Debunking myths on genetics and DNA

Friday, February 21, 2014

Converging genes reveal how plagues have shaped our genome

Evolution is shaped by numerous factors. Selection is one of such factors, but, contrary to popular belief, it is not the only force acting on genomes. I cringe when I hear the expression "this gene has been selected for" because most of our alleles (we all have the same genes, but each gene can have different alleles across different ethnic groups/populations) haven't been selected at all. Things change even without any selection pressure from the environment, a phenomenon known as random drift. every new generation is a (more or less) random sample from the previous generation, and this constant resampling ensures a background change in allele frequencies, even without any selection pressure from the environment.

Because selection is not the only factor that shapes evolution, it is hard to look at how our genome evolved and pin point what changes were due to selection and which ones weren't. However, there are some rare situations where scientists get lucky. One such example is the Rroma people, also known as Gipsies. This ethnic group originated from Northern India and migrated to Europe around 1,000-1,500 years ago. Because throughout the centuries they remained a homogeneous group and rarely mingled with the local population, when looking back at some of the historical plagues that swept through Europe, the Rroma offer a unique snapshot of a distinct population undergoing the same selection pressure as the locals.

Here's the logic: alleles found in the Rroma population but not in their Indian ancestors must have risen recently in the Rroma population. If those alleles are also found in the local population, which are not related to the Rroma, then these alleles must have risen independently in the two populations. But how, if the two populations did not intermerry? Well, if you think about it, the part of our body that's most certainly under selection pressure is the immune system: a strong immune system enables the survival of not just one individual, but also of his/her offspring if they inherit the right alleles. Historical plagues that swept through Europe exerted a strong selection pressure on the immune system at the population level. Individuals with favorable alleles were able to survive these plagues, whereas the others succumbed. So, when the researchers found alleles that had risen independently in the Rroma and in the local population, they concluded
that they had been selected by severe epidemics in Europe.

The study, published in PNAS last week [1], aimed at finding "convergent evolution" between the two coexisting but genetically distinct populations. Convergent evolution means that, under selection pressure (such as for example a widespread epidemic), distinct genomes are forced to converge independently to the same allele because that particular allele confers protection against the epidemic.
"We hypothesized that despite their different ethnic and genetic backgrounds, the strong infectious pressure exerted by the major epidemics of the last millennium (of which epidemics of plague are probably the most significant) has led to convergent evolution: specific immune genes, selected during these European epidemics, become signatures that differ from those found in the Northwest Indian populations from whom the Rroma have derived [1]."
Laayouni et al. [1] found several gene clusters under positive selection, of which one in particular (TLR1, TLR6, and TLR10) code for receptors that modulate responses to Yersinia pestis, the bacterium responsible for the bubonic plague.

Hafid Laayounia,1, Marije Oostingb,c,1, Pierre Luisia, Mihai Ioanab,d, Santos Alonsoe, Isis Ricaño-Poncef, Gosia Trynkaf,2, Alexandra Zhernakovaf, Theo S. Plantingab, Shih-Chin Chengb, Jos W. M. van der Meerb, Radu Poppg, Ajit Soodh, B. K. Thelmai, Cisca (2014). Convergent evolution in European and Rroma populations reveals pressure exerted by plague on Toll-like receptors PNAS DOI: 10.1073/pnas.1317723111

Sunday, February 16, 2014

This season's flu helps inform next season's vaccine

Last year I described the arm's race happening between virus and immune system during an HIV infection: as the immune system starts mounting its defense against the virus, the virus mutates trying to evade the attack. This is what pushes the virus to constantly evolve new strains, not just in HIV, but also in the flu virus, which evolves a new strain roughly every year. The HIV virus evolves within the same host to evade the host's immune response. On the other hand, the flu virus evolves more slowly: contrary to HIV, healthy individuals can clear the flu virus, and in doing so they acquire immunity against future infections from the same virus. This exerts pressure on the flu virus to evolve new strains capable of evading the population acquired immunity.

The process by which viruses constantly evolve new strains in order to evade immune responses is called antigenic drift.

