Meeting Minutes - November 8, 2012

Office of AIDS Research Advisory Council
Thirty-fifth Meeting
November 8, 2012

National Institutes of Health
U.S. Department of Health and Human Services
5635 Fishers Lane Conference Center
Rockville, MD

Members Present: Dr. Sharon Hillier (Chair), Dr. Jack Whitescarver (Executive Secretary), Dr. Judith Auerbach, Dr. Stefano Bertozzi, Dr. David B. Clifford, Dr. Myron S. Cohen, Dr. Steven Deeks, Dr. Carrie E. Foote, Dr. Lisa Jacobson, Dr. Rochelle Walensky, Dr. Judith Wasserheit, and Dr. Craig M. Wilson

Ex Officio Members Present: Dr. Maggie Czarnogorski for Dr. Victoria Davey

Invited Speakers and Guests: Dr. Kjersti Aagaard, Dr. Jean Anderson, Dr. Charlene Dezzutti, Dr. J. Gerardo Garcia-Lerma, Dr. Mimi Ghosh, Dr. Craig Hendrix, Dr. Betsy C. Herold, Dr. Thomas J. Hope, Dr. Gail H. Ironson, Dr. Rupert Kaul, Dr. Charu Kaushic, Dr. H. Clifford Lane, Dr. Christine K. Mauck, Dr. Alice K. Pau, Dr. Barbara Shacklett, and Dr. Gregory T. Spear

Welcome and Meeting Overview

The National Institutes of Health (NIH) Office of AIDS Research Advisory Council (OARAC) convened its thirty-fifth meeting on November 8, 2012 at 8:30 a.m. at the Fishers Lane Conference Center in Rockville, Maryland. 

Dr. Sharon L. Hillier served as chair.  She welcomed the OARAC members, invited speakers, and guests.  The minutes of the April 10, 2012, OARAC meeting were approved as submitted.

Director’s Report

Dr. Jack Whitescarver, Director of the Office of AIDS Research (OAR), welcomed everyone to this meeting of OARAC. 

NIH AIDS Budget Update
Dr. Whitescarver reported that NIH is operating under a continuing resolution until the end of March 2013 so we do not have a final budget for fiscal year (FY) 2013.  He explained that this means NIH is operating at the FY 2012 level.  He noted that OAR is in the process of developing the trans-NIH FY 2014 AIDS budget that will be presented by the President to Congress in early 2013.  

International AIDS Conference
Dr. Whitescarver reported that the NIH played a major role as the scientific partner for the International AIDS meeting held in Washington, D.C. in July 2012.  OAR had a significant role in developing the overall meeting and coordinating and managing the NIH’s presence at the conference.  NIH conducted several satellites, workshops, and grantsmanship sessions, three of which included participation by Dr. Francis Collins.  An OAR-coordinated exhibit showcased NIH AIDS research.  NIH also sponsored the Hispanic Pavilion in the Global Village that served as a common meeting place for  representatives from the many Spanish-speaking countries who attended the conference and provided HIV education and information exchange.

Update on International Collaborations
Dr. Whitescarver reported that there are a number of new bilateral funding initiatives with China, Russia, and India.  International partners have been solicited to form an alliance to conduct research towards a cure for HIV.  The Chinese government is actively involved and has planned a workshop on this topic that will include a number of OARAC members.  Progress has been made in the initiation of research collaborations with Russia.  Current RFAs for grant supplements and R21 grant applications support collaborations between U.S. and Russian scientists.  Research collaborations with India are ongoing.  

CONFLICT OF INTEREST STATEMENTS

Dr. Whitescarver asked Council members to review and sign the conflict of interest statement provided to them.  He reminded the Council members of the importance of this process.

UPDATE ON OARAC WORKING GROUPS FOR TREATMENT AND PREVENTION GUIDELINES 

Dr. Alice Pau, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), provided an overview of the recent updates of the five treatment and prevention guidelines.  The Panels that develop the guidelines are convened under the auspices of the OARAC, and the Guidelines are posted on AIDSinfo.nih.gov.  

Dr. Pau reported that the Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents were revised in March 2012.  A key revision was the recommendation that the fixed dose combination drug of elvitegravir and cobicistat plus tenofovir/emtricitabine could be used as an alternative regimen for antiretroviral (ART)-naïve patients.  This fixed drug combination was recently approved by the Food and Drug Administration.  She noted that concerns about drug-drug interactions, the function of cobicistat as a “pharmacokinetic enhancer”, and renal toxicity prevent this drug combination from being a preferred regimen.  There are limited data on the use of this combination in women and HIV-infected patients with advanced disease.

Dr. Pau noted that other sections of the Guidelines are currently being revised based on recent clinical findings including:

• the co-receptor tropism testing section that will include new genotypic assays;

• the “When to Start Therapy” section will incorporate the elvitegravir      recommendations;

• the “Acute Recent” (now called “Early HIV infection”) section will be extensively revised to include treatment in patients with acute and early stage HIV infection;

• the drug interaction section will include cobicistat; and

• a description of cobicistat as a pharmacokinetic enhancer.

Dr. H. Clifford Lane, Clinical Director, NIAID, and Dr. Pau commented that the Panel is revising the Conflict of Interest Guidelines in view of the recent Institute of Medicine Report.  The Panel is clarifying who can vote and who should be recused from certain Panel discussions.  In addition to financial disclosures, other conflicts, such as principal authorship or co-authorship and investigator status, must be resolved for Panel members involved in studies supported by pharmaceutical companies.

Dr. Pau reported that the Pediatric Panel recently revised the Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infectionand these were posted online in November 2012.  The key revisions included:  a new section on diagnosis of HIV in children; use of pediatric drugs in “When to Start” based on specific age ranges and CD4+ cell counts; “What Antiretroviral Drugs to Start” in treatment-naïve children; and new pediatric antiretroviral drug information that includes new FDA-approved formulations for children.  

Dr. Pau summarized other changes to the Pediatric Guidelines including:  the recommendation for protease inhibitor-based regimens for children under three years of age; use of tenofovir for children older than two years of age based on the Tanner scale; and use of tenofovir only for treatment-experienced children who are failing therapy.

