19. Molecular approaches to vaccine design: problems and solutions Flashcards
1
Q
What can vaccines control?
A
- Some infections disease can only be controlled by vaccines.
- Antibiotics can only be so effective in treating infections and for resistant infections vaccines could be the only solution.
- Vaccination and disease prevention could be the ultimate solution to AMR.
2
Q
What motivates most vaccine development?
A
- Diseases that kill young people or people in their prime
- This is why meningitis is such a focus of vaccine development
3
Q
What is an infection that really need good vaccines?
A
- Meningitis and the associated sepsis.
- You can show symptoms of sepsis and be dead in a few hours and antibiotics won’t help.
- For meningococcal disease you are at the mercy of rapid diagnosis, getting antibiotics and hoping for the best.
4
Q
What makes a good vaccine?
A
- Immunogenic
- Easy to manufacture, administer and transport.
- Stable a room temp
- Induces a long lasting immune response
- targets a conserved antigen throughout the species.
- Bioavailable target - makes sure the antigen of expressed and accessible in vivo.
- Effective at preventing mild and severe disease.
- Ideally want to prevent onwards transmission too.
- Doesn’t select for an escape strain or resistance.
- Safe with minimal side effects
5
Q
How do vaccine need to be immunogenic?
A
- They need to induce a strong protective immune response.
- This is different for different diseases.
- To do this you need to understand the appropriate correlate of protection for that disease.
6
Q
Why do vaccines need to be safe?
A
- To protect and help people
- To keep people trusting the vaccines and taking them.
- This is important for keeping herd immunity and protecting the whole population
7
Q
What can some capsular vaccines do?
A
Prevent onwards transmission
8
Q
Are vaccines hard to develop?
A
- Yes very hard
- They takes years and lots of money to develop.
- Includes pre-clinical and clinical research.
9
Q
What is pre-clinical development of vaccines?
A
- Pre-clinical research is carried out in lab assays and on animal models.
- It identifies and understands the relevant antigens you want in the vaccine.
- Creation of the vaccine concept.
- Evaluation of vaccine efficacy in test tubes and animals
- Manufacture of the vaccine to Good Manufacturing Practice standards.
10
Q
How could AI be used to speed up vaccine development?
A
- It can help screen and identify antigens that could be included in the vaccine.
11
Q
What is clinical development of vaccines?
A
- This is when the vaccine is tested in humans for the 1st time.
- It is high cost and highly regulated.
- Includes phase 1, 2, and 3 trials and later phase 4 trials.
12
Q
What are phase 1 clinical trials?
A
- They are small with less then 100 participants.
- It is to look for adverse side effective that weren’t picked up in animal models.
- It is all about safety of the vaccine.
- Animal models have differences to humans so you are looking to see if the vaccine is also safe in humans.
13
Q
How are animal models used in pre-clinical vaccine development?
A
- Mice or Murine models are used to see if the antibodies are effective at providing protection.
- It is testing the immunogenicity and that the right type of antibody targeting the right epitope is produced.
- Some times this doesn’t work well due to the difference between mice and humans.
- You can also use slugs to show the mucosa won’t over react to the vaccine but this is less common.
14
Q
What are phase 2 clinical trials?
A
- Involve 1000s of people
- Start to look at vaccine efficacy in humans.
- These are quite easy for common diseases like rhinoviruses as it is easy to find enough people that will get the disease.
- For rarer diseases it is harder as you can’t predict who will and won’t get the disease.
- These need to be designed and tailored to the disease.
15
Q
How are phase 2 vaccine clinical trials run for rare diseases?
A
- This is for diseases of around 20 cases per 100,000 or less.
- You can’t say who and how many people will get the disease.
- Data takes much longer to collect as the disease occurs less often.
- Efficacy data is collected a lot later when enough people have got the disease and you can see if the vaccine induced a protective response.
- These trials take much longer
16
Q
What are phase 3 clinical trials?
A
- This is just a scale up of numbers.
- usually 100,000s across multiple sites and countries.
- You are confident in the vaccines safety and efficacy at this point.
- You are looking at the safety across the population and the efficacy under natural disease conditions.
- look at diverse populations to see if the safety and efficacy is the same as the phase 2 trial.
- Can identify rarer side effects.
