Progress 02/01/07 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1. Characterize mucosal immunity induced by natural infection and vaccination with both high and low pathogenicity AIV to identify innate and adaptive immune indicators of protection. 2. Characterize the cellular and humoral immune responses to mucosal vaccination, and develop improved methods for identifying cells, cytokines and antibody from mucosal sites. 3. Assess immune variability of the host, both in different poultry species and lines, and non-poultry avian species, by examining the frequency of genetic polymorphism in host genes related to innate and adaptive immunity (toll-like receptors, cytokines, chemokines). 4. Using reverse genetics approaches for avian influenza and Newcastle Disease Virus, examine the role of individual viral genes in host gene response. Approach (from AD-416) New vaccine approaches for controlling avian influenza virus (AIV) in poultry will be developed following application of antigen via different routes of exposure to the mucosal system. Characterization of the mucosal immune response following vaccination and challenge will be used to extend the understating of the role of local immunity against AIV. Novel mucosal vaccines will be developed and tested with and without adjuvants to enhance immunological response to vaccination. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds followed by challenge. The role of cell mediated immunity against avian influenza will be determined following adaptive transfer of isolated lymphocyte fractions from birds previously exposed to live avian influenza and provided to na�ve birds prior to challenge. The pathogenomic basis of protection will be delineated by genomic characterization through sequence analysis of immune regulatory factors, including cytokines and toll-like receptors, from immunologically competent and na�ve birds. Reverse genetics will be used for vaccine development and pathogenesis studies with AIV by replacement or inactivation of genes involved in virulence and evasion of the host immune response. A comparison of pathogenesis from recombinant viruses will allow characterization of proteins and motifs involved with establishment of viral infection on mucosal surfaces which may be targets for vaccine development. The Mucosal Immunology project has been active both nationally and internationally to meet the objectives and milestones of the project. Accomplishments included: 1) the development of new techniques that allow for more thorough evaluation of avian influenza vaccines, 2) the protection of poultry from avian influenza infection through application of anti-viral molecules, and 3) the study of host markers for increased innate resistance to avian influenza viruses. Collaborative research continues with national and international partners to continue to study the immune response of poultry to avian influenza. University partners include, but not limited to, the University of Georgia, the University of Delaware, the University of Arkansas, and the Ohio State University. Collaborative work with industry has included projects with CEVA Biomune, and Goldsboro Milling Company. Internationally, collaboration with the U. S. Department of Agriculture, Office of International Research Programs and the All Russian Research Institute for Animal Health has continued to support the development of mucosal vaccines and immunology against avian influenza. Accomplishments 01 Reduction of pandemic H1N1 avian influenza growth with use of chicken interferon. Interferons represent one of the first lines of immune defense against influenza virus infection. The protective potential of chicken interferon-alpha was applied to chicken, duck, and turkey cell cultures prior to infection with the pandemic H1N1 virus. Results demonstrate that chicken interferon significantly reduced virus replication in chicken, turkey, and duck cells. These studies demonstra that chicken interferon is biologically active against the pandemic H1N1 virus, is active in other avian species, and may be useful as therapy against avian influenza infection. This information is critical for avi influenza control programs by offering an alternative to vaccine usage a can be applied to provide immediate protection. 02 Vaccine protection of chickens against Indonesian H5N1 highly pathogenic avian influenza (HPAI). In Indonesia, ongoing outbreaks of HPAI strains remain a threat to poultry and human health. Current vaccines and vaccination strategies are currently being developed to protect birds an decrease transmission of these viruses. The objective of this study is evaluate the efficacy of recombinant avian influenza vaccines (H5 subtyp against challenge with a recent Indonesian H5N1 isolate. Following challenge with a lethal dose of H5N1 HPAI, most all birds receiving the recombinant avian influenza (AI) vaccine survived. These results indica this type of recombinant vaccine can be used as an aid during AI eradication efforts in Indonesia. This information will be used by commercial poultry growers in the field as a tool to prevent avian influenza H5N1 in Southeast Asia. 03 Pathogenicity of recent Egyptian H5N1 HPAI viruses in domestic ducks demonstrates disease with high mortality. Domestic ducks have been implicated in the dissemination of H5N1 HPAI viruses. This increase in pathogenicity in ducks observed with H5N1 HPAI viruses has implications for the control of the disease since vaccinated ducks infected with high virulent strains shed more virus and for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds. This information will be used by commercial poultry growers to develop vaccine programs in ducks that wil minimize the risk of transmission and disease which will result in substantial money savings.
Impacts
(N/A)
Publications
- Spackman, E., Gelb, J., Preskenis, L., Ladman, B., Pope, C., Pantin Jackwood, M.J., Mckinley, E.T. 2010. The pathogenesis of low pathogenicity H7 avian influenza viruses in chickens, ducks and turkeys. Virology Journal. 7:331.
- Ewald, S.J., Kapczynski, D.R., Livant, E.J., Suarez, D.L., Ralph, J., Mcleod, S., Miller, C. 2011. Association of Mx1 Asn 631 variant alleles with enhanced resistance and altered cytokine response in chickens infected with a highly pathogenic avian influenza virus. Immunogenetics. 63(6):363-375.
