American Foundation for AIDS Research, July 2002
Bob Roehr

Introduction

Developing an effective microbicide "is one of the most important things that we can do" to reduce the spread of HIV, Peter Piot told the opening session of the Microbicides 2002 conference in Antwerp, Belgium, on May 12. The conference reviewed the latest research advances in the development of topical agents to block the vaginal and rectal acquisition of sexually transmitted diseases such as HIV. "People are looking for a magic bullet," he said. "We will never have a perfect microbicide… [rather, we are seeking] a constellation of different prevention techniques whose collective effect will be to reduce HIV transmission."

The executive director of UNAIDS called the development of microbicides "a classic case of market failure. The profit incentive for a first generation microbicide simply isn't there." Nor did he wish to underestimate the scientific obstacles that have impeded their development. But budding alliances between a number of players and the commitment of "real money" by foundations and governments to the field have heartened Piot. He said that the key to success is not to fall into "a lockstep approach" but to simultaneously develop multiple options.

There was consensus in the conference that a first-generation microbicide likely will not afford a very high level of protection. But as Charlotte Watts (London School of Hygiene and Tropical Medicine, UK) said, even a low efficacy microbicide used by a large number of women could have "substantial public health benefits."

But Peggy Johnson (NIAID, USA) feared that introduction of a partially effective microbicide or vaccine might lead to riskier sexual activity that could diminish or negate any benefit from the microbicide. Since HIV prevention trials include safe-sex counseling, Johnston was "concerned that if we take that vaccine or microbicide and put it into the field without the behavioral intervention, the effectiveness that we see could be very much diminished from what we saw in the trial."

Johnston also wants to "learn from the treatment arena…. The most success against this virus has come from attacking it at multiple points using combinations of agents, and preparing for the worst – emergence of mutant strains." This entails looking at combinations of microbicides that have "multiple mechanisms of action."

Menstrual Influences on Immunity

The proportion of immune cells within the total cell population can vary tremendously in the female reproductive tract, from 20% to 25% in the fallopian tubes down to 10% in the vagina. Composition and architecture of that immune cell population can vary with the stage of the reproductive cycle, said Charles R. Wira (Dartmouth Medical School, USA, Microbicides 2002 abstract A-269).

Perhaps most important for vulnerability to HIV infection, the number of CCR5 and CXCR4 receptors on CD4 T-cells changes over the course of the reproductive cycle. HIV depends on these "coreceptors" along with the CD4 receptor when it attempts to infect new cells. Coreceptor expression is low prior to ovulation, elevates as ovulation occurs and then declines. The CD4 T-cell population itself is elevated during the first half of the menstrual cycle, and then it too declines.

Nonspecific innate immunity also appears to change with the menstrual cycle, as demonstrated through laboratory assays of secretory leukocyte protease inhibitor (SLPI) production by vaginal epithelial cells. SLPI is a powerful virucide that can inactivate HIV. Innate immunity also changes with the stage of life. Wira said, "Secretions from premenstrual premenopausal women are capable of killing both gram-positive and gram-negative bacteria, but postmenopausal epithelial cells do not share this biological property." This observation strongly suggests that there is an as yet unidentified hormonal influence on vaginal innate immunity.

Candidate Microbicides

Wira also was "struck by the paradox that unprotected sexual intercourse with an HIV-infected individual has a risk of 1 in 1,000 or 1 in 10,000" of spreading that infection, depending on the site of exposure. He urged researchers "to think about the presence of endogenous microbicides," – defensins and other components of the innate immune system that may help keep these ratios low – and how they might be strengthened.

Rhesus monkeys produce circular antimicrobial peptides called defensins, but defensins in humans turn out to be problematic. Human bone marrow expresses a pseudogene that is 88.9% identical to a rhesus defensin. A crucial difference is a stop codon in the gene that prevents production of the human variation, reported Alexander Cole (UCLA School of Medicine, abstract A-048).

Cole synthesized the peptide that would be produced if the human stop codon were absent or inoperative and named it retrocyclin. He found that, in vitro, retrocyclin has a remarkable ability to inhibit proviral DNA gene formation, suggesting that it could block HIV at some point during the virus's cell entry process. Rhesus defensins offered some but more limited protection, suggesting the importance of species specificity of the peptides. Ongoing research is attempting to determine the retrocyclin's molecular targets.

