Development of a Comprehensive Human Immunodeficiency Virus Type 1 Screening Algorithm for Discovery and Preclinical Testing of Topical Microbicides

Abstract

Topical microbicides are self-administered, prophylactic products for protection against sexually transmitted pathogens. A large number of compounds with known anti-human immunodeficiency virus type 1 (HIV-1) inhibitory activity have been proposed as candidate topical microbicides. To identify potential leads, an in vitro screening algorithm was developed to evaluate candidate microbicides in assays that assess inhibition of cell-associated and cell-free HIV-1 transmission, entry, and fusion. The algorithm advances compounds by evaluation in a series of defined assays that generate measurements of relative antiviral potency to determine advancement or failure. Initial testing consists of a dual determination of inhibitory activity in the CD4-dependent CCR5-tropic cell-associated transmission inhibition assay and in the CD4/CCR5-mediated HIV-1 entry assay. The activity is confirmed by repeat testing, and identified actives are advanced to secondary screens to determine their effect on transmission of CXCR4-tropic viruses in the presence or absence of CD4 and their ability to inhibit CXCR4- and CCR5-tropic envelope-mediated cell-to-cell fusion. In addition, confirmed active compounds are also evaluated in the presence of human seminal plasma, in assays incorporating a pH 4 to 7 transition, and for growth inhibition of relevant strains of lactobacilli. Leads may then be advanced for specialized testing, including determinations in human cervical explants and in peripheral blood mononuclear cells against primary HIV subtypes, combination testing with other inhibitors, and additional cytotoxicity assays. PRO 2000 and SPL7013 (the active component of VivaGel), two microbicide products currently being evaluated in human clinical trials, were tested in this in vitro algorithm and were shown to be highly active against CCR5- and CXCR4-tropic HIV-1 infection.

Over 40 million people worldwide are currently infected with human immunodeficiency virus type 1 (HIV-1), and an estimated 12,000 new infections occur each day (UNAIDS, [http://www.unaids.org/en/HIV_data/epi2006/default.asp]). In 2002, women for the first time accounted for half of the new HIV infections in adults worldwide. The majority of HIV-1 infections occur in sub-Saharan Africa and Asia, where heterosexual intercourse is the predominant route of transmission (UNAIDS, [http://www.unaids.org/en/HIV_data/epi2006/default.asp]). Extensive efforts are being undertaken to accelerate the licensure of an HIV vaccine. However, due to the lack of data demonstrating clinical effectiveness, no HIV vaccine has yet received marketing approval (23). Thus, at the present time, the only options for prevention of HIV-1 infection are abstinence or the use of condoms (29). Proper use of male condoms has proven effective for decreasing sexual transmission of HIV-1, but women rely on the willingness of their partners to use this protective measure and female condoms are not widely accessible in many countries (29). Consequently, women are at high risk for acquiring HIV-1 during unprotected consensual or nonconsensual intercourse with infected partners (13, 41).

Topical microbicides are products designed for vaginal and/or rectal application to prevent acquisition or transmission of HIV and other sexually transmitted infections (STIs). It has been shown that STIs, such as gonorrhea, Chlamydia, and herpes simplex virus (HSV), increase the risk of HIV transmission by weakening mucosal barriers and by stimulating an inflammatory response that may activate or recruit HIV target cells to the portals of viral entry (14). The major advantage of microbicides over male condoms is that the product application is controllable by women, which can be an important factor in some cultural settings. Therefore, microbicides are considered one of the female-controlled counterparts to the male condom and offer, if applied consistently, the potential to drastically reduce rates of STI transmission, including HIV (32, 42). Thus far, the majority of efforts to develop effective microbicides have focused on products for vaginal use. However, mucosal surfaces of both the urogenital and the gastrointestinal tracts provide possibilities for entry of sexually transmitted pathogens, including HIV-1, and therefore, both mechanisms of viral transmission need to be considered in the microbicide development process (9).

Ideally, a microbicide should be colorless, odorless, inexpensive to manufacture and purchase, safe to use more than once a day and for long periods of time, effective against multiple STIs, fast-acting, undetectable to either partner, and available in contraceptive and noncontraceptive forms without a prescription (42). Products also must be nontoxic since disruption of the mucosal tissues and normal flora has been associated with increased rates of HIV-1 acquisition and shedding (2, 18). The best approach for development of microbicides against HIV-1 is to select for compounds that intervene with the virus replication cycle before integration into the host genome. Thus, viral targets to consider include prebinding (virucidal), binding/attachment, entry/fusion, uncoating, reverse transcription, and integration (42). Currently, more than 200 topical microbicide candidates, including those designed to prevent transmission of STIs other than HIV-1, are in preclinical development (www.microbicideportfolio.org), including both contraceptive and noncontraceptive agents. In addition, the following compounds are in clinical trials for the purpose of future marketing approval: VivaGel (SPL7013, phase 1), UC-781 (phase 1), Tenofovir/PMPA (phases 1 and 2), Dapivirine (TMC120, phases 1 and 2), Invisible Condom (phases 1 and 2), PRO 2000 (phases 2, 2B, and 3), BufferGel (phases 2 and 2B), and Carraguard (phase 3) (www.microbicide.org). Savvy (C31G) and Ushercell (Cellulose Sulfate) have entered phase 3 clinical trials that were stopped due to lack of evidence for effectiveness of the products against HIV (www.microbicide.org).

Based on the need for expansion of preclinical topical microbicide candidates with mechanisms of actions underrepresented in the current pipeline, a cell-based in vitro screening algorithm was established in partnership with the Division of AIDS/National Institute of Allergy and Infectious Diseases (NIAID) and was later incorporated into the NIAID Topical Microbicide Strategic Plan. The efficacy and toxicity assays chosen for the topical microbicide algorithm were based on the best scientific knowledge to date and the need to identify safe and efficacious microbicide candidates. Generally, compounds submitted for testing have shown prior anti-HIV-1 activity in an antiviral assay, but drug substances of unknown antiviral activity also may be screened. The described algorithm serves the purpose of evaluating compounds from government, academic, or private sector sources and provides guidance for the sponsors regarding which compounds should be pursued further. Once the data for a microbicide clinical trial have been fully analyzed, this information will be utilized to determine how in vitro methods could be modified to more closely predict clinical outcomes. Since mucosal HIV-1 infection is primarily mediated through the CCR5 coreceptor (19), the current algorithm uses two primary CCR5-tropic screening assays that mainly select for early targets of HIV. Compounds found to be active in at least one of the primary assays proceed to the second phase of the testing algorithm, which is comprised of a variety of mechanistic assays using both CCR5- and CXCR4-tropic HIV-1. Promising compounds that appear to be highly active and show no negative effect on cell viability or growth of commensal lactobacilli can be advanced to testing in animal models for safety and efficacy.

To conduct proof-of-concept testing in the in vitro algorithm, PRO 2000 and SPL7013, two highly promising microbicide candidates currently being evaluated in clinical trials were tested in all primary, confirmatory, and secondary assays. PRO 2000 is currently being evaluated in a phase 2/2B clinical trial using an active ingredient concentration of 0.5% in combination with BufferGel and in a phase 3 clinical trial using concentrations of 0.5 and 2%. The dendrimer SPL7013, the active component of VivaGel, is currently in phase 1 clinical trials in a formulation containing 3% (wt/wt) of the active ingredient (24).

