What defines cured for HIV?

http://news.sciencemag.org/scienceinsider/2012/06/evidence-that-man-cured-of-hiv.html?ref=hp

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.

Follow me on Twitter @christinamfarr

A Scientists' Worst Nightmare

http://blogs.nature.com/news/2012/06/brains-thaw-at-harvard-repository.html

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.

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).

Rubber Ducky Cupcakes

There’s been a lot of cool science news recently, so I’ve fallen behind on posting new cupcake recipes. I thought I’d post instructions on how to make your cupcakes look like rubber ducks. (This is based on the design from the book Hello Cupcake, What’s New, which I love!) You get a huge sugar high from eating one of these, and an even bigger one if you snack on the starbursts and M&Ms that are the wrong colour, lick the frosting off your fingers, and eat the extra Timbits, marshmallows, or starburst molding failures J

Decorating Supplies:

Vanilla frosting (I use Betty Croker, do NOT use whipped)
Yellow food colouring
Mini M&M’s (brown, blue and green)
Marshmallows
Orange starbursts         
Plain Timbits
Vanilla cupcakes in yellow or white liners (start with more than you want because it takes some practice to get them looking the way you want)

 Prep Stuff:

Bake the cupcakes as described and let them cool completely.

Cut the marshmallows in half diagonally.

Cut the starbursts in half and microwave for a few seconds at a time until you can mold them into beaks (or feet). I found it helpful to make a pointy end that could be stuck into the Timbit.

Assembling the Duckies

1.      Once they have cooled, ice the cupcakes and place the marshmallow at one end and the Timbit at the other to make the head and tail of the duck. Use the frosting like glue to help hold them in place.

2.      Put them in the freezer for about 15 minutes.

3.      Microwave the frosting until it is runny and use the yellow food colouring to dye the frosting.

4.      Carefully dip the cupcake into the frosting and let excess frosting drip off.

5.      Carefully add the starburst beak and M&M eyes now OR wait until the frosting has hardened then add them at this point, whatever works out better for you. You may need to use a dab of frosting to hold the M&Ms in place. I stick the pointy end of the beak into the Timbit to help it stay in place

To make upside down duckies:

Place the marshmallow in the opposite orientation from the original ducky. Once it has been dipped, carefully place the starburst feet on either side of the marshmallow tail. You may need a small dab of icing to help them stick in place.

The placement of everything is really important, and it took several tries to get it right. The cupcakes look really good when they are done, but there were a lot of casualties in the process!

BLT Mice

As I’ve alluded to in my recent posts, HIV is kind of difficult to work with. Working with the virus in Petri dishes and cell culture flasks isn’t too bad . . . except for the fact that it’s a level 3 pathogen in Canada, which means I have to work with it in a special facility with restricted access and tons of safety features. Not all research institutions have this type of facility available, so I’m lucky to be able to do all the experiments at my research institute.

Once I complete all the studies I can do with cells, I will have to do my next group of studies using animals. This is done to test the safety in a living organism (cells can only tell me so much) and provides a more real world study system. With no idea on the safety of my compounds, I can’t do any work using humans, which is kind of a problem when studying HIV. As stated in its’ name, HIV is the HUMAN immunodeficiency virus. This means that HIV only infects humans. There are other types of immunodeficiency viruses (like simian immunodeficiency virus), but using SIV in primates, or a hybrid SIV/HIV in primates has not had good translation to work with HIV in humans, because there are a lot of differences between SIV and HIV. This has been a challenge many researchers have tried to solve, and recently success has been observed with humanized mouse models of HIV.

Before I go further I should warn you that these mice involve some very controversial stuff. First off, working with mice, which is always a touchy area. To humanize a mouse you need to perform a minor surgery on them and carefully monitor the mice post-surgery to make sure they don’t develop infections or are in pain. And, to humanize the mouse you use human fetal tissue, which can only be obtained from aborted fetuses that are donated to research. If you have any issues with any of these things, I would suggest you stop reading now.

Each humanized mouse has to be created individually. This is a very lengthy process. You begin with a mouse that, through a combination of genetic defects, does not have an immune system. There are several different types of immunodeficient mice you can use for humanization. I will be using the NOD/SCID/IL2rgamma-/- mouse. That is a fancy name for a mouse that has a severe immune deficiency and is unable to make any functional immune cells. You have to be very careful with these mice, because if they get any sort of infection their body can’t fight it so they will likely die. Their water and food is specially sterilized, their lungs are monitored for infection every other month, and sentinel mice are used to monitor for any other infections.

