Marla J. Keller

Human α- and β-Defensins Block Multiple Steps in Herpes Simplex Virus Infection

This study examined the ability of nine human defensins (HD) to protect against herpes simplex virus infection. Noncytotoxic concentrations of all six α-defensins (HNP1–4, HD5, and HD6) and human β-defensin (hBD) 3 inhibited HSV infection. Two other β-defensins, hBD1 and 2, lacked this protective activity. Synchronized assays revealed that HNP-4, HD6, and hBD3 acted primarily by preventing binding and entry, whereas HNP1–3 and HD5 also inhibited postentry events. Even when added several hours after entry, substantial reduction in viral gene expression ensued. Human cervical epithelial cells incubated with HNP-1 or HD5 accumulated the peptides intracellularly. Surface plasmon resonance studies revealed that HNPs 1, 2, 3, and HD5 bound HSV glycoprotein B (gB) with high affinity, but showed minimal binding to heparan sulfate, the receptor for attachment. In contrast, HNP-4 and HD6 bound heparan sulfate, but not gB. HBD3 bound both gB and heparan sulfate, but hBD1 and hBD2 bound neither. Admixture of HD5 with hydroxyethylcellulose significantly protected mice from a viral challenge lethal to controls receiving an inactive peptide or hydroxyethylcellulose alone. These findings demonstrate that HDs act at multiple steps in the HSV life cycle and support the development of defensins or defensin-like peptides as microbicides.

PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity

Objective:Vaginal microbicides should protect against infection without disrupting the mucosal environment or its mediators of host defense. The objective of this study was to examine the effect of 14 daily applications of 0.5% PRO 2000 or placebo gel on mediators of mucosal immunity and intrinsic antimicrobial activity. Design and methods:A randomized, prospective, double-blind, placebo-controlled study was conducted among 24 healthy, abstinent women. Levels of cytokines, chemokines, defensins, and other protective factors and intrinsic antimicrobial activity were determined in cervicovaginal lavage samples collected on study days 0, 7, 14, and 21. Results:No increase in pro-inflammatory cytokines was observed. Rather cytokines and protective factors including interleukin (IL)-1 receptor antagonist, immunoglobulins and human beta-defensin 2 were lower in the drug compared with the placebo group. All of the mediators returned towards baseline on day 21. Women who were cycling had lower levels of most proteins on study days 7 and/or 14 compared with women on oral contraceptives; however, the magnitude of decline was greater in women who received PRO 2000 compared with placebo gel. The reduction in protective factors was not associated with a loss in the intrinsic anti-viral (HIV or herpes simplex virus) activity or anti-bacterial activity (Escherichia coli or Staphylococcus aureus). Conclusion:In contrast to experience with nonoxynol-9, PRO 2000 did not trigger an inflammatory response in cervicovaginal secretions. There was a modest reduction in mucosal immune mediators, but this loss was not associated with a reduction in intrinsic antimicrobial activity.

Disruption of Tight Junctions by Cellulose Sulfate Facilitates HIV Infection: Model of Microbicide Safety

BACKGROUND The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety. METHODS Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with human immunodeficiency virus (HIV) and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber. RESULTS Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl]adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, CS triggered nuclear factor kappaB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells. CONCLUSIONS Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of N-9 and CS. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes.

Candidate Sulfonated and Sulfated Topical Microbicides: Comparison of Anti-Human Immunodeficiency Virus Activities and Mechanisms of Action

ABSTRACT Poly(styrene 4-sulfonate), cellulose sulfate, polymethylenehydroquinone, and PRO 2000 are sulfated or sulfonated polymers (SPs) under development as topical microbicides. They are presumed to work through similar mechanisms of action, although to date there has been no extensive comparison of their anti-human immunodeficiency virus activities. To determine whether any of these candidate microbicides offers a potential advantage, their in vitro activities, mechanisms of action, stabilities in biological secretions, and toxicities were compared. All four compounds were found to be active against X4, R5, and dualtropic primary isolates and against X4 and R5 laboratory-adapted strains in CD4+ T cells, macrophages, and single-coreceptor cell lines. Our single-cycle experiments using pseudotyped virus suggest that all four SPs function at the binding and entry stages of the viral life cycle but differ in degree of postentry effect. Surface plasmon resonance analyses demonstrate that SPs bind to X4 and R5 monomeric glycoprotein 120 with similar high binding affinities. When mixed with cervicovaginal lavage fluid, SPs maintain inhibitory activity at concentrations achievable in formulations.

Candidate Topical Microbicides Bind Herpes Simplex Virus Glycoprotein B and Prevent Viral Entry and Cell-to-Cell Spread

ABSTRACT Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides.

Seminal Plasma Reduces the Effectiveness of Topical Polyanionic Microbicides

The objective of this study was to test the activity of microbicides against herpes simplex virus type 2 (HSV-2) introduced in seminal plasma. We found that seminal plasma interfered with the activity of PRO 2000 and of cellulose sulfate, increasing by 100-fold the concentration of drug required to inhibit 90% of viral plaque formation. Seminal plasma competitively inhibited binding of the microbicides to the HSV-2 envelope. Most of the interference was found in a high molecular-weight fraction; tandem mass spectrometry identified the proteins as fibronectin-1 and lactoferrin. In a murine model, the interference translated in vivo into a loss in protection. We found that 2% PRO 2000 gel protected 100% of mice challenged intravaginally with HSV-2 introduced in PBS, whereas only 55% of mice were protected if virus was introduced in seminal plasma (P=.0007, log rank test). If these findings are reflective of what occurs in humans, modifications to microbicides to ensure that they retain activity in the presence of seminal plasma are indicated.