MATERIALS AND METHODS

Cells and viruses.

GHOST(3) X4/R5 cells, H9, MOLT4-CCR5 cells, HeLa CD4 long terminal repeat (LTR) β-galactosidase (β-Gal) (MAGI) cells, MAGI-CCR5 cells, HL2/3 cells, HIV-1_IIIB and HIV-1_Ba-L, HIV-1 primary strains, and the molecular clone HIV-1_JR-CSF were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. JR-CSF virus was produced by transfection of HeLa cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The origin of the HIV-1_SK-1 strain has been described (5, 8). ME-180 cells and Lactobacillus jensenii and L. crispatus were obtained from the American Type Culture Collection (Manassas, VA). The HeLa-R5-16 cells were a gift from Roche, Palo Alto, CA (20). All cell lines were maintained in the recommended culture media with or without selection antibiotics.

All efficacy and cytotoxicity determinations were performed with a minimum of 6 log₁₀ or half log₁₀ dilutions in triplicate. Flat-bottom 96-well plates were used for all assays with adherent cell lines and the cervical explant model, and round-bottom plates were used for the peripheral blood mononuclear cells (PBMC) assay. Compound toxicity was assessed concurrently by cell viability measurement using a commercially available MTS {[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]} dye reduction assay (CellTiter 96 reagent; Promega). A brief description of all assays including control compounds with activity ranges is provided in Tables 1 to 3.

TABLE 1.

Comparison and summary of methods for microbicide efficacy assays

Parameter	CD4-dependent cell-free HIV-1 entry assay	CD4-dependent cell-associated HIV-1 transmission assay	CD4-independent HIV-1 transmission assay	HIV-1 fusion assay	HIV-1 cervical explant assay
Virus	IIIB (X4) or Ba-L (R5)	SK-1 (X4)-infected H9 cells or JR-CSF (R5)-infected MOLT4/CCR5 cells	SK-1 (X4)-infected H9 cells	None	Ba-L
Cell or tissue type	MAGI or MAGI-CCR5	GHOST(3) CD4-X4/R5	ME-180	HL2/3 cells (X4) fused with MAGI or HeLa R5-16 cells (R5) fused with MAGI-CCR5	Cervical tissues from surgery
Cell origin	Cervical cancer	Osteosarcoma	Cervical cancer	Cervical cancer	Primary cervical
Compound dilutions	6 log10 or half log10	6 log10 or half log10	6 log10 or half log10	6 log10 or half log10	2 to 4 log10 or half log10
No. of replicates per compound dilution	3	3	3	3	3-4
Time of compound incubation	2 h (X4) or 3 h (R5)	4 h	4 h	48 h	1-h preincubation with compound plus 4 h with virus
Assay duration	48 h	24 h	7 days	48 h	14 to 16 days
Endpoint	β-Galactosidase reporter expression (chemiluminescence)	Gag p24 of cell lysate	Gag p24 of cell supernatant	β-Galactosidase reporter expression (chemiluminescence)	Gag p24 of cell supernatant
Control compounds	AMD3100, TAK-779	AMD3100, TAK-779	Dextran, dextran sulfate	T20, Chicago Sky Blue, AMD3100, TAK-779	UC-781, medium only
Variations	+/− pH transition	+/− pH transition	None	None	None
	+/− 12.5% seminal plasma	+/− 25% seminal plasma

TABLE 3.

Acceptable IC₅₀ values of positive control compounds

Assay	Positive control drug	na	Acceptable IC50 values (nM)	No. of outliers
CCR5-tropic viral entry assay	TAK-779	111	≤4.2	1
CCR5-tropic viral entry assay with 12.5% seminal plasma (SP)	TAK-779	13	≤7.3	1
CCR5-tropic viral entry assay with pH transition	TAK-779	16	≤7.6	1
CCR5-tropic cell-associated HIV transmission assay	TAK-779	34	≤220	2
CCR5-tropic cell-associated HIV transmission assay with 25% SP	TAK-779	5	≤680	0
CCR5-tropic cell-associated HIV transmission assay with pH	TAK-779	7	≤240	1
CXCR4-tropic cell-associated HIV transmission assay	AMD3100	12	≤7.3	0
CXCR4-tropic fusion assay	AMD3100	21	≤6.5	1
CXCR4-tropic viral entry assay	AMD3100	23	≤5.6	1

^an, number of assays.

Sources, handling, and storage of test materials.

AMD3100 and TAK-779 were obtained as powders, and T20 was obtained as lyophilized material from the NIH AIDS Research and Reference Reagent Program. Dextran (D6924; molecular weight 9,000 to 20,000) and dextran sulfate (D6001; molecular weight >500,000) were obtained from Sigma-Aldrich (St. Louis, MO). Chicago Sky Blue (NSC 631567), UC-10 (NSC 645129), and UC-781 (NSC 675186) were obtained from the National Cancer Institute chemical repository. Quinobene was synthesized by Southern Research Institute according to a described previously method (15). Compounds were solubilized in 100% dimethyl sulfoxide and stored at −80°C until tested, unless alternative solvents and storage conditions were specified by the sponsor. The final dimethyl sulfoxide concentration was ≤1% at the highest test concentration, which has no effect on antiviral activity (unpublished data). T20 was dissolved in sterile phosphate-buffered saline (PBS).

PRO 2000, obtained from Indevus Pharmaceuticals, was resuspended in H₂O at a stock concentration of 40 mg/ml, and spectrophotometric evaluation of a 0.1-mg/ml solution at 290 nm yielded an absorbance of 2.023. SPL7013, obtained from Starpharma Pty, Ltd., was dissolved at a concentration of 40 mg/ml in H₂O. Dissolved compounds were stored at −80°C until use.

Sterile seminal plasma was obtained through Tennessee Blood Services (Memphis, TN) in compliance with all relevant federal guidelines and institutional policies from HIV-1-seronegative healthy individuals with no recent history of STIs, no urogenital abnormalities, and abstinence from sexual intercourse for 48 h prior to collection. The semen samples were collected in a sterile container, diluted 1:1 with serum-free AIM-V medium (Invitrogen), and centrifuged to separate the seminal plasma from the sperm cells. The seminal plasma was shipped on wet ice overnight to the testing facility. Since no patient identifier was provided, the received samples are exempt from Institutional Review Board Approval. Upon arrival, the seminal plasma from 5 to 10 donors was pooled and either used immediately in the assay or stored in aliquots at −80°C until use.

CCR5- and CXCR4-tropic CD4-dependent cell-associated HIV-1 transmission inhibition assays.

