Week 9 Part 2 - Vaccine Development

Question 1

What was Smallpox Caused By?

Answer 1

Virus called variola
From Poxviridae family
Genus orthopoxviruses

Question 2

How to Generate Live Attenuated Vaccines

Answer 2

Pathogenic virus isolated from patient and grown in human cultured cells
Cultured virus used to infect monkey cells
Virus acquires many mutations that allow it to grow well in monkey cells
Virus no longer grows well in human cells (attenuated) and can be used for vaccines

Question 3

Vaccine Generation and Examples - Live Attentuated

Answer 3

Pathogen adapts to new growth conditions
Less infectious to the original host
E.g. measles, mumps and rubella vaccine

Question 4

Vaccine Generation and Examples - Toxoid

Answer 4

Toxins cause disease
Toxin inactivated
Antibodies block the toxin
E.g. Tetanus and diphtheria

Question 5

Vaccine Generation and Examples - Subunit

Answer 5

Defined antigens
Purified or recombinant
Excellent safety
Not as immunogenic as whole organism
Need a strong adjuvant
E.g. HPV vaccine

Question 6

Vaccine Generation and Examples - Conjugate Vaccines

Answer 6

Bacteria protected by polysaccharide coat
Poorly immunogenic
Synthesise polysaccharides conjugated to immunogenic protein
E.g. HiB, meningitis C and pneumococcal vaccines

Question 7

DNA and RNA Vaccines

Answer 7

Used for gene therapy
Can contain multiple antigens, cheap and quick to develop

Question 8

Vectored Vaccines

Answer 8

Antigens vectored into host cells by replication deficient viruses
E.g. Adenovirus

Question 9

Reverse Vaccinology

Answer 9

Use genome sequencing to select immunogenic antigens

Question 10

Coronavirus Structure

Answer 10

Medium-sized virus
Largest mRNA genome
mRNA encased in nucleocapsid
Lipid bilayer (soap disrupts this)
Corona = Crowns for Spikes
- Glycoprotein Spike (S)
- enable attachment to tissues by ACE-2 Receptors

Question 11

ACE-2 Receptor Tissue Expression

Answer 11

Type 2 alveolar cells
Bronchial epithelia
Tongue > buccal epithelia
Upper Intestinal epithelia
Myocardial cells
Kidney proximal tubule cells
Bladder urothelial cells

Question 12

What are Nanoparticles

Answer 12

Small objects that behave as a whole unit in terms of transport and properties
Dimensions are 1-100 nanometres = 8,000th of a human hair in width
Occur naturally in clay, volcanic ash, ocean spray, milk, etc.
Can be manufactured

Question 13

How are Nanoparticles used in Vaccines?

Answer 13

As antigen delivery systems
As immune adjuvants
Nanoparticles can be entrapped with antigens such as:
- proteins
- peptides
- DNA, RNA, etc
= Control the release of vaccine antigens
Can optimise the immune response by selective targeting of the antigen to antigen presenting cells
Possible to target specific APCs, in particular dendritic cells (DCs)
Induce a CD4+ and CD8+ T cell response plus a B cell (antibody) response

Question 14

What Biodegradable and Biocompatible Polymers can Prepare Nanoparticles?

Answer 14

Poly(lactide-co glycolide) (PLGA)
Poly(amino acid)s
Polysaccharides

Question 15

Liposomes

Answer 15

Closed lipid bilayer vesicles that spontaneously form in water = a fatty capsule
Consist of a phospholipid bilayer shell with an aqueous core
Can be:
1. Unilameller - single phospholipid bilayer
2. Multilameller - several concentric phospholipid shells separated by layers of water
Can be tailored to incorporate:
- hydrophilic molecules (e.g. antigens) into the aqueous core
- hydrophobic molecules within the phospholipid bilayers

Question 16

Disadvantages of Liposomes

Answer 16

Short circulation time in blood stream
Unstable in human body
Lack selective targeting