- If this is successful the vaccine can apply for License.
17
Q
What are phase 4 clinical trials?
A
- These happens once the vaccine has been licensed and introduced across a population.
- You are looking at the global picture of the vaccine.
- It is very low risk.
- The aim is to assess long term efficacy and identify rare side effects.
- This assess the level of risk acceptable across the population in order to protect the population.
18
Q
What are the unresolved questions for bacterial vaccine development?
A
- Which gene products are required and expressed during natural infection?
- How does this change over time and space?
- How can we determine what goes on at a global level?
19
Q
Why is it hard to determine what gene products are required and expressed in natural infection?
A
- There are big difference in gene expression of bacteria in vitro and in vivo.
- This causes different phenotypes and different results.
20
Q
How do proteins interactions change over space and time in bacterial infections?
A
- Both antigenic and phase variation can occur during infection.
- If you are basing a vaccine on a specific strain you may not target the variation across the population.
21
Q
What older technologies can be used to identify virulence genes?
A
- Studying 1 gene at a time through knockouts.
- Signature tagged mutagenesis (STM) and In vivo expression technology (IVET).
- STM and IVET can identify genes expressed in vivo that are necessary for pathogen survival in infections but they cannot identify genome scale expression changes like complex adaptation.
22
Q
What is now used to identify virulence genes?
A
- Comparison transcriptomics, proteomics and genomics.
- Traditionally this used microarrays but now we use RNAseq
23
Q
What do microarrays and RNAseq measure?
A
Gene expression
24
Q
Why is bacterial mRNA harder to purify then human and viral mRNA?
A
- Human and viral mRNA has a poly A tail. Bacterial mRNA doesn’t.
- The lack of poly A tail makes it harder to separate the mRNA from DNA and other RNAs.
25
Q
What do microarrays and RNAseq assume?
A
1a. The gene makes mRNA which makes a proteins and the protein will function and go where it should.
1b. This basically always happens in viruses as the proteins are made by the human cell but there are conditions and circumstances for bacteria where this is not the case.
1c. eg Temperature can influence how much of a protein it made and if it folds correctly.
2. It also assumes the more mRNA expression = more protein expression which is not always the case.
26
Q
What is the regulation of gene expression key for?
A
Adaptation to changes in the environmental conditions and for survival.
27
Q
What is transcriptomics?
A
- The study of the complete set of mRNA in a cell (the transcriptome) produced from the genome or plasmids.
- It describes the regulation of gene expression on a genome-wide scale.
28
Q
What is transcriptomics used for?
A
- It is a useful starting point for looking at whether a gene is expressed.
- This can be in certain conditions or certain time
- You do this by mimicking conditions or taking samples from human or closely related animal models.
- You can look at the subsets of genes transcribed in carriage vs invasive infection.
29
Q
What does the transcriptome link?
A
- It is the dynamic link between the genome, the proteome and gene expression.
- The gene expression determines the cellular phenotype.
- the genome and the proteome are linked by the mRNA produced.
30
Q
What does transcriptomics show?
A
The potential protein profile for that cell.
31
Q
What techniques are used in transcriptomics?
A
- DNA microarrays which require sequence knowledge
- RNAseq which can determine mRNA expression without sequence knowledge
32
Q
Why are DNA microarrays a closed system?
A
- They require sequence knowledge.
- So you cannot detect gene expression of a gene you don’t know.
33
Q
How do DNA microarrays work?
A
- They are glass slides or membranes containing many copies of individual genes spotted and fixed onto a grid.
- They then use either PCR amplified genes or oligonucleotides synthesised directly onto the array.
- You are testing and comparing genes transcribed under 2 chosen conditions.
34
Q
What can microarrays or RNAseq be used to compare?
A
- Bacterial gene expression in vitro vs in vivo.
- You need a control (in vitro) condition and experimental conditions (in vivo).
- You identify expression in culture, in infection and in both,
- This tells us what is happening to protein expression.
- You need to check the change in mRNA levels is reflected in the change of protein levels.
35
Q
Why does proteomics need to be used alongside transcriptomics?
A
- Transcriptomics measures the genomic response not whether the protein has been generated.
- Other factors may stop the mRNA being translated into a protein or a functional protein.
