The Bioinformatics CRO Podcast

Transcript of Episode 27: Micah Luftig

Disclaimer: Transcripts may contain errors.

Grace Ratley: [00:00:00] Welcome to The Bioinformatics CRO Podcast. My name is Grace Ratley and I am the editor of the podcast and today I’m joined by Micah Luftig. Micah is an Associate Professor and vice chair of Molecular Genetics and Microbiology at Duke University. Welcome, Micah.

Micah Luftig: [00:00:16] Thanks for having me.

Grace Ratley: [00:00:17] No problem. So let’s start off with a little bit of the work that you’re doing now. So you work with Epstein-Barr virus, which is one of the nine herpes viruses to infect humans. So what is your research focused on?

Micah Luftig: [00:00:30] Sure. We’ve been studying EBV here at Duke for the last 14 years, and I actually did my PhD studying EBV so I’ve got a long history with this virus. For the most part, what we study is understanding how the virus takes over cells. We’re really a cell biology and cancer biology lab using the virus as a tool to guide our understanding of cell biology. EBV is a common pathogen. Actually, it’s virtually all adults worldwide have been infected and are latently infected with the virus, like 95% of us and in most people doesn’t cause much disease. But that’s because you’re infected when you’re really young and it doesn’t cause much disease. You make a really strong immune response to the virus. It goes latent. And then there’s a balance for your lifetime of a little bit of reactivation of the virus. And then the immune system deals with it and then the virus stays latent in your blood cells if you’re infected when you’re older. So in the second decade of life that actually most primary infections can lead to infectious mononucleosis. So that’s what causes mono. And a lot of folks don’t know that and don’t think about that too much. But EBV, not really through the infection process, but more the immune response is a little more exuberant in adolescence and post adolescent individuals. And so that causes the fatigue and other symptoms of mono usually resolves over weeks, sometimes months.

Micah Luftig: [00:02:00] But any event, then you wind up like the rest of us. And, well, I didn’t have mono, but many people did. Usually when I ask in a classroom how many folks have either had mono or know somebody who has, everybody raises their hand. In any event, when you’re past that phase, the virus latently infecting your B cells just like an asymptomatic carrier. But where the virus really becomes a problem is in the immune suppressed. So either transplant patients or HIV infected individuals, or even as we age in our immune system weakens, EBV actually can cause lymphomas in those patients at really high levels. So you have about 100 fold increase risk of an EBV lymphoma in the setting of HIV co-infection, for example. So the path that EBV uses to cause cancer in those settings is something that you can actually recreate in the lab. And so when you infect primary human B cells, the lymphocytes that EBV finds itself in, in vivo in the lab, EBV will immortalize the B cells and they become these lymphoblastoid cell lines or LCLs that mimic the initiation process of lymphomagenesis in these immune suppressed patients because you don’t have an immune system in the tissue culture dish in the lab to prevent those cells from growing out so many labs in the field. We have as well used this model to study the virus host interaction. And so it turns out to be super interesting.

[00:03:29] The virus expresses 8 or 9, depending on how you count them, latency, proteins and also actually about 44 microRNAs and some other non-coding RNAs. And together all of these gene products really take over the cell. So they infect a resting B cell, which is out of the blood otherwise would just sit there and then over a couple of days would die. The virus infects these cells and then within about 2 or 3 days, once you start making all of those viral proteins, the virus turns on the cell cycle. The cells get going and proliferating, and they’re constitutively activated through a number of pathways that the viral proteins mimic in terms of what those cells would normally see in the body. And they just keep going indefinitely. And so we and others study how that process works. So what the viral proteins are, what they do, how they interface with cellular proteins, how they regulate transcription, how they regulate cell survival, proliferation, metabolism, the immune system, you name it. That’s the bulk of what we do. And there are obviously more details beyond that. But in recent years expanded to studying other aspects of EBV biology. So the virus also infects epithelial cells. So what I didn’t mention earlier is that the virus spreads by saliva. So that’s why mono is often called kissing disease. The virus is always spread by saliva, whether it’s your mom or dad kissing you when you’re a kid or however saliva is exchange when you’re younger.

[00:05:00] That’s how the virus moves. And so what we know is that actually epithelial cells in the oral mucosa are a site of high level lytic replication, like the amplification of the virus. And that’s the reservoir for transmission. This biphasic life cycle between B cells and epithelial cells is really interesting. And the virus that comes out of a B cell is better at infecting an epithelial cell and the virus comes out of an epithelial cell is better than infecting a B cell because of the glycoproteins that put on the surface of the virus. And moreover, the ability to infect an epithelial cells increases about a thousand fold 100 to 1000 fold when a B cell is physically touching an epithelial cell. So it’s thought that these latently infected B cells get reactivated when they’re interfacing with an epithelial cell and transfer virus directly that way, rather than just virus that’s floating around in the saliva or something infecting new epithelial cells. So that’s super cool biology, which we study a little bit of. But it turns out the consequences of epithelial infection, while is normally just virus replication different from B cells where it goes latent typically as a default mechanism. When that goes awry for one reason or another, the virus can’t replicate and it has to go latent.

