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 E_max 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 {"type":"clinical-trial","attrs":{"text":"NCT00594373","term_id":"NCT00594373"}}NCT00594373     

MTN-001: Randomized Pharmacokinetic Cross-Over Study Comparing Tenofovir Vaginal Gel and Oral Tablets in Vaginal Tissue and Other Compartments

Background

Oral and vaginal preparations of tenofovir as pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection have demonstrated variable efficacy in men and women prompting assessment of variation in drug concentration as an explanation. Knowledge of tenofovir concentration and its active form, tenofovir diphosphate, at the putative vaginal and rectal site of action and its relationship to concentrations at multiple other anatomic locations may provide key information for both interpreting PrEP study outcomes and planning future PrEP drug development.

Objective

MTN-001 was designed to directly compare oral to vaginal steady-state tenofovir pharmacokinetics in blood, vaginal tissue, and vaginal and rectal fluid in a paired cross-over design.

Methods and Findings

We enrolled 144 HIV-uninfected women at 4 US and 3 African clinical research sites in an open label, 3-period crossover study of three different daily tenofovir regimens, each for 6 weeks (oral 300 mg tenofovir disoproxil fumarate, vaginal 1% tenofovir gel [40 mg], or both). Serum concentrations after vaginal dosing were 56-fold lower than after oral dosing (p<0.001). Vaginal tissue tenofovir diphosphate was quantifiable in ≥90% of women with vaginal dosing and only 19% of women with oral dosing. Vaginal tissue tenofovir diphosphate was ≥130-fold higher with vaginal compared to oral dosing (p<0.001). Rectal fluid tenofovir concentrations in vaginal dosing periods were higher than concentrations measured in the oral only dosing period (p<0.03).

Conclusions

Compared to oral dosing, vaginal dosing achieved much lower serum concentrations and much higher vaginal tissue concentrations. Even allowing for 100-fold concentration differences due to poor adherence or less frequent prescribed dosing, vaginal dosing of tenofovir should provide higher active site concentrations and theoretically greater PrEP efficacy than oral dosing; randomized topical dosing PrEP trials to the contrary indicates that factors beyond tenofovir’s antiviral effect substantially influence PrEP efficacy.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00592124","term_id":"NCT00592124"}}NCT00592124     

In Vitro and Ex Vivo Testing of Tenofovir Shows It Is Effective As an HIV-1 Microbicide

Background

Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.

Methods and Findings

Formulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.

Conclusions

These studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.     

Increasing the effectiveness of vaginal microbicides: a biophysical framework to rethink behavioral acceptability

Background

Microbicide candidates delivered via gel vehicles are intended to coat the vaginal epithelium after application. The coating process depends on intrinsic biophysical properties of the gel texture, which restricts the potential choices for an effective product: the gel first must be physically synthesizable, then acceptable to the user, and finally applied in a manner promoting timely adequate coating, so that the user adherence is optimized. We present a conceptual framework anchoring microbicide behavioral acceptability within the fulfillment of the product biophysical requirements.

Methods

We conducted a semi-qualitative/quantitative study targeting women aged 18–55 in Northern California to assess user preferences for microbicide gel attributes. Attributes included: (i) the wait time between application and intercourse, (ii) the gel texture and (iii) the trade-off between wait time and gel texture. Wait times were assessed using a mathematical model determining coating rates depending upon the gel''s physical attributes.

Results

71 women participated. Results suggest that women would independently prefer a gel spreading rapidly, in 2 to 15 minutes (P<0.0001), as well as one that is thick or slippery (P<0.02). Clearly, thick gels do not spread rapidly; hence the motivation to study the trade-off. When asked the same question ‘constrained’ by the biophysical reality, women indicated no significant preference for a particular gel thickness (and therefore waiting time) (P>0.10) for use with a steady partner, a preference for a watery gel spreading rapidly rather than one having intermediate properties for use with a casual partner (P = 0.024).

Conclusions

Biophysical constraints alter women''s preferences regarding acceptable microbicide attributes. Product developers should offer a range of formulations in order to address all preferences. We designed a conceptual framework to rethink behavioral acceptability in terms of biophysical requirements that can help improve adherence in microbicide use ultimately enhancing microbicide effectiveness.     