The yearly evolution of the flu virus is closely monitored: surveillance data is collected throughout the flu season and, based on the data, a prediction is made on which strains will be most likely to reappear during the next season -- this step is important for flu vaccine design. The vaccine needs to be available prior to the start of the new flu season. Therefore, researchers have to make an educated guess on what the evolved flu virus will be like in order to make the appropriate vaccine.
"Due to the fast evolution of the influenza virus, the components of the influenza vaccine are changed for many flu seasons. Even though the vaccine is usually redesigned to match closely the newly evolved influenza virus strains, there occasionally has been a suboptimal match between vaccine and virus [1]."
The surveillance data comes from the World Health Organization Global Influenza Surveillance Network (GISN), a network of 136 national influenza centers scattered in 106 different countries. The data focuses on one influenza gene in particular, the hemagglutinin (HA) because the protein it codes seems to drive the antibody response.

The HA protein coats the outer surface of the influenza virus. It enables the virus to recognize and bind target cells. Once bound to the surface of the cell, the virus is engulfed in a sac called endosome. This is a mechanism by which cells engulf extraneous objects and then try to destroy (digest them through enzymes) while inside the endosome. However, the influenza virus uses the endosome to get inside the cell and once there the HA protein undergoes a conformational change (triggered by a drop of pH) and becomes a "hook" that breaks the endosome and frees the virus into the cytoplasm. Without the HA protein the flu virus is unable to bind to the target cell or break the endosome. Therefore, antibodies that bind to the HA protein successfully clear the virus, which is why it is vital for the virus to evolve mutations that enable it to escape those antibodies.

In order to anticipate the next flu strains, researchers need to understand how well the population is responding to the current strains. The flu vaccine usually carries three different strains, selected from the most predominant and geographically spread ones so that the resulting immunity is reactive to a wide range of flu strains. How "different" any two strains are is measured by a quantity called "antigenic distance," which, in layman terms, measures how well current immune responses ("animal antisera raised against the same or related strains, [3]") are able to block those strains. Viruses with a high antigenic distance will be poorly blocked by the current immunological responses and therefore are more likely to diverge from the current flu strains and spread into the following season.

In [3], Smith et al. reconstruct the antigenic map of the influenza A virus starting from 1968. The map retraces the genetic evolution of the virus, showing that strains tend to form clusters that last 2-3 years and then evolve into a new cluster (a new strain that requires a new vaccine). New surveillance techniques are being developed based on this concept of antigenic distance and antigenic maps in order to help inform future vaccine selection.

[1] Pan K, Subieta KC, & Deem MW (2011). A novel sequence-based antigenic distance measure for H1N1, with application to vaccine effectiveness and the selection of vaccine strains. Protein engineering, design & selection : PEDS, 24 (3), 291-9 PMID: 21123189

[2] Cai Z, Zhang T, & Wan XF (2012). Antigenic distance measurements for seasonal influenza vaccine selection. Vaccine, 30 (2), 448-53 PMID: 22063385

[3] Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, & Fouchier RA (2004). Mapping the antigenic and genetic evolution of influenza virus. Science (New York, N.Y.), 305 (5682), 371-6 PMID: 15218094

Thursday, February 13, 2014

Another interview... with me this time!

Self-portrait - by EEG

Last month I had the privilege of doing my very first cover art for the amazingly talented Jack L. Pyke, an erotic romance/BDSM author from the U.K. The premise of her thriller Lost in the Echo intrigued me -- I'm a sucker for suspense -- so I hired a model and set off to work. Creating an image based on a premise is something I started doing last year and I confess I enjoy it very much. It's completely different than planning a landscape or a portrait shoot. When you are given a theme you have to think ahead, jot down a bunch of ideas and be ready to try out many different things until you nail the one you love. I loved working for Jack, and after a few iterations we ended up with an image we both loved for her thriller.

And guess what I got as a thank you? My very first interview! I enjoy interviewing authors here on the blog, but I confess I was also very flattered when Jack asked me to be the interviewee for a change!

Here's an excerpt of the interview:
JLP: Besides providing the image for Lost in the Echo, I have a few of your prints here that show the scope of your talents. This shape shifting, surreal effect to the print below is one of my favourites. I know you’re a photographer from these, but can you tell us a little bit about you? Who’s the person behind the camera?

