LifeGuide [Treatment Horizons]
Blocking the First Step
A silk-protein film aims to become an HIV prevention method
by Chael Needle
When we think of HIV prevention in sexually active individuals nowadays, the possibilities have moved beyond creating a latex barrier to pharmacological prophylaxis. For those living with HIV, this might mean suppressing the virus to undetectable levels with antiretroviral regimens to prevent transmission. For those who are HIV-negative, it might mean starting an antiretroviral-based regimen as pre-exposure prophylaxis (or post-exposure). And then there is a range of topical microbicides as prevention candidates. One of the latest developments is a silk-protein film.
Patricia LiWang, PhD, Professor of Biochemistry at the School of Natural Sciences, UC Merced, is leading a team of researchers who have zeroed in on a handful of chemokines—signaling proteins that are secreted by cells, whose normal function is pro-inflammatory—that prevent the virus from binding to cells, but which are not successful enough to stop infection as they exist naturally. The laboratory’s research has been dedicated to studying proteins, and their mutants, at the molecular level—their structure and function, and the relationship between the two—and part of the team’s work has focused on biochemically enhancing a few of those within the chemokine family of proteins that have displayed HIV-1-inibition qualities in order to prevent infection.
As part of this project, the P. LiWang Group is studying 5P12-RANTES (a variant of human protein RANTES) and Griffithsin (a protein derived from algae), which were initially discovered and/or designed by other research groups (now working in partnership with LiWang’s lab), as well as 5P12-RANTES-linker-C37 and Griffithsin-linker-C37, which LiWang and her team developed. The linker-C37 is what improves the inhibitors.
In collaboration with researchers at three other universities, LiWang and her team are focusing on creating biochemical changes in the chemokines to power-up their inhibitory qualities and deliver them to sites of initial infection, namely, the reproductive and digestive tracts. Ingestion is not ideal, so a different delivery system was needed. Partnering with a bioengineer at Tufts University, LiWang designed a silk-protein film that would be delivered in an easy-to-use suppository.
Explaining the choice of silk, Dr. Patricia LiWang tells A&U: “Silk has been used in medical situations for years (like in sutures) and it has been found to not cause any problems when introduced to patients. So there would be little worry about the silk protein causing inflammation or not breaking down properly.”
Once delivered, these HIV-inhibiting chemokines would also then work in support of the body’s natural defenses during attempts at infection. “Our work is mostly geared toward preventing initial infection, so, if we can keep the virus out of any cells, the virus will die and the body will clear it away without infection,” says Dr. LiWang.
As with treatment, adherence is a concern with any prevention method. Adherence depends of course on many factors, including any prevention method’s ease of use, durability, and the frequency and timing of application, among other factors. Something that works well in a controlled environment, like a laboratory or study site, may not translate to everyday life. “The real world is so important to consider, but it is not easy for a scientist to think ‘outside the lab’ and make sure that the results are going in that right direction,” notes Dr. LiWang. “Luckily, the NIH has stressed the importance of this for a while, and colleagues in the social sciences have been making this point, so that we can actually end up with something that can help end this terrible disease.” In comparison to other prevention methods, some of the silk-protein film’s qualities may theoretically improve adherence.
Across all of the collaborative partnerships, the researchers have many steps and studies to complete—testing for inflammation in animal studies and working on the time-release factor to make application biweekly or monthly are just two examples—but they are excited about making a contribution to the science of fighting AIDS.
Recently, LiWang was awarded a four-year, $2.3 million grant by the National Institutes of Health to pursue the silk-protein film as a prevention candidate. A&U had corresponded with Patricia LiWang about her exciting research, and, in particular, the basic science of improving HIV inhibition, using combination entry inhibitors, and the design of the delivery system.
Since the late 1980s, researchers have established that some chemokines were able to suppress HIV. But to do this, the chemokines need to bind to the chemokine receptors on the surface of cells—do what these particular type of chemokines normally do. However, HIV takes over the receptors, needing a CD4 molecule and co-receptors (CXCR4 and CCR5, for example), as well as other binding and fusion steps, to gain entry into the cell. When the chemokine receptor is being used by a chemokine or if it is defective (or mutated), then HIV is blocked.
In terms of the basic science of HIV inhibition, then, the silk protein’s mechanism of action is a workaround for our natural chemokines’ potentiality but ultimate failure to inhibit HIV. With the film, the enhanced, HIV-inhibiting proteins, once delivered, bind to the cell and prevent HIV from infecting it.
“Natural chemokines are not nearly as good as we would need them to be in order to stop infection,” notes Dr. LiWang about why the naturally occuring chemokines need enhancement. “There may be several reasons for this, including some evidence that natural chemokines only bind to one of several conformations of the chemokine receptors, so they are not able to ‘sit there’ all the time to efficiently block HIV every time it comes into contact with the cell….Also, circulating concentrations of chemokines may be low because their natural role is to cause immune cells to travel throughout the body…and the body does not always need high amounts of these chemokines.”
The enhancements of the chemokines aim for as much potency as possible. “Some very good protein chemists (the Hartley and Offord groups) randomly mutated the chemokine RANTES until they found a variant that showed much improved HIV inhibition over the normal chemokine. This [modified] chemokine is also [no longer] pro-inflammatory, so there is little worry about this causing inflammation if it were used as a drug (which would be the worry if a normal chemokine were used).” (Studies have shown that persistent inflammation leads to negative health effects.)
