Computationally optimized broadly reactive antigens for influenza

Abstract

The development of a computationally optimized influenza HA protein that elicits broadly reactive immune response to all H5N1 influenza virus isolates is described. The optimized HA protein was developed through a series of HA protein alignments, and subsequent generation of consensus sequences, for clade 2 H5N1 influenza virus isolates. The final consensus HA amino acid sequence was reverse translated and optimized for expression in mammalian cells. Influenza virus-like particles containing the optimized HA protein are an effective vaccine against H5N1 influenza virus infection in animals.

1 . An influenza virus-like particle (VLP) comprising an influenza hemagglutinin (HA) polypeptide, wherein the amino acid sequence of the HA polypeptide is at least 99% identical to SEQ ID NO: 2. 
     
     
         2 . The influenza VLP of  claim 1 , wherein the amino acid sequence of the HA polypeptide comprises SEQ ID NO: 2. 
     
     
         3 . The influenza VLP of  claim 1 , further comprising an influenza neuraminidase (NA) protein and an influenza matrix (M1) protein. 
     
     
         4 . The influenza VLP of  claim 3 , wherein the amino acid sequence of the influenza NA protein is at least 95% identical to SEQ ID NO: 4. 
     
     
         5 . The influenza VLP of  claim 3 , wherein the amino acid sequence of the influenza M1 protein is at least 95% identical to SEQ ID NO: 6. 
     
     
         6 . A composition comprising the influenza VLP of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         7 . A method of eliciting an immune response to influenza virus in a subject, comprising administering the VLP of  claim 1 , thereby eliciting an immune response to influenza virus. 
     
     
         8 . The method of  claim 7 , further comprising administering an adjuvant. 
     
     
         9 . A method of immunizing a subject against influenza virus, comprising administering to the subject a composition comprising the VLP of  claim 1  and pharmaceutically acceptable carrier. 
     
     
         10 . The method of  claim 9 , wherein the composition further comprises an adjuvant. 
     
     
         11 . The method of  claim 9 , wherein the composition is administered intramuscularly. 
     
     
         12 . The method of  claim 9 , wherein the composition comprises about 1 to about 25 μg of the VLP. 
     
     
         13 . A collection of plasmids comprising:
 (i) a plasmid encoding an influenza NA   (ii) a plasmid encoding an influenza M1; and   (iii) a plasmid encoding a codon-optimized influenza HA, wherein the nucleotide sequence encoding the codon-optimized influenza HA is at least 94% identical to SEQ ID NO: 1.   
     
     
         14 . The collection of  claim 13 , wherein the influenza NA is codon-optimized. 
     
     
         15 . The collection of  claim 14 , wherein the nucleotide sequence encoding the codon-optimized influenza NA is at least 95% identical to SEQ ID NO: 3. 
     
     
         16 . The collection of  claim 13 , wherein the influenza M1 is codon-optimized. 
     
     
         17 . The collection of  claim 16 , wherein the nucleotide sequence encoding the codon-optimized influenza M1 is at least 95% identical to SEQ ID NO: 5. 
     
     
         18 . The collection of  claim 13 , wherein:
 (i) the plasmid encoding influenza NA comprises SEQ ID NO: 8;   (ii) the plasmid encoding influenza M1 comprises SEQ ID NO: 9;   (iii) the plasmid encoding influenza HA comprises SEQ ID NO: 10; or   (iv) any combination of (i) to (iii).   
     
     
         19 . A method of generating an optimized influenza virus polypeptide sequence, comprising:
 (i) obtaining the amino acid sequences of the polypeptide from a group of influenza virus isolates, wherein the influenza virus isolates are from the same subtype;   (ii) organizing the amino acid sequences of the polypeptide from the group of influenza virus isolates by clade or sub-clade and then by geographical region within each clade or sub-clade;   (iii) aligning the amino acid sequences within each geographical region to generate primary consensus sequences, wherein each geographic region is represented by a primary consensus sequence;   (iv) aligning the primary consensus sequences to generate secondary consensus sequences, wherein each clade or sub-clade is represented by a secondary consensus sequence; and   (v) aligning the secondary consensus sequences to generate the optimized influenza virus polypeptide sequence.   
     
     
         20 . The method of  claim 19 , further comprising:
 (i) reverse translating the optimized influenza virus polypeptide sequence to generate a coding sequence; and   (ii) optimizing the coding sequence for expression in mammalian cells.