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>> Good morning everyone welcome to NanoFANS Forum series and in this series we'll be focusing on nanotechnology in vaccine delivery hope you may recall last spring we covered nanotechnology in infectious diseases and so within a year with various activities going on around the world on vaccine delivery we thought it's appropriate to talk about this press special topic on nano technology in lax in their degree and we have been very fortunate to have 4 speakers Dr Phil sometimes it'll wolves will be speaking today and we have Dr Evan Anderson and Dr n.g. friend and Dr becoming a part of scar they'll be speaking in the upcoming You know very harsh this month so now fans will be know series is sponsored by Southeastern nanotechnology infrastructure card are in short scenic scenic is one among the 16 sites under a special program sponsored by National Science Foundation called national $100.00 technology quantitative infrastructure and the goal for this program is that they knew most of these were being funded under this program may be able to open up their infrastructure capabilities to extra No users so they have their own internal on campus users accessing these resources but because of this program that enables them to open up their infrastructure capabilities to off campus researchers both from academia as well as industry So in short scenic is a nanoscale fabrication and characterization resource it's a partnership between 2 academic centers in the southeastern United States one is us Institute for electronic Sonata technology or George attack and our partner joint school of nano science and I know engineering.

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That is an academic collaboration between university of not Carolina Greensboro and not Kernighan to university so what we do seen it provides easy user access to a wide variety of micro none of fabrication and characterization sets and process capabilities all along with staff expertise to researchers in nanotechnology Escalus micro technology or any researchers who may be able to access these tools set for their own research.

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So more information is available at the website scenic dot get ignored it edu extremely users may be able to access the we have 2 prominent access modalities one is on site very research is from off campus maybe able to go to our campus and get trained on a piece of to your tool that they desire to use and use the tools themselves also they may be able to send samples and we have dedicated staff members process staff members who may be able to do the work for them those are very prominent crease access modalities we have in this program and in short scenic is all about a one stop shop for your nanotechnology research needs some more information again is available in scenic dark got picked out of the view there are other programs that you may be able to relate to and you may be able to make use of our facility but that said I would like to give a quick promo on coming events as they said they enter a month of May On Wednesdays that Levon we have.

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Very Norse focusing on nanotechnology Maxime delivery and if you have not signed up or if you have other colleagues or students who may be interested in this topic feel free to forward them this link that this tiny u.r.l. dot com slash non-offensive in ours we also have another very interesting.

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I mean our this afternoon at 4 pm e.d.t. it's on computation talk the Malaysian software next door and it's by Dr gets caught with a Leica She's a professor of electrical engineering and I had another son a state a new city she would be having one of these very been our sounder and n.c.i. seminar series so with that said it's a pleasure to invite.

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Professor for Santander law will be talking to was and still is a professor in the Wallace **** department of biomedical engineering he graduated from Polytechnic University in 1991 with a b.s. in aerospace engineering in 1980 opened a speech Dean in. Engineering from the University of California at Davis under Dr Ian Kennedy on the development of laser based diagnostics for multi-phase reacting Jetson droplet streams Dr Sandra followed Spears to with a post or 2 from ship at Sandia National happy in Livermore California under Christopher Shaddix and position in industry at Micron Affleck's in Atlanta Georgia next door percent annual return to academia as a bone structure fellow and then as a research faculty member at Georgia Tech under Professor Gangle in 2070 started as an assistant professor in the m.e. and was promoted with tenure in 2013 to associate professor and now is a professor.

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Of biomedical engineering Dr Sandeep Santander most current research focuses on the development of imaging and detection technology for the Study of orany regulation and pathogens as of our new viruses but that said it's a pleasure and a privilege to invite professors and Angelo to speak to us on this topic.

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So I'm going to talk a little bit today about how to Marty based vaccines so my lab does actually a fair amount of work I would say actually of the focus of a lot of our work early is on m r n a base there puting x. and we're doing more vaccine work interesting we now have some of the data I'm going to show you is actually from over a year ago because it was all pretty coded what we were doing some of our in a base vaccine work in particular and then Kobe kind of changed things a little bit but.