[00:06:20] So you can envision cells that have an amplified antiviral sensing mechanism or are not fully differentiated and don’t provide the proper milieu of cellular factors to allow virus replication. So something keeps the virus from replicating or maybe there’s a deletion in the viral genome. That could lead to a latent infection of epithelial cells and turns out again, the virus is really good at persisting and keeping cells alive. And so that can cause epithelial cancers. So EBV is associated with Nasopharyngeal carcinoma and also about 10% of gastric cancer, stomach cancer. So we have a couple of projects in the lab looking at how EBV causes stomach cancer and de novo infection models and corroborating that with clinical specimens from our collaborators in Singapore at Duke-NUS, where they’re studying changes in tumor cells that are infected or not with EBV in vivo. And we’re doing that in vitro and trying to understand mechanistically how that works. Part of that collaboration and thinking a little bit more translationally has opened up other translational projects in the lab also. So we have a couple of collaborations now where we study new antivirals that are being developed for EBV with small biotech companies that are starting up and interested in understanding more mechanistically about how they work and also what clinical correlates they might use to predict outcomes in patients that have these EBV lymphomas or carcinomas. So that’s what we do.

Grace Ratley: [00:07:54] Yeah, that is some really fascinating biology. So when are people who have Epstein-Barr virus infectious? Can you be infectious when you’re latent?

Micah Luftig: [00:08:03] Yeah, you’re basically always shedding virus. So yeah, there have been a couple of studies looking at this in people in this guy’s lab where they sampled saliva and everybody sheds a different amount of virus and it’s stable over time. It’s kind of fascinating, but it oscillates. It’s like the set point is stable. Like you’re kind of a high shedder or your medium shed or what have you. But like at different times during the days, over the course of weeks, you might be shedding more or less. So it’s really fascinating and it makes sense given how successful EBV is in transmitting across the world. It’s interesting when you think about things from that perspective where if a goal, let’s say, clinically, was to prevent infectious mononucleosis, what would be the best way to get there? Would it be a vaccine against EBV that prevents infection or that prevents symptoms somehow or would it be just getting infected naturally earlier? And how would you do that? It makes you think of chickenpox parties that happened in the maybe 70s and 80s. And to make sure all the kids get infected, which is another herpes virus. And maybe not the smartest approach, but gets the job done. I mean, it’s nuanced. In any event, there are vaccines now obviously for that virus. There are vaccines in development for EBV as well.

Grace Ratley: [00:09:31] Yeah. And how does it EBV differ from most of these other herpes viruses?

Micah Luftig: [00:09:36] Sort of what I alluded to earlier about the tropism, the cells that it infects. So the dogma in the herpes virus field is based on the genetics of these viruses. So you mentioned nine, which I appreciate because there’s 1, 2, 3, 4, 5, 6A and 6B, which the aficionado’s say are two different viruses, 7 and 8. So they branch out phylogenetically. Their genetics tell us that there are alpha, beta and gamma. So these three distinct groups of viruses and the dogma in the field, which is I think true, is that the alpha herpes viruses tend to have a broader tropism so they can infect more cell types and they can infect more different organisms. So a virus like herpes simplex virus that causes cold sores or genital herpes, herpes simplex 1 and 2 can infect lots of different cell types, certainly in the lab and I think also in vivo, it’s found in a number of cell types and also again in the lab you can infect mice or rabbits or monkey cells or what have you. And then the thought is that as you go towards beta and gamma herpes viruses, so beta an example is cytomegalovirus, Gammas are EBV and another oncogenic herpes virus KSHV associated with Kaposi sarcoma. The gamma is tend to be more narrow in terms of their tropism, so they infect less cell types. So for example, EBV primarily B cells and epithelial cells, although there are always exceptions to the rule and also much more narrow in terms of host range. So EBV cannot infect mice or rabbits or other organisms, so humans and then some nonhuman primates can be infected by EBV. Those would be what make EBV distinct from these other viruses.

Grace Ratley: [00:11:21] Yeah, it sounds like that might make EBV pretty difficult to study. Are there other model organisms?

