Modulation of replicative fitness by deoptimization of synonymous codons

Abstract

Methods of producing a pathogen with reduced replicative fitness are disclosed, as are attenuated pathogens produced using the methods. In particular examples, the method includes deoptimizing one or more codons in a coding sequence, thereby reducing the replicative fitness of the pathogen. Methods of using the attenuated pathogens as immunogenic compositions are also disclosed.

We claim: 
     
         1 . A method of attenuating a bacterium, comprising preparing a bacterial nucleic acid molecule comprising at least twenty deoptimized codons in a coding sequence of the bacterium by replacing each of the at least twenty codons in the coding sequence with a synonymous codon less frequently used in the bacterium, thereby generating an attenuated bacterium comprising a deoptimized bacterial nucleic acid molecule. 
     
     
         2 . The method of  claim 1 , wherein the bacterial nucleic acid molecule comprises replacement of at least 50-2000 codons with synonymous codons less frequently used in the bacterium. 
     
     
         3 . The method of  claim 1 , wherein replicative fitness of the attenuated bacterium is reduced by at least 20% as compared to an amount of replicative fitness by the bacterium having a coding sequence with a native codon composition. 
     
     
         4 . The method of  claim 1 , wherein replicative fitness of the attenuated bacterium is reduced by 10-98% as compared to replicative fitness of the bacterium with a native codon composition. 
     
     
         5 . The method of  claim 1 , wherein the deoptimized bacterial nucleic acid molecule comprises replacement of at least 50% of the coding sequence with synonymous codons less frequently used in the bacterium. 
     
     
         6 . The method of  claim 1 , wherein the deoptimized bacterial nucleic acid molecule alters the number of CG dinucleotides, the number of TA dinucleotides, or the number of CG dinucleotides and TA nucleotides in the coding sequence by at least 20%. 
     
     
         7 . The method of  claim 6 , wherein the deoptimized bacterial nucleic acid molecule increases the number of CG dinucleotides or TA dinucleotides in the coding sequence by at least 100%. 
     
     
         8 . The method of  claim 1 , wherein the deoptimized bacterial nucleic acid molecule comprises a coding sequence having an increased number of CG dinucleotides, TA dinucleotides, or CG dinucleotides and TA nucleotides in the coding sequence, wherein the CG or TA dinucleotides fall across codon boundaries. 
     
     
         9 . The method of  claim 1 , wherein the deoptimized bacterial nucleic acid molecule is introduced into the bacterial genome. 
     
     
         10 . The method of  claim 1 , wherein the bacterium is a gram-negative bacterium. 
     
     
         11 . The method of  claim 10 , wherein the gram-negative bacterium is  Escherichia coli, Shigella dysenteriae , or  Vibrio cholerae.    
     
     
         12 . The method of  claim 11 , wherein the bacterium is  E. coli  and wherein at least 20 codons in an ArgS or TufA coding sequence are deoptimized or the bacterium is  Shigella dysenteriae  and at least 20 codons in a RdsB coding sequence are deoptimized. 
     
     
         13 . The method of  claim 12 , wherein the deoptimized coding sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 48 or SEQ ID NO: 51. 
     
     
         14 . The method of  claim 13 , wherein the deoptimized coding sequence comprises a sequence shown in SEQ ID NO: 48 or SEQ ID NO: 51. 
     
     
         15 . The method of  claim 1 , wherein the bacterium is a gram-positive bacterium. 
     
     
         16 . The method of  claim 15 , wherein the gram-positive bacterium is  Bacillus anthracis, Staphylococcus aureus , pneumococcus, gonococcus, or streptococcal meningitis. 
     
     
         17 . The method of  claim 16 , wherein the bacterium is  Staphylococcus aureus  and at least twenty codons in an RplB or FusA coding sequence are deoptimized. 
     
     
         18 . An immunogenic composition comprising an attenuated bacterium, wherein the attenuated bacterium comprises at least twenty deoptimized codons in a coding sequence, wherein the attenuated bacterium is made by the method of  claim 1 . 
     
     
         19 . The immunogenic composition of  claim 18 , wherein the attenuated bacterium comprises a number of CG dinucleotides or TA dinucleotides that is at least 30% greater than a bacterium of the same species with a native coding sequence 
     
     
         20 . The immunogenic composition of  claim 18 , further comprising an adjuvant. 
     
     
         21 . The immunogenic composition of  claim 18  wherein the attenuated pathogen is a bacterium comprising at least 15 deoptimized codons in an ArgS or TufA coding sequence. 
     
     
         22 . A method of eliciting an immune response against a pathogen in a subject, comprising introducing into the subject a immunologically effective amount of the immunogenic composition of  claim 18 , thereby eliciting an immune response in the subject.