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One of the questions I think that's important that I'm raising in in the title which has lots do with the local station of these m.r.s.a. base vaccines vaccines and specifically in non-human primates and I'm going to talk about a multimode ality approach to this and this work was performed by Jared Byers or from SARS early in my lab and with my contributions to though.

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I want to talk about some fundamental questions in m.r. Navy's vaccines and I should mention this so when we talk about m.r.s.a. based vaccines it's kind of a comp it's a very subject in many respects because every formulation is different so the cargo is different the m.r. in a is different all the different manufacturers of r.n.a. coding my own lab we use different parts and pieces to make the r.n.a. in a tube there are differences in purification there are differences in sequences in the T.R.'s and so there are differences but fundamentally many of us are using m r n a s Now there's another class of vaccines that also use this replica on the m. R.N.A.'s that that cells amplify over time those are slightly they're actually quite different they're much bigger usually in size and they have different characteristics but all these vaccines the the way that they're delivered now or formulated is in the lifted nanoparticle and clean is going to talk at the end of the month much more about with the nano particles so I'm not going to give you.

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Much data and info on on that list now particles but they are pretty much the way that m.r.i. Nasar and capsulated in formulated for Vaccines and they contain 4 parts to them and so there's an eye nice with all of it there's a peg limpid there's usually a helper limpid and cholesterol and so you have the m r n a that encodes for an agent and you have these 4 components and they're typically formulated in a microfluidic device they don't have to be but that's typically what's done in that way you get I would say the size is the actual size the particles are a little smaller they're usually.

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Somewhere between $700.00 nanometers in size and their apologists versity it's fairly low actually if you formulate them in that in that manner but there's a lot about how we administer them so when you would minister an m.r.i. a base vaccine is there one way to do it and the quest.

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I mean the answer to that is no people have their up in some studies where they've been given by I am they've been given by so interim muscular route they've been given intradermal ie not affect a lot of the early work on m.r. in a vase vaccines one was on injured intradermal and administration and actually not in the next day it would be kind of came a little bit later on and the reason for the l.n.p. was that when folks moved from intradermal to injury muscular some of the formulations they were using for id didn't work anymore and they really don't work very well I am and the l.n.p. did and that's really why the n.t. I would say took off is that the expression was much higher for I am delivery using an l. and b. and you have to get above a certain level of expression of that engine for you to have a proper immune response but in general the rod is Administration matters and I think there's a lot about that route and its effect on vaccine performance that we still don't understand and so this is one of the variables that we were interested in the other issue is how cell and tissue uptake and l. and p. trafficking affect the performance that there's not a lot of data on it all and part of the reason is that whole body maps of of where m.r.s.a. where the m.r. in a no and he actually localized to and functions in general have not been generated and certainly not in non-human primates so our 1st from my perspective scientifically we wanted to be able to develop a methodology that would allow us to track where m.r.s.a. Ellen people related vaccines would go in vivo and so we've actually done a fair amount of work on labeling r.n.a. in a way that does not inhibit its function and we had a paper now a number of years ago in nucleic acids research where we looked at the labeling strategy and then more recently a paper in Nature be anemia and some of what we used in the age of the any tape or the tools we were actually going to I'm going to talk about today which is a modification so one of the keys was that we needed to make our pro.

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Moves that we use on the r.n.a. to label it to be able to see where it goes we had to make them smaller so we made some small or modified our in a probe that would allow us to see the the m r n a and we put those typically in the untranslated regions but we've shown more recently you can stick them in the coding region or even in the Poly a tail.