A Mixed Methods and Triangulation Model for Increasing the Accuracy of Adherence and Sexual Behaviour Data: The Microbicides Development Programme

Background

The collection of accurate data on adherence and sexual behaviour is crucial in microbicide (and other HIV-related) research. In the absence of a “gold standard” the collection of such data relies largely on participant self-reporting. After reviewing available methods, this paper describes a mixed method/triangulation model for generating more accurate data on adherence and sexual behaviour in a multi-centre vaginal microbicide clinical trial. In a companion paper some of the results from this model are presented [1].

Methodology/Principal Findings

Data were collected from a random subsample of 725 women (7.7% of the trial population) using structured interviews, coital diaries, in-depth interviews, counting returned gel applicators, focus group discussions, and ethnography. The core of the model was a customised, semi-structured in-depth interview. There were two levels of triangulation: first, discrepancies between data from the questionnaires, diaries, in-depth interviews and applicator returns were identified, discussed with participants and, to a large extent, resolved; second, results from individual participants were related to more general data emerging from the focus group discussions and ethnography. A democratic and equitable collaboration between clinical trialists and qualitative social scientists facilitated the success of the model, as did the preparatory studies preceding the trial. The process revealed some of the underlying assumptions and routinised practices in “clinical trial culture” that are potentially detrimental to the collection of accurate data, as well as some of the shortcomings of large qualitative studies, and pointed to some potential solutions.

Conclusions/Significance

The integration of qualitative social science and the use of mixed methods and triangulation in clinical trials are feasible, and can reveal (and resolve) inaccuracies in data on adherence and sensitive behaviours, as well as illuminating aspects of “trial culture” that may also affect data accuracy.     

No Evidence for Selection of HIV-1 with Enhanced Gag-Protease or Nef Function among Breakthrough Infections in the CAPRISA 004 Tenofovir Microbicide Trial

Background

Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial.

Methods and Results

We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis.

Conclusion

Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.     

A Phase 1 Randomized,Double Blind,Placebo Controlled Rectal Safety and Acceptability Study of Tenofovir 1% Gel (MTN-007)

Objective

Rectal microbicides are needed to reduce the risk of HIV acquisition associated with unprotected receptive anal intercourse. The MTN-007 study was designed to assess the safety (general and mucosal), adherence, and acceptability of a new reduced glycerin formulation of tenofovir 1% gel.

Methods

Participants were randomized 1∶1:1∶1 to receive the reduced glycerin formulation of tenofovir 1% gel, a hydroxyethyl cellulose placebo gel, a 2% nonoxynol-9 gel, or no treatment. Each gel was administered as a single dose followed by 7 daily doses. Mucosal safety evaluation included histology, fecal calprotectin, epithelial sloughing, cytokine expression (mRNA and protein), microarrays, flow cytometry of mucosal T cell phenotype, and rectal microflora. Acceptability and adherence were determined by computer-administered questionnaires and interactive telephone response, respectively.

Results

Sixty-five participants (45 men and 20 women) were recruited into the study. There were no significant differences between the numbers of ≥ Grade 2 adverse events across the arms of the study. Likelihood of future product use (acceptability) was 87% (reduced glycerin formulation of tenofovir 1% gel), 93% (hydroxyethyl cellulose placebo gel), and 63% (nonoxynol-9 gel). Fecal calprotectin, rectal microflora, and epithelial sloughing did not differ by treatment arms during the study. Suggestive evidence of differences was seen in histology, mucosal gene expression, protein expression, and T cell phenotype. These changes were mostly confined to comparisons between the nonoxynol-9 gel and other study arms.

Conclusions

The reduced glycerin formulation of tenofovir 1% gel was safe and well tolerated rectally and should be advanced to Phase 2 development.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01232803","term_id":"NCT01232803"}}NCT01232803.     

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.     

Prevention of Vaginal SHIV Transmission in Macaques by a Coitally-Dependent Truvada Regimen

Background

Daily pre-exposure prophylaxis (PrEP) with Truvada (a combination of emtricitabine (FTC) and tenofovir (TFV) disoproxil fumarate (TDF)) is a novel HIV prevention strategy recently found to prevent HIV transmission in men who have sex with men and heterosexual couples. We previously showed that a coitally-dependent Truvada regimen protected macaques against rectal SHIV transmission. Here we examined FTC and tenofovir TFV exposure in vaginal tissues after oral dosing and assessed if peri-coital Truvada also protects macaques against vaginal SHIV infection.