Micah Luftig: [00:11:27] Yeah. So the way to address that is twofold. So one is what we do basically is use human cells. So we infect primarily human B cells for all of our work. Recently, in the last decade or so, humanized mice have been developed where in an immune deficient background you can reconstitute a human immune system by providing hematopoietic stem cells that then differentiate into lymphocytes and other cell types in the blood that allow you to do infections with EBV. It turns out those models often favor B cells. You have more B cells in the blood than you would normally in a human. If anything, it helps things out. They’re really great for studying early infection and B cell expansion and replication of the virus and tumorigenesis. There is some immune response. There is a bona fide immune response to the virus. It’s not quite as broad and complex as the immune response that humans have, but it is restrictive. And so if you eliminate T cells or natural killer cells in that humanized mice model, EBV can cause tumors 100% of the time on infection. So it’s a pretty good model for studying viral genetics and signaling pathways and the kinds of things that we have studied in vitro.

Micah Luftig: [00:12:47] There are other ways of approaching this. Very expensive way is to use the rhesus lymphocryptovirus, which is very similar to EBV in lots of ways. A couple of labs have developed that model and shown that you can recapitulate a lot of human EBV biology in the rhesus macaque. But again, they’re super expensive to work with. And then the other way that folks go and I think is good to understand basic viral host interactions, but may not tell us a lot about EBV, but is to use mouse viruses like MHV68, is a virus that phylogenetically looks a little bit more like KSHV, but it’s definitely a gamma herpes virus and can recapitulate various aspects of B cell latency and reactivation and has some of the same signaling proteins that EBV and KSHV has. But I think has been a really great model for understanding gammaherpesvirus pathogenesis. But it may not be as specific to the unique biology of EBV as, for example, the rhesus lymphocryptovirus as an animal model.

Grace Ratley: [00:13:55] Now I’d really like to get back to talking a little bit about how viruses can cause cancer in people. I was wondering if you could give us a sense of how common that is or maybe give some other examples of viruses that can cause cancers in humans.

Micah Luftig: [00:14:12] At present, the viruses that we understand are associated with or cause cancer, it’s about 20% of all human cancers. And I say at present, because one of the major components of that is human papillomavirus, which I think most people know causes cervical cancer and also causes a range of other cancers, including anal cancer, penile cancer and some head and neck cancers. And there are also papillomavirus is associated with certain skin cancers, but human papillomavirus. Now there’s a vaccine against to prevent cervical cancer. And it’s amazing. It was an amazing breakthrough by Doug Lowy and John Schiller at the NIH for developing those papillomavirus like particles to immunize and acquire what became sterilizing immunity to prevent infection with these viruses. And so it prevents those cancers. I think most teenagers get this vaccine. The problem is it has been vaccine uptake. And so the reason I say at present is that it’s hopefully we’ll reach a point where the vaccine uptake is much higher. And cervical cancer, for example, is a thing of the past, or at least viral associated, which is the vast majority of it. And these other cancers that I mentioned. But there are about 8 or 9 depending on how you call them, human rather oncogenic viruses. We classify those as direct and indirect. So the direct cancer causing viruses would be papillomavirus and EBV, KSHV, where there are viral oncoproteins that are expressed in the tumors that are driving the process, and that if you were to eradicate the virus, you would treat the cancer or prevent the cancer. And then there are others which are more what we would call indirect oncogenic viruses, and that includes viruses like HIV and hepatitis C virus and hepatitis B virus, although there are certainly direct oncoproteins there.

[00:16:09] But HCV is an example where the constant infection and the inflammation in the liver that’s induced by HCV is thought to drive the oncogenic process. But it’s not necessarily that the virus is in every tumor cell driving the liver cancer. Those are some examples of oncogenic viruses in humans and how we think about dealing with them. And so vaccines is obviously a great approach if one can develop those. There are also now T cell based therapies. So the idea would be that and this has been demonstrated in EBV for many years in the setting of transplant, that if you develop specific and potent T cells against the viral antigens and it’s been done as an autologous process, which means you take the patient’s T cells out and you expand them against the viral antigens and then you re-infuse them. That has been shown to cure EBV lymphomas in the setting of stem cell transplant associated lymphoma. But really it’s not boutique, but only certain centers have the ability to do that. And so now with the advent of cellular therapies for cancer much more broadly, CAR T cells and so forth, the logic of simply developing viral specific T cells and expanding them and whether they’re autologous or allogeneic, I think is a super exciting area of research and development that’s ongoing certainly for the transplant population and also for some of these other viral associated cancers.

Grace Ratley: [00:17:45] Do you have any other technologies that you’re really excited about that maybe make your job studying the virus a lot easier? I know that you recently used single cell sequencing, which is something we do a lot here.

