Universal Influenza Vaccine Via Consensus Designed Neuraminidase in an Adenoviral Vector
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
- [00:00:10.230]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
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
- [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
- [00:03:53.650]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
- [00:05:17.500]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
- [00:05:45.880]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
- [00:09:01.890]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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