Methods

The pharmacokinetic profile of emtricitabine (FTC) and tenofovir (TFV) was evaluated at first dose. FTC and TFV levels were measured in blood plasma, rectal, and vaginal secretions. Intracellular concentrations of FTC-triphosphate (FTC-TP) and TFV-diphosphate (TFV-DP) were measured in PBMCs, rectal tissues, and vaginal tissues. Efficacy of Truvada in preventing vaginal SHIV infection was assessed using a repeat-exposure vaginal SHIV transmission model consisting of weekly exposures to low doses of SHIV162p3. Six pigtail macaques with normal menstrual cycles received Truvada 24 h before and 2 h after each weekly virus exposure and six received placebo. Infection was monitored by serology and PCR amplification of SHIV RNA and DNA.

Results

As in humans, the concentration of FTC was higher than the concentration of TFV in vaginal secretions. Also as in humans, TFV levels in vaginal secretions were lower than in rectal secretions. Intracellular TFV-DP concentrations were also lower in vaginal tissues than in rectal tissues. Despite the low vaginal TFV exposure, all six treated macaques were protected from infection after 18 exposures or 4 full menstrual cycles. In contrast, all 6 control animals were infected.

Conclusions

We modeled a peri-coital regimen with two doses of Truvada and showed that it fully protected macaques from repeated SHIV exposures. Our results open the possibility for simplified PrEP regimens to prevent vaginal HIV transmission in women.     

Women with Pregnancies Had Lower Adherence to 1% Tenofovir Vaginal Gel as HIV Preexposure Prophylaxis in CAPRISA 004, a Phase IIB Randomized-Controlled Trial

Background

Antiretroviral prophylaxis may be a critical strategy to reduce periconception HIV transmission. Maximizing the benefit of periconception pharmacologic HIV risk-reduction requires an understanding of the links between pregnancy and adherence to this prevention strategy.

Methods

We assessed study gel adherence among women with pregnancies compared to women without pregnancies enrolled in the CAPRISA 004 phase IIB trial of 1% vaginal tenofovir gel. Pregnancy was assessed with monthly urine tests. Adherence was measured monthly and defined as proportion of sex acts covered by two returned, used applicators based on pre- and post-coital dosing. High adherence was defined as a median adherence score of >80%, that is, more than 80% of sex acts were covered by two applications of study gel. A multivariate generalized estimating equations (GEE) model with a binomial distribution was used to assess covariates associated with high adherence (>80%) over time. Median adherence before and after pregnancy was compared using Wilcoxon signed rank test.

Results

Among 868 women, 53 had at least 1 pregnancy (4.06 per 100 woman years, 95% CI: 3.04, 5.31). Women with pregnancies had lower median adherence compared to women without pregnancies (50% [IQR: 45–83] vs. 60% [IQR: 50–100], p = 0.02). Women with pregnancies also had a 48% lower odds of high adherence compared to women without pregnancies when adjusting for confounders (aOR 0.52, 95%CI: 0.41–0.66, p<0.0001). Among women with pregnancies, adherence before and after pregnancy was not different (50% [IQR: 46–83] vs. 55% [IQR: 20–100], p = 0.68).

Conclusions

Women with pregnancies were less likely to have high adherence to study gel compared to women without pregnancies. Understanding these differences may inform findings from HIV prevention trials and future implementation of antiretroviral prophylaxis for at-risk women who choose to conceive. The protocol for the parent trial is registered on ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT00441298","term_id":"NCT00441298"}}NCT00441298, http://www.clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT00441298","term_id":"NCT00441298"}}NCT00441298.     

A Phase 1 Randomized,Open Label,Rectal Safety,Acceptability, Pharmacokinetic,and Pharmacodynamic Study of Three Formulations of Tenofovir 1% Gel (the CHARM-01 Study)

Objectives

The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study.

Methods

Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial.

Results

All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/10⁶ cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection.

Conclusions

All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection.