- To see the true response to the environmental condition you also need to use proteomics under the same conditions to see if the mRNA is translated.
- Test if the protein is expressed and if it still functioning.
36
Q
What is proteomics?
A
- The study of the function, composition and structure of proteins within a cell.
- This permits visualisation of the protein content of cells under different growth conditions.
37
Q
What is genomics?
A
- The study of genes and their function.
- It aims to understand the structure of the genome.
- It gives a good base starting point to look across strains and sub-strains to see what is conserved across species.
- This shows how prevalent a protein is across a species.
- It examines molecular mechanisms and interplay of genetic and environmental factors in disease.
- This is useful in disease prevention.
38
Q
What are most bacterial vaccines made up of?
A
- Multiple components from multiple strains of a species.
- This is required to ensure good coverage of a disease.
39
Q
What does genomics give us?
A
- A broad picture of what could be happening in a cell.
- Gives an idea of what might need to change in a vaccine
40
Q
What is functional genomics?
A
The characterisation of genes and their mRNA and protein products
41
Q
What is comparative genomics?
A
Studying the evolutionary relationships between genes and proteins of different strains or species.
42
Q
What is an example of using comparative genomics?
A
- Fusobacterium nucleatum is a bacteria that is closely related to the progression of colorectal cancer.
- Only certain strains make CRC more likely to spread and be drug resistance.
- The differences in strains and sub strains of F. nucleatum to see which surface or metabolic factors contribute to this progresiion.
- This was done with comparative genomics.
- It also considered the microbiome and what happens to bacteria in different circumstances and genetic backgrounds
43
Q
What is pharmacogenomics?
A
- Studying new biological targets and new ways to design drugs and vaccines.
- It can help determine whether a patient is going to be susceptible to treatment.
44
Q
What is structural genomics?
A
The dissection of the architectural features of genes and chromosomes.
45
Q
What is epigenomics?
A
- The study of the modifications of the genome.
- This includes DNA methylation patterns and DNA packaging.
46
Q
What do all branches of genomics inform?
A
Good decisions about vaccine and drug development
47
Q
What was the 1st genome ever sequenced?
A
- H. influenzae
- In 1995 and took millions of pounds and years.
48
Q
How can genome evolution studies help design vaccines?
A
- They look at the whole genome and compare between strains for good design of vaccines
- It helps find unique pathogen traits.
- eg The region of different 1 in M. tuberculosis which is missing from BCG and therefore the vaccine.
49
Q
How can genome evolution studies help design drugs?
A
- It can look at the genome of bacteria and humans and identify the difference.
- This helps to find specific drug targets that are very different from humans.
- eg Gentamicin which specifically targets bacterial ribosomes
- Also solve complex biosynthesis problems which involved lots of genes and important in new antimicrobial design.
50
Q
What are the types of immune protection?
A
- Passive immunisation
- Active immunisation
51
Q
What is passive immunisation?
A
- This was mostly used before we understood the immune system and vaccines.
- It involved giving someone antibodies from someone the seems to not get the disease then the other person will also be protected.
- Low risk by short lived protection.
- Maternal antibodies are passive immunity.
52
Q
What is active immunisation or vaccination?
A
- Deliberate exposure to antigen to mimic natural infection without you getting the infection.
- There is a risk of disease like with the live polio vaccine.
- There needs to be good stimulation of protective immunity.
- Need to be careful of the risk of autoimmunity, hypersensitivity and other side effects.
53
Q
How can the polio vaccine cause a polio infection?
A
- The live attenuated vaccine can revert back to actual polio.
- This can happen as it replicates and mutates in the body.
- The risk is low
- It is the cheaper vaccines so it is often favoured and the risk accepted.
54
Q
What are the types of vaccines?
A
- Inactivated
- Attenuated
- Subunit
- Conjugate
- Recombinant
- DNA/RNA vaccines
55
Q
What are inactivated vaccines?
A
- Whole pathogen vaccines that have been killed with formaldehyde.
- Need to ensure all formaldehyde is removed otherwise it can cause side effects.
56
Q
what are attenuated vaccines?
A
- Whole pathogen vaccines that have gone through repeated sub cultures in vitro.
- This causes a loss of virulence genes and weakens it
57
Q
What are subunit vaccines?
