Title:  Method of specifying vaccine components for viral quasispecies

United States Patent:  6,337,181

Appl. No.:  217293

An algorithm for determining the viral antigenic protein variants to be used to construct vaccines designed to immunize against variable viral populations (quasispecies) is described. The method entails analyzing multiple nucleotide sequences of viral proteins and identifying those variants that provide selective advantage to the virus. Examples are given for influenza A hemagglutinin 3 and HIV-1 gp120.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of determining the advantageous variants found in a viral population given aligned nucleotide sequences of antigenic proteins or protein regions of that viral population. Once these advantageous variants are identified, they may be used drug targeting and in vaccine design applications.

The algorithm used to identify the advantageous variants is as follows for each amino acid position: 1) Identified as an advantageous variant of the viral population is the most common (consensus) amino acid. 2) Replacement variants, those viral variants that differ in the amino acid sequence from the consensus, that are found to have significantly high replacement to silent mutation ratios are determined to be advantageous to the virus. 3) Conversely, replacement variants with significantly low replacement to silent mutation ratios are recognized as providing selective disadvantage to the virus and so are excluded from further consideration. 4) Replacement variants where the nucleotide replacement to silent mutation ratio is unable to classify the variant as significantly advantageous or disadvantageous are provisionally identified as advantageous variants; the selective advantage or disadvantage of these variants cannot be determined with the given sequence data set, so advantage or disadvantage must be determined experimentally. A reasonable subset of variants may be selected by including the 2^H+.sigma. most common variants (where H is the Shannon information content and .sigma. is its standard error of its estimation).

The identified advantageous viral variants may then be used for purposes including but not limited to: 1) specifying components of vaccines to be used in conjunction with appropriate vaccination vectors and techniques know in the art; 2) identifying appropriate targets for small molecule or other anti-viral compounds; 3) using constructed viral variant panels to screen for broadly neutralizing monoclonal antibodies, screen for broadly neutralizing anti-viral compounds, and/or determine the neutralization spectrum of anti-viral compounds or antibodies.

Claim 1 of 16 Claims

What is claimed is:

1. A method of determining which naturally occurring amino acid variants of a protein, protein subregion, or antigenic site of a virus are selectively advantageous to said virus, said method comprising the following steps:

aligning multiple nucleotide sequences of said protein subregion, or antigenic site to each other (multiple sequence alignment);

for each aligned amino acid position (nucleotide codon), identifying as selectively advantageous to said virus the consensus (most common, mode) amino acid;

for each aligned amino acid position (nucleotide codon), determining the replacement to silent ratio of each amino acid replacement mutation (observed R:S);

for each aligned amino acid position (nucleotide codon), determining the replacement to silent ratio that would be expected if nucleotide mutation were neutral (expected R:S);

for each aligned amino acid position (nucleotide codon), comparing said observed R:S to said expected R:S by means of a statistical test;

for each aligned amino acid position (nucleotide codon), identifying as selectively advantageous to said virus non-consensus amino acid replacement variants that are determined by said statistical test to have a said observed R:S significantly higher than said expected R:S;

whereby identifying which naturally occurring amino acid variants of said viral protein, protein subregion, or antigenic site are selectively advantageous to said virus.

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