Special Topic: Vaccines

Question 1

Coronavirus

Answer 1

• coronavirus take their name from the protein spike with rounded tips in their envelopes

Coronaviruses infect numerous species

Coronavirus usually cause typical cold symptoms such as sore throat, cough and stuffy nose

Coronavirus are quite common

most people have antibodies to them

Question 2

ACE2

Answer 2

Normally functions to counteract the activity of ACE which controls blood pressure through vasoconstriction

Question 3

SARS-CoV2

Answer 3

envelope spike proteins mediate coronavirus entry into host cell by first binding to ACE2 virus receptor on the host cell

ACE2 is expressed in the plasma membrane of cells located in the lungs, arteries, heart kidneys and intestine

zThe virus envelope fuses with the plasma membrane of the cell, or endosomal membrane, and the virus genome is released into the cytoplasm of the cell

The S protein is the obvious target for vaccines

There are several epitopes on this protein

Question 4

Vaccines

Answer 4

Vaccinations are the most successful medical approach to disease prevention and control

Vaccinations have saved millions of lives by introducing protective immunity before the pathogen infects host

Developmental and production of new human vaccines has been quite slow since the golden era

some vaccines induced T cell mediated and B cell mediated immunity (eg live virus vaccine eg measles )

Others include only B cell immunity eg tetanus and influenza

Question 5

Golden Era of vaccine development

Answer 5

1955-1990 - when polio, measles, mumps, hepatitis B viruses were developed

Still no vaccines for

• HIV
• Hep C
• Respiratory syncytial virus

Question 6

Ideal attributes for vaccines

Answer 6

Safety - for both recipient and close contacts

Efficacy/effectiveness - establishes a good strong protective immune response

Low cost

Easy to vaccinate individuals - quick procedure, minimal equipment needed

Minimal side effects

Rapid, reliable production protocols - need to make millions of doses

High yields during production - relates to cost availability

Stability/distribution - storage condition ea

Question 7

Vaccine for Influenza Virus

Answer 7

each year new vaccines prepared for seasonal influenza virus

A single dose is administered - most vaccines are designed to generate IgG type antibodies that will neutralize virus
- slow release of antigen from injection site over time

Most antibodies are generated against the hemagglutinin protein which is what attached to the surface of host cells

Several pharmaceutical companies produce vaccines that contain strains of circulating

these strains are identifies by WHO based on the dada collected

Question 8

Composition of inactivated influenza vaccines

Answer 8

The different vaccine format is do have differences in antigen components and immunity

WIV = whole inactivated virus

Virosomes = reconstituted influenza virus envelopes

Split = disrupted with detergent, contains all virus proteins, the virus’ DNA is usually lost

Subunit - puridied H and N after splitting

Question 9

The main problem with the conventional approach

Answer 9

Vaccines are usually propagated in eggs which raises problems:

• the need for cell/egg based technologies
• potential shortage of supplies can impact production
• tend to be a much slower process and more costly in term of producing million doses of vaccines
• tend to need dedicated production facilities

While mRNA vaccines are promising alternative
- though shortages in things like glass, vial, dry ice could create vaccine shortages and distribution challenges

Question 10

Influenza Virus Vaccine

Answer 10

The vaccine production process for influenza is complicated

All vaccines are formulated to include a standardized amount o Hemagglutinin protein from the influenza virus

production starts sometimes around Feb with the identification of strains

The vaccine is ready for distribution in Oct/noV
- vaccine that we know how to makes, what works what doesn’t, established production.distribution protocols - still take this long

Question 11

Advantages of mRNA vaccine over attenuated/killed/subunit/DNA vaccine

Answer 11

mRNA is non infectious- no chance of illness (like with attenuated)

mRNA is non integrating - no chance of insertional mutagenesis 9with DNA vaccines)

mRNA is degraded by normal cellular processes - it is transient
- modifications can be made, the mRNA is more stable and highly translatable

mRNA can be formulated with carrier molecules for in vivo delivery - allows for rapid uptake and expression in cytosol

mRNA vaccines can be administered repeatedly - no concert for immunity to vector like recombinant vaccines

mRNA vaccines have potential for rapid, inexpensive, and scalable manufacturing, mainly owing to the high yield of in vitro transcription reactions