One remarkable aspect of retrocyclin is that "it is also active if administered up to 24 hours post-infection ...with a 2 to 3 log [99% to 99.9%] reduction of p24 [HIV envelope protein]," Cole said. Retrocyclin protects CD4 T-cells from HIV that targets either CCR5 or CXCR5. Data from a close analog, RC-101, indicate that the protein forms patchy aggregates on the surface of CD4 T-cells. Cole is conducting further research, and UCLA is looking for a sponsor to develop a commercial product using retrocyclin.

Determining the crystal structure of gp120 and the process of the conformational changes that HIV undergoes during the process of binding with cell coreceptors has allowed development of a novel neutralizing agent, sCD4-17b. Edward Berger (NIAID, USA, abstract A-275) reported on the research. This synthetic molecule consists of a single-chain protein containing a soluble region of human CD4 cell receptor (sCD4) attached to a human monoclonal antibody, MAb 17b. MAb 17b targets an area in gp120 that binds to coreceptors. The agents by themselves have little activity. Berger said, "They derive their potency from simultaneous binding to gp120."

Initial lab work has determined that the sCD4-17b is effective against both CCR5- and CXCR4-seeking HIV, with potency at least ten-fold higher than broadly cross-reactive anti-HIV-1 neutralizing antibodies. However, it does not appear to bind equally well across all primary isolates. Berger speculated that perhaps a longer linking molecule is needed between the two active binding sites in order to be effective with some isolates.

There is a collaborative effort with Osel Inc. to bioengineer the sCD4-17b into a lactobacillus species that forms part of the healthy vaginal environment. The bacteria would either secrete the fusion protein or express it on their surface to form a kind of "viral flypaper" that ensnares HIV. The goal is a commercial product that will provide durable protection from HIV sexual transmission in the vaginal tract.

UC-781 is an NNRTI that caused quite a stir at the first microbicides conference two years ago. Poor absorption and bioavailability killed its chances for development as a systemic drug, but those qualities were perfect for topical use. The owner of the compound did not want to pursue development of a microbicide, and it took a year to transfer those intellectual property rights. At Microbicides 2002, Joseph Romano (Biosyn, Inc. USA, abstract A-254) represented the company that is now developing the compound.

The molecule tightly binds any form of HIV-1's reverse transcriptase 1, but it has no activity against HIV-2. There is also a "memory effect," where the compound can be applied to a cell culture and then washed away. "Pretreating cells can protect them against subsequent infection," Romano claimed. Candidate formulations of UC-781 (abstract A-255) are being evaluated for preliminary human trials.

Learning from the N-9 Experience

Nonoxynol-9 (N-9) was the straw that advocates had grasped years ago in hopes of an immediate HIV prevention tool. The spermicide had been widely available for decades, was presumed safe, and demonstrated activity against HIV in the lab. But the closer one looked, the more flimsy those hopes became.

Work by David Phillips (Population Council, USA) presented at the Microbicides 2000 conference demonstrated that use of N-9 in the rectum results in rapid shedding of large sheets of epithelial cells from that tissue. Also in 2000, the results of an N-9 trial with 1,000 African commercial sex workers found that N-9 use put women at increased risk of acquiring HIV, probably because the compound caused vaginal lesions. These trial data prompted the CDC to warn against using N-9 as a microbicide.

The National Institutes of Health sponsored a symposium at Microbicides 2002 to review the lessons learned from N-9 as we move to evaluate other microbicide candidates.

The initial lab work on N-9 was not very sophisticated, said Robin Shattock (St. George's Hospital Medical School, UK). In reviewing more current literature, he concluded, "N-9 only displays antiviral activity at doses that are cytotoxic" to the cells of the host. It appears to modulate the permeability of epithelial cells in a manner correlating with concentration and duration of exposure. There is a threshold effect "beyond which cells cannot recover." That suggests a difficult future for other products using the same "detergent" mechanism of action.

It is important to understand whether genital lesions and inflammation are the product of infection or are induced by a product, said Sharon Hillier (University of Pittsburgh, USA). "Looking is not as good as measuring," she said. Conducting a product's initial safety studies will require intense high tech methods performed on a limited number of female volunteers. Vaginal and rectal mucosal tissue repairs itself very quickly, so timing of measurement is critical and generally precludes research on an outpatient basis.

News that the FDA is considering removing the "generally regarded as safe" labeling of N-9 products stimulated animated discussion. The consensus was that more information is needed in order for consumers to make informed choices. Still, as Shattock noted, "I think it is going to be very hard to market a microbicide with labeling that says, 'you should only use it if you have sex infrequently.'"

