Pedro M. M. Mesquita

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

BACKGROUNDnThe 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.nnnMETHODSnChanges 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.nnnRESULTSnExposure 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.nnnCONCLUSIONSnEpithelial 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.

Intravaginal ring eluting tenofovir disoproxil fumarate completely protects macaques from multiple vaginal simian-HIV challenges

Significance Topical prevention of HIV is designed to pharmacologically interrupt sexual transmission at the genital mucosa. Attempts at preventing transmission in women using vaginal gels have yielded disappointing results in part because of poor rates of adherence. Controlled topical drug delivery using intravaginal ring technology should improve efficacy and adherence by providing sustained mucosal delivery of antiretrovirals. In this paper, we describe a reservoir intravaginal ring that delivers tenofovir disoproxil fumarate (TDF) for 1 month. The ring protected pigtailed macaques from weekly vaginal simian–human immunodeficiency virus challenges for 4 mo. The sterilizing performance of this drug delivery system supports the concept that an intravaginal ring delivering TDF could be an effective tool for prevention of HIV sexual transmission in women. Topical preexposure prophylaxis interrupts HIV transmission at the site of mucosal exposure. Intermittently dosed vaginal gels containing the HIV-1 reverse transcriptase inhibitor tenofovir protected pigtailed macaques depending on the timing of viral challenge relative to gel application. However, modest or no protection was observed in clinical trials. Intravaginal rings (IVRs) may improve efficacy by providing long-term sustained drug delivery leading to constant mucosal antiretroviral concentrations and enhancing adherence. Although a few IVRs have entered the clinical pipeline, 100% efficacy in a repeated macaque vaginal challenge model has not been achieved. Here we describe a reservoir IVR technology that delivers the tenofovir prodrug tenofovir disoproxil fumarate (TDF) continuously over 28 d. With four monthly ring changes in this repeated challenge model, TDF IVRs generated reproducible and protective drug levels. All TDF IVR-treated macaques (n = 6) remained seronegative and simian-HIV RNA negative after 16 weekly vaginal exposures to 50 tissue culture infectious dose SHIV162p3. In contrast, 11/12 control macaques became infected, with a median of four exposures assuming an eclipse of 7 d from infection to virus RNA detection. Protection was associated with tenofovir levels in vaginal fluid [mean 1.8 × 105 ng/mL (range 1.1 × 104 to 6.6 × 105 ng/mL)] and ex vivo antiviral activity of cervicovaginal lavage samples. These observations support further advancement of TDF IVRs as well as the concept that extended duration drug delivery devices delivering topical antiretrovirals could be effective tools in preventing the sexual transmission of HIV in humans.

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 (ru200a=u200a0.6, 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

Intravaginal ring delivery of tenofovir disoproxil fumarate for prevention of HIV and herpes simplex virus infection

OBJECTIVESnA safe and effective topical prevention strategy will likely require sustained delivery of potent antiviral drugs and a delivery system that simultaneously maximizes drug distribution and overcomes the behavioural challenges related to adherence. Activity against HIV and herpes simplex virus (HSV) would be advantageous, given the epidemiological link between the two pathogens. We hypothesize that tenofovir disoproxil fumarate (tenofovir DF), a prodrug of tenofovir, may be more potent than tenofovir and ideal for sustained intravaginal ring (IVR) delivery.nnnMETHODSnThe anti-HIV and anti-HSV activity of tenofovir and tenofovir DF were assessed in cell and explant models. Cumulative tenofovir DF release and stability from polyether urethane (PEU), ethylene-co-vinyl acetate (EVA) and silicone IVRs were compared, and the activity and safety of drug released were evaluated in cervical explants and in a polarized dual-chamber model.nnnRESULTSnTenofovir DF inhibited HIV and HSV at ≈ 100-fold lower concentrations than tenofovir and retained activity in the presence of semen. PEU rings delivered >1 mg/day of tenofovir DF for 30 days. Pre-treatment of cervical explants with 10 μg/mL tenofovir DF or eluants from PEU minirings resulted in >90% inhibition of HIV and reduced HSV-2 yields by 2.5 log. Tenofovir DF and eluants did not prevent cell growth or polarization, or have any deleterious effects on an epithelial barrier.nnnCONCLUSIONSnThe findings support the development of a PEU tenofovir DF ring, which may provide potent and sustained protection against HIV and HSV.