When the mice are 8 weeks old they have a surgery to implant human fetal liver and thymus under the kidney capsule. They also receive an IV injection of human fetal stem cells (which can be derived from the liver). The human stem cells will allow the development of all the human immune cells, and the liver/thymus allows the T cells of the immune system to mature properly. Because you use bone marrow (the stem cells), liver and thymus, these mice are called BLT mice. Twelve weeks after the surgery you take blood samples from the mice and check that all the immune cells are present. If so, the mice can be used for infection with HIV.

The BLT mice have been shown to be susceptible to vaginal infection with HIV, and the infection course is very similar to what is observed in humans. Whenever I’ve presented my research I’ve been asked how exactly you give a mouse HIV vaginally. The mouse is put under anesthesia, then a small instrument is inserted into the vagina and HIV in saline is deposited. The mouse is held in an inverted position so the saline doesn’t drip out. After a few minutes, the mouse can be woken up from anesthesia. Blood samples can be taken each week to determine HIV infection.

Today I am heading to a collaborators’ lab for 2 weeks to receive the surgical training I need to be able to make BLT mice. These mice will provide me with a lot of information about the “real world” uses of my compounds. I have to do everything perfectly for this mouse experiment, because it is really expensive. With the costs of my training trip, buying the mice, housing the mice, buying reagents and materials, and equipment rental costs, the 45-60 mouse cohort I can make from one set of donor organs will cost $20,000 or more.

Christina

PrEP for HIV Prevention

Another blog post on HIV . . . it’s sort of my thing :p (and the focus of my thesis!)

On May 10^th the Antiviral Drugs Advisory Committee of the US FDA debated whether an anti-HIV drug currently used as an antiretroviral treatment should be approved as a prevention method for uninfected people. It was recommended to the FDA that the drug, Truvada (made by Gilead Sciences), be approved for pre-exposure prophylaxis (PrEP).

PrEP means that the drug is taken routinely by uninfected people at high risk of becoming infected with HIV. It has been shown that taking the drug daily can lower risk of infection by more than 90% in two target groups – men who have sex with men; and uninfected heterosexual (men) with a long-term, HIV positive partner. However, clinical trials of the drug in heterosexual women have failed. (Also, in reading another article about this, it was claimed risk was lowered only by 42% and 73% in separate trials...)

PrEP (like birth control) would need to be taken daily to be effective. And, if adherence is low this could have disastrous consequences, particularly if a backup method of prevention (condoms) is not used. As I mentioned in my last HIV post, the virus replicates really quickly, and doesn’t make perfect copies of itself, leading to mutated forms of the virus. Some of these mutants could be resistant to Truvada. So, if you are not using PrEP as prescribed and have a partner that is HIV positive, it is likely that you will become infected with the virus. If you continue to take the PrEP irregularly, you are putting yourself at risk of being infected with drug-resistant HIV, which would likely make treatment more difficult. Of particular note is that HIV treatment with Truvada must be undertaken in combination with other antiretroviral drugs to prevent resistance.

During the clinical trials participants were monitored monthly for HIV infection, using the most sensitive test to detect HIV, so that PrEP could be stopped upon infection to prevent resistance. A few cases of infection were seen in the trial participants, and just over half of them developed resistance. This testing should be continued for those that are taking Truvada for PrEP, but this testing was not included in the recommendation to the FDA. Without regular testing, it is possible that HIV could be transmitted to someone taking PrEP (remember, it isn’t 100% effective) and they wouldn’t know about it, so they would keep taking the antiretroviral. This will lead to development of a resistant virus, which will create a lot of problems for further treatment once the virus is detected.

I did a PubMed search and wasn’t able to find any clinical trial papers about Truvada, which I would really like to be able to read before forming an opinion on this, particularly since Science and Nature report two different statistics for protection levels. I think this approval of Truvada could drastically lower HIV prevention rates, if used properly (and if affordable!), however, it could also be disastrous if used improperly (particularly without regular HIV screening). Condoms are more effective at preventing HIV transmission and I highly recommend them because they also protect against other STI’s and pregnancy. But, for those who are unable to use condoms for various reasons (particularly women in developing countries), Truvada represents a new hope for HIV prevention.

