Expression system for modulating an immune response
The present invention discloses methods and compositions for modulating the quality of an immune response to a target antigen in a mammal, which response results from the expression of a polynucleotide that encodes at least a portion of the target antigen, wherein the quality is modulated by replacing at least one codon of the polynucleotide with a synonymous codon that has a higher or lower preference of usage by the mammal to confer the immune response than the codon it replaces.
1. A method for constructing a synthetic polynucleotide from which a polypeptide is producible to confer a stronger or enhanced immune response to a target antigen in a mammal than that conferred by a parent polynucleotide that encodes the same polypeptide, wherein the immune response is selected from the group consisting of (1) a humoral immune response, (2) a cellular immune response and (3) a cellular immune response and a humoral immune response, wherein the polypeptide corresponds to at least a portion of the target antigen, the method comprising constructing the synthetic polynucleotide so that it is distinguished from the parent polynucleotide by replacement of at least 5% of codons which correspond to the target antigen in the parent polynucleotide with synonymous codons, wherein individual codon replacements are carried out by: (a) selecting a first codon of the parent polynucleotide for replacement with a synonymous codon according to TABLE 3:
TABLE 3
First Codon
Synonymous Codon
Ala GCG
Ala GCT
Ala GCA
Ala GCT
Ala GCC
Ala GCT
Arg CGG
Arg CGA
Arg CGG
Arg CGT
Arg CGG
Arg AGA
Arg AGG
Arg CGA
Arg AGG
Arg CGT
Arg AGG
Arg AGA
Glu GAG
Glu GAA
Gly GGC
Gly GGA
Gly GGT
Gly GGA
Gly GGG
Gly GGA
Leu TTA
Leu CTA
Leu TTA
Leu CTT
Leu TTA
Leu TTG
Leu TTG
Leu CTA
Leu TTG
Leu CTT
Phe TTC
Phe TTT
Pro CCG
Pro CCT
Pro CCA
Pro CCT
Ser AGT
Ser TCG
Ser AGT
Ser TCT
Ser AGT
Ser TCA
Ser AGC
Ser TCG
Ser AGC
Ser TCT
Ser AGC
Ser TCA
Ser AGC
Ser TCC
Ser TCC
Ser TCG
Ser TCA
Ser TCG
Ser TCT
Ser TCG
Thr ACT
Thr ACG
Thr ACT
Thr ACA
Thr ACA
Thr ACG
Thr ACC
Thr ACG
Val GTA
Val GTT
and (b) replacing the first codon with the synonymous codon.
2. The method according to claim 1 , further comprising (i) selecting a second codon of the parent polynucleotide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits a higher immune response preference than the second codon in a comparison of immune response preferences; and (ii) replacing the second codon with the synonymous codon, wherein the comparison of immune response preferences of the codons is represented by TABLE 4:
Second Codon
Synonymous Codon
Ala GCG
Ala GCT
Ala GCG
Ala GCC
Ala GCA
Ala GCT
Ala GCA
Ala GCC
Ala GCC
Ala GCT
Arg CGG
Arg CGA
Arg CGG
Arg CGC
Arg CGG
Arg CGT
Arg CGG
Arg AGA
Arg AGG
Arg CGA
Arg AGG
Arg CGC
Arg AGG
Arg CGT
Arg AGG
Arg AGA
Asn AAT
Asn AAC
Asp GAT
Asp GAC
Cys TGT
Cys TGC
Glu GAG
Glu GAA
Gly GGC
Gly GGA
Gly GGT
Gly GGA
Gly GGG
Gly GGA
Ile ATA
Ile ATC
Ile ATA
Ile ATT
Ile ATT
Ile ATC
Leu TTA
Leu CTG
Leu TTA
Leu CTC
Leu TTA
Leu CTA
Leu TTA
Leu CTT
Leu TTA
Leu TTG
Leu TTG
Leu CTG
Leu TTG
Leu CTC
Leu TTG
Leu CTA
Leu TTG
Leu CTT
Leu CTT
Leu CTG
Leu CTT
Leu CTC
Leu CTA
Leu CTG
Leu CTA
Leu CTC
Phe TTC
Phe TTT
Pro CCG
Pro CCC
Pro CCG
Pro CCT
Pro CCA
Pro CCC
Pro CCA
Pro CCT
Pro CCT
Pro CCC
Ser AGT
Ser TCG
Ser AGT
Ser TCT
Ser AGT
Ser TCA
Ser AGT
Ser TCC
Ser AGC
Ser TCG
Ser AGC
Ser TCT
Ser AGC
Ser TCA
Ser AGC
Ser TCC
Ser TCC
Ser TCG
Ser TCA
Ser TCG
Ser TCT
Ser TCG
Thr ACT
Thr ACG
Thr ACT
Thr ACC
Thr ACT
Thr ACA
Thr ACA
Thr ACG
Thr ACA
Thr ACC
Thr ACC
Thr ACG
Tyr TAT
Tyr TAC
Val GTA
Val GTG
Val GTA
Val GTC
Val GTA
Val GTT
Val GTT
Val GTG
Val GTT
Val GTC .