Micah Luftig: [00:17:57] That’s what I was going to bring up, if that’s okay. Yeah, I think single cell biology is really taking over a lot of the way. Most folks think about their experimental systems. I mean, certainly in virology, the idea of what the heterogeneity of a response to viral infection looks like is something we think about all the time. And now we can see it. And whether it’s single cell RNA seek guiding the way or some of these new beautiful spatial transcriptomic approaches to see what that looks like in tissue. It’s definitely a revolution that I think a number of folks are embracing. And we’ve gotten our start with some single cell RNA seek on EBV infected cells in the context of latent infection. We published that recently, and then now we’re doing that also in the context of lytic infection. So it’s really cool because we can basically see cells that are infected but do not replicate the virus and we can ask, Well, why is that? What’s their gene expression program that’s potentially preventing that from happening? And likewise, they may get started along the way and then stuck. They don’t make it all the way to full virus replication. Well, who’s expressed in those cells and what might be responsible for that? So it’s a beautiful hypothesis generating tool to then allow us to go back in and say, well, if we now knock out or overexpress these genes, how does that influence the outcome of infection? And you can imagine directly identifying therapeutic targets for EBV in our case, but certainly any virus by using an approach like that and I know a number of investigators have been doing that for their favorite virus, basically.

Grace Ratley: [00:19:37] Yeah, just reading all of the single cell papers that have come out in the last few years feel so lucky to be entering science at this point in time because I feel like there’s so much going on and so many places to apply these new things. So viruses are very popular right now. I feel a lot of people have taken up interest in virology and as a result of the pandemic, having a virologist on the show, I’m obligated to ask you questions about COVID-19. So how has COVID-19 affected your research overall, how you conduct it, and whether you’ve made any transition to helping out COVID-19 research efforts?

Micah Luftig: [00:20:19] That’s a great question. I think like most of us, the pandemic obviously shut things down for a while. There was quite a bit of uncertainty in March, mid-March, and Duke shut down pretty readily. We had about a two week runway before we had to essentially evacuate the lab. So everybody had to get their lab shut down, protocols in place and approved and then just do it. And for us, it was not too bad because we don’t have mice and we don’t have. Primarily we have a lot of primary cultures, but things that we can viably freeze. So for us, we took advantage of the shutdown in some ways by knowing we’d be out for a little while, by basically finding as many RNA samples as we had in the lab to do a massive RNA-Seq experiment so that people could analyze it over the break, not knowing how long the break would be. So we ended up sending 96 samples to Novogen for sequencing. And I was in the lab. We had evacuated and I was the last man standing. And I was literally pipetting from everybody’s tubes into the samples, packaging the box and sending it out while everything was shutting down around us. And we managed to get it out. And it was pretty chaotic on their end, too.

[00:21:37] They kept things open, but it was like, we’re going to ship it to the West Coast. No, we might ship it to China to sequence. No, we can’t ship it to China now so it was crazy. So we shut everything down and everybody went home and we got into this mode of learning computational biology. So I think a lot of folks probably did that, but luckily I’d recruited a computational postdoc to come to the lab. I think he started in March, February or March, it was right around then. And so he was brilliant in teaching everybody Python and basic programming and then some of the computational tools that he knew and helped us to analyze the data that we were getting back three or four weeks after that. So it was a lot of uncertainty, obviously, across the board for the first month or two there. And then by about, jeez, I can’t remember May, June, July time frame, we started to get the indication that we could come back. So we didn’t do any COVID research directly or indirectly. And so we weren’t prioritized in the labs, but a number of colleagues here did. And it was actually pretty exciting to see the Vaccine Institute dive in. And Duke is lucky to have a BSL-3 facility.

[00:22:46] So we had the ability to work with the virus in a number of those suites right off the bat, and everything from testing on campus developed by some of the folks in the Vaccine Institute to a colleague of mine upstairs, Nick Keaton, who works on flu, had a big contract from DARPA that they basically said, we want you to shift some of this to COVID. And all of a sudden, he generated all the tools and reagents to shift to do some screening and developing assays for COVID infection in cells and in animals. And there were a couple of other labs in our department and on campus that I think, again, either diagnostics and screening or really doing some biology very early on. And so a lot of that work has come to fruition and been published, a lot of really exciting studies at all those levels actually. We were one of the earliest places to start doing pool testing of COVID samples and so on campus for undergraduates and ultimately the research staff, whoever was going to be on campus, they found they could pool actually up to ten, but I think they ended up with five samples per and really reduced cost and kept sensitivity and specificity high. So there was a lot of efforts on that front. And then a lot of the the research that was ongoing while all of us were at home on our computers, there was quite a bit being done still at Duke.

[00:24:07] And so then over the summer time, they open up the research labs with distancing and masking and started at 15 foot distances. And not long after that I think got to six. And so we were lucky we had pretty decent sized lab space and so we were able to accommodate most folks back in the lab full time. But tissue culture was a little tight. A lot of labs struggled with density and so they had to have shifts for their scientists coming back in. So that really, I think, compromised a lot of the return to the lab for folks. But like I said, we were lucky in that regard, just space wise. So things other than the fact that everyone’s wearing masks and distancing and there’s a lot of uncertainty for the next 6 to 8 months, things were basically back to normal. We were able to ramp up and get our experiments going again. I think the other big hiccup has been just ordering, just getting supplies in. Because lab supplies have been in high demand, obviously, over the course of the pandemic. And so right early on, we donated our gloves and our masks and everything that we had to the hospital to help them keep up with the PPE needs.

