TWiV 1024: The Chumakoviridae

this week in virology the podcast about viruses the kind that make you sick from microbe TV this is twiv this weekend virology a special episode recorded on June 1st 2023. I'm Vincent rackinello and you're listening to the podcast all about viruses my guest today is formerly the lab head and head of lab of methods development and associate director of research in the office of vaccines and research and review at the FDA and also Adjunct professor University of Maryland and George Washington University question chumakov welcome to twiv yeah thank you Vincent a lot of titles well you accumulate over your life quite a few uh you were last on two of 604 I don't know if you remember at the beginning of the pandemic right you were trying to get opv tried as a temporary uh way of preventing uh kovid right yes you talked about that you never got a trial done all right no not really I mean it's Race So Many objections and all the polio eradication people were up in arms and just said no no no no no no yeah so it was a kind of quite uh it was upsetting but I can understand yeah you know so you you just retired from the FDA so and last week we had a symposium in your honor which was very nice and I wanted to talk about your life not that it's over anything but uh it's a good time to review it so let's start all the way in the beginning because you were born in USSR right what year was that roughly yeah well it was 1952. and and I was born in a family of two virologists and my father was Mikhail chumakov and my mother was Marina veracilova they were both virologists neurovirologists mostly studying neuro infections actually my father was became famous when he was a co-discover of tick-borne encephalitis in 1937. there was a I live zilber who was the kind of more senior Soviet virologist who headed this Expedition so my father participated in it and he actually got infected with stick board Encephalitis in this Expedition um and he he got sick but he recovered but he lost the movement in his right arm and lost Hearing in his one of his ears and the five percent hearing remained in the other so he was handicapped for the rest of his life and it happened when he was 27 years old so I mean it's quite was a kuwaitan a life-changing event so I was born into the family of virologists and do two virologists who did they train with well my father trained with the olive zilber okay he graduated from the medical school and he was interested in microbiology so he went to work for Lev Silber where was that it was in Moscow it was there was an Institute actually that's close to Moscow it was a sort of a military military biological Institute uh we're actually actually my grandmother also my mom's mom also worked there so they knew each other okay so so he knew my mother since she was little and then she also graduated from medical school and um in after the World War II there was a big outbreak of polio in Germany in the East Russian occupied Soviet occupied part of Germany so my father was sent there and then my mom also was in that expedition she was younger of course and then were there kind of romance started oh I see so basically she was his graduate student and then they started a family and I was a third of four kids so but of course it was probably a destiny to become a very religious even though I resisted all I could oh really yeah I never wanted to follow in the footsteps of anybody I mean it's just you know you know like teenagers are yeah sure because my father always used to say oh the only profession you should choose is medical profession go to a medical school and so on and well I said no no never ever so now in retrospect I think he was right because in our field of study I think having medical education is by far more appropriate than than any other education because I mean we're dealing with the human health you know viruses I mean we're not dealing with plant viruses or Marine viruses we're dealing with human viruses so you need to understand the physiology and all of that can I ask you about the history of Soviet virology so I I was told years ago that the father of Soviet virology was Victor zidanov is that correct no no it's not I mean he was a prominent virologist but the father of virology not Soviet biology was Dmitry ivanovsky who was the first to discover and to discover tmovic virus because he I think when at least that what is written in the you know textbooks that he was the first to filter this or infect plants he came up with this concept of virus okay I mean that's how it happened where was he working oh I wish I remember it was was he in Russia do you think oh no he he was in the what it was a Russian Empire it was at the end of 19th century right so it was kind of way back was jidennov a contemporary of your father yeah he was he was I think he was a bit younger maybe a couple of three years younger so they were friends and here is done of was quite an interesting guy he he became a deputy minister of Health at the age of in his early 30s so which was remarkable I don't know how he made this career he was a bright guy and then after being basically a high-level bureaucrat yeah he all of a sudden decided he wants to be a scientist so he quit the job and became the director of The ivanovsky Institute of virology Institute that my father used to be director of at the end of 1940s so and he actually done experience himself right I've seen photographs of him carrying Auto centrifuge rudders and everybody was wearing white coats and he were black you know like a blue collar guy so no he was quite an interesting scientist or very interested in in virology and he was one of the people who spearheaded the smallpox eradication okay along with Dia Henderson so he was one of I I think he may even suggested that this is possible and so he was one of the um you know brain Powers behind this whole effort so both your parents had medical MDS yes they were both MDS so I have in front of us a picture that I think vadima goal showed last year right which so Moscow around 1960 yeah why don't you describe what's in this picture well I mean the thing is that after my parents both of them uh traveled to the United States at the end of 1955 when the Institute of polio myelitis was established that was in Moscow it was in Moscow yeah it was well it's Moscow server suburbs okay so they were both sent to the United States for a few months along with anatolis Maroons of in other prominent Russian virologists from Saint Petersburg at the time it was Leningrad and he was actually he worked with polio even before my father started doing it so he was uh really a polio virologist and there was three of them plus one KGB officer who was accompanying them so I have this all in the stories from Alexis Sherlock of who actually was assigned by the state department to accompany them in their travel so their how they became friends uh Alexis was of Russian extraction but he never lived in Russia he were he was born in China into the Russian expat who fled Soviet Soviet Union during the revolution but he became a prominent virologist himself uh worked in the perimet Canal Zone and then he worked for a few other institutions in the United States so he was assigned to accompany them in their travel and of the United States so they visited all Labs that were doing poly research and mind you it was 1956 early 1956 so it was a height of the Cold War so it was quite an unusual circumstance so that's where they met uh Jonas Salk and Albert Sabin and everybody else who is who in polio virology and they became friends and when they came back uh they started this institute and organized production of ipd first uh and it quite quickly I mean it's took them maybe a year a year and a half to start producing ipv but then he was actually impressed by the results of Albert Sabin and your father Yes yes and so my father decided that he wants to start making oral poly vaccine so they basically interacted with the Sabin and Saban started visiting Soviet Union and then I think maybe in 1957 58 they started organizing a simple polio symposia in Moscow so everybody would come it was an amazing thing so all scientists from the United States and from Europe anybody who was interested in polio would be coming to Moscow for because I mean for for some period of time Moscow became the focus of popular vaccine product because it was the first and the only at the time maker of opv right because the US would not was not interested right I mean there was a quite a lot of resistance from my former agency you know I mean for a good reason because I mean as everybody knows perhaps this roll out of the first inactivated vaccine was not without a glitch right this Infamous car incident so there was enough trouble with ITV so but then giving live virus to kids I mean it was a kind of foreign concept at the time so and of course there were a lot of questions about how stable it is and even back then it was known that uh attenuated strains are not very stable they can't change and say they knew that they changed but he developed the strategy of how to use this vaccine despite the genetic instability uh so that's that's why having my father on his side you know as a collaborator was a very I think it was a kind of collaboration that was uh really made it have it the only thing that could make it happen so your father invited Saban to come and help with the opv production yes well he basically said look I mean you have your strengths uh let's try it right so the first thing that Saban brought this vaccine in his suitcase you know just a few bottles of vaccines so they formulated I think in Moscow and then they had a small clinical trial and it the results were excellent and so then after that my father started you know developing the production scheme you know all these things that you need for mass production and they very quickly rolled out the first batches of vaccine and started large-scale clinical trials that were also triumphant you know it was just basically it was mostly done in the Baltic republics you know no it's independent states Estonia Latvia and Lithuania I think they started with Estonia and their folder was almost gone instantly so it was really a huge success so that's when they started immunizing the entire country uh and uh actually Soviet Union was the first country that demonstrated this vaccine can can prevent polio so so they immediately started exporting this vaccine into I think at the height of this you know whole uh folio campaign more than 100 countries so they start of course all this you know satellite countries such as Eastern European countries and Cuba Cuba was the first country to eradicate polu in 1962. because I'm using opv opv because I mean it's the totalitarian country so they could immunize everybody if you want or you don't want nobody cared so they immunized so and until now they do it in campaigns exactly as Sabin wanted them to do because Sabin realized that the vaccine is unstable the only way to prevent uh cases of what we now call cvdpv is to immunize the entire population at once so that everybody gets this immunity at the same time so the virus that is shed the genetically modified you know virus that regained virulence would not really transmit to those who didn't get the vaccine okay so what about the recipients wasn't there any Vapin recipients uh possibly but it's such a rare occurrence so it's not really easy to even to figure out I mean it's not until the molecular methods became available it was possible even to establish that there was a vaccine Associated poly and Sabin until his last days he actually he never accepted this concept I I probably deepened his