Question 12

In the vitro synthesis of mRNA vaccine

Answer 12

Using a cell based system, the desired mRNA would have to be purified from the rest of the cell of the RNA - RNA is very susceptible to RNase which is everywhere

Cell free, in vitro transcription reaction allows for synthesis of a single species of RNA

the dsDNA template can be linearized plasmid or PCR DNA product

the mRNA product can be purified from the reaction

Question 13

In the vitro synthesis of mRNA vaccine

Answer 13

modified nucleosides such as 1-methyl phosphouridine in the in vitro transcription mix improves the stability of mRNA

Replace rare codons with more frequently used synonymous codons - more abundant cognate tRNA in the cytosol

add specific caps and the poly A tails to improve translation

Introduce specific mutation in the ORP to generate more stable protein product

Question 14

Two main types of mRNA vaccines

Answer 14

1. conventional (non-replicating) mRNA vaccines

contains ORF of the target antigen, flanked by the 5’ and 4’ UTR and an optimal length of poly-A tail
- the current mRNA vaccines result in transient antigen expression

2. Self-replicating mRNA vaccines - these replicons contain the RNA replication machinery (RDRP) and the ORF of the target antigen in place of the virus’ structural genes

Self-replicating mRNA vaccines generate multiple copies of the antigen encoding mRNA
- in many ways, it mimics the production of mRNA in a natural infection

Question 15

What happens when you get vaccinated with mRNA vaccine

Answer 15

With an RNA vaccine, your cells have to synthesize the protein antigen before your immune system can respond to it

The expressed protein of interest ar generated as secreted, trans-membrane or intracellular protein

Question 16

Efficient in vivo mRNA delivery is critical to achieving therapeutic relevance

Answer 16

Exogenous mRNA must penetrate the plasma membrane in order to reach cytoplasm to be translated to a functional protein

mRNA is too big and too charged to go through the lipid bilayer of the membrane

2 approaches:

1. Ex vivo delivery of mRNA to dendritic cells (transection efficiency) followed by re-infusion of dendritic cells
   - control of the cell type, the mRNS enters into transfection efficiency
   - time consuming labor intensive, expensive

2) Injection of RNA - with or without carriers
- fast, inexpensive
- may not control the cell type the mRNS enters into

Question 17

Transfection

Answer 17

Transfection is the process of artificially entering nucleic acids into eukaryotic cells, utilizing means other than viral infection

the introduced nucleic acid may exist in the cells transiently such that it is only expressed for a limited period of time

The introduced nucleic acid may be stable and integrate into the genome of the recipient’s DNA, replicating when the host genome replicates

someways of getting nucleic acid into cell

• electrophoresis
• lipid base

Question 18

Getting RNA into cells: Electroporation

Answer 18

Nucleic acid is added to solution of cells and an electric field i applied

the electric field introduced small gaps in the plasma membrane and pulls the nucleic acid into one electrode

When the plasma membrane heals, the nucleic acid is trapped inside the cell

Question 19

Getting RNA into cells: Electroporation in vivo

Answer 19

1) inject the nucleic acid into the tissue

2) an electrode is applied to the tissue and electric field is applied to move the nucleic acid into the tissue

Question 20

Getting RNA into cells: Lipid nanoparticles

Answer 20

LPNs have become one of the more commonly used mRNA delivery tools - can consist of

• ionizable cationic lipid - promotes self assembly into particles and allows for endosomal release of mRNA
• lipid linked polyethylene glycol (PEG) which increases half life of formulation
• cholesterol - stabilizing agent
• naturally occurring phospholipids, which supports lipid bilayer structure

Question 21

mRNA Vaccines for covid

Answer 21

First RNA vaccines for use. It does not contain an antigen –instead, it instructs our cells to synthesize the vaccine
• Moderna’svaccine: mRNA-1273
• Pfizer’s vaccine: BNY162b2

other are nucleoside-modified* RNA that encodes a prefusion stabilized** spike (S) protein that is found on SARS-CoV-2

** the stabilization would have been achieved by inserting specific mutations (translation efficiency was also improved by inserting specific mutations)