EEG: The story of my life in a nutshell: I was born in the UK, grew up in Tuscany (Italy), and lived in 4 different European countries and 4 different states in the U.S. before settling in beautiful New Mexico. As for “what” I am, I’m first and foremost a scientist -- that’s how my brain works. But I wouldn’t be who I am without my two creative outlets: writing and photography.

JLP: There’s a lot of scope for inspiration in all of the places you have lived, and it’s sparked a writing talent too. How long have you been a photographer? What influenced you to first pick up a camera?

EEG: I’ve been drawing and painting since I could hold a pencil and paintbrush. My first camera was a Sony point and shoot, which I used mainly to take pictures of things and people I wanted to paint. Painting is a demanding activity, though: you not only have to find the time to do it, you also need to have the right space. It’s a lot easier when you can afford a studio where you can keep up your work in progress for as long as you need. In my case, life took over: for about a decade we kept moving every other year, and the paintbrushes and paints ended up in a box, and, well, they stayed there. All those pictures I was still taking never turned into paintings. It finally dawned on me that I might as well perfect the one thing I was already doing: photography! I bought a used DSLR from a colleague, and once I discovered the freedom of being able to choose my own settings (aperture, ISO, exposure time, etc.) I felt like I had a whole new world to unravel. I started doing landscapes. I live in a place (New Mexico, USA) that offers staggering views and incredible skies, so it was the natural thing to do. I progressively moved on to macros, portraits, and now I’m doing the one thing I enjoy the most: photo composites, images that I create combining different pictures and backgrounds. It’s as if the cycle closed back, because compositing allows me to do what I used to do with my paintbrushes, except now my canvas is a JPEG file and I no longer need a studio. All I need is my laptop.

JLP: Along with your photography skills, you’re also an agented author. What kind of novels do you write?

EEG: Thrillers. I love action and I love to get my characters in a lot of trouble. I think it makes up for my long days spent in a cubicle at work.

JLP: Can you tell us about the novel you’re working on?

EEG: I just finished writing the first book in a new series set in the future. It features a murderous and sexy computer hacker, the biothreat federal agent who’s after her, an eccentric medical examiner, and a deadly pathogen. The world building was daunting at first. Even when I’m writing fiction, I research everything (location, people, history, etc.), but here I actually had to make up a whole society on my own. I solved the conundrum by doing both: I researched all the current state-of-the-art technology and then tried to imagine what it would look/be like one hundred years from now. I actually ended up having lots of fun with it. My agent just started shopping the book around, I’ve got fingers and toes tightly crossed!

JLP: I can hear the scientist and a love for facts in there. Do you think you’re more comfortable with writing from a male or female point of view, or doesn’t the gender of the MC hold any barriers for you?

EEG: The real “barrier” (though it doesn’t stop me from writing, so more than a barrier I would call it a challenge) is the voice more than the POV. No matter whether it’s male or female, young or old, I strive to give my characters their own voice. Success comes when you can tell the gender from the voice without knowing the character’s name. My first book series is written in first person, and the main character is a male LAPD detective. One of the agents who offered representation made me the best compliment ever: she said she had no idea I was a woman until she read my full name. At the time, I’d read all the Philip Marlowe books and loved the voice. Like Marlowe, my detective is also from Los Angeles, so I wanted to continue in the noir tradition with the first person, witty and sarcastic narrative. I guess the ultimate judges will be the readers.

JLP: Knowing you’re both photographer and author raises a curious conundrum. Do you find there’s anything you can express more in photography than you can in writing, or vice versa?

EEG: That’s an interesting question. There are instances when I can picture the setting of a scene very vividly in my head but I struggle to find the right words to describe it. I find myself thinking, “If only I could take a picture...” But for the most part it’s the other way around. Photography has made me very aware of the light around me, and it does affect the way I write. I’m often describing light sources in my scenes and how the light falls on my characters and how it affects their vision.

JLP: The idea for Lost in the Echo came from a photo supplied by a reader through a reader challenge on Goodreads. It’s a very intimate and annual author-reader challenge on Goodreads. Have you ever used any of your own prints to inspire your writing?

EEG: Not my own pictures, but I do browse images to inspire my writing all the time. For example, when I was researching the world building for my last book, I browsed a lot of futuristic buildings and architecture and I had a lot of fun doing that. Whenever my brain draws a blank on a particular setting for a scene I go to Google images, type a few keywords, and then inspiration suddenly comes to my screen in the form of beautiful images.
You can see the sparkly new cover I made for Jack and read the full interview on Jack's blog.