- Kapczynski, D.R., Liljebjelke, K.A., Kulkarni, G., Hunt, H.D., Jiang, H., Petkov, D. 2011. Cross reactive cellular immune responses in chickens previously exposed to low pathogenic avian influenza. Biomed Central (BMC) Genomics. 5(4):S13.
- Belisle, S.E., Tisonciki, J.R., Korth, M.J., Carter, V.S., Proll, S.C., Swayne, D.E., Pantin Jackwood, M.J., Tumpey, T.M., Katze, M.G. 2010. Genomic profiling of TNT-alpha receptor and IL1 receptor knockout mice reveals a link between the TNF-alpha signaling and increased severity of 1918 pandemic influenza virus infection. Journal of Virology. 84(24):12576- 12588.
|
Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) 1. Characterize mucosal immunity induced by natural infection and vaccination with both high and low pathogenicity AIV to identify innate and adaptive immune indicators of protection. 2. Characterize the cellular and humoral immune responses to mucosal vaccination, and develop improved methods for identifying cells, cytokines and antibody from mucosal sites. 3. Assess immune variability of the host, both in different poultry species and lines, and non-poultry avian species, by examining the frequency of genetic polymorphism in host genes related to innate and adaptive immunity (toll-like receptors, cytokines, chemokines). 4. Using reverse genetics approaches for avian influenza and Newcastle Disease Virus, examine the role of individual viral genes in host gene response. Approach (from AD-416) New vaccine approaches for controlling avian influenza virus (AIV) in poultry will be developed following application of antigen via different routes of exposure to the mucosal system. Characterization of the mucosal immune response following vaccination and challenge will be used to extend the understating of the role of local immunity against AIV. Novel mucosal vaccines will be developed and tested with and without adjuvants to enhance immunological response to vaccination. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds followed by challenge. The role of cell mediated immunity against avian influenza will be determined following adaptive transfer of isolated lymphocyte fractions from birds previously exposed to live avian influenza and provided to na�ve birds prior to challenge. The pathogenomic basis of protection will be delineated by genomic characterization through sequence analysis of immune regulatory factors, including cytokines and toll-like receptors, from immunologically competent and na�ve birds. Reverse genetics will be used for vaccine development and pathogenesis studies with AIV by replacement or inactivation of genes involved in virulence and evasion of the host immune response. A comparison of pathogenesis from recombinant viruses will allow characterization of proteins and motifs involved with establishment of viral infection on mucosal surfaces which may be targets for vaccine development. The Mucosal Immunology project has been active both nationally and internationally to meet the objectives and milestones of the research project. Major progress has been achieved in all four objectives. Progress includes: 1) the development of mucosal vaccines to protect poultry against avian influenza viruses, 2) the study of cell mediated immunity induced following infection with avian influenza of poultry, 3) the study of host markers for increased innate resistance to avian influenza viruses, and 4) the use of reverse genetics technology to study pathogenesis of avian influenza viruses in poultry and waterfowl. Collaborative research continues with national and international partners to continue to study the immune response of poultry to avian influenza. University partners include, but not limited to, the University of Georgia, the University of Delaware, the University of Arkansas, and the Ohio State University. Collaborative work with industry has included projects with CEVA Biomune, and Goldsboro Milling Company. Internationally, collaboration with the US Department of Agriculture, Office of International Research Programs and the All Russian Research Institute for Animal Health has continued to support the development of mucosal vaccines and immunology against avian influenza. Accomplishments 01 REACTIVE CYTOTOXIC T LYMPHOCYTES AGAINST HOMOLOGOUS AND HETEROLOGOUS AVI INFLUENZA SUBTYPES. Numerous reports have implicated a role of genetic resistance to bacterial infection and viral diseases. However, little i known about the role of genetics of chickens in generating protective immunity following avian influenza (AI) infection. In these studies, genetically-defined chickens were infected with a recent H9N2 AI isolate and thymus (T)-derived lymphocytes were analyzed for cross reactivity against different AI viruses. Results indicate cells isolated from H9N2 infected chickens displayed lysis of lung cells infected with many AI isolates. Removal of a specific portion of these cells, the CD8+ population, removed the specific immunological cross reactivity. Taken together, these studies provide insight into the cross reactive nature o avian T lymphocytes against AI viruses. 02 EFFICACY OF RECOMBINANT HERPESVIRUS-OF-TURKEYS VACCINE AGAINST MEXICAN- LINEAGE AVIAN INFLUENZA H5N2. In Mexico, outbreaks due to low pathogenic (LP) avian influenza virus (AIV) H5N2 strains started in 1993-1994 and several highly pathogenic (HP) strains emerged in 1994-1995. Although t HP strains were contained and have not been reported since 1996, LP strains remain endemic in Mexico despite an extensive vaccination progra The objective of this study is to evaluate the efficacy of turkey herpesvirus (HVT) vectored AIV vaccines (H5 subtype) against challenge with Mexican lineage H5N2 AIV strains. The avian influenza (AI) HA gene from two different H5 AIV was cloned into a HVT vaccine. Following challenge with a lethal dose of H5N2 HPAI, most all birds receiving eith of the HVT-AI vaccine survived. These results indicate this type of recombinant vaccine can be used as an aid during AI eradication efforts. 