ONE HEALTH UNIVERSAL SWINE INFLUENZA VACCINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025867
• Grant No.: 2021-67015-34464
• Project No.: NEB-99-917
• Proposal No.: 2020-06448
• Program Code: A1221
• Project Start Date: Jul 1, 2021
• Project End Date: Jun 30, 2024
• Grant Year: 2021

Project Director
Weaver, E.

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
School of Biological Science

Non Technical Summary
Problem: Swine influenza virus infections have a significant impact on the pork industry, with one study estimating over $700,000,000 in losses in the USA. In addition, pigs are susceptible to human, avian, and swine influenza and are excellent mixing vessels for reassorted influenza strains leading to increasing genetic diversity. Therefore, zoonosis events can lead to human infection with variant swine influenza which happens more frequently than most people realize. Since 2005 there have been more than 400 swine variant influenza infections in the United States. The 2009 H1N1 Swine flu pandemic that infected ¼ of the global population is a perfect example of this zoonosis. In March of 2009, an H1N1 swine influenza emerged in Mexico and the United States in both California and Texas. This novel Swine flu circulated the globe, caused worldwide panic, forced the closing of State Fairs and infected 24% of the world's population. Unfortunately, the current commercial swine influenza vaccines have limited strain coverage and are infrequently updated. Mismatched vaccines provide little or no protection and, in the case of swine influenza, can lead to vaccine-associated enhanced respiratory disease (VAERD).Solution: A universal swine influenza vaccine that would reduce the economic impact of swine influenza on the pork industry, along with reducing the probability of emergent zoonotic reassorted influenza viruses into the human population. We propose the use of optimized vaccine proteins that have been designed to induce broader cross-reactive immunity against divergent virus strains as compared to wildtype proteins. These optimized vaccine strategies may be instrumental in the production of a universal vaccine. In this proposal, we describe the production of foundational Epigraph influenza proteins for use as universal swine influenza vaccines. Our vaccine target is the hemagglutinin protein (HA), which is the primary target for neutralizing antibodies of influenza. We will test these Epigraph vaccine proteins for their ability to induce protection against divergent H3N2 Swine Influenza A viruses and evaluate their potential as a universal vaccine. H3N2 was selected due to significant agricultural burden, high genetic diversity, and increasing number of zoonotic events recently documented. Results from this proposal could further be applied to H1 swine influenza viruses in future studies. Our preliminary data from mice AND swine indicates that Epigraph vaccines induce superior protection against mismatched challenge viruses as compared to wildtype HA vaccines and commercial vaccines.

Animal Health Component

100%

Research Effort Categories

Basic

20%

Applied

80%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1090 - Immunology;

Keywords

epitope-optimized

swine influenza

universal

vaccine

virus

Goals / Objectives
The overarching goal of this research is the prevention of swine influenza virus infections. Influenza is a rapidly evolving virus, making vaccination strategies challenging. For this reason, human influenza vaccines are updated yearly based on global surveillance. Although swine influenza also evolves at a rapid rate, commercial vaccines are updated far less frequently. This falls short of covering many circulating strains which necessitates pig farmers to create autogenous vaccines to protect their heard. Therefore, the goal of this project is to develop computationally designed HA immunogens that confer the most cross-reactive immune responses in order to serve as a universal swine influenza vaccine. This project's objective directly fits the USDA's Program Priority of management of Diseases of Agricultural Animals. Objective 1: Dose titration of vaccine candidates. Since these are larger animal models, we will take a stepwise approach to narrowing down the effective vaccine dose. This will allow us to determine whether higher doses of vaccine are necessary to provide adequate protection. A reduction of the vaccine dose that is required to induce protective immunity will increase vaccine productivity and reduce vaccine production costs.Objective 2: Evaluate the vaccine efficacy in swine against a panel of representative divergent swine H3 viruses. Our preliminary data indicates that a single dose of 1011 vp per pig (4 weeks old) of the Ad-Epigraph vaccine induces HI titers ≥40 to 9 out of 12 (75%) of the swH3 strains. The most effective method of determining vaccine efficacy is the challenge study. We will test the Ad-Epigraph vaccines for the ability to protect against multiple divergent influenza challenges. The panel of swine influenza challenge viruses twill include: CladeI: A/swine/Texas/4199-2/1998; Clade II: A/swine/Colorado/23619/1999; Clade IV-A: A/swine/Wyoming/A01444562/2013; CladeIV-F: A/swine/Kansas/11-110529/2011.Objective 3: Determine the rapid onset of immunity and the duration of immunity. Vaccines need to work quickly after administration and, ideally, they should provide long-term immunity. Therefore, we will determine 1) how quickly vaccination induces immunity (onset) and 2) how long that immunity last (duration).The successful completion of these goals could lead to long-range impacts on the agricultural industry. Our preliminary data suggests that epitope optimized synthetic immunogens induce the greatest degree of cross-protective immune responses against mismatched influenza challenges. Successful completion of the goals described in this study could lead to significant changes in how Swine Influenza vaccines are designed and delivered with the ultimate goal of preventing endemic infections, reducing economic impacts and potential zoonotic infections.