3. A method for constructing a synthetic polynucleotide from which a polypeptide is producible to confer a weaker or reduced immune response to a target antigen in a mammal than that conferred by a parent polynucleotide that encodes the same polypeptide, wherein the immune response is selected from the group consisting of (1) a humoral immune response; (2) a cellular immune response; and (3) a cellular immune response and a humoral immune response, wherein the polypeptide corresponds to at least a portion of the target antigen, the method comprising constructing the synthetic polynucleotide so that it is distinguished from the parent polynucleotide by replacement of at least 5% of codons, wherein individual codon replacements are carried out by: (a) selecting a first codon of the parent polynucleotide for replacement with a synonymous codon; and (b) replacing the first codon with the synonymous codon to construct the synthetic polynucleotide, wherein the first and synonymous codons are selected from the TABLE 5:
First Codon
Synonymous Codon
Ala GCT
Ala GCG
Ala GCT
Ala GCA
Ala GCT
Ala GCC
Ala GCC
Ala GCG
Ala GCC
Ala GCA
Arg CGA
Arg AGG
Arg CGA
Arg CGG
Arg CGC
Arg AGG
Arg CGC
Arg CGG
Arg CGT
Arg AGG
Arg CGT
Arg CGG
Arg AGA
Arg AGG
Arg AGA
Arg CGG
Asn AAC
Asn AAT
Asp GAC
Asp GAT
Cys TGC
Cys TGT
Glu GAA
Glu GAG
Gly GGA
Gly GGC
Gly GGA
Gly GGT
Gly GGA
Gly GGG
Ile ATC
Ile ATA
Ile ATC
Ile ATT
Ile ATT
Ile ATA
Leu CTG
Leu CTA
Leu CTG
Leu CTT
Leu CTG
Leu TTG
Leu CTG
Leu TTA
Leu CTC
Leu CTA
Leu CTC
Leu CTT
Leu CTC
Leu TTG
Leu CTC
Leu TTA
Leu CTA
Leu TTG
Leu CTA
Leu TTA
Leu CTT
Leu TTG
Leu CTT
Leu TTA
Leu TTG
Leu TTA
Phe TTT
Phe TTC
Pro CCC
Pro CCT
Pro CCC
Pro CCA
Pro CCC
Pro CCG
Pro CCT
Pro CCA
Pro CCT
Pro CCG
Ser TCG
Ser TCT
Ser TCG
Ser TCA
Ser TCG
Ser TCC
Ser TCG
Ser AGC
Ser TCG
Ser AGT
Ser TCT
Ser AGC
Ser TCT
Ser AGT
Ser TCA
Ser AGC
Ser TCA
Ser AGT
Ser TCC
Ser AGC
Ser TCC
Ser AGT
Thr ACG
Thr ACC
Thr ACG
Thr ACA
Thr ACG
Thr ACT
Thr ACC
Thr ACA
Thr ACC
Thr ACT
Thr ACA
Thr ACT
Tyr TAC
Tyr TAT
Val GTG
Val GTT
Val GTG
Val GTA
Val GTC
Val GTT
Val GTC
Val GTA
Val GTT
Val GTA .
4. A method for constructing a synthetic polynucleotide from which a polypeptide is producible to confer a weaker or reduced immune response to a target antigen in a mammal than that conferred by a parent polynucleotide that encodes the same polypeptide, wherein the immune response is selected from the group consisting of (1) a humoral immune response; (2) a cellular immune response; and (3) a cellular immune response and a humoral immune response, wherein the polypeptide corresponds to at least a portion of the target antigen, the method comprising constructing the synthetic polynucleotide so that it is distinguished from the parent polynucleotide by replacement of at least 5% of codons, wherein individual codon replacements are carried out by: (a) selecting a first codon of the parent polynucleotide for replacement with a synonymous codon; and (b) replacing the first codon with the synonymous codon to construct the synthetic polynucleotide, wherein the first and synonymous codons are selected from TABLE 6:
First Codon
Synonymous Codon
Ala GCT
Ala GCG
Ala GCT
Ala GCA
Ala GCT
Ala GCC
Arg CGA
Arg AGG
Arg CGA
Arg CGG
Arg CGT
Arg AGG
Arg CGT
Arg CGG
Arg AGA
Arg AGG
Arg AGA
Arg CGG
Glu GAA
Glu GAG
Gly GGA
Gly GGC
Gly GGA
Gly GGT
Gly GGA
Gly GGG
Leu CTA
Leu TTG
Leu CTA
Leu TTA
Leu CTT
Leu TTG
Leu CTT
Leu TTA
Leu TTG
Leu TTA
Phe TTT
Phe TTC
Pro CCT
Pro CCA
Pro CCT
Pro CCG
Ser TCG
Ser TCT
Ser TCG
Ser TCA
Ser TCG
Ser TCC
Ser TCG
Ser AGC
Ser TCG
Ser AGT
Ser TCT
Ser AGC
Ser TCT
Ser AGT
Ser TCA
Ser AGC
Ser TCA
Ser AGT
Ser TCC
Ser AGC
Thr ACG
Thr ACC
Thr ACG
Thr ACA
Thr ACG
Thr ACT
Thr ACA
Thr ACT
Val GTT
Val GTA .
