Progress 02/01/17 to 01/31/18

Outputs
Target Audience:Scientists in the field of influenza, medical and animal virology, immunology & vaccinology, infectious diseases; swine industry; poultry industry; medical industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported thetraining of 1 undergraduate student, 2 Ph.D students, and two post-docs. One research associate was also involved. In addition to training on the discipline area (virology, immunology, and infectious diseases), students and post-docs presented the results at the local, national, and international meetings and also published the manuscripts in peer-reviewed journals. How have the results been disseminated to communities of interest?The results were presented at the international, regional and local meetings. Six manuscripts has been published or are under review for publication. In addition, the results were shared with the private, state and government and research lab people working for poultry and swine industry. What do you plan to do during the next reporting period to accomplish the goals?Based on the results we have obtained so far and also based on the results from ongoing animal experiments, we will select the best combination of different vaccines, adjuvants, vaccination route, and vaccination regime for each species. In swine model, we will combine the nanoparticle delivery of conserved epitope, mycobacterium adjuvant, live attenuated vaccine and the respiratory route of vaccination. As mentioned, the swine model is being used to develop human flu vaccine in addition to swine itself, and the study is designed with such consideration. In chicken model, we will prime vaccinate the birds with live attenuated vaccine via intranasal route and boost vaccinated with M2e-PP & HA2-PP combined with inactivated vaccine via subcutaneous route. Depending on the availability of the high biosecurity (BSL-3) facility, protective efficacy against highly pathogenic avian influenza virus will be determined. We will also continue to determine the immune correlates of protection of birds and mammals vaccinated with different vaccine formula with focus on cross-reactive immune responses.

Impacts
What was accomplished under these goals? New chimeric P particles with different epitopes linked with M2e were successfully constructed and tested in different vaccine formulations in mice. 1) We developed and tested several chimeric P particle constructs with more M2e copies, different insertion sites, as well as two T cell epitopes linked with M2e. Our study showed that incorporation of Tetanus universal T cell epitope (Tet830) and use of MPLA enhanced both humoral and cell mediated immune response in mice. 2) We have incorporated the M2e and two different sizes (55aa and 130aa) of HA2 protein in the P particle representing avian and swine influenza virus consensus sequences. In addition, we have developed new construct expressing HA2 protein (HA2-AtCYN). We have demonstrated strong antibody responses using these constructs in mice, chickens, and pigs. Currently, protective efficacy studies using different vaccination regimes are on-going. Immunogenicity and protective efficacy of different vaccine formulations in chickens 1) Chimeric M2e P particle (M2e-PP) is immunogenic in chickens when administered by subcutaneous route with commercial oil adjuvant. No detectable IgG or IgA response was observed after IN vaccination in chickens. 2) M2e-PP immunization consistently reduced virus shedding against 3 low pathogenic avian influenza (LPAI) virus challenges in chickens. 3) Supplementation of inactivated vaccine with M2e-PP significantly enhanced HI antibody titers against homologous and heterologous viruses. Combined vaccination of M2e-PP with inactivated vaccine complemented the efficacy in reducing virus shedding against heterologous challenges. 4) Mechanistically, we demonstrated that M2e antibodies are capable of recognizing the native M2e epitopes exposed on the surface of influenza virus-infected MDCK cells and whole virus. We also showed that M2e antibodies have a role in blocking viral replication by conducting 5) In addition to recombinant subunit vaccine (M2e-PP, HA2-PP, etc.), we also tested protective efficacy of live attenuated influenza vaccine (LAIV) in combination with M2e-PP against heterosubtypic H5N2 and heterologous H7N2 challenge viruses. The heterosubtypic H5N2 challenge virus replicated to high titers in mock-vaccinated birds at 4 days post challenge. On the contrary, birds from the LAIV-only and LAIV combined with M2e-PP groups shed significantly lower viral titers (2-3 logs lower titers) compared to the mock-vaccinated controls. The results shows that both LAIV and M2e-PP can be good components of universal influenza vaccine. Immunogenicity and protective efficacy of different vaccine formulations in pigs 1) We showed a mild reduction in gross lesion and virus shedding by intramuscular (with oil adjuvant) or intranasal (with MPLA or TET830 adjuvant) M2e-PP vaccination. We also observed specifically proliferated PBMCs in HA2-PP, M2e-PP+HA2-PP, intranasal M2e-PP with MPLA vaccinated groups suggesting the presence of specific immunogen recognition. Additional studies are on-going to evaluate the enhancement of the specific immunity and the protective efficacy of the immunogens. 2) We also showed that biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP)-entrapped conserved H1N1 influenza virus peptides vaccine induces peptide specific T cell response in pigs. Conserved peptides in NP vaccine induced either CTL, T-helper, or T-helper/ memory specific response in the lungs. Detectable replicating challenged virus was absent in PLGA-NP peptides cocktail vaccine received pig lungs. 3) Inactivated swine influenza virus (SwIV) H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared and confirmed the induction of maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/ memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways was observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-gamma secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross protective cell-mediated immune response in a pig model. 4) We also used inactivated whole H1N2 virus encapsulated in polyanhydride (CPTEG:CPH; 20:80) nanoparticles to enhance the breadth of protection. Majority of Poly-KAg vaccine particles were around 200 nm size. Poly-KAg vaccination: 1) rescued pigs from fever; 2) reduced gross lung pathology; 3) significantly reduced lung antigenic mass; and 4) reduced viral load in SwIV challenged pig lungs. In addition, Poly-KAg induced better cellular immune response and comparable humoral immune response to that of soluble antigen. antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity. Impacts: The experimental universal vaccines/vaccine formulations have been primarily tested in mice. Our study provides direct evidence showing that the mouse model may not reflect vaccine efficacy in humans and swine can be a better model for human flu vaccine development. Specifically, our pig study and the obtained data confirm the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans. Since swine and poultry are the two species being significantly affected by the flu and sporadically transmit the virus to humans, we get a dual benefit of public and animal health from this research. In addition, we have tested array of novel vaccines/vaccine formulations (recombinant and live vaccines, adjuvants, etc.) in different animal models. This wealth of knowledge will be used to optimize and define best vaccine combinations and vaccination approach for each species that can provide broadly reactive protective immunity against emerging and re-emerging influenza viruses.