Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides

Aquateric (the micronized form of CAP containing ≈ 66% CAP and ≈ 34% of Poloxamer and distilled acetylated monoglycerides) was obtained from the FMC Biopolymer Corporation, Philadelphia, PA. The following polymers were obtained from commercial sources different from proprietary products being developed as microbicides: carrageenans κ and λ (Sigma, St. Louis, MO; mixed at a 1:1 (w/w) ratio in all experiments); cellulose sulfate (Across Organics, Piscataway, NJ); poly(napthalene sulfonate) (BASF, Parsippany, NJ); and polystyrene-4-sulfonate (Polysciences, Inc., Warrington, PA).

The HIV-1 non-nucleoside reverse transcriptase inhibitors, UC781 and TMC120 were obtained by custom synthesis from Albany Molecular Research, Inc., Albany, NY. Zinc finger inhibitors #89 and #247 were a gift from Dr. Ettore Appella and Dr. Marco Schito (National Cancer Institute, Bethesda, MD). Stabilite SD60 Polyglycitol, hydroxypropyl methylcellulose E4M and Avicel PH 105, respectively, were from SPI Polyols, New Castle, DE, Dow Chemical Co., Midland, MI and the FMC Biopolymer Corporation, Philadelphia, PA. SP was purchased from Vital Products, Inc., Boynton Beach, FL. HIV-1 IIIB and BaL were from Advanced Biotechnologies, Inc., Colombia, MD. HeLa-CD4-LTR-β-gal, MAGI-CCR5 and TZM-bl cells were obtained from the AIDS Reagent and Reference Reagent Program (operated by McKesson BioServices, Rockville, MD) and contributed by Drs. M. Emerman, J. Overbaugh and J. C. Kappes and X. Wu (Tranzyme, Inc.), respectively. Dulbecco's modified Eagle medium (DMEM) was from GIBCO Invitrogen Corporation, Carlsbad, CA. The Galacto-Light Plus chemiluminescence reporter assay for β-galactosidase was from Applied Biosystems, Foster City, CA.

Seventy μl of serially two-fold diluted compounds in DMEM medium (final concentrations after dilution: 1.25 to 10,000 μg/ml) were mixed with 70 μl of HIV-1 IIIB and BaL, respectively, and 70 μl of either SP or DMEM medium. The mixtures were added to TZM-bl indicator cells (in 96-well plates) which can be infected by both X4 and R5 HIV-1, enabling quantitative analysis of HIV-1 infection using either β-galactosidase (β-gal) or luciferase as a reporter [19]. After 90 min at 37°C, the virus (and SP) containing supernatants were removed from the cells. The cells were washed once with DMEM medium, supplemented with the same medium, and incubated for 48 hr at 37°C. The removal of SP after 90 min was necessary to avoid problems caused by the cytotoxicity of SP evident after prolonged incubation [20‐22]. Finally, the culture supernatants were removed and the cells were washed once with phosphate buffered saline, pH 7.2 (PBS). Subsequently, 50 μl of lysis buffer from the Galacto-Light Plus kit were added to the wells for 1 hr at 20°C. Aliquots (20 μl) of the cell lysates were transferred into wells of 96-well microplates and β-gal was quantitated using the Galacto-Light Plus System chemiluminescence reporter assay in a MicroLight ML 2250 luminometer (Dynatech Laboratories, Inc. Chantilly, VA). The concentration of viruses was selected so as to provide a readout of ≤ 70 in the absence of SP and drug, respectively. The percentage of inhibition was calculated using the Microsoft Excel computer program. ED₅₀ values were calculated using an online computer program [23]. The inhibitory activity of the compounds in the absence of SP was measured also at a lower (1/6) virus dose (readout ≤ 20) to determine whether or not this would result in higher ED₅₀ values. This was not the case, confirming that the presence of SP (causing partial virus inactivation or inhibition) truly diminishes the inhibitory activity of the polymers under investigation.

The following two formulations were tested for virucidal activity against HIV-1 IIIB and BaL, respectively, in the presence of SP: (1) Aquateric, 18%; glycerol, 58%; Stabilite SD60, 20%; Hydroxypropyl methylcellulose E4M 1%, Avicel PH 105, 3%; and (2) 18% Aquateric in a universal placebo gel [24].

Two-fold serial dilutions of HIV-1 IIIB treated with Aquateric formulations, and separated from these polymers by precipitation with 3% PEG or by centrifugation at 14,000 rpm for 1 h, and control virus (100 μl), respectively, were added to HeLa-CD4-LTR-β-gal cells which had been plated a day before infection in 96-well plates at 1 × 10⁴ cells/well in 100 μl of DMEM medium containing 10% fetal bovine serum (FBS). After incubation at 37°C for 48 h, the culture supernatant fluids were removed and the cells washed once with PBS. β-gal in the cells was measured as described above. The infectivity of treated and control HIV-1 BaL was measured by the same method except that MAGI-CCR5 cells were used.

Measurement of HIV-1 infectivity in seminal fluid is impeded by the cytotoxicity of SP [20, 21] contributed to by spermine [22]. Therefore, HIV-1 was separated from SP ingredients by precipitation with 3% PEG or centrifugation (14,000 rpm for 1 h) and the infectivity of the resuspended pellets was measured. Such cytotoxic effects were minimized by using for titrations of virus infectivity pellets after precipitation with 3% PEG or after centrifugation (14,000 rpm for 1 h).

CAP in the form of a complex with ruthenium red was quantitated spectrophotometrically [25].