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Without affecting function and these probes give us the ability to put a floor force on the r.n.a. so you can use them in a fluorescent House say in addition t. later is so why he later is the reason is that if we're going to image whole in a whole body manner where they are any goes we want the greatest sensitivity for seeing more than r.n.a. goes head imaging is really ideal and so imaging positron emission tomography allows us to get a whole body view and it's really the most sensitive way and we've been doing pet imaging in my lab now for a number of years so we had all of the tools to do that and as I said if you look at our nature be any paper some of those tools are actually highlighted in that in that particular paper but this is a little bit different because the formulation that we looked at in the nature of being here was not an l And it was slightly different and so this is exciting because we're going to look at what happens when l.n.b. Ok so what we're interested in we did some things in terms of optimizing the labeling approach and how we hybridised our probes to the our innate self and then analyze it by age people see and one of the things you can see in this is that the centrally with our smaller probes which are in the gray or the yellow You can see that the they really run along with the r.n.a. they're not affecting the size of the r.n.a. in a significant manner in a way that it runs differently on this amount of graft and then here one of the things we looked at we obviously looked at function in terms of inhibiting translation and then our new methodology which is in this darker gray here is very similar to the r.n.a. So we saw no statistical difference in the expression So these probes are are small they can be they can be incorporated into an l. and p. very nicely and they allow us to detect the r.n.a. and they allow us to they don't inhibit r.n.a. function but we did some other other analyses on the g.s. pm r.n.a. to see if when they were labeled and you can see actually the little white dots are are actually r.n.a. in this and then you can see green is the expression of g.p. and actually.

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Not all the cells that receive our National Express and that's a whole different story but the point of Hatter is that when we compared our different methodologies for labeling we didn't see any statistical differences in protein buy in you know fluorescents either we also wanted to see if we could use this to examine where the could you see m.r. again in the in an end to sound could you see that released over time and so they are bright enough that you can see the individual are in a zone which are in red and the green is actually markers for the basically a general General markers for the NDAA so more compartment because Ellen Pease do you are taken up by understood ptosis and the army is eventually released and so in this case what we wanted to see could we actually see where they go and what you're seeing here is a time course in.

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Of the l.n.p. and this is a time course of life in fact I mean which is a common transsexual agent that's used in vitro I should note though that we were doing this mostly so that we could be sure that using my cross could be that we could detect the are in a while and we could also see it relative to the and it's almost system unfortunately Ellin piece and he function in vitro does not we capitulate l.n.p. function in vivo and so on which is work that we've done with the with James James Collins lab and so James has been spearheading that sought for quite a long time and so we've been supporting him in those efforts but there's no doubt that in vitro you go don't really Coralie but we wanted to be sure that we could use these with current My Cross we technology that we would be sensing no sensitive enough to take the our name and you can see that can we did some initial studies just to see if we could see a release from the end so over time and certainly like effect I mean is released very rapidly these Ellen piece interact very slowly but over time you'll see them being taken up they'll actually you'll see release over time I can tell you this doesn't mimic at all what happens in vivo but it was just a matter of could we actually detect this in The point is and it does allow us to detect it over time but let's get the pet now so.

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In these experiments again the goal was to actually understand where the r.n.a. goes where does it end up so we label the r.n.a. with both one guided to highlight fixating which is a near or die and then we also had 2 later as for copper 64 and so the m. morning was incorporated in Alan p. the l. and p. though was that after it was after the r.n.a. formulated mix with copper 6437 c. there is some spin filtering through move any excess copper but essentially the l.n.p. take up the copper and the r.n.a. he laid cold this actually to the r.n.a..

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And so in this case I'm just going to talk about 2 mechanics they were injected with about pointing to this $8.00 mix parkade which is fairly low but again this is to simulate a vaccine given it I am and they were image to point 53 in 24 hours so there might be one that we took out 48 of our number correctly but this is what we get from a pet image and so I'm just showing you a single slice from the animals but because the half life of copper 64 is about 12.7 hours you can actually image the are in a 4 for days actually by not much more than probably $2.00 to $3.00 days but you can image it over time and you can do the.

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Testings you can actually image it in animal in the same animal over time and that I think is actually really important so what you're seeing here at Point 5 hours is actually the the mass of I have the the gains kicked up a bit so you can see all of the r.n.a. if possible but here what you're seeing is the bulk of it's in the muscles so this was given in the leg not the arm probably should have done the arm I think in the future will do some on but this was in the leg and the thought a muscle and at half an hour really it's predominantly in the muscle what you see by 3 hours though is that you'll see some of the muscle and this is the same animal but I'm showing you different slices through the animal pet c.t. imaging is 3 dimensional and you get all of that 3 dimensional information unfortunately though I could show you a 3 d. movie of them spinning around that kind of thing but honestly it's most it's easiest just to show different slices so this is different slices out of the same animal and so you can see it still in the muscle you can see some iliac lymph node at 3 hours you can see some in the liver and you're starting to see some in the small bowel and a little bit in the kidneys 2 by 24 hours you actually see still some in the muscle but you're seeing some actually again quite a bit more in the liver and you're seeing a distribution in this is the is that.

