Patent Publication Number: US-5153129-A

Title: Membrane protein having proteolytic activity obtainable from plasmodium falciparum blood schizonts and merozoites

Description:
This application is a continuation-in-part of Ser. No. 07/376,926, filed Jul. 10, 1989 now U.S. Pat. No. 5,032,397 which was a continuation of Ser. No. 07/045,220 filed Apr. 29, 1987 now abandoned, which was a continuation of Ser. No. 06/644,727 filed Aug. 23, 1984, now abandoned. 
    
    
     Further investigation in the Plasmodium falciparum membrane proteins showed that they comprised a polypeptide having a molecular weight of 76,000 daltons (or 76 Kd polypeptide) which can be released in the form of an enzymatically active protease from the membranes of intact merozoites or isolated blood-schizont membranes, when the latter are treated with a phospholipase, such as the phosphatidylinositol-specific phospholipase, C (PI-PLC), particularly a PI-PLC of Staphylococcus aureus. It can also be released from the parasites cells in the form of a water soluble enzyme by osmotic shock. 
     This PI-PLC induced 76 Kd protease is a water soluble enzymatically active protease, more particularly a serine-protease whose activity is inhibited by PMSF (phenylmethyl sulfonyl fluoride), chymostatin, leupeptin, yet it is not inhibited by pepstatin and EDTA (ethylenediamine tetracetate). It is also recognized by monoclonal antibodies which recognize inactive 76 Kd polypeptide, e.g. monoclonal antibody 31c13 disclosed in PERRIN L., DAYAL R. ; Immunological review, 61 : 245-270 (1982). 
     The invention relates to immunogenic compositions containing the above-mentioned enzymatically active 76 Kd protease, extracted from malarial parasites, said protease being also characterized by its capacity to react with protective antibodies originating from monkeys resistant to human parasites of malaria and particularly to species of human Plasmodium falciparum. 
     Particularly, the invention relates to immunogenic compositions containing the enzymatically active 76 Kd protease, 
     which induces particularly in monkey, and more particularly Saimiri Sciureus monkey, active antibodies against malaria parasites, more particularly Plasmodium falciparum or parasites which present the same essential biological characteristics. 
     which is recognized by sera or other immunoglobulin compositions originating from animals, particularly Samiri Sciureus monkeys, resistant to parasites, these sera or other compositions containing the corresponding immunoglobulins being capable, by in vivo passive transfer to animals sensitive to the parasite, to protect them against said parasite. 
     More particularly, the 76 KD protease according to the invention is characterized by the fact that it is recognized by protective antibodies which are contained in a protective serum obtained from immunized Saimiri Sciureus monkey as a result of previous infection by FUPC I-212 P. falciparum strain, said serum having been collected within 10 to 90 days from the end of the acute infection. 
     The invention also relates to a process for obtaining such immunogenic compositions. This process comprises treating a preparation which has been obtained from a malarial infectious parasite, particularly of the plasmodium type, such a Plasmodium falciparum, particularly at the merozoite or blood schizont stage of the parasite, with a phospholipase, such as the Staphylococcus aureus PI-PLC, or Trypanosoma brucei PI-PLC, or alike, liable to induce the release of the 76 Kd protease in the soluble fraction, separating the soluble fraction from the membrane pellet, and recovering the 76 kd protease. 
     The recovery of the 76 kd protease can be obtained by an appropriate method, such as gel filtration, electrophoresis or alike, and particularly by contacting the solution, resulting from the treatment of the parasite with the phospholipase, with antibodies recognizing said protease, which antibodies have been previously fixed to a non-soluble support, removing the non fixed proteins, such as by washing, dissociating the complex formed between the fixed antibodies and the 76 Kd protease, and recovering said 76 Kd protease. 
     For instance, the above-mentioned antibodies consist of protective antibodies originating from monkey, or monoclonal antibodies such as the above-cited monoclonal antibody 31c13. 
    
    
     Additional characteristics of the invention will appear again in the description which follows of examples of production of the enzymatically active 76 Kd protease and of the biological properties thereof. 
     1) The FUP strain of P. falciprum (GYSIN et al, J. Parasitol, 66, 1003-1009 (1980)) is cultivated as described (BRAUN-BRETON et al, Molec. Biochem. Parasitol, 20, 33-43 (1986)). Synchronization is performed by incubation of the cells in 2 volumes of 0.3M alanine, 10 mM Hepes for 3 minutes at 37° C. Schizonts are purified on Percoll-Sorbitol gradients as described (ALEY et al, J. Exp. Med., 160, 1585-1590 (1984)).  35  S methionine labeling and cell fractionation were previously described (BRAUN-BRETON et al, Molec. Biochem. Parasitol, 20, 33-43 (1986)). The membrane fraction is resuspended in phosphate-buffered saline (PBS) and is incubated for 1 hour at 37° C., with 1 to 5 μg ml -1  S. aureus PI-PLC. For immunoprecipitation, the membrane suspension is diluted in 1 volume of 20 mM Tris pH 7,5-20 mM EDTA -1 M Na Cl - 2% Triton X100, incubated for 30 minutes at room temperature and centrifuged 5 mn at 12000 rpm. The 31 c13 immunoglobulins were purified on Affigel-protein A -MAPS II (Biorad) under these conditions the IgG were depleted of the proteases present in the ascitic fluid. Purified IgG are used for immunoprecipitation as described ((BRAUN-BRETON et al, Molec. Biochem. Parasitol, 20, 33-43 (1986)) with the following modifications : incubation with antigen for 1 hour at room temperature in the absence of protease inhibitors. For the last wash each sample is divided into 2 equal volumes in such a way that half of the immunocomplexes are resuspended in Laemli sample buffer for polyacrylamide gel electrophoresis (SDS-PAGE) (LAEMLI et al, Nature, 277, 680-682 (1970)) and half resuspended in solution P (2,5% SDS, 2% sucrose, 0,1% bromophenol blue) for analysis of proteases. Electrophoretic analysis of proteases in gels containing SDS and 0,1% gelatin was previously described (HEUSSEN et al, Anal. Biochem., 102, 196 (1980)). 
     The specific monoclonal antibody 31c13 immunoprecipitates an enzymatically active 76 Kd protease from PI-PLC treated membranes and an inactive 76 Kd polypeptide from detergent treated membranes, e.g. SDS treated membranes. Detergent extraction of membrane proteins does not solubilize an enzymatically active 76 Kd protease. Detergent does not irreversely inhibit the protease activity since the PI-PLC induced activity is resistant to identical detergent conditions, and induction of the protease activity by PI-PLC can be performed on detergent extracted membrane proteins. 
     2) Inhibition of protease activity by protease inhibitors 
     
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                       Pepstatin                                          
                              Leupeptin                                   
       PMSF    EDTA    50 μg.                                          
                              5/50 μg.                                 
                                      Chymostatin                         
inhibitors                                                                
       10 mM   10 mM   ml.sup.-1                                          
                              ml.sup.-1                                   
                                      50 μg. ml.sup.-1                 
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76 Kd  -       ++      ++     +-/-    -                                   
protease                                                                  
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 Schizont membranes were treated with 5 μg. ml.sup.-1 S. aureus PIPLC, 
 resuspended in solution P, and analyzed for protease activity as         
 abovedescribed. Protease inhibitors were added in the O, 1M Glycin byffer
 in which the gels were incubated after electrophoresis.                  
 - no detectable protease activity,                                       
 +- faint protease activity,                                              
 + detectable activity but lower than control (without inhibitor),        
 ++ protease activity comparable to the control.