5. The method according to claim 4 , further comprising (i) selecting a second codon of the parent polynucleotide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits a lower immune response preference than the second codon in a comparison of immune response preferences; and; (ii) replacing the second codon with the synonymous codon, wherein the comparison of immune response preferences of the codons is represented by TABLE 7:
TABLE 7
Second Codon
Synonymous Codon
Ala GCT
Ala GCG
Ala GCT
Ala GCA
Ala GCT
Ala GCC
Ala GCC
Ala GCG
Ala GCC
Ala GCA
Arg CGA
Arg AGG
Arg CGA
Arg CGG
Arg CGC
Arg AGG
Arg CGC
Arg CGG
Arg CGT
Arg AGG
Arg CGT
Arg CGG
Arg AGA
Arg AGG
Arg AGA
Arg CGG
Asn AAC
Asn AAT
Asp GAC
Asp GAT
Cys TGC
Cys TGT
Glu GAA
Glu GAG
Gly GGA
Gly GGC
Gly GGA
Gly GGT
Gly GGA
Gly GGG
Ile ATC
Ile ATA
Ile ATC
Ile ATT
Ile ATT
Ile ATA
Leu CTG
Leu CTA
Leu CTG
Leu CTT
Leu CTG
Leu TTG
Leu CTG
Leu TTA
Leu CTC
Leu CTA
Leu CTC
Leu CTT
Leu CTC
Leu TTG
Leu CTC
Leu TTA
Leu CTA
Leu TTG
Leu CTA
Leu TTA
Leu CTT
Leu TTG
Leu CTT
Leu TTA
Leu TTG
Leu TTA
Phe TTT
Phe TTC
Pro CCC
Pro CCT
Pro CCC
Pro CCA
Pro CCC
Pro CCG
Pro CCT
Pro CCA
Pro CCT
Pro CCG
Ser TCG
Ser TCT
Ser TCG
Ser TCA
Ser TCG
Ser TCC
Ser TCG
Ser AGC
Ser TCG
Ser AGT
Ser TCT
Ser AGC
Ser TCT
Ser AGT
Ser TCA
Ser AGC
Ser TCA
Ser AGT
Ser TCC
Ser AGC
Ser TCC
Ser AGT
Thr ACG
Thr ACC
Thr ACG
Thr ACA
Thr ACG
Thr ACT
Thr ACC
Thr ACA
Thr ACC
Thr ACT
Thr ACA
Thr ACT
Tyr TAC
Tyr TAT
Val GTG
Val GTT
Val GTG
Val GTA
Val GTC
Val GTT
Val GTC
Val GTA
Val GTT
Val GTA .
6. A method of making a chimeric construct, comprising constructing a synthetic polynucleotide according to the method of claim 1 and operably linking a regulatory polynucleotide to the synthetic polynucleotide.
7. The method according to claim 6 , further comprising constructing the chimeric construct so that it includes the coding sequence of an adjuvant.
8. The method according to claim 7 , wherein the adjuvant is selected from: detoxified mutants of bacterial ADP-ribosylating toxins, diphtheria toxin, pertussis toxin, cholera toxin, Escherichia coli heat-labile toxins, Pseudomonas endotoxin A, Clostridium botulinum C2 and C3 toxins, toxins from C. perfringens, C. spiriforma and C. difficile ; and protein-destabilizing elements.
9. The method according to claim 7 , wherein the adjuvant is a protein-destabilizing element selected from a destabilizing amino acid at the amino-terminus of the polypeptide, a PEST region or an ubiquitin.
10. The method according to claim 6 , further comprising formulating the chimeric construct for transcutaneous administration, epidermal administration, dermal administration, intradermal administration, biolistic delivery, microneedle delivery or intradermal injection.
11. A synthetic polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 82.
12. A chimeric construct comprising the synthetic polynucleotide of claim 11 operably linked to a regulatory polynucleotide.
13. The method of claim 1 , wherein the target antigen is a herpes simplex virus antigen.
14. The method of claim 13 , wherein the herpes simplex virus antigen is a glycoprotein D.
15. The method of claim 13 , wherein the herpes simplex virus antigen is gD2.