The CD4-dependent cell-associated HIV-1 transmission inhibition assays use the CD4-positive GHOST(3) X4/R5 cell line as target cells (26). The effector cells (i.e., virus transmitting cells) are either H9 or MOLT-4/CCR5 cells chronically infected with the SK1 isolate of HIV-1 (CXCR4-tropic assay) or with the HIV-1_JR-CSF molecular clone (CCR5-tropic assay), respectively. At 24 h prior to the assay, the target cells are seeded in 96-well flat-bottom microtiter plates at 10⁴ cells per well. On the day of assay, effector cells are treated with mitomycin C (200 μg/ml; Sigma-Aldrich) for 60 min at 37°C in order to induce cell death without impairing virus transmission and then washed once with complete Dulbecco modified Eagle medium (cDMEM). Test compounds are added to the target cells followed by 500 (CXCR4-tropic) or 1,000 (CCR5-tropic) effector cells. The effector and target cells are incubated with drug for 4 h, followed by three washes with cDMEM (cDMEM). Twenty hours after assay initiation, the assay medium is removed, and the cells are lysed by adding 100 μl of lysis buffer containing 20 mM Tris (pH 7.5), 50 mM NaCl, and 0.5% Triton X-100 with one cycle of freeze-thaw prior to testing the lysate in the HIV-1 Gag p24 enzyme-linked immunosorbent assay (ELISA). TAK-779 and AMD3100 are used as control compounds to demonstrate specificity for the CCR5 or CXCR4 coreceptor (Table 1).

CCR5- and CXCR4-tropic HIV-1 entry assay.

This assay is designed to identify compounds that inhibit cell-free HIV-1 entry into HeLa CD4 LTR β-Gal (MAGI) or MAGI-CCR5 cells (7, 22). The CCR5-tropic HIV-1 entry assay is one of the primary screening assays and is performed as described previously, with the modifications outlined below (25).

At 24 h prior to the assay cells were plated at 5 × 10³ per well for the CXCR4-tropic or at 10⁴ per well for the CCR5-tropic HIV-1 entry assay. The medium was removed, and the compound diluted in medium was placed on the cells and immediately incubated for 15 min at 37°C. Ten 50% tissue culture infective doses of the IIIB or Ba-L strains of HIV-1 were then added to the wells, and incubation was continued for 2 h for the CXCR4-tropic or 3 h for the CCR5-tropic viral entry assay. At the end of the incubation, the wells were washed once with cDMEM, and the culture was continued for 40 to 48 h at 37°C. At termination of the assay, the medium was removed, and the β-Gal enzyme expression was determined by chemiluminescence using Tropix Gal-Screen (Applied Biosystems) according to the manufacturer's instructions. TAK-779 and AMD3100 were used as control compounds to demonstrate specificity for the CCR5 or CXCR4 coreceptor (Table 1).

Seminal plasma and pH transition assays.

The cell-associated and cell-free CD4-dependent HIV-1 transmission inhibition assays were modified to include human seminal plasma or a pH transition from 4.0 to 7.0 (Table 1). Cell-free virus or effector cells in seminal plasma were added to target cells, which were preincubated for 15 min with drug, yielding a final concentration of 12.5 or 25% seminal plasma, respectively, for the cell-free or cell-associated assays. The compound Quinobene (15) served as an additional assay performance control since it is consistently inactivated at a concentration of 100 μg/ml in the presence of seminal plasma.

The pH transition assay mimics the pH change that occurs during sexual intercourse. It has been shown that the pH in the vaginal tract of postpuberal, premenopausal women is ∼4.0 (4) and that the pH of semen is ∼7.7 (33). The target cells, representing the vaginal tract, were incubated with compound in assay medium at pH 4.0, and the effector cells, representing the ejaculate, were resuspended in assay medium at pH 7.7. The addition of the effector cells to the target cells resulted in a neutral pH. Since the exposure time of the target cells to the low pH was short, the effect on cell viability was negligible (data not shown).

CD4-independent cell-associated HIV-1 transmission inhibition assay.

The assay was performed similarly to the CD4-dependent assay described above, except that ME-180, a CD4-negative cervical cell line that contains human papillomavirus DNA, was used as the target cell (16, 36). ME-180 cells were plated at a density of 5 × 10³ cells per well 48 h prior to the assay. On day 2, serially diluted compound and 5 × 10⁴ mitomycin C-treated H9/HIV-1_SK1 cells were added to each well. After a 4-h incubation, compound and effector cells were removed by washing the plate three times with PBS. Additional washing steps were performed at 24 and 48 h postinfection. On day 6, supernatants were collected and evaluated for cell-free HIV-1 Gag p24 antigen expression by ELISA. Dextran sulfate was used as the positive control compound, and dextran was used as the negative control compound (Table 1).

CCR5- and CXCR4-tropic fusion assays.

The fusion assays assess the ability of compounds to block cell-to-cell fusion mediated by HIV-1 Env expressed on one cell line that interacts with the CD4 receptor and the CXCR4 or CCR5 coreceptors expressed on a separate cell line. Upon fusion of the two cell lines, HIV-1 Tat was introduced from the effector into the target cell (MAGI or MAGI-CCR5), leading to the transactivation of the LTR attached to the β-Gal reporter gene and expression of the β-Gal enzyme. This assay is sensitive to inhibitors of gp120 to CD4/CCR5/CXCR4 binding and inhibitors of gp41-mediated fusion. The CXCR4-tropic assay was performed as described previously (25). The CCR5-tropic fusion assay was set up in the same manner except that 1.5 × 10⁴/well MAGI-CCR5 cells (expressing CD4 and CCR5) were used as target cells and 7.5 × 10³/well HeLa R5-16 cells (expressing HIV-1 Tat and CCR5-tropic Env from HIV-1 92US715, GenBank accession no. U08451 (20) were used as effector cells. For the CCR5-tropic assay, the effector cells were plated 1 day before the assay. Target and effector cells with compound were incubated without intermediate washing steps for 40 to 48 h, after which fusion was monitored by the measurement of β-Gal enzyme expression, detectable by chemiluminescence (Tropix Gal-Screen β; Applied Biosystems). TAK-779 and AMD3100 were used as control compounds to demonstrate specificity for the CCR5 or CXCR4 coreceptor (Table 1). T20 and Chicago Sky Blue served as additional control compounds.

Human PBMC-based assays and combination drug assays.

For testing topical microbicide candidates against the clinically more relevant primary HIV-1 isolates, an assay using primary human PBMCs was established. For the PBMC-based assay, phytohemagglutinin (PHA; Sigma-Aldrich)-stimulated cells from at least two normal donors were pooled and plated in 50 μl at 5 × 10⁴ cells/well. Each plate contained no-compound control wells (cells plus virus) and experimental wells (compound, cells, and virus) for two different test compounds, evaluated in triplicate wells at nine different concentrations. Primary HIV-1 strains were obtained predominantly from the NIH AIDS Research and Reference Reagent Program. A portion (50 μl) of virus stock at a predetermined titer was added to each test well. The PBMC cultures were incubated at 37°C and 5% CO₂ for 7 days after infection, and subsequently cell-free supernatant samples were collected for analysis of reverse transcriptase activity (5) or HIV-1 Gag p24 antigen.

For combination testing of topical microbicides, the CCR5-tropic HIV-1 antiviral assay in MAGI cells was used. The assay procedure is similar to the HIV-1 CCR5-tropic viral entry assay except that the virus and drug were left in the culture for 48 h. For each two-drug combination assay, five concentrations of drug 1 (e.g., a U.S. Food and Drug Administration-approved drug) were tested in all possible combinations with eight concentrations of drug 2 (e.g., the test compound under development). As part of the assay, each compound was also tested in a single dose-response determination for evaluation of the individual compound antiviral activities. The combination data analysis was performed using the MacSynergy II 3-D model developed by Prichard and Shipman (37, 38), which determines antagonistic, additive, or synergistic effects of the drug combination. The positive antagonism control of stavudine in combination with Ribavirin was included into each experiment in order to validate the assay system.