03 DEVELOPMENT OF ENZYME LINKED IMMUNOSPOT ASSAY (ELISPOT) TO DETECT AVIAN INFLUENZA SPECIFIC ANTIBODY-SECRETING B CELLS IN CHICKENS. Vaccines rema a cost-effective means to protect animals from infectious disease by establishing immunity following application. To evaluate next generation vaccines, new methodologies are needed to expand analysis of antibody producing cells in the host after vaccination. A novel ELISPOT method wa developed which allows detection and enumeration of antibody producing cells from chickens against avian influenza (AI). Inactivated and label AI virus was incubated with lymphocytes from AI-vaccinated chickens and the number of cells producing antibodies against AI determined. With thi method one can identify and enumerate both the total number of antibody secreting cells and those secreting antibodies to AI in a highly sensiti manner and at the cellular level. In this way, AI vaccines and adjuvant inducing higher numbers of antigen-specific antibody secreting cells may be developed and compared to currently available vaccines. 04 DIFFERENTIAL GROWTH CHARACTERISTICS OF AVIAN INFLUENZA VIRUSES IN PRIMAR CELL CULTURE. Low pathogenic avian influenza (LPAI) viruses cause varyi pathogenicities when inoculated into chickens. Infection with some LPAI isolates results in no overt signs of clinical disease, while others cau respiratory distress, weight loss and diarrhea. In these studies the growth characteristics of four different LPAI isolates was compared in primary chicken cell cultures. Results indicate two H5 isolates grew poorly in chicken embryo liver (CEL) cells, compared to either a H7N2 or H9N2 LPAI isolate. In contrast, all four LPAI viruses grew to higher titers in chicken embryo kidney (CEK) cells. The presence of trypsin in the growth media increased the titers of the LPAI viruses in the CEL cel but had no effect on titer in the CEK cells. Taken together these results shed insight into the varying growth characteristics displayed b LPAI viruses of differing pathogenicities. 05 INTRANASAL ADMINISTRATION OF ALPHA INTREFERON REDUCES MORBIDITY ASSOCIAT WITH LOW PATHOGENIC AVIAN INFLUENZA INFECTION. Type I interferons, including interferon alpha (IFN-alpha), are expressed rapidly after vira infection, and represent a first line of defense against avian influenza Following infection of chickens with avian influenza virus (AIV), transcription of IFN-alpha is quickly up regulated along with a myriad o other immune-related genes. In these studies, we assessed the protectiv potential of IFN-alpha applied to birds prior to exposure to low pathogenic AIV. Intranasal application with IFN-alpha prior to and durin active AIV infection reduced clinical signs of disease, including weight loss and fever, compared to phosphate-buffered saline (PBS) treated controls. In addition, the incidence of viral shedding and viral titers from oral swabs was significantly reduced in IFN-alpha treated birds. Taken together, these studies show that IFN-alpha can protect chickens from disease associated with low pathogenic AIV and reduce the risk of transmission through decreased shedding. 06 ADAPTIVE TRANSFER OF LYMPHOCYTES FROM AVIAN INFLUENZA INFECTED CHICKENS PROTECTS FROM OVERT CLINICAL SIGNS OF DISEASE FOLLOWING INFECTION WITH H9N2 LOW PATHOGENIC AVIAN INFLUENZA. Immunity against avian influenza (A is largely based on the induction of neutralizing antibodies produced against the hemagglutinin, although host lymphocytes have been reported critical for clearance of virus from infected cells. In these studies, chickens were infected with a recent H9N2 AI isolate, and lymphocytes fr those birds adaptively transferred to immunologically na�ve birds. The birds receiving the lymphocytes produced against AI were then challenged with the H9N2 virus. Results indicate lymphocytes from infected birds could protect na�ve birds from overt clinical signs of disease. In contrast, control birds had significant decreases in body weight and higher body temperatures following H9N2 infection. These studies demonstrate the protective nature of cell mediated immunity of chickens against AI viruses. 07 STUDYING THE EFFECT OF NS1 GENE EXCHANGE ON THE PATHOGENICITY OF H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES IN DUCKS. Until 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses caused only mild respiratory infections in ducks. Since then, new viruses have emerged th cause clinical disease and high mortality in ducks and other waterfowl. However, there is no clear explanation of why the pathogenicity of some H5N1 HPAI viruses has increased. The NS1 influenza virus protein is know to suppress immune responses in virus-infected hosts, consequently affecting the virus pathogenicity. In order to determine if the NS1 protein contributes to the increased virulence in ducks, single gene reassortant viruses were generated. The NS1 gene from a virus that produces mild disease in ducks and from a very virulent virus for ducks were exchanged for the NS1 of a moderately pathogenic virus in ducks. Exchanging the NS1 gene had minimal effect on the pathogenicity of the virus, and suggests that other viral genes, or combination of genes, are most likely contributing to the increased virulence of H5N1 HPAI viruses in ducks. 08 GENETIC AND AMINOACID COMPARISONS OF PANDEMIC H1N1 TO U.S. H1N1 AVIAN INFLUENZA VACCINE ISOLATES. In 2009, a pandemic influenza A H1N1 (pH1N1 virus was isolated in swine in Canada in June, and later in turkey breeders in Chile, Canada, and the U.S. The pH1N1 virus consists of gen segments of avian, human and swine influenza origin and raises the potential for infection in poultry following exposure to infected humans or swine. In these studies, the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to U.S. H1N1 AI isolates used as inactivate vaccines in commercial turkeys was determined. Genetic analysis indicat U.S. H1N1 AI vaccine isolates contained between 76- 92 % nucleotide sequence similarity to the pH1N1 virus. However, comparison of amino acids found at antigenic sites of the hemagglutinin (HA) protein indicat major differences were found between pH1N1 and the U.S. H1N1 vaccine isolates. Taken together these results suggest limited cross reactivity between U.S. H1N1 vaccines and the pH1N1 virus. Current vaccines used i turkey breeders against circulating H1N1 viruses should be updated and tested to ensure adequate protection for field exposure.