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Colon and you can see that here quite nicely this is the same animal and again at half an hour again you see in the muscle here this is just a different slice there's a little I mean you touch a signal in the liver and in the kidneys here very small again by 3 hours more and you can actually start to see the kidneys pretty easily see more signal in 24 hours in the kidneys you're seeing some of it also again in the liver and some in the sending it's in the colon actually missing and so we can quantify in 3 dimensions the percent of uptake in different organs and what you can see is that really it's 30 minutes it's mostly in the obviously at the injection site of the eye and site and what you see over time is it comes out and so a lot of it by 24 hours is really most of the signal is out of the out of the muscle at this point and so.

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Over 6 short period fiber a lot of it has end up trafficking to to the liver so that's what you're seeing here too so it does end up in the bloodstream and obviously some of it as I mentioned isn't the only our Cliff Notes We'll talk more about that a 2nd but you're also seeing trafficking the whole body trafficking are actually in full liver so once it gets in the liver obviously it's I mean for it to get through liver it has to be in the bloodstream and so there is a fair amount of it that's actually circulating.

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And this is the distribution across the animal over time so we looked at the iliac lymph node as I mentioned by 3 hours you actually can see that signal quite quite strongly which decays over time you're seeing uptake again in the sigmoid colon the ascending colon be seen across the transverse and small bowel and so there is a fair amount of it getting there and you also see some course in the kidneys too and so we did see it cross we did see actually very little in the spleen at least pipette which is kind of interesting thing is we do see some staining.

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Just the same. It is in a separate animal it was pretty similar in terms of what we were seeing again iliac lymph node and seeing it cross the gut and then some in the kidney to. We did verify it so since this is pretty much the 1st time so that would be done.

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In take tissues from these animals and we look specifically for expression of the energetic and so wasn't Covidien this is Hawk time Griego it wasn't spike but the point of that was you could see it in the muscles we were able to sustain the muscle in and both of these animals and you can see it in the muscle very easily so these are individual muscle fibers and you can see expression along them on the edges of the fiber or actually within the fiber itself and this is a control and so this gave us a pretty good we we did expect that that wasn't a big surprise so we would see it in the in the coal in the in the muscle but just the same it's good to see the liver this particular l.n.p. is not terribly prone to hitting about a sites and the dose was extremely low and so you do see mostly Cooper cells actually taking it up in the liver somehow the sites but mostly here some staining in presence but really it's a small number again livers very large in these animals and just to confess all of take itself is enough to.

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Account for are seen by pet but there is expression actually in the in the liver. Then the splitting We did see some now this is kind of strange we didn't see this much we did see some. Some signal in the can by pet but we didn't really catch as much of it but we did see some staining in the spleen and we certainly saw a staining in these sending Colin the transverse colon and there's a whole story about this that we're we're really interested in that we're that Jared is working on now but I think we have explanations for why we're seeing in the polling and some of the and why why this is happening in mechanistically how it's happening I'm talking about that we did see standing in the Lachlan snow then again his liver from another animal mostly in the cells and this is just more in the colon so the reason I'm sure this is that this is a pretty This was an unusual finding and again we think we have mechanistic understanding of this now and will be more of the story coming out in the future from them also we did look at flow cytometry of again because we have a fluorescent marker on the r.n.a. we can easily look at the percentage of cells in the left and right muscle obviously they were given the right and so what did we see in terms of cells infiltrating in the muscle and there were a couple things that are interesting to note here is that pretty leaves monocytes So I mean my slaves were clearly dominating I would say both the mouths of I'll take some conventional d.c.s. but I would say most of the uptake was certainly in in monocytes and what's interesting is that this is similar to what's been seen.

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By Karen Lori and I mean ologist Carolyn's and she did a study. Which I published of reference to but that was funded by me and with some of the Madonna Ellen piece and she saw similar I would say the data is pretty similar and that was predominantly in in monocytes So just keep that in mind and I think there's a lot here.