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Elaish M, Ali A, Xia M, Ibrahim M, Ngunjiri JM, Jang H, Hiremath J, Dhakal S, Helmy YA, Jiang X, Renukaradhya GJ, Lee CW. Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens. PLoS One. 12(2):e0171174. 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dhakal, S, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, K. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B. Narasimhan, C.W. Lee and G.J. Renukaradhya. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs. J Control Release, 247:194-205. 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dhakal, S, J. Goodman, K. Bondra, Y.S. Lakshmanappa, J. Hiremath, D. Shyu, K. Oyuang, K. Kang, S. Krakowka, M.J. Wannemuehler, C.W. Lee, B. Narasimhan and G.J. Renukaradhya. Polyanhydride nanovaccine against swine influenza virus in pigs. Vaccine, 35(8): 1124-1131. 2017.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Dhakal, S, X. Cheng, J. Salcido, S. Renu, K. Bondra, Y.S. Lakshmanappa, C. Misch, S. Ghimire, N.F. Ruiz, B. Hogshead, S. Krakowka, K. Carson, J. McDonough, C.W. Lee and G.J. Renukaradhya. Liposome based influenza conserved peptides vaccine and monosodium urate adjuvant elicits protective immune response in pigs. Under Revision.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Hyesun Jang, Mohamed Elaish, Mahesh KC, Michael C Abundo, Amir Ghorbani, Chang-Won Lee. Efficacy and Synergy of Live-attenuated and Inactivated Influenza Vaccines in Young Chickens. Under Review.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhakal, S, K. Bondra, D. Shyu, K. Tabynov, C.W. Lee and G.J. Renukaradhya. Intramuscular route of delivery of PLGA-nanoFlu vaccine improves antibody response in pigs. OARDC Research Conference, The Ohio State University, Columbus, Ohio. April 20, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhakal, S, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, K. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B. Narasimhan, C.W. Lee and G.J. Renukaradhya. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs. Abstract # 263, Immunology 2017, AAI meeting, Washington Convention Center, Washington DC, May 12-16, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhakal, S, R. Sankar, S. Ghimire, Y.S. Lakshmanappa, B. Hogshead, N.F. Ruiz, S. Krakowka, C.W. Lee and G.J. Renukaradhya. Chitosan delivery of inactivated influenza vaccine improves heterologous protection by enhancing antibody and cellular immune responses in pigs. AAVI mini-symposium and 98th Annual CWRAD meeting, Chicago, IL. December 3-5, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jang H, Ngunjiri JM, Elaish M, Lee CW. Efficacy and Synergy of Live-attenuated and Inactivated Influenza Vaccines in Young Chickens. 98th Annual CWRAD meeting, Chicago, IL. December 3-5, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Amir Ghorbani, John M. Ngunjiri, Mohamed Elaish, Hyesun Jang, Mahesh KC, Michael C. Abundo and Chang-Won Lee. Enhanced protection against heterosubtypic avian influenza virus challenge conferred by M2e-PP subunit vaccination in SPF chickens previously primed with live attenuated influenza vaccine. OARDC Research Conference, The Ohio State University, Columbus, Ohio. April 20, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mohamed Elaish, John M. Ngunjiri, Hyesun Jang, Amir Ghorbani, Mahesh KC, Michael C. Abundo, Chang-Won Lee. Potential mechanism of protection by M2e-based vaccine in chickens. American Society for Virology 36th Annual Meeting. Madison, WI. June 24-28, 2017.
• Type: Other Status: Published Year Published: 2017 Citation: Lee CW. UNIVERSAL FLU VACCINE BY A NOROVIRUS P PARTICLE PLATFORM. 2017 USDA/NIFA Animal Health and Animal wellbeing Project Director Meeting. Chicago. IL. December 1, 2017.
• Type: Other Status: Other Year Published: 2017 Citation: Lee CW. Toward the Development of a Universal Influenza Vaccine. September 14, 2017. Department of Veterinary Medicine, University of Maryland.
• Type: Other Status: Other Year Published: 2017 Citation: Lee CW. Development of a Broadly-Reactive Influenza Vaccine. November 8, 2018. Department of Pathobiology, University of Illinois at Urbana-Champaign.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Hyesun Jang, John N Ngunjiri, Mohamed Elaish and Chang-Won Lee. Application of NS1-truncated variants as live attenuated influenza vaccine. OARDC Research Conference, The Ohio State University, Columbus, Ohio. April 20, 2017.

Progress 02/01/16 to 01/31/17

Outputs
Target Audience:Scientists in the field of influenza, medical and animal virology, immunology & vaccinology, infectious diseases; swine industry; poultry industry; medical industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This projects support training of 2 Ph.D. students and two post-docs. One research associate was also involved. In addition to training on the discipline area (virology, immunology and infectious diseases), students and post-docs presented the results at the local and national meetings and also published the mansucripts in peer-reviewed journals. How have the results been disseminated to communities of interest?The results were presented at the international, regional, and local meetings. Four manuscripts has been published or accepted for publication and another manuscript was submitted. In addition, the results were shared with the private, state and government and research lab people working for poultry and swine industry. What do you plan to do during the next reporting period to accomplish the goals?Based on the results we have obtained so far and also based on the results from ongoing animal experiments, we will select the best combination of different vaccines, adjuvants, vaccination route, and vaccination regime for each species. In swine model, we will combine the nanoparticle delivery of conserved epitope, mycobacterium adjuvant, live attenuated vaccine and the respiratory route of vaccination. As mentioned, the swine model is being used to develop human flu vaccine in addition to swine itself, and the study is designed with such consideration. In chicken model, we will prime vaccinate the birds with live attenuated vaccine via intranasal route and boost vaccinated with M2e-PP & HA2-PP combined with inactivated vaccine via subcutaneous route. Depending on the availability of the high biosecurity (BSL-3) facility, protective efficacy against highly pathogenic avian influenza virus will be determined. We will also continue to determine the immune correlates of protection of birds and mammals vaccinated with different vaccine formula with focus on cross-reactive immune responses.