Dr. Pau stated that the Recommendations for Use of Antiretroviral Drugs in Pregnant HIV-1-Infected Women for Maternal Health and Interventions to Reduce Perinatal HIV Transmission in the United States were last updated in July 2012.  The guidelines now include a section that addresses reproductive options for HIV serodiscordant couples reflecting the data from the HPTN 052 Treatment as Prevention Study.  The guidelines recommend the use of PrEP prior to conception in the HIV-uninfected partner and providing antiretroviral therapy (ART) to the HIV-infected partner at a CD4+ cell count of 550 cells/ml or less with the aim of decreasing the infected partner’s viral load and the risk of HIV acquisition in the uninfected partner.

The Perinatal Guidelines also include a number of changes in the recommendations for ARVs used during pregnancy: 

• Atazanavir and boosted Atazanavir are preferred drugs.  Drugs or drug combinations are designated as preferred for use in pregnant women when clinical trial data in adults have demonstrated optimal efficacy and durability with acceptable toxicity and ease of use; pregnancy-specific PK data are available to guide dosing; and no evidence of teratogenic effects or established association with teratogenic or clinically significant adverse outcomes for mothers, fetuses, or newborn are present.

• Boosted ritonavir is an alternative treatment.  Drugs or drug combinations are designated as alternatives for initial therapy in pregnant women when clinical trial data in adults show efficacy, but any one or more of the following conditions apply:  experience in pregnancy is limited; data are lacking on teratogenic effects on the fetus; or the drug or regimen is associated with dosing, formulation, administration, or interaction issues.

• Women using efavirenz prior to pregnancy or in early pregnancy can continue that treatment if viral suppression is adequate and the patient does not wish to switch to another regimen.  However, the alternative regimens without efavirenz should be considered for women planning to become pregnant.

• Intrapartum intravenous AZT will no longer be required if the mother is on ART and has complete viral suppression.

• Neonatal ZDV should now be dosed twice daily instead of three times per day.

Dr. Pau reported that the Guidelines for the Prevention and Treatment of Opportunistic Infections among HIV-Exposed and HIV-Infected Children have been updated and are undergoing final clearance.  The revised Guidelines will be posted online in early 2013.

Dr. Pau stated that the Guidelines for Prevention and Treatment of Opportunistic Infections in HIV-Infected Adults and Adolescents also are being revised.  Due to the length of this material (500 pages, covering 30 topics), future revisions will only be available online at https://clinicalinfo.hiv.gov/en/guidelines.

DISCUSSION

OARAC members asked several questions in follow-up to Dr. Pau’s presentation about the relationship between these treatment and prevention guidelines and those of the World Health Organization, that made recommendations based on availability and affordability of treatments.  Dr. Pau commented that the Guidelines are evidence-based and recommend only FDA-approved medications.  She explained that the OARAC Working Groups’ guidelines are developed for use only in the U.S., and issues of international cost-effectiveness are not considered.  OARAC members also asked if the guidelines include recommendations for the use of Pre-exposure Prophylaxis (PrEP).  
Dr. Pau noted that PrEP is not included because the current guidelines are for treatment of HIV-infected individuals while the current use of PrEP is for individuals who are HIV-uninfected.   The Centers for Disease Control and Prevention have issued guidelines for the use of PrEP.

INTRODUCTION TO THE MEETING TOPIC

Dr. Hillier introduced the topic of the meeting -- HIV and women’s risk and prevention with a focus on basic research.  She noted that the presentations would focus on the anatomic, physiologic, immunologic, and histologic parameters involved in HIV acquisition and disease progression in women.  The specific issues presented at this OARAC meeting included basic research on female genital tract immunology and microbiology, the role of semen and cervical mucus, animal and tissue models of HIV acquisition, impact of exogenous hormones on HIV risk, application of metagenomics to study the female genital tract microbiome, and novel approaches and strategies to measure antiviral distribution, drug levels, and pharmacokinetics in the female genital and anal tract.  The purpose of the meeting is to ensure that NIH priorities continue to address the scientific opportunities and needs in this area of research.

ANATOMY AND HISTOLOGY OF THE FEMALE GENITAL TRACT: HIV RISK AND PREVENTION ACROSS THE LIFECYCLE

The first speaker, Dr. Jean Anderson, Professor of Gynecology and Obstetrics, and Director of the Division of Gynecologic Specialties, Johns Hopkins University, described the changes in the anatomy and histology of the female genital tract (FGT) over the lifespan.  These changes include:  the increase in the ratio of uterus to cervix size; modifications in cervical tissue from single layer columnar epithelium to squamous epithelium; and development of the squamo-columnar junction at the opening of the cervix that contains HIV target cells.  She noted that these changes are controlled by many factors including:  the interplay and changes in central and local hormones over the life span; changes in adaptive and innate immune responses of the mucosa; and changes in the vaginal bacterial flora.  Each of these factors can affect a woman’s degree of vulnerability to acquisition of HIV and other pathogens.

Dr. Anderson stated that HIV infection can occur anywhere along the female genital tract (FGT); however, the endocervix is a preferential site for initial infection, perhaps due to the higher concentration of HIV target cells in that area.  While studies disagree whether cervical ectopy increases susceptibility to HIV infection, ectopy is associated with increased risk for other sexually transmitted diseases, higher levels of pro-inflammatory cytokines, and detectable genital tract HIV RNA in HIV-infected women.

Dr. Anderson noted that there is increased risk for HIV acquisition at certain times during the lifecycle:  1) in early adolescence there is greater cervical ectopy and thinner vaginal mucosa; 2) in the luteal phase of the menstrual cycle at ovulation when progesterone levels have reached their peak and begin to fall resulting in vaginal thinning, as well as thinning of the cervical mucus, which alters its barrier function and may facilitate passage of sperm and pathogens; 3) during pregnancy with elevated progesterone levels, alterations in vaginal immune function, and up-regulation of HIV co-receptor expression; and 4) in post-menopause when hormone levels have decreased and vaginal tissue is thinner, less pliable, and more easily damaged, vaginal pH is increased and the production of cervical mucus is decreased.