Jack L. Pyke is the author of the BDSM thriller Don't, a rainbow award honorable mention, nominated for the 2013 Goodreads best debut novel and best BDSM. The sequel, Antidote, is to be released in April. You can find Jack on Amazon and on Goodreads.

Sunday, February 9, 2014

Gene therapy for the heart

My post today is about state-of-the-art gene therapy that delivers genes straight to the heart, where the genes activate proteins critical in restoring cardiac tissue in people affected by heart failure. The technique, developed at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, is undergoing clinical trial.

Cardiovascular disease is the leading cause of death worldwide. Heart failure--a condition by which the heart weakens and no longer pumps blood efficiently throughout the body--is one of the manifestations of cardiovascular disease. According to the CDC, heart failure affects about 5.1 million people in the US, and about half of the people who develop heart failure die within 5 years of diagnosis.

A lot is going on at the cellular level when muscles contract and release. Calcium ions work like a "switch" that allows the contraction to start. Therefore, it is of vital importance, for the correct functioning of the muscle, that the calcium ions are released at the right time and then reabsorbed at the end of the contraction. When this flow of calcium ions is impaired heart failure can occur.

Calcium is normally stored in an organelle of the cell called sarcoplasmic reticulum. Muscle contraction is carried on thanks to the interaction of two proteins, actin and myosin. At rest, these two proteins are separated by a molecule called troponin. When the neurons send a stimulus to the muscle to contract, calcium is released from the sarcoplasmic reticulum into the cytoplasm where it binds to the troponin molecule, shifting the conformation of the complex, and making actin and myosin interact and initiate the contraction. Upon termination, calcium pumps regulate the uptake of calcium back into the sarcoplasmic reticulum. Troponin gets back between actin and myosin and the contraction stops.

Therefore, muscle cells need to (1) store large amounts of calcium ions, and (2) make sure the calcium ions are free to flow during release and uptake. The release, uptake and intake of calcium ions in the cells of cardiac muscle is regulated by two proteins, SUMO-1 and SERCA2a. Reduced levels of SUMO-1 cause SERCA2a levels to drop too, and low levels of both proteins have been associated to heart failure. The genes that encode these two proteins are down-regulated in patients suffering from heart failure, causing calcium ions to "linger" in the cells instead of flowing in and out as required for proper muscular contractions.

Researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai have been studying this process in animal models and demonstrated that heart function can be substantially restored through a single dose of SUMO-1 and/or SERCA2a gene transfer [1]. Following these promising results in animals, a clinical trial started and, according to a press release from last November, the single dose gene therapy is already showing very promising results:
"The new long-term follow-up results from their initial Calcium Up-Regulation by Percutaneous Administration of Gene Therapy In Cardiac Disease (CUPID 1) clinical trial found a one-time, high-dose injection of the AAV1/SERCA2a gene therapy results in the presence of the delivered SERCA2a gene up to 31 months in the cardiac tissue of heart failure patients. In addition, study results show clinical event rates in gene therapy patients are significantly lower three years later compared to those patients receiving placebo. Also, patients experienced no negative side effects following gene therapy delivery at three-year follow-up."
The one dose gene therapy is delivered directly to the heart through a catheter, and the SERCA2a genes are inserted inside a modified adeno-associated virus (AAV). I've discussed viral vectors for gene therapy in the past (see this post and this one). What I didn't know at the time is that there's a new family of viral vectors fine tuned for cardiac gene therapy: they are called cardiotropic vectors [2].

AAV has been historically used in gene therapy because it is found in 80% of the human population and it is often asymptomatic, meaning that it is well tolerated in the population (basically, it is harmless). This makes it a safe means to deliver genes. However, it preferentially transfers genetic material to the liver, not the heart. Among the various things that can make gene therapy go wrong is of course, delivering the genes to the wrong target. In [2] authors Yang and Xiao discuss how by introducing specific mutations to the AAV genome they were able to construct an AAV mutant specific to the cardiac muscle tissue. These techniques make use of bioinformatic methods to reshuffle the AAV genes and introduce mutations according to prediction models to generate new variants that are then tested in mice models for organ specificity. This is quite exciting as we can foresee a future where we will have a vector for every possible tissue we need to target with gene therapy.

