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Making a yeast-based candidate vaccine against Porcine Epidemic Diarrhea Virus (PEDV)

Periodic Reporting for period 1 - PIGYVAX (Making a yeast-based candidate vaccine against Porcine Epidemic Diarrhea Virus (PEDV))

Reporting period: 2016-06-13 to 2018-06-12

Porcine epidemic diarrhea virus (PEDV) is an economically important pathogen of swine. It is a coronavirus, like the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). However, it infects pigs only, causing acute and severe watery diarrhea that leads to severe dehydration and up to 100% mortality in newborn piglets and pre-weaning pigs. Despite previous vaccination programs, new variants of PEDV have been causing devastating outbreaks throughout Asia since 2010 before spreading rapidly to North America in 2013 [1,2]. Within one year of its first outbreak in the U.S. PEDV had wiped out 8 million piglets, resulting in an estimated loss of 280 million dollars alone from the death toll [3]. Furthermore, other infected pigs exhibited slower growth while infected sows that survived the infection had lower farrowing rates, causing a decrease in overall production. The swine export industry was likewise strongly affected, conferring an overall economic burden of the disease in the range of 900 million to 1.8 billion dollars [4].
In Europe, PEDV was first recognized in 1971 in the UK [5]. Since then, there have been sporadic outbreaks in small farms all over Europe [6,7,8] . While PEDV has yet to cause major economic issues in Europe, there were many cases reported in Germany and France in 2014 as well as in Belgium and Ukraine in early 2015 [9,10,11] . These strains appear to be more closely related to the U.S. INDEL strain OH851 than the previous European strain CV777 [12]. This suggests that either the INDEL strain has been recently introduced to Europe from America or it has been circulating in Europe before its introduction to the US. Without sufficient preparation, the emergence of highly virulent strains can cause devastating losses, as seen in Asia and America. Therefore, effective preventative measures for PEDV outbreaks are urgently needed.
Institut Pasteur, France, and the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, have joined forces by combining the vaccine platform strategies of Institut Pasteur and BIOTEC’s expertise in PEDV pathogenesis to produce a vaccine that can stimulate immunity in sows and fully protect piglets from the disease through colostrum. All vaccine candidates are aimed at neutralizing the function of the Spike membrane protein of PEDV (Spedv) in order to prevent virus entry into the cells. We have tested two vaccine platforms developed and patented by Institut Pasteur: 1) a yeast-based vaccine carrying a small subunit of Spedv fused to the measles virus (MV) nucleoprotein (Nmv), and 2) A recombinant MV-based vaccine vector (rMV) delivering Spedv.
The yeast-based platform uses whole cells of Pichia Pastoris yeast (BOX 1) to produce and deliver Spedv fused onto Nmv, forming nanoparticles that enhance recognition of the target protein to immune cells. Moreover, P. pastoris yeast, commonly present on fruits, is a certified safe microorganism for animal feeds, rendering its combination with feed a most practical and convenient way to deliver vaccine to large-scale farms.
The rMV is well–established and extensively studied vaccine platform with a well-characterized animal model based on the live attenuated MV vaccine, one of the safest and most effective vaccines available. This rMV has demonstrated proof-of-principle in humans and a preclinical track record of rapid adaptability and effectiveness for a variety of pathogens [13]. The rMV can directly deliver the targeted protein to the major immune cells due to the nature of its infection and stimulation of anti-viral signalling molecules, resulting in a strong immune response. For the PIGYVAX project, we aim to develop MV-Spedv to deliver the Spedv antigen into sows, which should help prevent piglets from succumbing to PEDV infection through passive antibody transfer from sow colostrum.
The success of this project will not only reshape the animal vaccination landscape, but also will provide proof–of-concept for further development of vaccines against other coronavirus outbreaks in both humans and animals.

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13 Frantz PN., et al., Microbes Infect. 2018 Feb 1. pii: S1286-4579(18)30038-8
Even though the yeast-based platform was found to be limited by the size of the targeted protein, we successfully constructed the proposed yeast-based vaccine candidate. Unfortunately, mouse immune responses were too weak and demonstrated no specific inhibition of PEDV. Nevertheless, the platform was shown to increase antigen immunogenicity by over a thousand fold, demonstrating its effectiveness.
The MV antigen-delivery system was chosen in place of the yeast platform as it can accommodate the larger size of the Spedv antigen. We created rMV carrying the Spedv gene, which will produce Spedv protein during infection. This vaccine candidate was tested in mice to see whether it can stimulate specific immune responses to the Spedv protein. Unfortunately, only a weak immune response was detected due to poor antigen presentation on the cell surface. We have therefore redesigned Spedv to improve surface localization. The new MV-Spedv vaccine candidate (figure 1 and 2) is in the process of being tested to determine its ability to stimulate strong an protective immune responses in mice and pigs.
This project has been an important stimulus for generating linkages in the global research community. The PIGYVAX vaccines were initially a product of combining the vaccine platform strategies of Institut Pasteur and BIOTEC’s expertise in viral pathogenesis. With the Vaccines for Enteric Diseases (VED) 2017 conference, we initiated a collaboration with Ghent University in Belgium to conduct pig experiments in their facility, where they will provide insights into swine immunity and experimental design. In addition, we also gained a collaborator from Huazhong Agricultural University, China, during the International Symposium on Human and Veterinary Viruses in Thailand in 2018. Because the yeast-based and the MV platforms are designed to be plug-and-play systems applicable to both humans and animals, this interdisciplinary group embodies the “One Health” concept in its efforts to develop fast and effective vaccination responses to mitigate devastating outbreaks in the future. As an outgrowth of these collaborations, we are now further collaborating in another EU project, SAFE Aqua under the RISE funding scheme. All these active linkages underline the strong intercontinental network that has built up between the European and Asian scientific communities as a direct result of this project.