Source: OHIO STATE UNIVERSITY submitted to

UNIVERSAL FLU VACCINE BY A NOROVIRUS P PARTICLE PLATFORM

Sponsoring Institution National Institute of Project Status TERMINATED

Food and Agriculture Funding Source AFRI COMPETITIVE
GRANT
Reporting Frequency Annual Accession No. 0232147
Grant No. 2013-67015-20476 Project No. OHO01103-SS
Proposal No. 2012-04042 Multistate No. (N/A)
Program Code A1241 Project Start Date Feb 1, 2013
Project End Date Jan 31, 2020 Grant Year 2013

Project Director
LEE, C. W.

Recipient Organization Performing Department

OHIO STATE UNIVERSITY Food Animal Health Research Program
1680 MADISON AVENUE
WOOSTER,OH 44691

Non Technical Summary

Influenza virus continues to evolve due to its genetic nature and the emergence of novel strains is inevitable.
The 2009 H1N1 pandemic is a good example demonstrating that predicting when and where the next pandemic
strain will arise is almost impossible even in 21st century. This poses a number of problems in designing timely
preventive measures. The recent outbreaks of highly pathogenic H5N1, traced initially to avian species, and the
2009 pandemic of H1N1 in humans, most closely related to swine influenza viruses, speak to the necessity of
developing vaccines with greater efficacy and protection against a broader range of antigenic variants and
subtypes. However, commercially available vaccines are in general directed specifically towards the strain
contained in the vaccine and careful selection of vaccine strain should be made every few years so that they
match with the circulating strain. Thus, it is of high veterinary and public health importance to explore novel
ways to develop more broadly reactive vaccines without compromising their safety. In this study, we will
develop a broad-spectrum subunit vaccine against flu viruses using our newly discovered P particle of norovirus
as a vaccine platform to present the highly conserved protein (M2e epitope) of flu viruses. In addition to the
structural advantage as a vector, the P particle is highly immunogenic, easily produced in E.coli and extremely
stable which may enable reducing production costs and less dependence on the cold - chain distribution which
is critical for vaccination programs in remote areas and developing countries. We will use our readily available
mice, poultry (chicken) and swine challenge models to study the mechanisms of the immune enhancement and
develop polyvalent M2e-based vaccines to maximize the protection spectrum against all avian, swine and
human flu viruses.

Animal Health Component 50%

Research Effort Categories

Basic 50%
Applied 50%
Developmental (N/A)
Classification

Knowledge Area (KA) Subject of Investigation (SOI) Field of Science (FOS) Percent

311 3299 1040 20%

311 3299 1090 15%

311 3299 1101 15%

311 3599 1040 20%

311 3599 1090 15%

311 3599 1101 15%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other; 3599 - Swine, general/other;

Field Of Science
1090 - Immunology; 1101 - Virology; 1040 - Molecular biology;

