James E. Cummins

Preclinical Testing of Candidate Topical Microbicides for Anti-Human Immunodeficiency Virus Type 1 Activity and Tissue Toxicity in a Human Cervical Explant Culture

ABSTRACT A human cervical explant culture was utilized for the preclinical assessment of anti-human immunodeficiency virus type 1 (HIV-1) activity and tissue toxicity of formulated, candidate topical microbicides. Products tested included cellulose acetate 1,2-benzene dicarboxylate (CAP), a carrageenan-based product (PC-515), a naphthalene sulfonate polymer (PRO 2000), a lysine dendrimer (SPL7013), a nonnucleoside reverse transcriptase inhibitor (UC781), and an antimicrobial peptide (D2A21), along with their placebos. Cervical explants were cultured overnight with HIV-1 with or without product, washed, and monitored for signs of HIV-1 infection. HIV-1 infection was determined by p24gag levels in the basolateral medium and by immunohistochemical analysis of the explant. Product toxicity was measured by the MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] assay and histology. CAP, PRO 2000, SPL7013, and UC781 consistently prevented HIV-1 infection in all explants tested. PC-515 and D2A21 prevented HIV-1 infection in 50% or fewer of the explants tested. Placebos did not prevent infection in any of the explants tested. With the exception of PRO 2000 (4%), the MTT assay and histological analysis of the other products and placebos showed minimal toxicity to the epithelium and submucosa. Collectively, these data suggest that this culture system can be used for evaluating the safety and efficacy of topical microbicides designed for vaginal use.

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.

Cervical and Prostate Primary Epithelial Cells Are Not Productively Infected but Sequester Human Immunodeficiency Virus Type 1

Primary prostate and cervical epithelial cells and epithelial cell lines were examined for human immunodeficiency virus type 1 (HIV-1) infection or transmission to peripheral blood mononuclear cells (PBMC). Neither cell-free nor cell-associated HIV-1 infected primary epithelial cells or cell lines. Pretreatment of HIV-1 to enhance CD4-independent entry did not augment infection. Cell surface expression was detected for galactosyl ceramide but not for CC-chemokine receptor 5, CXC-chemokine receptor 4, or CD4. The ability to transfer HIV-1 to resting or activated PBMC was tested by culturing with rinsed or trypsinized and replated HIV-1-exposed epithelial cells. Virus was not recovered from the rinsed or replated cocultures with resting PBMC; however, activated PBMC recovered HIV-1 from rinsed epithelial cells and rarely from replated epithelial cells. Although urogenital epithelial cells are not infected, these data suggest that they can transfer virus to activated immune cells and have implications for sexual transmission of HIV-1.

A Human Colorectal Explant Culture to Evaluate Topical Microbicides for the Prevention of HIV Infection

A human colorectal explant culture was developed to assess the safety and efficacy of topical microbicides proposed for use in humans. Because any product marketed for vaginal application will likely be used for anal intercourse, it is important to evaluate these products in colorectal explant tissue. Microbicides tested included cellulose acetate 1,2-benzenedicarboxylate (CAP), PRO 2000, SPL7013, Vena Gel, and UC781, along with their accompanying placebos. Colorectal tissues were exposed to microbicides overnight and either fixed in formalin to evaluate toxicity by histological analysis or placed in 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) to quantitatively determine tissue viability. Histological analysis showed minimal toxicity for CAP, UC781, and Vena Gel. Shedding of epithelium with intact lamina propria occurred for the PRO 2000 and SPL7013 products, and shedding of epithelium and necrosis of the lamina propria occurred in explants cultured with nonoxynol-9. The MTT assay confirmed these results for PRO 2000 (4% and 0.5%), SPL7013 (and placebo), and nonoxynol-9 but also demonstrated reduced viability for CAP. However, viability of tissues treated with all products was not significantly different from that of the medium control. Efficacy of the microbicides was evaluated by measuring human immunodeficiency virus type 1 (HIV-1) infection of explants in the absence or presence of products. All microbicide formulations tested were highly effective in preventing HIV infection. However, explants treated with some of the placebo formulations also exhibited a lower level of infection. Most of the products developed for vaginal application showed minimal toxicity and were effective in reducing HIV-1 infection in colorectal tissues. These results suggest that this model is useful for evaluating the safety and efficacy of topical microbicides when used rectally.

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.