heart he realized it but he never openly admitted that this is possible yeah but and in fact the only way to prove that this periodic case was caused by polio myelitis was to isolate the virus and at the time it was not that easy and then to prove that this virus is vaccine derived Okay so until there were molecular methods to do it then it was so basically when you're doing Mass campaigns you can't distinguish vaccine paralysis from naturally occurring paralysis yeah well look I mean even because a polio technically it's acute flaccid paralysis right so this is a condition that is not only caused by polio and not only by viruses but many other you know it's a basically a symptom so to prove that this particular case of acute flaccid paralysis is caused by polio and much less polio derived virus you need to sequence it and to prove that this is really derived genetically from from the vaccine virus so it's only started happening in the 1980s when it was become clear so this photo you were about eight years old right so it's probably yeah it was eight or nine percent that's you in the photo right yes that's me yeah then your parents my parents and Albert said yeah it was taken at the river cruise terminal in Moscow because they're one at one of these conferences and it's always was accompanied by some social events so and so we went on a short you know day river cruise from himke this is a place north in North part of Moscow and of course my parents took me with them so at this time uh they must have been doing the trials of opv already right yeah they already were producing it and I think it started mass campaigns so it was those a year do you remember any of the the the interaction between saving and absolutely I remember maybe not at this age but I remember remember that trip and actually this picture was given to me by Albert himself well already when I lived in Washington and so we we met quite often and he gave me this he pulled it out from his archive and he even signed the back of this photographs a very nice word to Coast to chumakov in memory of our you know long friendship for the great scientists he became such you know flowery words so you know Saban tells the story that he went to your father and asked him to do the trials and then went back with the data to the U.S and said here it's a beautiful vaccine and then they had to use it is that fair you think well I mean it's not not that simple because actually there is a very good historical account of all this Saul Benson historian of science historian he wrote a very nice nice uh review uh quite a long it's not a book but it's a chapter in some book I'm very detailed with the excerpts from their correspondence letters that Satan and all the kind of politics around it very interesting and they have a copy of this sure absolutely I can send you I would love that yeah and also Albert Sabin himself he wrote an article saying the history of my collaboration with Soviet scientists so you may have read it that one I've read yeah yeah and that actually was in that paper there is no controversy but actually there was a controversy about whose vaccine this was because some people in the Soviet Union then unfortunately including uh anatolis maronians of they claimed that this is a Russian Soviet vaccine so and there was a newspaper article saying that Soviet scientists a great success and so on and so forth and uh both my father and Satan were quite upset and Saban once went to a meeting in who where the deputy Minister was present Soviet Minister was present and he raised and he was quite you know direct I mean he never missed words and he said I want to ask your point blank do you claim that this is your vaccine and so on in this minister of Health had the courage to stand up and say no Dr Sabin I apologize on behalf of the Soviet government that this is misinformation so so they forced basically to retract this stories and and actually I remember that on the packages on the cartons with the vials that my father produced at his Institute that was said from savings trains this was clearly marked that it was because that story was really a kind of yeah it could have ruined their friendship but then I think in the mid 1980s my father he was already kind of his health was failing but he managed to submit a nomination to the Soviet government to give Sabian the order of the Friendship of Nations or something that was kind of ordered what they would give to some figures that promoted International you know collaboration and so on so and then Sabin wrote this um it I think it was his acceptance speech you know basically he made into this article when he described in his own words uh how this whole thing unraveled so let's now move to your career so you're in this photo you're very young but you probably don't want to be a virologist yet yeah no no no when did you change your mind well look I mean I I never wanted to be even a biologist I mean because it's not I mean I never liked you know biology because I thought it was kind of you know catching butterflies and you know collecting Mouse poop in the forest you know all this stuff it was never appealed to me I mean I was more like a kind of uh I actually when I was maybe at the age between 10 and 14 I I probably wanted to be a mathematician so I actually I liked math and it I think I did quite well but my two older brothers they wanted to be biologists my older brother was just you know absolutely I mean he raised some plans and he loved or he knew all the animals and everything so he was a and I of course you know teenagers they are contrarians so they I didn't want to have anything to do and my second brother who was much uh one year older than me he also wanted to be a biologist so I I didn't but then what happened uh actually I read a book written by an American scientist and I keep for I forgot the name that was called something from molecules to cells or something in which and I was probably like 14 or 15 years old and he described first of all the structure of atom you know hydrogen atom all the you know electrons and uh nuclei or whatever and then uh Theory theory of chemical bond and then inorganic molecules and then it going to organic molecules and then DNA wow biology is a science it's based and I started to be a little more interested so maybe I wanted to be a molecular biologist never bought any store you know studying a real life and then what happened when I was 14 or about to turn 15 in the Soviet Union um in high school actually I called it high schools so that the American audience can understand what I'm talking about because in the Soviet Union it was just one school I mean from the first grade to the 10th grade kids started in the same building almost the same teachers changing of course year to year but still it was one continuous process so in the and it was 10 grades in the ninth and 10th grades I would call the Junior and senior we were supposed to acquire some trade profession you know there was some trade education so our school was assigned to the uh our boys were supposed to learn auto mechanic trade you know and girls I forgot what something like tailors or maybe cooks or I I forgot what girls were doing because it was separate at the time uh so the first uh we were studying the automobile the you know how it's done and then starting a winter semester we were supposed to go to the garage of it was a garage of the Soviet Academy of Sciences and it was Winter Moscow wind Chile I mean smells gasoline and all the you know all this mechanics were running around you know muttering and so on it was just not experiencing the 14 year boy from a good family I would want to and actually my brother and I we were in the same grade and so we complained to our parents and my mother decided that she will arrange a special kind of exemption for us so she went to the school directory and she took him into she said look I mean at our Institute we have a school of technicians virology technicians so I promised that if you let them attend this school we will give them the diploma of virology technician and that's what happened so that's how I got into the I remember the day it was March 1st 1967. so uh both my brother and I we were brought to the Institute and first started in the histology lab so we'll learn how to make sections of tissues and it was of course monkey brains and then stained them hematoxylin eosine and then look at the microscope and so on and to evaluate you know lesions I mean that was amazing for a 14 year old boy then we went to the laboratory of Immunology we did learn how to grow virus how to do precipitation and so on and so forth and then we got to I think it may have been it was kind of a rotation and so when we got into vadima Gold's lab which was perhaps maybe April or May of that year uh and that's how our collaborations with vadima goals started 1967 so which is like what is it 16. 70 something no 50 something years almost so and then your parents must have known vadima God oh yeah of course because he worked he worked in their Institute here at the time he was in his late 30s and he was the head of the biochemistry lab but he stubbornly refused to study viruses I mean at first he studied whatever his scientific interest was in uh I think oxidative for civil relation some kind of Hardcore biochemistry but then at the same point he decided to to begin working with viruses and that's where the time when I joined his lab so we were doing sucrose gradients and so on so I mean we learned all this technique and after Vadim vadima Gold's lab and we went to electron microscopy lab so learn how to work on this Japanese electron microscope because Institute was quite well equipped because it was a time when Napoleon was a big problem so a Soviet government I mean never saved on this so it was well equipped so that's how I learned and I loved viruses you know I I realized that this is exactly what I mean because it's a very simple thing so and and it's so easy because I'm I'm afraid of complex systems I mean I'm only comfortable in something that I really understand so then eventually you got a PhD with vadima gork yeah yeah it was later because I mean when I I graduated from from the school I was six I just turned 16 years old and of course I got into Moscow University and the first year we're studying whatever some general biological things uh but then starting from the second uh second uh second grade when whatever you would call sophomore yeah yeah so we had to split to different departments so I of course choose Department of virology because that's where my team was he was one of the co-organizers of the this department in I think in 1963 or when Moscow University decided that they need this new Department of virology but I started working with him even on the in the first year I would after the uh you know all the classes were over I would travel to the Institute first and then he actually assigned me to his one of his associates to in the just newly built building in Moscow University that was called molecular building so it was an interesting building because there were people from chemical Department physical Department biology department and mathematicians so it was a kind of multi-discipline Institute it was not called Institute at the time but it was called the lab but it was a quite a big six-story building where you know it was basically all experts from different you know walks of science they could collaborate so it was a very very one of the best in the Soviet Union the places to work in molecular biology so it was just built and it was not half empty but there was a lot of free space so he gave me a room on the sixth floor with a