Testing PCP-Consensus Proteins for Multivalent Dengue Vaccine Design 2nd Stage

mosquito acquires blood meal

Principle Investigator: Catherine Schein, PhD

 

Objective: to produce a physicochemical properly (PCP)-consensus sequence of domain III of the envelope (EIII) protein of DENVs that will stimulate a neutralizing antibody response against all four Dengue virus (DENV) types.

 

Background: Flaviviruses include many dangerous pathogens. Mosquito control programs essentially eliminated yellow fever virus (YFV) and dengue viruses (DENV) in the US, but recent outbreaks due to West Nile virus (WNV) in the US, and DENV in Puerto Rico, show the need for antivirals and vaccines. Development of a vaccine for dengue has been complicated by the necessity of simultaneously inducing protective immunity against four related but distinct virus types. In the first stage of our project, we used annotated flavivirus genome and polyprotein sequences archived in our Flavitrack database to design physicochemical property (PCP)-consensus sequences of the EIII of DENVs (conEIII). A first conEIII based on 600 sequences was expressed from a synthetic gene, purified from E. coli, and shown to bind antibodies specific for 3 of the 4 types. The next stage of the project is to isolate a 2nd generation conEIII that accounts better for the distinct properties of the fourth DENV type and will generate neutralizing (i.e. protective) antibody response against all DENV.

 

Methods: Our PCP-consensus program automatically determines the amino acid that, at each column of an alignment of related protein sequences, most closely matches the properties of all others at that position. Synthetic genes have been obtained for PCP-consensus sequences for domain III of the Envelope proteins (E-con domIII), recloned and the proteins expressed in E. Coli. As with the first conEIII, the ability of our 2nd generation PCP-consensus protein to bind anti-DENV antibodies to all four virus types (monoclonal and polyclonal mouse sera, and characterized DENV patient sera) will be assayed, and the best protein further characterized structurally and for its ability to generate a neutralizing antibody response in mice and to protect cultured cells from DENV infection. The fold of the purified recombinant protein will be characterized with circular dichroism (CD), and preliminary NMR (15N-HSQC).

 

Anticipated results: As our first consensus protein bound antibodies in a fashion similar to that of three of the four individual serotypes of the proteins used to design it, we anticipate that the proteins designed to be closer to DENV4 in sequence will be tetravalent, and able to induce a neutralizing antibody response. Demonstrating that one of our PCP-consensus proteins can act as a multivalent antigen, and fold correctly will make the PCP-consensus protein an important candidate for novel vaccines.

 

We have already been able to add some of our initial data to other NIH applications in progress. Subsequent studies would undertake further sequence optimization and assessment of platform technologies (i.e. adjuvants, immunostimulatory fusion partners) to enhance vaccine performance.