Biomarkers of Cervicovaginal Inflammation for the Assessment of Microbicide Safety

The human cervicovaginal mucosa is the primary target of HIV-1 infection during male to female transmission. This tissue contains the the full spectrum of cell types and immune modulators that comprise both the innate and adaptive arms of the immune system. Mounting evidence indicates that mucosal epithelial cells are sentinels of the female reproductive tract, producing innate immune mediators that control the vaginal microflora under normal conditions. Recent studies, however, indicate that certain factors secreted in response to another pathogen or after exposure to a vaginal product may in fact enhance infection by HIV-1. Mucosal inflammation and CD4 cell activation as well as disruption of TLR function and epithelial integrity represent potential causes for such effect. It is therefore important to make sure that vaginal products, including microbicides, do not disrupt the structure or function of the cervicovaginal mucosa. Although a number of biomarkers have been linked to microbicide-induced cervicovaginal inflammation and many of these markers have been measured in preclinical and clinical assays, there are currently no data that demonstrate a correlation between any one marker and susceptibility to HIV-1 infection in humans. To date, the lack of a validated biomarker of cervicovaginal safety represents a gap in the knowledge base that hinders the rational and expeditious selection of microbicide candidates entering clinical trials. Currrent discovery efforts and preclinical assessment of microbicide safety use an integrated sequential evaluation system that includes cell-based models, explant-based models, and animal-based models. Relevant research in these areas is yielding new assays and biomarkers that, if validated, will be essential to the rational selection of microbicide candidates for efficacy trials.

In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories.

ABSTRACT The first product to be clinically evaluated as a microbicide contained the nonionic surfactant nonoxynol-9 (nonylphenoxypolyethoxyethanol; N-9). Many laboratories have used N-9 as a control compound for microbicide assays. However, no published comparisons of the results among laboratories or attempts to establish standardized protocols for preclinical testing of microbicides have been performed. In this study, we compared results from 127 N-9 toxicity and 72 efficacy assays that were generated in five different laboratories over the last six years and were performed with 14 different cell lines or tissues. Intra-assay reproducibility was measured at two-, three-, and fivefold differences using standard deviations. Interassay reproducibility was assessed using general linear models, and interaction between variables was studied using step-wise regression. The intra-assay reproducibility within the same N-9 concentration, cell type, assay duration, and laboratory was consistent at the twofold level of standard deviations. For interassay reproducibility, cell line, duration of assay, and N-9 concentration were all significant sources of variability (P < 0.01). Half-maximal toxicity concentrations for N-9 were similar between laboratories for assays of similar exposure durations, but these similarities decreased with lower test concentrations of N-9. Results for both long (>24 h) and short (<2 h) exposures of cells to N-9 showed variability, while assays with 4 to 8 h of N-9 exposure gave results that were not significantly different. This is the first analysis to compare preclinical N-9 toxicity levels that were obtained by different laboratories using various protocols. This comparative work can be used to develop standardized microbicide testing protocols that will help advance potential microbicides to clinical trials.

Inhibition of HIV-1 Replication by Amphotericin B Methyl Ester SELECTION FOR RESISTANT VARIANTS

Membrane cholesterol plays an important role in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. Here, we have investigated the target and mechanism of action of a cholesterol-binding compound, the polyene antifungal antibiotic amphotericin B methyl ester (AME). We found that AME potently inhibited the replication of a highly divergent panel of HIV-1 isolates in various T-cell lines and primary cells irrespective of clade or target cell tropism. The defects in HIV-1 replication caused by AME were due to profoundly impaired viral infectivity as well as a defect in viral particle production. To elucidate further the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Mutations responsible for AME resistance mapped to a highly conserved and functionally important endocytosis motif in the cytoplasmic tail of the transmembrane glycoprotein gp41. Interestingly, truncation of the gp41 cytoplasmic tail in the context of either HIV-1 or rhesus macaque simian immunodeficiency virus also conferred resistance to AME. The infectivity of HIV-1 virions bearing murine leukemia virus or vesicular stomatitis virus glycoproteins was unaffected by AME. Our data define the target and mechanism of action of AME and provide support for the concept that cholesterol-binding compounds should be pursued as antiretroviral drugs to disrupt HIV-1 replication.

Multisite Comparison of Anti-Human Immunodeficiency Virus Microbicide Activity in Explant Assays Using a Novel Endpoint Analysis

ABSTRACT Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

Amphipathic DNA polymers exhibit antiviral activity against systemic Murine Cytomegalovirus infection

BackgroundPhosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated.ResultsIn vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC50 of 0.045 μM and 0.16 μM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC50 of 0.05 μM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers.ConclusionThese studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.