[00:25:23] So then there’s this phase of the vaccine. We just watch the data come rolling in. And there were trials here and things were approved and like, next thing you know, the whole lab is vaccinated and it’s just like, wow, here we are. Like, we’re in this pseudo post-pandemic world. We’re still masking in the lab, obviously. So there’s that and distancing is the same. But everyone feels a bit more comfortable with being together in the lab. So everyone’s basically back on the same routine. And we’ve had a couple outdoor lab meetings together and everybody’s really looking forward to getting off a zoom. And that’s the upshot is like the day that we can have in-person lab meeting again and then ultimately seminar speakers and folks on campus. I mean, everybody’s really looking forward to that. In April of last year when we were shut down, Duke really had a number of investigators and a number of different fields coming together to work on COVID, ranging from the testing to vaccine development and testing to developing animal models and screening in cell lines. So this whole enterprise of COVID research just manifested over literally weeks or maybe a couple of months.

[00:26:45] Once the sequence came out, everybody knew what we were dealing with. And so even if you weren’t a card carrying corona virologist, you knew what a polymerase looked like and you knew what a spike protein looked like and you knew what the length of the RNA was and the structures of the RNAs. And so the whole Duke Research enterprise really took a serious look at this virus and started to make some headway. And so we organized a symposium about mid-April that brought together all of these folks, everything from Coronavirus 101 basics for everyone to therapeutic ideas to vaccine approaches. And it was just Duke investigators. And we had over 1000 people on the Zoom call on campus. It was restricted to Duke. I mean, it was astounding. An all day symposium on this new virus. I have goosebumps thinking about it like it was such a well attended event. And it just was organized, great discussions. It was fantastic. And it spearheaded a lot of the efforts on campus to study this virus. And so that’s been maintained. They have a, I think, biweekly colloquia on COVID research and again coming from all these different fields. And so it’s cool to see what’s happened on campus research wise and screening wise for COVID.

Grace Ratley: [00:28:10] Yeah, that is incredible. I just find all the collaboration that has come out of COVID to be so exciting. Obviously within a university like collaboration between departments. And then there’s been a lot of collaboration between biotech and academia to develop the vaccines and a lot of collaboration internationally. Now that we have all of this infrastructure in place to really communicate long distance from home. And to me, it’s really exciting and I hope that the collaborative atmosphere is something that is maintained even after we go back to doing things in person.

Micah Luftig: [00:28:45] One of the things that I hope it showed the public is just how collaborative globally science really is. I mean, we were lucky to have Zoom and Skype and everything else in place to be able to do it, but we’ve been collaborating with people around the world the whole time. And so this idea that a virus that’s identified in China, that the sequence is posted and researchers in Germany and Cambridge and everywhere else around the world can just immediately start working on it and sharing information and sharing reagents that the community would come together, really rallying around this common enemy. I think it’s great for science. I actually think that despite all of the challenges that we have in this country, at least with regard to science as truth or not, I think it showed those that are willing to listen that there’s actually a lot of really great collaborative work that’s been done where there was an infrastructure there and then new pathogen inserted. And then here you go. We can all work together for this common enemy. But for the first three or four months, six months, maybe, my wife who’s not a scientist was listening to Twib every week. This week in virology and like, really getting into the details of mutations and Spike and the T-cell responses and what’s the neutralizing antibody and all this stuff. And The New York Times and all of these news media outlets have just educated the public broadly about how viruses work, how the immune system works. And so it’s really cool to have this collective education happening in real time on this pandemic. I hope that we can retain that level of engagement with the public in terms of communicating how these pathogens work and how scientists work and all of that stuff.

Grace Ratley: [00:30:47] Certainly I’m sure that it’ll inspire a new generation of young scientists, especially those interested in virology and biology at large. Speaking of inspiration, I figure this is a good time to try and transition to what led you to become a scientist. So I guess you probably didn’t have a global pandemic to really spark your interest in Epstein-Barr virus. But tell us a little bit about what got you into science.

