Development of universal influenza vaccines based on influenza virus M and NP genes

Infection

Volumn 42, Issue 2, 2014, Pages 251-262

  Zheng, M ^a   Luo, J ^a   Chen, Z ^a,b,c  

^a SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS   (China)

^b WUHAN INSTITUTE OF VIROLOGY   (China)

^c HUNAN NORMAL UNIVERSITY   (China)

Author keywords

Broad spectrum; Influenza virus; Matrix protein; Nucleoprotein; Universal vaccine

Indexed keywords

ALUMINUM HYDROXIDE; ALUMINUM POTASSIUM SULFATE; AVIAN INFLUENZA VACCINE; CHITOSAN; CPG OLIGODEOXYNUCLEOTIDE; EPITOPE; GAMMA INTERFERON; IMMUNOLOGICAL ADJUVANT; INFLUENZA VACCINE; MATRIX PROTEIN; MIFAMURTIDE; N 8295; PHOSPHORYL LIPID A; QS 21; TREHALOSE; UNCLASSIFIED DRUG; UNIVERSAL INFLUENZA VACCINE; VAX 102; VIRUS NUCLEOPROTEIN; CORE PROTEIN; M1 PROTEIN, INFLUENZA A VIRUS; M2 PROTEIN, INFLUENZA A VIRUS; NP PROTEIN, INFLUENZA A VIRUS; RNA BINDING PROTEIN;

CELLULAR IMMUNITY; CLINICAL TRIAL (TOPIC); CYTOKINE RELEASE; IMMUNITY; INFLUENZA; INFLUENZA VACCINATION; INFLUENZA VIRUS; INFLUENZA VIRUS M; MEDICAL RESEARCH; NONHUMAN; NUCLEOTIDE SEQUENCE; REVIEW; VIRUS GENE; ANIMAL; CHICKEN; GENETICS; HUMAN; IMMUNOLOGY; INFLUENZA IN BIRDS; INFLUENZA, HUMAN; METABOLISM; MOUSE; ORTHOMYXOVIRIDAE INFECTIONS; ORTHOMYXOVIRUS; VIROLOGY;

ANIMALS; CHICKENS; HUMANS; INFLUENZA IN BIRDS; INFLUENZA VACCINES; INFLUENZA, HUMAN; MICE; ORTHOMYXOVIRIDAE; ORTHOMYXOVIRIDAE INFECTIONS; RNA-BINDING PROTEINS; VIRAL CORE PROTEINS; VIRAL MATRIX PROTEINS;

EID: 84897541173     PISSN: 03008126     EISSN: 14390973     Source Type: Journal    
DOI: 10.1007/s15010-013-0546-4     Document Type: Review

References (85)