Inhibition of HIV-1 IIIB (a virus utilizing CXCR4 as cellular coreceptor = X4 virus) and HIV-1 BaL (a virus utilizing CCR 5 as cellular receptor = R5 virus) [8, 26] infection, respectively, of TZM-bl cells [19] by polymeric candidate microbicides in the absence or presence of SP (final concentration 33.3%) was investigated. The polymers included: carrageenan, poly(naphthalene sulfonate), cellulose sulfate, cellulose acetate 1,2-benzene dicarboxylate (CAP) and polystyrene sulfonate. Dose response curves for inhibition of HIV-1 IIIB infection (Fig. 1) show the suppressive effect of SP on the virus inhibitory activities of the polymers. Similar results were obtained for HIV-1 BaL (data not shown). The results are summarized in Table 1 showing values for 50% inhibition of infection (ED₅₀) corresponding to the distinct polymers. The ED₅₀ values are consistently higher for HIV-1 BaL than for HIV-1 IIIB. This is consistent with lower positive charges on V3 loops of R5 gp120 envelope glycoproteins as compared with X4 gp120, leading to diminished binding of negatively charged polymers [27, 28]. The presence of SP led to a ≈ 4 to ≈ 73-fold increase of ED₅₀, resulting in ED₅₀ values for HIV-1 BaL (a representative of more frequently sexually transmitted R5 viruses [8, 26]) > 100 μg/ml. This raises the question whether or not sufficient concentrations of the respective inhibitors can be reached in the vaginal environment at which cells playing a major role in the virus replicative cycle (dendritic cells, macrophages and CD4+ T cells) occur [11].

In contrast with the polymeric candidate microbicides, the anti-HIV-1 activities of reverse transcriptase inhibitors UC781 and TMC120, and of zinc finger inhibitors #89 and #247, respectively, were not affected by the presence of SP (data not shown). This will support confidence for results of efficacy tests for these antiretroviral drugs in animal model systems for male to female virus transmission. The presence of SP alone (33.3 to 80%) resulted in 4- to 22-fold, and 2- to 10-fold inhibition of HIV-1 IIIB and BaL infection, respectively, in different experiments.

Anionic polymeric candidate microbicides are thought to interfere with HIV-1-cell interactions involving cellular receptors either directly in productive infection (CD4 and CXCR4/CCR5) or in cell to cell transfer of virus (DC-SIGN etc.) [11]. However, some polymers rapidly (≤ 5 min) inactivate HIV-1. Under these circumstances, there is no need for the antiviral compounds to reach cells involved in virus dissemination or infection, the virus being rendered non-infectious before encountering cellular/tissue surfaces. Among the aforementioned polymers, cellulose sulfate, polystyrene sulfonate, poly(naphthalene sulfonate) (at a concentration of 10 mg/ml; at concentrations ≤ 1 mg/ml the compounds may not be virucidal [29]), CAP (micronized), as well as the acidifying formulation, BufferGel, inactivate not only X4 viruses but also the R5 virus, HIV-1 BaL [30]. Poly(naphthalene sulfonate) and CAP, respectively, rapidly elicit in the virus envelope the formation of "dead-end" gp41 six-helix bundles rendering the virus irreversibly incapable to fuse with susceptible target cells, a prerequisite for infection [30]. CAP being the only candidate microbicide provided in a water insoluble micronized form, and therefore, very unlikely to penetrate into tissues or cells [31], was selected for further studies regarding the effect of SP on its virus inactivating properties.

CAP formulations (expected to contain about 240 to 480 mg CAP per dose for human use) have a good acidic buffering capacity, are non-toxic to vaginal and cervical explants or reconstituted tissues [32], and provide a pH ≤ 5.5 if the volume of SP is ≤ 1.8 ml per 100 mg CAP (Fig. 2). CAP remains water insoluble under these conditions (Fig. 3) and will remain in the form of particles about 1 micron in diameter. Changes in pH and CAP solubility during the course of addition of SP to formulated CAP (≈ 120 mg/ml) are shown in Fig. 4. It is evident that even at an excess of SP over the volume of the formulation used (3:1), the pH remains < 5.5 and the maximum concentration of CAP becoming soluble is ≤ 7 μg/ml. This concentration is sufficient for partial inhibition of infection by the laboratory strain HIV-1 IIIB [33] but insufficient for inhibition of most other HIV-1 strains [34]. Thus, the predominant contribution to anti-HIV-1 activity of formulated CAP is expected to be attributable to the insoluble micronized particles even at an excess of SP. These particles adsorb both X4 and R5 viruses, elicit 6-helix bundle formation in their envelope glycoproteins and render the viruses non-infectious [30]. Both HIV-1 IIIB and BaL are inactivated within 5 min at 37°C by CAP in micronized form even in the presence of a volume excess of SP (Table 2). Similar results were obtained with an Aquateric formulation in a "universal placebo" gel [24] (Fig. 5). Virucidal activity against HIV-1 BaL was still detectable at relatively high dilutions of the formulation in SP (resulting in pH ≥ 7.0), not expected to occur in vivo. The virucidal activity of CAP was similar to that observed in the absence of SP (see Fig. 2 in our earlier publication) [35]. In contrast, acidic pH alone is much less virucidal than CAP in micronized form, the half-life for virus infectivity being 6, 10 and >120 min at pH 3.5, 4.0 and 4.5, respectively [36] and fails to elicit "dead-end" six-helix bundle formation in HIV-1 gp41 (data not shown). Thus, combination of a low pH buffering system with a compound targeted to functionally important sites on HIV-1 envelope glycoproteins (e.g. CD4 and/or CXCR4/CCR5 coreceptor binding sites, and gp41 regions involved in 6-helix bundle formation) provides an attractive approach for development of virucidal microbicides. In the case of CAP, the low-pH buffering capacity is an inherent property of the polymer.