Special Topic: Vaccines Flashcards
Coronavirus
- 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
ACE2
Normally functions to counteract the activity of ACE which controls blood pressure through vasoconstriction
SARS-CoV2
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
Vaccines
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
Golden Era of vaccine development
1955-1990 - when polio, measles, mumps, hepatitis B viruses were developed
Still no vaccines for
- HIV
- Hep C
- Respiratory syncytial virus
Ideal attributes for vaccines
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
Vaccine for Influenza Virus
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
Composition of inactivated influenza vaccines
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
The main problem with the conventional approach
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
Influenza Virus Vaccine
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
Advantages of mRNA vaccine over attenuated/killed/subunit/DNA vaccine
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
In the vitro synthesis of mRNA vaccine
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
In the vitro synthesis of mRNA vaccine
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
Two main types of mRNA vaccines
- 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
- 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
What happens when you get vaccinated with mRNA vaccine
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
Efficient in vivo mRNA delivery is critical to achieving therapeutic relevance
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:
- 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
Transfection
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
Getting RNA into cells: Electroporation
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
Getting RNA into cells: Electroporation in vivo
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
Getting RNA into cells: Lipid nanoparticles
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
mRNA Vaccines for covid
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
mRNA Vaccines for covid
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
Efficacy vs Effectiveness
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
Efficacy
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$
