Porcine Epidemic Diarrhea Virus (PEDV) was first detected in Europe in the 1970s, but did not emerge in the United States until 2013. When it arrived, it ran rampant due to the lack of previous exposure, causing the death of 7-8 million neonatal piglets and $900 million to $1.8 billion in losses to the U.S. pork industry in 2013 and 2014. This virus causes diarrhea and vomiting which leads to dehydration and in extreme cases, death. Neonatal piglets rely heavily on passive lactogenic immunity from their mother's milk, thus making them especially vulnerable to this disease. Within 2-3 days of infection during the initial outbreak, there was a 90-95% mortality rate among these weaning piglets. Additionally, this virus is highly contagious, with high rates of fecal shedding during infection. To control the outbreak, the USDA had approved two emergency-relief vaccines, but both have proved to be ineffective at preventing disease or reducing fecal shedding. These vaccines are still available today. As such, it is necessary to develop a vaccine that will be effective at preventing illness and viral shedding. PEDV is a single-stranded RNA virus made of four major subunits: a structural spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The one most studied and of particular interest is the S protein as it facilitates the virus' attachment and entry into the host cell. The S protein is made of two domains, the S1 domain which allows for protein interactions between the virus and the host cell, and the S2 domain which allows for membrane fusion. Because of the S1's role in protein interaction, it is often the target of potential vaccines. Within the S1 domain, it's C-terminal domain encodes for the receptor binding domain (RBD), which is why the S1 CTD is the target of this study. In this study we focused on the expression, purification, and immunogenicity testing of the CTD protein using T7 Express E. coli as the expression host. We used PCR, gel electrophoresis, Sanger Sequencing, western blots, and mass spectrometry to ensure that the protein was being expressed properly. The future goal is to use this protein as the antigen in a future nanoparticle-based PEDV vaccine. / Master of Science / In 2013, Porcine Epidemic Diarrhea Virus (PEDV) emerged in the United States, causing an estimated $900 million to $1.8 billion in damages to the pork industry and the death of 7 to 8 million newborn piglets in just one year. This virus causes diarrhea and vomiting which causes dehydration and death, and newborn piglets are particularly vulnerable. During the initial outbreak, two emergency-relief vaccines were approved but have not been proven effective against the disease. Thus, it is of great importance to develop a vaccine that is both effective and safe. Therefore, our task was to express, purify, and test the immunogenicity of a segment of the PEDV spike protein to be used as the antigen of a future nanoparticle-based vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two of the most prevalent swine pathogens that have impacted the global swine industry for decades. Both are RNA viruses with increasing heterogeneity over the years, making a vaccine solution ever so challenging. Modified live-attenuated vaccines (MLVs) have been the most common approach, but the long-term safety regarding their potential for pathogenic reversion still needs to be addressed. Subunit based vaccines have been the focus of numerous development studies around the world with renewed interest in their promising prospects in both safety and efficacy. Our lab has developed a unique approach to use hepatitis B virus core capsid protein (HBcAg) as a vaccine delivery vehicle for either PRRSV or PEDV viral epitope antigens. Recombinantly produced HBcAg forms an icosahedral capsid virus-like particle (VLP) that has 240 repeats in a single assembled particle. By inserting different epitope antigens from these porcine pathogens into the particle, we can achieve repetitive antigen presentation to the host's immune system by taking advantage of the polymeric nature of VLP. The first animal study evaluated the efficacy of 4 VLP based vaccine candidates against PRRSV in mice. These 4 vaccines incorporated 2 B-cell epitopes (61QAAIEVYEPGRS72 and 89ELGFVVPPGLSS100) and 2 T-cell epitopes (117LAALICFVIRLAKNC131 and 149KGRLYRWRSPVIIEK163) from PRRSV structural proteins GP3 and GP5 respectively. Candidate GP3-4 was able to stimulate a significant viral neutralizing response in mouse sera against two PRRSV strains, one being heterologous, demonstrating its potential of cross-protection against PRRSV. The second animal study took an optimized VLP vaccine candidate against PEDV from previous development studies in mice, and assessed its efficacy through a comprehensive pregnant gilt vaccination and neonatal piglet challenge model. The vaccine candidate incorporated B-cell epitope 748YSNIGVCK755 from the PEDV spike protein. It was able to elicit significant viral neutralization antibody titer in gilt milk at 3 days post-farrowing (DPF), and provided nursing piglets with clinical relief in terms of morbidity, viral shedding, small intestinal lesions, and 10 days post-challenge (DPC) survival rate. / Doctor of Philosophy / Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two pathogens that infect pigs, resulting in immense economic losses to the global pork production industry every year. Both viruses have large diversity with various strains due to mutations that have occurred over the years. This makes vaccine development that aims at combating the pathogens even more challenging. One common vaccine strategy has been immunizing animals with modified live viruses with decreased pathogenicity. Naturally, long term safety of this option has been a concern. A much safer vaccine approach that is purely protein based has attracted renewed interest around the world. Protein based vaccines lack genetic materials from the viruses and are not able to replicate inside the host. Our lab has developed a platform that uses protein-based particles (VLPs) originated from the hepatitis B virus (HBV), and incorporates short pieces of proteins from either PRRSV or PEDV to train host's immune system to recognize these pathogens, and hopefully to prevent future infection. For the first animal study, we tested 4 VLP vaccine candidates against PRRSV in mice and discovered that mouse serum from one candidate GP3-4 was able to prevent infection of 2 distinct PRRSV strains in petri dishes, paving the way for further development. For the second animal study, we took an optimized VLP vaccine candidate against PEDV from previous mouse studies, and evaluated its performance in pigs. We immunized pregnant mother pigs with the vaccine before they gave birth, then experimentally infected newborn piglets with the virus. Piglets from the vaccinated mothers showed improved clinical signs and faster recovery from the infection.