Keywords
influenza vaccine norovirus p particle m2e
immunity swine poultry

Goals / Objectives
The goal of this proposal is to develop universal influenza vaccines with greater efficacy and protection against
a broader range of antigenic variants and emerging strains. We hypothesize that M2e-based influenza vaccine
using P particle of norovirus as a vaccine platform is economical, extremely stable, highly immunogenic, and
provide greater efficacy and protection against a broader range of antigenic variants and subtypes. We will test
our hypothesis following four specific aims: 1) Optimization of M2e-P particle vaccines for higher efficacy; 2)
Further enhancement of M2e-P particle vaccines in swine and chickens by combining the administration of the
M2e vaccine with prototype vaccines which target conserved HA regions; 3) Delineation of the mechanism of
protective immunity conferred by M2e-P particle vaccines; and 4) Development of polyvalent consensus M2e
vaccines for a broad protection against a wide range of avian, swine and human flu viruses. The M2e vaccines
have been almost exclusively studied in mice model and we are proposing to logically extend the studies to
generate useful information for development of flu vaccine and prevention of influenza for swine, chicken and
human. We expect to 1) demonstrate the effectiveness of M2e-based vaccine in swine and chicken, where
limited effort has been made to prove the efficacy of the M2e-based vaccine, using the new norovirus P particle
platform; 2) determine the effect of M2e copy number, insertion of immune stimulatory molecule, and
adjuvants in enhancing the immunogenicity and immune response while minimizing the vaccine dose; 3)
enhance the cross-protective immunity through synergistic effect of M2e and HA-based vaccines. Since M2e-
and HA-based immune mechanism is rather distinct, we expect the effect will be complimentary and provide
broadened immune response. Thus our study will provide ample information toward the development of a
universal vaccine against one of the most important diseases of humans and many animals, influenza.
Project Methods
1) Optimization of M2e-P particle vaccines for higher efficacy: We will continue to improve the M2e-P particle
vaccines for better immunogenicity and protective efficacy through increasing the copy number of M2e epitope
per P particle by inserting repeated M2e peptide sequence to all three surface loops and the N- and C-terminus.
We will also construct M2e-P particle expressing immune enhancement epitopes to induce T cell-based
immunity. Different formulations (powder form) and adjuvants for intranasal immunization will also be tested.
2) Further enhancement of M2e-P particle vaccines in swine and chickens and combining the administration of
the M2e vaccine with prototype vaccines which target conserved HA regions: Initially, we will test the protective
efficacy by generation of avian- and swine-consensus M2e-P particles, respectively, that match with the
challenge strains. Once the best M2e-P vaccine candidate and optimal regime is determined, we will expand the
study to validate the protective efficacy against 3-4 different challenge strains including the highly pathogenic
H5N1 strains. In addition to M2e-based vaccine approach, two practical strategies will be employed to
incorporate the HA-based vaccine into the M2e-based vaccine to maximize the cross-protective efficacy of the
vaccine. First, we will incorporate optimized M2e-P into commercial HA-based vaccines. Second, we will
incorporate conserved HA epitope into P-particle vaccine platform. 3) Delineation of the mechanism of
protective immunity conferred by M2e-P particle vaccines: Although previous studies support that protection
induced by M2e-based vaccine depends largely on anti-M2e antibodies, recent findings also suggested other
mechanisms of protection and the important role played by cell-mediated immunity. Type of protective
immunity may differ among vaccine type and formulation. Based on the results from previous aims, we will
select intranasal vaccine candidates and perform detailed immunological studies to seek additional
understanding of the mechanisms of the protective immunity conferred by the M2e-P particle vaccine. 4)
Development of polyvalent consensus M2e vaccines for a broad protection against a wide range of avian, swine
and human flu viruses: We will take advantage of the multiple surface loops of the P particles and the N- and C-
terminus of the P domain to design a polyvalent M2e vaccine by insertion of degenerated M2e with major
consensus of the M2e epitope for a maximal spectrum of protection against all avian, swine and human flu
viruses. We will perform sequence alignments of additional M2e sequences of representative flu strains from the
GenBank to design degenerated consensus M2e to fulfill our goals. We will test the consensus M2e for cross
reactivity in mice and then for potential cross-species protection in chickens and pigs.
Progress 02/01/13 to 01/31/20

