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United States Department of Agriculture

Agricultural Research Service

Research Project: H1n1 Influenza a Virus in Swine Supplemental Research Program
2011 Annual Report


1a.Objectives (from AD-416)
1. Identify swine influenza virus strain specific antigenic epitopes to support the development of serological assays for surveillance in swine. 2. Compare pathogenesis and transmissibility of selected isolates of the wild-type novel A/HINI virus from animals (e.g., Canadian, Chilean and Argentinean viruses) and correlate with genetic and antigenic changes. 3. Determine efficacy of conventional inactivated swine influenza vaccines against emerging isolates of pandemic A/H1Nl from animals (e.g., Canadian, Chilean and Argentinean viruses). 4. Evaluate a reverse genetics-derived modified live vaccine in pigs and other susceptible animal hosts against pandemic A/H1N1. 5. Evaluate an adenovirus-vectored influenza virus vaccine in pigs.


1b.Approach (from AD-416)
1. Conduct immunological investigations of influenza A virus components that lead to immune responses against specific epitopes that may enable serological surveillance for the 2009 A/H1N1 in swine and determine whether heterologous immunity against endemic swine influenza viruses interferes with serological surveillance methods. 2. Conduct an animal study to determine the pathogenesis and transmissibility of selected isolates of the wild-type novel A/HINI virus from animals and correlate with genetic and antigenic changes. 3. Conduct an animal study to determine the efficacy of conventional inactivated swine influenza vaccines against emerging isolates of pandemic A/H1Nl from animals. 4. Conduct an animal study utilizing a reverse genetics-derived modified live vaccine in pigs and other susceptible animal hosts against pandemic A/H1N1. 5. Conduct an animal study evaluating an adenovirus-vectored H1N1 influenza virus vaccine in pigs.


3.Progress Report

We developed and tested a novel live attenuated influenza vaccine that is safe and efficacious for use in swine. Vaccination of pigs with the vaccine candidate resulted in sterilizing immunity following intratracheal challenge with the 2009 H1N1 pandemic virus.

This past year we constructed and evaluated the efficacy of a single intranasal vaccination with adenovirus vectors expressing a codon optimized hemagglutinin (HA) from the pandemic virus A/California/04/09 (H1N1) against live virus challenge with the same pandemic strain or an endemic H1N2 swine influenza strain, A/Swine/MN/02011/08 (H1N2). Efficacy was compared with an inactivated vaccine prepared from the A/Swine/MN/02011/08 (H1N2) virus. Pigs challenged with the parent virus used in the vaccines had complete protection as expected. However, pigs vaccinated with a single dose of the adenovirus vectored vaccines also had some cross-protection against challenge with the unrelated delta-cluster A/Swine/MN/02011/08 (H1N2) virus. Pigs vaccinated with the inactivated A/California/04/09 (H1N1) virus and subsequently exposed to live delta-cluster A/Swine/MN/02011/08 (H1N2) virus had enhanced pneumonia as a result of a mismatch between the vaccine strain and the challenge virus. This same effect was not observed when the adenovirus vector vaccine was used.


4.Accomplishments
1. Developed and tested a novel live attenuated influenza vaccine that is safe and efficacious for use in swine. Influenza A virus causes a respiratory disease in swine similar to that in humans and is considered one of the three most important swine respiratory pathogens. We have previously shown the advantages of live attenuated influenza vaccines in pigs. Emergence of the 2009 A/H1N1 pandemic virus in several species around the world highlights the need for development of a vaccine that is effective in multiple animal species. Because the new pandemic strain is a triple reassortant virus, ARS scientists at the National Animal Disease Center, Ames, IA, in collaboration with scientists at the University of Maryland chose a swine-like triple reassortant virus isolate of the H3N2 type, to introduce genetic modifications (predicted to be temperature-sensitive mutations in 2 genes involved with viral replication) with the goal of producing live attenuated influenza vaccines. In the lab, this genetically weakened virus had impaired ability to grow at elevated temperatures that mimic the normal pig, thus indicating attenuation. Then using this virus backbone, we substituted the H3 and N2 genes for an H1 and N1 gene and tested this attenuated virus. The H1N1 vaccine candidate generated using the attenuated virus backbone was confirmed as highly attenuated in mice as indicated by the absence of signs of disease, limited virus growth and minimal tissue damage in the respiratory tract. A single immunization with the attenuated vaccine conferred complete protection against a lethal 2009 pandemic H1N1 infection in mice. More importantly, when tested in pigs we found sterilizing immunity against the 2009 H1N1 pandemic virus. Our studies highlight the safety of the temperature sensitive mutant vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.