Micah Luftig: [00:31:16] Sure. This one’s actually easy. It’s a dirty little secret. My dad’s a virologist, so that’s what got me started. My dad studied viruses since the 60s. He worked on phage in graduate school and his postdoc, and he used the electron microscope to study the structure of viruses and moved to retroviruses in the 70s and 80s and discovered the retroviral proteases that are important for maturation of retroviruses including HIV, and also worked a little bit on adenovirus structure and function. And so when I grew up in Louisiana, he was the chair of microbiology at LSU Medical School in New Orleans and had started to already wind his research lab down and was doing more mentorship and administrative work, but really was someone who inspired me to think about viruses and challenging biological questions. So when I was a kid, I would go to American Society for Virology meetings in college campuses in the summers, met virologists there and started to go to talks when I was about 14 or 15 and started asking questions when I was about 16 or 17, and they would say little Luftig in the back. What’s your question? And so, yeah, I just loved it. I really thought viruses were such fascinating little machines. I guess what I wanted to do, though, as I went through high school and college and started thinking about really what I wanted to study was I was always interested in Math pretty heavily. So quantitative biology was an interest. And then I have always been really interested in cancer. It’s just one of those great challenges. And so my dad didn’t work on cancer viruses, although I did say HIV was an indirect one, but he didn’t work on any of these oncogenic viruses. And I wanted to do something different. And I had worked in a herpes simplex virus lab when I was in college at LSU, and then I had an opportunity to go to the CDC when I was between my junior and senior year in college, and we were working on this new virus that had been discovered called KSHV Kaposi sarcoma associated herpesvirus. So I guess there was my own little pandemic or at least viral discovery moment. And so we were characterizing glycoproteins of KSHV, and I went to the CDC and worked in the herpes virus section there, and I learned a ton of basic virology and biochemistry and viral characterization. And that was on an oncogenic herpes virus. So I thought this is pretty cool. But it wasn’t clear that any of the tools were really in place to study this virus yet. And so I wasn’t quite ready to jump into that for graduate school. But in talking with my mentors about my interest in oncogenic viruses and being in a great environment and just the challenges of these complex viruses, I landed on EBV as the one to go after it was discovered in 1964, and we knew all about the genome structure and the RNAs that are expressed and all these crazy latencies and the B cell immortalization and all this stuff. And so I basically wrote to a handful of EBV Labs and said, I’d like to work with you. And then I applied to those schools and wound up going to Harvard and working with Elliott Kieff, who is one of the real pioneers in EBV molecular virology. So that was the backstory of how I got interested in viruses.

Grace Ratley: [00:34:56] Yeah. And then after your PhD, you did your postdoc in Italy. Tell us a little bit about that. How did you wind up there?

Micah Luftig: [00:35:03] Yeah, that was a pretty non-traditional decision. So when I was a graduate student, we were required to do two rotations, not three. And I really knew I wanted to work with Elliott Kieff, so I had to do another rotation. So I met with folks and I really enjoyed meeting Don Wiley, who is a crystallographer, who had solved the structure of the influenza haemagglutinin protein and also MHC Class 1 to show what the peptide presentation looked like. Really preeminent structural biologist and virologist. And I met with him and I was telling him about our work on glycoproteins of herpes viruses, and he said we were given these herpes virus glycoproteins to solve. And I don’t know anything about herpes viruses, but it sounds like you do. So he said, Well, why don’t you come work in the lab as a rotation student and you work with this postdoc I have named Andrea Carfi. He’s a great crystallographer, but he doesn’t really know anything about viruses either. So why don’t you guys work together? And I think you’d be good for him and you’d learn some things from him. And I thought, Wow, Don Wiley thinks I’d be good at anything. That’s cool, Let’s do it. And it was just a really amazing, amazing experience. So Don Wiley and Steve Harrison, who was one of the first to crystallize the structure of whole viruses. They had shared a joint lab since the 70s at Harvard, and they actually had one lab in Cambridge, one in Boston at the medical school.

[00:36:33] And so I rotated with Don technically or with Andrea, this postdoc in the lab in Boston. And I went in the summer before graduate school because they had an opportunity for us to do that and spent three months full time, knee deep or head deep whatever, into studying or really into learning biochemistry and crystallography at a very basic level. And it was awesome. I mean, it was beautiful. We purified proteins that we expressed in baculovirus. I was challenged constantly. It was the kind of thing where all of the basic knowledge I had from undergrad, which I thought was pretty good, I had purified viruses and run gels and done all kinds of techniques and I would be challenged. Andrea would say, Well, how does this work? Well, how do you think these proteins actually fold or how do you think this? I just didn’t really know. And I had to do this exploratory sort of self, not totally self driven, he was making me do it. But I had to learn all this stuff. Not just practical stuff about how you purify proteins, but why do you do it this way then that way. And it was just a transformative experience being in Don’s lab and with Andrea and the folks that were in that lab at the time and have always been are now world leaders in virology and infectious disease biology. It’s amazing that I had that opportunity.