Trial Registration

ClinicalTrials.gov NCT01575405     

Strong Relationship between Oral Dose and Tenofovir Hair Levels in a Randomized Trial: Hair as a Potential Adherence Measure for Pre-Exposure Prophylaxis (PrEP)

Background

Pre-exposure prophylaxis (PrEP) trials using tenofovir-based regimens have demonstrated that high levels of adherence are required to evaluate efficacy; the incorporation of objective biomarkers of adherence in trial design has been essential to interpretation, given the inaccuracy of self-report. Antiretroviral measurements in scalp hair have been useful as a marker of long-term exposure in the HIV treatment setting, and hair samples are relatively easy and inexpensive to collect, transport, and store for analysis. To evaluate the relationship between dose and tenofovir concentrations in hair, we examined the dose proportionality of tenofovir in hair in healthy, HIV-uninfected adults.

Methods

A phase I, crossover pharmacokinetic study was performed in 24 HIV-negative adults receiving directly-observed oral tenofovir tablets administered 2, 4, and 7 doses/week for 6 weeks, with a ≥3-week break between periods. Small samples of hair were collected after each six-week period and analyzed for tenofovir concentrations. Geometric-mean-ratios compared levels between each pair of dosing conditions. Intensive plasma pharmacokinetic studies were performed during the daily-dosing period to calculate areas-under-the-time-concentration curves (AUCs).

Results

Over 90% of doses were observed per protocol. Median tenofovir concentrations in hair increased monotonically with dose. A log-linear relationship was seen between dose and hair levels, with an estimated 76% (95% CI 60–93%) increase in hair level per 2-fold dose increase. Tenofovir plasma AUCs modestly predicted drug concentrations in hair.

Conclusions

This study found a strong linear relationship between frequency of dosing and tenofovir levels in scalp hair. The analysis of quantitative drug levels in hair has the potential to improve adherence measurement in the PrEP field and may be helpful in determining exposure thresholds for protection and explaining failures in PrEP trials. Hair measures for adherence monitoring may also facilitate adherence measurement in real-world settings and merit further investigation in upcoming PrEP implementation studies and programs.

Trial Registration

ClinicalTrials.gov +{"type":"clinical-trial","attrs":{"text":"NCT00903084","term_id":"NCT00903084"}}NCT00903084.     

Acceptability of Carraguard vaginal microbicide gel among HIV-infected women in Chiang Rai, Thailand

Background

Few studies of microbicide acceptability among HIV-infected women have been done. We assessed Carraguard® vaginal gel acceptability among participants in a randomized, controlled, crossover safety trial in HIV-infected women in Thailand.

Methodology/Principal Findings

Participants used each of 3 treatments (Carraguard gel, methylcellulose placebo gel, and no product) for 7 days, were randomized to one of six treatment sequences, and were blinded to the type of gel they received in the two gel-use periods. After both gel-use periods, acceptability was assessed by face-to-face interview. Responses were compared to those of women participating in two previous Carraguard safety studies at the same study site. Sixty women enrolled with a median age of 34 years; 25% were sexually active. Self-reported adherence (98%) and overall satisfaction rating of the gels (87% liked “somewhat” or “very much”) were high, and most (77%) considered the volume of gel “just right.” For most characteristics, crossover trial participants evaluated the gels more favorably than women in the other two trials, but there were few differences in the desired characteristics of a hypothetical microbicide. Almost half (48%) of crossover trial participants noticed a difference between Carraguard and placebo gels; 33% preferred Carraguard while 12% preferred placebo (p = 0.01).

Conclusions/Significance

Daily Carraguard vaginal gel use was highly acceptable in this population of HIV-infected women, who assessed the gels more positively than women in two other trials at the site. This may be attributable to higher perceived need for protection among HIV-infected women, as well as to study design differences. This trial was registered in the U.S. National Institutes of Health clinical trials registry under registration number {"type":"clinical-trial","attrs":{"text":"NCT00213044","term_id":"NCT00213044"}}NCT00213044.     

Non-compliance with growth hormone treatment in children is common and impairs linear growth

Background

GH therapy requires daily injections over many years and compliance can be difficult to sustain. As growth hormone (GH) is expensive, non-compliance is likely to lead to suboptimal growth, at considerable cost. Thus, we aimed to assess the compliance rate of children and adolescents with GH treatment in New Zealand.