1. Kamps BS, Hoffmann C, Preiser W, editors. Influenza report 2006. ISBN 3-924774-51-X. http://www.influenzareport.com.
2. Slepushkin VA, Katz JM, Black RA, Gamble WC, Rota PA, Cox NJ. Protection of mice against influenza A virus challenge by vaccination with baculovirus-expressed M2 protein. Vaccine. 1995;13:1399-402.
3. Fiers W, De Filette M, El Bakkouri K, Schepens B, Roose K, Schotsaert M, Birkett A, Saelens X. M2e-based universal influenza A vaccine. Vaccine. 2009;27:6280-3.
4. Treanor JJ, Taylor DN, Tussey L, Hay C, Nolan C, Fitzgerald T, Liu G, Kavita U, Song L, Dark I, Shaw A. Safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125) in healthy young adults. Vaccine. 2010;28:8268-74.
5. Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, Jacobs A, Liu G, Huang Y, Desai P, Maksymiuk G, Takahashi V, Umlauf S, Reiserova L, Bell R, Li H, Zhang Y, McDonald WF, Powell TJ, Tussey L. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs. PLoS One. 2008;3:e2257.
6. Ambrozaitis A, Groth N, Bugarini R, Sparacio V, Podda A, Lattanzi M. A novel mammalian cell-culture technique for consistent production of a well-tolerated and immunogenic trivalent subunit influenza vaccine. Vaccine. 2009;27:6022-9.
7. Kemble G, Greenberg H. Novel generations of influenza vaccines. Vaccine. 2003;21:1789-95. (Pubitemid 36398375)
8. Sui Z, Chen Q, Fang F, Zheng M, Chen Z. Cross-protection against influenza virus infection by intranasal administration of M1-based vaccine with chitosan as an adjuvant. Vaccine. 2010;28:7690-8.
9. Sui Z, Chen Q, Wu R, Zhang H, Zheng M, Wang H, Chen Z. Cross-protection against influenza virus infection by intranasal administration of M2-based vaccine with chitosan as an adjuvant. Arch Virol. 2010;155:535-44.
10. Guo L, Zheng M, Ding Y, Li D, Yang Z, Wang H, Chen Q, Sui Z, Fang F, Chen Z. Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein. Arch Virol. 2010;155:1765-75.
11. Kim TJ, Kim KH, Lee JI. Stimulation of mucosal and systemic antibody responses against recombinant transferrin-binding protein B of Actinobacillus pleuropneumoniae with chitosan after tracheal administration in piglets. J Vet Med Sci. 2007;69:535-9. (Pubitemid 46883912)
12. Helenius A. Unpacking the incoming influenza virus. Cell. 1992;69:577-8.
13. Ye ZP, Baylor NW, Wagner RR. Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides. J Virol. 1989;63:3586-94. (Pubitemid 19205427)
14. Adler S, Reay P, Roy P, Klenk HD. Induction of T cell response by bluetongue virus core-like particles expressing a T cell epitope of the M1 protein of influenza A virus. Med Microbiol Immunol. 1998;187:91-6. (Pubitemid 28495514)
15. Plotnicky H, Cyblat-Chanal D, Aubry JP, Derouet F, Klinguer- Hamour C, Beck A, Bonnefoy JY, Corvaïa N. The immunodominant influenza matrix T cell epitope recognized in human induces influenza protection in HLA-A2/K(b) transgenic mice. Virology. 2003;309:320-9. (Pubitemid 36579225)
16. +) T cells with specificity for a new major histocompatibility complex class II-restricted influenza A virus matrix protein epitope in peripheral blood of influenza patients. J Virol. 2000;74:8740-3.
17. Goodman AG, Heinen PP, Guerra S, Vijayan A, Sorzano CO, Gomez CE, Esteban M. A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus. PLoS One. 2011;6:e25938.
18. Lamb RA, Lai CJ, Choppin PW. Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci USA. 1981;78:4170-4. (Pubitemid 11010301)
19. Ito T, Gorman OT, Kawaoka Y, Bean WJ, Webster RG. Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins. J Virol. 1991;65:5491-8.
20. Zebedee SL, Lamb RA. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol. 1988;62:2762-72.
21. Treanor JJ, Tierney EL, Zebedee SL, Lamb RA, Murphy BR. Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J Virol. 1990;64:1375-7. (Pubitemid 20087784)
22. Saelens X. The influenza matrix protein 2 as a vaccine target. Future Virol. 2008;3:167-78. (Pubitemid 351385981)
23. Schotsaert M, De Filette M, Fiers W, Saelens X. Universal M2 ectodomain-based influenza A vaccines: preclinical and clinical developments. Expert Rev Vaccines. 2009;8:499-508.