Dr. Anderson described the tissue types in key areas of the FGT and the numerous factors that can compromise epithelial integrity and increase HIV risk including:  early age sexual debut and activity; sexual assault; female circumcision; genital infections; douching; use of vaginal drying agents, intravaginal medications, spermicides, or tampons; hormonal contraception; and smoking.

Dr. Anderson commented that there is a need for additional research in all of these areas to better understand women’s risk for HIV acquisition.

ANIMAL MODELS FOR HIV RISK AND PREVENTION IN WOMEN

Dr. J. Gerardo Garcia-Lerma, Research Microbiologist, National Center for HIV and AIDS, Viral Hepatitis, STDs, and TB Prevention, Centers for Disease Control and Prevention, discussed the use of animal models, including non-human primates and humanized mice, to study prevention of HIV transmission. 

Dr. Garcia-Lerma described the utility of animal models in testing biomedical interventions to prevent HIV infection.  Non-human primates and other animal models can be used in studies to: 

• demonstrate proof of concept of the efficacy of multiple intervention strategies;

• identify the most promising candidates for PrEP or as microbicides;

• evaluate different drugs, dosing strategies, and formulations;

• evaluate interactions between biological cofactors that may increase susceptibility to infection;

• determine if interventions can protect against different routes of virus exposure; and

• determine the relationship between plasma and tissue drug levels and levels of protection against HIV.

Dr. Garcia-Lerma stated that animal models can be used to explore relationships between different types of HIV exposure (intravenous, oral, vaginal, rectal) and the efficacy of potential treatments.  These models also are useful to assess different intervention regimens (daily or intermittent) and to evaluate whether the same interventions can be used to prevent both vaginal and rectal HIV transmission. 

Dr. Garcia-Lerma described the importance of using the appropriate animal model for the scientific questions being asked.  He provided the example of using pigtail macaques as a model to answer questions about HIV infection in women because these non-human primates have menstrual cycles, including changes in hormone levels, similar to those seen in humans.  He noted that animal models have been used to evaluate biological and virological cofactors that may modulate the effectiveness of microbicides and PrEP.  He described several studies that used the macaque model to better understand the relationships between vaginal epithelial thinning with physiologic elevations of progesterone during the luteal phase of the menstrual cycle and HIV risk related to the exogenous administration of depot-medroxyprogesterone acetate (DMPA).  These models were used to determine the dose and tissue levels of DMPA needed to cause vaginal thinning and also demonstrate adequate protection with PrEP despite DMPA administration.  He noted that these animal models also will allow researchers to explore how other cofactors, including sexually transmitted infections, in addition to DMPA may modulate PrEP and microbicide efficacy.  He suggested that animal studies conducted to predict the effects of DMPA in humans should use dosing that mimics human levels for the data to be predictive.

Dr. Garcia-Lerma stated that studies using validated animal models are important in advancing HIV prevention research. 

TISSUE MODELS TO STUDY HIV RISK AND PREVENTION IN WOMEN

Dr. Charlene Dezzutti, Associate Professor, University of Pittsburgh School of Medicine Department of Obstetrics, Gynecology, and Reproductive Sciences, and Associate Professor of Infectious Diseases, Graduate School of Public Health, Magee-Women’s Research Institute, described the use of tissue models to study the mechanisms of HIV transmission and approaches to prevent HIV transmission.

Dr. Dezzutti described two models for preparing tissues specimens – 1) the non-polarized methods that were first developed using ectocervical tissue and 2) the polarized methods.  While the non-polarized method does not allow for the evaluation of transmission events or drug formulations, the polarized model allows for the addition of virus and prevention products in a way that is more biologically relevant.  She described several limitations and the different utility for each tissue model in studying drug and formulation safety, drug permeability, and tissue viability.  She noted that these tissue models may have potential as surrogates for clinical trial efficacy.  Dr. Dezzutti presented the results of a study using the polarized model that demonstrated the need to reformulate initial versions of tenofovir gel and placebo gels when histology showed epithelial fracturing and sloughing.  The findings from this study showed the tenofovir and placebo gels were hyperosmolar.

Dr. Dezzutti noted that tissue models also can be used to evaluate tissue permeability of drug and microbicide candidates, test drug levels required for protection, and determine the susceptibility of different tissues to infection with HIV.  She noted that the goal of ongoing tissue culture research is to provide a more biologically appropriate system for assessing the safety and efficacy of microbicide candidates using cervical, vaginal, and rectal explants.  Current research using tissue models is evaluating antibody development that will be useful to inform vaccine development.  She described some of the barriers to tissue model research, including the limited availability of fresh tissue, and difficulties in using cryopreserved specimens that can result in the loss of tissue architecture and the inability to infect this tissue with HIV.

Dr. Dezzutti identified several scientific opportunities and needs in using tissue models in HIV research including better methods to:  optimize tissue preservation for future use; demonstrate tissue viability and drug safety; determine effective drug levels that minimize toxicity; identify and validate effective ex vivo challenge models; demonstrate HIV infection; and determine the time course for virus integration.

DISCUSSION

OARAC members and speakers discussed the effects of hyperosmolar tenofovir preparations that had not been recognized in earlier studies.  They noted that the HPTN-059 study showed no toxicological effects with tenofovir gel.  They suggested that this may underscore the need to determine optimal drug concentrations compared to drug safety in clinical studies.

OARAC members and speakers suggested that the tissue models may be useful to test potential microbicide candidates and eliminate compounds that show no promise.  They also discussed whether menstrual cycle information was collected from the donors at the time of tissue harvest and whether those time-points affected how the tissues reacted in various assay systems.  Dr. Dezzutti noted that the human serum in which the tissues grow in culture may come primarily from men, adding endogenous hormones to the system.  This represents a factor that requires further study.  

OARAC members and speakers also discussed the similarities and differences between male and female genital tract tissues.  They noted that differences in commensal microflora are present in varied locations along the genital tract.

FUNCTIONAL IMMUNOLOGY AND GENITAL TRACT INFLAMMATION

Dr. Mimi Ghosh, Assistant Professor, Department of Epidemiology and Biostatistics, George Washington University School of Public Health and Health Services, described the immunology of the female reproductive tract, genital tract inflammation, and the interaction of the two that may affect HIV acquisition and transmission.  She suggested that a better understanding of the basic biology of the female genital tract may inform the differences between animal models and what is seen in humans.  She noted that many studies using in vitro systems and animal models have not predicted what is seen in clinical trials.