Cervical explant assays.

All tissues were acquired through the National Disease Research Interchange (NDRI; Philadelphia, PA). NDRI complies with all federal and institutional regulations concerning privacy, informed consent, and the shipping and handling of biomaterials. Normal ectocervix was obtained from premenopausal women undergoing routine hysterectomy. All donors were tested for HIV seropositivity, and a pathology report was provided with every shipment, allowing exclusion of tissue with abnormal pathological findings. Tissues were transported on ice in L15 (Leibovitz) medium (Invitrogen) containing 10% fetal calf serum, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. The cervix was washed twice with PBS. The cervical explant assay was performed essentially as described previously (10). Briefly, dermal biopsy punches (3 mm; Miltex Instrument Company, Inc., Bethpage, NY) were used to cut full-thickness tissue specimens. Typically, 15 to 30 punches can be obtained from one donor, allowing for testing of one compound's efficacy in a dose-response curve in four concentrations with four replicates. The remainder of the punch biopsies was used for toxicity evaluation. If extensive toxicity testing is required and more tissue is needed, tissues from another donor were utilized.

Each explant was placed in a 96-well plate (submerged, not embedded in agarose) and cultured in a final volume of 0.25 ml of DMEM containing 10% human AB serum, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and nonessential amino acids. Tissues were cultured at 37°C with 5% CO₂ in a humidified incubator.

In some instances, explants were activated for 2 days in cDMEM containing 4 μg of PHA/ml and 100 U of human interleukin-2 (IL-2; Roche, Indianapolis, IN)/ml. If control or experimental compound was added, it was prepared at two times the high test concentration in a 0.125-ml volume of cDMEM and added to the explant tissues. After a 1-h incubation, 0.125 ml of HIV-1_Ba-L (containing 2.5 × 10⁴ 50% tissue culture infective doses) in complete medium was added to the epithelium and incubated overnight (∼18 h). Afterward, the epithelium was washed five times with PBS to remove residual virus, and the culture medium was replaced with cDMEM containing IL-2 without PHA. If tissue activation was not used, the same virus quantity was added on day 0 in cDMEM without IL-2, and the same infection procedure as described above was followed. Culture medium (∼0.25 ml) was harvested every 3 to 4 days over a 14- to 16-day period and stored at −80°C. Viral replication was determined by using a HIV-1 Gag p24 ELISA.

To minimize donor-to-donor variability, each test compound was evaluated in tissues from at least two donors. Topical microbicide candidates were typically only tested as single compounds and not in combination with other compounds. To account for variability within donor tissues, four replicates were used per drug concentration. The positive control for the assay was the non-nucleoside reverse transcriptase inhibitor UC-781 (NSC 675186), and the negative control was medium alone. Toxicity using MTT was tested concurrently with efficacy as described below on tissues from the same donor.

HIV-1 Gag p24 antigen ELISA.

ELISA kits were purchased from Perkin-Elmer (Shelton, CT). Detection of supernatant or cell-associated p24 antigen was performed according to the manufacturer's instructions.

CellTiter 96 staining for cell viability and MTT assay of explant tissues.

In all cell-based assays, cell viability and 50% toxic concentrations (TC₅₀s) were derived by using a commercially available soluble tetrazolium-based MTS reagent kit (CellTiter 96 AQueous One Solution cell proliferation assay), which uses a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity, or chemosensitivity assays (Table 2). The assay was performed according to the manufacturer's instructions.

TABLE 2.

Comparison and summary of methods for microbicide toxicity assays

Parameter	Lactobacillus growth inhibition assay	MTS assay	MTT assay	Live/dead cytotoxicity assay	Vybrant cytotoxicity assay
Manufacturer		Promega	Sigma	Molecular Probes/Invitrogen	Molecular Probes/Invitrogen
Cell type	Lactobacillus jensenii or L. crispatus	All mammalian cells	Cervical explant tissue	All mammalian cells	All mammalian cells
Medium	Lactobacillus MRS broth (Difco/Fisher Scientific, Pittsburgh, PA)	DMEM complete medium without phenol red	DMEM with phenol red	DMEM complete medium without phenol red	DMEM complete medium without phenol red
Assay principle	Growth inhibition of bacteria	MTS tetrazolium compound that is bioreduced by cells into a colored formazan product (mitochondrial function)	MTT is bioreduced by cells into a colored formazan product (mitochondrial function)	Measurement of intracellular esterase activity and plasma membrane integrity with two fluorescent probes	Release of the cytosolic enzyme G6PD from damaged cells
Compound dilutions	6 log10 or half log10	6 log10 or half log10	2 to 4 log10 or half log10	6 log10 or half log10	6 log10 or half log10
Replicates per compound exposure	3	3	3-4	3	3
Time (h) of compound dilution	24	2 to 4	5	3	3
Assay duration	24 h	50 h	14 to 16 days	49 h	49 h
Endpoint	OD490	OD490/650	OD570	Fluorescence	Fluorescence
Control compound(s)	Penicillin-streptomycin	Triton X-100, nonoxynol-9	Triton X-100	Triton X-100, nonoxynol-9	Triton X-100, nonoxynol-9

The viability of the explants and assessment of compound toxicity was quantified by the reduction of the tetrazolium salt, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), by viable tissue into a methanol-soluble formazan product (Table 2). Cervical explants were incubated overnight (∼18 h) with or without product in cDMEM. For comparison, a tissue control was used that was incubated in medium alone. After the explants were washed five times in PBS, tissues were immediately cultured in cDMEM containing MTT (250 μg/ml) for an additional 2 h at 37°C. Tissues were then placed in 200 μl of absolute methanol for a minimum of 24 h (protected from light). Tissue viability was determined by dividing the optical density at 570 nm (OD₅₇₀) of the formazan product by the dry weight of the explant. The effect of each microbicide on tissue viability was determined by comparing the viability of the treated explants to the untreated tissue control. Tissues from a minimum of two donors were tested for each product.

Live/Dead and Vybrant cytotoxicity assays.

The Live/Dead cytotoxicity assay (Invitrogen) provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of live and dead cells with two probes that measure two recognized parameters of cell viability: intracellular esterase activity and plasma membrane integrity. The Vybrant cytotoxicity kit (Invitrogen) monitors the release of the cytosolic enzyme glucose 6-phosphate dehydrogenase (G6PD) from damaged cells into the surrounding medium. Both assays were performed according to the manufacturer's instructions.

Lactobacillus toxicity assays.

Lactobacillus crispatus and L. jensenii were grown in Lactobacillus MRS broth (Difco/Fisher Scientific, Pittsburgh, PA). This medium allows efficient growth of the Lactobacillus species under facultative anaerobic conditions. Bacterial stocks were produced and frozen in 15% glycerol at −80°C. To assess the effect of compounds on L. crispatus and L. jensenii growth, 10 ml of MRS medium was inoculated with a stab from the glycerol bacterial stock, and the culture was incubated for 24 h at 37°C. After the incubation the bacterial density was adjusted to an OD₆₇₀ of 0.06, corresponding to a 0.5 McFarland turbidity standard or ca. 10⁸ CFU/ml (21). Compounds were plated at the appropriate concentrations into 96-well round-bottom plates in a volume of 100 μl, and the diluted Lactobacillus spp. were also added in a volume of 100 μl. Commercially available penicillin-streptomycin solutions at maximal test concentrations of 1.25 U/ml and 1.25 μg/ml, respectively, were used as the positive toxicity controls. The plates were incubated for 24 h at 37°C in a GasPak CO₂ bag, and bacterial growth was determined by measurement of the OD₄₉₀ using a 96-well spectrophotometric plate reader (Table 2).

