Wednesday, 20 June 2012

What defines cured for HIV?

This is a follow-up to my post on the man that was cured of HIV with a bone marrow transplant. A presentation made at a scientific meeting in early June provides data that indicates he still harbours some HIV, which brings into question what constitutes “cured” when you’re talking about HIV? This patient stopped taking antiretroviral drugs after he had the transplant, the virus never returned and his doctors pronounced him cured from HIV.

Steven Yuki (UCSF), who works in Joseph Wong’s lab made the presentation at the meeting. They found some signals of HIV in the man’s body, but are unsure if these are real or from contamination. Yuki used a technique called polymerase chain reaction (PCR) to look for any signs of HIV. PCR allows you to amplify small quantities of (specified sequences) nucleic acid to determine if what you are looking for is present. When they performed PCR on the man’s cells they detected bits of viral nucleic acid, but a collaborator in a different lab didn’t detect any. This is highly suggestive that contamination of Yuki’s sample occurred, but the possibility can’t be ruled out that the man still harbours some HIV. Even more puzzling, another collaborator found signs of the virus, but it was unable to make copies of itself, suggesting it is harmless or defective genetic pieces of HIV. What makes this even more complicated is that the bits of virus don’t match each other, or the virus that he was infected with before the transplant. This provides stronger evidence that contamination of the samples may have occurred. Alain Lafeuillade (General Hospital of Toulon, France) wasn’t involved with the new study, but has issued a press release and a blog post with his interpretation of the results. He questions whether the man was reinfected with HIV and is still infectious to others. This could be possible because the virus detected doesn’t match the original virus the man had.

This new information calls into question what defines being “cured” when you’re discussing HIV. If you can’t detect the virus using p24 assays (the most common detection method), does that mean you’re cured? What if the p24 assay is negative but HIV nucleic acid is present – are you considered infected? Despite the question about whether he has been cured or not, the man has been off antiretrovirals for 5 years and is healthy. I think he, and others with HIV, would consider that cured . . . no matter what scientists decide.

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Monday, 18 June 2012

A Scientists' Worst Nightmare

There are several things that can make up a scientists worst nightmare, ranging from lack of funding for your research, being “scooped” (someone else publishes the same thing you’ve been working on before you can, so your research is no longer novel), and a myriad of factors within your experiments. So far, the worst that has happened to me is an undergraduate student in the lab accidently contaminating the cancer cells I was working on – I didn’t know the contamination had happened and the cells don’t show any signs that there is a problem for several weeks, so I put the cells in some mice for a 12-20 week long experiment, then found out about the problem. There was no way to tell if the cells that went into the mice were contaminated, so I had to wait out the whole course of the experiment before finding out that it didn’t work. I then had to repeat the experiment, which took an additional 24 weeks (about 6 months!), and this was the last experiment I needed for my MSc thesis, so I finished 6 months later than I wanted, 1 week before my PhD program started across the country, and I had to give up the month-long trip to Europe I had been planning and saving up for over the previous year.

All of this pales in comparison to what happened recently at Harvard’s brain bank – 147 brains were lost when a freezer failed, and a third of those brains were donated from deceased people that had autism. This was one of only a few repositories in the US that distributes autism brain tissue to researchers around the world. A significant source of brain tissue is no longer available, which can hinder all sorts of future research into autism. A bunch of things just seemed to happen all at once to cause this loss to occur. Normally the brains are spread among a bunch of different freezers, but they had been consolidated into one freezer for a visit from the Autism Tissue Program. The freezer went down and the two sensors that monitor temperature and send out an alarm also went down. From the sounds of it, no one knew anything was wrong until the freezer (normal temperature -80C) was opened and it didn’t feel cold inside. The odds of all these things failing during the time so many brains were in one freezer seems highly unlikely, and an investigation is being undertaken.

Something like this is always a fear for a scientist, particularly when you have “precious” samples (especially from human donors, more so when the samples can only be obtained after death) because it can take a lot of time to replace what was lost. If your research relies on these samples, months of time could be lost before enough samples are obtained to resume experiments.