Methods

This was a national survey of GH compliance, in which all children receiving government-funded GH for a four-month interval were included. Compliance was defined as ≥85% adherence (no more than one missed dose a week on average) to prescribed treatment. Compliance was determined based on two parameters: either the number of GH vials requested (GHreq) by the family or the number of empty GH vials returned (GHret). Data are presented as mean ± SEM.

Findings

177 patients were receiving GH in the study period, aged 12.1±0.6 years. The rate of returned vials, but not number of vials requested, was positively associated with HVSDS (p<0.05), such that patients with good compliance had significantly greater linear growth over the study period (p<0.05). GHret was therefore used for subsequent analyses. 66% of patients were non-compliant, and this outcome was not affected by sex, age or clinical diagnosis. However, Maori ethnicity was associated with a lower rate of compliance.

Interpretation

An objective assessment of compliance such as returned vials is much more reliable than compliance based on parental or patient based information. Non-compliance with GH treatment is common, and associated with reduced linear growth. Non-compliance should be considered in all patients with apparently suboptimal response to GH treatment.     

MABGEL 1: First Phase 1 Trial of the Anti-HIV-1 Monoclonal Antibodies 2F5, 4E10 and 2G12 as a Vaginal Microbicide

Background

Monoclonal antibodies (mAbs) which potently neutralize a broad range of HIV isolates are potential microbicide candidates. To date, topical application of mAbs in humans and their stability in vaginal secretions has not been studied.

Objectives

To assess the pharmacokinetics and safety of the mAbs 2F5, 4E10 and 2G12 when applied vaginally in women.

Design

A randomized, double-blind, placebo-controlled phase 1 trial.

Methods

Twenty-eight healthy, sexually abstinent women administered 2.5 g of gel daily for 12 days containing either 10 or 20 mg/g of each mAb (MABGEL) or placebo. Main clinical evaluations and sampling occurred at baseline, 1, 8, and 24 hours post-1^st dose and 12 and 36 hours post-12^th dose.

Results

After adjustment for dilution factors, median levels of 2F5, 4E10 and 2G12 in vaginal secretions at 1 hour post high-dose MABGEL were 7.74, 5.28 and 7.48 mg/ml respectively. Levels of 2F5 and 4E10 declined exponentially thereafter with similar estimated half-lives (4.6 and 4.3 hours). In contrast, 2G12 levels declined more rapidly in the first 8 hours, with an estimated half-life of 1.4 hours during this period. There was no evidence of systemic absorption. There were no significant differences in local or systemic adverse event rates or vaginal flora changes (by qPCR) between active and placebo gel arms. Whilst at least 1 adverse event was recorded in 96% of participants, 95% were mild and none were serious.

Conclusions

Vaginal application of 50 mg of each mAb daily was safe over a 12 day period. Median mAb concentrations detected at 8 hours post dose were potentially sufficient to block HIV transmission.2G12 exhibited more rapid elimination from the human vagina than 4E10 and 2F5, likely due to poor stability of 2G12 in acidic human vaginal secretions. Further research is needed to develop mAb-based vaginal microbicides and delivery systems.

Trial Registration

ISRCTN 64808733 UK CRN Portfolio 6470     

"One teabag is better than four": Participants response to the discontinuation of 2% PRO2000/5 microbicide gel in KwaZulu-Natal, South Africa

Introduction

The Microbicides Development Programme evaluated the safety and effectiveness of 0.5% and 2% PRO2000/5 microbicide gels in reducing the risk of vaginally acquired HIV. In February 2008 the Independent Data Monitoring Committee recommended that evaluation of 2% PRO2000/5 gel be discontinued due to futility. The Africa Centre site systematically collected participant responses to this discontinuation.

Methods

Clinic and field staff completed field reports using ethnographic participant observation techniques. In-depth-interviews and focus group discussions were conducted with participants discontinued from 2% gel. A total of 72 field reports, 12 in-depth-interviews and 3 focus groups with 250 women were completed for this analysis. Retention of discontinued participants was also analysed. Qualitative data was analysed using NVivo 2 and quantitative data using STATA 10.0.