Project Methods
The research described in this project has undergone rigorous preclinical evaluations in mice and swine. Therefore, the results of the studies described here will be the most informative and definitive of all.Methods for Dose Titration: Previous studies have shown that 2×1010pfuof recombinant adenovirus provided excellent protection against homologous Swine influenza virus challenge. We will confirm this protection by immunizing groups of 6 pigs with 109 and 1010 pfu of Ad-H3-TX/98. A single dose vaccine is ideal, however a prime/boost strategy may be more effective at inducing protection. The sera from the vaccinated pigs will be screened by HI assay. If the overall HI titers are not above the standard protective titer of 40, we will boost the pigs with a second equivalent dose of rAd vaccine. The pigs will then be intratracheally challenged with 7x105TCID50of the homologous A/Swine/TX/4199-2/1998 virus at 4 weeks post-vaccination as previously reported (59) and necropsied at day 5 post-challenge. A challenge control and blank control group (6 pigs/group) will be included. Clinical signs and body temperature will be monitored daily. These studies will determine the optimal vaccine dose and schedule for the remaining studies.Methods for Evaluating Vaccine Efficacy Against a Wide Array of Divergent Swine Influenza viruses: We will test the Ad-Epigraph vaccines for the ability to protect against multiple divergent influenza challenges. Groups of 6 pigs 4 weeks of age will be intramuscularly immunized with 1011 vp (or optimal dose) per pig of the Ad-epigraph vaccine or mock-immunized with PBS as challenge controls. A group of 6 pigs will receive the commercial WIV vaccine FluSure XP at the recommended dosing and schedule as a vaccine control. Four weeks post vaccination, each pig will be intratracheally challenged with 106 TCID50 of a single H3 swine influenza virus as depicted in Table 2. Clinical symptoms (including lethargy, coughing, and nasal discharge) and body temperature will be monitored daily throughout the experiment. Blood samples will be collected before vaccination, at 2 and 4 weeks post vaccination, and at necropsy day. Nasal swabs will be collected on days 0, 1, 3, 5 post-challenge. All 6 pigs in each group will necropsied at day 5 post challenge. During necropsy, the percentage of gross lesions on each lung lobe will be scored by a single experienced veterinarian. Bronchioaveolar lavage fluid (BALF) samples will be collected by flushing a lung with 50 ml of MEM. Virus titer of BALF and nasal swab samples will be determined by TCID50 on MDCK cells in 96-well plates. In addition, qPCR on BALF and nasal swabs will be performed as a secondary measure of viral titers. Tissue samples from the nasal turbinate, trachea, and the right cardiac lung lobe will be collected and fixed in 10% buffered formalin for the pathological examination. Lung sections will be examined by a veterinary pathologist in a blinded fashion and given a score of 0 to 3 to reflect the severity of bronchial epithelial injury as described previously. The serum samples collected at each time points will be used for HI and NI assays to further assay the cross-reactive antibody responses prior to challenge. The PBMC collected before challenge can be used for ELISPOT assay. These post-challenge samples will also act as a replicate of our previous immune correlate studies in order to confirm reproducibility and minimize animal usage.In order to determine whether the universal vaccines can provide cross-protective immunity across different clusters of the H3 subtype, we will challenge with A/Swine/Texas/4199-2/1998 (Clade I), A/Swine/Colorado/23619/1999 (Clade II), A/swine/Wyoming/A01444562/2013 (Clade IV-A), and A/swine/Kansas/11-110529/2011 (Clade IV-F). Viruses within Cluster IV and subclusters are the dominant genotype H3 virus circulating in swine in the USA. These viruses have been well characterized