[00:38:02] So I spent about almost a year in Don’s lab rotating, crystallize the herpes simplex virus gD glycoprotein D bound to its entry receptor nectin-1, went on many trips to synchrotrons to collect diffraction data on crystals almost. But didn’t quite get to solve the structure. We didn’t have high enough resolution data. You’re probably wondering at this point, what does this have to do with Italy? Except you did hear an Italian name along the way. So I moved to Elliot’s lab and started my thesis work. I actually came back to Don’s lab to do some stuff on some other glycoproteins that he had acquired from a collaborator. And so I still have my feet in the structural biology world a little bit. I really loved it. And then sadly, actually, Don passed away in 2001, and I was in Elliot’s lab a couple of years at that point and just really sad situation. And so Andrea, the postdoc I was working with had been applying for jobs and he was recruited to be the head of structural biology at a Merck research lab site in Rome. And it was this really beautiful opportunity for him because the place was actually a hybrid institute where they had students and postdocs, but they also had Merck funding and Merck pharmacology and chemistry and drug development and everything else. And he was recruited to publish well and to do Merck work. It was kind of a hybrid situation.

[00:39:35] So he goes there and then I wrapping up my PhD, I had a short list of folks I wanted to do a postdoc with more in cancer biology. I did my PhD with Elliott mostly on NF-kappa B signaling from this viral protein, LMP1 and EBV. And that was really exciting. But I wanted to also do broader cancer biology signaling and so forth. But I don’t know exactly what it was. My wife and I went to Italy for the last year of graduate school and just really fell in love with it. We backpacked around for about a month and saw Andrea, spent some time with him and connecting and when it came down to deciding what to do, we were in touch. And I said, What if I came to work with you? And he said, Why would you come work with me? You could go anywhere in the world with your credentials. And I said, Well, because I know you and I know our relationship and I think we could do amazing things. We could really solve some cool structures. And I said maybe what I’ll do is I’ll come there, we’ll solve some cool structures, and then I’ll go do a postdoc in cancer biology and David Baltimore’s lab or something at Caltech, if David’s ever listening to this now he knows that story.

[00:40:46] Anyhow, I convinced him that that was a good idea. He would be happy to have me. So he wrote up. We wrote up an Italian fellowship. I got it. And we packed our bags. And my wife, who was five months pregnant, we got on a plane and we went to Rome and moved into an apartment provided by the institute initially. And I started working. I was thinking I would do when I got there was to solve the structures of HDAC histone deacetylases, which had not been solved at that point. And there was a big biochemistry effort there and there was a lot of enthusiasm around HDAC inhibitor called SAHA that had been developed and Merck was in the process of acquiring that company and getting into stocks and a lot of biochemistry and chemistry and cool stuff was going on at that site. But I knew that they were also doing viral stuff. They were working on Hepatitis C virus, they were working on HIV, and Andrea had brought some of the herpes projects with him from Don’s lab. So I get there and rather than HDAC as the project, they say, Well, we just identified this new neutralizing antibody against HIV. And this was a time where in the field there were less than a handful of what are called broadly neutralizing antibodies, which was thought to be the holy grail for HIV vaccine design. And frankly, it’s still the holy grail for HIV vaccine design if you could elicit these.

[00:42:11] And so they said, we have this new antibody that we discovered. We’ve got the heavy and light chain plasmids. You want to make this thing and see if you can express it and crystallize it with Gp41, which is one of the HIV fusion proteins. I said, okay my dad worked on HIV, so I really don’t want to do this for a living. But I’ll give it a go here if this is new and exciting. And really, I wanted to cut my teeth with an exciting crystallography project more than anything. Things just worked. Things just went really quickly. Like we made the antibody, I purified it. Marco Mattu made the Gp41. I made the complex. I purified it. I crystallized it and in a total of four months, I had a 1.9 angstrom structure of this complex. And it was amazing. It was beautiful. Like it actually told us something unexpected. But also that corroborated some of the genetics that the other groups were doing. They hadn’t even published the identification of this antibody yet before we had the structure. So obviously I made a lot of friends on the Merck research video conferences, both in Rome, but in New Jersey and West Point where they were calling in from. And so we had a really great time developing that story and our understanding of HIV neutralization from that work. And I followed up a little bit on that, trying to make the antibody even better at neutralizing by predicting mutations that one could make to improve the structural interface and whatnot.

[00:43:41] But really what I had an amazing opportunity to do though, was to switch gears and I actually won an EMBO fellowship. So that’s given to postdocs that are moving from one country to another, probably the only one maybe ever that went from the US to Italy to do it. But nevertheless, I was a part of this cohort, which is a really cool group of, it’s probably 100 or 200 every year or something, but mostly Europeans either going to the US or other countries. But then you get together at meetings and stuff like that. And so I had this EMBO fellowship so they weren’t paying for me. And like I said, it’s an institute where there are students and postdocs and it’s a really cool environment. And I just started to think about what I wanted to do next. What I thought I wanted to do was cancer still. But I wanted to bring the structural biology background that I had been developing to a problem in cancer biology. And so just around this time since about 2004 or 2005, there was a paper published in Nature reviews cancer called the Census of Cancer Genes so the first shot at cataloging all the genes that were mutated in cancer. And there was something like 200 genes, and it had the functions of the proteins and where the mutations were and what cancers they were in and all this stuff.