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James Baldwin I have a new project we're going to be looking at different Ellen P.'s and how this changes the uptake if we can skew the uptake to different cell types how does that change the the function of. Of the of the vaccine can we improve it and so that's something we're going to be looking at in the near future but I think that you know all the tools that we bring to bear will help us understand how these vaccines work and how to make them even better and I think there is definitely room for improvement and.

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Maybe to talk about that in just a 2nd the other thing we did is we looked at some of the. I would say markers for for activation and one thing I want to note is that you know in b. cells and t. cells you see some some improvements in in in these activation markers as you go you compare the left and right muscle you do see the right muscle go up but what's really interesting is that in monocytes it was pretty clear obviously you see almost 90 percent of them are activated but when you look at the ones that have r.n.a. in them you really see the difference in these activation markers so having our name label we think is really quite critical for understanding looking at the right cell populations and so this is certainly one of the ways that I think it distinguishes and why these approaches I think are helpful is that we really need to be able to find the cells that actually receive the r.n.a. more than anything else and in the end I think we have some new markers that will allow us to see not just who receive the r.n.a. inflow but also who are making protein.

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In flow also and so I think that combining all of these tools will allow us to really understand how we are basically affecting the immune response and so this is just a snippet of data put it in but it is from a marking and it certainly showed that the tool is giving us this kind of information.

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And so you can still fairly short talk but I'll comment on a few things stack so we were able to see the m r n l n p s express not just localized but expressing the muscle draining lymph nodes liver and spleen. Certainly monocytes seem to of really dominate the uptake in the muscle which is similar to some previous work.

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Again how that compares to what you're going to see if you were targeting other cell types I think is unclear the are any positive ones are certainly the ones you want to look at regarding activation and so and I think that's a big issue with a lot of these studies in that if you just go Well 1st of all if you're going to pull lymph nodes so one of things you notice is that there was a prominent iliac lymph you know that you see the r.n.a. there are plenty of other iliac lymph nodes and one of the things that we showed in our nature b m e paper was that depending on how the Army is formulated it makes shift nodes and so you get some nodes that were see that some nodes that won't and so you have to be able to pick the right ones if you just randomly pick No if you even missed the node than actually received the r.n.a. And I think that that's one of the real problems with these studies is that and some of the studies are even going on now is that is that they don't they're doing this blindly and I think using imaging really allows us and labeling tools allows us to get at what's actually happening you know pull the right to shoes because in a mouse you might be able to get away with it because there are far less less lymph nodes but in a monkey there are a lot of lymph nodes and so really getting into correct lymphoid tissue is really important and this allows us to do that and so one of the other issues I think of all of us is posed this is that we clearly saw expression in the gut and so gut is part of the mucosal mucosa part of the coastal immune system and a big player in coastal immunology and so one of the questions I have is that is that possibly why these vaccines are working so well for the rest for a system in that we're getting the expression because and so is expression in the gut actually helping because the.

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System both in the long cut and in fragile is all connected to each other and rectal and so there's connections between them you know logically and so I think you know I think that's an important finding and again I think there's more work to be done to understand localization and whether or not it impacts the function of these vaccines.

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I think it's an it was certainly an important finding in these animals but there's more Stay tuned for her more information on this I think. I think that's all I really had to say today about m.r. Anyways back scenes and so I hope it's a fairly short part but I hope that you found it interesting but I think these tools are really going to be important and in just a plug for dark Dom's Labview I mean I think James is tools for using basically developing single cell or any speak methods coupling those to the image imaging tools that we've been developing will be really quite I think helpful for us to really decode how the formulation how they are in a and there are the ministration and how that actually influences the outcomes and I think there's there's a lot of work to be done to understand that.

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I'll stop there. Sandra actually listen to. This time for questions seriously having questions feel free to quickly. If you can text it think I have some text here to read for. You and the 1st one is here yes feel free to it's from same different it's from the tracking cells that get the m.r. name makes sense can you then home and on cells receiving secondary signals from the m.r. nutritive selfs.

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Yeah so I think we can I mean. Given that we kind of know where to look now if you're looking for things like cytokines or looking at other cell specially immune cells markers we do have a sheet. For looking at it again. For detecting cytokines and for detecting other immune cells and their changes in function in Bebo So that is something that we we do have ways to do that and certainly again knowing where to look also helps us in terms of just adding flow cytometry and other methodology is to look at so.