Herpes Simplex Virus Downregulates Secretory Leukocyte Protease Inhibitor: a Novel Immune Evasion Mechanism

ABSTRACT Secretory leukocyte protease inhibitor (SLPI), an anti-inflammatory mediator of mucosal immunity, inhibits human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cell culture. Epidemiological studies demonstrate that higher concentrations of SLPI in mucosal secretions are associated with a reduced risk of HIV transmission. The current studies were designed to test the hypothesis that HSV triggers a loss of SLPI to evade innate immunity and that this response may contribute to the increased risk of HIV infection in the setting of HSV infection. Exposure of human cervical epithelial cells to HSV-1 or HSV-2, but not HIV or vesicular stomatitis virus, triggered a significant and sustained reduction in SLPI levels. The reduction persisted when cells were infected in the presence of acyclovir but not following infection with UV-inactivated virus, indicating that viral gene expression, but not replication, is required. Reverse transcriptase PCR studies demonstrated that the loss of SLPI is mediated by downregulation of gene expression. SLPI downregulation was associated with activation of NF-κB signaling pathways and upregulation of proinflammatory cytokines, consistent with the known inhibitor effects of SLPI on NF-κB pathways. The downregulation mapped to viral early-gene expression, as variants impaired in expression of the ICP4 or ICP0 immediate-early gene failed to downregulate SLPI or activate NF-κB. Together, these results identify a novel role for HSV immediate-early-gene expression in regulating mucosal immune responses.

Postcoital Bioavailability and Antiviral Activity of 0.5% PRO 2000 Gel: Implications for Future Microbicide Clinical Trials

Background The pharmacokinetics and pharmacodynamics of vaginal microbicides are typically assessed among sexually abstinent women. However, the physical act of sex may modulate gel distribution, and preclinical studies demonstrate seminal plasma interferes with the antiviral activity of several microbicides. This study compared the biological activity and concentration of PRO 2000 in cervicovaginal lavage (CVL) collected in the absence or following coitus. Methods CVL samples were collected from ten heterosexual couples at baseline, after sex, after a single dose of 0.5% PRO 2000 gel and sex, and after gel application without sex. The impact of CVL on HIV-1 infection of TZM-bl cells and HSV-2 infection of CaSki cells was monitored by luciferase and plaque assay, respectively. PRO 2000 concentrations were measured by fluorescence. Results CVL collected after PRO 2000 application significantly inhibited HIV-1 and HSV-2 (pu200a=u200a0.01). However, the antiviral activity was reduced following sex and no significant protective effect was observed in postcoital CVL obtained in the presence compared to the absence of PRO 2000 for HIV (pu200a=u200a0.45) or HSV-2 (pu200a=u200a0.56). Less PRO 2000 was recovered in postcoital CVL, which, in conjunction with interference by seminal plasma, may have contributed to lower antiviral activity. Conclusions Postcoital responses to PRO 2000 differ from precoital measures and the results obtained may provide insights into the clinical trial findings in which there was no significant protection against HIV-1 or HSV-2. Postcoital studies should be incorporated into clinical studies before embarking on large-scale efficacy trials.

Griffithsin Protects Mice from Genital Herpes by Preventing Cell-to-Cell Spread

ABSTRACT Griffithsin, which binds N-linked glycans on gp120 to prevent HIV entry, has the most potent HIV-1 inhibitory activity described for any antiviral lectin and is being developed for topical preexposure prophylaxis. The current studies were designed to further assess its potential by exploring its activity against herpes simplex virus 2 (HSV-2), a cofactor for HIV acquisition, in vitro and in a murine model. Safety was evaluated by examining its impact on epithelial barrier integrity in polarized cultures and testing whether repeated intravaginal dosing potentiates the susceptibility of mice to genital herpes. Griffithsin displayed modest inhibitory activity against HSV-2 if present during viral entry but completely blocked plaque formation if present postentry, reduced plaque size, and prevented cell-to-cell spread. These in vitro findings translated to significant protection against genital herpes in mice treated with 0.1% griffithsin gel. Griffithsin, but not placebo gel, prevented viral spread (visualized with a luciferase-expressing virus), significantly reduced disease scores, and resulted in greater survival (P < 0.05, log rank test). Protection persisted when HSV-2 was introduced in seminal plasma. Although griffithsin triggered a small decline in transepithelial electrical resistance in polarized cultures, this did not translate to any significant increase in the ability of HIV to migrate from the apical to the basolateral chamber nor to an increase in susceptibility to HSV-2 in mice treated with griffithsin gel for 7 days. These findings demonstrate that griffithsin inhibits HSV-2 by a unique mechanism of blocking cell-to-cell spread and support its further development for HIV and HSV-2 prevention.