Dr. Ghosh stated that the mucosal immune system of the female reproductive tract has a unique role in maintaining a finely-regulated, fine-tuned balance of protection against pathogens versus allowing passage of allogenic sperm and supporting the fetus.  She noted that the rate of vaginal HIV transmission is low (~ 1:222,000) and the estimated protective efficacy of the vaginal mucosal barrier is calculated to be over 99 percent.  She commented that rectal HIV transmission rates are high in comparison to vaginal rates.  While most studies are focused on the lower tract and use sperm and other pathogens as a model, the upper tract also is potentially exposed to HIV.  She noted that the upper and lower genital tract cells secrete mucosal fluid rich in soluble immune mediators.

Dr. Ghosh described the two major components of genital tract immunity -- the innate and the adaptive immune systems.  She noted that the innate immune system has an immediate and nonspecific response to pathogens.  The adaptive immune system develops slowly over time and generates a specific response to pathogens.  Dr. Ghosh outlined the multiple levels of protection in the female reproductive tract including:  1) the barrier function - the first point of contact between the pathogen and the host with epithelial cells of the reproductive tract forming tight junctions with electrical resistance across the barriers; 2) mucus - that changes throughout the menstrual cycle as fertility levels change and serves to trap pathogens and prevent entry; 3) pH – that is highly acidic in the vagina; 4) Lactobacillus species - that colonize the lower reproductive tract and protect from harmful bacteria; 5) pattern recognition receptors -  that are expressed by cells of the reproductive tract capable of recognizing pathogen-associated molecular patterns and initiating a signal transduction event and antiviral pathways that inhibit pathogen growth or replication; 6) antimicrobial peptides  - that are secreted in the mucosal fluid and have specific anti-HIV activity; 7) cytokines and chemokines; 8) interferons  - that supply an active pathway to inhibit the virus; 9) proteases and anti-proteases  - that activate and deactivate a number of the antimicrobial peptides; and 10) other immune cells - including T-cells, B-cells, macrophages, dendritic cells, NK cells, and neutrophils and antibodies.

Dr. Ghosh noted that lifecycle changes associated with puberty, pregnancy, and menopause also have an effect on the female reproductive tract and result in alterations in the immune profile.  She described the anatomy of the female genital tract in relationship to viral entry and highlighted the transition zone between the ecto- and endocervix as the possible point of entry of HIV and other sexually transmitted pathogens.  She noted that beneath the epithelial layer there are dendritic cells or Langerhans cells that can capture the virus and pass it to immune target cells.  

Dr. Ghosh described the functional immunology in the female genital tract and reported on studies using cervical vaginal lavage fluid to identify factors that enhance/inhibit HIV infection and/or replication.  She outlined the causes of genital tract inflammation resulting from co-infection, including pre-existing sexually transmitted infections and sexual trauma that can enhance HIV infection and replication.  Dr. Ghosh noted that during shedding of genital tract virus, local changes may affect HIV transmission and acquisition.  She also stated HIV and pro-inflammatory cytokines can decrease trans-epithelial resistance and allow HIV to breach the natural epithelial barrier in the genital tract.  This action facilitates the ability of HIV to come in contact with target cells and leads to infection.

Dr. Ghosh outlined several key factors associated with genital tract immunity that should be considered for preventing HIV infection in women.  These include:  the multi-layered complexity of genital tract mucosal immune function; cytokines, chemokines, antimicrobials, and immunoglobulins; genital tract proteases and protease inhibitors; and hormones that regulate the actions and interactions of genital tract immune modulators.   She also described endogenous microbicides that can be protective by stimulating immune function or antagonistic resulting in inhibition of immune function and enhancing HIV infection.

Dr. Ghosh identified several areas of genital tract immunology that require further study.  She suggested that additional research is needed on:  local mucus; changes and differences in the genital tract immune function across the lifecycle including adolescence, pregnancy, and menopause; and the influence of semen on genital tract immunity.  She also suggested that studies are needed to elucidate the balance between beneficial immune factors that prevent HIV acquisition and detrimental factors that enhance HIV acquisition.

THE ROLE OF CERVICAL MUCUS IN HIV RISK AND PREVENTION

Dr. Thomas J. Hope, Professor, Department of Cell and Molecular Biology, Northwestern University Medical School, described the role of cervical mucus in HIV acquisition and prevention.  He noted that cervical mucus serves as the first barrier of defense in the female reproductive tract and in the gastrointestinal tract.  He also explained that cervical mucus changes over the course of the menstrual cycle and its role in covering potentially fragile surfaces.   

Dr. Hope commented that the science of cervical mucus evolved as methods were developed to clearly see virions in tissue.  He described the two types of mucus (cervical and cervico-vaginal mucus) and noted that each functions differently from the other.  He explained how cervical mucus was collected and tested in an experimental system that compared the movement of HIV delta envelope viral particles to movement of nanoparticles through both types of mucus.  Dr. Hope reported these studies demonstrated that cervical mucus allowed similar movement of HIV delta envelope particles and experimental nanoparticles while cervico-vaginal mucus showed more efficient movement of the nanoparticles, but delayed movement of viral particles.  These studies also showed that a component of cervical-vaginal mucus has an antiviral activity directed at the HIV envelope.  Dr. Hope discussed the interactions of mucus and antibodies; specifically, IgA antibodies that interact with mucins.  He also provided data to support the different actions of IgA and IgG in mucus. 

Dr. Hope suggested that a better understanding of HIV infection in women requires additional research on the role of mucus using tissue explant and cell culture systems, as well as animal models.  He described several studies using non-human primates to answer scientific questions on how the changes that occur over the course of the menstrual cycle effect HIV acquisition and infection.  The results indicate that macaques exposed to virus in the follicular phase, luteal phase, and at mid-cycle demonstrate that virus gets into tissue during the luteal phase and mid-cycle with the luteal phase emerging as the time of greatest risk for HIV infection.