in the pig model by Dr. Ma and colleagues at MU. The selected viruses used for evaluation of efficacy of vaccine candidates will be amplified in MDCK cells or eggs in order to prepare virus stocks, then will be titrated in MDCK cells using either plaque assay or TCID50 assay per milliliter. Most of these viruses have already been amplified and characterized in swine.Methods to Determine the Rapid Onset of Immunity and the Duration of Immunity: Groups of 6 pigs 4 weeks of age will be intramuscularly immunized with 1011 vp per pig of the Ad-epigraph vaccine or mock-immunized with PBS as challenge controls. A group of 6 pigs will receive the commercial WIV vaccine FluSure XP at the recommended dosing and schedule as a vaccine control. Since performing challenge studies against all swine influenza viruses is not within the scope of this study, we will analyze immune correlates against a large panel of swine H3 influenza strains that belong to different clusters and subclusters. The viruses form a highly comprehensive panel of swine H3 influenza that covers the majority of viral diversity. These viruses have already been amplified to high titer in eggs and used in our preliminary immune correlate studies. All candidate vaccine responses will be compared to the commercial WIV FluSure XP (Zoetis) per the manufacturer's recommendations. All vaccinations will be administered intramuscularly.In order to quantitate the rapid onset of immunity, we will collect sera every 3rd day for HI assays. We will collect sera for three weeks after the prime immunization and for three weeks post-boosting. In order to determine the durability of immunity we will collect sera monthly for 1 year which will be analyzed by HI assays. We will also collect PBMCs in order to quantitate the levels of antigen-specific cellular immunity. Cellular immunity will be evaluated by a standard ELISpot assay using overlapping peptide libraries that represent diverse wildtype flu strains. This data will allow us to extrapolate the true breadth and strength of the anti-influenza immunity induced by the various vaccines. We will analyze both cellular and humoral immunity as both play critical roles in protection and clearance.Hemagglutination Inhibition (HI) Assay. Since we cannot evaluate all swine influenza in pig challenge models, the HI assay will allow us to fully evaluate the cross-protective antibodies induced by the immunogens against a comprehensive panel of representative viruses. This assay will be perform according to the World Health Organization standard protocols. Briefly, sera is treated with receptor destroying enzyme (RDE) and tested for non-specific agglutinins. Sera is then two-fold diluted and incubated with 4 hemagglutination units of the assay swine H3 virus. After incubation for 30 min, 0.5% red blood cells (RBC) are added, and the agglutination pattern is read. Neutralization (NI) Assay. In order to determine the functional neutralization of the sera against homologous and heterologous sera, microneutralization assays will be performed. Briefly, heat inactivated sera will be serially diluted and incubated with 50 TCID50 before being plated on MCDK cells. The next day, the media will be replaced with trypsin containing growth media (FBS negative). After a 4 day incubation, 0.5% RBC are added and agglutination patterns read. Cellular Immunity (ELISpot). To evaluate the breadth and magnitude of the cellular immunity induced by the vaccine immunogens, we will quantitate the absolute number of interferon secreting cells by IFN-g ELISpot as previously described (50). Peripheral blood mononuclear cells (PBMCs) will be obtained from each of the pigs by centrifugation of blood on a discontinuous gradient. This is followed by isolation of the buffy coat, washing with PBS, and quantification of the PBMCs. ELISpots will then be performed with overlapping peptide libraries and spots per million will be quantified.