2. Demonstrated the time course of lung lesion development and pro-inflammatory cytokine response in pigs with vaccine-associated enhanced respiratory disease (VERD). Influenza A virus causes a respiratory disease in swine similar to that in humans and is considered one of the three most important swine respiratory pathogens. Inactivated vaccines work well when pigs are exposed to influenza viruses represented in the vaccine. However, vaccine efficacy is reduced when pigs are infected with new strains. In fact, vaccinating with an inactivated influenza virus can increase the severity of disease when pigs are exposed to a new strain of influenza virus compared to pigs that are not vaccinated. ARS scientists at the National Animal Disease Center, Ames, IA, have now shown that VERD in pigs vaccinated with a human-origin influenza virus (first introduced into pigs from humans around 2003) is evident as little as 24 hours after challenge with the pandemic human influenza A virus (2009). Pigs that were vaccinated with a mismatched virus strain exhibited greater percentages of pneumonia compared to non-vaccinated pigs, and the microscopic character of the pneumonia was more severe with distinct types of lung damage. Elevated immune factors associated with inflammation and disease were detected in the lungs at all time points tested. Active surveillance and monitoring of the quality of match between vaccine strains and strains infecting swine herds is necessary to prevent vaccine mismatch in the swine population. Future vaccines that stimulate improved immune responses across differing influenza viruses will be important to prevent infection and clinical disease, and reduce the burden of this economically important disease. Since influenza viruses from swine may infect people, controlling influenza in the swine population has important implications to human health as well.

3. Detected cellular immunity to inactivated swine influenza virus vaccine in pigs. Influenza A virus causes a respiratory disease in swine similar to that in humans and is considered one of the three most important swine respiratory pathogens. Inactivated vaccines work well when pigs are exposed to influenza viruses represented in the vaccine. However, vaccine efficacy is reduced when pigs are infected with new strains. In fact, vaccinating with an inactivated influenza virus can increase the severity of disease when pigs are exposed to a new strain of influenza virus compared to pigs that are not vaccinated. The introduction of the 2009 A/H1N1 virus into the U.S. swine herd posed a significant risk in this context to the health of U.S. pigs vaccinated with unrelated endemic swine influenza strains. Using a combination of influenza viruses known to cause vaccine-associated enhanced respiratory disease, ARS scientists at the National Animal Disease Center, Ames, IA, in collaboration with scientists at Iowa State University have now shown a strong cellular immune response against the vaccine virus (an endemic swine influenza strain) and a significant cellular immune response to the antigenically unrelated 2009 A/H1N1 pandemic virus were detected, especially in a subset of T cells thought to be memory T-cells. The immunology method used to measure cellular immune responses to influenza virus was found to detect an immune response against a wider range of viral antigens than what is detected with antibody immune responses. This data provides insights in how to design safer and better cross-protective vaccines against recently emerged strains of influenza A viruses.

4. Production of a novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice. The surface hemagglutinin (HA) protein of the 2009 pandemic H1N1 viruses (pH1N1) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through reassortment and incorporation of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains have undergone substantial antigenic drift. ARS scientists at the National Animal Disease Center, Ames, IA, in collaboration with scientists at the University of Maryland developed of a novel monoclonal antibody that shows high neutralization activity against not only pH1N1, but also against representatives of several swine-lineage H1 influenza viruses. Mice receiving a single intranasal dose of the novel monoclonal antibody were protected against lethal challenge with either pH1N1 or cH1N1 virus. These studies highlight the potential use of this monoclonal antibody as an effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections. Since development of vaccines against a pandemic virus strain usually takes several months, passive antibody immune therapy represents a possible alternative antiviral strategy and is the strategy of choice for the prevention and treatment of other viral diseases in children at high risk of contracting disease. This is the first report demonstrating a monoclonal antibody with broad cross-reaction against multiple (but not all) swine H1 influenza lineages.