Dr. Hope proposed that additional research is needed on the effects of hormonal contraception on the mechanisms of HIV acquisition.  He suggested that the dose of Depoprovera used in animals in future studies should more closely resemble the effective dose and progesterone levels in humans. 

THE GENITAL TRACT MICROBIOME AND HIV RISK AND PREVENTION

Dr. Gregory T. Spear, Professor of Immunology and Microbiology, Rush University Medical Center, described the genital tract microbiome and its relationship to HIV risk and prevention.  He discussed the differences in the microbiota normally present in the genital tract compared to the polymicrobiota associated with bacterial vaginosis (BV).

Dr. Spear noted that bacterial vaginosis has been associated with increased susceptibility to HIV infection, female-to-male transmission, and increased HIV shedding.  He described several methods that can be used to examine genital tract bacteria, including traditional culture and Gram staining, as well as new molecular biology methods involving high-throughput sequencing (pyrosequencing).  The latter can be used to identify thousands of bacterial sequences in one subject and facilitates the identification of bacteria that can be difficult to culture, especially anaerobes.  Polymerase chain reaction (PCR) specific for different types of bacteria also can be used to quantify the types of bacteria in lower genital tract samples. 

Dr. Spear stated that molecular biology techniques also can assist in determining the patterns of vaginal microbiota and their role in HIV risk.  An understanding of the specific bacteria or patterns of bacteria associated with HIV infection or HIV shedding may lead to prevention interventions. He commented that bacterial vaginosis is highly recurrent, and it is important to prevent BV relapse and to promote healthy microbiota.  Dr. Spear described several recent studies that utilize molecular biology techniques to understand the vaginal microbiome including a longitudinal study of microbiota over time in women from the NIH-sponsored Women’s Interagency HIV Study.  The goal of that study is to determine if the presence of BV can be associated with HIV incidence or progression.  He also described several studies using Rhesus and pigtail macaques to investigate the microbiota in lower genital tract infections that are similar to BV in humans. 

Dr. Spear proposed that there are microbiologic differences between the human female genital tract and that of macaques; however, the macaque could serve as a model, with some microbiologic manipulations, for future studies on human microflora in the female genital tract and bacterial vaginosis.

SEMEN AND SEX

Dr. Betsy Herold, Professor, Department of Pediatrics, and Division of Infectious Diseases, Microbiology, and Immunology, as well as Obstetrics, Gynecology and Women’s Health, Albert Einstein College of Medicine, described the role of semen and sex on HIV transmission and acquisition.  She described several preclinical and clinical studies on the mechanisms of HIV infection, as well as several preclinical studies testing PRO 2000 that considered sex and semen including:

• initial studies that showed a dose-response prevention of HSV infection;

• a recent study using seminal fluid instead of cell culture media and the same dose of drug that showed a one log loss of viral infection; and

• several studies that showed proteins in seminal plasma, in particular fibronectin 1, that interfered with the ability of PRO 2000 to bind to viruses. 

Dr. Herold also described the results of several clinical studies that demonstrated a “real life” understanding of the effects of semen and sex on mechanisms of HIV infection.  These studies showed endogenous anti-HIV activity in cervico-vaginal lavage specimens that was diminished in the presence of semen.  She commented that there is some variation in endogenous anti-HIV activity among women -- some women demonstrated no inhibition of HIV infection, while other women demonstrated an enhancement of HIV infection.   
 
Dr. Herold summarized how tenofovir works to prevent HIV infection.  She commented that the role of sex, semen, or mucus in this transport process is unknown.  Similarly, it is not known what effect(s) sex, semen, or mucus may have in drug permeability, drug metabolism, and product concentration.  She noted that semen has a number of functions that may affect HIV acquisition including:  buffering the acidic pH of genital tract secretions; interfering with the ability of mucus to trap HIV; and inducing the expression of inflammatory markers by genital tract epithelial cells.  Dr. Herold stated that semen contains proteins, cytokines, and chemokines that can either augment or inhibit HIV infection, as well as protease inhibitors and proteases that can interfere with the activity of anti-microbial peptides.

Dr. Herold described several microbicide clinical studies that examined the effects of sex and semen on antiretroviral pharmacokinetics and pharmacodynamics.  She suggested that sex and semen may increase HIV risk via multiple mechanisms.

Dr. Herold proposed that additional research is needed on mucus, semen, genital tract secretions, the epithelium, and the microbiota of the genital tract.  She commented that findings from these studies would potentially impact PrEP and microbicide efficacy. 

DISCUSSION

The OARAC members and presenters commented on the utility of the macaque model for studies on bacterial vaginosis and the human vaginal microflora.  They also discussed the challenges of planning and conducting studies that model real life settings.  They discussed additional research needed on the effects of endogenous and exogenous hormones on the microbiota of the female genital tract and cervicovaginal mucus.

THE IMPACT OF EXOGENOUS HORMONES ON HIV RISK

Dr. Christine Mauck, Medical Consultant, CONRAD, described the impact of exogenous hormones in the form of contraceptive products on HIV risk.  She discussed the results from several clinical studies using injectable contraception, specifically Depo-Provera (DMPA).  

Dr. Mauck described the results from the secondary analysis of data from the Partner’s PrEP study that demonstrated the use of DMPA increased the risk of HIV acquisition and transmission by nearly two-fold.  She described the strengths of the study that included frequent follow-up and a high retention of study participants as well as a known HIV exposure with genetic linkage to the HIV-infected partner.  She also noted the limitations of the study:  1) the secondary analysis was not designed to answer the question of contraception and HIV risk; 2) there were very few seroconversions; 3) the study participants were not randomized to the contraceptive method; and 4) the use of contraceptives and condoms was captured by self-reporting. She also discussed data from previous clinical trials that yielded discrepant data about DMPA and increased HIV risk.

Dr. Mauck discussed the report from the World Health Organization Expert Panel meeting in 2012 on the use of DMPA among women at high risk of HIV.  Based on the inconsistency of the association between DMPA and HIV risk, the Panel recommended that women using progesterone-only injectable contraception should be strongly advised to always use male or female condoms, and other HIV-preventive measures.  The Panel also stated that expansion of contraception method mix and further research on the relationship between hormonal contraception and HIV infection was essential.

