antibodies. For coating with antibodies, wells were first coated with protein A (1 μg/well) in 0.1 M Tris buffer, pH 8.8 for 2 h at 20°C followed by either mAbs (1 μg/well) or rabbit antisera diluted 1:100 in phosphate buffered saline (PBS) for 1 h at 20°C. Subsequently, the wells were washed and postcoated for 1 h at 20°C with bovine serum albumin (BSA) and gelatin (1 and 0.1 mg/ml in 0.14 M NaCI, 0.01 M Tris, pH 7.0 [TS]). Chicken serum (10%) in PBS (Ch-PBS) was used instead in experiments with HIV-1 BaL. The wells were washed with TS and stored at 4°C. HIV-1 virus particles suspended in PBS were added to the wells for 5 h at 4°C. Subsequently, the wells were washed 10 × with ice cold PBS or 1:50 anti-p24 mAb in Ch-PBS for HIV-1 BaL, and then treated with lysis buffer (1% Nonidet P40 [NP40], 100 μg/ml BSA in PBS) for 30 min at 37°C. The supernatants were removed and tested for p24 antigen using ELISA kits from Coulter Immunology and following the manufacturer's protocol. In other experiments, wells were coated with CAP (1 μg/ml) in 25 mM sodium acetate, pH 6.0 and postcoated as described above.

To detect binding of CD4 to CAP treated and control gp120, wells coated with graded amounts of gp120 and postcoated as described above were reacted with biotinylated sCD4 (1 μg/well) in PBS containing 100 μg/ml BSA for 18 h at 4°C, washed with TS and the bound biotinylated CD4 was determined after adding HRP-streptavidin (1 μg/ml) in TS containing 0.25% gelatine and 0.05% Tween 20 for 30 min at 37°C. The wells were washed and bound HRP was detected using the test kit from Kirkegaard and Perry and the absorbance read at 450 nm.

Flow cytometry

To determine the binding of gp120–biotinylated CD4 complexes to HIV-1 coreceptor expressing cells, CAP treated and untreated gp120 (5 μg) and biotinylated CD4 (2.5 μg) in PBS containing 100 μg/ml of BSA were mixed for 1 h at 20°C and then added to 10⁶ MT-2 cells in RPMI-1640 medium containing 100 μg/ml of BSA. Biotinylated CD4–gp120 complexes were not added to control cells. After 30 min at room temperature, the cells were washed 3 times with PBS containing 100 μg/ml of BSA, and PE-streptavidin (0.1 μg) was added. After 20 min at 20°C, cells were washed and fixed in 1% formaldehyde in PBS. Flow cytometry analysis was performed in a FACSCalibur flow cytometer (Becton Dickinson Immunocytometric Systems, San Jose, CA). Similar experiments were done with peripheral blood lymphocytes (PBL) isolated by the Isopaque-Ficoll technique [20].

Quantitation of CAP–gp120 binding

Forty μg of gp120 were mixed with 40 μg of CAP in 1 ml TS or 1 M NaCI, 0.01 M Tris, pH 7.0 and incubated at 20°C for 30 min. The mixtures were transferred into a Centricon centrifugal ultrafiltration device with a M_w cut-off of 100,000 and centrifuged at 3,500 × g for 30 min. The filtrates were transferred to a similar device with a M_w cut-off of 50,000 and centrifuged under the same conditions. CAP retained on top of the filter was quantitated as a complex with ruthenium red [21]. The retentate on the 100,000 M_w cut-off filter was dissolved in 1 ml of 2 M guanidinium hydrochloride and CAP released from the CAP–gp120 complex and retained on the 50,000 M_w cut-off filter was quantitated by the same method.

Molecular Modeling

A cellulose chain consisting of one cellotetraose unit (composed of four 1,4-linked β-D-glucose units) was created in Quanta [22] and 50% of the hydroxyl groups at positions 2- and 3- were modified to acetyl ester and 25% of the hydroxyl groups at position 6 were modified to phthaloyl ester [1,23]. The acetylated and phthaloylated cellotetraose structure (CTAP) was minimized by the steepest descent method followed by the adopted basis Newton-Raphson (ABNR) method. The energy change of 0.05 Kcal/mol between two successive structures was used as the termination criterion in both the steps.

The crystal structure of gp120 (1gcl) [13] was retrieved from the pdb (http://www.rcsb.org) and the V3 loop, created by homology modeling {(based on the nmr structure of the V3 loop from 1ce4) using the SWISS-MODEL [24] automated comparative protein modeling server (http://www.expasy.ch/SWISS-MODEL.html} was attached to the gp120 crystal structure using Quanta's protein design module.

The docking simulation of CTAP onto the entire gp120 protein surface was performed by the Dockvision program [25]. A grid box (125 Å × 125 Å × 125 Å) with grid stepsize of 0.5 Å was created to cover the entire protein surface with enough area for the ligand to dock. The default forcefield (Research Potential Function) was used to perform 1000 Monte Carlo runs for the docking. Both ligand and the target protein were kept rigid. Intermolecular energy criteria were used to identify the best possible dockings of CTAP.