magnificent view of Moscow downtown it's just I mean you could first of all that's a high it's called the time it was called linen Hills it was a high Bank of Moscow River and and the blue was just magnificent so I would at night I would turn over the lights and just look at this beautiful Skyline but I started working in my first project was to I worked with the in several of myocarditis virus Mouse virus and then my first assignment was to develop guanidine resistant or go any insensitive for dependent strains which of course failed because it only works with polio virus for for EMC it never worked but this was my first science project uh so you're a college student still right yeah I'm on the front I'm a freshman freshman wow yeah I'm 16 years old and I'm running my own project so it was and working with Vadim was very interesting because I mean I I would call it a negative selection because I mean he really gave specific ideas he would give I think you should study this and then you're on your own you have to go and read literature and you have to come up with some experiments you have to then report what you want to do he would criticize he would always criticize oh no no it's not going to work okay yes it will work sorry and I know that for a lot of people it didn't work because some people were intimidated so some people didn't have enough I guess energy or creativity to so basically or people who actually succeeded in his lab were really really creative because I mean anybody who who somebody who needs some guidance in Daily supervision I mean would never survive in his lab so that that's I guess the success of his um you know school and you know that there are how many people from coming from his lab actually became really sure excellent scientists so that's a kind of lesson maybe for a younger professors how you teach your I don't know if this can be adapted to the American education system but that's how it was with me and Vadim yes sir all right so it was a lot of um throughout the your whole College time you worked in this in his in this lab on your guidance right and then you worked on various Pokemon viruses right emcv well it was exclusively at the time it was exclusively emcv yeah uh and um I think the the main reason why if I didn't chose this model uh maybe also as a contrarian if I'm in the Institute of polio I should say something else or maybe but primarily because of it was so easy to work with much easier just like Anne palmenberg told that the Symposium last week it was really really easy because to grow enough polio virus for biochemical studies uh you need a huge amount of volumes of cell culture it was at the time it was only monolayer culture so you need you know you know you know dozens of huge flasks and so on so it's a very labor intensive so EMC we grew EMC in the ascitis culture it was called crabs too it was propagated on mice so you would inject 0.5 mL of the cystitis fluid from a previous Mouse and then next week you have a mouse with a huge um you know belly full of up to 10 mils of and the concentration was I think uh three to the 10 to the eight sales per mil so it was enormous about Charlie then you spin it down and so on you can get you know uh dozens or maybe hundreds of grams of cells so and the virus could be grown in cultures as I recall attempt to the two to the 10 to the seven per mil suspension culture so you could you could grow tons of virus and then do whatever milligram quantities of RNA or if you study virus itself it was a so easy to work with so that's why we were studying it and uh I think my graduate project was my undergraduate because actually well it's hard to tell because in in the Soviet Union the university education was five years so it was somewhere between Mastery and Bachelor and master I mean Moscow University at least when I came to the United States it was um accepted as a master degree in some other university maybe not that prominent sometimes they interpret it as a bachelor but nevertheless so we had to do experimental project and write a sort of a thesis on this so my project was to study the uh reason why doubles Trend that RNA is infectious and you know this was a mystery uh because uh double-stranded RNA is close to how can it initiate infection so it was a big problem so the idea was that we will see what um strand of this double-stranded molecule is important so my task was to separate the strands and activate them separately with UV light and then real reconstituting the double stranded and see which one will work and it turned out that it's not easy to separate the strands so we somehow will manage by using the metalsulfoxide and some other tricks and the the result was of course that the positive strength was and that was my uh you know undergraduate thesis but then since we had such a hard time uh separating strands we couldn't understand why and then I think it was 1974 or 75 Peter felner who I think is mostly famous for this ribosomal RNA sequencing even before the sequencing it was done by some kind of uh technique with the oligonucleotide maps and so on he generated a sequence I think that was 30 percent error rate something like this but then all of a sudden um nature we found the paper he just published in nature that uh EMC is a funny virus that instead of pulley a at the three prime and that's all normal guys have this virus has fully see um and since I was trying to separate strings using uh oligod DT immobilized on cellulose so I knew that there is a poly a I mean this is there was no question that there was a fully a for me because we separated plus Trends we fished out on this uh oligidity cellulose and so I remember it was Friday and uh the redeem came before he went on uh on a weekend uh he said well what are your plans what are you going to do I said well I came up with the scheme how to how to localize this policy because I I I'm sure that it's not that the Supreme end he said oh of course John don't don't even bother you know because they already figured it out so I'm sure that his second paper is already in press and so on and so forth and I said well no I will I will still do it so I basically I uh I crushed the um they are in a molecule by boiling it for like 10 minutes yeah so it was fragmented into small pieces uh randomly random length and then I separated those that contained pulley a or absorbing on the oligidity cellulose and then whatever I eluded from the cylinders I absorbed on all the good Di that would bind policy so and then put it on the sucrose gradient and to my surprise they uh for all the fragments were distributed across the entire sucrose gradient but those that pass through the two colors were sharp full length molecules so it was just an unambiguous result showing that policy is on the five Prime and not three prime that's right it was so shock wow that was so easy just one weekend you said you worked all weekend to show him on Monday right yeah on Monday he he came to the lab and I shot him and he said okay so where do we send this paper so I was very proud and you you were an undergraduate at this point uh uh yes yes I was undergraduate yeah and this was my PhD thesis because I worked for a few more years because wedding wouldn't let me actually defend my PhD he said no no a little more a little more just get a little more results but then I broke my arm I fell um uh slipped on the ice and broke my uh arm and I couldn't work in a lab so I said I I will probably I will use this month that I'm still in the cast um I will start trading my PhD thesis I said okay all right that's how it happened but but the the result was generated in one weekend and actually this paper was because we we um we're trying to rush to be published yeah it was early 1976. in this in the Soviet and of course we wanted to publish it in a decent Journal uh something that will be fast but still English language not in the Soviet because nobody would read it we wanted to be first before Peter Peter filner would publishing the results sorry and and it was not easy in the Soviet Union there was a system that you were First supposed to publish your findings in Russian and then if you want to send it to America to you know English language or foreign Journal you will have to uh pass through the special uh committee that would review this and you will have to fill out the form in which you state exactly that this paper contains no new information and therefore it can be published in in the foreign language it was absurd but the post office would not accept the manuscript for shipping there was no internet you couldn't upload it to a PDF so so there was basically the only way to publish it fast was to smuggle it out of the country and fortunately at the time Carlton Geiger was visiting Moscow it was the the end of May 1976 and he's I told him that we have this problem I have a manuscript ready for publication and he said well I can help you because I'm going to Paris now and I will meet Jack Manor and he is the editor of the biophysics and biochemistry research communication dbrc so he took the paper and it was published so uh Jacques mono submitted it on May 30th because I have the letter from him May 30th 1976 on May 31st he passed away so it was the last paper that he submitted wow so it was kind of it was so weird I I only learned that he passed away a few months later and said let me look at the letter of acceptance and it was kind of the last the last uh the last day of his life that's a good story yeah so during this time also I understand Eugene coonan and Sasha gorbalenya were then the lab and working with you is that right well uh yes I think Sasha joined in 1975 at the time I graduated in 1973 and so he actually came to the Institute of polio where I formally where I never worked there you know kind of I was never an associate of The Institute but I formally I worked in the Moscow University but the best lab was redeem's lab was in The Institute and Vadim said well I think it's time for you to start your group I mean and there is a guy who was sent to us from novices biersk and at the time Sasha graduated from Nova cebersky University and he was sent to work to this new uh newly formed Institute that's called Vector it was an Institute That was supposed to be involved in uh military develop I mean some kind of uh by weapons development so maybe not exclusively but the large part of it was um but since there was no building yet so they were only started construction so all the new Associates who just got from college they were sent to different places in Moscow to basically to learn and to for training so and that's how Sasha and I started working together and then Eugene Cooney just um uh graduated from uh from our department of virology also and so he started working also about the same time so there was a three of us working in the same room and it was kind of it was a very good time so you're you're getting Independent by this time oh yeah no I was always independent uh I mean that that's the thing about uh vadim's lab uh so I mean because uh he or he treated all his associates uh kind of uh it was not like there was a hierarchy so he I I was helping Sasha and Eugene uh um so they were sort of in my group but they also interacted with Vadim directly so it was not like in some labs when you basically a lab Chief doesn't really know what the kind of everybody is doing so it was not a big lab I think there was a maybe 10 or maybe sometimes 15 people so it was not a huge lab so that's we started working initially it was um uh exclusively a lab work because uh Sasha's first project that I came up with because I mean when I was told that Sasha will be working with you and William said