SBIR-STTR Award

Swine morbididty and mortality caused by porcine reproductive and respiratory syndrome virus (PRRSV) is estimated to cost pork producers in the U.S. nearly $600 million annually. With current vaccine shortfalls and a national eradication effort being proposed, the need for a second generation vaccine that is effective and safe is crucial. This project explores the feasability and effectiveness of a novel PRRSV vaccine. OBJECTIVES: The objectives of this proposal seek to determine the feasibility of producing and using autogenous replicon particle (ARP) vaccines in pigs. Preliminary work by the PD and collaborators has shown that RP vaccines are capable of inducing an immune response in pigs to a foreign antigen. Additional work by the PD and collaborators has also shown the importance of the GP5-M heterodimer in protection from PRRSV. It is unknown how an ARP vaccine containing the GP5 and M genes compares to traditional vaccines (killed and MLV) in terms of efficacy and, in the case of traditional killed autogenous, preparation time. Our objectives are to 1)determine if pigs vaccinated with these ARP vaccines and challenged with virulent virus are protected from disease via comparison to currently available vaccines in a vaccination-challenge model and as measured by clinical signs, lesions, and viral load, and 2) determine the feasibility and timeline of producing ARP vaccines via construction of an ARP vaccine from a clinical sample originating on an Iowa swine farm. APPROACH: This project will allow us to determine the feasibility of making an ARP vaccine from a virus infected tissue received from a pig farm. It is anticipated that PCR can be performed on a sample to both provide a positive diagnosis of PRRSV and also generate cDNA of the genes needed to make the ARP vaccine. It is anticipated that this entire process can be performed in less time (1 month) than it generally takes for a pig producer to get a traditional autogenous vaccine made and delivered(3 months). However, even if production time were to take longer than the anticipated 1 month, the ARP vaccine still has the advantages of being safe, strain specific, and compatible with diagnostic tests used in an eradication program. Secondly, this research will determine the effectiveness and safety of the PRRSV ARP vaccine via comparison to currently available vaccines for PRRSV including MLV and traditional autogenous killed. Based on preliminary studies it is anticipated that the PRRSV ARP vaccine will induce an immune response and protect as well and likely better than other current vaccines in a vaccination-challenge model and as measured by clinical signs, serology, lesions, and viral load

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) costs the swine industry approximately $560M annually through reproductive loss in mature females and respiratory disease in growing pigs. There is a need for new vaccines for PRRSV to battle the strain evolution and heterogeneity of this RNA virus. Currently the only available US commercial product is a Modified Live Virus (MLV) from BI-Vetmedica. Use of a MLV PRRSV vaccine poses the risks of reversion to virulence and recombination with field viruses and thus is not compatible with an eradication program. Traditional killed autogenous vaccines for PRRSV are also available but are slow to produce / prepare (up to 3 months) and because they contain the whole virus they are not compatible with differential diagnostic tests. In a recent meeting at the University of Illinois attended by renowed scientists and chaired/organized by Dan Rock a white paper was produced which concluded that MLV and inactivated whole virus vaccines were not efficaciouis under field conditions. Unfortunately, Live Virulent Virus Inoculation (LVI) has been thought of as a last resort which leads to dramatic losses of animals and profits of an affected farm. As a result a number of requirements were presented in the white paper for the identification of an effective vaccine; 1 ? Rapid induction of immunity 2 ? Cross-protection against most currently prevalent strains 3 ? No adverse outcomes to swine health 4 ? DIVA (Differentiate Infected from Vaccinated Animals) 5 ? Simplicity of administration to ensure compliance. Based on these set criteria we believe that PRRS-RP technology not only has a potential significant advantage over traditional vaccines but also meets each of these requirements as a new effective PRRS vaccine. The objective of the proposal is to show the PRRS-RP vaccine candidate is not only able to significantly reduce viremia but is safe and efficacious. We aim to accomplish this by; determining if vaccinated pigs shed PRRS-RP and if so do non-vaccinated co-mingled pigs become infected. In response to this data we will determine if a non-target species is capable of shedding the PRRS-RP and if so can it infect other comingled animals. To aid in potency testing we aim to build on the current potency assay (IFA based) by developing a qPCR assay to quantify genome number (total particles / total genomes) giving a reproducible measure of complete PRRS-RP per dose hence aid in the determination of an effective dose. To determine the PRRS-RP product safety profile a study will be performed. These studies will position Harrisvaccines, Inc. to effectively pursue a USDA licensed product, to combat and alleviate the effects of PRRSV on the swine industry. In addition, this newly licensed product will not induce antibodies currently detected in the standard ELISA diagnostic test for PRRSV (DIVA) which will be essential in a national eradiction program, as endorsed by the AASV and producers. OBJECTIVES: Objectives: 1. Determine the ability of PRRSV-RP to shed and spread from vaccinated pigs to comingled non-vaccinated pigs. 2. Determine if a non-target species (mice) inoculated with PRRS-RP is capable of shed/spread to non-inoculated animals comingled with the source animal. 