Dr. Mauck described the possible mechanisms for the effect(s) of DMPA on the female genital tract and risk of HIV acquisition including:  1) changes in the epithelia architecture including thinning, increased cervical ectopy, and disruption of the epithelium; 2) loss of protective antimicrobial peptides; 3) increased inflammation, which increases target cells, target cell activation, and viral replication; and 4) changes in vaginal flora including the loss of protective lactobacilli and the presence of herpes.  She summarized that hormonal contraceptive methods could cause both protective effects with changes in some biological factors and increased risk with changes in other factors.

Dr. Mauck described the Hormonal Contraception (HC) – HIV Study, the HPTN 035 sub-analysis study, and the CAPRISA 004 sub-analysis study that were designed to determine what hormonal factors increase HIV risk.  These studies include data on the pre-infection genital tract of women who acquired HIV infection and compared it to matched study controls who did not acquire HIV.  She proposed that this methodology can be applied to contraception studies.

Dr. Mauck outlined important differences between endogenous and exogenous hormones.  She noted that Medroxyprogesterone acetate (MPA) was developed as a more potent form of endogenous progesterone to provide more stable and longer lasting blood levels.  MPA binds to the glucocorticoid and progesterone receptors with greater affinity than progesterone.  The glucocorticoid receptor may also affect HIV replication by interacting with the long terminal repeat.  She suggested this may explain why some data showed a more consistent association between DMPA and the risk of HIV.

Dr. Mauck described the mixed effects of combined oral contraceptives (COC) that have been demonstrated in several studies to be associated with:

• an increase in cervical ectopy and no change in epithelial thickness;

• a decrease in beta-defensin with an increase in soluble pro-inflammatory factors;

• an increase in CCR5- positive CD4+ cells; and  

• no change in the number of women with lactobacilli, but a small decrease in the number of women with high counts of lactobacilli. 

She suggested that this may be the result of the combined effects of the different hormones in COCs.  The hormonal effects may balance each other out to some extent and may explain why studies do not demonstrate a consistent effect on HIV risk in users.

Dr. Mauck described the ramifications of DMPA-associated HIV risk.  She suggested that HIV prevention studies and safety studies should examine study groups and samples for equal distribution of HC users or stratify entry criteria to include HC users and non-users, all HC users, or all non-users.  She suggested that the same factors that increase HIV acquisition, like epithelial thinning, could also impact the pharmacokinetics of microbicides and the outcomes of microbicide clinical studies. 

Dr. Mauck suggested that:  1) additional research is needed to test various contraceptives in cell and tissue systems for the purpose of looking at changes in immune mediators and HIV risk; 2) studies using non-human primates should be conducted to evaluate the impact of hormonal contraception on HIV susceptibility; 3) studies are needed to compare DMPA and norethisterone enanthate (NET-EN) in tissue and explant assays as well as in clinical studies; and 4) standardization of laboratory methods for mucosal collection and data analyses systems are needed to allow comparisons across studies.

DISCUSSION

The OARAC members and presenters discussed scientific and bioethical issues associated with conducting a clinical trial to compare contraceptives and HIV risk in which all of the study arms are using a contraception method.  This type of study provides a hierarchy of HIV risk comparing the contraceptives to each other, but it would not provide information on absolute risk.  Determining absolute risk would require a control group that uses condoms only. 

ANAL/RECTAL BIOLOGY AND HIV RISK

Dr. Barbara Shacklett, Associate Professor, Department of Medical Microbiology and Immunology, University of California Davis School of Medicine, discussed the relationship of anorectal biology to HIV risk and acquisition.  She provided an overview of anorectal histology and HIV susceptibility, as well as potential HIV target cells and potential transmission mechanisms.  She also described the T-cells and mucosal antibodies that are active in the gastrointestinal tract.

Dr. Shacklett outlined several possible routes of HIV transmission in the anal/rectal tract including: 1) transcytosis via epithelial cells; 2) standard epithelial cells; 3) micofolded cells; and 4) gut dendritic cells.  She also described the anorectal mucosal immune defenses.  She noted that the risk of HIV transmission during anal sex is significantly higher than it is via vaginal transmission due to tearing and breaching of the epithelium that can occur during anal intercourse.  It is estimated between 2 and 30 percent of women have had anal intercourse; however, the prevalence of anal intercourse among heterosexual women in the U.S. is underestimated.  The rate of anal intercourse has the potential to impact the power of microbicide studies designed with the vaginal intercourse paradigm.  She suggested that anal intercourse can be a confounder and should be carefully assessed in prevention studies. 

Dr. Shacklett identified several areas for additional research to determine:

• How the loss of specific T-cell subsets contribute to pathogenesis
• The role of gut CD4 cells in specific subsets and/or other gut cells as a potential reservoir for HIV in patients on suppressive antiretroviral therapy   
• The mechanisms by which mucosal antibodies can block viral entry, which is critical for vaccine development
• How to induce strong, broad mucosal T-cell responses similar to those seen in HIV elite controllers and antibody responses.

She also proposed that it is important to support the development, formulation, and evaluation of rectal microbicides, as well as to design prevention trials that include the potential for anal intercourse among heterosexual women. 

STRESS, IMMUNE FUNCTION, AND HIV RISK

Dr. Gail Ironson, Professor, Department of Psychology and Psychiatry, University of Miami School of Medicine, described how stress and psychosocial factors predict HIV risk and disease progression.  She also addressed the behavioral and biological mechanisms by which stress affects HIV disease progression, as well as the role for stress-management interventions as part of HIV prevention.

Dr. Ironson characterized abuse in women who are HIV-infected or at risk.  She noted that the overall rates of adult and childhood trauma and abuse are higher in HIV-infected than in HIV-uninfected women.  These issues affect mental health and quality of life. They also are associated with poorer health outcomes and higher HIV-risk behaviors.  She commented that the majority of studies on stress and disease progression before and after ART are mostly in men who have sex with men and show conflicting data.  The one small study on women pre-ART demonstrated that trauma predicted decreased CD4/CD8 ratios.  She noted that that there are few post-ART studies on this topic.  She reported that some studies have demonstrated that experiencing greater lifetime violence or trauma increases all-cause mortality, AIDS-related deaths, and greater increases in viral load.

