New methods for evaluation of consistency of vaccines and cell substrates used for their production is the main theme of this program. The methods include new molecular approaches for assessment of genetic stability of live viral vaccines both in the course of their production, in vaccine recipients, and during possible circulation of vaccine strains in environment. Another group of methods under development is designed to assess potency, purity, and consistency of vaccine products and are based on fine immunological profiles of the vaccine products. Finally improved methods for assessment of efficacy of vaccines in preclinical studies, clinical trials, and postmarketing surveillance, including analysis of adverse reaction cases, are being developed. The methods include rapid immunological tests and analysis of individual and population immunity, as well as characterization of vaccine-derived strains causing adverse reactions. Molecular assay for neurovirulent revertants in live poliovirus vaccine. There was no research activity on this project because the WHO-sponsored collaborative study on evaluation of MAPREC for type 1 and type 2 OPV is underway. The results will be discussed at the November 2002 meeting of the WHO to determine future direction of this project. Molecular test for revertant mutations in live virus vaccines other than poliovirus vaccine. The goal of this project is to develop molecular methods for consistency monitoring of live viral vaccines. In our previous studies we have shown that stocks of Urabe AM9 strain used for production of Mumps vaccine live differed in two companies (SKB and Chiron SpA). The product of one company was withdrawn from the market, while the other did not show any signs of increased adverse reactions. During this year we have continued to work with Jeryl Lynn strain, and have sequences its derivatives that were de-attenuated by passaging in cell cultures. We have also determined sequences of a wild-type strain and its partially attenuated derivative, and identified several mutations that presumably lead to the change in virulence. We have also initiated a project on evaluation of yet another vaccine strain (L3) that has a history of genetic instability. We are now sequencing its genome in attempts to identify markers suitable for consistency monitoring of vaccine production. Another branch of the project involves development of consistency monitoring approaches for Yellow Fever Vaccine. Previously we have identified one mutation, which occurs during virus passaging in chicken embryos. Currently we are working on creation of more sensitive methods of mutational screening based on micro array hybridization. DNA microarray hybridization for genotyping of microorganisms and evaluation of vaccines. DNA microarray hybridization is used to identify different viruses and viral strains. During this year it has been used to study the following groups of microorganisms: In our previous studies we have used this technology for genotyping of Rotaviruses and Orthopoxviruses, including smallpox, monkeypox, vaccinia, rabbit pox, etc. We have also developed a method for identification of pathogenicity factors in enteric bacteria (in collaboration with CFSAN). A new computer program for selection of optimal oligonucleotide probes and PCR primers was created and used for automated design of oligonucleotide micorarrays. The most recent new developments in this project include a method for testing for the presence of antibiotic-resistance factors in pathogenic bacteria, and for studies of genetic stability of attenuated Poliovirus. We have improved the microchip for genotyping of recombinant poliovirus. The new version provides much more detailed physical maps of the genome of vaccine-derived poliovirus. A large-scale screening of VDPV isolated in different parts of the world has been initiated. A new method for screening for mutations in virual genomes and for evaluation of divergence of vaccine-derived viral strain was developed. This method was used for molecular epidemiology studies with VDPV, and also projects on using this new technology for assessment of divergence of Hepatitis C virus and emergence of drug-resistant HIV viruses were initiated. Another sub-project deals with genotyping of reassortant strains of Influenza B virus by using specially developed oligonucleotide microarrays. This method is being used for rapid screening of prospective vaccine strains developed by the Influenza lab at DVP. Methods for evaluation of immunogencity and efficacy of inactivated poliovirus vaccine. Currently, enhanced inactivated poliovirus vaccine (eIPV) are produced using wild-type poliovirus strains as substrates. The strains possess high pathogenic potential and if they were to be accidentally released from manufacturing areas into communities (and such an accident has actually occurred) they might be responsible for outbreaks of poliomyelitis. After eradication of poliomyelitis is completed, work with wild-type polioviruses will require strict containment measures, which will demand serious changes in the manufacturing process to protect surrounding communities, and to prevent potentially catastrophic consequences of poliovirus release. An attractive alternative approach to safety of eIPV production would be to switch to using non-pathogenic strains of poliovirus. Direct evaluation of efficacy of these new IPV as well as of combination vaccines containing IPV component is not possible, and therefore any regulatory actions should rely on surrogate immunogenicity data. In our previous studies we have shown that wild-type MEF-1 strain used fro production of IPV differs significantly from Sabin 2 strain used for OPV production, suggesting that these vaccines may have different immunogenicity. The Tg-mouse immunization - challenge test showed that conventional IPV provides good protection against challenge with both wild-type strain and with vaccine-derived virus, while IPV prepared from Sabin 2 strain only protects against challenge with homologous virus. In vitro ELISA test also confirmed that MEF-1 has a broader immunogenicity than Sabin 2 strain, suggesting that should IPV be produced from Sabin 2 strain, its immunization regimen may need to be adjusted for maximum efficacy. We have also developed an improved ELISA test for potency testing of IPV, as well as for consistency monitoring of IPV production. Respective reference reagents were developed. The first method is based on direct ELISA with polyclonal IgG and biotin-avidin conjugates. The consistency test takes advantage of block-ELISA test with monoclonal antibodies, and allows us to determine epitope profiles of inactivated vaccines as well as of strains isolated from vaccine-associated cases caused by OPV. The future directions of this project will include studies of IgA antibodies in saliva as a rapid method for evaluation of immunogenicity of poliovirus vaccines, and further development of epitope profiling. This project incorporates FY2002 projects 1Z01BK008005-06, 1Z01BK008010-05, and 1Z01BK008011-05.