There are two mutations in the gene that cause the S protein to adopt a shape that stimulates the production

Question 22

mRNA Vaccines for covid

Answer 22

Moderna’s mRNA vaccine is formulated as RNA lipid nanoparticles composed of the proprietary ionizable lipid and 3 commercially available lipids

Pfizer’s mRNA is also formulated as a RNA lipid nano particle (mixture of different lipids including ALC - 0315 a type of PEG200, cholesterol and phosphatidulcholine)

Both vaccine need to be kept very cols, mRNA

Question 23

Efficacy vs Effectiveness

Answer 23

Efficacy - when a study is carried out under ideal conditions such as clinical trials
Effectiveness when the study is carried out in less than perfectly controlled conditions

Vaccine efficacy is calculated by comparing the percept reduction in cases in the vaccine arm versus the control arm

formula = (% infected in control group - % infected in vaccinated group)/ % infected in control group

Question 24

Efficacy

Answer 24

for vaccine that reports 90% efficacy - the numbers might look like

• 6487 control subjects with 60 infections in this group (0.9%)
• 38955 vaccinated subjects - 34 infections in this group - 0.09%

(0.9-0.01)/0.9 = 90%

As trial continues there may be more infections in both groups and the % efficacy might decrease

A vaccine of less than 50% efficacy ins not good and not approved by FDA

seasonal fly vaccines typically 40-60$

Question 25

How do we get an immune response

Answer 25

For both mRNA and recombinant vaccines involve the host cell synthesizing antigen to be targeted by the immune system
- this is quite unlike unconventional vaccines where the antigen is present in vaccines

So how do we get an immune response to the spike proteins made by the host cell?

• the vaccine replies on immature dendritic cells in the vicinity taking up lipid nanoparticles or adenovirus vectors
• non-immune cells such as those in muscle can take up the lipid nanoparticles or adenovirus vectors too, but they seem to take it up less efficiently than dendritic cells

The dendritic cell displays spike proteins on their surface

The peptides in the MHC class I would be from spoke proteins condemned to proteasome

the peptides on MHC class 2 is from spike proteins being recycled from the plasma membrane

Question 26

How do we get an immune response

Answer 26

If the host cell that took up the vaccine is a dendritic cell, it would migrate to the lymph node

when the dendritic cell enters the lymph nodes from the incoming lymphatics it passes through the B cell zone

Any b cells that has. BCR that recognizes the spike protein expressed on the plasma membrane of dendritic cell is activated

the B cell might pull out a spoke protein out to present to TH cells

Question 27

How do we get an immune response?

Answer 27

the dendritic ells then move from b cell into the T cell zone

any T cell with TCR that recognizes a peptide from spike protein expressed on the MHC proteins of a dendritic cell is activcated

Both CD4 T cells and CD8 T cells would be activated in the dendritic cells are presenting peptides on MHC 1 and 2

Question 28

Advantages and disadvantages of Vaccines

Answer 28

All versions have + and - but all are valuable and may be more suitable for different areas

mRNA +:

• quick to product
• does not rely on egg or cell baed technology
• can quickly modify nucleic acid sequence if needed

mRNA -:
- requires extremely cold storage which can be challenging for distribution

Cell based vaccine +

• track record for production
• distribution and storage

disadvantages:

• slow to produce
• costly

Question 29

Moderna and Pfize mRNA

Answer 29

lipid nanoparticles fuses with plasma membrane releasing mRNA into cytoplasm of the cell

the mRNA is translated by ribosome to make spike protein

spike protein processed throguh golgi and are inserted into lasma membrane

some spike protein ar broken down throgh proteosome and presented on MHC

Question 30

Recombinant vaccines for SARS-CoV-2

Answer 30

This technology has been used in other vaccines

• Johnson & Johnson’s vaccine: JNJ-78436795 or Ad26.COV2.S
• Oxford-AstraZeneca vaccine: ChAdOx1 nCoV-19 or AZD1222

Both vaccines are developed with a replication-defective adenovirus

Adenoviruses have dsDNA genomes and replicated/express their genomes in the nucleus of the host cell

The vaccine developers have added the gene for Coronavirus spike protein into the adenovirus DNA