Outputs
Target Audience:Scientists in the field of influenza, medical and animal virology, immunology &
vaccinology, and 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 the training of undergraduate student, Ph.D students, research associates and post-docs.
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. Manuscripts havebeen published
in timely manner and we expect to publish two morepublications in peer reviewed jounrals. In addition, the
results were shared with the scientists wroking at academic, private, state and governmentresearch labs and
also with people working atpoultry and swine industries. What do you plan to do during the next reporting
period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Poultry and pig farmers all know that the flu is nothing to sneeze
at. In combating the flu, researchers have often turned to vaccines. However, they tend to be effective
against only a few particular strains of the virus. Our project was focused on developing universal flu vaccine
which can provide broader protection against new and emerging influenza viruses. The project also used
swine as a model to develop novel influenza vaccines for humans against animal influenza. Overall
achievement: The experimental universal vaccines/vaccine formulations have been primarily tested in mouse
model. To our knowledge, this is the first time the protective efficacy of broadly reactive vaccine (aka
"universal vaccine") has been extensively evaluated in farm animals. Our team has developed several new
candidate vaccine formulations, and evaluated their immune responses and protective efficacy administered
in combination with different adjuvants, delivery systems, and vaccination approaches in mice, chickens and
pigs. Our comparative studies demonstrated clear differences in immune response and efficacy of influenza
vaccines in food animals compared to mice, and justify the need of species-specific design of the vaccines
and vaccination approaches. This study also provided direct evidence that swine can serve as a better animal
model for human flu vaccine development than relying on mice studies. We also confirmed the efficacy of our
candidate vaccines and vaccination regimen against highly pathogenic avian influenza virus infection in high
containment biosecurity level 3 facility. The 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. Construction of recombinant vaccine platform
(Objective 1 linked to other objectives). We developed novel chimeric P particle constructs with different
number of M2e (conserved epitope of influenza virus) copies at different insertion sites, as well as two T cell
epitopes linked with M2e. We also have incorporated two different sizes of HA2 protein (55aa and 130aa),
another conserved epitope of influenza virus with capability to induce neutralizing antibody, in the P particle
representing consensus sequences of both avian and swine influenza viruses. Furthermore, we have
developed a new construct expressing HA2 protein and Arabidopsis thaliana cyanase protein (HA2-AtCYN).
We have demonstrated that these constructs induce strong immune responses in mice, chickens, and pigs.
Immunogenicity and protective efficacy of different vaccine formulations and regimes in chickens (Objective
2, 3, and 4). Chimeric M2e P particle (M2e-PP) is immunogenic in chickens when administered by
subcutaneous route with commercial oil adjuvant. M2e-PP immunization consistently reduced virus shedding
against several low pathogenic avian influenza (LPAI) virus challenges in chickens. Supplementation of
inactivated vaccine with M2e-PP significantly enhanced the efficacy in reducing virus shedding against
heterologous challenges. Mechanistically, we demonstrated that M2e-PP vaccine-induced antibodies are
capable of recognizing the native M2e epitopes exposed on the surface of influenza virus-infected MDCK cells
and on whole virus particles. We also showed through plaque-reduction assay that M2e antibodies in sera
obtained from M2e-PP vaccinated chickens have a role in blocking viral replication. HA2-AtCYN vaccine was
shown to induce various levels of HA2-specific IgA in tears as well as serum IgG, which were associated with
partial protection of chickens against tracheal shedding of a challenge virus. Furthermore, intranasal
administration with a combination of HA2-AtCYN and M2e-PP vaccines resulted in enhanced protection
compared to each vaccine alone. In addition to recombinant subunit vaccine, we also tested the protective
efficacy of NS1-truncated live attenuated influenza vaccine (LAIV) alone or in combination with M2e-PP
against H5N2 and H7N2 challenge viruses in chickens. Both vaccination regimens resulted in significant
reduction of virus shedding independently of the subtype. The results show that both LAIV and M2e-PP can
be good components of a universal influenza vaccine, and LAIV priming and M2e-PP boosting strategy could
be utilized as one of vaccination regimens in the field. We also showed that LAIV can broadly block shedding
and transmission of antigenically distant heterologous HPAI viruses. Immunogenicity and protective efficacy
of different vaccine formulations and regimes in pigs (Objective 2, 3, and 4). We showed a partial 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,
and intranasal M2e-PP with MPLA vaccinated groups, suggesting the presence of specific immunogen
recognition. To improve the vaccine efficacy and delivery of the vaccine through intranasal route, polylactic-
co-glycolic acid (PLGA), which is a biodegradable FDA approved polymer, was used to encapsulate M2e-PP
and highly conserved flu peptides (epitopes).We showed that PLGA nanoparticle vaccine resulted in induction
of peptide specific T cell responses including cytotoxic T cells (CTLs), T-helper, and T-helper/memory
responses in the lungs. The PLGA nanoparticle peptide cocktail vaccine was able to reduce the replication of
the heterologous challenge virus to undetectable levels in lungs. We also used PLGA to encapsulate the
inactivated flu vaccines. The PLGA coated vaccine reduced the clinical disease in pigs challenged with
heterologous strains and induced cross-protective cell-mediated immune response. We further used liposome
nanoparticle-based subunit flu vaccine containing ten encapsulated highly conserved B and T cell epitope
peptides together with monosodium urate (MSU) crystals as an adjuvant and co-administered the vaccine
formulation as an intranasal mist to pigs. Liposome peptide flu vaccine delivered with MSU adjuvant improved
the serum IgG and mucosal IgA antibody response, enhanced the frequency of peptides and virus-specific T-
helper/memory cells and IFN-γ response, improved protection from flu-induced fever and pneumonic
lesions, and reduced the nasal virus shedding and viral load in the lungs. Overall, our flu vaccine studies in
pigs show great promise for using liposome and different adjuvant formulations for intranasal vaccination and
the data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control
flu in humans. Exploiting the elite interferon-inducing Influenza viruses to engineer next-generation live
vaccines. During this project, we realized that live attenuated influenza vaccines (LAIV) has a great potential
to be one of practical options as a universal vaccine in the future. We showed that the effectiveness of our
LAIV vaccine was linked to its ability to induce higher levels of interferons and further demonstrated that the
immunogenicity of live influenza vaccines can be improved by selecting the viral clones with magnified
interferon-inducing capabilities. Our study highlights that interferon-based screening of influenza virus
populations will result in vaccine candidates with elite innate immunostimulatory properties. Our approach
can be utilized to develop next-generation live attenuated vaccines against not only influenza viruses but also
other interferon-sensitive pathogens.