24. Yewdell JW, Bennink JR, Smith GL, Moss B. Influenza A virus nucleoprotein is a major target antigen for cross-reactive antiinfluenza A virus cytotoxic T lymphocytes. Proc Natl Acad Sci USA. 1985;82:1785-9. (Pubitemid 15085262)
25. Thomas PG, Keating R, Hulse-Post DJ, Doherty PC. Cell-mediated protection in influenza infection. Emerg Infect Dis. 2006;12:48-54. (Pubitemid 43143535)
26. + mediated cellular immune response to recombinant influenza nucleoprotein. Vaccine. 2002;20:3731-8.
27. Okuda K, Ihata A, Watabe S, Okada E, Yamakawa T, Hamajima K, Yang J, Ishii N, Nakazawa M, Okuda K, Ohnari K, Nakajima K, Xin KQ. Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene. Vaccine. 2001;19:3681-91. (Pubitemid 32522766)
28. Chen Q, Kuang H, Wang H, Fang F, Yang Z, Zhang Z, Zhang X, Chen Z. Comparing the ability of a series of viral protein-expressing plasmid DNAs to protect against H5N1 influenza virus. Virus Genes. 2009;38:30-8.
29. Heinen PP, Rijsewijk FA, de Boer-Luijtze EA, Bianchi AT. Vaccination of pigs with a DNA construct expressing an influenza virus M2-nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus. J Gen Virol. 2002;83:1851-9. (Pubitemid 34846448)
30. Tompkins SM, Zhao ZS, Lo CY, Misplon JA, Liu T, Ye Z, Hogan RJ, Wu Z, Benton KA, Tumpey TM, Epstein SL. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg Infect Dis. 2007;13:426-35. (Pubitemid 46348126)
31. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med. 1999;5:1157-63. (Pubitemid 29474420)
32. De Filette M, Ramne A, Birkett A, Lycke N, Löwenadler B, Min Jou W, Saelens X, Fiers W. The universal influenza vaccine M2e-HBc administered intranasally in combination with the adjuvant CTA1-DD provides complete protection. Vaccine. 2006;24:544-51. (Pubitemid 43017819)
33. Frace AM, Klimov AI, Rowe T, Black RA, Katz JM. Modified M2 proteins produce heterotypic immunity against influenza A virus. Vaccine. 1999;17:2237-44. (Pubitemid 29225675)
34. Fan J, Liang X, Horton MS, Perry HC, Citron MP, Heidecker GJ, Fu TM, Joyce J, Przysiecki CT, Keller PM, Garsky VM, Ionescu R, Rippeon Y, Shi L, Chastain MA, Condra JH, Davies ME, Liao J, Emini EA, Shiver JW. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine. 2004;22:2993-3003. (Pubitemid 39037199)
35. Mozdzanowska K, Feng J, Eid M, Kragol G, Cudic M, Otvos L Jr, Gerhard W. Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. Vaccine. 2003;21:2616-26. (Pubitemid 36561354)
36. Ernst WA, Kim HJ, Tumpey TM, Jansen AD, Tai W, Cramer DV, Adler-Moore JP, Fujii G. Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 2 epitope vaccines. Vaccine. 2006;24:5158-68. (Pubitemid 43818194)
37. Denis J, Acosta-Ramirez E, Zhao Y, Hamelin ME, Koukavica I, Baz M, Abed Y, Savard C, Pare C, Lopez Macias C, Boivin G, Leclerc D. Development of a universal influenza A vaccine based on the M2e peptide fused to the papaya mosaic virus (PapMV) vaccine platform. Vaccine. 2008;26:3395-403.
38. Eliasson DG, El Bakkouri K, Schön K, Ramne A, Festjens E, Löwenadler B, Fiers W, Saelens X, Lycke N. CTA1-M2e-DD: a novel mucosal adjuvant targeted influenza vaccine. Vaccine. 2008;26:1243-52.
39. De Filette M, Martens W, Roose K, Deroo T, Vervalle F, Bentahir M, Vandekerckhove J, Fiers W, Saelens X. An influenza A vaccine based on tetrameric ectodomain of matrix protein 2. J Biol Chem. 2008;283:11382-7.
40. Ionescu RM, Przysiecki CT, Liang X, Garsky VM, Fan J, Wang B, Troutman R, Rippeon Y, Flanagan E, Shiver J, Shi L. Pharmaceutical and immunological evaluation of human papillomavirus viruslike particle as an antigen carrier. J Pharm Sci. 2006;95:70-9.
41. Bessa J, Schmitz N, Hinton HJ, Schwarz K, Jegerlehner A, Bachmann MF. Efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design. Eur J Immunol. 2008;38:114-26.
42. Huleatt JW, Nakaar V, Desai P, Huang Y, Hewitt D, Jacobs A, Tang J, McDonald W, Song L, Evans RK, Umlauf S, Tussey L, Powell TJ. Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin. Vaccine. 2008;26:201-14. (Pubitemid 50008089)