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So I guess the point is that yes I think adding tools to the tool box to really get a complete picture of what's going on is something that absolutely has to be done so let me just answer before I get to Cindy there was a cup there were a couple of other ones Dr Henderson had do you think the distribution of taking the intestine is just metabolisms Grecian liver No Wait a minute.

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Just metabolism excretion by the liver No not exactly and what we have I would be happy to follow up with you once we have a little more data so I don't think that's true completely I do think it is a the the liver have had a bit earlier it's basically the head of delivery system is involved but I can update you really soon we have some ongoing experiments where we're looking at that in great because I think it's not just excretion as we can talk about that in the near future definitely.

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Does monocycle talk with D.C.'s or other. Is what most of these Swedes and your position. Do monocytes Cross Talk of the Seas Yeah absolutely I mean there's Yes And so absolutely and I don't know what happens to some of these monocytes once they're out of the. Outside of the muscle either so I think there's Yeah there's quite a lot to be understood about monocytes presentation who they're presenting to supposedly they are presenting cells and humans and cells and so they can present and so I think there's yeah yeah a lot more to understand about their role I was actually surprised I thought we were going to see a lot more D.C.'s but we didn't but again it matched some of the data from Karen Lawrie And so how we shift that distribution or not is something that you can be really interesting to see what the results for.

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Let me see in city looking at other routes of history and yeah absolutely I mean long term certainly into something. Held is another whole another whole story and so we are developing both with M.G.'s lab and with changes lab ways to. Inhale. Are in a base they are formulated r.n.a. that's that's in the works we've published some of that and some of that's coming.

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I would say there is we have some of my lab has an intern is a war coming up soon to that's fun to the Gates Foundation so we're going to be looking at that and see how well that look how that works both for preventatives and for. And possibly as vaccines that that's coming to and so and we're even looking at d.c. based vaccines with a group from Yale so I would actually say that the route of administration is long and and there's a lot there's a lot to go look at there I think one of the questions you know what what we've been working on in terms of inhalation is mostly not provoking immune responses so far of therapies that are in the long we've been attempting to create.

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I guess I'll call it I mean a silent methodology for delivering r.n.a. and. Along So if we are going to deliver a vaccine though we may want some inflammation we may want to drive a little bit but you don't drive too much for that would be really bad so I think that's one thing about inhaled.

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I would say any kind of unhealed vaccine that gets actually deep in the long has to be careful about that I think it's intraday Zal and it only stays in the upper airway inflammation in the Nolte's would not be bad or in your upper airway would be Ok briefly I think probably would be fun but it would be Ok And so I think that that's another possibility it's really a matter though are we getting strong enough expression are we getting and actually as a matter of fact I think James and I are now I think about you know one of our vaccine projects coming up we will be doing some intranasal delivery now that I think so that is coming and we've done some l.a.p.d. internees a livery in the past and you can definitely get expression but you can also get too much inflammation so I think dialing in the the amount of in the imaging to see so to speak with the inflammatory effects of the l n p is going to be important I think what's interesting about vaccines is that I think about the way James has a you know it has fantastic ways for seeing where Ellen pees or discovery when he go to different parts of the body and I think mostly looking at function the function being to find his expression I think when we start doing these vaccine projects together which we have a few Now we're going to be adding these other variables you know I mean you're adding this is not just the function being to deliver the aria functional r.n.a. but also how much basically how much algae an effect did that l n p have or not and then also what were the best characteristics for the m.r.s.a. you know does it need to be as clean as it normally is for a therapeutic if we actually have some action in fact from the r.n.a. and that a problem or not in the past many actually published a few papers on this but we have tools for looking at the edge and in fact the r.n.a. we had a paper from Emmeline Blanche was the 1st author and molecular therapy nuclear Cousens on tools for looking at what the r.n.a. is turning on and I think initially a lot of work from Cure back and even mature in our early on the arteries were dirtier and so you certainly saw you know certainly I would say.