Data analysis and quality control protocol.

Defined criteria for quality control and pass/fail decisions were established for all assays. For example, the assays failed if the HIV-1 Gag p24 measurements for the virus control were less than 200 pg/ml for the CD4-independent (ME-180) cell-associated assays and less than 500 pg/ml for the CD4-dependent (GHOST) cell-associated assays. The viral entry and fusion assays failed if the relative light units were less than 20,000 for the virus control wells. These cutoff values were chosen based on the analysis of historical data. Additional quality control parameters included confirmation that the negative control compound is inactive and that the the positive control is active. Activity ranges of the positive controls are provided in Table 3. For the toxicity assays, the plates were developed after the addition of the colorimetric reagent until the ODs exceeded 0.5. Subjective observations (e.g., visual inspections) were also made during each assay. At the beginning of the experiment, the wells containing the high-test concentrations of drugs were inspected microscopically for evidence of compound precipitation. Similarly, at the end of the experiment, the plates were quickly monitored to verify that the cell pellets or monolayers were uniform in size or confluence.

Controls included with each assay (e.g., background signal controls, compound color controls, etc.) were used to normalize the data. Replicate values were then averaged, and the percentages of control values were calculated for antiviral efficacy (percent virus control) and cytotoxicity (percent cell control viability). Standard deviations were calculated from replicate values, with 20% serving as a cutoff for quality control. The dose-response data were analyzed to compute the 50 and 90% inhibitory concentrations (IC₅₀ and IC₉₀, respectively) and toxic concentrations (TC₅₀) by interpolation between the two data points surrounding the value to be calculated. The ID₅₀ (i.e., the concentration inhibiting 50% growth of the lactobacilli) was determined by the same method used for the IC₅₀ computation. The therapeutic indices were calculated by dividing the appropriate toxicity values by the efficacy values (i.e., TC₅₀/IC₅₀).

For each assay, raw data for antiviral activity and toxicity with a graphical representation of the data were provided in a printout summarizing the individual compound activity.

RESULTS

Utility of the in vitro algorithm for topical microbicide efficacy testing.

An algorithm consisting of a variety of in vitro assays was developed for preclinical evaluations of the activity and cytotoxicity of compounds with antiviral activity against HIV-1 that may have potential for development as topical microbicides (Fig. 1). The algorithm serves as a mechanism for preclinical testing of proprietary topical microbicide candidates that are submitted from academic, government, and private sector sources to the NIAID. Generally, compounds submitted for testing have shown prior anti-HIV-1 activity in an antiviral assay, but compounds of unknown antiviral activity also may be screened if there is a strong scientific rationale supporting why they would be expected to demonstrate inhibitory activity as a microbicide (e.g., structural analogs of a known active compound). Every compound is initially tested in two primary microbicide assays, a cell-associated CCR5-tropic CD4-dependent HIV-1 transmission inhibition assay and a cell-free CCR5-tropic CD4-dependent HIV-1 entry assay. Figure 2 shows a representative graph for the CCR5-tropic viral entry assay. CD4/CCR5-tropic assays were chosen because it has been shown that, in an ex vivo human tissue system, HIV-1 entered CD4⁺ T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion (19). The assays were designed with removal of compound and virus 3 to 4 h after infection to select for mechanisms of action that target early steps of the HIV-1 life cycle (attachment, fusion, uncoating, and reverse transcriptase inhibitors). The β-Gal reporter assay is also sensitive to inhibitors of the Tat/TAR interaction and suppressors of the β-Gal enzyme, which is considered an artifact of the assay. Since intense color could be a deterrent to the use of a microbicide, a standard was established for advancing active compounds on the basis of color. According to this scheme, colorless compounds with a therapeutic index of >10 or colored compounds with a therapeutic index of >50 based on a high-test of 100 μg/ml or 100 μM are considered active. The threshold for the therapeutic index is set low to identify a wide variety of active compounds that will be narrowed down by the results in the secondary and specialized assays. In addition, for compounds with low activity, analogs are identified and evaluated in the algorithm's primary assays. Whenever possible, analogs that have been tested previously and found to have HIV-1 antiviral activity were selected for building structure-activity relationships. However, analogs of unknown activity also may be screened in the primary assays since these assays can effectively identify antiviral compounds that have the potential for development as microbicides. Analogs that were found to be active were advanced for confirmatory and secondary testing in the topical microbicide algorithm.

FIG. 1.

Current NIAID topical microbicide algorithm. Every compound that is submitted for testing through the NIAID microbicide program is subjected to two primary assays: a cell-associated CCR5-tropic CD4-dependent HIV-1 transmission assay and a cell-free CCR5-tropic CD4-dependent viral entry assay. If the compounds are active in the primary assays, they are advanced to confirmatory testing. If a compound is confirmed to be active, it is moved to secondary screening, which evaluates the compound under more physiological conditions and delineates its mechanism of action. Highly active compounds are also tested in the specialized assays. TI, therapeutic index.

FIG. 2.

Representative data and graph for the CCR5-tropic HIV-1 entry assay. The assay is typically performed at six drug concentrations in triplicate. The mean and percent standard deviations of the triplicate data were calculated and are displayed graphically. Cytotoxicity was evaluated in parallel with the efficacy. The IC₅₀, TC₅₀, and IC₉₀ were calculated by linear regression analysis, and the therapeutic index (i.e., the TC₅₀ divided by the IC₅₀) is indicated. TI, therapeutic index.

Confirmatory tests of the compounds consist of a repeat of the same primary assay in which it was active. In rare circumstances, it occurs that the activity of the primary assays cannot be confirmed, possibly due to compound instability or lack of reproducibility for technical reasons. If activity cannot be confirmed, the compounds do not progress to testing in the secondary assays.

Compounds confirmed to be active in either of the two primary assays (therapeutic index of >10) were tested in the following secondary assays: the CXCR4-tropic cell-free HIV-1 entry assay, the CXCR4- and CCR5-tropic fusion assays, the cell-associated CXCR4-tropic CD4-dependent HIV-1 transmission inhibition assay, the cell-associated CXCR4-tropic CD4-independent HIV-1 transmission inhibition assay (ME-180 cell assay), and the Lactobacillus toxicity assays (Fig. 1). The secondary assays were developed to demonstrate whether the topical microbicide candidates could be classified as entry or fusion inhibitors and show activity against CCR5- and CXCR4-tropic viruses. In addition, the ME-180 assay selects for compounds that inhibit HIV-1 in a CD4-independent manner. To eliminate falsely active compounds, a cytotoxicity plate was evaluated in parallel using an MTS-based system. If compounds reached a TC₅₀ below 100 μg/ml or 100 μM, they were flagged as potentially toxic. Finally, if a compound was shown to be toxic to beneficial Lactobacillus species, it was considered not suitable as a topical microbicide and was not evaluated further.