Explains Dr. LiWang: “We tried to build on their modified RANTES, with the result that our chimeric protein is more potent under most conditions as well as able to inhibit a wider variety of HIV strains.”
5P12-RANTES targets a common receptor that HIV uses to gain entry: CCR5. To 5P12-RANTES, the P. LiWang Group added C37, a fusion inhibitor of gp41, an envelope protein of HIV needed for infection. The combination entry inhibitor utitlized in the silk-protein film, 5P12-linker-C37 (as well as 5P14-linker-C37, another variant), showed strong anti-HIV activity against R5-tropic viruses and was 100 times more potent than 5P12-RANTES alone or when combined with unlinked C37. The researchers also saw that C37’s activity against X4-tropic viruses was also preserved, making an even more potent inhibitor a possibility.
“There are many different strains of HIV, even in the body of one patient, so it is necessary to be able to inhibit as many as possible in order to prevent more cells from being infected or to prevent infection of another person,” she says about the premise of combination entry inhibition.“We have found that if we covalently join inhibitors with two different mechanisms of action, we often get an improvement, either in potency or in breadth of HIV viral strains that are inhibited, or both.”
Why is a combination of inhibitors potentially more efficacious than a single inhibitor? “There are a couple good reasons for having an entry inhibitor that is comprised of a combination of inhibitor types. For one thing, there is no guarantee that a single inhibitor will be able to ‘cover’ all of its natural target sites. Also, we have found overall more potency with covalently linked inhibitors. But probably more important is the fact that HIV mutates very quickly, so any single inhibitor may lose potency if HIV happens to mutate in a way to overcome it. This has happened to many HIV drugs in the past. With a combination inhibitor, if HIV mutates to become less sensitive to one component, it is very unlikely that it would also be insensitive to the other component.”
The work of the proteins, however, greatly depend on a viable delivery system: Notes Dr. LiWang: “The silk film that we work on is the result of the necessity that any material to prevent the spread of HIV should be cheap, easy to use, and [should] not require refrigeration, because it may be heavily used in the developing world. Our collaborators in the Kaplan lab at Tufts University have great expertise in stabilizing biologically active molecules, and so we decided to combine our expertise. We provide very potent and broadly-acting inhibitors, and they encapsulate them into thin films made of silk proteins.
“So far, we have found that our proteins remain fully active after months of incubation at 50 °C (122 °F), so we think it can handle the heat in sub-Saharan Africa.”
She adds: “The next step is to give our insertable films the ability to slowly release inhibitor. In this way, a person could conceivably insert a little film and have protection for days or weeks. This would be preferable to some kind of daily dose that a person might forget or not have time for.”
Researchers do not quite know yet why the inhibitors stay put in the targeted areas rather than migrating away, but, LiWang says, it’s a question “we will be investigating when we start to work with animal models in collaboration with Dr. Dandekar at UC Davis. So we don’t have good data for this yet, but I can tell you what we are thinking and hoping. There is a lot of precedent for insertion of medical devices into the vaginal tract, and we have hope that a film will gradually release inhibitor over the course of days, slowly allowing the body’s own moisture to propagate the inhibitor over the whole area (and possibly slowly dissolving the film). The digestive tract (for people of both genders that engage in anal sex) may be more difficult. There is a larger volume and area to cover, and the question is, will a small sheet of filmy substance be able to provide protection to the whole area? Right now we don’t know. Our collaborators have used silk proteins to make a large variety of delivery systems (films, gels, solids, etc.) so we are hoping to look into this question as soon as possible.”
But, if the “very selective” and “very potent” chimeric inhibitors show anti-viral activity, why is prevention the focus and not treatment? “The main reason is twofold: First, physicians have some really great treatment options to use now (although admittedly, they are always looking for more and more effective strategies). Second, our protein inhibitors are not very well suited to be taken as treatment, because they would be digested if they were formulated as a pill and swallowed. The other option for proteins is injection, but that is not considered appealing by patients at all, especially compared to the pills that are now available.
“So we have been focused on prevention of the sexual spread of HIV by using directly applied inhibitors to the various affected areas. Other researchers have found problems with gels and similar types of delivery systems for a variety of reasons. Probably the most important reason was that in clinical trials people simply didn’t use them as directed. The gels often required daily dosing (or dosing at a certain time in relation to sexual activity) and this did not work for many of the study participants.
Says Dr. LiWang: “This is why the NIH has become very insistent that any delivery system be ‘user acceptable’: You can have the best inhibitor out there, but if people won’t use it, you won’t save any lives. Some people didn’t like the inconvenience, and some complained about drippiness. This may sound silly to a reader of your article, but in reality it is very serious. On a day-to-day basis, people (especially young people) aren’t thinking about catching a deadly disease. So it may make sense in the moment for them to not use the preventative measure, and that may be the instance that changes the course of their life. We have many similar situations that we all understand. We know that about half of all pregnancies in the U.S. are unplanned, and the vast majority of those people ‘could’ prevent a pregnancy if they were careful every single day. But of course, that is not the way it happens, and the goal of prevention is to ‘meet people where they are’ to design the most effective method that will work in real life.”
Chael Needle wrote about an NNRTI candidate in the December 2014 issue.