Impaired infectivity of CAP treated HIV-1

Results of earlier studies indicated that HIV-1 infection of cells is inhibited in the presence of CAP (ED₉₀ = 5 to 10 μg/ml for HIV-1 IIIB, i.e. < 200 nM) [2]. However, the mechanism involved in the inhibitory activity and the possibility of virus inactivation by CAP have not been explored. Results shown in Fig. 1 indicate that CAP in a dose dependent manner rapidly inactivates at 37°C HIV-1 IIIB, an X4 virus and HIV-1 BaL, an R5 virus, the latter appearing relatively more resistant.

CAP retention on the surface of treated HIV-1 particles

Evidence for CAP–HIV-1 binding was obtained from results of solid phase immunoassays in which attachment of HIV-1 IIIB and BaL virus particles, respectively (detected by subsequent ELISA of p24 antigen released from virus particles by detergent treatment) to wells precoated with CAP was measured (Fig. 2A). CAP treated HIV-1 IIIB and BaL viruses, unlike control viruses, bound to wells coated with antibodies against phthalate (Fig. 2B). These results indicate that treated HIV-1 particles, utilizing either CXCR4 or CCR5 as coreceptors, retain CAP on their surface and suggest that this is responsible for the altered properties and impaired infectivity of the treated viruses.

Identification of CAP binding sites on HIV-1 envelope glycoproteins

The attachment sites on the surface of HIV-1 IIIB for CAP were determined from binding of control and CAP treated HIV-1, respectively, to distinct ligands (Fig. 3). The quantities of control virus and treated virus in these experiments were identical, as determined from the content of p24 antigen in preparations of detergent-disrupted virus particles. CAP binding to HIV-1 IIIB particles most profoundly affected their binding to the V3 loop specific mAbs 9284 [26] and 9305 [27] and to the coreceptor CXCR4, while binding to sCD4; to virus neutralizing mAbs specific for the CD4 binding site on gp120, b12 [28] and 588D [29]; to mAb 17b specific for a discontinuous conserved epitope proximal to the binding site for both CD4 and anti-CD4 binding site antibodies [30]; to mAb 2G12 specific for an epitope centered around the C3/V4 domain of gp120 also involving N-linked glycans [31] and to the gp41 specific mAb 2F5 [32] was less affected. In order to determine whether R5 viruses are similarly affected by CAP, the binding of HIV-1 BaL as a representative of this virus group to anti-V3 BaL and CCR5, respectively, before and after CAP treatment was studied. The results shown in Fig. 3 indicate that the CAP-treated HIV-1 BaL bound to these ligands much less than did untreated virus.

The binding of control and CAP-treated HIV-1 IIIB to antibodies against peptides derived from gp120/gp41 [16] was also determined. In agreement with the results obtained using mAbs, CAP treatment resulted in most pronounced decreases of virus binding to antibodies against peptide 303–338 (= V3 loop) and the adjacent peptide 280–306 (Fig. 4).

Pretreatment with CAP impairs gp120 binding to coreceptors

Data reported so far suggest that the lethal hit to HIV-1 caused by CAP treatment involves the coreceptor binding site on gp120. To further support this conclusion, the binding of labeled gp120–CD4 complexes to coreceptor [11,12,33] expressing cells was studied. First, it was determined from quantitative binding studies using CAP staining with ruthenium red [21] that pretreatment with CAP resulted in binding of 0.90 ± 0.13 CAP molecules/gp120 in TS. The binding appeared augmented by ~ 30% in 1 M NaCl. The binding capacity of gp120 for sCD4 was preserved after CAP treatment (Fig. 5). On the other hand, gp120–sCD4 complexes containing CAP treated gp120 bound to coreceptor expressing cells to a much lesser extent than similar complexes containing untreated gp120 (Fig. 6).

Negatively charged sulfated polymers were reported to have anti-HIV-1 activity and are being considered for development as topical microbicides. They include: dextran sulfate [34,35], carrageenans [35,36], dextrin-2-sulfate [37,38], cellulose sulfate [39] and naphthalene sulfonate polymer (PRO 2000; [40]). Except for dextrin-2-sulfate and PRO 2000, which appear to have anti-HIV-1 inhibitory activities similar to that of CAP, the other sulfonated polymers are less inhibitory [34,35,41]. HIV-1 may develop resistance to the inhibitory effect of dextran sulfate and R5 viruses were reported not to be inhibited by this polymer [42,43]. Dextran sulfate was shown to bind to gp120 and to interfere with gp120/CXCR4 interactions but it did not bind to gp120 of R5 viruses [43].

CAP in micronized form, providing an acidic environment, causes disintegration of HIV-1 leading to loss of infectivity (2). Here we have shown that CAP directly inactivates HIV-1 at neutral pH since treated virus particles after removal of excess CAP had reduced or no infectivity (Fig. 1), in agreement with recent observations [44,45]. Virus inactivation can be attributed to strong binding of CAP to HIV-1, preventing the access to virus particles predominantly of antibodies against the gp120 V3 loop, known to be involved in coreceptor binding and specificity