Compounds confirmed to be active in the primary assays were also tested in the presence of seminal plasma or a pH transition, two conditions, which could negatively influence compound activity. Although the concentration of seminal plasma is potentially higher than 12.5 to 25% during sexual intercourse, this is the maximal concentration that can be achieved using the current assay conditions. If the activity of the compound is diminished in the presence of seminal plasma or a pH transition, repeat testing at higher concentrations (e.g., 1 mM or 1 mg/ml high-test concentrations) can be performed based on the overall testing results for a particular compound. This repeat testing is sometimes useful to demonstrate whether the higher compound concentration can overcome the reduced activity under these modified assay conditions.

Control compounds routinely used for the individual assays are shown in Tables 1 and 2. The acceptable maximum IC₅₀ values for the control compounds were calculated by adding two standard deviations to the mean of historical data (Table 3). All historical IC₅₀ data fell within this range, with the exception of a maximum of two outliers per control compound. Figure 3 shows the distribution of 111 IC₅₀ values of TAK-779 in the CCR5-tropic HIV-1 entry assay. All but one value fell within two standard deviations of the mean (Fig. 3A) and were normally distributed (Fig. 3B).

FIG. 3.

(A) One-hundred eleven historical IC₅₀ values for TAK-779 in the CCR5-tropic viral entry assay were evaluated to determine whether they fall within two standard deviations of the mean. Only one outlier was found. (B) The frequency distribution of raw IC₅₀s of TAK-779 in the CCR5-tropic viral entry assay is shown.

Selected compounds identified as highly active and not toxic to cell lines or Lactobacillus species with a preferred mechanism of action were advanced for further testing beyond the secondary assays. These specialized assays included, but were not limited to, testing in the human cervical explant model; clade panel testing against representative HIV-1 strains of the M, N, and O groups; and combination drug assays.

Human cervical explant assays were used for highly active compounds to confirm activity in a tissue-based setting. Since only CCR5-tropic viruses produce a reliable infection of the explant tissues, the assay routinely uses HIV-1_Ba-L. As shown in Fig. 4, the thiocarboxanilide non-nucleoside reverse transcriptase inhibitor UC-781 suppressed virus replication completely at concentrations of 50 and 100 μM in the explant model, and the furanyl-containing analog of oxathiin carboxanilide, UC-10, suppressed virus replication entirely at 100 μM but not at 5 μM.

FIG. 4.

Inhibition of HIV-1_Ba-L by UC-781 and UC-10 in the human cervical explant model. The arithmetic mean of four replicates for each compound concentration or control is shown. UC-781 suppressed virus replication completely at concentrations of 50 and 100 μM. UC-10 suppressed virus replication entirely at 100 μM but not at 5 μM. Both compounds are analogs of oxathiin carboxanilides.

The purpose of testing compounds in primary PBMC is to demonstrate whether the compounds have general activity against primary CCR5- or CXCR4-tropic isolates and to evaluate efficacy against subtypes other than B that are prevalent in countries where HIV-1 is endemic. Combination testing to evaluate synergistic, additive, or antagonistic properties is needed if microbicide candidates are designated to be applied in combination with other products. Of particular importance is the identification of antagonistic or toxic combinations. Finally, drug resistance assays test for the ability of the topical microbicide candidates to combat single or multidrug resistant isolates of HIV-1.

Elimination of falsely active compounds by cytotoxicity testing.

In addition to the MTS assay, the specialized toxicity assays, Vybrant and Live/Dead (Invitrogen), were performed for highly active compounds. The three different cytotoxicity assays complement each other by assessing cell viability as a function of different aspects of cell metabolism. The MTS assay was routinely performed in parallel with the efficacy assays to distinguish between activity and cytotoxicity. The Vybrant and Live/Dead assays were used for extended cytotoxicity testing on high priority topical microbicide candidates.

PRO 2000 and SPL7013 were highly active in the topical microbicide testing algorithm.

To demonstrate how two microbicide candidates currently in clinical trials perform in the topical microbicide testing algorithm, PRO 2000 and SPL7013 were tested in the described in vitro assays. The naphthalene sulfonate polymer PRO 2000 is currently being evaluated in a phase 2/2B clinical trial at a concentration of 0.5% in combination with BufferGel and in a phase 3 trial at concentrations of 0.5 and 2%. The dendrimer SPL7013, the active component of VivaGel, is currently in phase 1 clinical trials in a formulation containing 3% (wt/wt) active ingredient (24).

PRO 2000 was active in the cell-associated and cell-free CCR5-tropic HIV-1 transmission assays, with mean therapeutic indices of >86 and >128, respectively (Table 4). It therefore met the criteria for an active compound (therapeutic index of >10). It showed a 5-fold loss of activity in the CCR5-tropic cell-free pH transition assay, an 18-fold loss of activity in the CCR5-tropic cell-free seminal plasma assay, a 19-fold loss of activity in the CCR5-tropic cell-associated assay with pH transition, and a 28-fold loss of activity in the CCR5-tropic cell-associated assay with seminal plasma. PRO 2000 was also highly active in the CXCR4-tropic cell-associated assay and CXCR4-tropic cell-free viral entry assay, with mean therapeutic indices of >303 and >270, respectively. It was active in the CCR5-tropic and CXCR4-tropic fusion assays but not in the CD4-independent ME-180 assay. The compound was also not toxic to L. jensenii and L. crispatus.

TABLE 4.

Activity of PRO 2000 in the in vitro topical microbicide algorithm

Assay type	No. of repeats (description)	Mean concn (μg/ml) ± SD			TIa	Comment
		IC50	IC90	TC50
CD4-dependent CCR5-tropic	5 (standard)	1.16 ± 0.64	7.17 ± 2.01	>100	>86	Active
cell-associated HIV-1 transmission assay	3 (pH transition)	21.57 ± 17.64	74.07 ± 16.14	>100	>5	Avg of 19-fold-reduced activity
	3 (25% seminal plasma)	32.23 ± 26.12	>100	>100	>3	Avg of 28-fold-reduced activity
CD4-dependent CCR5-tropic	7 (standard)	0.78 ± 0.61	18.34 ± 36.12	>100	>128	Active
viral entry assay	3 (pH transition)	4.28 ± 1.52	43.59 ± 31.06	>100	>23	Avg of 5-fold-reduced activity
	3 (12.5% seminal plasma)	13.89 ± 16.24	59.60 ± 34.72	>100	>7	Avg of 18-fold-reduced activity
CD4-dependent CCR5-tropic fusion assay	4	1.80 ± 0.60	7.09 ± 0.51	>100	>56	Active
CD4-dependent CXCR4-tropic cell-associated HIV-1 transmission assay	3	0.33 ± 0.22	35.01 ± 56.32	>100	>303	Active
CD4-dependent CXCR4-tropic viral entry assay	3	0.37 ± 0.09	1.08 ± 0.31	>100	>270	Active
CD4-dependent CXCR4-tropic fusion assay	6	0.63 ± 0.08	4.91 ± 0.60	>100	>159	Active
CD4-independent assay	3	52.87 ± 40.83	>100 ± 0	>100	>2	Not active
Lactobacillus crispatus	1	NAb	NA	>100	NA	Not toxic
Lactobacillus jensenii	1	NA	NA	>100	NA	Not toxic

^aTI, therapeutic index.

^bNA, not applicable.