Impacts
What was accomplished under these goals? To develop universal influenza vaccine platform with greater efficacy against a broader range of antigenic variants and emerging strains, we continued to optimize new vaccine constructs and evaluated their immunogenicity and protective efficacy in combination with new adjuvants, delivery systems, and vaccination approaches in mice, chickens and pigs. Key findings and accomplishments are listed below. New chimeric P particles with different epitopes linked with M2e were successfully constructed and tested in different vaccine formulations in mice. We developed and tested several chimeric P particle constructs with more M2e copies, different insertion sites, as well as two T cell epitopes linked with M2e. Our study showed that incorporation of Tetanus universal T cell epitope (Tet830) and use of MPLA enhanced both humoral and cell mediated immune response in mice. We have incorporated the M2e and two different sizes (55aa and 130aa) of HA2 protein in the P particle representing avian and swine influenza virus consensus sequences. In addition, we have developed new construct expressing HA2 protein (HA2-AtCYN). We have demonstrated strong antibody responses using these constructs in mice, chickens, and pigs. Currently, protective efficacy studies using different vaccination regimes are on-going. Immunogenicity and protective efficacy of different vaccine formulations in chickens Chimeric M2e P particle (M2e-PP) is immunogenic in chickens when administered by subcutaneous route with commercial oil adjuvant. No detectable IgG or IgA response was observed after IN vaccination in chickens. M2e-PP immunization consistently reduced virus shedding against 3 low pathogenic avian influenza (LPAI) virus challenges in chickens. Supplementation of inactivated vaccine with M2e-PP significantly enhanced HI antibody titers against homologous and heterologous viruses. Combined vaccination of M2e-PP with inactivated vaccine complemented the efficacy in reducing virus shedding against heterologous challenges. Mechanistically, we demonstrated that M2e antibodies are capable of recognizing the native M2e epitopes exposed on the surface of influenza virus-infected MDCK cells and whole virus. We also showed that M2e antibodies have a role in blocking viral replication by conducting plaque reduction assay to determine neutralizing capability of sera obtained from M2e-PP vaccinated chickens. In addition to recombinant subunit vaccine (M2e-PP, HA2-PP, etc.), we also tested protective efficacy of live attenuated influenza vaccine (LAIV) in combination with M2e-PP against heterosubtypic H5N2 and heterologous H7N2 challenge viruses. The heterosubtypic H5N2 challenge virus replicated to high titers in mock-vaccinated birds at 4 days post challenge. On the contrary, birds from the LAIV-only and LAIV combined with M2e-PP groups shed significantly lower viral titers (2-3 logs lower titers) compared to the mock-vaccinated controls. The results shows that both LAIV and M2e-PP can be good components of universal influenza vaccine. Immunogenicity and protective efficacy of different vaccine formulations in pigs We showed a mild reduction in gross lesion and virus shedding by intramuscular (with oil adjuvant) or intranasal (with MPLA or TET830 adjuvant) M2e-PP vaccination. We also observed specifically proliferated PBMCs in HA2-PP, M2e-PP+HA2-PP, intranasal M2e-PP with MPLA vaccinated groups suggesting the presence of specific immunogen recognition. Additional studies are on-going to evaluate the enhancement of the specific immunity and the protective efficacy of the immunogens. We also showed that biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP)-entrapped conserved H1N1 influenza virus peptides vaccine induces peptide specific T cell response in pigs. Conserved peptides in NP vaccine induced either CTL, T-helper, or T-helper/ memory specific response in the lungs. Detectable replicating challenged virus was absent in PLGA-NP peptides cocktail vaccine received pig lungs. Inactivated swine influenza virus (SwIV) H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared and confirmed the induction of maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/ memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways was observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-gamma secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross protective cell-mediated immune response in a pig model. We also used inactivated whole H1N2 virus encapsulated in polyanhydride (CPTEG:CPH; 20:80) nanoparticles to enhance the breadth of protection. Majority of Poly-KAg vaccine particles were around 200 nm size. Poly-KAg vaccination: 1) rescued pigs from fever; 2) reduced gross lung pathology; 3) significantly reduced lung antigenic mass; and 4) reduced viral load in SwIV challenged pig lungs. In addition, Poly-KAg induced better cellular immune response and comparable humoral immune response to that of soluble antigen. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity. Impacts: The experimental universal vaccines/vaccine formulations have been primarily tested in mice. Our study provides direct evidence showing that the mouse model may not reflect vaccine efficacy in humans and swine can be a better model for human flu vaccine development. Specifically, our pig study and the obtained data confirm the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans. Since swine and poultry are the two species being significantly affected by the flu and sporadically transmit the virus to humans, we get a dual benefit of public and animal health from this research. In addition, we have tested array of novel vaccines/vaccine formulations (recombinant and live vaccines, adjuvants, etc.) in different animal models. This wealth of knowledge will be used to optimize and define best vaccine combinations and vaccination approach for each species that can provide broadly reactive protective immunity against emerging and re-emerging influenza viruses.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Elaish M, Ali A, Xia M, Ibrahim M, Ngunjiri JM, Jang H, Hiremath J, Dhakal S, Helmy YA, Jiang X, Renukaradhya GJ, Lee CW. Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens. PloS One. In Press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hiremath J, Kang KI, Xia M, Elaish M, Binjawadagi B, Ouyang K, Dhakal S, Arcos J, Torrelles JB, Jiang X, Lee CW, Renukaradhya GJ. Entrapment of H1N1 influenza virus derived conserved peptides in PLGA Nanoparticles enhances T cell response and vaccine efficacy in pigs. PLoS One. 11(4):e0151922. 2016.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Dhakal S, Hiremath J, Bondra K, Lakshmanappa YS, Shyu D, Oyuang K, Kang KI, Binjawadagi B, Goodman J, Tabynov K, Krakowka S, Narasimhan B, Lee CW, Renukaradhya GJ. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs. Journal of Controlled Release. In Press.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Dhakal S, Goodman J, Bondra K, Lakshmanappa YS, Hiremath J, Shyu D, Oyuang K, Kang KI, Krakowka S, Wannemuehler MJ, Lee CW, Narasimhan B, Renukaradhya GJ. Polyanhydride nanovaccine against swine influenza virus in pigs. Vaccine. In Press.