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Anyway differences in terms of. I lost my train of thought but anyway there's a lot of prosecuting on one piece and how they're driving inflammation or not I'm sorry I actually got distracted by Dr Anderson's. Comment about the tablet. And more Nicole said The think is the return of vaccine it's proper predominates each one buys response which I'm not surprised like is that's pretty typical for our neighbors vaccines which may suggest more class one presentation in.

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We have not looked at C.D.'s or cd for response is a presentation to different cell types at all I think that that's connecting that is on the in a see one of the projects we have with James that is one of things will definitely be looking at and how different Alan P's are basically skewing those responses or changing those responses so I'll be able to update the near future I would say but I think the the one of the issues about you know the analysis of the current vaccines is that you really only see the current.

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So you're seeing you know one l n p one r.n.a. one l n t one r.n.a.. Well there's a whole host of Ellen pieces that could be used that you know differences in m.r. an age that could be used I think the variables This gives you a window into what you're seeing but is is you know in terms of variable space it's actually quite large so I think there's a lot more for us to figure out.

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So you have a question like we've seen that you know. These doses are well expressed and. In particularly got to you mentioned and that's happened to be very kind of effective but how do you actually optimize these doses like how do you decide basically how much to administer technically it's a very lengthy study to optimize that or I would say you know I mean I mean those responses I think you know in most cases people are looking at you know in a body level so you give a range of doses early on and they look for in a body level's basically initially I think most of the early studies world were predominant not looking at salvia.

[00:36:30]
Sponsors But I mean they follow usually with somebody but the 1st thing is to get that antibody response up as. You know usually they if you noticed most of the early studies they were comparing to convalesce So folks who had been infected and what were there in a body levels and were infected then recovered what were their end of body levels like and could you match that a lot of the initial data from the during and Pfizer we're comparing with with and by levels of people that had been infected so they were trying to match that and so that's why in 2 doses I mean and maybe I'm sure Dr Anderson could comment on this more than I can but you know the single why do we have 2 doses I mean I think in general it's because they want to get those levels up a bit higher and even though your 1st post goes up quite a bit over time that boost kind of pushes it up a bit more and it's a bit more robust so I think in general you get more robust and a body response is hence the 2nd dose and so even though I think in many folks the 1st dose is sufficient after even a fairly short period of time to be protective but they wanted to get that level up to a certain level I mean the problem with any vaccine is that there are some vaccines that we actually know good i'm glad he's going to talk.

[00:37:41]
To dose is good and so it's perfect. I will say that. You know there's there's still a bit for us to figure out in terms of how how could you dial it in so you only would be one dose or not I think that that's something that still needs to be examined in more detail and what's what how do you dial in the l. and p. and the r.n.a. so that you can just have one but I think that's that's really the key I mean I guess what I was going to say is that there are some vaccines that we know the correlates of protection really well we know exactly what the vaccine needs to do and order to be protected and I don't get I don't think I was completely clear upfront with coding and so it seems like to be that folks what they used comes in Syria as kind of one way of gauging that and but I don't think you know for a lot of actions we don't know I mean each of these a great example that you know what's the correlates of protection for HIV seems to depend on who you talk to whether it's in a body and see for responses or not and how much and in what balance and you need both and should you want to know and I think it kind of they go through different.

[00:38:51]
Different phases in terms of the science on that but I think both are always best I think investments cases but sometimes one is just enough and fine so I think that it just comes down to you know if you don't know they try to give you the broadest you know the price response you can possibly get but I think that's that's something new but I think now that we kind of know we have a lot more data now in terms of what's protective and what's not these these vaccines could be refined in the in the near future and I think actually all of the human data that's being produced will really really be helpful for refining.

[00:39:25]
This approach in the near future so. That's. So feel I have a couple of questions disappear from memory. Here Meister one quick presentation questions on Am I don't have platforms you have talked when you were here are usually the outbreak of covert talked about comparing a modern era.

[00:39:47]
Very intriguing findings that you so I'm trying to save most in the illiac to just shoot questions steady just him thinking they'd win the most and then hold close to signal presenting if you followed more than 24 hours and my interest is no so the answer to all of that is is that depending on the formulation we do see differences in terms of an old versus iliac and sometimes you do see him pop up in the other and in these animals with this formulation we saw more iliac than he will definitely.