Impacts
(N/A)
Publications
- Sylte, M.J., Suarez, D.L. 2009. Influenza neuraminidase as a vaccine antigen. In: Compans, R.W., Orenstein, W.A., editors. Vaccines for Pandemic Influenza. New York, NY: Springer. p. 227-242.
- Kapczynski, D.R., Swayne, D.E. 2009. Influenza vaccines for avian species. In: Compans, R.W., Orenstein, W.A., editors. Vaccines for Pandemic Influenza, Current Topics in Microbiology and Immunology. Berlin: Springer- Verlag. p. 133-152.
- Layton, S.L., Kapczynski, D.R., Cox, M.M., Higgins, S., Higgins, J., Wolfenden, A.D., Liljebjelke, K.A., Bottje, W.G., Swayne, D.E., Berghman, L.R., Kwon, Y.M., Hargis, B.M., Cole, K. 2009. Vaccination of chickens with recombinant salmonella expressing the M2e and CD154 increase protection and decrease viral shedding following low pathogenic avian influenza challenge. Poultry Science. 88(11):2244-2252.
- Petkov, D., Linnemann, E., Kapczynski, D.R., Sellers, H. 2007. Full-length sequence analysis of four IBDV strains with different pathogenicities. Virus Genes. 34(3):315-326.
- Petkov, D.I., Linneman, E.G., Kapczynski, D.R., Sellers, H.S. 2009. Identification and characterization of two distinct bursal B-cell subpopulations following infectious bursal disease virus infection of White Leghorn chickens. Avian Diseases. 53(3):347-355.
- Liljebjelke, K.A., Petkov, D., Kapczynski, D.R. 2010. Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza. Vaccine. 28(27):4430-4437.
- Avellaneda, G.E., Mundt, E., Lee, C., Jadhao, S., Suarez, D.L. 2010. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus. Avian Diseases. 54:278-286.
- Sarmento, L., Wasilenko, J.L., Pantin Jackwood, M.J. 2010. The effects of NS gene exchange on the pathogenicity of H5N1 HPAI viruses in ducks. Avian Diseases. 54:532-537.
- Suarez, D.L. 2010. Avian Influenza: Our current understanding. Animal Health Research Reviews. 11(1):19-33.
- Jadhao, S.J., Lee, C., Sylte, M.J., Suarez, D.L. 2009. Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza in chickens. Vaccine. 27:6247-6260.