Current Clinical Trial Issues

Zeda Rosenberg (International Partnership for Microbicides, USA) reviewed the status of compounds in clinical trials. Phase III trials "are going to have to be very large trials in order to pick up very small variations," she said. Care must be taken to build community support and minimize dropouts if researchers are to accomplish this task. It is imperative that measurements, and to the greatest extent possible designs, be standardized so that results can be compared across trials.

Robin Shattock (St. George's Hospital Medical School, UK) was encouraged by the growing findings from basic research, "but until we finish a successful phase III trial, we are unable to validate existing work" in the lab and in animal models.

He expressed concern that the wave of five polysaccharide entry inhibitors now entering phase III trials focuses on a single mechanism of action –charged sulfate regions on the polysaccharides stick to oppositely charged regions on HIV. It will be four years before those trial sites will have significant available capacity to evaluate products with other mechanisms of action.

This is a particularly important public question because government and nonprofit entities will support most phase III microbicide trials – unlike most phase III trials, which are conducted and funded by industry.

Note on rectal microbicides: Several speakers at Microbicides 2002 summarized issues raised last summer at a workshop focused on this issue. For a detailed look at preventing HIV transmission through anal sex, see the amfAR monograph “Creating a Research and Development Agenda for Rectal Microbicides that Protect against HIV Infection” (May 2002). This monograph was distributed at Microbicides 2002 and is available on the Web at http://www.amfar.org/binarydata/AMFAR_PUBLICATION/download_file/28.pdf.

Men's Odds of Spreading HIV

Stephen Taylor (University of Birmingham Edgbaston, UK, Microbicides 2002 abstract A-302) presented "the token semen talk." He explained the variety of factors affecting drug penetration and action within tissue compartments. His group determined that the drug levels of nevirapine in semen reach about 60% of the levels seen in blood throughout the course of the day. Efavirenz attained only 10% of blood concentrations due to its high level of binding to blood proteins.

The nucleoside analogs d4T and abacavir were seen at similar levels in semen and blood, whereas AZT and 3TC achieve semen concentrations three to five times higher than in blood. Of the protease inhibitors, only indinavir penetrated to levels equivalent to plasma, while other drugs in the class were present only in sub-therapeutic concentrations. Given those findings of what can be called "compartmental monotherapy," he was not surprised to find evidence of viral escape in semen. Drug-induced viral load reductions in the blood do not automatically translate into similar reductions in the semen. For that reason, they do not render an HIV-positive person free of transmission risk.

Turning to "seminal super shedders" of HIV, Taylor found a clear correlation with an active STD. Among patients who were on potent anti-HIV therapy, those with undetectable viral load in semen generally remained undetectable despite the presence of urethritis. Successful treatment of the STD brought a reduction in seminal viral load in patients who had experienced an increase.

A pilot study examined six men with urethritis and detectable seminal HIV while on therapy. Four of the six had multiple drug resistance-associated mutations in HIV isolated from both their blood and semen. Taylor concluded that a man who is on an incompletely suppressive anti-HIV regimen and has a sexually transmitted infection carries a significantly enhanced risk of transmitting resistant virus.

The R5/X4 Conundrum

Why does sexually transmitted HIV almost always have an affinity for the CCR5 coreceptor rather than CXCR4? Leonid Margolis (NICHD, USA, abstract A-313) set out to answer that question using an ex vivo system that maintains human lymphoid morphology and architecture. He found that both CCR5- and CXCR4-seeking HIV infect lymphoid tissue "equally well, so it is not at the level of lymphoid tissue that this selection occurs."

Margolis discovered that CXCR4-tropic HIV severely depletes CD4 T-cells so that "less than 20% remain in the tissue after two weeks." CCR5-oriented HIV depletes CD4 T-cells cells only slowly. He traced this to the fact that within lymphoid tissue, 80% of the T-cells express CXCR4, with many of the remaining cells expressing both receptors. The cellular activity in the tissue and the cytokines and chemokines that cells produce to regulate other cells' activity determines which receptors are more likely to be expressed and hence which virus is the most efficient killer.

Margolis believes that initial infection likely is by "a swarm of virus" that reacts with other pathogens and perhaps with each other. Using real-time PCR to measure in vivo, he found that HHV-6 coinfection up-regulates the chemokine RANTES, which inhibits the R5 but not the X4 type of HIV. "Pathogens talk to each other in our body" through chemokines, he said. This "may play a profound role in the R5-to-X4 switch." He suggested that perhaps this communication could be tapped, using particular microbes as a microbicide to modulate chemokines production to prevent HIV infection.

Microbicides in Clinical Trials

This article has been reprinted from the Treatment Directory at amfAR.org

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