Dr. Ironson described several mechanisms by which stress affects health.  She reported  on a study in women that included a healthy lifestyle component and addressed different risk factors, as well as anxiety, depression, self-efficacy, coping, social support, and quality of life.  The study findings showed increases in self-efficacy significantly predicted a decrease in viral load and an increase in CD4+ cell counts.   Dr. Ironson summarized the findings from several other studies that demonstrated:  stress has a significant, but small effect on disease progression; there are larger effects for cumulative stress, depression and for coping; there are potential biological pathways for stress effects on health; stress management interventions can improve psychological well-being, but have a small effect on biology; and past trauma should be taken into account in risk reduction interventions. 

Dr. Ironson suggested that additional research is needed to study the effects of stress and other psychological factors on immune function and HIV risk.

SEXUAL VIOLENCE AND THE BIOLOGY OF HIV RISK

Dr. Charu Kaushic, Associate Professor, Department of Pathology and Molecular Medicine, McMaster University, discussed the biology of how sexual violence could influence HIV risk.  She provided an overview of the recent Greentree meeting on this important topic that was sponsored by NIH including the OAR, UNAIDS, and the Social Science Research Council.

Dr. Kaushic reported that sexual violence, which is categorized as rape or sexual abuse, occurs in as many as 15 percent of women over their lifetime.  She stated that while there is some data to support increased HIV rates among sexual violence victims, this has not been shown consistently in numerous studies.  Dr. Kaushic reported mathematical modeling of this issue has been recently performed that included factors that cannot be considered in observational studies.  The findings predicted that a rape survivor’s individual risk for HIV may increase by as much as 2 to 27 fold.  On a population basis, this mathematical modeling showed that the incidence of HIV could actually increase by as much as 10 percent in women who experience rape.  She reported the recommendations from the Greentree meeting underscored the need to better understand the intersection of basic, clinical, social, and behavioral sciences and the role of genital/anal injury in HIV transmission, acquisition, and pathogenesis. 

Dr. Kaushic discussed the role of the genital epithelium in HIV infection.  She described the histology of the upper and lower female reproductive tract.  The upper tract includes the cervix, endometrium, and fallopian tubes and is comprised of columnar epithelium. The lower reproductive tract includes the vagina, which is composed of the pseudo- squamous epithelium, and the transformation zone between the endocervix and the ectocervix which is particularly susceptible to HIV due to the high concentration of CD4+ cells and the presence of cervical ectopy.

Dr. Kaushic described a model of pure genital epithelial cells in primary culture that was used to study the mechanisms of HIV infection.  She reported the study findings that showed:

• HIV crosses the epithelial barrier and induces an intracellular signaling response that leads to the production of proinflammatory cytokines and TNF that can  disrupt tight junctions;

• transcytosis;

• barrier breach that allowed immune activation that correlates with microbial translocation; and

• local inflammation with TNF in the microenvironment.  

Dr. Kaushic summarized the effects of various hormones using this epithelial cell culture system.  The study tested the uptake of HIV in normal physiological concentrations of estradiol, progesterone, and medroxyprogesterone acetate (MPA).   She reported that MPA appears to enhance the uptake of HIV in this assay system.

Dr. Kaushic suggested that additional research is needed on blocking epithelial disruption or inflammation as a mechanism to protect against HIV infection. 

LEVERAGING GENITAL AND ANAL TRACK BIOLOGY FOR HIV PREVENTION

Dr. Rupert Kaul, Associate Professor in the Departments of Medicine and Immunology,  University of Toronto, discussed mucosal immunity of the female genital tract and 
vaginal immunology.  His presentation addressed mucosal target cells and how these cells become infected during HIV transmission. 

Dr. Kaul described the mechanisms of infection.  He noted that within hours of exposure, the virus enters target cells, and within a few days the virus disseminates systemically.   He stated that local replication in the mucosa is critical to HIV acquisition and requires having sufficient numbers of target cells in the mucosa for HIV replication to occur.  He suggested that biology and the mechanisms of infection may play a role in the decline in HIV incidence reported in some populations that has previously been attributed to only changes in behavior.

Dr. Kaul summarized several studies on cervical inflammatory cytokines and other innate immune defense factors associated with subsequent HIV acquisition.  He reported research findings that show a direct correlation between increasing concentration of RANTES in cervical-vaginal lavage specimens and an increasing number of CD4+ T-cells collected on a cervico-vaginal cytobrush.  He noted that the cells that are infected in the genital tract are different than the cells that are found in blood and differ by site of infection (cervix compared to the anus or penile foreskin).  The differences include variability of CD4+ cell types, CCR5 expression, and the expression of other mucosal homing markers.  Dr. Kaul described several models that his laboratory is developing to study cellular infection that can be applied to other types of mucosal cell subsets, such as dendritic cells, that may be important in risk for HIV infection.  

Dr. Kaul proposed that additional research is needed to better understand T-cell subsets that are responsible for HIV transmission.  The findings from these studies may inform the development of combination prevention strategies against two or three key mucosal targets. 

GENITAL AND ANAL TRACT PHARMACOKINETICS

Dr. Craig Hendrix, Professor of Medicine, Pharmacology, and Molecular Sciences and Epidemiology, Johns Hopkins University Bloomberg School of Public Health, discussed the pharmacokinetics of antiretroviral drugs in the genital and anal tracts.  He emphasized the importance of these studies in the development of microbicide candidates.   

Dr. Hendrix discussed the distribution of HIV in cells in the genital tract.  He described recent studies on the distribution of topical microbicides to various tissues in order to see if these agents reached HIV-infected cells.  He noted the limitations on available data since large, randomized clinical trials cannot predict infection.  Dr. Hendrix described several biological models of vaginal and rectal infection using an HIV surrogate and suggested that these models can be used to determine virus concentration over distance and time.  These models can then be used to facilitate the development of microbicide candidates that distribute to the genital and anal locations where the virus is located.
                            
Dr. Hendrix summarized several molecular level studies of tenofovir that showed a membrane transporter is needed for the drug to enter cells.  He reported that these membrane transporters are expressed in various amounts in different tissue types and are hormonally responsive.  He noted that these transporters are not well described and require further study.  He commented that intracellular phosphorylation of tenofovir occurs with anal and genital distribution of drug, but cellular activation can inhibit phosphorylation of tenofovir and make it less effective for prevention. 