|
Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) 1. Characterize mucosal immunity induced by natural infection and vaccination with both high and low pathogenicity AIV to identify innate and adaptive immune indicators of protection. 2. Characterize the cellular and humoral immune responses to mucosal vaccination, and develop improved methods for identifying cells, cytokines and antibody from mucosal sites. 3. Assess immune variability of the host, both in different poultry species and lines, and non-poultry avian species, by examining the frequency of genetic polymorphism in host genes related to innate and adaptive immunity (toll-like receptors, cytokines, chemokines). 4. Using reverse genetics approaches for avian influenza and Newcastle Disease Virus, examine the role of individual viral genes in host gene response. Approach (from AD-416) New vaccine approaches for controlling avian influenza virus (AIV) in poultry will be developed following application of antigen via different routes of exposure to the mucosal system. Characterization of the mucosal immune response following vaccination and challenge will be used to extend the understating of the role of local immunity against AIV. Novel mucosal vaccines will be developed and tested with and without adjuvants to enhance immunological response to vaccination. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds followed by challenge. The role of cell mediated immunity against avian influenza will be determined following adaptive transfer of isolated lymphocyte fractions from birds previously exposed to live avian influenza and provided to na�ve birds prior to challenge. The pathogenomic basis of protection will be delineated by genomic characterization through sequence analysis of immune regulatory factors, including cytokines and toll-like receptors, from immunologically competent and na�ve birds. Reverse genetics will be used for vaccine development and pathogenesis studies with AIV by replacement or inactivation of genes involved in virulence and evasion of the host immune response. A comparison of pathogenesis from recombinant viruses will allow characterization of proteins and motifs involved with establishment of viral infection on mucosal surfaces which may be targets for vaccine development. Significant Activities that Support Special Target Populations The Mucosal Immunology project has been active both nationally and internationally to meet the objectives and milestones of the research project. Major accomplishments have been achieved in all four objectives. Accomplishments included: 1) the development of mucosal vaccines to protect poultry against avian influenza viruses; 2) the construction of novel recombinant vaccine technologies for control of avian influenza in poultry; 3) the use of reverse genetics technology to study pathogenesis of avian influenza viruses in poultry and waterfowl; and 4) the study of genomic markers for increased innate resistance to avian influenza viruses. Collaborative research continues with national and international partners to continue to study the immune response of poultry to avian influenza. University partners include, but not limited to, the University of Georgia, the University of Delaware, the University of Arkansas, and the Ohio State University. Collaborative work with industry has included projects with CEVA Biomune, and Goldsboro Milling Company. Internationally, collaboration with the U.S. Department of Agriculture, Office of International Research Programs and the All Russian Research Institute for Animal Health has continued to support the development of mucosal vaccines and immunology against avian influenza.
Impacts
(N/A)
Publications
- Swayne, D.E., Kapczynski, D.R. 2008. Strategies and challenges for eliciting immunity against avian influenza virus in birds. Immunological Reviews. 225:314-331.
- Swayne, D.E. 2009. Avian influenza vaccines and therapies for poultry. Comparative Immunology Microbiology and Infectious Diseases. 32:351-363.
- Kapczynski, D.R., Gonder, E., Liljebjelke, K.A., Lippert, R., Petkov, D., Tilley, B. 2009. Vaccine induced protection from egg production losses in commercial turkey breeder hens following experimental challenge with a triple reassortant H3N2 avian influenza virus. Avian Diseases. 53:7-15.
- Sarmento, L., Afonso, C.L., Estevez, C., Wasilenko, J.L., Pantin Jackwood, M.J. 2008. Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses. Veterinary Immunology and Immunopathology. 125:291-302.
- Lipatov, A.S., Kwon, Y., Pantin Jackwood, M.J., Swayne, D.E. 2009. Pathogenesis of H5N1 influenza virus infections in mice and ferret models differs according to respiratory tract or digestive tract system exposure. Journal of Infectious Diseases. 199:717-725.
- Wasilenko, J.L., Sarmento, L., Pantin Jackwood, M.J. 2009. A single substitution in amino acid 184 of the NP protein alters the replication and pathogenicity of H5N1 avian influenza viruses in chickens. Archives of Virology. 154:969-979.
- Pillai, S.S., Pantin Jackwood, M.J., Jadhao, S.J., Suarez, D.L., Wang, L., Yassine, Y., Saif, Y., Lee, C. 2009. Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys. Vaccine. 27:819-824.
- Pantin Jackwood, M.J., Swayne, D.E. 2009. Pathogenesis and pathobiology of avian influenza virus infection in birds. OIE Scientific and Technical Review. 28(1):113-136.
|
Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) 1. Characterize mucosal immunity induced by natural infection and vaccination with both high and low pathogenicity AIV to identify innate and adaptive immune indicators of protection. 2. Characterize the cellular and humoral immune responses to mucosal vaccination, and develop improved methods for identifying cells, cytokines and antibody from mucosal sites. 3. Assess immune variability of the host, both in different poultry species and lines, and non-poultry avian species, by examining the frequency of genetic polymorphism in host genes related to innate and adaptive immunity (toll-like receptors, cytokines, chemokines). 4. Using reverse genetics approaches for avian influenza and Newcastle Disease Virus, examine the role of individual viral genes in host gene response. Approach (from AD-416) New vaccine approaches for controlling avian influenza virus (AIV) in poultry will be developed following application of antigen via different routes of exposure to the mucosal system. Characterization of the mucosal immune response following vaccination and challenge will be used to extend the understating of the role of local immunity against AIV. Novel mucosal vaccines will be developed and tested with and without adjuvants to enhance immunological response to vaccination. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds followed by challenge. The role of cell mediated immunity against avian influenza will be determined following adaptive transfer of isolated lymphocyte fractions from birds previously exposed to live avian influenza and provided to na�ve birds prior to challenge. The pathogenomic basis of protection will be delineated by genomic characterization through sequence analysis of immune regulatory factors, including cytokines and toll-like receptors, from immunologically competent and na�ve birds. Reverse genetics will be used for vaccine development and pathogenesis studies with AIV by replacement or inactivation of genes involved in virulence and evasion of the host immune response. A comparison of pathogenesis from recombinant viruses will allow characterization of proteins and motifs involved with establishment of viral infection on mucosal surfaces which may be targets for vaccine development. Significant Activities that Support Special Target Populations This project falls within the 2005-2010 Animal Health National Program (NP103) Action Plan, Component 1 - Bio-defense research (Foreign and Emerging Animal Diseases) and Component 4 - Countermeasures to prevent and control respiratory diseases. More specifically the research addresses Agency Performance Measure 3.2.1 (Provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health) and 3.2.3 (Develop and transfer tools to the agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health). The research generated during FY2008 addressed each of the four objectives above. In objective one, Scientists performed multiple experiments aimed at comparing the route of delivery of inactivated vaccines and examined protective efficacy following challenge. Overall most of the mucosal vaccines tested induced an immune response, however only a few were able to confer any protection against highly pathogenic avian influenza (AI) challenge. We also examined the influence of adjuvants in the formulation of mucosal AI vaccines and demonstrated that a number of them were able to enhance the immune response. Both of the milestones associated with this objective were fully met. Milestones for objective two were fully met, as numerous bird studies were completed in chickens and ducks that examined the cytokine responses in lung, spleen, bursa or Peyers patch with real-time reverse transcriptase-polymerase chain reaction (RRT-PCR). Overall these results are extending our knowledge of how different bird species recognize and respond to AI. In objective three, we have completed some sequencing of the Mx and TLR7 genes from bird species. Our findings to date indicate a single polymorphism in the Mx protein can extend the mean death time in commercial chickens. Although the birds are still susceptible to high pathogenicity avian influenza (HPAI), the observations that a single change in one protein can have such a dramatic effect in the birds is promising. We plan to continue the work in this area to examine polymorphisms in other avian immune response genes. In objective four, we performed comparisons with three different microarray platforms with tissues taken from chickens infected with HPAI H5N1. The results suggest an unregulated proinflammatory response, coupled with a delayed interferon response may play a role in the inability of the birds to overcome infection. Future studies are planned in subsequent years. Technology Transfer Number of New/Active MTAs(providing only): 1
Impacts
(N/A)
Publications
- Toro, H., Tang, D.C., Suarez, D.L., Sylte, M.J., Pfeiffer, J., Van Kampen, K.R. 2007. Protective avian influenza in ovo vaccination with non- replicating human adenovirus vector. Vaccine. 25:2886-2891.
- Swayne, D.E., Avellaneda, G.E., Mickle, T.R., Pritchard, N., Cruz, J., Bublot, M. 2007. Improvements to the hemagglutination inhibition test for serological assessment of recombinant Fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals. Avian Diseases. 51:697-704.
- Pantin Jackwood, M.J. 2008. Immunohistochemical staining of avian influenza viruses in tissues. In: Spackman, E., editor. Avian Influenza Virus. Methods in Molecular Biology. Humana Press, Totowa, NJ. p. 77-83.
- Kapczynski, D.R. 2008. Evaluating the cell mediated immune response of avian species to avian influenza viruses. In: Spackman, E., editor. Avian Influenza Virus. Totowa, NJ: Humana press, Inc. p. 108-121.
- Kapczynski, D.R., Kogut, M.H. 2008. Measurement of avian cytokines with real time RT-PCR following infection with avian influenza. In: Spackman, E. , editor. Avian Influenza Virus. Totowa, NJ: Humana Press, Inc. p. 122-129.
- Toro, H., Tang, D.C., Suarez, D.L., Shi, Z. 2008. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine. Vaccine. 26:2640-2646.
- Goetz, S., Spackman, E., Hayhow, C., Swayne, D.E. 2008. Assessment of reduced vaccine dose on efficacy of an inactivated avian influenza vaccine against an H5N1 high pathogenicity avian influenza virus. Journal of Applied Poultry Research. 17:145-150.
- Joseph, T., Mcauliffe, J., Lu, B., Vogel, L., Swayne, D.E., Jin, H., Kemble, G., Subbarao, K. 2008. A live attenuated cold adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets. Virology. 378(1):123-132.
- Steel, J., Burmakina, V., Thomas, C., Spackman, E., Garcia-Sastre, A., Swayne, D.E., Palese, P. 2008. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus. Vaccine. 26:522-531.
- Swayne, D.E., Kapczynski, D.R. 2008. Vaccines, vaccination and immunology for avian influenza viruses in poultry. In: Swayne, D.E. editor. Avian Influenza. Ames, Iowa: Blackwell Publishing. p. 407-451.
