Vaccines P2 (Fouty)

Additional material (Lecture)

DNA/RNA vaccines are based on _______ not polypeptides or polysaccharides

nucleic acids

mRNA vaccines

DNA vaccines

Nucleic acid vaccines are based on ______ DNA technology

Recombinant

Ways recombinant DNA technology is used

Replication defective adenovirus is used to carry the recombinant DNA into the host cells in first side

Nucleic acid vaccines induce a _____ and ____ cell response

B and T 

How nucleic acid vaccines produce B and T cell response

DNA Vaccines Require _______ Before Translation, Whereas mRNA Vaccines Do Not

transcription

DNA vaccines vs mRNA vaccines 

Traditional vs RNA/DNA vaccines

Attenuated or kill infectious agent or a subunit/antigen of infectious agen

Immunogen of traditional vaccine

Nucleic acid encoding an antigen of an infectious agent

Immunogen of RNA/DNA vaccine

Safety concerns when studying/manufacturing vaccines

safety of traditional vaccines 

No Infectious risk even for high-risk organisms (HIV, Influenza, Malaria)

Safety of RNA/DNA vaccines

Mainly antibodies

(Antibody and T cell response for live-attenuated/VLP)

Traditional vaccines mechanism of immune response

Antibody and T cell response.

RNA/DNA mechanism of immune response

Manufacture/storage comparisons 

Traditional vaccines: 6 month prep time, Cold Storage/Room Temp

DNA/RNA vaccines: 1 month prep time, Room Temp (DNA), Cold Store (RNA)

First mRNA tested in animals in 1991

Unstable

Activated innate immunity

Minimally immunogenic

2005 Suppression of RNA recognition by ____ : Impact of nucleoside modification and evolutionary origin of RNA

TLRs

Uridine has been replaced by N1-methyl-pseudouridine

In mRNA vaccines

Activated innate immunity activated dendritic cells release 

TNF alpha 

Some types of RNA activate _____ (in vitro/mito/bacterial) cells, whereas most _____ RNA does not

dendritic

mammalian

modified RNA nucleosides showed almost no ______ and reduced activations of CD80 and CD86

TNF alpha

Modifying RNA nucleosides decreased activation of _____ immunity by RNA transcribed in vitro

innate

without impairing translational efficiency 

2011 Purifying in vitro transcribed RNA using HPLC decreased activation of ____ immune system and improved protein _________

Innate 

translation 

Even modified RNA still activated DC to some extent, but purified and modified almost 0 activation

Green shows expression: greener more robust

after 2013 Development of Lipid nanoparticle delivery system (LNP) to improve mRNA ____ into cells

entry

1.Phospholipids

–Membrane fusion and Endosome escape

2.Cationic- ionizable amino-lipids

–Condense with nucleic acids

3.PEG lipids

–Steric stabilization of LNP

4.Cholesterol

–Vesicle stability

Components of Lipid nanoparticles

Membrane fusion and endosome escape 

Phospholipids

Condense with nucleic acids

Cationic-ionizable amino lipids

Steric stabilization of LNP

PEG lipids

Vesicle stability

Cholesterol

Using modified uridine couple with LNP yields highest ______ __________

protein expression 

2003 ___ identified as a functional receptor for SARS coronavirus

ACE2 (bind to cell_

2011 Identification of serine protease TMPRSS2 as a key enhancer for SARS ______

entry into cells

SARS-CoV-1 requires molecule _____ (TMPRSS2) to fuse to membrane and enter cell

Scissors 

2012 ____ identified with high mortality

MERS middle eastern respiratory syndrome

member of betacoronavirus family with spike protein but receptor if DPP4 not ACE2

2017 Recognition that the SARS/MERS ____ protein in the pre-fusion configuration is more ____ than when it is in the post-fusion configuration

spike

immunogenic

Adding extra ____ to the hinge region of the spike protein stabilized the ______ configuration

prolines

Pre-fusion

January 10th 2020, _______ for severe acute respiratory virus Coronavirus 2, Wuhan-Hu-1 isolate was publish 

sequence 

30,000 + ssRNA

DNA template for moderna vaccine was generated within ___ hours of SARS-CoV-2 sequence being published

72

1. mRNA vaccine was codon optimized to eukaryotic

2. Vaccine was synthesized using modified uridine

3. Vaccine incorporated 2 prolines to stabilize translated spike protein in the pre-fusion state

4. DNA Template Was Synthesized And mRNA Transcribed Using Modified Nucleosides (⍦) In Cell-Free Conditions

5. Purified (HPLC) to remove DNA template, enzymes, dsRNA, truncated transcripts

6. Complexed with Lipid Delivery System (LNP)

Moderna Vaccine Manufactured __ Days After SARS-CoV-2 Sequence Was Published

25

Moderna Vaccine Shipped To Clinic __ Days After SARS-CoV-2 Sequence Was Published

42

Plans To Improve On The Beta Version Of Nucleic Acid Vaccines:

Use of self-amplifying mRNA

Use of mRNA that can generate a Virus-Like Particle

Addition of more Prolines to further stabilize Spike protein in pre-fusion state

Updates on virus variants