Dr. Hendrix reported the distribution of tenofovir applied topically to the vaginal lumen distributes to the blood at the lowest concentration, vaginal tissue at a higher concentration, and cervical vaginal fluid at a much higher concentration.  He noted that there is no difference in the accumulation of drug with single compared to multiple doses.  He also described a crossover design study of daily oral versus vaginal dosing of TNF in women that demonstrated a much lower serum concentration of drug after vaginal dosing.  Dr. Hendrix also reported that vaginal dosing resulted in measurable rectal fluid levels, and rectal dosing achieved vaginal fluid concentrations.  These trans-compartment concentrations are greater than would be expected through serum absorption, circulation, and redistribution.  He suggested that this is the result of direct diffusion across the rectal-vaginal septum.  He noted that in the FemPrEP and iPREX studies the data showed low drug tissue concentrations in the vagina and rectum are related to low levels of protection from tenofovir. 

Dr. Hendrix proposed that decisions to initiate large clinical trials of potential microbicides should be based on pharmacokinetic and pharmacodynamic data from preclinical studies.

APPLYING NEW TECHNOLOGY TO UNDERSTAND HIV RISK AND PREVENTION: METAGENOMICS OF THE FEMALE GENITAL TRACT

Dr. Kjersti Aagaard, Associate Professor, Department of Obstetrics and Gynecology, Baylor College of Medicine, discussed the use of metagenomics as a new technology to better understand HIV risk and prevention.  She described metagenomics and microbiome research in the female genital tract and its applicability to HIV research.

Dr. Aagaard discussed the utility of metagenomics methods to provide a better understanding of susceptibility to HIV infection.  She stated that metagenomics of the microbiome can be applied using phylogenetic arrays to outline which microbial species are present including their composition, abundance, and gene catalogues.  While traditional identification of microbes relied on culture and catalogue techniques, many microorganisms were missed since they could not be easily cultured.  The metagenomics methodology allows for a more complete determination of the microbiome in a specimen, the ability to determine the function of the microbiome species, and permits the identification of community clusters to determine the differences between at risk groups who acquire HIV and those who do not based on difference in the microbiome.  Dr. Aagaard suggested that this approach also differentiates the host DNA from microbial DNA allowing for the determination of which microbes may cluster with which host DNA.  She noted that while there are differences in the microbiome between individuals, the greatest differences exist between areas of the body.

Dr. Aagaard described the results of several studies that examined the vaginal microbiome using metagenomics.  These studies demonstrated that the microbiome varies within the upper, middle, and lower regions of the vagina.  She suggested that these differences may affect the reliability of “self-collected” vaginal samples.  This methodology also has been applied to pregnancy and the effect that the mode of delivery has on the infant microbiome.  She commented that a better understanding of this microbial association may provide critical information on maternal-infant HIV transmission and horizontal transmission.  Dr. Aagaard suggested that vaginal microbiome differences could potentially serve as a biomarker for HIV susceptibility and disease progression.

Dr. Aagaard proposed that metagenomics technology could be applied to other areas of AIDS research to:  elucidate genomic susceptibility, host or microbe drug response, drug resistance, and viromics; generate reference genomes to better understand how microbial communities change based upon microbial abundance; and design and conduct global research projects to examine a very large population base. 

DISCUSSION

OARAC members and speakers discussed the applicability of metagenomics for AIDS research.  They commented on the practicalities of using this technology and the large number of individuals who would need to be studied in order to characterize HIV susceptibility differences.  They also discussed the differences found between data obtained with culture methodology compared to metagenomics.  These differences were attributed to the self-collection approach used in studies that examined cultures.

OARAC members and speakers discussed the importance of additional research on cell and viral variants, as well as the need to study mucosal protective mechanisms including neutralizing antibodies.  They expressed concerns about the discrepancies between the inefficiency of HIV transmission described in cell-based research and the increasing incidence of HIV infection in women.

OARAC members proposed that additional research is needed in the following areas:  1) the factors that influence women’s risk across the lifecycle; 2) the biology of the female genital tract for HIV risk and prevention; 3) the factors that drive the efficiency of HIV transmission in women with a particular focus on understanding the earliest stages of HIV infection; 4) the effects of hormones including pregnancy and age-related endogenous hormones, and exogenous hormones on HIV risk; 5) the effects of systemic or endogenous hormones on immune function in gut immunology; 6) the role of sex and semen in facilitating infection; 7) the role of local and systemic drugs for the prevention of infection; 8) the role of anal intercourse and women’s risk for HIV; 9) the differences in rectal immunology between men and women; and 10) the contribution of sexual violence and other types of trauma to HIV risk.

OARAC members also encouraged additional research on the development and validation of animal and in vitro models that are predictive of HIV acquisition and drug concentration, pharmacokinetics, and pharmacodynamics. They also proposed the standardization of methodologies in specimen collection, assays, and data analyses to allow comparisons of data between various studies.  OARAC members suggested that standardized protocols should be developed to determine which prevention agents should be advanced in the pipeline; biospecimens in repositories should be used to answer scientific questions whenever possible; and treatment, adherence, and social factors should be incorporated into studies on the biology of HIV risk and prevention.

PUBLIC COMMENTS

No members of the public requested time to comment.

CLOSING COMMENTS

Dr. Hillier thanked the OAR staff for organizing the meeting. She also thanked the members, speakers, and guests for their broad consideration of research on HIV and women’s risk and prevention. Dr. Whitescarver emphasized the importance of this meeting in helping OAR make decisions about priorities in the area of research on women and HIV. He also thanked the speakers for their stimulating presentations and the OARAC members for their thoughtful comments. The topic for the next OARAC meeting will be HIV-associated neurological manifestations and disorders. 

ADJORN

The meeting was adjourned at 5:00 p.m. on November 8, 2012.

/Jack Whitescarver, Ph.D./
Jack Whitescarver, Ph.D., Executive Secretary

/ Sharon L. Hillier, Ph.D./
Sharon L. Hillier, Ph.D., Chair