[1] Tilemann L, Lee A, Ishikawa K, Aguero J, Rapti K, Santos-Gallego C, Kohlbrenner E, Fish KM, Kho C, & Hajjar RJ (2013). SUMO-1 gene transfer improves cardiac function in a large-animal model of heart failure. Science translational medicine, 5 (211) PMID: 24225946

[2] Yang L, & Xiao X (2013). Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution. Virology journal, 10 PMID: 23394344

Friday, February 7, 2014

Book deals, crime fiction, and French castles: a chat with Mark Pryor, author of the Hugo Marston mysteries

My guest today is not new to the blog: district attorney and author of the Hugo Marston mysteries, Mark Pryor has visited CHIMERAS before to discuss fingerprint evidence and DNA evidence in court.

Last time we talked, Mark had just signed a three-book deal with Seventh Street Books for his mystery series. That was two and a half years later, so I thought I'd check back with Mark to see how things are going with him and his books. Well, guess what? Mark has happily signed his second three-book deal with his publisher for three more Hugo Marston novels. has said of Mark's books: "Once you've had a bit, you can't wait for more"; and the Portland Book Review defined the series "A leaf out of a classic Agatha Christie novel, mixed with the modern world of crime."

Mark has also published a true crime book, As she lay sleeping, about a cold case he prosecuted. Mark appeared in the CBS show 48 Hours to discuss the case, how cool is that? Yes, I do love to brag about my friends. :-)

EEG: Congratulations, Mark: so many achievements in such a short time. How does it feel? Are you still floating twelve inches above ground, like last time we talked?

MP: It feels great, quite honestly, I still pinch myself. I have people asking me to give talks or meet with their books clubs, and when I agree they act like I'm doing them a favor. Little do they know! When people ask me what I do for a living, I feel good about saying, "I'm an author." Yet it still sounds weird to me...!

I don't know if it's changed me greatly, though. I feel more confident in my abilities as a writer, I suppose. But the thing about being a writer is that you're only as good as your last book, and the next one takes you to the precipice all over again. You peek over and wonder whether you've gone too high, and whether anyone will care if you fall. Scary.

The truth is (and don't tell anyone!) I've come further than I thought I would. All I wanted was a publisher for THE BOOKSELLER, to get that story out there. And now... an on-going series. So I'm still feeling fortunate and, hopefully, it's not changed me. I'll add, by the way, that as a result of the books being published I've met a lot of big-name authors and without exception, they all seem very down to earth, very generous with their time and advice. So maybe that says something about authors more generally, I don't know.

EEG: One thing I really loved from The Bookseller are the descriptions of Paris. What are your thoughts on writing about a place you don't live in? I know you travel to Paris often, but I'm just curious if when you go, you take notes/pictures and stuff like that. Did you find it challenging at first and then became easier as you went, or was it the opposite?

MP: I should mention I've been to Paris a dozen times by now. Thereabouts, anyway. I think there's a difference in the way I look at a place nowadays. Initially, I'd wander aimlessly and gaze at the monuments and famous icons of Paris. Now, I wander aimlessly and make a point of looking for the small things. For me, it's the little things that let you create atmosphere. For example, a year ago my wife and I were in Paris and we stumbled across this wonderful little street, almost a movie-set street with its restaurants and cafes, its cheese and flower shops. And, while we were enjoying it, a pretty girl on a bicycle came riding towards us, a smile on her face and pulling a wheeled, carry-on suitcase behind her. It was awesome, and so I plucked that out of reality and put it in the new book.

And that's my technique, if you can call it that. It's taking the small things and using them as color, rather than taking pains to describe the Eiffel Tower or other landmarks. Sure, they come into the stories because they're there, but the real flavor of a place is in the small things.

I'm curious if I can do a decent job with Barcelona, where book five will be set. I took the family there for ten days, and did the same thing: wandered around looking. I did have a notebook, yes, I have a different one for each novel. A couple of times we'd be strolling along and then I'd hear "Daddy!" from a hundred yards ahead of me. I'd stopped to write down a thought or idea, sparked by something I'd seen there.

