Designing proteins that reflect the natural variability of a pathogen is 
essential for developing novel vaccines and drugs. Flaviviruses, including 
Dengue (DENV) and West Nile (WNV), evolve rapidly and can "escape" neutralizing 
monoclonal antibodies by mutation. Designing antigens that represent many 
distinct strains is important for DENV, where infection with a strain from one 
of the four serotypes may lead to severe hemorrhagic disease on subsequent 
infection with a strain from another serotype. Here, a DENV physicochemical 
property (PCP)-consensus sequence was derived from 671 unique sequences from the 
Flavitrack database. PCP-consensus proteins for domain 3 of the envelope protein 
(EdomIII) were expressed from synthetic genes in Escherichia coli. The ability 
of the purified consensus proteins to bind polyclonal antibodies generated in 
response to infection with strains from each of the four DENV serotypes was 
determined. The initial consensus protein bound antibodies from DENV-1-3 in 
ELISA and Western blot assays. This sequence was altered in 3 steps to 
incorporate regions of maximum variability, identified as significant changes in 
the PCPs, characteristic of DENV-4 strains. The final protein was recognized by 
antibodies against all four serotypes. Two amino acids essential for efficient 
binding to all DENV antibodies are part of a discontinuous epitope previously 
defined for a neutralizing monoclonal antibody. The PCP-consensus method can 
significantly reduce the number of experiments required to define a multivalent 
antigen, which is particularly important when dealing with pathogens that must 
be tested at higher biosafety levels.