Universal Influenza Vaccine Via Consensus Designed Neuraminidase in an Adenoviral Vector

Enzo LaMontia-Hankin Author

04/05/2021 Added

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UCARE 2021 Presentation over Universal Influenza Vaccine

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[00:00:00.540]Hello, my name is Enzo LaMonte Hankin. Today,
[00:00:02.880]I'll be talking to you about my UCare project,
[00:00:04.860]universal influenza vaccine by consensus designed neuro nascent and adenoviral
[00:00:09.090]vector.
[00:00:10.230]The goal and premise of this project is to create a universal influenza vaccine
[00:00:14.520]using your competent adenovirus that has a consensus nerve and its antigen
[00:00:18.480]inside of it.
[00:00:19.950]This is generally done by inserting a consensus neuraminidase gene sequence
[00:00:24.750]into an adenoviral vector. This was the primary goal of this project, uh,
[00:00:29.760]Dr. Weaver.
[00:00:30.330]And I believe that I'm maybe able to successfully clone this together by the end
[00:00:34.860]of this semester. Thankfully,
[00:00:37.590]I have actually recently just gotten to this point and I should be able to move
[00:00:41.220]past it a touch. I'm sure most of you are familiar with influenza as an idea.
[00:00:46.500]It is generally a seasonal virus that circulates around every year and infects,
[00:00:50.670]you know, several people usually resulting in a mild cold, however,
[00:00:54.900]more severe cases of influence are quite common within 2018 and 2019,
[00:00:59.070]there being nearly half a million hospitalizations due to influenza and about
[00:01:03.330]34,000 deaths beyond just the daily normal
[00:01:08.040]seasonal influenza, there is quite a big pandemic potential with an influenza,
[00:01:12.660]something I'm sure is on most people's minds. Nowadays,
[00:01:17.160]most notably the 1918 Spanish flu is a prime example of how
[00:01:21.690]much of a potential pandemic influence it can be.
[00:01:25.320]Even more recently in 2009 with the swine flu has shown that influence is still
[00:01:30.240]evolving and can still post significant issues to human health.
[00:01:34.440]Even hauntingly H five and one strains have been identified to infect humans.
[00:01:40.320]Thankfully from 2003 to 2016, a span of about 13 years,
[00:01:43.980]there's only been about 856 recorded case of this,
[00:01:47.130]but almost half of them have resulted in depths.
[00:01:52.200]If H five N one is able to become more common within circulating
[00:01:56.730]influenza, we could have a significant problem on our hands
[00:02:01.620]beyond just the need for potential pandemic prevention.
[00:02:04.890]We should be improving our influenza vaccine.
[00:02:07.200]The current approaches only reduce infection rates about 40 to 60%
[00:02:12.720]are trivalent vaccines,
[00:02:13.830]pick a probable representative sample of circulating influenza. In most years,
[00:02:18.690]scientists are able to predict what kinds of, what kinds of influence will,
[00:02:22.260]will be most common within a certain area. However,
[00:02:25.680]2009 with the newly emerging swine flu was so different
[00:02:30.450]than what we had predicted would be around that. We had some issues.
[00:02:35.220]So if scientists are able to make a universal vaccine for influenza,
[00:02:39.300]we may be able to vaccinate people much more effectively,
[00:02:41.970]even towards unpredictable influenza strains,
[00:02:47.280]the general procedure,
[00:02:49.230]in order to design a universal influenza vaccine,
[00:02:53.190]the antigen must be designed in a way that represents many different kinds of
[00:02:56.670]influence of antigen. To do this. We use a consensus design,
[00:03:00.280]which essentially takes thousands of known sequences of neuro remedies,
[00:03:05.380]and it uses them to construct a single representative
[00:03:10.300]antigen foreigner imitate, hopefully this average.
[00:03:14.740]So to speak of all of these different known strains of influenza
[00:03:19.930]will allow the body to build lasting immunity towards any influence of that.
[00:03:24.670]It may come across also uniquely in this project,
[00:03:28.960]we're targeting neuraminidase for a vaccine target neuraminidase is the
[00:03:33.880]end within the H number and number naming system.
[00:03:36.490]Most people are familiar with it is a cyclic acid-base and it AIDS and
[00:03:41.200]influences ability to enter cells by cleaving silent acid on human cell
[00:03:45.190]services. Most vaccines today,
[00:03:47.920]target hemagglutinin also known as ha hemagglutinin is in
[00:03:52.840]fact,
[00:03:53.650]the viral protein that allows influence it to enter
[00:03:58.360]ourselves. This is why hemagglutinin is a popular target for vaccines nowadays.
[00:04:04.180]That's all well and good,
[00:04:05.380]but this leaves no remedies as an under studied element of potential influence
[00:04:09.970]of vaccine strategies.
[00:04:13.840]The general procedure of this is fairly straightforward.
[00:04:17.200]You take the consensus gene,
[00:04:19.300]which will be referred to as anyone con into an adenoviral vector.
[00:04:24.190]Um, it's just some cloning subs. It doesn't sound too difficult,
[00:04:26.380]but it does take quite a lot of time. Given the consensus,
[00:04:29.350]no remedies in a puck 57 plasmas by Dr. River,
[00:04:33.910]then anyone con was cut out of the puck 57 using restrictions
[00:04:38.500]restriction enzymes. Then it was gated into a linearized shuttle.
[00:04:42.460]Plasmin this shuttle plasmin serves as the important intermediary between