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Xia M, Fang H, Tan M, Zhong W, McNeal M, Lee CW, Jiang X. Optimization of a P particle-M2e chimeric vaccine candidate against influenza virus and norovirus. Submitted.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jang H, Ngunjiri J, Lee CW. Correlation between interferon response and protective efficacy of NS1-truncated mutants as influenza vaccine candidates in chickens. American Veterinary Medical Association (AVMA) Meeting, San Antonio, TX. August 6-9, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Elaish M, Ngunjiri JM, Jang H, Lee CW. Evaluation of in vitro biological role of ectodomain of the influenza virus matrix protein 2 (M2e) specific antibodies in chickens. Conference of Research Workers in Animal Diseases (CRWAD) Meeting. Chicago, IL. December 46, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jang H, Elaish M,, Ngunjiri JM, Lee CW. Application of NS1-truncated variant as live attenuated influenza vaccine for early protection and its complementary use with inactivated vaccine in chickens. Conference of Research Workers in Animal Diseases (CRWAD) Meeting. Chicago, IL. December 46, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ghorbani A, Ngunjiri JM, Jang H, Elaish M, Lee CW. Protective efficacy of NS-1 truncated live attenuated influenza vaccine combined with or without M2e subunit vaccine against heterologous and heterosubtypic challenges. Conference of Research Workers in Animal Diseases (CRWAD) Meeting. Chicago, IL. December 46, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal S, Hiremath J, Bondra K, Lakshmanappa YS, Shyu D, Oyuang K, Binjawadagi B, Kang K, Goodman J, Krakowka S, Narasimhan B, Lee CW, Renukaradhya GJ. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous protection through cell-mediated immunity in pigs. OARDC Research Conference, The Ohio State University, Wooster, Ohio. April 21, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bondra K, Dhakal S, Krakowka S, Kang K, Lee CW, Renukaradhya GJ. Nanoparticle delivered inactivated swine influenza virus vaccine reduces the lung pathology in heterologous virus challenged pigs. OARDC Research Conference, The Ohio State University, Wooster, Ohio. April 21, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal S, Hiremath J, Bondra K, Lakshmanappa YS, Shyu D, Oyuang K, Kang K, Binjawadagi B, Goodman J, Tabynov K, Krakowka S, Narasimhan B, Lee CW, Renukaradhya GJ. Poly-lactic-co-glycolic acid nanovaccine against swine influenza virus in a pig model. Nanovaccine Research Initiative, Iowa State University, Ames, Iowa. May 22  24, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal S, Goodman J, Bondra K, Lakshmanappa YS, Hiremath J, Shyu D, Oyuang K, Kang K, Binjawadagi B, Krakowka S, Lee CW, Narasimhan B, Renukaradhya GJ. Polyanhydride nanoparticle based vaccine against swine influenza virus in pigs. Conference of Research Workers in Animal Diseases (CRWAD) Meeting. Chicago, IL. December 46, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal S, Hiremath J, Bondra K, Lakshmanappa YS, Shyu D, Oyuang K, Binjawadagi B, Kang K, Goodman J, Tabynov K, Krakowka S, Narasimhan B, Lee CW, Renukaradhya GJ. PLGA nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous protection through cell-mediated immunity in pigs. Conference of Research Workers in Animal Diseases (CRWAD) Meeting. Chicago, IL. December 46, 2016.

Progress 02/01/15 to 01/31/16

Outputs
Target Audience:Scientists in the field of influenza, medical and animal virology, immunology & vaccinology, infectious diseases; swine industry; poultry industry; medical industry Changes/Problems:Ourresultshighlight species difference in vaccine efficacy and immune reponse. Thus, we will optimize the vaccine and vaccination strategy based on species. What opportunities for training and professional development has the project provided?This projects support training of 2 Ph.D. students and two post-docs. In addition, one undergraduate student was involved as part of the summer research program. Students and post-docs presented the results at the local and national meetings. How have the results been disseminated to communities of interest?The results were presented at the international (ASV, CRWAD, APPC) and regional (NCADC) meetings. One manuscript has been published, another manuscript was submitted, and two additional manuscripts will be submitted within a month. In addition, the results were shared with the private, state and government and research lab people working for poultry and swine industry. In 2015, our results were presented at the key international influenza meetings including 9th International Symposium on Avian Influenza and 3rd International Symposium on Neglected Influenza Viruses and also at the premier vaccine conference (2015 World Vaccine Congress held at D.C.). What do you plan to do during the next reporting period to accomplish the goals?We will continue to 1) fine tune the vaccine constructs, select the best adjuvant, delivery route and method, and application strategy for each species, 2) identify the immune mechanism and correlates of protection for each species, and 3) establish swine model for human vaccine development. Based on the results we have obtained so far and also based on the results from ongoing animal experiments, we will combine M2e, HA2, and T and B cell epitopes into a single P particle platform to maximize the efficacy as necessary. In swine model, we will combine the nanoparticle delivery and MPLA adjuvant via the respiratory route of vaccination that showed promising results in swine. As mentioned in overall goal, the swine model is being used to develop human flu vaccine in addition to swine itself, and the study is designed with such consideration. In chicken model, we will test the protective efficacy of HA2-PP in different combinations. We already showed this year as a preliminary study that HA2-PP induces antibody response in a dose-dependent manner in chickens. Protective efficacy and immune response will be evaluated. We will also continue to determine the clear role and functional capabilities of M2e antibodies in protection of birds and mammals against different influenza viruses with focus on cross-reactivity among different strains. The specific role and extent of the function of these antibodies in protection will be assessed by in vitro plaque reduction assay and other methods.