well think about what what he will do and I said well I mean now we know that this policy is on the five Prime men let's figure out the function so let's try first see what what proteins binded so and so Sasha's project was to use all this um oligar DC all the good di oligody a cellulose is to basically uh to do Affinity selection of proteins that bind it so both in various affected cells and normal cells so that's how he would uh pass the protein extractors through the cellulose and then wash it off and then around gels uh one dimensional two-dimensional and so on and look at this basically here uh spots and would compare and look at the difference and I was impressed how perceptive he was he was a look at this you see this tiny spot here it's not there oh really but he really was so focused and he had this ability to see Tiny differences yeah so that's and we published this I think in virology some of these findings and then Sasha was sent back to the vector Institute where he uh basically refused to sign the security clearance because I mean of course they required because it was a special kind of Institute and so he was he he didn't want to have anything to do with this so he was sent to the basement to open crates with Furniture I mean they couldn't fire him but since he was not uh he didn't get the security clearance he couldn't work in the lab and so and that's where it was the time when you and Eckhart published polio nucleotide sequence in Sasha during the lunch break he would look at the sicknesses and here with another guy Vladimir blinov they would basically but in without any computer they would build a gaze the sequence and see some things for instance they saw something that resembled protease and uh that then at some point um uh Sasha was able to come back to vadim's lab uh and basically demonstrate that indeed this protein that this p52 a PPA 22 of EMC is indeed a protease so it was I mean I was shocked because I mean that probably it was the first example at least that I know of that you find something in the sequence and then prove the biological function just by looking at the structures looking at the motives yeah and it was 1977 or something it was really early I mean there were no computers in the Soviet Union maybe even the United States they only started yeah that's right only mainframes yeah so last week Eugene coonan told the story of his working he tried to teach him to do a biology experiments and he couldn't do it so you you had told him to work on a computer right no no I didn't tell him to work on the computer you know because look I mean not everybody is the same you know Eugene is brilliant absolutely I mean and I enjoyed working with him because I mean uh one of his first projects was he a cell free system for any RNA replication so and so we worked uh together on this because he was still sort of under my wing so to speak uh and we quickly realized that nucleoside phosphates are Incorporated much faster than triphosphates and first we will say what's going on here because I mean it's it's counter-intuitive and even one of us face and were Incorporated better than triphosphates which is country intuitive because everybody knows this triphosphates are used by as a substrate buyer in a polymer by any polymerase but then we came across a similar finding for DNA phages that actually were interpreted that there is all the enzymes kinases that generate triphosphates from monophosphates diphosphates triphosphates they are probably organized in a certain pipeline physical pipeline so that to get to the active Center of polymerase you need to enter as a monophus fate because for triphosphates it's very hard to get into this because it's just structure release so I think it was an interesting observation so that that RNA polymerase complex of epicornoviruses are organized in such an interesting and complex way so basically a viral polymerase is integrated with cellular kinases so I mean it's I think it's a fact that it's not really appreciated that the internal structure of a cell is not just it's not a bag with enzymes that are just that's right floating around so they're structured yeah that's right so but and you just so he could do experiments but then when the sort of you know nucleotide sequences started rolling in and then when such a success with this discovery of protease and the analysis of of sequences here got so much interested in this so and at that at the same time I also drifted into into sort of bioinformatics even though there was no such word at the time because they they they think it the way it happened was also funny because there was a guy in The Institute of polio who studied the virus and he thought it was a virus and he even said it's it's a it's probably antivirus because it's about 30 nanometer uh 30 nanometers in size capsid and it's filterable and it was isolated from a case of villus encephalitis so it was a mysterious illness in Siberia when people started had symptoms similar to Kuru the slow virus infection that is now believed to be caused by prions and so of course there was an expedition sent to this place and they isolated quite a few viruses that most of them were completely relevant but there was one virus or agent they called it kpn as initials of the victim of this Village Encephalitis so they started about for 10 years so they would propagate it in cell cultures and uh they couldn't figure out what it is because in under the microscope uh they say well it's a virus that causes such a dramatic change in the cellular morphology I mean the the cells are unrecognizable uh so but they couldn't figure out so at the time I was running gels like every day and so this um uh Andre uh caravanov he asked me do you want to look at this because you're working with the enteroviruses it will be probably easier for you so the first gel then I ran was weird because it was clear I've never seen such pictures there was a three closely located bands of RNA about 20s there was a sharp bend on top that looks like DNA DNA always run there's a sharp Bend and down at the bottom uh there was a kind of diffuse bands something looked like tyranny and there was a sharp Bend of something looked like 5x70 I say well it's not a virus it's like or if it's a virus it's a weird one because it has both RNA and all this collection of things so I decided to sequence this 5S RNA because it was easy and I sequenced it there was a method that was developed by Deborah PT it was similar to Sanger Gilbert and Sanger methods but slightly different chemistry and when I got this sequence I had no computer at the time so but there was a issue of uh nucleic acid research that published every year all known sequences it was it was not a very sick issue so I basically opened the chapter verse 5S RNA and I typed on the Strip of paper uh this nucleotide sequence and I was basically moving it you know and if I found the sequence that was 100 match all 121 nucleotides matched exactly and it was on the side it was called acante niba castillani it is amoeba it's a free living amoeba that occasionally causes a neurological disease so I mean it was just amazing I was so and then then of course when I started reading about it turned out that the 20s 28 srna is split in half so that's why it has three bands of so it was everything explained beautifully and then when I took this virus supposedly to electron microscopies they um to The Institute of parasitology they say where did you get such an excellent sample of honey but can I use it for my lectures so it was classical and the dramatic transformation of the cell morphology was very simple because it was amoeba yeah it was not a cell culture and even if you you know try to use this Focus you know fine focus you can see that the amiibo were sitting on top of the culture so it grew perfectly so it used cell culture as a substrate but it was not inside the cell but on top of the cells though so I was shocked that it was so easy by sequencing to resolve something the mystery that existed for um for uh 10 years and while I was still kind of unsure what it is before I sequenced I started collecting samples from all over Moscow all um kind of bacteria and chlamydia or all other organisms and since it was so easy to sequence I accumulated a huge database of FS RNA I think at some point I'm sequenced more firefest RNA than anybody else combined so I had a huge database and of course I didn't know what how to analyze them I started asking around does anybody have any software to do it and first of all there were no computers there were mainframes because Soviet Union was under embargo there was no way you can import anything at the time there was none there was not even Macintosh I think that Steve Jobs was still in high school I mean it was a time when there were no personal computers uh uh but there was a guy working in the Institute uh uh who actually they did have uh they did have a personal computer uh it was called Why cat it was before before way before Microsoft or apple it was Unix based computer that was smuggled in again I mean by some Swedish salesperson who sold them some equipment but and there was there was some change that he owned them and so he asked me what can I bring you they said bring us a computer so he just brought it in a suitcase so nobody asked questions so so I I was I had access to this computer and I met a mathematician who and said that he he was a topologist Sergey yushmanov and he said well he just got his PhD for some mathematical work on graph Theory and I said I I visited his seminar and he was talking about the phylogenes and so on and reconstruction uh and I actually after the seminar I came up to him and said look can you write a program because I have a computer you know the algorithms so he's a short let's work together he was about my age so we were all in the early 30s or enthusiastic and so on and so he we started writing he wrote a computer code that wouldn't work and he would work from home you know mathematicians they don't want to go to offices very often so I would call him and say you know look I mean it doesn't work it gives me such an error message oh I'm so sorry I mean online 156 there is a comma and there shouldn't be a daughter period I I actually go back and I said okay I replace it and it works so I started looking at the coordinator well it's almost in English if four and all this and so I started doing it myself so at the end of the day I learned how to do this computer programming and I wrote this substantial Suite of programs myself including uh reconstruction of phylogenes using different algorithms and Sergey and I we came up with some new algorithms that Eugene mentioned last week so we published but only in the Russian literature because I mean we didn't have time to you know write papers and so on so but we created this uh Suite of software products and used it quite efficiently because I mean then by by then Eugene got interested and we used my programs to reconstruct all these phylogenes as polymerases and so on that's how these papers there we published together came about and then Leonid Brodsky a mathematician who worked at the same that's the beauty of this place I mean you can find any anybody somebody who had a smuggled in computer somebody who is a mathematician and so on uh so once we met and he said well I heard you're writing as a biological software he said let's maybe try to sell them let's start a company at the time it was called cooperatives I mean it wasn't just very strict it just started so they allowed some kind of private Enterprises I said wow who would need this is is there ideas for my research only yeah so but nevertheless we ended up forming