Antigen selection and purification
- The developer must decide whether the vaccine aims to elicit an antibody response, or CD8 T cell response. This may depend on the stage in the pathogen’s life cycle being targeted (e.g. for TB or liver stage malaria, the vaccine aims to elicit a T cell response, but for blood stage malaria antibodies are desirable). Sometimes, the vaccine TPP may stipulate both T cell and antibody responses should be induced (e.g. desirable for HIV or Flu, and there is evidence from Ebola that both cellular and humoral responses are important)
- Draw up a list of candidates, which may be a single candidate for a simple virus:
- For an antibody response, is there a major (or a few major) proteins on the surface of the pathogen?
- Is the pathogen related to others for which successful vaccines have been developed?
- Look at published data for clues to best antigen – in particular, has anyone published T cell or antibody responses to pathogen proteins, and do they correlate with protection against disease (either vaccinated individuals or natural immunity)? This can be in animals or, better still, in humans
- Select antigen that is as conserved as possible across all known strains of the pathogen, and ideally related species
- Select antigen that is vital for pathogenicity e.g. not part of a redundant cell entry pathway (so for example, has data been published on knock outs of the gene in question, and does the antigen play a vital role in pathogenicity?)
- Remember for some pathogens one antigen may not be enough – consider options eg fusion proteins, viral vector expressing two or more antigens. Also for pathogens with >1 strain eg Ebola, a vaccine covering surface glycoproteins from two or more strains/species would be extremely useful
Antigen purification (required for point 3 above):
- Try expression of antigen as a) protein, or b) in a viral vector. For soluble proteins, it is typical to try a) mammalian cells usually HEK293, b) E. coli, and c) Yeast, usually Pichia pastoris or Saccharomyces cerevisiae. If these fail other expression systems are available eg insect cells, cell free systems. Test expression on SDS PAGE gel, with W blotting if a detection antibody is available.
- Purification is usually performed initially using an affinity tag – typically His tag, the Jenner is now using the C-tag from BAC.
- It’s important to consider whether glycosylation is important – if yes, then the best expression system is mammalian. Malaria antigens are not typically glycosylated in vivo (during natural infection) as they are made by parasite-specific enzymes
- Antigen QC is important to check correct conformation – ultimately this is done by testing immunogenicity in mice and looking for activity of the antibodies in inhibiting pathogencitiy.
- Selecting the right antigen or list of antigens to test
- Having an appropriate assay/model in which to test the antigens
- Being able to express the antigen in its native conformation
The time taken to select and purify the antigen may take 6 months to several years (6 months for simple virus). Bottlenecks in this process include:
- Developing a model in which to test antigen suitability/efficacy (either an in vivo model or an in vitro model of infectivity). This may, as for malaria, involve generating transgenic parasites.
- Expressing antigens –some antigens are notoriously difficult to express, for example if they are cysteine-rich
- Collaboration with other groups who have developed models, or (as for Ebola) are able to test infection of the native pathogen
- Development of models which can be easily adapted to new pathogens eg the lentivirus surrogate infectivity assay that was developed for Ebola (which could be applied to other virus antigens)
- Developing centres of excellence for protein expression and purification
- Develop high throughput methods for testing multiple antigens/vaccines
Resource is generally most rate limiting (money to fund the research is needed, and laboratory space).
Time is also rate limiting in the context of outbreak situations (it takes a long time to a) get funding in place, and b) carry out vaccine design, production and testing in the pre-clinical phase, then moving into GMP manufacture). Generally in vivo data is important and this is, by definition, time consuming.