Impacts
What was accomplished under these goals? Based on the data we obtained in Year 1 and 2, we extended the study to validate the vaccine constructs (M2e-PP: M2e - P particle & HA2-PP: HA2-P particle vaccines) and test new delivery methods (delivery vehicles and routes) and adjuvants. Immunogenicity and protective efficacy of different vaccine platforms in pigs. The study had three goals: (1) evaluation of newly constructed HA2-PP immunogenicity, (2) testing the combined effect of M2e-PP and HA2-PP vaccination, and (3) mucosal immunization with M2e-PP. Three groups of seven to eight 3-week-old colostrum-deprived piglets were immunized intramuscularly with M2e-PP, HA2-PP, or both (i.e., M2e-PP + HA2-PP) vaccines mixed with a commercial oil adjuvant. Two groups of pigs received intranasal M2e-PP vaccinations adjuvanted with monophosphoryl lipid A (MPLA) or a universal human tetanus toxin T-cell epitope (TET). One group of pigs served as a mock vaccine group. After three times of vaccination at 2-week intervals, all groups were challenged via intratracheal route with an H1N2 influenza virus. Pigs were monitored daily and necropsied at day 5 post challenge (DPC). Key findings include: Pathological evaluation showed a mild reduction of gross lesions in both intranasal M2e-PP vaccine groups (M2e-PP with MPLA and M2e-PP with TET). Specific proliferation of PBMCs was detected in intramuscular HA2-PP and M2e-PP + HA2-PP, and intranasal M2e with MPLA vaccine groups suggesting the presence of specific immunogen recognition. The number of PBMCs from HA2-PP, M2e-PP + HA2-PP, and M2e with MPLA groups was significantly increased by M2e stimulation at 5 DPC. Moreover, PBMCs from HA2-PP vaccinated pigs were also increased by inactivated whole virus stimulation, indicating antigen specific cell responses were generated. Further analyses regarding virus replication and shedding, microscopic lesion formation, and specific humoral immune responses will be conducted to evaluate the level of specific immunity and protective efficacy of the immunogens. PLGA nanoparticle-entrapped conserved H1N1 influenza virus peptides vaccine induces peptide specific T cell response in pigs. Apart from being an adjuvant, biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticle (PLGA-NP) acts as a vaccine delivery vehicle which can cross-present antigens (Ags) to naïve T cells. In this study, M2e-PP and conserved H1N1 peptides of pandemic 2009 and classical human and avian viruses were entrapped in PLGA-NPs. In addition, we prepared and evaluated non-pathogenic mycobacterial WCL derived from Mycobacterium vaccae, which was used earlier as an adjuvant in rodent models. Briefly, a cocktail of selected two each of conserved T & B cell peptides and M2e-PP were entrapped in PLGA nanoparticles and co-administered with M. vaccae WCL intranasally, and the immune correlates were evaluated in a heterologous challenge pig model. Key findings include: Detectable replicating challenged SIV was absent in PLGA-NP peptides cocktail vaccine received pig lungs PLGA-NP entrapped conserved H1N1 peptides vaccine predominantly elicited epitope specific T-cell response in lungs Conserved peptides in NP vaccine induced either CTL, T-helper, or T-helper/memory specific response in the lungs Continued efforts to optimize the constructs and testing in mice and chickens.We made a number of new constructs in Year 1 and 2 which have 1) insertion of M2e in different loops of P particle, at either the N- or C-terminus, 2) multiple copies of M2e in tandem repeat, and 3) incorporation of T cell epitopes. We also tested the use of Monophosphoryl Lipid A (MPLA) adjuvant. In our ongoing efforts based on in vivo data, we are combining the useful epitopes we found into single vaccine constructs with optimal expression. These constructs are initially being tested in mice and chicken before testing in swine model which requires more resources and time. Continued efforts to Understanding the protective immune mechanism. In addition to potential protective mechanism mentioned above, extensive efforts are being made to understand both M2e specific antibody and T cell immunity in chickens and pigs. We showed that M2e-specific antisera strongly reacted with M2e peptide but mild to moderately with whole viruses in ELISA while the opposite results were obtained with antisera prepared with whole viruses. M2e-PP immune sera from all three animals efficiently bind to influenza virus infected MDCK cells as demonstrated in FACS analysis and immunofluorescence assay. However, the binding intensity was much weaker with M2e-PP antisera compared to that of whole-virus antisera. In addition, antisera generated with M2e-PP in different animals cross reacted with influenza viruses from different animal species. Future study will focus on determining the magnitude and mechanism of inhibition of influenza replication by the M2e-specific sera. In chickens, in contrast to results obtained from pig experiment, antigen specific cell response was not observed despite extensive analysis of PBMCs after each vaccination. This result further emphasize the species difference and additional study is required to delineate species specific immune correlates. Impact Addition of M2e-PP to inactivated vaccine conferred improved protection compared to single regime vaccination suggesting a possible approach to modify traditional vaccination strategy. Addition of M2e-PP also enhanced the hemagglutination inhibition (HI) antibody response induced by inactivated vaccine, which correlated with protective efficacy. The study will continue to determine the most practical and cost-effective approach for poultry industry. Given their genetic and physiologic similarity to humans, pigs are a useful animal model for human vaccine study and success in pigs can be a better predictor of success in humans. The recent avian flu outbreak reemphasized the benefits and urgency to develop a universal vaccine. We never thought this could happen in the US. The situation has changed because of the involvement of wild birds, making the potential role of a vaccine in preventing outbreaks even more important. The success of this groundbreaking research would be of enormous benefit for the health of humans and animals alike, addressing a challenge that has cost countless lives and dollars.