this software company that was called gin B uh and we would go to another city to some biological Institute or give a seminar and then sell it so that's why the last few years of our life in the Soviet Union was kind of interesting it was a spin-off yeah basically so that that's how this whole bioinformatics thing started with Sasha and with Eugene but I actually very soon in in a year or two I moved to the United States and then started doing very different things so why did you why did you come to the US what's that story well the story was that now I told you that like when I was studying this microbial phylogeny and classification and at the time uh of course Carl Lewis was you know developing this ribosomal RNA based phylogeny and most microbiological and uh you know journals they stopped accepting papers describing a new species uh without phylogeny yeah rightfully so I mean at the time it was kind of absolutely a requirement uh so um and uh since the rumors about me doing this spread around Moscow I was asked by there was an Institute of microbiology um and the Soviet Academy of Sciences so the The Institute was famous for a lot of expeditions they would do to some remote places and finding microbes here and there and everywhere and but they had a problem with publication so they needed somebody who would do it so I would first help them to do it and then the Institute director changed so they it used to be a kind of a guy in his like maybe late 80s I mean completely I mean from a totally different era he wouldn't understand much about molecular biology at all so he was a kind of well anyway so he basically they managed to get him out and then appointed a new a director who was in his I think maybe early 50s much more you know kind of modernized westernized and kind of quite reasonable so they talked I mean all the associates they talked him into inviting me to come to their Institute and at the time I was basically I was doing all the different things Vadim didn't like it at all because he said well I mean you're not doing virology anymore you're doing all this phylogeny you're running you're doing computer programs or whatever you're not interested in a virology anymore so so basically I was still in his in in his Department in Moscow University but sort of our relationships we basically split ways for for a while he was doing his thing I was helping him a lot for instance I wrote for their they worked with all in queue at the time on Galino lipsky and all in they worked on this oligonucleotide maps to distinguish vaccine derived viruses from Wild viruses and so on so they needed basically they would run this two-dimensional jails with spots telling them the difference but it was not easy for them to kind of match each spot with the sequence so I it was for me it was easy I just wrote a computer code that would display on the screen the theoretically derived oligonucleotide map so that basically you put in a sequence and you get the map on a computer so for them it was very very uh important to have this kind of software so that was the only collaboration I had with Vadim at the time so and finally I got this invitation uh to build to become a lab Chief in the this instance of microbiology it was a huge honor because the previous chief of this lab was academician you know well established and he was a former director and so on so for me it was like 35 years old uh it was a huge honor I mean it's just to become a lab Chief in the such a prestigious place so but so I accepted this offer uh but I quickly realized that it was not what was bargaining for because they had no money it was the end of the Soviet Union everything was falling apart Mikhail Ivanov who was this new director of The Institute he promised to me good funding uh so that we could have no problem but we had not even a Furniture we we got a new um three rooms without the furniture and he told me well you can hire 10 Associates so I basically I invited um Everybody whom I respected of my former students including Eugene Cooney who was my Deputy at the time so we were very enthusiastic I was the oldest 35 years old the rest of the bunch were in their late 20s so we were so excited that we have such a young lab I mean we will you know basically move mountains and so on but then we were sitting in the bare walls I mean unfortunately we had some chairs that we could have seminars but and also fortunately each of us retained some connections to their old places where we came from and including myself I had I still had the lab in the Moscow University and it lasted for a year and a half and I realized that I'm not going to spend my most productive years you know basically sitting in this bare walls with no furniture and fighting windmills I mean it was just not so I started thinking about uh going to the United States I tried many times before and I was never a eckered women invited with me twice you know a few years back when during the Soviet Union before perestroika I was never allowed to travel probably I was not a member of a Communist party I probably told too many jokes about brush knifers I mean I'm I I would love to see my KGB file but it probably was extensive okay so I was never allowed to travel but then the time have changed and I was already a lab Chief so on so it was easier so I thought maybe I will try so I I wrote a letter to Carlton geidusik who was a good friends with my parents and also we became friends so I mean so and he's sure absolutely no problem because I mean yeah I've seen your paper about this kpn agent being an amoeba so why don't you bring the samples with you and we'll we'll start this we'll continue this research um and then at the same time I I actually was introduced to a lady in S11 book who was also a Russian expat so she immigrated from the Soviet Union she used to work at The Institute of teresevich Institute which is a counterpart of cyber in Russia so it was a controlled Institute of control of biological products uh and but she immigrated in 1979 and she started working for a receiver so she it was the first time that she came back from to Moscow from the United States because of course they left forever because then there was no way I mean if somebody emigrated from the Soviet Union they would never allowed to be to come back but during the curvature of time it was relaxed so she came back and we were introduced to each other we spoke for five hours and obviously we liked each other and she said well look if you have somebody if you have a kind of a young student who would be interested in coming because I have a interesting project that I want to start so if you have a candidate just give me his or her CV so I gave him her a CV of one of my young Associates and she left and then I went on vacation it was August or and when I came back from vacation I found two telegrams uh you know the old-fashioned telegrams with the strips of Costa and I found the good I am I found a good position for you not for your guy but for you it's a two-year position at FDA so please come and so on let's start working on this and I when I went to my uh Institute director he said oh no no no you cannot go for two years you're a lab Chef you must be here so but you can go for a couple of months and just you know make contacts and just look around and so on so okay so in months later I was in the here and the first day I walked in the lab I said no I'm not going back I'm not stupid because I mean it was and or the business was Carlton it directs for so long and he was traveling here and there so it never actually materialized so that's how I ended up in sieber what year was that it was 1979 the the very end of 1989 sorry uh so and then of course the project was on polio and it was the first time I touched polio so it was ironic I I tried to escape this fate for all my life and ended up doing things that my parents did so it was so when you were here now in the Washington area Albert Sabin was here right oh yeah yeah so you must have been in contact with him right or very often yeah he lived in New Mexico Avenue so I I would be a conduit between my father and him my father would send me letters in Russian I would translate them in English and I would go to him and meet his wife Louisa and we would spend time they would invite us for dinner and we wouldn't write them and so yeah no we interacted so he he was a very nice guy I mean personally he was very uh I remember once he invited me to a cosmos collab a club that you visited recently right yeah that's uh last night yeah yeah yeah was it last night uh yeah wait today is Thursday right yeah yeah it was I gave a talk at the Cosmos Club here his picture is on the wall because yeah yeah he was a member absolutely that's right yeah so let's talk about some aspects of what you did uh um at here at the FDA one of them I mean you got involved in working on polio vaccines it was opv mainly right at first yes you know and um you developed and this is how I got to to meet you through this test that you developed for looking at the the five Prime non-coding region changes and and making sure that production lots of vaccines had the right it was called map wreck right yes um muted analysis by PCR and restriction enzyme cleavage so tell us what is what was the General problem that you were trying to solve well the problem is that the strains there of um you know oral polio vaccine Sabian strains they are genetically unstable so when you grow them because I mean look they are attenuated meaning they're their Fitness is reduced intentionally so that they don't cause disease so they're basically attenuated uh so but uh all live organisms they want to reproduce so they actually like to increase Fitness so this is a general biological law that all organisms they want to increase their Fitness and live as much progeny as they can so of course uh this happened and Albert saving knew this I mean because he tested his vaccine I think that he he used like 10 000 monkeys before he actually was sure that this vaccine can be used in children so he was quite thorough in his experiments and he knew that the virus if it's grown under inappropriate conditions and virus that passed through the gut that is excreted has a higher virulence than the original strain so it was known well before molecular biology but of course at the time nobody knew what was the you know molecular basis for this de attenuation so that's why he required and he demanded that anybody who produces a vaccine from his train must do the monkey neurons test meaning that each lot of uh vaccine which is consists of three stereotypes right each of the serotypes must be tested in uh in monkeys so the first test was they inoculated intraspinally and into cerebrally at different virus concentrations and then they would sacrifice monkeys make sections of the CNS and they look at the lesions and so on and compare results for a group of monkeys inoculated with the test vaccine with a group of monkeys inoculated with the reference vaccine so then after a collaborative study that was conducted in the early 1980s they came up with an improved method uh which streamlined the process but the principle was the same you have to inoculate a lot of monkeys with vaccine and the reference train and compare the results they developed a special scoring system uh grading lesions and inflammation on the scale of one to four and then averaging get out I mean it was a very cumbersome test but it was first of all inhumane uh using so many monkeys because for one lot of uh trivalent vaccine you would need 200 monkeys and in many countries the test was supposed to be run by a manufacturer and by the regulatory Authority so I mean