Micah Luftig: [00:45:06] And I just spent weeks studying this census thinking about which gene am I going to land on and couple the genetics and signaling and cancer biology with structural biology. And it’s going to be beautiful. We’re going to understand how cancer works, at least for this one, protein or one system. And I landed on a gene called ATM, which is a DNA damage response kinase, many mutations in cancer, breast cancers, lymphomas, lots of different cancers and ataxia telangiectasia where the gene comes from is actually a cancer predisposing syndrome. And so there was a lot of cool stuff to do and there was really great signaling work that had been done on how ATM works. And so I started writing to people and asking for constructs and developing, the tools to study the signaling and start doing structural biology on my own. I had the blessing of Andre and the Institute director, and I thought it was like a brilliant project that I had written up and started applying to jobs, academic jobs in the United States at top notch places with this set of ideas and some preliminary data in mind. And it was fairly naive frankly of me to think that I would be competitive for those jobs or ambitious, maybe you could say.

[00:46:29] And so I started getting feedback from some colleagues that were in the field and they said, this is all great, but you’re not working in this field. You’re not pedigreed in cancer biology right now or in ATM signaling. It’s great you have some new data, but that’s not how it works. And so the advice I got was interesting, which was go back to the virus. You’re not that far out of your PhD, go back to your virus that you started with your cancer virus. What are the most exciting and interesting questions there that haven’t been answered around the same time? As I tell the story, I realize how serendipitous it is. But around that time, Antonio Lanzavecchia comes to our institute, who’s a famous immunologist who studies B cell, T cell interactions, and he was really interested in identifying neutralizing antibodies to SARS 1. At the beginning of his talk, he said, I wanted to use EBV immortalization of memory B cells to expand them to identify neutralizing antibodies. But it turns out it’s really inefficient like only 1% of the infected cells grow out and so we can’t screen anything. So he said, but we found a trick to get EBV to immortalized 100% of the cells. And the trick he used was to add an agonist of the toll like receptor 9. And together with EBV, he actually got a much more robust outgrowth that then allowed him to identify neutralizing antibodies to SARS.

[00:48:04] And I thought I’d never heard anybody say EBV immortalization wasn’t efficient. And so I look back at the literature and it turns out two papers, 1977 Journal of Virology, the efficiency of EBV transformation is about 1% of infected cells. And I thought, Well, jeez, that sounds like a problem. What’s going on in 99% of the cells? And how does that work? To cut a long story short, at the same time in 2005, there were a series of papers in nature showing, of all things, the DNA damage response, ATM-Chk2 and ATR-Chk1 were responsible for sensing the earliest events in oncogenic stress. So when an oncogene is activated, induces replicative stress. The DNA damage response gets activated and that prevents the cells from continuing to grow, either by causing apoptosis or cell cycle arrest. And so if you eliminate those pathways genetically or however, you get growth of tumors more robustly, and that also reconciles the cancer predisposing nature of an ATM. And so all this stuff was coming together and I thought, geez, I wonder if EBV is inducing the DNA damage response early in infection. And that’s why most of the cells don’t grow out. The answer is yes. We started scratching the surface of that, but I wrote the Proposal for Jobs on this idea of EBV and the DNA damage response that I then ultimately got an interview at Duke and a couple other places and landed here in Durham and started my lab.

[00:49:46] And over the next maybe two or three years, we worked it out in primary B cells. That was our first big paper in 2010 we published to show the DNA damage response was indeed a barrier or a repressor of transformation. Probably 5 to 10 fold of that 100 fold restriction, the DNA damage response is responsible for that. The cool twist is that that actually collaborates with TLR9 agonists and can make efficiency even better. And so you can imagine there’s a practical application, which is if you want to identify antibodies to SARS 2 or HIV or flu or anything else, you take memory B cells from some convalescent patient, put them in culture with EBV. But now not only do you activate TLR9, but you inhibit the DNA damage response and you get a much more robust outgrowth and you can screen many more wells for possible antibodies that are being made. And so now, I would guess most vaccine institutes and companies use this approach. I know that our Vaccine Institute does. I know that James Crowe at Vanderbilt does. I know lots of other people, because when we made that discovery, we published it and we shared that information with all those folks. And so it really helped improve the efficiency of that process and help to identify antibodies for lots of different pathogens.

Grace Ratley: [00:51:10] Wow. Well, we have made it full circle. And what an amazing story. It’s been wonderful talking with you today Micah. Thank you so much for coming on the podcast. I learned so much and it was just really awesome to get to talk to you today.

Micah Luftig: [00:51:22] Thank you for having me. It has been a lot of fun too.