|
Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) 1. Characterize mucosal immunity induced by natural infection and vaccination with both high and low pathogenicity AIV to identify innate and adaptive immune indicators of protection. 2. Characterize the cellular and humoral immune responses to mucosal vaccination, and develop improved methods for identifying cells, cytokines and antibody from mucosal sites. 3. Assess immune variability of the host, both in different poultry species and lines, and non-poultry avian species, by examining the frequency of genetic polymorphism in host genes related to innate and adaptive immunity (toll-like receptors, cytokines, chemokines). 4. Using reverse genetics approaches for avian influenza and Newcastle Disease Virus, examine the role of individual viral genes in host gene response. Approach (from AD-416) New vaccine approaches for controlling avian influenza virus (AIV) in poultry will be developed following application of antigen via different routes of exposure to the mucosal system. Characterization of the mucosal immune response following vaccination and challenge will be used to extend the understating of the role of local immunity against AIV. Novel mucosal vaccines will be developed and tested with and without adjuvants to enhance immunological response to vaccination. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds followed by challenge. The role of cell mediated immunity against avian influenza will be determined following adaptive transfer of isolated lymphocyte fractions from birds previously exposed to live avian influenza and provided to na�ve birds prior to challenge. The pathogenomic basis of protection will be delineated by genomic characterization through sequence analysis of immune regulatory factors, including cytokines and toll-like receptors, from immunologically competent and na�ve birds. Reverse genetics will be used for vaccine development and pathogenesis studies with AIV by replacement or inactivation of genes involved in virulence and evasion of the host immune response. A comparison of pathogenesis from recombinant viruses will allow characterization of proteins and motifs involved with establishment of viral infection on mucosal surfaces which may be targets for vaccine development. Accomplishments UNDERSTANDING THE INTERACTIONS BETWEEN AVIAN INFLUENZA AND HOST IMMUNE RESPONSE. The interferon response in chickens plays an important role in the control of avian influenza virus. The better understanding of the immune response of chickens to avian influenza virus is important to help us better target vaccines for control of the disease. Two variants of influenza, one that had a full length NS1 protein and the other with only a partial length NS1 protein, were compared and it was demonstrated that the virus with a short NS1 protein induced higher levels of interferon that resulted in poor growth of the virus in chickens. The NS1 protein had previously been shown to be an important interferon blocker in mammals, but this study showed it has the same role in chickens and expanded our understanding of the pathogenesis of the virus. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. DEVELOPMENT OF A BACTERIAL VACCINE EXPRESSING THE HEMAGGLUTININ GENE OF AVIAN INFLUENZA. A bacterial vaccine expressing the hemagglutinin gene of avian influenza was developed for protection of chickens. Vaccines can be a valuable tool for the control of avian influenza, but current vaccines are costly and difficult to administer. We developed a recombinant bacterial vector expressing the HA gene of H5N1 AI which can be delivered to mucosal surfaces in poultry, providing the potential for a mass administered vaccine. Birds orally immunized with the bacterial vaccine exhibited increased antibody response to the HA protein of AI, and following experimental challenge of vaccinated birds, complete protection from clinical disease was observed. The bacterial expression system to make vaccines for avian influenza can potentially be mass administered, be compatible as a DIVA (differentiate infected from vaccinated animals) approach, and be produced at a reasonable cost. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. COMPARISON OF THE INNATE IMMUNE RESPONSE TO H5N1 AVIAN INFLUENZA IN CHICKENS AND DUCKS. Comparison of the innate immune response in chickens and ducks to H5N1 avian influenza show a markedly different response between species. The innate immune response is responsible for detecting invading microorganisms during the initial stages of infection, which is a crucial determinant of disease resistance or susceptibility. These studies were designed to examine the role of the innate immune response in protection from disease by measuring cytokine expression immediately following infection. The results indicate differential cytokine expression between chickens and ducks following exposure with H5N1 viruses isolates recovered from Southeast Asia in different years. Ducks generally displayed increased cytokine expression and resistance to challenge, while chickens exhibit decreased cytokine expression. These studies emphasize the importance of innate immunity in birds and correlate increased pathogenicity of recent H5N1 viruses for wild waterfowl with an enhanced suppression of the host immune response. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. USE OF REVERSE GENETICS TO EXAMINE THE CONTRIBUTIONS OF INDIVIDUAL GENES TO HOST IMMUNE RESPONSE AND PATHOGENICITY. A reverse genetics approach was used to examine the contributions of individual genes to host immune response and pathogenicity. Avian influenza viruses are extremely variable in sequence, and it is difficult to correlate specific genetic changes with virulence. The H5N1 viruses from Southeast Asia appear to be increasing in pathogenicity; we used reverse genetics to generate single-gene recombinant viruses to examine their contributions in stimulating an innate immune response in chickens and ducks. Reassortants combining genes from different H5N1 isolates of Asian lineage demonstrated that exchanging the hemagglutinin and polymerase genes considerably affected pathogenicity, and this was reflected in increased mortality and increased viral replication and spread in tissues. The NS gene also had an effect on viral replication; however, no effect on mortality was observed. The reverse genetics system is allowing us to target specific genes associated with virulence in avian influenza virus, and this tool will improve our understanding of the role of the innate immune response on the outcome of infection. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. DIFFERENCES IN GROWTH CHARACTERISTICS OF AVIAN INFLUENZA IN DIFFERENT BIRD SPECIES. Differences are observed in the growth characteristics of avian influenza viruses in different bird species. Ducks and chickens infected with AI viruses display clear differences in disease manifestation. To understand the mechanisms responsible for these differences we have determined the ability of several AI isolates to replicate in primary tracheal epithelial cells and fibroblasts from both species. Results suggest differences in the capacity of the host species to support viral replication, and a varied ability of different avian influenza viruses to infect identical cell types. Transcriptional host responses were analyzed using a microarray of the complete chicken genome, and several patterns of host response were seen with different viruses. These studies indicate that some isolates are better at evading the host response than others, and this information will help us to identify additional factors in how avian influenza virus persist in the host. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. H5 INACTIVATED VACCINES PROTECT CHICKENS FROM H5N1 HIGH PATHOGENICITY (HP) AVIAN INFLUENZA (AI) VIRUSES. Avian influenza viruses change rapidly, and there is concern that currently available vaccines will lose their effectiveness after long term use. Vaccination efficacy studies were performed with commercially available and experimental vaccines using a 2005 Vietnam H5N1 highly pathogenic avian influenza virus as a challenge strain. The vaccine protected chickens from clinical signs and death when challenged with high dose of the H5N1 HPAI virus, and vaccinated birds excreted less virus than controls. These data indicate the killed AI vaccines tested still provided good protection from the 2005 Vietnam strain of H5N1 HPAI virus, but monitoring of vaccine efficacy needs to continue in the future. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. ANTIGENIC VARIABILITY BETWEEN DIFFERENT H5 AVIAN INFLUENZA VIRUSES. Antigenic variability between different H5 avian influenza viruses were compared with sequence information. Antigenic variability of different avian influenza viruses is an important factor to consider when comparing vaccine efficacy. A panel of antibodies to H5 HA subtype AI viruses from North America, Europe, and Asia was used to evaluate the antigenic relatedness among H5 viruses from around the world. Gene sequence from the HA proteins of the target viruses were also analyzed. It was shown that there is wide variation among H5 viruses in antigenic structure and that geographical origin is often a predictor of relatedness, however percent sequence identity does not always correspond to antigenic relatedness. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. PATHOGENICITY OF ASIAN H5N1 HPAI VIRUSES IN PEKIN DUCKS: EFFECTS OF AGE. The age of Pekin ducks affects the observed pathogenicity of Asian H5N1 HPAI viruses. The age of an animal can affect how the animal responds to different viral infections, and little data is available on how age affects avian influenza virus infection in ducks. Experiments were conducted to determine the differences between young and older ducks in their response to AIV infection: what genes/cytokines are involved in the increased resistance to infection observed in older ducks; and the differences observed in response to different AIV strains. Microarray analysis was used to understand and compare gene expression following infection. Differences in cytokine expression found between younger and older ducks highlights the importance of the innate immune response on the outcome of infection. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. UNDERSTANDING THE DIFFERENCES IN PATHOBIOLOGY OF H5N1 HPAI VIRUSES IN CHICKENS AND DUCKS. Differences in the pathology produced by highly pathogenic H5N1 virus infection were observed between chickens and ducks. Characterizing the pathologic lesions seen in animals is important in helping understand viral pathogenesis. Our pathologic comparisons showed that the lesions in the lung were more severe in chickens, and virus replication was observed in vascular endothelial cells which were not observed in the ducks, explaining in part the differences observed in the presentation of the disease. Innate responses differed also between chickens and ducks. In general, cytokine expression in chickens was suppressed following infection when compared to controls. This information provides insights into the pathobiology of AIV in different avian species. This accomplishment is in the National Program 103, Animal Health (100%). Component 1: Biodefense Research, Problem Statement 1A: Foreign Animal Disease, and Component 4: Countermeasures to Prevent and Control Respiratory Diseases, Problem Statement 4C: Poultry Respiratory Diseases. Technology Transfer Number of New CRADAS and MTAS: 5 Number of Non-Peer Reviewed Presentations and Proceedings: 7
Impacts
(N/A)
Publications
- Suguitan, A.L., Mcauliffe, J., Mills, K.L., Jin, H., Duke, G., Lu, B., Luke, C.J., Murphy, B., Swayne, D.E., Kemble, G., Subbarao, K. 2006. Live attenuated influenza A H5N1 candidate vaccines provide broad cross- protection in mice and ferrets. PLoS Medicine. 3(9):e360.
- Cauthen, A.N., Swayne, D.E., Sekellick, M.J., Marcus, P.I., Suarez, D.L. 2007. Amelioration of influenza pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene. Journal of Virology. 81(4):1838-1847.
- Pantin Jackwood, M.J., Swayne, D.E. 2007. Pathobiology of Asian highly pathogenic avian influenza H5N1 virus infection in ducks. Avian Diseases. 51:250-259.
- Kash, J.C., Tumpey, T.M., Proll, S.C., Carter, V., Perwitisari, O., Thomas, M.J., Basler, C.F., Palese, P., Swayne, D.E., Taubenberger, J.K., Garcia- Sastre, A., Katze, M.G. 2006. Genomic analysis of increased host immune and cell death responses by 1918 influenza virus. Nature. 443(7111):578- 581.
- Sylte, M.J., Hubby, B., Suarez, D.L. 2007. Influenza neuraminidase antibodies provide partial protection for chickens against high pathogenic avian influenza infection. Vaccine. 25:3763-3772.
|
|