5. Evaluated the matrix protein of the novel 2009 A/H1N1 virus as a potential diagnostic target for differentiating pigs exposed to the 2009 A/H1N1 pandemic virus versus contemporary North American swine influenza isolates. Influenza A virus causes a respiratory disease in swine similar to that in humans and is considered one of the three most important swine respiratory pathogens. Following emergence of the 2009 A/H1N1 pandemic virus there was no serum-based test available to determine if pigs had been exposed to the pandemic virus. Although it is known that influenza viruses have high mutation rates and any such diagnostic test might quickly become less effective, an attempt was made to develop such a test against one of the least variable proteins of influenza virus, the matrix (M1) protein. Although the M1 protein is not a structural protein, antibodies to M1 are produced during an influenza infection. ARS scientists at the National Animal Disease Center, Ames, IA, in collaboration with scientists at Iowa State University and the University of Georgia identified antigenic sites unique in the novel 2009 A/H1N1 virus matrix (M1) protein based on available virus sequences and developed a test designed to differentiate exposure to the pandemic H1N1 strains from previously circulating swine influenza strains. Unfortunately, cross-reactivity at varying levels was found with several contemporary swine influenza strains containing the traditional North American matrix gene found in nearly all strains of swine influenza widely circulating in North American prior to the emergence of the 2009 A/H1N1 pandemic virus. These data indicate such a diagnostic approach is not possible as it will always be fraught with varying degrees of cross-reactivity and loss of specificity as the 2009 A/H1N1 pandemic virus continues to mutate over time.

6. Demonstrated efficacy of a single dose intranasal vaccine made using an adenovirus vector containing an optimized hemagglutinin (HA) gene from the 2009 A/H1N1 pandemic virus. Influenza A virus causes a respiratory disease in swine similar to that in humans and is considered one of the three most important swine respiratory pathogens. Rapid deployment of new vaccines against novel emerging strains is critical to control disease caused by influenza viruses. Adenovirus vectors are made from a virus not capable of creating a new infectious virus, but the vectors are able to infect the host and induce strong immunity like a natural infection. ARS scientists at the National Animal Disease Center, Ames, IA, assessed the efficacy of an adenovirus-vectored HA vaccine in pigs against the 2009 A/H1N1 pandemic strain or an unrelated group of swine H1 viruses that originated from humans around 2003. Efficacy was compared with a killed vaccine prepared from the 2009 H1N1 pandemic virus. Pigs challenged with the pandemic virus had complete protection as expected. However, pigs vaccinated with a single dose of the adenovirus vectored vaccine also had some cross-protection against challenge with the unrelated swine H1N1 virus. Surprisingly, following vaccination, none of the pigs vaccinated with the adenovirus vectored vaccines developed detectable serum antibody titers against the virus in the vaccine. Pigs vaccinated with the killed 2009 pandemic virus and subsequently exposed to the swine H1N2 virus had enhanced pneumonia as a result of a mismatch between the vaccine strain and the challenge virus. This same enhanced disease effect was not observed when the adenovirus vector vaccine was used. Results of this study indicate that circulating antibodies may not be required for protective immunity against influenza virus and that mucosal antibodies or cell-mediated immunity may be what is necessary and sufficient for protective immunity.


Review Publications
Pena, L., Vincent, A.L., Ye, J., Ciacci-Zanella, J.R., Angel, M., Lorusso, A., Gauger, P.C., Janke, B.H., Loving, C.L., Perez, D.R. 2011. Modifications in the polymerase genes of a swine-like triple reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 Viruses. Journal of Virology. 85(1):456-469.

Platt, R., Vincent, A.L., Gauger, P.C., Loving, C.L., Zanella, E.L., Lager, K.M., Kehrli, Jr., M.E., Kimura, K., Roth, J.A. 2011. Comparison of humoral and cellular immune responses to inactivated swine influenza virus vaccine in weaned pigs. Veterinary Immunology and Immunopathology. 142(3-4):252-257.

Pena, L., Vincent, A.L., Ye, J., Ciacci-Zanella, J.E., Angel, M., Lorusso, A., Gauger, P.C., Janke, B.H., Loving, C.L., Perez, D.R. 2011. Safety and efficacy of a novel live attenuated influenza vaccine against pandemic H1N1 in swine. Influenza and Other Respiratory Viruses. 5(S1):341-344.

Shao, H., Ye, J., Vincent, A.L., Edworthy, N., Ferrero, A., Qin, A., Perez, D. 2011. A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice. Virology. 417(2):379-384.

Last Modified: 11/25/2014
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