SPL7013 showed a pattern of activity similar to that of PRO 2000. It was highly active in the two primary assays, with mean therapeutic indices of >81 and >370, respectively (Table 5). It showed a 3-fold loss of activity in the CCR5-tropic cell-free pH transition assay, a 9-fold loss of activity in the CCR5-tropic cell-free seminal plasma assay, an 8-fold loss of activity in the CCR5-tropic cell-associated assay with pH transition, and a >81-fold loss of activity in the CCR5-tropic cell-associated assay with seminal plasma. In the CXCR4-tropic cell-associated HIV transmission assay, it had a mean therapeutic index of >333. SPL7013 was also highly active in the CXCR4-tropic viral entry and fusion assays, with mean therapeutic indices of >238 and >192, respectively, and in the CD4-dependent ME-180 assay with a mean therapeutic index of >74. It was also active in the CCR5-tropic fusion assay and was not toxic to L. jensenii and L. crispatus.

TABLE 5.

Activity of SPL7013 in the in vitro topical microbicide algorithm

Assay type	No. of repeats (description)	Mean concn (μg/ml) ± SD			TIa	Comment
		IC50	IC90	TC50
CD4-dependent CCR5-tropic	5 (standard)	1.23 ± 0.55*	16.28 ± 12.62	>100	>81	Active
cell-associated HIV-1 transmission assay	3 (pH transition)	9.25 ± 6.57	71.70 ± 14.12	>100	>11	Avg of 8-fold reduced activity
	3 (25% seminal plasma)	>100	>100	>100	1	Avg of >81-fold-reduced activity
CD4-dependent CCR5-tropic	7 (standard)	0.27 ± 0.05	1.33 ± 1.23	>100	>370	Active
viral entry assay	3 (pH transition)	0.74 ± 0.09	5.98 ± 0.09	>100	>135	Avg of 3-fold-reduced activity
	3 (12.5% seminal plasma)	2.48 ± 0.49	21.22 ± 20.51	>100	>40	Avg of 9-fold-reduced activity
CD4-dependent CCR5-tropic fusion assay	4	2.14 ± 0.28	7.34 ± 0.19	>100	>47	Active
CD4-dependent CXCR4-tropic cell-associated HIV-1 transmission assay	3	0.30 ± 0.11	5.36 ± 6.19	>100	>333	Active
CD4-dependent CXCR4-tropic viral entry assay	3	0.42 ± 0.06	0.90 ± 0.05	>100	>238	Active
CD4-dependent CXCR4-tropic fusion assay	6	0.52 ± 0.06	3.41 ± 1.06	>100	>192	Active
CD4-independent assay	3	1.35 ± 0.31	68.85 ± 6.15	>100	>74	Active
Lactobacillus jensenii	1	NAb	NA	>100	NA	Not toxic
Lactobacillus crispatus	1	NA	NA	>100	NA	Not toxic

^aTI, therapeutic index.

^bNA, not applicable.

Since both compounds showed decreased activity during a pH transition and in the presence of human seminal plasma, they were retested at a high-test concentration of 1 mg/ml to evaluate whether the compounds are still active at increased concentrations relevant to clinical doses. The clinically applied concentrations are 30 mg/ml (3%) for SPL7013 and 5 or 20 mg/ml (0.5 or 2%) for PRO 2000, values which greatly exceed the concentrations used for the in vitro assays. The results demonstrated that PRO 2000 inhibited virus replication by at least 87% and SPL7013 inhibited virus replication by at least 82% relative to the no-compound control at a concentration of 1 mg/ml in the presence of 25% human seminal plasma (Fig. 5 and 6) or during a pH transition from 4.0 to 7.0 (data not shown). Therefore, PRO 2000 and SPL7013 are likely active in the environment of the vaginal vault at concentrations used in the ongoing clinical trials.

FIG. 5.

Evaluation of PRO 2000 at a maximum concentration of 1 mg/ml in the CCR5-tropic HIV-1 entry assay (A) and in the CCR5-tropic HIV-1 cell-associated transmission assay (B) in the presence of seminal plasma. The data points represent the averages and standard deviations of three replicates of each drug concentration. The increase above 100% virus control at the lower compound concentrations reflects the inherent variability of the assay and not the true infectivity enhancement of the compound.

FIG. 6.

Evaluation of SPL7013 at a maximum concentration of 1 mg/ml in the CCR5-tropic HIV-1 entry assay (A) and in the CCR5-tropic HIV-1 cell-associated transmission assay (B) in the presence of seminal plasma. The data points represent the average and standard deviation of three replicates of each drug concentration. The increase above 100% virus control at the lower compound concentrations reflects the inherent variability of the assay and not the true infectivity enhancement of the compound.

DISCUSSION

Preclinical evaluation of candidate microbicides prior to selection for clinical trials provides considerable savings in costs and time, given the expense and length of time required for formal efficacy trials (40). Selection criteria include high activity against cell-free and cell-associated virus in mucosal explant models, low irritation potential based on a range of preclinical toxicity assays, high in vitro activity in the presence of semen, and effectiveness in animal models using a challenge virus that is relevant to sexual HIV transmission. Thus, one of the goals of the microbicide testing algorithm is to find in vitro markers of efficacy and toxicity that correlate with clinical outcomes. The presented algorithm is a prioritization screen for topical microbicide candidates based on the best estimate of how in vitro assays relate to complex in vivo phenomena.

The compounds that have been or are currently in phase 2 or 3 clinical trials represent a limited number of chemical classes. Three of these products (PRO 2000, Carraguard, and Ushercell) are anionic polymers and inhibit HIV-1 infection by preventing virus-cell entry mechanisms (1, 43), predominantly through charge-based interactions with the V3 loop of gp120 (6, 27, 30). The other two microbicides tested in phase 2 or 3 trials are a buffering gel (BufferGel) containing the carbopol 974P polymer, and a product based on the novel surfactant C31G (termed Savvy) (11). SPL7013, which is currently being evaluated in a phase 1 trial, is a dendrimer possessing a highly branched three-dimensional structure. Thus, another aim of the in vitro screening algorithm is to expand the microbicides pipeline by identifying compound classes that are currently underrepresented. Potentially, all compounds that inhibit HIV before integration, including virucidal agents, attachment and fusion inhibitors, products that prevent uncoating, and reverse transcriptase inhibitors, are topical microbicide candidates of first choice. Integrase inhibitors and compounds that act after virus integration can be added to a combination microbicide that includes mainly compounds acting before integration. The two primary screening assays were designed to identify compounds that prevent HIV infection by inhibition of targets up to the reverse transcription process. However, the reporter-based CCR5-tropic cell-free viral entry assay also identifies compounds as actives that inhibit the Tat-TAR interaction. The confirmatory assays verify the results obtained in the primary assays. Secondary assays test the compounds' robustness in the presence of a pH transition from 4 to 7 or human seminal plasma. The rationale for testing the compounds in the pH transition assay is that the secondary and tertiary structure of some compounds, in particular proteins and antibodies, can be affected by an acidic or alkaline pH, changing the activity profile of the compound. In addition, seminal plasma has the potential to inactivate compounds due to its substantially higher osmolarity than blood plasma (354 mosm); slightly alkaline pH (7.7); and high protein, glucose, fructose, and ion (citrate, Ca, Cl, K, Mg, Na, and Zn) content (33). In particular, cations could neutralize the negatively charged polyanionic compounds, eliminating their charge-based mechanism of action, and direct protein binding to charged sulfated polyanions could also result in reduced activity. In this regard, it has been reported that carrageenan and cellulose sulfate are 57- and 24-fold less active, respectively, against CCR5-tropic infection in 33% seminal plasma (31). The current decision-making process requires that a microbicide candidate should still be highly active in the presence of seminal plasma and stable at pH 4 for up to 2 h to warrant further preclinical testing. The activity change of the test compounds in relation to exposure time at pH 4 is currently under investigation. However, some formulated products, such as VivaGel, have a pH of 4.5 to 5.5, so they have the required long-term chemical stability under these acidic conditions.