[00:04:46.690]getting our gene of interest to anyone con into the adenoviral vector
[00:04:51.640]P shuttle CMV,
[00:04:53.230]and anyone con were cut using restriction enzymes in order to
[00:04:57.910]linearize them. Then through ligation reaction,
[00:05:00.550]you refuse together to create one single plasmin PCL CMV,
[00:05:04.000]anyone con this was then confirmed using KPN one in Hindi through restriction
[00:05:08.620]digests. At this point,
[00:05:10.990]we had a P shuttle plasmin with anyone caught inside of
[00:05:15.880]it.
[00:05:17.500]Here is the difficult and time consuming part of this project using the
[00:05:21.850]adenoviral vector from the ad easy adenoviral vector system from Agilent,
[00:05:27.160]we needed to incorporate our,
[00:05:28.300]anyone caught inside of this in order to do this P shuttle,
[00:05:31.960]anyone con was co transformed into competent equally BJ five, one eight,
[00:05:36.340]three with the P at easy one plasma,
[00:05:40.090]and then using the cells ability for re homologous
[00:05:44.320]recombination.
[00:05:45.880]Anyone con was inserted into the P at easy plasmin,
[00:05:51.010]as it turns out,
[00:05:52.090]putting two different plasmids inside of a single equalized cell and having that
[00:05:57.410]successfully homologous only recombine them is quite difficult.
[00:06:02.060]After several tries, we were able to obtain a single colony of BJ five,
[00:06:07.010]one 83 e-coli that had a homologous,
[00:06:10.220]the recombined P at easy one with anyone con this was
[00:06:15.200]conf confirmed via PAC one restriction digest.
[00:06:20.000]By this point, taking that plasmid we were able to re insert it into XL.
[00:06:24.500]One competent you call ourselves and they were saved. And the glycerol stock,
[00:06:29.390]this slide outlines the overall process of this code transformation.
[00:06:33.950]As you can see here,
[00:06:34.770]the P shuttle vector that would contain our gene and the P add easy vector
[00:06:40.160]or electroporated into a single cell.
[00:06:43.010]And this is the process of homologous recombination here with similar left and
[00:06:47.210]right arm homologies our gene of interest was successfully inserted inside of
[00:06:51.860]our add plasma. And then from here on out,
[00:06:56.450]we successfully have recombinant adenoviral viral genome,
[00:07:00.470]which can then be transfected into HEC two 93 cells,
[00:07:04.370]and hopefully recover and adeno virus that expresses our
[00:07:09.020]recombinant or remedies.
[00:07:12.770]The original goal of this project was to successfully clone the
[00:07:17.990]anyone con inside of the adenoviral vector because cloning takes
[00:07:22.730]time. We felt it was a reasonable expectation to have this cloning done.
[00:07:26.450]However, with it being early April, and we have, and us having the step done,
[00:07:31.070]we can hopefully move forward currently this week. In fact, um,
[00:07:35.780]me and the other undergraduates in my lab are going to be transecting
[00:07:41.210]our recombinant DNA virus into heck two 93 cells to hopefully recover our
[00:07:45.860]recombinant DNA virus. Any steps from here would be testing.
[00:07:49.370]If this vaccine is successful, potentially moving towards infection in mice
[00:07:55.640]at the end of it,
[00:07:56.630]this project should hopefully point out what works or what may not work within
[00:08:01.430]the realm of universal influence of vaccines is entirely possible that a
[00:08:05.660]consensus remedies may be a poor vaccine target,
[00:08:09.140]but is important for us to understand what works and what doesn't.
[00:08:12.980]So hopefully if there is a pandemic potential
[00:08:18.110]influenza that could be circulating,
[00:08:19.730]hopefully we can be prepared to make a vaccine quickly and efficiently to
[00:08:24.350]prevent another pandemic similar to the COVID-19 pandemic
[00:08:29.330]for knowledge. And so my project, um,
[00:08:31.220]I would not have been able to do this by myself first and foremost,
[00:08:34.310]Christine and Lee,
[00:08:35.570]the two other undergraduates in my lab have worked with me every single day
[00:08:38.990]throughout this project to help me accomplish it.
[00:08:41.180]They had previous experience working on similar projects and their time
[00:08:45.680]effort and knowledge were absolutely essential to me.
[00:08:49.640]I would also like to thank Dr. Weaver for his assistance in this project.
[00:08:53.300]Not only did he give me the opportunity to work in his lab,
[00:08:56.490]he also gave me the reassurance and guidance required to actually work in it
[00:09:00.600]effectively.
[00:09:01.890]He was always there to give me advice and reassure me when things aren't
[00:09:06.090]working. And I really appreciate his confidence. Finally,
[00:09:10.260]I'd like to thank the graduate students in the lab, Bria, Bridget, Erica,
[00:09:13.920]and Matt, Bridget,
[00:09:16.170]and Bria were very helpful towards me in making a coherent procedure and giving
[00:09:20.730]me guidance on what to do next.
[00:09:23.820]And Erica and Matt were essential with having me talk through problems that were
[00:09:26.940]going on and just helping me in generally being good friends. As I mentioned,
[00:09:31.770]I could not have done this project without all of their combined help.
[00:09:34.080]And I'm very thankful to have had this opportunity inside the Weaver lab.
[00:09:38.220]Thank you very much.

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Universal Influenza Vaccine Via Consensus Designed Neuraminidase in an Adenoviral Vector

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