A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection

Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.

[1]  A. Jegerlehner,et al.  Influenza A Vaccine Based on the Extracellular Domain of M2: Weak Protection Mediated via Antibody-Dependent NK Cell Activity , 2004, The Journal of Immunology.

[2]  Aaas News,et al.  Book Reviews , 1893, Buffalo Medical and Surgical Journal.

[3]  V. Jestin,et al.  Importance of a Prime–Boost DNA/Protein Vaccination to Protect Chickens Against Low-Pathogenic H7 Avian Influenza Infection , 2007, Avian diseases.

[4]  D. Swayne.  laboratory manual for the isolation and identification of avian pathogens , 1998 .

[5]  L Döhner,et al.  [Genetics of influenza viruses]. , 1978, Archiv fur experimentelle Veterinarmedizin.

[6]  Y. Kwon,et al.  Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge. , 2009, Poultry science.

[7]  S. Raman,et al.  Structure-based design of peptides that self-assemble into regular polyhedral nanoparticles. , 2006, Nanomedicine : nanotechnology, biology, and medicine.

[8]  K. Lohman,et al.  Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes , 2002, Journal of Clinical Microbiology.

[9]  Sang-Jeom Ahn,et al.  Innovative vaccine production technologies: The evolution and value of vaccine production technologies , 2009, Archives of pharmacal research.

[10]  D. Swayne.  Avian influenza vaccines and therapies for poultry. , 2009, Comparative immunology, microbiology and infectious diseases.

[11]  P. S. Kim,et al.  A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. , 1993, Science.

[12]  T. Brüning,et al.  A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. , 2002, Molecular and cellular probes.

[13]  C. Sweet,et al.  Mechanism of immunity to influenza: maternal and passive neonatal protection following immunization of adult ferrets with a live vaccinia-influenza virus haemagglutinin recombinant but not with recombinants containing other influenza virus proteins. , 1989, The Journal of general virology.

[14]  W. Fiers,et al.  Universal influenza A vaccine: optimization of M2-based constructs. , 2005, Virology.

[15]  Walter Fiers,et al.  A universal influenza A vaccine based on the extracellular domain of the M2 protein , 1999, Nature Medicine.

[16]  R. Kammerer,et al.  Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer , 2001, Nature Structural Biology.

[17]  S. B. Hitchner,et al.  A Laboratory Manual for the Isolation and Identification of Avian Pathogens , 1990 .

[18]  R. Zinkernagel,et al.  Role of Repetitive Antigen Patterns for Induction of Antibodies Against Antibodies , 1997, The Journal of experimental medicine.

[19]  W. C. Gamble,et al.  Protection of mice against influenza A virus challenge by vaccination with baculovirus-expressed M2 protein. , 1995, Vaccine.

[20]  W. Marsden.  I and J , 2012 .

[21]  A. Klimov,et al.  Modified M2 proteins produce heterotypic immunity against influenza A virus. , 1999, Vaccine.

[22]  Ueli Aebi,et al.  A Nonadjuvanted Polypeptide Nanoparticle Vaccine Confers Long-Lasting Protection against Rodent Malaria1 , 2009, The Journal of Immunology.

[23]  R. Zinkernagel,et al.  The influence of antigen organization on B cell responsiveness. , 1993, Science.

[24]  D. Pérez,et al.  A New Generation of Modified Live-Attenuated Avian Influenza Viruses Using a Two-Strategy Combination as Potential Vaccine Candidates , 2007, Journal of Virology.

[25]  R. Lamb,et al.  Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. , 1991, Virology.

[26]  J. Gelb,et al.  Virulence of Low Pathogenicity H7N2 Avian Influenza Viruses from the Delmarva Peninsula for Broiler and Leghorn Chickens and Turkeys , 2008, Avian diseases.

[27]  I. Capua,et al.  The use of vaccination to combat multiple introductions of Notifiable Avian Influenza viruses of the H5 and H7 subtypes between 2000 and 2006 in Italy. , 2007, Vaccine.

[28]  D. Leclerc,et al.  Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization. , 2007, Virology.

[29]  W. Fiers,et al.  Universal influenza A M2e-HBc vaccine protects against disease even in the presence of pre-existing anti-HBc antibodies. , 2008, Vaccine.

[30]  R. Lamb,et al.  Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice , 1990, Journal of virology.

[31]  Y. Guan,et al.  Universal primer set for the full-length amplification of all influenza A viruses , 2001, Archives of Virology.

[32]  K. Mozdzanowska,et al.  Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity. , 1994, Journal of immunology.

[33]  R. K. Evans,et al.  Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin. , 2008, Vaccine.

[34]  K. Okuda,et al.  Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene. , 2001, Vaccine.

[35]  Huanchun Chen,et al.  Detection of Antibodies to the Nonstructural Protein (NS1) of Avian Influenza Viruses Allows Distinction Between Vaccinated and Infected Chickens , 2005, Avian diseases.

[36]  C. W. Lee,et al.  Avian influenza vaccination in North America: strategies and difficulties. , 2006, Developments in biologicals.

[37]  R. Stephenson.  A and V , 1962, The British journal of ophthalmology.

[38]  W. Fiers,et al.  A "universal" human influenza A vaccine. , 2004, Virus research.

[39]  K. Lohman,et al.  Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes , 2002, Journal of Clinical Microbiology.

[40]  R. Hodges,et al.  Peptide Nanoparticles as Novel Immunogens: Design and Analysis of a Prototypic Severe Acute Respiratory Syndrome Vaccine , 2008, Chemical biology & drug design.