When I was working on my MSc thesis I was always worried about my computer crashing, or losing my USB key, or something, so I had my thesis and all my data backed-up in different locations, and I practically slept with my laptop so I could easily grab it and run if there was a fire. I don’t worry too much about incubators, fridges and freezers in the lab right now, mostly because I don’t have anything that can’t be replaced if something happened. My biggest worry in the lab is that someone will use my sterile water and that will contaminate my experiment, which isn’t a big deal now, but will be a huge issue if I’m working with mice. Soon I will constantly worry about the BLT mice – they are so costly and one small thing could completely destroy months of work.

Tuesday, 12 June 2012

HIV Prevention Using Microbicides

HIV prevention is a “hot” research area. A lot of time and money is being spent to try to prevent HIV spread. There are several avenues of research for HIV prevention, but 2 of the most popular are vaccines and microbicides. A vaccine (with low production and distribution cost) for HIV would be ideal, particularly if it offers long term protection with no need for a booster. However, it has been very difficult to develop an efficacious vaccine. HIV mutates rapidly, which means that immunity to one version of HIV might not protect you from a different version of the virus (sort of like getting the flu even though you’ve had the flu vaccine). Due to the lack of success with vaccines, several research groups (mine included) are pursuing alternative prevention strategies, such as microbicides.

Now, you may wonder why we are focussing on coming up with new ways to prevent HIV infection when condoms can efficiently prevent HIV infection. There are lots of reasons why people don’t use condoms. In the developing world, where HIV burden is the highest, many women are not able to insist on condom use, due to cultural or societal norms. These women represent more than 50% of the new HIV infections that occur each year. Since the standard prevention options are not feasible for them, an alternative needs to be designed. This is why we are focusing on microbicides – ideally, women could use them prior to intercourse and their partner doesn’t need to know.

Microbicides are drug products that are can be topically applied to the vaginal or rectal tract to prevent infection with HIV. Many microbicides have been under development for HIV, but none have been brought to market yet. Several are undergoing clinical trials, and several have already failed in clinical trials. Tenofovir is one microbicide that I have been hearing a lot about recently. Tenofovir is an anti-retroviral that has been formulated into a microbicide gel. Tenofovir was successful in its’ first clinical trial, showing protection rates of 39%. However, the second trial was recently stopped because no difference was observed between the drug group and the placebo.

An emerging area of microbicide development is the use of engineered bacteria. Several research groups have focussed on Lactobacillus which is a normal component of the vaginal microflora. They hope that by engineering Lactobacillus to express or secrete an anti-HIV protein they can, with one application, provide long-term protection from HIV infection. Ideally, there would be no need to reapply the Lactobacillus microbicide, because the bacteria would be able to live in the vaginal tract, and should (in theory) continually express the protein. However, what do you do if some sort of side effect occurs? How do you kill the engineered bacteria without destroying the natural vaginal microflora? How do you know the bacteria is still expressing the protein?

There are a lot of issues that can arise from the use of Lactobacillus as a microbicide. My research is investigating an alternative engineered bacteria based microbicide system, using a bacteria called Caulobacter crescentus. My lab has developed a system to express a wide variety of different anti-HIV proteins on the surface of C. crescentus. These proteins are expressed at very high levels (20% of total cell protein would contain the anti-HIV protein) and we have had no issue with maintaining long term expression. My research is finding new anti-HIV proteins to put on the C. crescentus, and testing two of the most important factors of a microbicide, safety and efficacy. I have to ensure that topical application of the C. crescentus to the vaginal tract will have no adverse effects. I also have to determine how high protection from HIV infection is. My first round of testing indicates that I can decrease HIV infection rates by 85%! This is all done in a test tube, and I will eventually have to move on to testing this in an animal system (see BLT mice blog post). All of this testing (and some side projects that come up along the way) will compose my PhD thesis.

So far, the only downfall of the C. crescentus system is that the microbicide would need to be applied regularly (I haven’t figured out how often yet). The bacteria can’t survive in humans, so protection from infection will be transient. C. crescentus is cheap to grow, so this shouldn’t be cost prohibitive, but the need for reapplication may be a deterrent for clinical use. Indeed, it is believed that adherence to the dosing regime is the reason why the second clinical trial of tenofovir showed no effect.

The development of a microbicide for HIV has the potential to revolutionize the field of HIV prevention. However, there are still a lot of problems in translating laboratory success to clinical success. In the meantime, prevention strategies should focus on testing for infection, condoms, and pre-exposure prophylaxis (when applicable).