Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

Abstract

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.

Figure 1

Inactivation of HIV-1 by CAP. Cellulose acetate phthalate (CAP) (final concentrations between 10 and 0.078 mg/ml) was added to HIV-1 IIIB containing tissue culture medium and to HIV-1 BaL, respectively. After incubation for 5 min at 37°C, the mixtures were cooled on ice and a solution of polyethylene glycol 6000 (PEG) [Reference 53] was added to a final concentration of 3% to separate HIV-1 from CAP (which does not precipitate in 3% PEG). After 90 min at 4°C, the mixtures were centrifuged at 10,000 rpm, the supernatant fluids removed and the pellets washed twice with 3% PEG in PBS containing 10 mg/ml BSA. The final pellets were resuspended in tissue culture medium and titered for infectivity. The percentage of residual infectivity is shown in a probability scale.

Figure 2

Evidence for CAP binding to HIV-1 virus particles. A) Virus binding to CAP coated wells. B) Binding of CAP treated and untreated HIV-1 IIIB and BaL, respectively, to wells coated with antibodies against phthalate [reference 17]. CAP was added to a final concentration of 5 mg/ml to 50 μl of suspensions of purified HIV-1 IIIB (6.8 × 10⁹ virus particles per ml) and HIV-1 BaL (1.8 × 10¹⁰ virus particles per ml), respectively, in 0.1 M sodium acetate pH 7.0. CAP was not added to control virus preparations. After 5 min at 37°C, HIV-1 was separated from unbound CAP as described for Fig. 1. The pellets containing HIV-1 were resuspended in 50 μl PBS containing 100 μg/ml BSA. 200 μl of 5-fold diluted virus supensions containing equal amounts of virus particles for both CAP treated and control virus (as determined by quantitation of p24 antigen), were added to wells coated as indicated above and to control wells. Bound virus was quantitated by ELISA for p24 antigen.

Figure 3

Binding of CAP treated and untreated HIV-1 to distinct ligands. The binding of untreated and CAP treated HIV-1 IIIB to wells coated with sCD4 or with distinct mAbs and of HIV-1 BaL to anti-V3 BaL was measured as described for Fig. 2. The % of binding corresponding to CAP treated virus was calculated based on the formula (% residual binding = [absorbance corresponding to bound CAP treated virus ÷ absorbance corresponding to bound untreated HIV-1] × 100. Absorbance corresponding to p24 antigen (5-fold dilution of the sample) from untreated HIV-1 bound to the respective ligands was in the range of 0.56 to 1.54. Absorbance corresponding to virus captured onto wells coated with control IgG was 0.046. All experiments were done in triplicate. To measure virus binding to the coreceptor CXCR4, treated and control HIV-1 IIIB recovered after PEG precipitation was mixed with 10 μg of sCD4. After 5 min at 20°C, the respective samples were divided into 2 aliquots, each of which was added to 5 × 10⁵ GHOST CXCR4 cells suspended in 100 μl PBS containing 100 μg/ml of BSA. Similar experiments were carried out with purified HIV-1 BaL, except that GHOST CCR5 cells were used. After 1 h at 4°C, the cells were pelletted and washed with ice cold PBS containing 100 μg/ml BSA. The pelletted cells were lysed for 30 min at 37°C in PBS with 1% NP40. Serial 5-fold dilutions in PBS (1:5 to 1:4.9 × 10⁷) were tested by ELISA for the p24 antigen. Changes in HIV-1 binding were determined using calibration curves relating absorbance to virus dilutions.

Figure 4

Binding of CAP treated and untreated HIV-1 IIIB to wells coated with antibodies to peptides from gp120/gp41 [reference 16]. Experimental conditions were similar to those described in the legend for Fig. 3. The absorbance corresponding to untreated HIV-1 captured onto the wells was in the range of 0.09 to 0.37. The absorbance corresponding to controls (virus captured onto wells coated with Protein A followed by normal rabbit serum) was 0.014. Numbering of gp160 amino acid residues was the same as in reference 16. Decreases of CAP treated virus binding, as compared with binding of control virus, were plotted.

Figure 5

Binding of sCD4 to CAP treated and untreated gp120. Recombinant gp120 IIIB (5 μg in 400 μl of 0.1 M sodium acetate buffer pH 7.0) was treated with CAP (5 mg/ml) for 5 min at 37°C and then cooled to 0°C. CAP was omitted in control experiments. BSA was added to a final concentration of 25 μg/ml and the samples were filtered using a 2 ml Centricon centrifugal ultrafiltration device with a M_w cutoff of 100,000 and centrifuged at 3,500 × g for 30 min. gp120 retained on the filters was washed with PBS, resuspended in PBS, and serially diluted 5-fold. The diluted samples (corresponding to gp120 quantities indicated on the abscissa) were used to coat wells of 96-well polystyrene plates. Binding to the wells of biotin labeled sCD4 was detected from subsequent binding of HRP-streptavidin.

Figure 6

Binding of sCD4 complexes with CAP treated and control gp120, respectively, to HIV-1 coreceptor expressing cells. CAP treated or control gp120 IIIB or MN (prepared as described for Fig. 5) were mixed with biotinylated sCD4 and added to 10⁶ MT-2 cells (A) or PBL (B and C). The cells were washed with PBS, treated with phycoerythrin (PE)-labeled streptavidin, fixed in 1% formaldehyde and submitted to flow cytometry analysis.

Figure 7

Stereodiagram of two best docked modified cellotetraose units (CTAP) (marked as Dock1 and Dock2, respectively) on the x-ray crystal structure of gp120 (HXBc2 strain) with the V3 loop attached. The residues on gp120 nearest to the docked CTAPs are shown to indicate possible interaction patterns. The V3 loop (peptide 303–338) is indicated in yellow. Regions corresponding to peptides 113–142, 280–306, 361–392 and 393–417 (Fig. 4) are indicated in orange, green, red and purple, respectively. The coreceptor binding site is indicated in gray. The rest of the gp120 is in cyan. The figure was generated using the Sybyl program [54].