Modified immunogenic pneumolysin compositions as vaccines

Abstract

This invention relates to modified pneumolysin polypeptides that retain the immunogenic nature of pneumolysin but have reduced or undetectable hemolytic activity compared to native pneumolysin. The invention also provides a method for generating novel pneumolysin variants with these desired characteristic properties. The invention also provides immunogenic compositions useful as pharmaceutical compositions including vaccines in which non-toxic, modified pneumolysin is used to stimulate protective immunity against Streptococcus pneumoniae . The vaccines may be compositions in which the modified pneumolysin is conjugated to bacterial polysaccharides or may be carried on an attenuated viral vector. In addition, the invention also provides a method of using the non-toxic, modified pneumolysin toxoid in order to stimulate antibodies against Streptococcus pneumoniae in a treated individual which are then isolated and transferred to a second individual, thereby conferring protection against Streptococcus pneumoniae in the second individual.

1 - 15 . (cancelled)  
     
     
         16 . A recombinant nucleic acid molecule encoding a modified type 14 pneumolysin polypeptide comprising one or more amino acid substitutions in a wild-type pneumolysin polypeptide having the amino acid sequence of SEQ ID NO:3, wherein said one amino acid substitution occurs at a position selected from the group consisting of position  61 ,  148 , and  195 , or wherein said more than one amino acid substitution occurs at positions selected from the group consisting of  17 ,  18 ,  33 ,  41 ,  45 ,  46 ,  61 ,  63 ,  66 ,  83 ,  101 ,  102 ,  128 ,  148 ,  189 ,  195 ,  239 ,  243 ,  255 , and  257 , and wherein said modified pneumolysin polypeptide is soluble, elicits antibodies which are cross-reactive with wild-type pneumolysin, and has attenuated hemolytic activity.  
     
     
         17 . The recombinant nucleic acid molecule according to  claim 16  comprising the pneumolysin nucleic acid sequence of SEQ ID NO: 1, 
 and wherein said nucleic acid sequence comprises one or more of the nucleotide substitutions selected from the group consisting of:    A-50→G, G-54→T, T-181→C, A-196→T and T-302→C;    A-122→G, A-514→G, T-583→A and A-764→G;    A-187→T, T-380→A, A-382→C and T-443→A;    T-98→C, T-137→C, T-248→C, T-717→A and A-770→G;    T-134→C, A-305→G, A-566→G and T-583→G;    T-583→G;    T-583→A;    T-443→A;    and    T-181→C.    
     
     
         18 . The recombinant nucleic acid molecule of  claim 16  as contained in a vector.  
     
     
         19 . A microorganism comprising the nucleic acid molecule of  claim 16 .  
     
     
         20 . The microorganism according to  claim 19 , wherein the microorganism is selected from the group consisting of: bacteria, yeast, mammalian and insect cells.  
     
     
         21 . The microorganism according to  claim 20 , wherein the microorganism is  E. coli.    
     
     
         22 - 26 . (cancelled)  
     
     
         27 . A method for killing bacteria comprising contacting said bacteria with antibodies to an immunogenic molecule comprising modified pneumolysin comprising one or more amino acid substitutions in a wild-type pneumolysin polypeptide having the amino acid sequence of SEQ ID NO:3, wherein said one amino acid substitution occurs at a position selected from the group consisting of position  61 ,  148 , and  195 , or wherein said more than one amino acid substitution occurs at positions selected from the group consisting of  17 ,  18 ,  33 ,  41 ,  45 ,  46 ,  61 ,  63 ,  66 ,  83 ,  101 ,  102 ,  128 ,  148 ,  189 ,  195 ,  239 ,  243 ,  255 , and  257 , and wherein said modified pneumolysin polypeptide is soluble, elicits antibodies which are cross-reactive with wild-type pneumolysin, and has attenuated hemolytic activity in the presence of complement.  
     
     
         28 . The method according to  claim 27 , wherein the immunogenic molecule is a polysaccharide-polypeptide conjugate wherein the polysaccharide is a bacterial capsular polysaccharide.  
     
     
         29 . A method for immunization of mammals comprising administering a vaccine of comprising the modified pneumolysin polypeptide comprising one or more amino acid substitutions in a wild-type pneumolysin polypeptide having the amino acid sequence of SEQ ID NO:3, wherein said one amino acid substitution occurs at a position selected from the group consisting of position  61 ,  148 , and 195, or wherein said more than one amino acid substitution occurs at positions selected from the group consisting of  17 ,  18 ,  33 ,  41 ,  45 ,  46 ,  61 ,  63 ,  66 ,  83 ,  101 ,  102 ,  128 ,  148 ,  189 ,  195 ,  239 ,  243 ,  255 , and  257 , and wherein said modified pneumolysin polypeptide is soluble, elicits antibodies which are cross-reactive with wild-type pneumolysin, and has attenuated hemolytic activity and a pharmaceutically acceptable carrier to said mammals.  
     
     
         30 . A method for obtaining modified pneumolysin polypeptides, wherein said modified pneumolysin polypeptides have reduced hemolytic activity and suitable for eliciting an immunogenetic response which is cross-reactive with wild-type pneumolysin comprising the steps of: 
 (a) mutating a nucleic acid molecule encoding wild-type pneumolysin to produce mutated nucleic acid molecules encoding modified pneumolysin polypeptides, wherein the modified pneumolysin polypeptides comprise one or more amino acid substitutions in a wild-type pneumolysin polypeptide having the amino acid sequence of SEQ ID NO:3, wherein said one amino acid substitution occurs at a position selected from the group consisting of position  61 ,  148 , and  195 , or wherein said more than one amino acid substitution occurs at positions selected from the group consisting of  17 ,  18 ,  33 ,  41 ,  45 ,  46 ,  61 ,  63 ,  66 ,  83 ,  101 ,  102 ,  128 ,  148 ,  189 ,  195 ,  239 ,  243 ,  255 , and  257  and expressing the mutated nucleic acid molecules in host cells;    (b) assaying the modified polypeptide expressed by the host cells for hemolytic activity; and    (c) identifying the modified pneumolysin polypeptides having substantially similar molecular weight as native wild-type pneumolysin and which are refoldable.    
     
     
         31 . The recombinant nucleic acid molecule of  claim 16 , wherein the vector is selected from the group consisting of: a plasmid, cosmid, bacteriophage and yeast artificial chromosome.