Results

Participants responded initially with fear that discontinuation was due to harm, followed by acceptance after effective messaging, and finally with disappointment. Participants reported that their initial fear was exacerbated by being contacted and advised to visit the clinic for information about the closure. Operational changes were subsequently made to the contact procedures. By incorporating feedback from participants, messages were continuously revised to ensure that information was comprehensible and misconceptions were addressed quickly thereby enabling participants to accept the discontinuation. Participants were disappointed that 2% PRO2000/5 was being excluded as a HIV prevention option, but also that they would no longer have access to gel that improved their sexual relationships with their partners and assisted condom negotiations. In total 238 women were discontinued from gel and 185 (78%) went on to complete their scheduled follow-up period.

Discussion

The use of qualitative social science techniques allowed the site team to amend operational procedures and messaging throughout the discontinuation period. This proved instrumental in ensuring that the discontinuation was successfully completed in a manner that was both understandable and acceptable to participants.

Trial registration

Current Controlled Trials. ISRCTN64716212.     

Viral Load,Integration and Methylation of E2BS3 and 4 in Human Papilloma Virus (HPV) 16-Positive Vaginal and Vulvar Carcinomas

Objective

To investigate if viral load, integration and methylation of E2BS3 and 4 represent different ways of tumor transformation in vaginal and vulvar carcinoma and to elucidate its clinical impact.

Methods

Fifty-seven samples, positive for HPV16, were selected for the study. Detection of viral load was made with realtime-PCR using copy numbers of E6 and integration was calculated from comparing E2 to E6-copies. Methylation of E2BS3 and 4 was analysed using bisulphite treatment of tumor DNA, followed by PCR and pyrosequencing.

Results

Vaginal tumors were found to have a higher viral load (p = 0.024) compared to vulvar tumors but a high copy number (> median value, 15 000) as well as high methylation (>50%) was significantly (p = 0.010 and p = 0.045) associated with a worse cancer-specific survival rate in vulvar carcinoma, but not in vaginal carcinoma. Four groups could be defined for the complete series using a Cluster Two step analysis; (1) tumors holding episomal viral DNA, viral load below 150 000 copies not highly methylated (n = 25, 46.3%); (2) tumors harboring episomal viral DNA and being highly methylated (>50%; n = 6, 11.1%); (3) tumors with viral DNA fully integrated (n = 11, 20.4%), and (4) tumors harboring episomal viral DNA and being medium- or unmethylated (<50%) and having a high viral load (> total mean value 150 000; n = 12, 22.2%). The completely integrated tumors were found to be distinct group, whilst some overlap between the groups with high methylation and high viral load was observed.

Conclusion

HPV16- related integration, methylation in E2BS3 and 4 and viral load may represent different viral characteristics driving vaginal and vulvar carcinogenesis. HPV16- related parameters were found to be of clinical importance in the vulvar series only.     

Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota

Objectives

Bifidobacterium species are one of the major components of the infant''s intestine microbiota. Colonization with bifidobacteria in early infancy is suggested to be important for health in later life. However, information remains limited regarding the source of these microbes. Here, we investigated whether specific strains of bifidobacteria in the maternal intestinal flora are transmitted to their infant''s intestine.

Materials and Methods

Fecal samples were collected from healthy 17 mother and infant pairs (Vaginal delivery: 12; Cesarean section delivery: 5). Mother''s feces were collected twice before delivery. Infant''s feces were collected at 0 (meconium), 3, 7, 30, 90 days after birth. Bifidobacteria isolated from feces were genotyped by multilocus sequencing typing, and the transitions of bifidobacteria counts in infant''s feces were analyzed by quantitative real-time PCR.

Results

Stains belonging to Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium longum subsp. longum, and Bifidobacterium pseudocatenulatum, were identified to be monophyletic between mother''s and infant''s intestine. Eleven out of 12 vaginal delivered infants carried at least one monophyletic strain. The bifidobacterial counts of the species to which the monophyletic strains belong, increased predominantly in the infant''s intestine within 3 days after birth. Among infants delivered by C-section, monophyletic strains were not observed. Moreover, the bifidobacterial counts were significantly lower than the vaginal delivered infants until 7 days of age.

Conclusions

Among infants born vaginally, several Bifidobacterium strains transmit from the mother and colonize the infant''s intestine shortly after birth. Our data suggest that the mother''s intestine is an important source for the vaginal delivered infant''s intestinal microbiota.