A
- Vaccines that only contain purified antigen
- eg B. pertussis (Whooping cough)
58
Q
What are conjugate vaccines?
A
- These are polysaccharide capsules from bacteria fused to a protein.
- The proteins ensure a T dependent response is induced.
- eg Hib ad MenC
59
Q
What are recombinant vaccines?
A
- The genes are inserted into a vector.
- This leads to in vitro amplification and expression.
- This is then used a protein vaccine.
60
Q
What are DNA/RNA vaccines?
A
- The DNA/RNA is cloned and injected into muscle cells.
- These host cells express the pathogen protein and stimulate the immune system.
- Makes your cells do the work.
- interest in using these as cancer vaccines.
61
Q
What are the problems with developing meningococcal vaccines?
A
- The meningococcal surface is hypervariable so using the right antigen and capsule is tricky.
- There are several distinct serogroups based on capsule type.
- There are also serotypes and subtypes based on outer membrane proteins and immunotypes based on LPS structure.
- It is constantly undergoing phase and antigenic variation.
- This hyper-variability means is it hard to develop a universal vaccine.
62
Q
What was the tetravalent polysaccharide meningitis vaccine?
A
- Some N. meningitidis capsules are immunogenic in humans.
- Just used the capsule to induce serum bactericidal antibodies.
- These control meningococcal disease.
- ACYW135 licensed since 1981.
- Limited due to no long term immunity, immune memory, no T cell interactions, didn’t work in infants.
63
Q
How were polysaccharide vaccines improved?
A
By making them conjugated to proteins
64
Q
What are polysaccharide conjugate vaccines?
A
- The polysaccharide capsule is conjugated to a T cell dependent protein antigen.
- This is normally diphtheria toxoid or tetanus toxoid.
- This means that a T cell response is generated to the vaccine.
- first developed in 1929 but sidelined for antibiotics.
65
Q
What is the mechanism of conjugate vaccines?
A
- The conjugate is recognised by polysaccharide specific B cells.
- This is taken up and present to T cells.
- This causes plasma cell maturation and memory response
66
Q
What was the first effective conjugate vaccine?
A
- Hib
- Introduced in the 90s.
- Protected lasted 12 years so a booster was added.
- Now there is very low levels of circulation of disease.
67
Q
What is the meningococcal C conjugate vaccine?
A
- 1995-2000 there was a rise in MenC infections. It was the most common treatable Men in the UK.
- A conjugate vaccines was developed and the C capsule fuse to diphtheria toxoid.
- Given to the 2 main risk groups: babies and adolescents in 1999 introduce in UK.
- Protection lasted for 12 years so a booster was added in adolescence.
68
Q
What are the indirect effects of immunisation?
A
- Herd immunity.
- This is the immunity in a population resulting from vaccination of a sizeable subpopulation which protects unvaccinated individuals.
- This happens with vaccines that induce mucosal responses which prevent carriage and transmission.
- This means it is difficult to maintain a chain of infection for the disease.
- The more immune individuals present in a population, the lower the likelihood that a susceptible person will come into contact with an infected person.
69
Q
Why was there no vaccine for MenB?
A
- The polysaccharide capsule has a poly sialic acid which is exactly the same as the ones on NCAM on our nerve cells.
- This means if we did make a vaccine there is a very high risk of autoimmunity.
- This also means the natural immune response is low as the body sees the bacteria as self.
- Tolerance is important to consider when designing a vaccine.
70
Q
What is the trend of invasive meningococcal disease in England from 2005-2015?
A
- Due to the success of the Men ACWY vaccine there are very few cases of these.
- There have been some increases in Y.
- MenB is the most common by far but is on a general downward trend.
- This trend is due to people being more aware of symptoms and seeking healthcare earlier which reduces transmission.
- There is also the natural waxing and waning of disease over time.
71
Q
What are the different approaches people have taken to designing MenB vaccines?
A
- Live attenuated vaccine of N. meningitidis
- Sub capsular approaches like outer membrane vesicles (OMV), recombinant porin vaccines or other antigens (PorA, NspA or Opa).
- Purified OMV shows the surface of the bacteria and is full of LPS so very immunogenic but cannot replicate.