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Jagadish Hiremath; Kang Kyung-il; Xia M; Mohamed Elaish; Basavaraj Binjawadagi; Kang Ouyang; Santosh Dhakal; Jesus Arcos; Jordi Torrelles; X Jiang; Chang Won Lee. PLGA nanoparticle-entrapped conserved H1N1 influenza virus peptides vaccine induces peptide specific T cell response in pigs.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lee CW, Elaish M, Kang KI, Ngunjiri JM, Jang H, Ali A, Xia M, Jiang X. Efficacy of M2e-based vaccine in chickens in comparison to murine model. 9th International Symposium on Avian Influenza. April 12-15, Athens, GA. 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kang KI, Xia M, Elaish M, Ibrahim M, Ali A, Ngunjiri JM, Jang H, Jiang X, Lee CW. Immunogenicity and Protective Efficacy of Combined Vaccination with Recombinant M2e and Inactivated Influenza Vaccines in Pigs. 3rd International Symposium on Neglected Influenza Viruses , April 15-17. Athens, GA. 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lee CW. Approaches Toward the Developemnt of Universal Influenza Vaccines. 2015 World Vaccine Congress, Washing DC. April 7-9. 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lee CW. Current understanding on intercontinental HPAI: To vaccinate or not? Keynote address at the 96th CRWAD meeting. December 6-8. Chicago, IL. 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Renukaradhya GJ, Hiremath JM, Elaish M,Kang KI, Binjawadagi B, Ouyang K, Dhakal S, Lee CW. PLGA nanoparticle-entrapped conserved H1N1 influenza virus peptides vaccine induces peptide specific T cell response in pigs. Keystone Symposia: Immunity to Veterinary Pathogens: Informing Vaccine Development. Keystone Resort, Colorado. Jan 20-25. 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Renukaradhya GJ, Hiremath JM, Elaish M,Kang KI, Binjawadagi B, Ouyang K, Dhakal S, Lee CW. PLGA nanoparticle-entrapped conserved H1N1 influenza virus peptides vaccine induces peptide specific T cell response in pigs. European Veterinary Immunology Workshop. Vienna, Austria. Sept 2-4. 2015.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Elaish M, Ali A, Xia M, Kang Kl, Ibrahim M, Jiang X, Lee CW. Immunogenecity and protective efficacy of M2e-expressing norovirus P particle vaccine in chickens. Vaccine. 33:4901-9. 2015.

Progress 02/01/14 to 01/31/15

Outputs
Target Audience: Scientists in the field of influenza, vaccinology, virology, infectious disease, swine and poultry diseases; swine industry, poultry industry; vaccine company Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supports training of 2 Ph.D students and two post-docs. In addition to training on specific research, students and post-docs presented the results at the local and national meetings. How have the results been disseminated to communities of interest? The results were presented at the international (ASV, CRWAD, APPC) and regional (NCADC) meetings. One manuscript has been submitted and 3 additional manuscript will be submitted to peer-reviewed journals within two monthsto disseminate the findings from first two years. In addition, the results were shared with the private, state and goverment and research lab people working for poultry and swine industry. The results were also shared with the industry and veterinarians (from Ohio, Michigan, and Indiana) in the semiannual meetings. In 2015, our results will be presented at two key international influenza meetings (9th International Symposium on Avian Influenza & 3rd International Symposium on NeglectedInfluenza Viruses)and also at the 2015 World Vaccine Congress held at D.C. What do you plan to do during the next reporting period to accomplish the goals? Based on extensive data obtained in Year 1 and 2, we will continue to fine tune the vaccine constructs, select the best adjuvant and application strategy for each species, and identify the immune correlates and study the protective immune mechanism. Based on the results we obtained so far and also based on the results from ongoing animal experiments, we will combine the M2e, HA2, T and B cell epitopes into a single P particle platform to maximize the efficacy as necessary. We will also incorporate the epitopes or antigens into different locations of the platform and express the protein in different structure to develop the most efficacious vaccine (Objective 1). In chicken model, we will test the new P particle construct that express HA2 (HA2-PP) for their efficacy in comparison to M2e-PP. In addition, we will test M2e-PP that contains increased (3 times) number of M2e copies which is expected to enhance the humoral immune response with subcutaneous vaccine approach. We will also test if the use of strong mucosal adjuvant, Monophosphoryl Lipid A (MPLA) can enhance the T-helper 1 (Th-1) response which will further induce interferon gamma with other cytokines resulting in enhancement of both humoral and cellular immune responses. We are particularly interested in cost-effective intranasal vaccine approach with the MPLA adjuvant (Objective 2 and 3). In swine model, we will test the M2e-PP constructs with T and B cell epitopes that showed promising results in mouse model and also in preliminary study in pigs. We will also test the effect of MPLA as described in chicken study. As mentioned in overall goal, the swine model is being used to develop human flu vaccine in addition to swine itself, and the study is designed with such consideration (Objective 2 and 3). We will determine the clear role and functional capabilities of M2e antibodies in protection of birds and mammals against different influenza viruses. We will test the ability of M2e antibodies induced by vaccination in different species to recognize the native form of M2e expressed on the virus infected cells using immunofluorescence assay and flow cytometry. The specific role and extent of the function of these antibodies in protection will be assessed by evaluating the reduction in cell to cell spread and the reduction in size of plaque formation against different influenza viruses. We will also determine the magnitude and mechanisms of inhibition of influenza growth by assessing the antibody in supporting NK cell-dependent ADCC in vitro.