it was extremely expensive monkeys were at the time I remember a few thousand dollars per monkey because they were bread in a special colony in the United States in other countries they were imported from Africa or from Asia so it was really I mean something that everybody wanted to get rid of I mean but without this test it was not possible to control and make sure that the vaccine is safe because it is not a exactly a safety test but it's sort of indicative for virus stability so if a vaccine batch was prepared in an appropriate conditions the thinking was that it may not be safe because this we know that these mutations are increased virulence in monkeys and by inference it was decided that maybe it's also connected to ovariance in humans no we don't actually know because no one ever released a lot of no actually it's not yeah it's not true there were instances when vaccine lot was released and then later on upon retest in another country would be shown to be inappropriate in unacceptable in terms of monkey neural virulence so that actually helped us to develop this Maverick that lot would it cause more paralysis than you would expect yes yes no no it did not no it did not result in any single case of VAP which maybe tells us the monkey test is irrelevant for humans right well it's not irrelevant it's a consistency it's the consistency it is it may have nothing to do with virulence in humans because every single case of Epp was caused by a vaccine that passed the test and so vaccine-associated paralytic petroleum right right I mean all this unfortunate very rare unfortunate cases of vaccine Associated paralysis were caused by vaccine that passed the monkey test was flying colors I mean that's uh it is not a safety test but it's a consistencies test which is also important it's very important because every consistency is very it's a Cornerstone of good manufacturing practice I mean you have to be able to produce something consistently Time After Time After Time so but never and especially for knowing that this is a consistency and not a safety test of course everybody hated monkey tests and by then uh uh Phil Miner and Jeff almond and his and their group they have identified a mutation that was primarily responsible for this de attenuation it was in the five Prime utr and later other groups demonstrated that similar mutations in other stereotypes of polio because Finland and Jeff they worked with type 3. also are located in the same hairpin structure in the called domain five in the five Prime and translated region which is we know now that is part of Iris internal ribosome anthracite a very important element for virus life cycle for protein synthesis so in NASA came up with this idea so maybe there's lots that fail a monkey test maybe there is a increased content of revertence and she actually talked with her son who who also graduated from our department at that time I think he worked in MIT no no at the time he moved already he graduated at MIT from he worked with Alec rashevsky and then he got his own lab in the University of Chicago Illinois and Chicago and they actually discussed all this because he was a molecular biologist and he basically I think that he gave her this idea that maybe this this can be done so that was the project I was invited to work on and when I came very quickly we realized that this by then they had some experiments that demonstrated that this PCR actually the the the the way it was done that one of the primers was located next to the uh this 472 position and it was modified the three prime end so that it would create a restriction sign that would be toggled on and off by this mutation right so the uh normal vaccine sequence would be cut by in F1 and the mutant would be cut by MB on one so it was very easy so they have done some preliminary experiments and when I came and said well guys I mean we're never gonna get enough sensitivity unless we use um uh use radioactivity so we started labeling one of the primers with um gamma ATP and then around the gels and look at the X-ray films and so on so that's how we basically and and it took less than a year to to demonstrate that it first the method works and second fortunately we had uh close to 300 lots of vaccine made by different manufacturers so and we knew they were all tested in Monkey test and we we knew which one our fail built in which one are passed so that's why for us it was easy to tell them all and then plot this chart that Phil Miner showed that the Symposium last last week and it was a remarkable I mean realization that all batches that passed they were uh had the content of mutants less than less than one percent mm-hmm and vaccines that failed they were above one percent and there was a quite a clear cutoff line that was determined by the U.S neural Airlines reference not surprisingly because this was the reference that defined a pass uh or fail result uh so and we published and actually Albert saving um I I called him and I said well look I mean we have this paper would you consider it for publication in pnas uh and he said no question I'm so sorry I I decided I no longer will submit anything because I'm getting old and so on but please send me the paper I sent him the manuscript in a few days later he calls me question I read your paper and I decided I will make an exemption but but only on condition that you will accept all my edits all my changes that I will suggest I said sure I'll be honored and so on so I spent few days in his kitchen in his apartment um he would use this red pen and would cross out every instance of the word mutation and replace it nucleotide change nuclear pages okay all right that's not bad that's actually that bad that's probably in retrospect it's probably right it's right yeah I mean yeah I'm not much into semantics but I think he just wanted to avoid because that's why we call them Snips instead of mutations exactly because it's a polymorphism the polymorphism and he said well mutation has a negative connotation and in this case you don't know you don't have a phenotype I mean even so so he was great I mean he was so smart it's amazing uh he understood all this concepts of this causes this is way before anybody I mean even thought about this not even the term but in this that's how he selected his trains yeah I thought it was a kind of alchemy you know basically he passes here and there different cultures like purified I mean just like most people think it's a kind of empiric pro it was not empiric process he knew what he was doing and he explained to me how he came up with this idea he said that viruses are a mixture of different particles with different genetic potentialities as he called so there's always a mixture and it's a matter of selecting those that pre-exist there and I realized that when you pass such virus at a low multiplicity they retain their properties if you select at a high multiplicity do rapid passages they quickly lose their feet he didn't use the word Fitness but yes they lose their virulence so that's why he passes them 30 rapid passages meaning that he would infect with this quite a significant Moi multiplicity of infection and would Harvest 24 hours later yeah regardless of CP and then do the same essay and he passes 30 times and it worked so he selected the virus that contains and 472 U instead of C something that virus would love to get rid of and the same goes for this another mutation and VP vp1 nucleotide 6 yeah to 493. uh also I mean this is a mutation that is replaced reverts back like in an instant you know two passages and it's almost completely reverts back but somehow he selected it so I mean there was something to his reasoning so that's why and it worked for all three strains so why is if you're if your molecular Test shows that the amount of C correlates with monkey newer virulence why isn't it a safety test then because it's it's the amount of C will then predict what happens in people or we're not even sure about that well I I don't know I mean I think again this is a matter of consistency uh we also made an experiment that also puzzles me um it makes me think that there's something else in play because I mean for instance we would inject a monkey uh spinal cord uh with a perfectly good vaccine with a U at 470 yeah yeah absolutely quite an acceptable with low levels of all these mutations and then sacrifice them a day later like 24 hours later and would isolate virus from the spinal cord or just do PCR on the homogene it it would completely reward it means that if you if you inject into monkeys into spinal cord virus with hundred percent for 72c they would be paralyzed instantly but 24 hours later all virus that you can recover from the spinal cord is already mutated and they are not paralyzed so and in fact in kids and every kid who gets yeah within 48 hours it's reverted but only a few get paralyzed right in kids it's not 24 hours it's more like a week okay uh but it's uh it's a gut you know it's slightly different you know because uh virus polio virus rarely gets into the CNS at all but in this case it was spinal cord it was where the motor neurons are and it's well my interpretation now there was some innate immune response going on so that would virus would revert and because you cannot imagine that there will be a specific immune response you know uh 1924 as too soon but there was something that happened but after inoculation that would protect neurons or CNS in general from this virus that was breeding inside the CNS and nothing would happen and yeah so it's it's still still to be explored I guess well unfortunately I don't think we will be able to because people can't work on polio very readily anymore oh that's it yeah that's the end of the road it's a big problem because I think there are a lot of questions that need to be addressed and yeah absolutely well look Vincent I think that that was one of the reasons that I decided to move on because I mean it became so difficult it's just an uphill battle and this poly eradication is not helping I mean it's it's like I said last week I mean they did not eradicate the virus but they eradicated polio virologists because I mean a lot of people just voluntarily say okay the hell with it I mean we don't want to mess with this stupid containment which makes no scientifically makes no sense and it's now all in the hands of bureaucrats they don't really care about science or even public health because I mean this program I mean is been in the Dire Straits for many years but now it's 35 years later we're talking about the eradication eradication program and it's still it's like a and nobody wants to think a kind of stand back and think about what are we trying to accomplish so it's like a Flying Dutchman you know the ship is moving and nobody's on the ship it's moving and people are doing I mean a lot of people are involved and nobody's thinking about the strategy it's just it's a shame but here we are so I wanted to end on talking a bit about eradication first of all before I do that the map Rec test you did devise was that ever used and did it ever replace the monkey test no unfortunately not I mean it did not uh um we have like the first year when we developed we published a paper and then of course who got interested in a lot of companies also were excited about this possibility and so we started who started sponsoring collaborative studies with the participation of vaccine manufacturers and National control authorities and it lasted for almost 10 years because it was started with type 3 then type 1 and type 2 and so on and it was it's quite a lengthy process so we figured out a lot of we are basically identified a lot of glitches you know technical tricks that you