2010 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.
The objectives of this project are: 1. Develop adenoviral vectored mucosal adjuvants to enhance the immune response to replication-defective Ad-5 expressing the Foot-and-Mouth Disease Virus (FMDV) capsid proteins, 2. 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 3. Develop multiplexed microarray assays of biomarker signatures that will be diagnostic for vaccination and infection status in swine.
1. engineered four synthetic genes for expression in the replication defective Ad-5 vector, which were positively analyzed for protein expression 2. conducted initial experiments with guinea pigs to determine cross-reactive immunity against multiple sero and subtypes of FMDV. Some animals produced significant levels of antibodies, however many did not. These experiments will be conducted in FY 2011 using BALB/c mice 3. assessed the immunogenicity and protective efficacy of glycosylated and non-glycosylated forms of CSFV envelope proteins E1, E0, and E2 in swine. To assess the role of CSFV non-structural proteins in virulence, virus-host interactions have been studied by means of determining protein-protein interactions using a yeast two-hybrid system. Screenings showed numerous interactions between CSFV non-structural proteins and host proteins expressed in a swine primary macrophage library 4. infectious and non-infectious Vesicular Stomatitis Virus (VSV) particles have been tested using Surface plasmon enhanced fluorescence (SPEF) and Grating-coupled surface plasmon resonance imaging (GCSPRI). We are able to capture and detect VSV viral particles using either the antiserum or a commercial anti-VSV-G (peptide) monoclonal antibody. Serum sample testing has begun on pigs immunized with a synthetic FMDV A-12 peptide-carrier protein conjugate for the presence of FMDV-specific antibodies and levels of multiple cytokines by SPEF.
This project was monitored through email and telephone exchange as well as by site visits to ARS, PIADC and UConn.