Immunogenic formulation and process for preparation thereof
View Patent ↗A process is disclosed for preparation of a immunogenic peptide mixture in a single synthesis. The peptide mixture collectively represents the in vivo variability seen in immunogenic epitopes from a pathogen. The mixture is termed a hypervariable epitope construct (HEC). Immunization with a HEC evokes broadly reactive immunity against divergent strains of a pathogen upon which the HEC is based.
1. A process for preparation of an immunogenic peptide mixture comprising the steps of:
Obtaining from a database hypervariable immunogenic epitope sequences of a pathogen HCV or influenza virus, said immunogenic epitope sequences having a common residue region and at least one variable residue with which said sequences differ from each other;
determining the frequency with which different amino acids are found at the at least one variable residue of the immunogenic epitope sequences; the at least one variable residue comprising an amino acid selected from the amino acids most frequently found at the variable residue in the immunogenic epitope sequences of the pathogen, and found at the variable residue with a frequency greater than a threshold frequency of about 12%;
rounding the frequency with which an amino acid is found at a variable residue to the nearest 25%, wherein only those amino acids having a non-zero rounded frequency are included at the variable residue position in the peptides of the peptide mixture with the caveat that no more than four amino acids are selected for a variable residue; the frequencies of similar amino acids found individually at a variable residue below the threshold frequency being pooled to form a pooled frequency, the pooled frequency being assigned to the most frequently found of the similar amino acids to calculate the rounded frequency; and
synthesizing a peptide mixture comprising from 2 to about 64 different peptides, each peptide having the common residue region and having at a variable residue position an amino acid selected from those found at a variable residue of the immunogenic epitope sequences at a non-zero rounded frequency; the different amino acids appearing at the variable residue position being present relative to each other in proportion to the rounded frequency with which each different amino acid appears at the variable residue of the immunogenic epitope sequences.
2. The process of claim 1 wherein similar amino acids are amino acids belonging to a single classification selected from the group consisting of: aromatic amino acids; aliphatic amino acids; aliphatic hydroxyl side chain amino acids; basic amino acids; acidic amino acids; amide-containing amino acids, and sulphur-containing amino acids.
3. The process of claim 1 wherein the step of synthesizing is conducted using amino acid coupling, and the variable residue position is coupled by adding amino acids in proportion to the rounded frequencies determined in the step of rounding.
4. The process according to claim 1 wherein the pathogen is HCV.
5. The process according to claim 1 wherein the pathogen is influenza.