Impacts
What was accomplished under these goals? Based on extensive baseline data we obtained in Year 1 from animal studies in mice, chickens, and pigs using M2e P particle (M2e-PP) vaccines, we extended the study to further optimize the vaccine constructs, develop new constructs, and test their efficacy in animals in Year 2. The newly obtained results in this reporting period are summarized below. Immunogenicity and efficacy of new M2e-PP constructs in mice (Objective 1&2): We tested a number of new constructs which has 1) insertion of M2e in different loops of P particle, at either the N- or C-terminus, 2) multiple copies of M2e in tandem repeat, 3) incorporation of T cell epitopes, and 4) use of Monophosphoryl Lipid A (MPLA) adjuvant. Our study clearly showed that incorporation of Tetanus universal T cell epitope (Tet830) and use of MPLA enhance both humoral and cell mediated immune response in mice. Thus, we will further test these two options in chicken and swine model. We also constructed M2e-PP that contains the different size of HA2 from avian and swine flu viruses. This vaccine has been successfully expressed, purified, and prepared for the animal studies in Year 3. Effect of combined vaccination with M2e-P and inactivated influenza vaccine in chickens (Objective 2&3): We expanded the study initiated in Year 1 by testing different combination and vaccination regime of M2e-PP and inactivated vaccine and testing against heterologous and heterosubtypic challenge. We observed that the addition of M2e protein to inactivated vaccine conferred improved protection compared to single regime vaccination suggesting a possible approach to modify traditional vaccination strategy. Furthermore, addition of M2e vaccine enhanced the hemagglutination inhibition (HI) antibody response induced by inactivated vaccine, which correlate with protective efficacy. The study will continue to determine the most practical and cost-effective approach for poultry industry. Enhancement of M2e-PP vaccine efficacy in pigs by using new adjuvant and delivery system (Objective 2&3): Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticle (PLGA-NP) is a potent vaccine delivery system capable of presenting antigens to the immune system and also possess the adjuvant property. In Year 2, M2e-PP and highly conserved four T and B cell epitopes were entrapped in PLGA-NP by double-emulsion method. The PLGA-NP entrapped peptide vaccine received pigs had no fever in spite of comparable gross lung lesions to that of unentrapped peptides vaccine received virus-challenged animals. Interestingly, though the viral RNA copy numbers in BAL fluid was not significantly reduced in PLGA-NP peptide vaccine group compared to control animals, the replicating infective virus was undetectable in NP peptide vaccine received pigs. Immunologically, the difference in specific antibody, virus neutralizing and hemagglutination inhibition titers though higher in PLGA-NP vaccinated compared to control animals, the data was not statistically significant. But strikingly, the PLGA-NP vaccine received pigs had significantly increased frequencies of IFN-γ secreting CD3+CD4+CD8-, CD3+CD4-CD8+ and CD3+CD4+CD8+ cells in the lung mononuclear cells as analyzed by flow cytometry. This data was consistent with the secretion of increased amounts of IFN-γ in to the supernatant by in vitro stimulated lung mononuclear cells. Our data suggested that the PLGA-NP entrapped candidate peptide vaccine induced the viral epitope specific cell-mediated immune response in intranasally vaccinated pigs. In Year 3, we will extend the study by directly incorporating the 4 epitopes into M2e-PP and also optimize the vaccine program. Understanding the protective immune mechanism (Objective 3): The role of M2e-specific antibodies on virus-neutralization is unclear. It has been suggested that anti-M2e antibodies can bind to the M2e protein on infected host cells and reduce virus replication by interfering with virus budding and mediate the killing of infected cells by complement or by cells of the innate immune system. However, recent study by Swinkels et al. (Virol J. 2013) showed that in chickens, a vaccination with the full length M2 protein or synthetic M2e peptide in a tetrameric conformation proved to be immunogenic but the induced antibodies did not recognize M2 on the virus or on infected cells. Thus, we conducted the study to evaluate the binding ability of M2e-PP vaccine induced antibodies in mice, chickens, and pigs to their targets on virus and infected cells in order to evaluate their potential role in the prevention of virus infection. Our results clearly showed that 1) anti-M2e antibodies generated in mice, chickens, and pigs by immunization with synthetic M2e-PP efficiently bind to the target epitopes on cells and viruses and 2) antisera generated by M2e-PP in animals cross react with different influenza viruses. In summary, our study shows that M2e-PP induced antibodies produced in 3 different species effectively bind to the antigen on viral surface and virus infected cells which indicate their potential role in neutralization and antibody dependent infected cell killing. In year 3, we will directly assess the functional capabilities of M2e antibodies in limiting cell to cell spread of virus and supporting NK cell-dependent ADCC.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Elaish M, Kang K, Ali A, Awe O, Ibrahim M, Lee CW. Immunogenicity and protective efficacy of combined vaccination with norovirus P particle-M2e chimera and inactivated influenza vaccine in chickens. Annual Meeting. St. Paul: 65th North Central Avian Disease Conference. 2014.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Lee CW, Kang KI, Elaish M, Ngunjiri JM, Jang H, Ali A, Hiremath J, Dhakal S, Xia M, Jiang X, Gourapura R. Efficacy of M2e-based vaccine in murine, avian and swine models. 95th CRWAD Meeting. Chicago: Dec. 79. 2014.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Elaish M, Kang KI, Ibrahim M, Ali A, Awe O, Lee CW. Immunogenicity and protective efficacy of combined vaccination with recombinant M2e and inactivated influenza vaccines in chickens and pigs. Annual Meeting. Fort Collins: American Society of Virology. 2014.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Hiremath J, Kang KI, Elaish M, Binjawadagi B, Ouyang K, Dhakal S, Lee CW, Gourapura R. PLGA-Nanoparticle entrapped swine influenza virus peptides vaccine induces epitope specific cell-mediated immune response in pigs. 95th CRWAD Meeting. Chicago: Dec. 79. 2014.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Lee CW. Different Approaches Toward Universal Influenza Vaccines. In: Asia Pacific Poultry Conference Proceeding. Seoul, Korea, APPC. p61-63. 2014.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Elaish M, Ali A, Xia M, Kang Kl, Ibrahim M, Jiang X, Lee CW. Immunogenecity and protective efficacy of M2e-expressing norovirus P particle vaccine in chickens. Submitted.