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:We have reached local, regional, national and international audiences of both general interest and professional interests through the following presentations and publications: Manuscript #1 Petro-Turnquist E, Pekarek M, Jeanjaquet N, Wooledge C, Steffen D, Vu H and Weaver E (2023) Adenoviral vectored epigraph vaccine elicits robust, durable, and protective immunity against H3 influenza A virus in swine. Front. Immunol. Front. Immunol., 15 May 2023, Sec. Vaccines and Molecular Therapeutics, Volume 14 - 2023 https://doi.org/10.3389/fimmu.2023.1143451 Magazine Article #1 Erika Petro-Turnquist and Eric Weaver.Epitope Optimized Vaccine Provides Long-Lasting Immunity Against Influenza in Pigs. Nebraska Pork Talk.April/May/June,2023, Volume 55 No. 2, Page 16. National Presentations Erika Petro-Turnquist, Matt Pekarek, andEric Weaver. 2022. "Vaccination with epigraph immunogens induces broadly protective humoral immune responses against swine H1 influenza A viruses." American Society of Virology,Annual Meeting,Madison, WI. Erika Petro-Turnquist, Matthew Pekarek, Nicholas Jeanjaquet, David Steffen, Hiep Vu, andEric Weaver. 2023."Adenoviral-vectored epigraph vaccine induces robust and durable immunity against H3 Influenza A Virus in swine." 2023 Conference for Workers on Agricultural Diseases (CRWAD). Chicago, IL: January 20 - 24th, 2023. Erika Petro-Turnquist, Matt Pekarek, Nicholas Jeanjaquet, Cedric Wooledge, David Steffen, Hiep Vu, andEric Weaver. "Adenoviral-vectored epigraph vaccine induces robust and durable immunity against H3 Influenza A Virus in swine." World Vaccine Congress, April 2 -6, 2023, Washington DC. Local Presentations Erika Petro-Turnquist, Matt Pekarek, andEric Weaver. 2022. "Vaccination with epigraph immunogens induces broadly protective humoral immune responses against swine H1 influenza A viruses." 30th Annual Intercampus Virology Meeting, Lincoln, NE.Award- Best Poster. Erika Petro-Turnquist, Matt Pekarek, andEric Weaver. 2022. "Vaccination with epigraph immunogens induces broadly protective humoral immune responses against swine H1 influenza A viruses." Student Research Days, Lincoln, NE.Award- Top Graduate Student Presenter: Biological Sciences Division. Erika Petro-Turnquist, Brianna Bullard, Matt Pekarek, andEric Weaver"Adenoviral-Vectored Centralized Consensus Hemagglutinin Vaccine Provides Broad Protection against H2 Influenza A Virus." 31st Annual Intercampus Virology Meeting, Lincoln, NE. March 14th, 2022. Erika Petro-Turnquist,Matt Pekarek, Nicholas Jeanjaquet, Cedric Wooledge, David Steffen, Hiep Vu, andEric Weaver. "Adenoviral-vectored epigraph vaccine induces robust and durable immunity against H3 Influenza A Virus in swine." 2023 UNL Student Research Days, Lincoln, NE.Award- Best Poster: Biological Sciences Division. Erika Petro-Turnquist, Matthew J. Pekarek, Nicholas Jeanjaquet, Cedric Wooledge, David Steffen, Hiep Vu, andEric A. Weaver."Epitope optimized multivalent H3 swine flu vaccine induces broad and long-lasting immunity in swine."42ndAnnual Graduate Student Symposium, University of Nebraska - Lincoln, April 28th, 2023.Best Poster - 1stplace. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?There have been many opportunities for training and professional development. First, the students, faculty and staff involved directly and indirectly in these studies have presented and trained each other in various skill sets, such as large animal procedures, sample collection, virus challenges, etc. In addition, this research has been presented at many local and national meetings as previously described. How have the results been disseminated to communities of interest?We have reached local, regional, national and international audiences of both general interest and professional