Publications
Type: Journal Articles Status: Published Year Published: 2020 Citation: Ghorbani A, Ngunjiri JM, Lee
CW. Influenza A Virus Subpopulations and Their Implication in Pathogenesis and Vaccine Development.
Annu Rev Anim Biosci. 8:247-267. 2020.
Type: Journal Articles Status: Published Year Published: 2019 Citation: Elaish M, Xia M, Ngunjiri JM,
Ghorbani A, Jang H, Kc M, Abundo MC, Dhakal S, Gourapura R, Jiang X, Lee CW. Protective immunity
against influenza virus challenge by norovirus P particle-M2e and HA2-AtCYN vaccines in chickens.
Vaccine. 8;37(43):6454-6462. 2019.
Type: Journal Articles Status: Published Year Published: 2019 Citation: Ghorbani A, Ngunjiri JM, Xia M,
Elaish M, Jang H, KC M, Abundo MC, Jiang X, Lee CW. Heterosubtypic protection by live attenuated and
chimeric norovirus P-particle-M2e vaccines in chickens. Vaccine. 37(10):1356-1364. 2019.
Type: Journal Articles Status: Published Year Published: 2018 Citation: Dhakal S, Cheng X, Salcido J,
Renu S, Bondra K, Lakshmanappa YS, Misch C, Ghimire S, Feliciano-Ruiz N, Hogshead B, Krakowka S,
Carson K, McDonough J, Lee CW, Renukaradhya GJ. Liposomal nanoparticle-based conserved peptide
influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs. Int
J Nanomedicine. 13:6699-6715. 2018.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Amir
Ghorbani, John M. Ngunjiri, Michael C. Abundo, Kara Taylor, Hana Ji, and Chang-Won Lee. Targeted
selection of elite interferon-inducing viral subpopulations reveals live attenuated influenza vaccines with
enhanced in vivo immunostimulatory activities. 1st Midwest Virology Symposium, October 11-13, 2019,
Columbus, OH, USA.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Amir
Ghorbani, John M. Ngunjiri, Michael C. Abundo, Mohamed Elaish, Hana Ji, Kara Taylor, and Chang-Won
Lee. Improvement of an NS1-truncated live attenuated influenza vaccine by selection of viral
 subpopulations with enhanced interferon inducing capability. The American Association of Avian
Pathologists (AAAP) Annual meeting, August 2-5, 2019, Washington, D.C.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Amir
Ghorbani, John M. Ngunjiri, Michael C. Abundo, Hana Ji, Kara Taylor, Mohamed Elaish, and Chang-Won
Lee. Targeting Viral Subpopulations to Improve the Effectiveness of Live Attenuated Influenza Vaccine.
70th North Central Avian Disease Conference, March 12-14, 2019, Minneapolis, MN.
Type: Other Status: Published Year Published: 2017 Citation: Lee CW. Global health is for the birds:
Studying avian diseases to protect human health. Scientia. 113:132-135. 2017.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Amir
Ghorbani, John M. Ngunjiri, Kara Taylor, Michael C. Abundo, and Chang-Won Lee. Systematic
Development of Next-Generation NS1-Truncated Live Attenuated Influenza Vaccines by Targeting Viral
Subpopulations with Enhanced Interferon-Inducing Capacity. OPTIONS X for the Control of Influenza,
August 28 - September 1, 2019, Singapore.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: John M.
Ngunjiri, Michael C. Abundo, Kara Taylor, Hana Ji, Amir Ghorbani, Mahesh KC, and Chang-Won Lee.
Optimized NS1-Truncated Live Vaccines Provide Robust Protection from Highly Pathogenic Avian
Influenza Virus Infection. OPTIONS X for the Control of Influenza, August 28 - September 1, 2019,
Singapore.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Amir
Ghorbani, John M. Ngunjiri, Michael C. Abundo, Kara Taylor, Hana Ji, Chang-Won Lee. Viral
subpopulation-based selection reveals live attenuated influenza vaccines that induce robust immune
responses. 100th Conference of Research Workers in Animal Diseases, November 2-5, 2019, Chicago,
IL, USA.

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.