I have been reviewing the literature hoping to understand how to determine the amount of rebombinant protein is needed for one dose of vaccine and it seems quite variable (From 2um to 40g!)
Any suggestions of steps/protocols/technical books to follow?
Thanks
Dear Sir,
See:
Article Recombinant vaccines and the development of new vaccine strategies
In my experience the immunogenicity of a recombinant antigen depends on several factors including the quantity of recombinant protein, adjuvant (type as well as dose), route of immunization and number of booster immunizations.
These can be only tested empirically and there is no one formula that works for all the recombinant proteins.
I would suggest that you initially try two immunizations with 20ug protein mixed with alum (as adjuvant), by intramuscular route (in 50 ul) on days 0 and 21. Two weeks after the booster that is on day 35 from the first immunization, isolate sera and evaluate antibodies against the recombinant protein.
If the titer is more than 5 fold higher then you can proceed to challenge with the relevant pathogen to test efficacy of your vaccine.
If the titer is not enough then you may consider another booster on day 42 and follow antibody analysis two weeks later and subsequent challenge.
I believe you may get a lot of insight from one experiment that you can direct subsequent experiments.
By the way this dose and immunization schedule I am recommending for mice.
Good luck,
That is not a Q one can easily answer. It depends on your recombinant protein. Also on the disease your vaccine is trying to target. It would also depend on your vaccine formulation/delivery platform. When using conjugated polysaccharides, you need 10 times less polysaccharide (around 5 ug) as compared to when using non-conjugated polysaccharides (50 ug). When using a recombinant protein you may need to use 10 times more of the protein as opposed to if you had expressed the protein in a virus or VLP.
Identifying the vaccine dose (or the amount of API to be included in one vaccine dose) takes years and it is all about finding a dose that is effective/ immunogenic but at the same time safe. Once you have a vaccine candidate, you first do a dose response in a relevant in vitro model (usually cells). The aim is to identify a few doses where your vaccine candidate shows you the desired effect but is not causing cell death (or any undesired effect).
Once you know your vaccine candidate is effective and you have chosen a few doses that are safe, you then test these doses out in a relevant pre-clinical model (usually rodents). The idea here is to verify safety and efficacy in vivo. From here, you pick 1-2 doses where you see maximum effect but again do not cause any adverse events in the animals. If it makes sense, you then test a couple of doses in higher order animals (non-human primates) and see whether you vaccine works. Finally you do a phase 1 clinical trial (to assess safety) with a single dose that has been found effective in pre-clinical studies or a dose-escalation study if you are unsure what the ideal dose would be.
The simple answer would be to look at the literature and see if there are studies already done with the recombinant protein you are interested in (or similar proteins). Use that as a starting point. However, it is always a good idea to test multiple doses when you are starting out, as results can vary from lab to lab and depending on the model. You may also have to perform dose conversion when you move from species to species and there are guidelines out there to help you do this.
Thank you all!
Very helpful answers.
I did a compilation of data in the literature for the targeted animal species (pig) and it varies from 20-200ug, but as you mentioned, 50ug is a good start point.
Now I will choose the adjuvant from those who are more often used in pigs, considering all the factors you brought up here.
I agree with Sudeep and Julie. The only addition that I would make is that if you are working at developing a recombinant vaccine for animals, try using a better adjuvant than alum. MVP adjuvants (www.mvpadjuvants.com) has an array of them. Emulsigen, Emulsigen-D and Polygen have been used successfully with recombinant antigens. The stronger your adjuvant, the less antigen that you will require.
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