Christina

A cure for HIV?

According to the World Health Organization and UNAIDS, over 33 million people worldwide are infected with HIV, the virus that causes AIDS. Over 2 million people die AIDS-related deaths each year. HIV/AIDS has enormous social and economic impact, particularly in developing countries. Despite over 30 years of research into HIV, an effective method of prevention (besides condoms) and a cure have not been found. Currently, once someone become infected with HIV they can take a combination of anti-retroviral drugs on a strict regimen, which should suppress the virus for several years. Eventually, the patient develops AIDS which typically leads to death within 1 year. It is thought that antiretroviral therapy can increase survival time by 4-12 years, but the patient would still eventually develop AIDS.

It is really difficult to treat HIV for several reasons. The virus replicates very fast (in less than 2 days) and doesn’t always make a perfect copy when it replicates. This leads to mutations that can allow the virus to become resistant to the antiretrovirals, or to escape the immune system. All of these mutated virus also replicate quickly, so someone infected with HIV will have millions of copies of different versions of HIV. In addition, HIV infects (and kills) CD4+ T cells, which are a very important part of the immune system. So, while your immune system is trying to fight a constantly changing invader, the invader is also killing those cells that have a key role in fighting it. What is really cool is that some genetic combinations provide protection from infection or suppress HIV replication. One that is called Δ32 CCR5 deletion is present in a small percentage of Caucasians descended from Western Europeans, and also prevents infection with bubonic plague.

I have only found 1 case where someone was cured of HIV. An HIV-positive patient with acute myeloid leukemia was given a hematopoietic stem cell transplant (basically a bone marrow transplant). Since this patient had both AML and HIV there wouldn’t be very many (or maybe even any) treatment options available, so this situation provided the opportunity to try something new. The doctors decided to give them a hematopoietic stem cell transplant using cells from a donor that had the Δ32 CCR5 deletion. The transplanted cells would not be infected with HIV, and should be able to destroy any remaining HIV-infected cells in the patient. So far, HIV suppression has been observed in this patient, and they have stopped taking antiretrovirals!

Walker et al (http://jvi.asm.org/content/86/10/5719.abstract) were interested in creating a gene therapy vector that could provide protection from HIV infection. They created a gene therapy vector that would induce the Δ32 CCR5 deletion, in addition to expressing TRIM5α (shown to prevent HIV replication) and a TAR decoy (which should prevent HIV replication).

To determine if this gene therapy vector could be clinically relevant, it needs to be tested in an animal model, which is difficult for HIV. HIV is the HUMAN immunodeficiency virus, which means it can only infect humans. This has posed a huge problem for HIV researchers, but a pretty cool solution has been found – you can create mice that have a human immune system (there will be a blog about how to do this soon, because I’m going to learn how to make my own franken-mice). These mice can be infected with HIV and the infection is similar to what is observed in people.

Walker et al isolated CD34+ hematopoietic stem cells and infected them with the gene therapy vector, then injected the cells into mice. These stem cells developed into immune system cells, including the CD4+ T cells that HIV can infect. The CD4+ T cells appeared functionally normal and contained the gene therapy vector.

Once they verified everything was working as expected, they infected the mice with HIV and monitored the mice. They found that the mice that received the gene therapy vector had significantly enhanced survival of CD4+ T cells after HIV infection. However, the level of HIV virus in the blood was not changed. This suggests that the gene therapy vector was able to keep the CD4+ T cells alive, but didn’t stop HIV from replicating.

This study is a good start for using gene therapy as HIV prevention, but follow-up work on why the HIV levels in blood were unchanged should be undertaken. It is also important to realize that in this study they were sort of putting the cart before the horse – they gave the mice the gene therapy vector before HIV infection. To build on this study they should infect mice with HIV then give them a bone marrow transplant with hematopoietic stem cells that contain the gene therapy vector to see if the HIV infection is cured – it would be more clinically relevant that way. This sort of treatment would be a long way from the clinic, particularly since you’re dealing with a treatment option that could be highly controversial, since it involves stem cells and gene therapy. I am definitely going to keep my eyes on this field!

Christina