- OMV vaccines are successful in countries with only a single MenB strain is causing disease like New Zealand or Norway. This doesn’t work in the UK
72
Q
How is functional genomics and reverse vaccinology used for vaccine discovery?
A
- Functional genetics is used to associate phenotypes with genes so you are starting with the trait then finding the gene that causes it.
- Reverse genetics starts with an array of genes with unknown function and aims to mutate them to study the resulting phenotype.
- This needs to be done to a large number of genes to find their function.
- You analyse all the open reading frame to find a good vaccine target.
73
Q
How was reverse genetics/vaccinology used to make vaccines for MenB?
A
- They analysed the genome of N. meningitidis B MC58 for genes that may encode putative exposed surface proteins.
- These genes were cloned and expressed in E coli as recombinant proteins.
- These were immunised into animals to see if they generate a protective immune response and effective cause bacterial killing.
- The advantages is proteins expressed at low levels or only expressed in vivo can be identified.
- These proteins are less likely to encounter immunological selection and be conserved.
74
Q
How were new targets for Men B found?
A
- Reverse genetics identified 2158 open reading frames.
- 570 potential surface proteins
- 350 expressed as fusion proteins in e coli and used to immunise mice.
- 85 gave a strong antibody response
- 7 were conserved surface exposed proteins that were used as vaccine candidates.
- Genomics was also used to design attenuated pathogens to identify genes required for virulence
75
Q
What were the proteins identified for the MenB vaccine?
A
- Factor H binding protein
- Neisseria heparin binding antigen
- Neisserial adhesion A
- GNA2091
- GNA1030
76
Q
What was the development process for the 4CMenB vaccine?
A
- GNA2091 and GNA1030 were proteins with unknown functions and it was found they didn’t really induce immunity.
- NadA was expressed only in human disease so was almost not included but it is a strong immunogen.
- The vaccine containing the 5 proteins went to trial and didn’t work very well so OMV were added.
- OMV express all surface proteins and LPS.
- This produced better results with PorA being the dominant antigen but this can be strain specific.
- This lead to the 4CMenB or BEXSERO vaccine.
77
Q
What is the 4CMenB or BEXSERO vaccine?
A
- It contains factor H binding protein, Neisseria heparin binding antigen, Neisserial adhesion A and OMV.
- It worked and gave high levels of protection against the strain it was designed against.
- There were other studies to look at the effectiveness across strains.
- They found fHbp and NHBA was not as conserved as they thought and NadA is conserved.
- If PorA is expressed by the strain then the vaccine works very well.
- This means the effectiveness of the vaccine is very strain specific.
78
Q
How was the 4CMenB vaccine introduced in the UK?
A
- It received lots of praise in the press.
- In 2013 the JCVI looked at QALYs and concluded that it was not cost effective to introduce at any cost as it was not effective enough.
- In 2014 this was reversed for unknown reasons to roll out the vaccine to infants. This was on the condition there were carriage studies done but not much has come of it.
- The vaccine was rolled out in 2015.
79
Q
What is the effect of the 4CMenB on invasive meningococcal disease in the UK?
A
- 4CMenB was introduced in infants without an adolescent campaign despite recommendations.
- MenB continued on a downward trend but it hasn’t gone away and it hasn’t dropped post covid despite the vaccine.
- However it has caused invasive disease in infants to massively drop. This occurred in the 1-4 years old so it is helping.
- We need another 10 year to see if the protect lasts through the adolescent risk period or if a booster is needed.
80
Q
Is PorA in the 4CMenB vaccine?
A
- Yes
- It is expressed as part of the OMV
81
Q
What allowed the golden age of vaccine development?
A
- The ability to grow viruses in cell culture
- The ability to attenuate viruses by passing them through cells
82
Q
What was the 1st vaccine to be developed through genetic engineering?
A
Hepatitis B
83
Q
What important vaccine was developed through genetic engineering?
A
HPV
84
Q
What is the role of immunology in vaccine development?
A
- We need to understand was immune responses are protective to develop a vaccine.
- Usually this is serum or mucosal antibodies.
- But T cells are also important in vaccination as Th cells are essential for B cell memory and long lasting antibody responses.
- Systems immunology increases our knowledge of what genes correlate with a protective immune response and can aid our understanding of how to induce specific immune responses and develop vaccines.