[00:40:19]
Did you see anything in the Trinity Cliffs Notes at all because you obviously see stuff going into the colon and yes we've heard him call we didn't pull missing Terex for that experiment so we have another one coming up in about in a short period of time where we plan on pulling them to see and so there's no question that there is definitely more digging to understand where where you know where hole until craving happens right so because it's important for us to understand where look class myself in the terminal simply stop being triggered because the longevity of this human response obviously will be driven by think duction of dollars and Reddick Sackler always are extremely critical to understand.

[00:41:01]
That's why we developed a tool I mean that was that was exactly why we developed this you know I would say this was more of a proof of concept and I thought to be funded show you're serious there's many more studies and really looking at how the l. and p. is changing the Olympiad the r.n.a. is because they're really a package and I mean by that is that you know it's hard to separate the cargo from the l.n.p. in some respects too so I think that you know understanding how those the 2 and their properties how they when they change how that changes that is something that will be I mean it's part of a grant we have at the moment James and I have but at the same time I'm very happy to write many other grants to do our bit it's because I think there's a lot more to do and terms of what we can definitely.

[00:41:47]
Using the tools that we have pick out exactly what nodes and exactly what tissues that we need to look at and we can even some help with this you know with sorting because they are nice labeled in terms of looking at any positive in or any negative populations within those nodes within the lymphoid tissue I hope that offers this general comment.

[00:42:08]
On nasal spray seems to be good for colored vaccine from China I mean would you like to comment on it. Not much because I just don't I mean it seems like it makes sense I mean it it makes sense it really comes down to. I don't know much about what they've done or what they haven't done with Type of our what kind of vaccine I mean for m.r. in a base vaccine I know that if we do it internees all administration we get expression of the energy and I think depending on how it's formulated You know what cells take it out the know how for how long and how long suppression is we don't know yet so I would say stay tuned I think you know will be able to have more data on that but I think it can be pretty formulation dependent in terms of in terms of function I think it should work definitely but I I think we have to do it in order for us to really you know for us to understand but the response is look like for him or navy sexing field just to again at response to other comments initially about the issues that are involved if that makes you feel in a better suit with formulation we have documented that dominant cell type just like a car of say.

[00:43:17]
Good monocytes in the states can do a fantastic job of presenting and it doesn't have to be controlled access doesn't have to be my Lord of the National p.c. So I think the data is very consistent what we've seen but that's what formulation says well that's what is it should you think do you feel like in the whole conversation might be worth it to talk about this offline but do you think it would be if we target the d.c. state so with James we're going to be looking at different Ellen piece for I mean would we get a better response or would be better it's legal I think it's actually.

[00:43:47]
Independent definitely have a long conversation offline on this one I think monocytes did our country itself been documented to be a predominant Cuban and caption but having one thing actually meant so duck session it would go into you know the so-called well defined gentry excels of prosperous something decent etc That's neat that needs to be relooked at this monocytes clearly have a lot of for old yeah a lot of the infinitely surprised I thought we were actually when we 1st looked at it thought I'm going to do something wrong.

[00:44:14]
I don't know I'd like to get a super consistent puppy had been an issue we've been documenting in the lung tissue not with Colgate with battle with lymph nodes. With with blood I think monocytes of playing a central role so that next you know it's very consistent does it does and I think certainly in terms of you know what Ellen piece we decide to move forward with in the future.

[00:44:40]
You know we might want to I mean I think James and I are interested in testing that hypothesis in terms of which ones do we actually in other words if you pick one that was very you know. N.b.c. or conventional d.c. focused and you picked one that was more mana saved was picked up and expressed by monocytes what it was how does that has a change and about a response and how does that change somebody's response I mean I think that will be an interest it'll be interesting just to see what the results are but just the same I mean it that is what we saw predominate Sates and that is exactly what we saw what I'm pretty sure countless all with.

[00:45:16]
With an environmental and p formulation I don't know if it was the one that was in it's in people yet but at the same time that's what she saw the. Well there are no questions let's. Bowl one more time and fully appreciate your time. On a friends audience thank you for your coming and present in your garden threesomes and everyone have a great day and we'll meet next Wednesday at 11 am for our 2nd ever in our in this in this series thank you by.