3. Determine an effective and reliable assay for the quantification of complete replicon particles to allow accurate administration of a prescribed dose of vaccine by developing a qPCR assay which will quantify the number of genomes present in a given dose; when used in combination with the developed IFA (total particles / total genomes) will give us an accurate reproducible measure of complete PRRS-RP per dose. 4. Determine the effective dose of PRRS-RP in protecting pigs against PRRS. 5. Determine the safety profile of the PRRS-RP product by measuring any negative effects to the animal following ante- / post-mortem analysis. Expected Outcomes: Objective 1: it is anticipated that PRRS-RP's will produce an effective immune response within the vaccinated individuals; we also anticipate that seroconversion to the vaccine will not be found in the co-mingled controls and hence it will not be spread. It is also expected that no PRRS-RP will be found to shed from vaccinated individuals either via the respiratory or fecal route. Objective 2: in the correlating study (study 2) we expect the mice to readily develop an immune response to the vaccine followed by quick clearance from the animal and hence not show any tissue / organ tropism but staying localized to the injection site. This in combination with the fact the co-mingled non-vaccinates will not have any detectable vaccine or serconversion to the vaccine will prove that even in non-target species, it is unable to spread. Combined with the fact we do not expect to detect RP in the feces of any study animal, we believe this supports the fact the vaccine is not shed from non-target species. Objective 3: This study will give us the ability to accurately administer the same dose of PRRS-RP each and every time. Objective 4: This objective when complete will allow us to identify the optimal vaccine dose that can be administered to a given animal while potentially highlighting a lower and upper limit for the effective range of the vaccine. Objective 5: We anticipate that no toxic / negative effect will be observed at the 10-fold dose of the vaccine, instead we will observe a similar response as the comingled optimal dose group. We do not envisage any tissue / organ tropism in the target-species nor do we expect to see any tissue damage at the injection site which would lead to a lower meat quality or have an adverse effect on the animal's quality of life. Also, we do not expect to see any detectable vaccine at the termination of the study. APPROACH: Methods: RP Construction. The procedures used for producing PRRS-RP vaccines are based on modifications of published methods and previous experience with other RP vaccines. In brief, replicon plasmids containing the target genes (GP5 and matrix) combined with the two helper plasmids which contain the VEE capsid or glycoprotein genes will be prepared. RNA transcripts will be produced in vitro (RiboMAX T7 Express, Promega) from the replicon and helper plasmids, then purified by either spin-column (gel binding and elution) or size-exclusion chromatography, followed by agarose gel analysis to assess integrity, and quantification by ultraviolet (UV) absorbance. Specified (previously optimized) mass amounts of the replicon and helper RNAs will be mixed with USDA certified Vero cells in electroporation chambers and pulsed using previously optimized square wave electroporation parameters. Electroporated cell suspensions will be seeded into roller bottles containing serum-free medium and incubated at 37oC in 5% CO2 for 16-18 hours. RP will be harvested from culture fluids and the infectious titer of the RP preparation will be measured by antigen-specific IFA and tested in a cytopathic effect (CPE) assay to assure the absence of detectable replication-competent virus (RCV; see Characterization of RP below). RP will be purified by size exclusion / ionic exchange filtration. The potency (infectious titer) of the purified bulk RP will be determined by IFA and the preparation will be formulated and frozen at -80C. Virus Propogation. PRRSV is propagated in M145 cells grown in (DMEM, 10% CCS, 1% L-glutamine, 1% Antibiotic-Antifungal). Pig Trials. Pigs 2-3 weeks old will be obtained from a farm with no current history of PRRSV infection based on clinical signs and serology. Quantification of PRRSV-RP. A qPCR methd will be developed with PRRSV-RP specific primers. Analysis of Samples: GFP-RP Neutralization assay. This procedure was developed and validated by one of our collaborators (AlphaVax, Inc.) as an aid to detect and measure the anti-vector response of a given sample. Gross Pathological Lesions and histology. Assessment of lesions will be completed by Diagnostic Laboratory veterinarians with recognized PRRS expertise. Splenocyte immunological assay. Supernatants from splenocytes will be assayed for IL-4, 10, 12 and IFN. Assessment of Viremia. TCID50 and qPCR will be used. Serological Tests. ELISA and viral neutralization will be performed with the serum samples. Statistical analysis: Analysis of variance (ANOVA) will be used to analyze log-transformed results from gross lung lesion scoring, serum HI titers, and virus titers from nasal swabs and bronchial alveolar lavage fluid. Rectal temperature data will also be analyzed via ANOVA or Student's t test. All analyses will be conducted using SAS statistical software. Results of the studies will be disseminated in scientific journals and as presentations at scientific meetings