need to be aware of it's remarkable it was a very uh telling experience how they the research grade test differs from the you know regulatory grade tests I mean the rigor of evidence the level of certainty it must be much higher so but one way or another at the end of 1990s it was recommended by the expert committee on biological standardization as the preferred in vitra test because our arguments that it predicts uh almost 100 percent and the failure in in the monkey test uh fell on you know basically deaf ears uh because I mean at the time this committee was mostly composed of for old time you know uh biological standardization types they noon almost nothing about molecular biology and I remember when I was presenting somebody asked me are you telling me that by looking at this x-ray film on this black spots you can tell me that is safe to inject into my grandchild I said well you know I think I do oh no no no no no we still need you know we still need a uh monkey test but fortunately at the time in our lab in S11 book and Eugenia dragoonski they started working on using transgenic mice for as a replacement for monkeys and also they went through this rigorous and laborious and time-consuming collaborative study process to basically implement it so it was also accepted by Who as a replacement for monkey test in conjunctions with metric so basically these two methods that were developed in our lab became the gold standard for opv lot release and so now opv that's made I understand there's just one country in the world that makes opv is that on I'm not sure no I I know know that probably more than one because there's a lab in Indonesia Indonesia I I don't know if I am if Pastor mirror I mean the sanofi still makes it okay maybe they do it the jsk I think they they used to make it until recently maybe they're not anymore but I think that there's no they make it also Indonesia yes definitely Indonesia but I think also somewhere in China okay but they they use mapreck and transgenic mice no longer the monkey no I think there's some some companies still use monkeys because for some of them it's uh it's cheaper to you because transgenic mices are not easy to breed their um they're produced by in Japan by this Central Institute of experimental animals they're quite expensive so that's why uh I I think that some countries still use money okay got it oh yeah Russia Russia still produces and they use monkeys uh so yes I mean it was implemented it was implemented and now it's being used but now we're trying to get rid of it I mean Maverick yeah and because of course uh it's kind of it's a 20th century method and it's requires already active isotopsy you can use fluorescence but I think it's still uh preferable to use radioactive isotopes which is now difficult for uh regulatory purposes uh reasons yeah uh so I mean uh so that's why after we have uh done this with Maverick and it was one other actually I think more legitimate argument than just looking at the black spots uh was that your look at just one nucleotide no how can you tell us that there is no reversion at some other certain I mean that was a legitimate uh argument that's why we started exploring different methods uh mass spectrometry and microchip hybridization all all these new things that actually came along the way in the early 2000s and then until of course this new Next Generation sequencing came along then I instantly realized that this is the solution perhaps so and we published I think in 2010 we published a paper with my associate on the use of Next Generation sequencing as a replacement for Maverick and it was just remarkable I mean you can't quantify quantify the mutation at 472 C in type 3 opv and quantify it exactly get this exactly the same numbers as with maprick and uh but also you could identify the entire as we call it now SNP profile so you can measure the content of every type of mutant in every single nucleotide in the genome so it generated enormous amount for information so we immediately realized this this could be a solution and we proposed in that now 13 year old paper we proposed that it could be a replacement and then we started the collaborative studies again under the who auspices and it was accepted just last year A couple of years ago it was accepted as a replacement for maverepric because now it's much easier to do basically it's very well standardized method companies they don't want to mess with all this equipment and know how they can contract it out right I mean there are a lot of companies that will do it for living basically they have a highly proficient and so on so but now we're still working on uh on on replacing the monkey test right because they and we presented this concept last year at the expert committee on biological standardization at who suggesting that I'm looking at this SNP profile it tells them you're much more than the monkey test because monkey test is not very sensitive and it looks on just one phenotype but there may be some other things for instance mutations can disrupt antigenic sites and it's also a matter of consistency right so in the ecbs they were quite positive and they say you know it may be a good idea I should just go ahead and organize a collaborative study get everybody to learn how to do it and harmonize and so on so now we're in the process of doing this and hopefully this will be um I mean I think the the plan is currently like next year to wrap up the collaborative study and come up with the criteria for pest fail decisions because it's not trivial and it's again it's um it's a heavily bioinformatic task you know essentially yeah you have 7 500 roughly 7 500 nucleotides and each nuclear data can have three different types of mutations so we're talking about 21 000 measurements how do you make your decision based on 21 uh 21 000 analytes quantitative results so well you can use your viruses that pass their failed the monkey tests to generate some some baseline for that right not really because I mean every company makes their vaccine whether different profiles okay even though even though theoretically they come like two passages from the master seed virus that is stored at the who I mean this still you know conditions even one passage can change significantly so that's why one solution would be for each company to establish their own bass line for this profile and then stick to it but the question is how much deviation you can afford is acceptable what if a mutation comes up uh how to decide whether it's significant or insignificant when you have to switch to a new seed virus which happens because you run out of the old one uh you make a or you change conditions something you you're important you're installing new bioreactor with slightly different conditions how do you make sure that this is still acceptable so there are a lot of you know questions that maybe may look trivial for those who are not involved in this Regulatory Field but then somebody must have exact sop how to do it and the SOP for not only technical but how do you make your judgment about the acceptable unacceptable so and it's it's quite that's what they call regulatory science right answering all these questions uh for most people are mundane and uninteresting but without answering these questions you cannot make a biological products sure all right let's end up with eradication so 1988 who said we're going to eradicate polio and now today we have no more wild no more disease called by wild types two and three on the wild type 1 continues to circulate but we have replaced uh the types two and three with vaccine-derived viruses so and in fact there's more polio now than we had previously for many years so and it's all caused by vaccine derivatives so what is your uh outlook on the on eradication are we able to eradicate and there are two questions I mean one is can we eradicate polio myelitis and the other is can we eradicate poliovirus and I what was the original goal was it poliovirus or the disease to be eradicated well my favorite response to this is to say Bill Clinton it depends on what your definition of the word is is indeed I mean what does it mean to eradicate the virus actually there is a very clear definition uh that if you use vaccine and you control a disease at a certain level that is everybody thinks is actually there's a control then elimination means that you have no cases correct but you only can maintain this zero morbidity if you continue to apply measures of hygiene vaccination and so on so this is a situation when you basically have no disease but you must be vigilant and do everything to maintain the status eradication means entirely different thing it means that you eradicate you're uprooted completely you know basically there's no way virus can get back into circulation so it can stop taking protective measures you can stop vaccination because there's no virus and it will never come back that's small pox that's smallpox right even though in retrospect it's not so because there was a good reason why at the turn of this Millennium they had to develop a new vaccine and put it in a stockpile in case it comes back because it's still there not only it's there you know it can be easily synthesized now right so that just for this reason alone I think that in a strict sense of the world eradication of Polo is not possible uh but I'm not going into semantics and I'm not going to argue let's not let's erase this issue and say that it's not possible I think it is possible in a common sense uh sense of the word that eradication means there is no disease and actually when the wha World Health assembly resolved 35 years ago they resolved to eradicate polio they meant exactly that because if you go back and read this declaration that was I think it was signed on May 13 1988 35 years ago it says that we resolve to stop uh poliomyelitis in the context of the Improvement of vaccine delivery of all other vaccines so the idea was that we can eradicate poliomyelitis there was not a single mention of the eradicating the virus not even once ah by improving vaccine coverage to 80 percent and they even call the exact number eighty percent I think maybe now we should adjust it to higher than 80 percent but at the time their belief was that 80 should be enough so that there will be no polio uh but very quickly in the program on that was established This Global polar eradication initiative uh there was a mission creep they basically interpreted as a a hunt for viruses I see and they set the unattainable uh milestone for themselves basically their measure of success was no virus no circulation nothing you know basically Chase it down to the last virus particle and we know this is extremely difficult uh because the the disease itself is sort of I mean benign you know it just paralyzes one out of few hundred or maybe a thousand people for those who are perilous it's not benign at all I mean it's a sometimes deadly disease a nasty disease and leaving people paraways for life but in the public health sense of the word I mean it's not really a big problem it never was until the turn of the 20th century when it became pandemic with the vaccination programs it's also not a huge problem in countries that can deliver vaccine but in countries that most kids are not immunized of course it's a problem and this is a problem for public health authorities why don't we immunize everybody one why don't we improve vaccine delivery and instead chase this ghost of this eradication of one disease that maybe uh a few hundred cases a year and some years is even less this year is even less than I mean close to I mean we're not talking about a thousand cases in the entire world so but still the the objective of