Vaccine against intra-cellular pathogens
A pathogen and vaccine technology, applied in the direction of antibody medical components, bacterial antigen components, protozoan antigen components, etc., can solve problems that have not yet been prompted and improve the intracellular level of HSPs
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Embodiment 1
[0034] Example 1: Preparation of heat-induced SPs
[0035] Cells infected with M. bovis were washed in a serum-free medium such as RPMI (Sigma), then heat-shocked at 45°C for 0.5 hours, or at 42°C for 5 hours, and incubated overnight. Cells were then washed in serum-free medium followed by phosphate buffered saline (PBS). Cells were resuspended in homogenization buffer. This homogenization buffer can be a hypotonic buffer, such as containing 2mM MgCl 2 10mM phosphate, pH7.4. Cells are then disrupted with a cell homogenizer (eg Dounce or Potter homogenizer, Ultraturrax or Waring mixer). Alternatively, the homogenization buffer may contain a detergent, such as PBS with 0.5% Tween, at a concentration between 0.1-1%, and suitable for solubilizing cell membranes. Cell lysates are then processed by centrifugation, typically at 3-5000g for 5 minutes to remove nuclei and cell debris, followed by high speed centrifugation at 100000g for 15-30 minutes. The supernatant thus obtained...
Embodiment 2
[0039] Cell lines infected with Plasmodium are incubated in serum-free medium such as RPMI (Sigma) and incubated with TNF-[alpha] overnight. Typically, rat hepatocytes prepared by collagenase treatment of rat liver tissue were infected with Plasmodium Berghei by incubating rat hepatocytes with three times the number of parasite cells at 35°C for 5 hours. Cells were then incubated overnight with or without TNF-α. TNF-induced and control cells were washed in serum-free medium followed by phosphate-buffered saline (PBS). Example 3: Immunization with induced HSPs; immunity in vaccine recipients
Embodiment 3
[0040] Vaccine compositions containing HSP complexes were prepared as described in Example 1, and mice and rabbits were vaccinated by injecting 1-10 μg of stress protein-containing extracts in phosphate buffered saline, after an initial injection of one Booster with the same dose of vaccine 1 month later. Immunity induced by pathogens was analyzed by Western blot using total Plasmodium or M. bovis proteins. Antibody titers of 1:1-10000 are usually obtained, and cytotoxic T cell activity against pathogen-infected cells can also be detected in immunized mice. Challenge with immobilized Plasmodium or M. bovis at 6, 12 and 18 months after primary immunization resulted in good antibody responses with titers ranging from 1:1 to 10,000, indicating a good memory response in immunized animals. Example 4: Comparison of peptides bound in constitutive and induced SP complexes and their use as vaccines
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