I think this may be another aspect of why I'm lucky: being a writer, I'm forced to look hard at new places, or familiar ones, to try and see them in a new way, so that I can describe them with a modicum of originality, but also vividly.

EEG: That's so interesting. BTW, should you ever need a photographer to visually document these locations, I, er, happen to know one who would happily volunteer... Ahem. Anyways... when you signed your 3-book deal with Seventh Street you still had to write the third book in the series. Does it feel different to write a book on a deadline knowing that it's already slated to come out to the public on a certain day?

MP: Yes, it was very different. I tried to approach it the same way I had the first two, by just making up the story as I went along, but I plotted myself into a nasty corner. That's fine, no worries about starting over again except... that deadline! So THE BLOOD PROMISE, which was the third novel, became the first one I ever out-lined. I devised a system for plotting, actually, that I plan to use with future books so maybe all that stressing did me a favor.

Interestingly, I signed a second three-book deal with my publisher and the first of those books was already written. I know, it's weird, but it'll be a prequel and I wrote it about five years ago. It's set in London, but neither I nor my editor wanted to take Hugo back in time for the first three books. That was another one I plotted as I went along, so it'll be interesting to see if anyone notices a difference.

EEG: Can't wait to read it, Mark! What's next in your writing career? Are you working on more Hugo Marston novels or on something completely different?

MP: Yes, more Hugo. I have two more to write, which will bring the series to six. The London book (THE BUTTON MAN) will come out in September, and the next ones next year I expect. One will be set in Barcelona, and I haven't decided on the sixth. If they continue to attract readers, maybe we'll look at doing more, I certainly hope so.

I'm also working on non-Hugo stuff. Just this week I finished a novel set here in Austin, and I have another rattling around in my head that's asking to be let out. Both are crime fiction, I think I'm settled in the genre for good.

EEG: Who are your role models when it comes to writing?

MP: Oooh, that's tough. I don't know that I have any, to be honest. I mean, there are tons of writers whose books I admire but role model makes me think of the way someone lives their life. And I'm perfect, didn't you know that? No?

EEG: You told me "I'm not where I want to be just yet" -- where would you like to be as a writer?

MP: I'd like to be in a French chateau in the Loire Valley, behind the antique desk in my large study overlooking a manicured lawn and a winding river... seriously, that's where I'd like to be. :) For me, I suppose the ultimate goal would be to be able to write full-time and I can't do that now, not without my family making some significant sacrifices (that chateau in the Loire? Right now it'd be a shed on the outskirts of Marseille).

That said, I'm aware how far I've come and how lucky I am things have come together so quickly. It really goes back to your first question (nicely done!) in that my reality has changed, so have my ambitions, but I'm still feeling giddy about it all.

EEG: Haha, I'll settle for that shed outside of Marseille. Maybe in on of those Mediterranean pine groves right by the coast, how's that?

Thanks so much, Mark, for answering my questions and telling us about your writing process and your adventures in the world of publishing. Your success is very much deserved and I do wish you all the best with it. And if you do secure that castle on the Loire, please do come let us know. ;-)

You can find out more about Mark and his mystery books by visiting his blog, DA Confidential. Oh, and check out Mark's last book trailer, it's adorable !!

Sunday, February 2, 2014

Computer generated viruses

By "computer generated viruses" I don't mean bits of code that can harm your desktop. I mean actual viruses, objects that have the ability to infect and replicate, but were created in silico, by a computer algorithm. I know this is a concept that has the anti-vaxxers enraged, but in HIV it has become quite common to generate vaccine candidates through computer algorithms. Today I want to address two questions: why and how.

Candidate vaccines are made from virus isolates: you take a real virus, make it weaker, and inject it into the body so that it will elicit an immune response. Why hasn't this worked for HIV? One of the issues with HIV is that it is a highly variable virus. Think about the influenza virus: every year there's a new flu vaccine because the virus mutates into a new strain every year. HIV can reach that kind of diversity in one individual alone. So, you can't just take one strain of HIV and make a vaccine because it would only protect from one particular strain against millions of others.

These strains have evolved from one single common ancestor, one "patriarch" that jumped from monkeys to humans last century (see this post and the second part for a discussion of the papers that estimated when the HIV pandemic started). Since then, HIV has changed drastically and diversified in 4 major groups. Most HIV-infected people are infected with strains from group M, and within that group alone there are 9 distinct subtypes, plus "recombinants," strains that resulted from a "cross-over" of two or more subtypes.