interests through the following presentations and publications: Manuscript #1 Petro-Turnquist E, Pekarek M, Jeanjaquet N, Wooledge C, Steffen D, Vu H and Weaver E (2023) Adenoviral vectored epigraph vaccine elicits robust, durable, and protective immunity against H3 influenza A virus in swine. Front. Immunol. Front. Immunol., 15 May 2023, Sec. Vaccines and Molecular Therapeutics, Volume 14 - 2023 https://doi.org/10.3389/fimmu.2023.1143451 Magazine Article #1 Erika Petro-Turnquist and Eric Weaver.Epitope Optimized Vaccine Provides Long-Lasting Immunity Against Influenza in Pigs. Nebraska Pork Talk.April/May/June,2023, Volume 55 No. 2, Page 16. National Presentations Erika Petro-Turnquist, Matt Pekarek, andEric Weaver. 2022. "Vaccination with epigraph immunogens induces broadly protective humoral immune responses against swine H1 influenza A viruses." American Society of Virology,Annual Meeting,Madison, WI. Erika Petro-Turnquist, Matthew Pekarek, Nicholas Jeanjaquet, David Steffen, Hiep Vu, andEric Weaver. 2023."Adenoviral-vectored epigraph vaccine induces robust and durable immunity against H3 Influenza A Virus in swine." 2023 Conference for Workers on Agricultural Diseases (CRWAD). Chicago, IL: January 20 - 24th, 2023. Erika Petro-Turnquist, Matt Pekarek, Nicholas Jeanjaquet, Cedric Wooledge, David Steffen, Hiep Vu, andEric Weaver. "Adenoviral-vectored epigraph vaccine induces robust and durable immunity against H3 Influenza A Virus in swine." World Vaccine Congress, April 2 -6, 2023, Washington DC. Local Presentations Erika Petro-Turnquist, Matt Pekarek, andEric Weaver. 2022. "Vaccination with epigraph immunogens induces broadly protective humoral immune responses against swine H1 influenza A viruses." 30th Annual Intercampus Virology Meeting, Lincoln, NE.Award- Best Poster. Erika Petro-Turnquist, Matt Pekarek, andEric Weaver. 2022. "Vaccination with epigraph immunogens induces broadly protective humoral immune responses against swine H1 influenza A viruses." Student Research Days, Lincoln, NE.Award- Top Graduate Student Presenter: Biological Sciences Division. Erika Petro-Turnquist, Brianna Bullard, Matt Pekarek, andEric Weaver"Adenoviral-Vectored Centralized Consensus Hemagglutinin Vaccine Provides Broad Protection against H2 Influenza A Virus." 31st Annual Intercampus Virology Meeting, Lincoln, NE. March 14th, 2022. Erika Petro-Turnquist,Matt Pekarek, Nicholas Jeanjaquet, Cedric Wooledge, David Steffen, Hiep Vu, andEric Weaver. "Adenoviral-vectored epigraph vaccine induces robust and durable immunity against H3 Influenza A Virus in swine." 2023 UNL Student Research Days, Lincoln, NE.Award- Best Poster: Biological Sciences Division. Erika Petro-Turnquist, Matthew J. Pekarek, Nicholas Jeanjaquet, Cedric Wooledge, David Steffen, Hiep Vu, andEric A. Weaver."Epitope optimized multivalent H3 swine flu vaccine induces broad and long-lasting immunity in swine."42ndAnnual Graduate Student Symposium, University of Nebraska - Lincoln, April 28th, 2023.Best Poster - 1stplace What do you plan to do during the next reporting period to accomplish the goals?We have combined objective 1 and 2 into one series of immunizations and challenges. We combined the objectives into 4 seperate experiments that overlap very well and slightly reduced the animal numbers. Experiments 1 and 2 combine the dose titration and breadth of protection studies. the 3rd and 4th experiments only expand the breadth of the protection studies described in objective 2. This allows for control animals to be used in both objectives thereby reducing animal numbers and eliminating repetitive laborious procedures.We have completed experiment 1 and will be starting experiment 2 shortly. We expect to have all of objective 1 and 2 completed within the next year and ready for publication in early2024.