Progress 02/01/13 to 01/31/14

Outputs
Target Audience: Scientists in the field of influenza, virology, infectious disease, swine and poultry disease; swine industry, poultry industry, vaccine company Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supports training of2 Ph.D students andtwo post-docs. In addition to training on specific research, students and post-docs presented the results at the local and national meetings. How have the results been disseminated to communities of interest? The results were presented at the national (AAAP) and regional (NCADC) meetings and manuscripts are in prepartion topublish in peer-reviewed journals in timely manner to disseminate the novel findings toother scientists. In addition, the results were shared with the private, state and govermentand research lab people working for poultry and swine industry. The results were also shared with the industry and veterinarians (from Ohio, Michigan, and Indiana)inthe semiannual meetings. What do you plan to do during the next reporting period to accomplish the goals? Based on extensive data we obtained from animal experiment studies (refer to Accomplishment section), we will further optimize M2e-based P particle (M2e-P) vaccines and evaluate the new vaccine contructs as proposed in Objective 1 and 2. The animal study will include the testing of HA-based vaccine in combination with M2e-based vaccine (Objective 2). In addition to vaccine construction itself, vaccine dose, application, interval, etc. will be adjusted and optimized for animal experiments based on Year 1 studies. Furthermore,mechanism of protective immunity conferred by M2e-P particle vaccines will be further investigated using different immunological assays as proposed in Objective 3.

Impacts
What was accomplished under these goals? The efficacy of M2e-based vaccines has been primarily tested in mouse model. Our data in farm animals, especially swine which are anatomically, physiologically and immunologically similar to humans, provides more relevant data for human vaccine development. Since swine and poultry are the two species being significantly affected by the flu and sporadically transmit the virus to humans, we get a dual benefit from this research. M2e-based P particle (M2e-P) vaccines of different M2e consensus sequence have been generated and tested in animal models for a broad protection against a wide range of avian, swine and human flu viruses. The newly obtained results in this reporting periodare summarized below. Construction of new M2e-P & HA2-P vaccines (Objective 1): 1) For human M2e, we made a number of new constructs for the optimization of M2e-P chimeric vaccine by insertion of M2e in different loops, at either the N- or C-terminus, with multiple copies, and with the addition of T cell epitopes. Testing of these new constructs in mice for enhanced immunogenicity of M2e is ongoing; 2) Avian consensus M2e was incorporated into Norovirus P particle Loop-2 which was tested in chickens as described below. For swine, two consensus M2e-P chimeric vaccines were made for currently circulating swine influenza and one of the constructs are being tested in swine model. Immunogenicity and protective efficacy of M2e-P vaccine in chickens (Objective 2&3): 1) 1, 3 or 5ug of M2e-P vaccine per bird is highly immunogenic in chickens and induce M2e-specific IgG antibody when administered subcutaneously with commercial oil adjuvant; 2) M2e-P immunization significantly reduced virus shedding against three subtypes (H5N2, H6N2, H7N2) of low pathogenic avian influenza virus challenges; 3) Despite no IgG or IgA induction, intranasal immunization of birds without adjuvant also demonstrated protective efficacy in terms of reduction in virus shedding. We observed no difference among 3 different methods (intranasal drop, eyedrop, and microspray) of vaccination; 4) In addition to M2e-specific IgG, innate and/or other adaptive immune responses are being investigated. Effect of combined vaccination with M2e-P and inactivated influenza vaccine in chickens (Objective 2&3): 1) Chickens vaccinated with H7-inactivated vaccine and given M2e-P booster vaccination after 2 weeks or M2e-P mixed with inactivated vaccine demonstrated higher protective efficacy against H7N2 virus challenge compared to chickens receiving inactivated vaccine or M2e-P vaccine alone. The level of M2e specific antibody response was similar between combined vaccination group and M2e-P alone vaccinated birds; 2) The protective efficacy against heterosubtypic challenge (HA subtype that is different from inactivated vaccine) is being investigated and reduced amounts of inactivated vaccine will be tested. Immunogenicity and protective efficacy of M2e-P vaccine in pigs (Objective 2&3): 1) Both 50 and 200ug (per pig) of M2e-P vaccine via intramuscular (IM) route with oil adjuvant induced IgG antibody responses in pigs. As observed in chickens, intranasal M2e-P vaccination did not induce detectable IgG or IgA responses; 2) M2e-P vaccination via IM route showed reduction of virus titers both in the nasal swab and lungs in challenged pigs compared to unvaccinated-challenged pigs; 3) As in chickens additional study is on-going to investigate the protective immune mechanism and also to evaluate the effect of combined vaccination strategy. We have successfullycompleted extensive animal studies both in chickens and pigs to obtain baseline data to further optimize the M2e-based vaccines.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Ali A, Awe O, Shany SA, Tan M, Wang L, Xia M, Jiang X, and Lee CW. Immunogenicity and protective efficacy of the norovirus P particle-M2e chimeric influenza vaccine in chickens. 64th North Central Avian Disease Conference. March 11-12. 2013. St. Paul, Minnesota.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Lee CW, Ali A, Elaish M, Awe O, Xia M, Wang L, Tan M, Jiang X. Development of universal flu vaccine using M2e-P particle in chicken. 150th AVMA Annual Convention. July 2023, 2013. Chicago, Illinois.
• Type: Other Status: Other Year Published: 2013 Citation: Lee CW. Universal flu vaccine by a norovirus P particle platform. NIFA Project Directors Workshop. October 6-8, 2013. College Park, Maryland.