Susceptibility to genital herpes as a biomarker predictive of increased HIV risk: Expansion of a murine model of microbicide safety

BACKGROUNDnA crucial gap in the development of microbicides for HIV prevention is the absence of models predictive of safety. Previous studies have demonstrated an increased susceptibility to genital herpes in mice following repeated applications of nonoxynol-9 (N-9). This study was designed to explore the underlying mechanisms, focusing on the effects that N-9 has on genital tract epithelium and to apply this expanded model to evaluate the safety of microbicides that have been advanced to clinical trials.nnnMETHODSnMice were treated intravaginally with formulated 3.5% N-9, 1% tenofovir, 0.5% or 2% PRO 2000, hydroxyethylcellulose (HEC) placebo or no treatment and the effect on herpes simplex virus 2 (HSV-2) susceptibility, epithelial cell architecture, junctional proteins and inflammation were assessed.nnnRESULTSnMice treated with seven daily doses of N-9, but not tenofovir, PRO 2000 or HEC, were significantly more susceptible to challenge with low doses of HSV-2; confocal microscopy demonstrated increased numbers of viral particles deep within the genital tract. N-9 disrupted the epithelium with loss of tight and adherens junctional proteins. By contrast, the epithelium was relatively preserved following tenofovir, PRO 2000 and HEC exposure. Additionally, N-9, but not the other microbicides, triggered a significant inflammatory response relative to untreated mice.nnnCONCLUSIONSnThese findings indicate that disruption of the epithelium contributes to increased HSV-2 susceptibility and might provide a biomarker predictive of increased risk for HIV acquisition. The results are consistent with the safety outcomes of the recently completed Phase IIb clinical trial with 0.5% PRO 2000 gel, and predict that tenofovir gel will not adversely affect the genital tract.

Female Genital Tract Secretions and Semen Impact the Development of Microbicides for the Prevention of HIV and Other Sexually Transmitted Infections

Citation u2028Herold BC, Mesquita PM, Madan RP, Keller MJ. Female genital tract secretions and semen impact the development of microbicides for the prevention of human immunodeficiency virus (HIV) and other sexually transmitted infections. Am J Reprod Immunol 2011; 65: 325–333

Candidate Microbicide PPCM Blocks Human Immunodeficiency Virus Type 1 Infection in Cell and Tissue Cultures and Prevents Genital Herpes in a Murine Model

ABSTRACT A structurally novel candidate microbicide, PPCM, which is formed from the reaction of d,l-mandelic acid with sulfuric acid, provides activity against human immunodeficiency virus (HIV) and herpes simplex virus (HSV) and is not cytotoxic. The objectives of the current studies were to comprehensively evaluate the activity of PPCM in cell and explant cultures, explore the possibility of combining PPCM with HIV-specific reverse transcriptase inhibitors, and evaluate the efficacy of a formulated gel against genital herpes in a murine model. PPCM inhibited infection by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical and endocervical tissue explants exposed to HIV-1BaL in the presence of PPCM were protected (50% inhibitory concentrations [IC50] of 3.9 μg/ml for ectocervix and 3.1 μg/ml for endocervix), and transfer of virus to target T cells via migratory cells was significantly impaired (IC50 of 35.7 μg/ml for ectocervix and 54.6 μg/ml for endocervix). The drug also blocked infection by cell-associated virus. Combinations of PPCM with UC-781 or PMPA in vitro exhibited additive anti-HIV activity. PPCM was incorporated into stable, low-pH gel formulations at concentrations of 0.4% and 4%. Both gels prevented genital herpesvirus infection in mice, even when virus was introduced in human seminal plasma. The abilities of PPCM to inhibit primary HIV isolates, reduce infection by cell-associated virus, and transfer of HIV from migratory to T cells, combined with the complete protection provided by formulated gel against genital herpes, indicate that this drug is an excellent candidate for inclusion in a combination microbicide and would provide protection against both HIV and HSV.