In contrast to the results reported by Münch et al. (28), no enhancement of infection in the presence of seminal plasma was observed in the presented studies. One reason for the lack of enhancement may be that the high multiplicity of infection and/or infectivity of the chronically infected cells mask the effect of the semen-derived enhancer of virus infection. In the future, evaluating lead topical microbicide candidates in the presence of unprocessed sperm in comparison to seminal plasma will also be considered.

Future directions for the algorithm include incorporating vaginal secretions or simulant, which consists of ions, serum bovine albumin, lactic acid, acetic acid, glycerol, urea, and glucose (34), into the assay. All of these components have the potential to interfere with the activity of compounds against HIV and other sexually transmitted diseases. Historically, the algorithm included an assay with medium containing 150 μg of porcine mucin/ml to mimic the human mucin present in the vaginal tract, but only minimal differences in activity with or without mucin were observed (data not shown). Therefore, the mucin assay was removed from the algorithm.

In addition to the seminal plasma and pH transition assays, a secondary screen comprised of a variety of mechanistic assays is performed. Viral entry and fusion assays identify compounds that inhibit the early events of HIV-1 infection. The ME-180 assay identifies compounds that block HIV-1 transmission to a cervical cell line in a CD4-independent manner (36). TAK-779, AMD3100, T20, and most nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors were inactive in this assay, which uses a 4-h incubation of compound with virus and cells. Possibly, due to the slow infection kinetics in this assay, the compounds need to be present for a longer period of time to demonstrate activity. The mechanism of transmission to ME-180 cells has also been revealed as nonspecific transcytosis across cellular synapses (36), providing another explanation for the lack of activity of the coreceptor blockers. In the future, it is planned to expand the algorithm to incorporate other mechanistic assays; for example, the addition of a virucidal and reverse transcriptase inhibition assay should be considered.

In order to evaluate the effect of potential microbicides on beneficial probiotic flora in the vaginal tract, this testing algorithm currently evaluates the toxicity of compounds for dose-dependent growth inhibition of L. jensenii and L. crispatus since both species are common bacteria in the vaginal tract (17). Other test species for future consideration are L. vaginalis, L. gasseri, and L. acidophilus since they are also represented among the vaginal flora. To avoid selecting compounds with potential clinical toxicity, particular emphasis is placed on evaluating cytotoxicity in the in vitro testing algorithm. The three different cytotoxicity assays complement each other by assessing cell viability as a function of different aspects of cell metabolism. The cellular conversion of tetrazolium compounds to the UV-absorbing formazan product (for the MTS assay) has been demonstrated to be directly proportional to the number of viable cells for most commonly used assay procedures (Promega CellTiter 96, AQueous One Solution Cell Proliferation Assay Technical Bulletin TB245). However, it has been reported recently that there may not be a direct requirement for mitochondria or succinate dehydrogenase to accomplish the reduction of tetrazolium compounds in proliferation assays (3). Cellular metabolism resulting in the formation of reducing equivalents such as NADH or NADPH is thought to be responsible for the conversion of the tetrazolium compounds to the colored formazan products. Thus, if a microbicide candidate affects cell viability in this assay, it is not necessarily toxic to mitochondria but could instead affect the NADPH enzymatic pathway. The Live/Dead cytotoxicity assay evaluates intracellular esterase activity, and the Vybrant cytotoxicity kit monitors the release of the cytosolic enzyme G6PD from damaged cells into the surrounding medium, and thus both assays provide measurements for plasma membrane integrity.

PRO 2000 and SPL7013 both demonstrated promising activity against CXCR4- and CCR5-tropic HIV strains, similar to previous reports (1, 12, 39). It is important to mention that the chemical stability of both compounds is routinely monitored by using chromatography assays. Although PRO 2000 exhibited up to a 19-fold loss of activity and SPL7013 exhibited up to an 8-fold decrease of activity during short-term exposure at low pH, both compounds remain active (therapeutic index of >10). It is currently under evaluation whether this retention of activity holds true during longer-term exposure at low pH. Although both compounds exhibited an increase of the IC₅₀ values in the presence of seminal plasma, they were still >80% inhibitory to HIV-1 at increased test concentrations, indicating that seminal plasma does not affect the activity at clinically relevant concentrations. One limitation of the in vitro assays is that the compound concentration used in the clinical trials exceeds what can feasibly be applied in tissue culture and that formulated products may be too viscous to be tested in cell culture assays. However, Patel et al. (35) also demonstrated that a 2% PRO 2000 gel was less protective against HSV-2 infection in a mouse model in the presence of human seminal plasma. This study revealed that the formulated compound at clinical concentrations is still subject to the inactivating effects of seminal plasma.

While the NIAID topical microbicide testing algorithm concentrates mainly on the development of vaginal microbicides, the risk of rectal HIV transmission should also be considered in the topical microbicide development process. In North America, 44 to 46% of new infections can be attributed to sexual intercourse between men; this equates to almost 20,000 new infections per year through this route (UNAIDS, [http://www.unaids.org/en/HIV_data/epi2006/default.asp]). Therefore, assays that evaluate compounds in a colorectal HIV-1 transmission model should be part of an in vitro algorithm. It is also important to note that the algorithm focuses on prevention of HIV infection and does not address whether the lead topical microbicide candidates are spermostatic or spermicidal. Ideally, when several microbicides become available, users should be able to choose between contraceptive and noncontraceptive options.

The specialized assays described address general concerns that are commonly raised with anti-HIV compounds. In order to test the breadth of activity, PBMC-based assays allow the topical microbicide candidate to be evaluated against primary HIV isolates that are circulating in different HIV endemic regions. This is important since the core assays of the algorithm only use laboratory-adapted strains of the B subtype. To evaluate drug resistance of potential topical microbicide candidates, single-drug- or multidrug-resistant strains can also be tested in PBMC-based assays. Finally, combination assays are available to make predictions on the optimal combination of compounds with different mechanism of action.

The algorithm is restricted to measuring the activity and toxicity of compounds, however chemical and physical properties of a drug are also taken into account when the final selection is made. Compounds that require few synthesis steps are preferred over those that involve many steps. Also, the stability of the compounds factors into the decision making process. Since many microbicides will be used in developing countries, the compounds ideally should be heat and shear resistant. Activity and toxicity of the formulated product will be evaluated in animal models. Overall, many factors will be taken into account for the final selection of those topical microbicides that will enter clinical trials.

In summary, the topical microbicide algorithm provides a useful tool for evaluating the toxicity and efficacy of compounds in vitro and for detecting the potential problems of microbicide candidates prior to evaluation in animal models or clinical trials.