2009 Annual Report
1a.Objectives (from AD-416)
1. Develop novel approaches to inducing mucosal immune responses to FMDV vaccines with the capacity to cross-neutralize a broader array of virus sub-types. 1.A. Evaluate the efficacy of mucosal adjuvants, delivered via the replication-defective human adenovirus 5 (hAd5) vector system along with the hAd5-FMD vaccine, for augmenting immunity and protection following intranasal administration to swine. 1.B. Assess the cross-neutralization and cross-protection afforded by capsid-based vaccines engineered with chimeric VP1 G-H loops bearing immunogenic or toleragenic epitopes to broaden the specificity of the vaccine.
2. Determination of classical swine fever virus genetic determinants of virulence, immunogenicity and antigenicity. 2.A. Evaluate immunogenicity and protective efficacy of genetically modified CSFV glycoproteins. 2.B. Evaluate the role of non-structural proteins in CSFV virulence.
3. Develop and validate grating coupled surface Plasmon resonance imaging multiplexed microarray biosensor platform for the rapid detection of FMDV and CSFV, and the characterization of host responses to each pathogen.
1b.Approach (from AD-416)
1. Evaluation of mucosal adjuvants efficacy delivered through the ad5 platform, to induce mucosal immune responses to FMDV. Evaluate in vivo mucosal adjuvants alone or in combination with FMDV vaccine for induction of rapid protection in swine. Determine cross-neutralization and cross-protection provided by capsid-based vaccines engineered with chimeric VP1 G-H loops containing immunogenic or toleragenic epitopes. An epitope map will be created using anti-sera from murine bearing cross-reactive immune responses between FMDV types O and SAT3. Testing chimeric GH loop bearing hAd5 vectors in swine will be conducted to assess cross-neutralization. Challenge studies will be performed utilizing homo-typic and heterotypic virus.
2. Evaluate the role of non-structural proteins in CSFV virulence and protection against infection will be performed through; complete cloning of CSFV structural proteins into Baculovirus transfer vectors, completing the production of recombinant Baculovirus expressing parental CSFV structural proteins and of autonomous replication CSFV defective genomes, and by completing the immunogenicity studies in naïve swine with sera from infected swine.
3. Development and validation of the GCSPRI device will be done to use as rapid detection of FMDV and CSFV. Identify diagnostic reagents and develop host immune response characterization. Assay conditions and sensor chip configurations will be optimized to capture host leukocyte populations. In vivo virus detection will be tested through immune response by GCSPRI and by traditional bioassay.
This project is aimed to understand the molecular mechanisms involved in Classical Swine Fever Virus (CSFV) induction of disease, generalization of infection, shedding, host range, and induction of immune response. Understanding those mechanisms will permit a rational design of live attenuated CSF vaccines of unprecedented safety, efficacy and utility. The proposal is based on the hypothesis that CSFV determinants of virulence, antigenicity and immunogenicity are linked to patterns of glycosylation of viral envelope proteins. Thus, to further characterize the molecular basis of CSFV virulence in swine we are studying the role of N- and O-linked glycosylation of CSFV envelope proteins in antigenivcity, immunogenicity, and induction of protection. The approach involves the individual expression of wild-type and glycosylation site modified proteins in the Baculovirus sytem. Antigenivcity, immunogenicity, and induction of protection by these proteins are then analyzed in vivo in inoculated swine. In addition we will evaluate the role of non-structural proteins in CSFV virulence. The approach includes the construction of CSFV replicon system and analysis of replicative efficiency of CSFV non-structural protein mutants. Then, transfer of modified these modified genes into the genetic backbone of a full-length infectious cDNA clone for the production of recombinant viruses. These viruses will then in turn be analyzed for virulence in swine.
Cloned and expressed all CSFV strain Brescia wild-type and glycosylation site mutatnt structural proteins (E0, E1, and E2) in the Baculovirus-insect cells protein expression system. Twenty-His tagged proteins has been expressed and purified from Baculovirus infected insect cells. Wild-type and glycosylation deprived forms of E0, E1, and E2 CSFV proteins have been purified, combined with water-in-oil emulsion adjuvants, and used to assess immunogenicity in swine.
Activities in this project were monitored through email and telephone exchange, as well as site visits to ARS, PIADC and the University of Connecticut.