Progress 07/15/15 to 09/14/16
Outputs
Target Audience:- Swine veterinarians - Companies that manufacture and sell veterinary biologics - USDA, Center for Veterinary Biologics Changes/Problems:During this reporting period several changes occurred. First, the anticipated studies in pigs aimed at testing the immunogenicity of the HCAV formulations were delayed due to lack of available space in the animal facility to conduct these studies. For that reason, a no-cost-extension was requested to complete the second objective of the project. Likewise, for the first animal experiment, we followed the suggestion of the reviewers and tested only two adjuvants rather than all of five adjuvants as we had originally proposed. We believe that the information that was derived from testing these two adjuvants is sufficient to demonstrate the proof of principle for the parenteral version of the HCAV. We also decided not to use the interferon-alpha-encoding plasmid as we had originally proposed. Rather, we used a proprietary bacterial product as an adjuvant that we have reason to believe would be more effective, cheaper and easier to manufacture and use in the vaccine formulation. By reducing the cost of goods, we believe that this modification will increase the likelihood of Aptimmune's commercial success. These slight modifications did not change the goal of the project, and were based on company developments that occurred after the submission of the proposal, namely the access to the nanoparticle technology for the formulation of the HCAV as well as access to potent adjuvant technology that has been proven to work for the intranasal vaccination of swine. What opportunities for training and professional development has the project provided?Professional and technical staff were trained in conducting cell, virus and analytical methods for homologous cell autogenous vaccine preparation. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Swine morbidity and mortality caused by porcine reproductive and respiratory syndrome virus (PRRSV) causes losses to the U.S. pork industry exceeding $650 million per year, posing a significant threat to food security. For the last two decades, despite the use of live virus as well as well as inactivated virus vaccines against this pathogen, PRRS virus is still the leading cause of disease outbreaks in commercial swine operations. The purpose of this SBIR Phase I project was to examine the feasibility of developing the technology to manufacture an effective autogenous (inactivated) PRRSV vaccine. The development of such technology would have a major impact not only in mitigating the economic impact of PRRSV, but could also have broad application for production of vaccines against other viral pathogens that afflict the pork industry such as influenza virus. Based on the expertise and technology available to Aptimmune Biologics, as well as the success achieved in this phase I project, we believe that our approach offers great promise for the commercialization an effective autogenous vaccine that will increase the level of food security for the population of the USA. The first objective of the project was to determine the feasibility of completing, within 5 weeks from the time of initiating the effort to isolate the field virus from an bona fide clinical sample, all of the steps required for the production of an autogenous PRRSV vaccine. The first step in the process of the preparation of an autogenous virus vaccine is the reliable and expeditious isolation of the virus from a clinical sample from an infected pig at a farm afflicted by a viral disease outbreak. In the case of PRRSV, using conventional techniques, this critical step is met with failure in more than 50% of the cases. However, due the use of our proprietary porcine alveolar macrophage cell line, termed ZMAC, we demonstrate that the rate of successful isolation of PRRSV, from clinical samples certified by molecular techniques to contain this virus, is over 95%. Furthermore, in every case, the period required to determine that the virus isolation was successful was less than 60 hours after the initiation of the virus isolation attempt. In the subsequent steps, by defining the procedures for optimization of cell density and multiplicity of infection for infecting the ZMAC cells with virus isolates, filterability studies for concentrating the virus and establishing an inactivation protocol suitable for the production of large scale batches of virus stocks, we accomplished our goal to demonstrate the technical feasibility of manufacturing an autogenous PRRSV vaccine in no more than 5 weeks. Thus, with our novel approach, which utilizes the natural host cell for this virus, namely porcine alveolar macrophages, the turn-around time is significantly shorter than the traditional 8 to10-week period required for the production of the same biologic where simian cells are used to prepare the virus stock to manufacture the vaccine. The second phase of the project consisted of testing the immunogenicity of different formulations of the autogenous vaccine. Because the virus to be used as the source of the viral antigen to formulate the vaccine is being produced in the porcine-derived ZMAC cells, we have termed this product as a Homologous Cell Autogenous Vaccine (HCAV). Two different studies were carried out to demonstrate the ability of the PRRS HCAV to induce an immune response in pigs after parenteral or mucosal delivery. The first animal vaccination experiment was aimed at testing the immunogenicity of a parenteral version of the PRRS HCAV.For this experiment we formulated the vaccine by mixing the inactivated virus with either of two commercially available adjuvants from SEPPIC: 1) a water-oil-water (W/O/W) emulsion and; 2) a water soluble polymeric adjuvant. Each formulation was administered to two different groups of pigs by intramuscular injection. As a control, a third group received intramuscularly the identical dose of the inactivated virus but without the adjuvant. Pigs that were immunized with either of the two adjuvanted versions of the HCAVs exhibited the presence of PRRSV-specific antibodies in their serum by 3 weeks after the first vaccination and a significant increase in antibody titer after a booster immunization. In contrast, pigs immunized with the unadjuvanted HCAV remained seronegative throughout the monitoring period. The information from this experiment demonstrates that the HCAV prepared from viral antigen in produced in ZMAC cells administered parenterally is immunogenic in swine. For a mucosal HCAV, Aptimmune is focused in using nanoparticles to efficiently deliver the viral antigen to the respiratory mucosal surface and associated lymphoid tissues. Accordingly, two different types of nanoparticles were tested: i) liposomes and ii) solid nanoparticles (SN). The vaccine formulation consisted of mixing with inactivated PRRSV with the nanoparticles in combination with a proprietary bacterial component adjuvant. We performed an experiment in which the the mucosal HCAV was delivered intra-nasally to groups of 3 week-old pigs and its immunogenicity compared to that of the parenteral version of the HCAV consisting of the same viral antigen but mixed with the W/O/W adjuvant used in the first experiment and injected intramuscularly. As a placebo control, a fourth group of animals received vaccine diluent. The animals were immunized twice, three weeks apart, with the respective vaccine formulation and challenged two weeks later with the same wild-type PRRSV strain used to prepare the vaccine antigen. The analysis of the results showed that the pigs vaccinated with the SN formulated mucosal HCAV and the parenteral HCAV exhibited a secondary antibody response following the viral challenge. Notably, the animals vaccinated with the SN formulated mucosal HCAV exhibited statistically significantly reduced manifestation of clinical signs resulting from the virulent virus challenge as well as a greater mucosal antibody response than the animals in other treatment groups. In summary, this phase I project succeeded in demonstrating the feasibility of using our novel approach to manufacture a new class of PRRS Homologous Cell Autogenous Vaccine (HCAV) and deliver an immunogenic autogenous PRRSV vaccine to the pork industry. The mucosal HCAV formulated with the SNs displayed favorable results and is considered suitable to enter large scale vaccine manufacturing. Furthermore, the formulations tested are scalable and within the low cost of goods necessary to have an adequate profit margin to sustain a competitive business. A phase II project will be submitted focused on the optimization of the vaccine formulation and vaccine efficacy studies.
Publications
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