Impacts
What was accomplished under these goals? IMPACT Problem: The goal of this study was to evaluate our vaccine against the current commercial influenza virus vaccine, FluSure XP whole-inactivated vacine (WIV).Swine Influenza A Virus (IAV-S) is a significant pathogen that effects swine populations around the world and impresses a significant burden on the pork industry. Economic losses to the U.S. pork industry can cost nearly $700,000,000 per year, and are due to reduced weight gain, delayed production, and increased susceptibility to secondary infections leading to greater medical costs. At present IAV-S in swine is considered one of the three top health challenges to the swine industryand affects swine in all phases of production. ?Zoonotic emergence of IAV was recently named a top priority of the One Health workshop for disease prevention in the United States. More specifically, swine were considered a significant intermediate reservoir in IAV infections and pose the greatest risk of zoonotic transmission of IAV into humans. One such example occurred in 2009 and was termed the "swine flu" pandemic. This novel swine influenza isolate initially arose in Mesoamerica, but quickly spread and infected ~24% of the global population within the first year. IMPACT Solution: To address the need for improved vaccination methods in swine, our group has recently characterized the use of a replication-defective adenoviral-vectored Epigraph vaccine against swine H3 influenza A virus mice and swine. The Epigraph platform uses a computational algorithm to determine the frequency of potential T cell epitopes in a target population of sequences and incorporates the highest frequency epitopes into a synthetic immunogen for optimal T cell activation after immunization. Indeed, we have previously shown that this platform induces significantly higher cross-reactive antibodies, robust T cell activation, and protection against divergent swine and human H3 influenza challenge in mice compared to a WT immunogen and the commercial comparator WIV vaccine, FluSure XP. However, to the best of our knowledge, no studies have performed a longitudinal study analyzing the onset and duration of immune responses elicited after vaccination with an adenoviral-vectored vaccine and compared these results to a WIV vaccine in swine.Here, we evaluated the kinetics of antibody and T cell development after vaccination with an adenoviral-vectored Epigraph vaccine (Ad-swH3-Epi) and compare the responses observed to vaccination with a commonly used WIV vaccine, FluSure XP. We further characterize the differences in antibody class switching after vaccination with these different platforms. Finally, we assessed protection against challenge 6 months after the initial vaccination to evaluate the extent of the protective responses in a clinically relevant situation, as the average lifespan of standard market pig in the pork industry is 6-7 months of age. The data observed in this study support the use of Ad-swH3-Epi for robust and durable protection against H3 IAV-S in swine. The major impacts of these studies show that the Ad-Epigraph swine influenza vaccine is superior at protecting swine over a considerable length of time. The use of this vaccine should have considerable impacts on the swine production industry. In addition, the reduction of swine in our herds will reduce the likelihood that novel reassorted, and possibly pandemic, strains will be zoonotically transmitted to humans. We have fully completed objective 3 and the results have been published in Frontiers in Immunology.https://www.frontiersin.org/articles/10.3389/fimmu.2023.1143451/full The overall impacts have also been described in the Nebraska Pork Talk online magazine (Page 16).https://nepork.org/wp-content/uploads/2023/04/Pork-Talk-Apr-Jun-23-Web.pdf Objective 3:Determine the rapid onset of immunity and the duration of immunity.Vaccines need to work quickly after administration and, ideally, they should provide long-term immunity. Therefore, we will determine 1) how quickly vaccination induces immunity (onset) and 2) how long that immunity last (duration)? The results are described briefly here and in more detail in the abstract. The two main questions that were asked and answered were1) how quickly vaccination induces immunity (onset) and 2) how long that immunity last (duration)? It turns out that the viral vecctored Epigraph vaccine induces rapid immunity against swine influenza virus as compared to the commercial influenza vaccine, FluSure XP. In addition, the commercial influenza vaccine, FluSure XP only induced transient immunity as compared to our vaccine which showed protective levels of immunity for up to 6 months post-vaccination. Additionally, upon viral challenge our Ad-vectored Epigraph vaccine induced stronger levels of protectinon and lower level of pathogenesis as compared to the FluSure XP and DPBS control. Overall, our vaccine induced superior results as compared to the current commercial influenza vaccine. Abstract Current methods of vaccination against swine influenza A virus (IAV-S) in pigs are infrequently updated, induce strain-specific responses, and have a limited duration of protection. Here, we characterize the onset and duration of adaptive immune responses after vaccination with an adenoviral vectored Epigraph vaccine. In this longitudinal study we observed robust and durable antibody responses that remained above protective levels six months after vaccination. We further identified stable levels of antigen-specific T cell responses that remained detectable in the absence of antigen stimulation. Antibody isotyping revealed robust class switching from IgM to IgG, while the commercial comparator vaccine failed to induce strong antibody class switching. Swine were challenged six months after the initial vaccination, and animals immunized with the adenoviral vectored Epigraph vaccine demonstrated significant protection from microscopic lesion development in the trachea and lungs, reduced duration of viral shedding, lower presence of infectious virus and viral antigens in the lungs, and significant recall T cell responses following challenge. The results obtained from this study are useful in determining the kinetics of adaptive immune responses after vaccination with whole inactivated virus vaccines compared to adenoviral vectored vaccines and contribute to the continued efforts of creating a universal IAV-S vaccine.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: 1. Petro-Turnquist E, Pekarek M, Jeanjaquet N, Wooledge C, Steffen D, Vu H and Weaver E (2023) Adenoviral vectored epigraph vaccine elicits robust, durable, and protective immunity against H3 influenza A virus in swine. Front. Immunol. Front. Immunol., 15 May 2023, Sec. Vaccines and Molecular Therapeutics, Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1143451