PRO 2000 Gel Inhibits HIV and Herpes Simplex Virus Infection Following Vaginal Application: A Double-Blind Placebo-Controlled Trial

BACKGROUND Microbicides used to prevent the transmission of human immunodeficiency virus (HIV) are advancing to clinical trials on the basis of activity observed in vitro and in animal models. However, no data demonstrate activity of microbicides after application in humans. This study was designed to determine the antiviral activity in cervicovaginal lavage (CVL) samples collected after intravaginal application of 0.5% PRO 2000 gel (Indevus). METHODS A randomized, double-blind study was conducted to assess the anti-HIV and anti-herpes simplex virus (HSV) activity of PRO 2000 in CVL samples obtained at screening (48 hours before) and 1 hour after application of study or placebo gel. HeLa cells or human macrophages were inoculated with CVL samples spiked with replication-defective HIV containing a luciferase indicator gene and pseudotyped with an R5 envelope. Human cervical epithelial cells were inoculated with CVL samples and challenged with HSV-2(G), and the virus titer was then determined. RESULTS CVL samples obtained after application of PRO 2000 gel significantly inhibited HIV and HSV infection by at least 1000-fold, compared with CVL samples obtained at screening (P < .001). There were no differences in cytokine levels between the drug and placebo groups. CONCLUSIONS PRO 2000 gel (0.5%) is sufficiently bioavailable and retains substantial antiviral activity after intravaginal application. This strategy provides a mechanism for testing the efficacy of a microbicide before embarking on large-scale clinical trials.

Cervicovaginal Secretions Contribute to Innate Resistance to Herpes Simplex Virus Infection

Defining and preserving the innate antiviral activity found in cervicovaginal secretions is critical. Cervicovaginal lavage (CVL) samples were obtained from 20 healthy women and evaluated for anti-herpes simplex virus (HSV) activity. CVL samples reduced HSV-2 yields by 23-fold (median), and the anti-HSV activity of CVL samples correlated with the concentration of human neutrophil peptides (HNP)-1-3. Both CVL samples and HNP-1-3 interacted with virus and prevented entry after binding. Substantially less protective activity was observed in CVL samples obtained from 20 human immunodeficiency virus--infected subjects, but the addition of CVL samples from healthy subjects enhanced the antiviral activity. The significance of the innate activity was further demonstrated by showing that CVL samples prevented murine genital herpes. Fourteen of 15 mice were protected from genital herpes if they were challenged with HSV-2 pretreated with CVL samples from healthy subjects. In contrast, all 15 mice challenged with untreated HSV-2 died. These findings are evidence that cervicovaginal secretions contribute to innate resistance to HSV-2 and identify defensins as contributors to this activity.

A Comprehensive Murine Model to Evaluate Topical Vaginal Microbicides: Mucosal Inflammation and Susceptibility to Genital Herpes as Surrogate Markers of Safety

A critical gap in microbicide development is the absence of surrogate safety markers. The objective of the present study was to develop a murine model to examine the mucosal response to microbicides and to assess the functional implication of observed changes. Mice received 14 daily intravaginal doses of nonoxynol-9, PRO 2000, or placebo gel. Nonoxynol-9 induced an inflammatory response characterized by increases in levels of cytokines and chemokines, recruitment of neutrophils and monocytes into the genital tract, and activation of the transcription factors NF- kappa B and activator protein-1. Minimal inflammation was observed in response to 2% PRO 2000. Nonoxynol-9-treated mice were significantly more susceptible to challenge with a low dose of herpes simplex virus type 2; the response of PRO 2000-treated mice was similar to the response to placebo. These findings suggest that PRO 2000 has little deleterious effect on mucosal immunity and, if validated by clinical experiences, support the inclusion of this model in the preclinical evaluation of future candidate microbicides.

A randomized trial to assess anti-HIV activity in female genital tract secretions and soluble mucosal immunity following application of 1% tenofovir gel.

Background Preclinical and early phase clinical microbicide studies have not consistently predicted the outcome of efficacy trials. To address this gap, candidate biomarkers of microbicide pharmacodynamics and safety were evaluated in a double-blind, placebo-controlled trial of tenofovir gel, the first microbicide to demonstrate significant protection against HIV acquisition. Methods 30 women were randomized to apply a single daily dose of tenofovir or placebo gel for 14 consecutive days. Anti-HIV activity was measured in cervicovaginal lavage (CVL) on Days 0, 3, 7, 14 and 21 by luciferase assay as a surrogate marker of pharmacodynamics. Endogenous activity against E. coli and HSV-2 and concentrations of immune mediators were quantified in CVL as candidate biomarkers of safety. Tenofovir levels were measured in CVL and blood. Results A significant increase in anti-HIV activity was detected in CVL from women who applied tenofovir gel compared to their endogenous anti-HIV activity in genital tract secretions on Day 0 and compared to activity in CVL from women in the placebo group. The activity correlated significantly with CVL concentration of tenofovir (r = 0.6, p<0.001) and fit a sigmoid Emax pharmacodynamic model. Anti-HIV activity in CVL from women who applied tenofovir persisted when virus was introduced in semen, whereas endogenous anti-HIV activity decreased. Tenofovir did not trigger an inflammatory response or induce sustained loss in endogenous antimicrobial activity or immune mediators. Conclusions Tenofovir gel had no deleterious impact on soluble mucosal immunity. The increased anti-HIV activity in CVL, which persisted in the presence of semen and correlated with tenofovir concentration, is consistent with the efficacy observed in a recent clinical trial. These results promote quantified CVL anti-HIV activity as a surrogate of tissue pharmacodynamics and as a potential biomarker of adherence to product. This simple, feasible and inexpensive bioassay may promote the development of models more predictive of microbicide efficacy. Trial Registration ClinicalTrials.gov NCT00594373