the program is unchanged from 75 years for 35 years ago and you know the definition of Albert's of uh Albert Einstein what's Insanity it's doing the same thing and expecting a different outcome I mean I'm not you know saying that people who are doing this are insane but collectively we are insane I mean trying to do this over and over again so I mean we published a lot of editorial uh papers uh with Eckhart Wilmer earlier and filled by Jim mcaul recently with Bob gallo and Stanley Plotkin Christian priscio arguing that we need to go back to the drawing board and rethink I mean what we're trying to accomplish no response no response I mean it's just so that's why one of the reason why you think should I continue fighting windmills I mean it's well let's see I I wish it happens but I'm not optimistic because virus can hide in a lot of places there is also this phenomenon of Orphan lineages meaning that all in a area where folder was kind of declared eradicated and five years later virus is isolated with genetic uh genetically traceable to the lineage that was circulating here five years ago so I mean when you bring this argument those who are kind of arguing for this continuation of this the honor is just a failure of it's a kind of surveillance breach so what does it matter surveillance it can hide from us it's it can circulate undetected for five years and you say that after the last case of polio in three years we can stop immunization does it make sense how how you can make sure that there is no cryptic circulation okay you can look in the Wastewater uh in places and that have this sewage system and that's how this recent event unfolded in New York City in London and Israel but there are plenty of places on this Earth where there is no sewage system so how can you make sure there is no circulation and that's exactly where the virus is circulating it's impossible to do environmental surveillance uh worldwide at a quality that would be sufficient to make sure that there's not a single virus particle remaining in circulation so that's why I think that the solution is very simple just improve immunization vaccine delivery everywhere and fold this ipv or perhaps use this novel OPD that was developed into the routine immunization schedule depending on the local circumstances because if if everybody is immune who cares if the virus is circulating or not and if if there is a one case okay well it's very unfortunate but it's real life I mean you cannot really throw billions of dollars just to chase something that you can never accomplish I I don't know I mean I I wish them success I wish that happens one day or they declare it but again I don't know what it means I don't know if we want to do this do we really want to create a world where everybody is a hostage uh can you ever stop immunization I think no never ever I mean it's just unacceptable because if you stop immunization in five years time every child on the age of five will be susceptible and can you imagine the level the tragedy that will happen if some insane person would synthesize it from chemicals which can be easily done in a week right I mean can you rule it out no so okay should we make every child Hostage to the situation of course it will be just ethically unacceptable so that's why I think that immunization should continue indefinitely and when the current vaccines are not perfect I mean even recently we sadly we learned even this novel OPD that is by far more stable and genetically than the original one is those is also not perfect it also can mutate much at a much lower rate but still but there are so many new ideas new your technologies that can result in a much better vaccine so I think it would be unacceptable not to do it I mean I know that maybe economics is not there because with this number of with this number of cases it's probably nobody would want to develop a new vaccine but if you think strategically I think it will be it would be much better if we had a very inexpensive vaccine that would be comprehended induced comprehensive immunity but unfortunately I mean this is not a perfect world and we probably will have to settle for something but even the existing ipv if it's used in the combination with other antigens such as dtp this pentavalent hexavalent vaccine which now some companies are developing and already these vaccines are being used if we put more efforts into developing a worldwide infrastructure for vaccine delivery that would go much longer first of all it will promote immunization against that other diseases so it will be kind of win-win it will be a current of effect that would be not only on this polio which is doesn't really have a big economic impact right now I mean we're talking about 200 cases a year so again that's why I personally think that this eradication initiative is completely misguided I mean it was misinterpreted the original intent and now I I think that it would be it's it's a it's a good time to go back to the drawing board but who am I to uh well I I think you've thought about it a long time you are the right person but if I think your idea of developing new vaccines makes a lot of sense but we're being told to stop working on polio so can it be done in a containment world of course not of course not but the sad thing is that one initiative gets into the hands of bureaucrats that's the end of the story because I mean at who the left hand doesn't know what the right hand is doing I mean one part whichever participating I'm a member of The Polar research committee at who this is a group of people they understand all these problems and they do all their best to help development the uh this committee is reviewing Grant applications for some projects and vaccine development surveillance and everything so this is a group that is trying to do the right thing but there is another group that is actually doing this containment they deaf to all the arguments I mean they because they're trying to do their job their job is to stop all your research that's it right they want to stop it because they were told uh that it's not needed anymore somebody told them way back and now they're on a mission to stop and then it is who is not maybe the biggest problem because now it's delegated to each uh country and at CDC the same thing they they don't care if that's an important project they just on a mission to stop it so that's why I I'm not optimistic that it will ever happen uh and my pessimistic scenario is that they will declare education and then something really bad happens 10 years from now I mean so we will remember the covet outbreak as a kind of Cakewalk yeah if you stop vaccinating as you said that'll be a real problem yeah yeah it's not it's not candy it will be a problem so you uh despite retiring you're still involved with these questions right you said you're part of this who group right right yeah no I'm still involved because I mean I I strongly feel that this development of this next Generation sequencing approach it's it's not only important for polio because it's a kind of new paradigm for quality control of all biological I mean vaccine products because uh for for most vaccines you don't have such a costly animal testing on sale so I mean most vaccines are not tested but the issue of consistency is very important and not only for vaccine products but also for biotic and other biotechnology projects products such as for gene therapy and so on so I know that for some experimental development product development they they use something similar so I think that getting it through the entire regulatory approval pipeline is very important it will be a new paradigm for quality control so that's why I'm still involved and I want to help as much as I can even though I don't have a wet lab anymore but I have some expertise in uh both in bioinformatics and virology so I think that is important for developing of this new approach and interpreting results and kind of you know 33 years being at the FDA I learned a few things about circulation could you could one make a polio vaccine is not injected it is not infectious but which you would take orally and would immunize your intestines and generate immunity yeah I think it is possible it is possible there are different ways to do this I mean before my retirement we started for instance working on the RNA vaccine against polio and got very good results in vitro I mean we didn't finish it and hopefully somebody can continue it but we had excellent uh actually after a transfection of cells there was more antigen the antigen in cell culture than after virus infection so it's clearly can be done yeah so you you can actually if you can deliver this RNA then it will be a good vaccine I mean of course there's a long way to go I mean yeah another I think approach that I like very much is that George belloff proposed that he expressed uh polio capsids in indeed in Newcastle disease virus I mean there can be other virus but also with the very good results I mean good vlps with the antigenicity are expressed and can be delivered on mucosal surfaces so in theory you can make a vaccine that contains no live virus and that can most likely can can produce mucosal immunity similar to oral poly vaccine so there are many many ways to do it also a work on uh adulants for inactivated vaccine yeah at some point we worked on vitamin D3 that is known to redirect dendritic cells into peripheral lymph nodes we had good results in mice never tested in humans just because somebody else pursued it for a flu vaccine and yeah despite a good Mass Mouse results in humans that do nothing perhaps a difference in the vitamin D3 metabolism I mean there are many ideas so but without being able to work with Polo nothing would come out of it so it's so I have one more question for you if you if you had not been a scientist what would you have done I don't know it's it's very hard to I mean look and I'll tell you that I grew up in the Soviet Union I mean as the way I looked at it it was the only way um to live a kind of a relatively decent life and not make um moral and uh you know ethical sacrifices because what what else could you do in the Soviet Union what was the other career to become a party uh functioner or become a military guy or there wasn't when I was a little kid I I dreamed of becoming an airline pilot I mean but you know a boy is a dream about stupid stuff and maybe it's not such stupid but I was fortunate that I grew in a family that actually kind of subtly channeled my intellectual energy into something all right so all right that's a special episode of twiv you can find the show notes at microbe.tv twiv you can send questions or comments to twiv at microbe.tv if you enjoy our work of uh spreading the word about science to everyone please consider supporting us we exist on your financial support you can go to microbe.tv slash contribute my guest today question thank you so much fascinating career great stories and I think the polio story is not over yet yeah I hope so I hope so thank you very much for joining me thank you for having me I'm Vincent rack and yellow you can find me at virology.ws I'd like to thank the American Society for virology the American Society for microbiology for their support of twive Jolene for the timestamps and Ronald Jenkins for the music we've been listening to this week in virology thanks for joining us we'll be back next week another twive is viral

Channel: Vincent Racaniello

Views: 598

Keywords: virus, viruses, viral, virology, poliovirus, Maprec, neurovirulence, production consistency, Mikhail Chumakov, Marina Konstantinovna Voroshilova, CBER/FDA

Id: rnge4JXqges

Length: 120min 18sec (7218 seconds)

Published: Thu Jul 13 2023