The way we study the "history" of HIV is through phylogenetics. Imagine a room full of people, and imagine making groups based on similarity. Related people (brothers, sisters, parents) are going to form the closest subgroups. Zoom out one step and you are going to form larger groups based on physical characteristics: brunette dark-skin, brunette fair skinned, blonde fair-skinned, blonde dark skin. Next, you'll probably have ethnic groups. At the end of the process, you end up with a graphical depiction of the group of people: each person is a leaf, and the leaves closest together are on a branch (family) which comes from a larger branch, which in turn comes from a larger branch, until you get to the main big branches that are the ethnical groups and the trunk of the tree is the common mother we know lived in Africa many, many years ago.

We do the same with HIV. Each virus is a leaf. When we group the leaves into branches we see that the big tree that retraces the history of the main HIV group, group M, has 9 main branches (subtypes that are called "clades"). Even if you pick two viruses from the same clade, their envelopes (the proteins that form the outer shell of the virus) can differ up to 20% in amino acids, making it again impossible to use a single strain for a vaccine.

And yet all these strains are related. They all evolved from the same ancestor. So, wouldn't it be a good idea to try and use that ancestor as a vaccine candidate? The problem is that the ancestor is no longer found in present infections. In fact, we have no documentation of it because by the time we had the technology to genotype the virus, the population had already diversified. However, we can estimate the genome of the ancestor using the phylogenetic methods I described above. Every node in the tree represents a change in the genome. By walking "backwards in time" along the nodes of the tree, we can retrace the mutations that evolved from the ancestor. Distinct HIV subtypes can differ at as many as 35% sites. However, because of the way consensus viruses are constructed, they are on average closer to any given subtype and therefore they have the potential to elicit immune responses to more diverse viruses than just a one-clade vaccine.

A consensus virus is constructed using a computer algorithm that first creates the phylogenetic tree I described above, then estimates the genome of the root of the tree. Once the genome is estimated through the computer algorithm, viral proteins with that exact genome can be built in the lab. There are some issues associated with using an in silico virus in a vaccine. First of all, you need to prove that the viral proteins constructed in this manner are viable, meaning they retain their original functions. As it turns out, these "artificial" constructs replicate and infect like regular viruses.

One of such consensus viruses is called CON-S, and monkey studies have already shown very promising results when using it as an HIV candidate vaccine. In [2], some rhesus monkeys were vaccinated with CON-S and some with a single strain, B-clade vaccine. To assess how many and what kind of HIV strains the vaccinated monkeys were able to recognize, the researchers measured cellular responses against bits of HIV proteins taken from four major clades: A, B, C, and G. They found that the CON-S vaccine was able to elicit statistically significantly better (and more) response to clades A, C, and G, than the B-clade vaccine:
"We show that vaccine immunogens expressing the single centralized gene CON-S generated cellular immune responses with significantly increased breadth compared with immunogens expressing a wild-type virus gene. In fact, CON-S immunogens elicited cellular immune responses to 3- to 4-fold more discrete epitopes of the envelope proteins from clades A, C, and G than did clade B immunogens. These findings suggest that immunization with centralized genes is a promising vaccine strategy for developing a global vaccine for HIV-1 as well as vaccines for other genetically diverse viruses [2]".
This indicates that CON-S, being genetically closer to all clades is potentially able to protect better from viruses across clades, whether using a single clade strain would miss protecting from strains from other clades.

The other type of in silico viruses tested in HIV vaccine design are mosaic vaccines, which I will discuss next week.

[1] Gaschen B, Taylor J, Yusim K, Foley B, Gao F, Lang D, Novitsky V, Haynes B, Hahn BH, Bhattacharya T, & Korber B (2002). Diversity considerations in HIV-1 vaccine selection. Science (New York, N.Y.), 296 (5577), 2354-60 PMID: 12089434

[2] Santra S, Korber BT, Muldoon M, Barouch DH, Nabel GJ, Gao F, Hahn BH, Haynes BF, & Letvin NL (2008). A centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys. Proceedings of the National Academy of Sciences of the United States of America, 105 (30), 10489-94 PMID: 18650391