Human optimized Bacillus anthracis protective antigen

Abstract

The invention relates to a humanized nucleic acid construct from Bacillus anthracis protective antigen (PA) gene and method of modifying the gene. The humanized gene, and method of producing it, improves the structural fidelity of expressed protein product, when produced in mammalian host cells, to native, bacterially produced protein. The construct is useful in nucleic acid based vaccine formulations against B. anthracis.

1 . An immunogenic composition comprising a recombinant  Bacillus anthracis  protective antigen gene, wherein said composition is encoded by a nucleic acid sequence wherein regions of said sequence corresponding to regions of  B. anthracis  containing rare bacterial codons are replaced with rare mammalian codons and where the codons in the first 2 to 5% of the sequence are highly utilized human codons. 
     
     
         2 . The immunogenic composition of  claim 1 , wherein the mammalian codons are human. 
     
     
         3 . The immunogenic composition of  claim 1 , wherein the first 50 codons are highly utilized human codons. 
     
     
         4 . The immunogenic composition of  claim 1 , wherein said amino acid sequence is SEQ ID No. 1 and is encoded by nucleic acid sequence of SEQ ID No. 2. 
     
     
         5 . The immunogenic composition of  claim 1 , wherein said DNA is expressed from a DNA or viral expression system. 
     
     
         6 . The composition of  claim 1 , wherein protein product encoded from said recombinant protective antigen gene is induced via the TPA signal. 
     
     
         7 . A method of modifying a recombinant bacterial gene for expression in a mammalian host comprising:
 a. replacing highly utilized host codons in the first 2 to 5 percent of the total bacterial gene sequence;   b. replacing regions of the sequence where there are stretches of three or more rare bacterial codons with rare host codons;   c. evaluation said modified bacterial gene sequence to identify regions of complementarity that could result in RNA folding back on itself;   d. removing said regions of complementarity by altering the codons in these regions but retaining the same amino acid sequence of the expressed protein; and   e. evaluating for cryptic ribosomal splice sites in order to avoid regions of said gene being deleted by the host cell;   f. altering regions containing said splice sites to remove recognition sequences but retaining the original amino acid sequence.   
     
     
         8 . A method of inducing an immune response against  Bacillus anthracis  comprising:
 a. administering to the immunogenic composition of  claim 1 ;   b. Administering a second, boosting dose of said immunogenic composition.   
     
     
         9 . The method of  claim 8 , wherein said immunogenic composition is expressed from a DNA or viral expression vector.