The study used recombinatorial cloning to overproduce a large number of S. Typhimurium proteins. These were used to validate 2-D gel/ immunoblot screening. Production of antibodies to specific antigens was found to change during the course of animal infection and varied with mouse strain. Soluble proteins, seen as antigens during infection, failed to provide strong protective immunity on immunisation, even when appropriate antibody responses were elicited; adjuvants differed in degree of ability to elicit IgG2a (a TH1 indicator). Moreover antigens differed in ability to prevent weight loss during infection. This appeared linked with length of animal survival post-infection. Moreover some antigen combinations adversely affected weight loss, even when IgG2a was elevated. Pilot analysis showed that macroarrays have the potential for parallel serum screening of antigens and for monitoring kinetics of antibody development during infection, providing a useful tool to aid vaccine development.
Salmonella Typhimurium infection is a very common problem. It affects both humans and animals and is a problem for food production (particularly from poultry and cattle). Moreover Salmonella are often resistant to antibiotics. Vaccines provide an alternative to antibiotics for reducing or preventing disease. Live vaccines, which are crippled bacteria, exist but cannot be used, especially when people/animals are young or when health is reduced (e.g. by disease, pregnancy or when under chemotherapy). A vaccine based on bacterial proteins would be much safer, as it cannot cause infection. However, at the moment we know little about which proteins to use, as there have been very few studies on this topic.
Using blood (serum) taken from mice which have been infected with a live vaccine, we have been able to identify some of the bacterial proteins that are recognised by antibodies during the infection. Since the body responds to these proteins naturally during infection and recognises them as foreign, these same proteins might provide a way to stimulate antibody production if they are given artificially in an injection, and this might then protect against infection.
Using recombinant DNA technology we have artificially produced and extracted a number of these bacterial proteins, to see whether they can protect mice from S. Typhimurium infection. We were able to show that we could stimulate mice to produce antibodies of the right type to possibly give protection against infection. However, none of the antigens actually resulted in really strong protection when tested. However, we did find that some proteins reduced the amount of weight that mice lost during Salmonella infection and the mice also looked healthier; this is good because it suggest that it may be possible to reduce the side-effects of the illness (although more work is needed to understand how this happens). Unfortunately, the effect was sometimes lowered when several proteins were given together and it will be difficult to predict which proteins are likely to be most useful and which are not.
As a pilot study, we examined whether we could use robots to print proteins on a membrane. These proteins could then be screened all at once, to see if they are normally recognised by antibodies in the blood of infected animals. This should help speed up the process of finding proteins which might be useful for forming a vaccine, as antibodies are needed to protect against infection. Using the membranes we screen a small number of proteins and were able to tell which proteins produced antibodies quickest and also, which produced the strongest antibody responses.
When proteins are used as a vaccine, a compound called an adjuvant is also given to help boost the body’s reaction. The range of adjuvant s which can be used in humans or animals is very limited at the moment. As part of our studies we examined the ability of three different adjuvants to help boost antibody production. Each worked to an extent and the right type of antibody was produced to indicate that the body reacted properly but the levels of antibody produced were found to depend on the specific adjuvant. In the longer term, this should help us develop better vaccines.
1. Recombinatorial cloning allowed production of a large number of Salmonella typhimurium proteins which were screened by 2-D gel/ immunoblotting.
2. The timing of antibody production to specific antigens was found to change during the course of animal infection and varied with mouse strain.
3. Cytosolic proteins, seen as antigens during infection, failed to provide strong protective immunity, even when appropriate antibody responses were elicited.
4. Antigens differed in ability to prevent weight loss during mouse infection, and this correlated with length of animal survival post-infection.
5. Some antigen combinations adversely affected weight loss, even when IgG2a (a TH1 indicator; cell mediated immunity is a known requirement for protective
immunity) was elevated.
6. Adjuvants were found to differ in degree of ability to elicit IgG2a .
7. Pilot analysis showed that macroarrays have the potential for parallel serum screening of antigens and for monitoring kinetics of antibody development during infection, providing a useful tool to aid vaccine development