Question 17

Disadvantages of Liposomes and it’s Solution - Short Circulation Time in Blood Stream

Answer 17

Solution: Improve longevity by coating the surface with biocompatible inert polymers such as PEG
- should be undetected
- but some people experience severe allergic reactions to PEG

Question 18

Disadvantage of Liposomes and it’s Solution - Unstable in the Human Body

Answer 18

Solution: Develop stimuli responsive liposomes e.g. sensitive to to temperature and pH

Question 19

Disadvantages of Liposomes and it’s Solution - Lack Selective Targeting

Answer 19

Solution: Can place antibodies on the surface of the liposome

Question 20

Advantage of Liposomes

Answer 20

Good platform to deliver DNA and RNA
RNA, mRNA, siRNA and DNA difficult to deliver directly due to:
1. Negatively charged and hydrophilic
2. Prevents passive diffusion across plasma membranes
3. Susceptible to nuclease degradation - free mRNA breaks down quickly in the body
Solution: Use nanoparticles with synthetic positively charged (cationic) lipid
- nucleic acids are now more stable and resistant to nuclease degradation.

Question 21

Pfizer, Moderna and CureVac Vaccines - Overview

Answer 21

Liposomes with mRNA coding for the coronavirus spike protein
Pfizer and Moderna = 2 doses >90% successful at reducing infection and disease severity – a variant
CureVac = 47% effective at preventing disease = failure, must be > 50%

Question 22

Similarities between Pfizer and Moderna

Answer 22

Ionizable cationic lipid
PEGylated lipid
Cholesterol
Phospholipid distearoylphosphatidylcholine (DSPC)
Modified RNA = incorporate pseudouridine in place of uridine
- may help avoid inflammatory reactions to foreign mRNA
Pfizer = 30 micrograms of vaccine
Moderna = 100 micrograms of vaccine

Question 23

CureVac Vaccine

Answer 23

Dose & side effect problems
Uses normal uridine
- altered the mRNA sequence - does not affect the protein it codes for
- aimed to help the mRNA evade immune detection
- structural differences in the non-coding regions of the CureVac sequence
Higher storage temperature = might accelerate breakdown of mRNA

Question 24

How do RNA Vaccines Work?

Answer 24

1. mRNA enters the cell
2. Cell starts spike protein production
3. Spike protein is recognised by the immune system
4. Antibody production
5. Immune response + cytotoxic T cell response

Question 25

Adenovirus Vectors

Answer 25

Double-stranded DNA viruses
Cause mild respiratory and gastrointestinal tract infections
Isolated from many mammalian species, including chimpanzees and humans
Express two types of genes:
- early genes (E1A, E1B, E2, E3, and E4) = support viral replication inside host cells
- late genes = host cell lysis, viral assembly and virion release
Can use adenoviral vectors to transfer DNA into host cells to trigger an immune response

Question 26

Advantages of Adenoviral Vectors

Answer 26

Infect a wide range of dividing and nondividing cells = broad tissue tropism
High thermostability
Can grow to high titers
The risk of insertion mutagenesis believed to be low
- the viral genome ‘does not integrate’ with the host genome
Express pathogen-associated molecular patterns (PAMPs)
-activates the innate immune system
- induces CD4+ T cell and CD8+ T cell responses
Large well-characterised genome = easy to genetically manipulate
- insert a transgene cassette into the adenoviral backbone
- expresses the target antigen under the control of a promoter to sustain expression of the transgene

Question 27

ChAdOx1 nCoV-19 Vaccine

Answer 27

Adenoviral vector DNA SARS-Cov-2 vaccine
ChAdOx1 = a modified version of a chimpanzee adenovirus
Can enter human cells but not replicate
An unknown part of E1 replaced by coding sequence for the SARS-CoV-2 spike protein
- = replication defective
Expression of the spike glycoprotein is under the control of a CMV promoter
Still carries 28 kilobase pairs - particularly of late chimpanzee adenovirus genes