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:In this initial year of this project we are still generating data for publication. We expect that our first public reports of these projects will be completed late in 2022. We have a large study that we will publish in a scientific journal and we plan to present our results through outreach using University,local and regional news outlets. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?These research projects have allowed for the training of two graduate students in the processes and procedures that are involved in swine research. We have a very experienced and skilled staff in our animal facility. They have taken great care to train these students as these eperiments have progressed. 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?In the next reporting period, we will complete objective 1 and 3. These data will be published in a scientific journal and presented at the 2023 Conference of Research Workers in Animal Disease. In addition, we will perform preliminary studies with our swine influenza stocks in order to ensure that our challenge studies, that are described in objective 2, are successful.In addition, objective 2 will be started in the next reporting period, but may not be completed until the following reporting period.

Impacts
What was accomplished under these goals? Swine Influenza virus is widespread and results in an estimated $700 million dollar loss to the swine industry annually. Swine influenza also poses a great threat to public health due to its zoonotic potential. Since 2005, there have been >400 swine influenza variant infections in humans.The most notable variant was the 2009 swine flu pandemic that infected 24% of the global population. Based on our preliminary data we will explore an Epigraph immunogen approach as a universal vaccine for the prevention of swine influenza.We will evaluate Epigraph immunogens for their ability to drive cross-reactive immunity against divergent swine H3N2 influenza viruses in a swine model.First, Epigraph immunogens are designed to maximize potential T and B cells epitopes (PTBE).Second, Epigraph vaccines are designed to create a cocktail of immunogens.Our preliminary data in mice showedthat the Epigraph vaccine induces superior cellular and humoral immunity against a wide array of divergent swine influenza viruses. This data is again supported in swine studies, the target species.In swine, the Epigraph vaccine was again superior at inducing cellular and humoral immunity as compared to the wildtype and commercial vaccines. Therefore, we plan to evaluate this novel vaccine strategy to the existing traditional inactivated FluSure XP vaccines.We are confident that these universal vaccine antigens will offer superior protection against divergent Swine influenza as compared to wildtype antigens.Based on our preliminary data, we are optimistic that these universal vaccine immunogens will successfully control swine influenza and, will not only reduce the economic impact of this pathogen to the swine industry, but will also eliminate the zoonotic threat of reassorted pandemic influenza. Accomplishements (Year 1): Since objective three would require long-term and extensive studies, we started with this objective. Objective 3 will determine the rapid onset of immunity and the durations of immunity in vaccinated swine. This is important because protection induced by vaccinationshould work quickly and provide long-lasting immunity. These experiements were started in early 2022 and we will be completing the data collection from this study by the end of the summer. We have found that our Epigraph vaccine induces immunity against more diverse swine influenza strains as compared to the commercial FluSure XP vaccine. In addition, immunity was induced rapidly and to a higher degree than that induced by the FluSure vaccine. Finally, our data indicates that, not only are stronger immune responses generated by the Epigraph vaccine, the responsesare also longer-lasting than the inactivated commercial FluSure vaccine. The following figure is a part of the data collected so far for objective 3:? Finally, Dr. Wenjun and I have met several times in this reporting period and have finalized a plan to complete objective 2. He has agreed to travel to the University of Nebraska and assist us with the 4 swine influenza virus challenge studies.

Publications