43. Zhang GG, Li DX, Zhang HH, Zeng YM, Chen L. Enhancement of mucosal immune response against the M2eHBc+ antigen in mice with the fusion expression products of LTB and M2eHBc+ through mucosal immunization route. Vet Res Commun. 2009;33:735-47.
44. Ameiss K, Ashraf S, Kong W, Pekosz A, Wu WH, Milich D, Billaud JN, Curtiss R 3rd. Delivery of woodchuck hepatitis viruslike particle presented influenza M2e by recombinant attenuated Salmonella displaying a delayed lysis phenotype. Vaccine. 2010;28:6704-13.
45. Zhao G, Sun S, Du L, Xiao W, Ru Z, Kou Z, Guo Y, Yu H, Jiang S, Lone Y, Zheng BJ, Zhou Y. An H5N1 M2e-based multiple antigenic peptide vaccine confers heterosubtypic protection from lethal infection with pandemic 2009 H1N1 virus. Virol J. 2010;7:151.
46. Zhao G, Du L, Xiao W, Sun S, Lin Y, Chen M, Kou Z, He Y, Lustigman S, Jiang S, Zheng BJ, Zhou Y. Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant. Vaccine. 2010;28:7233-40.
47. Wraith DC, Vessey AE, Askonas BA. Purified influenza virus nucleoprotein protects mice from lethal infection. J Gen Virol. 1987;68:433-40. (Pubitemid 17007830)
48. Tamura S, Miyata K, Matsuo K, Asanuma H, Takahashi H, Nakajima K, Suzuki Y, Aizawa C, Kurata T. Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein. J Immunol. 1996;156:3892-900. (Pubitemid 26139942)
49. Wang W, Huang B, Jiang T, Wang X, Qi X, Gao Y, Tan W, Ruan L. Robust immunity and heterologous protection against influenza in mice elicited by a novel recombinant NP-M2e fusion protein expressed in E. coli. PLoS One. 2012;7:e52488.
50. Roose K, Fiers W, Saelens X. Pandemic preparedness: toward a universal influenza vaccine. Drug News Perspect. 2009;22:80-92.
51. Lo CY, Wu Z, Misplon JA, Price GE, Pappas C, Kong WP, Tumpey TM, Epstein SL. Comparison of vaccines for induction of heterosubtypic immunity to influenza A virus: cold-adapted vaccine versus DNA prime-adenovirus boost strategies. Vaccine. 2008;26:2062-72.
52. Lillie PJ, Berthoud TK, Powell TJ, Lambe T, Mullarkey C, Spencer AJ, Hamill M, Peng Y, Blais ME, Duncan CJ, Sheehy SH, Havelock T, Faust SN, Williams RL, Gilbert A, Oxford J, Dong T, Hill AV, Gilbert SC. Preliminary assessment of the efficacy of a T-cell-based influenza vaccine, MVA-NP+M1, in humans. Clin Infect Dis. 2012;55:19-25.
53. Xu K, Ling ZY, Sun L, Xu Y, Bian C, He Y, Lu W, Chen Z, Sun B. Broad humoral and cellular immunity elicited by a bivalent DNA vaccine encoding HA and NP genes from an H5N1 virus. Viral Immunol. 2011;24:45-56.
54. Pandey A, Singh N, Vemula SV, Couëtil L, Katz JM, Donis R, Sambhara S, Mittal SK. Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an ade-novirus-based H5N1 influenza vaccine. PLoS One. 2012;7:e33428.
55. Li Z, Gabbard JD, Mooney A, Chen Z, Tompkins SM, He B. Efficacy of parainfluenza virus 5 mutants expressing hemagglutinin from H5N1 influenza A virus in mice. J Virol. 2013;87:9604-9.
56. Boyd AC, Ruiz-Hernandez R, Peroval MY, Carson C, Balkissoon D, Staines K, Turner AV, Hill AV, Gilbert SC, Butter C. Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus. Vaccine. 2013;31:670-5.
57. Donnelly JJ, Friedman A, Ulmer JB, Liu MA. Further protection against antigenic drift of influenza virus in a ferret model by DNA vaccination. Vaccine. 1997;15:865-8. (Pubitemid 27287977)
58. Jeon SH, Ben-Yedidia T, Arnon R. Intranasal immunization with synthetic recombinant vaccine containing multiple epitopes of influenza virus. Vaccine. 2002;20:2772-80. (Pubitemid 34597137)
59. Zhou D, Wu TL, Lasaro MO, Latimer BP, Parzych EM, Bian A, Li Y, Li H, Erikson J, Xiang Z, Ertl HC. A universal influenza a vaccine based on adenovirus expressing matrix-2 ectodomain and nucleoprotein protects mice from lethal challenge. Mol Ther. 2010;18:2182-9.
60. Chen Z, Yoshikawa T, Kadowaki S, Hagiwara Y, Matsuo K, Asanuma H, Aizawa C, Kurata T, Tamura S. Protection and antibody responses in different strains of mouse immunized with plasmid DNAs encoding influenza virus haemagglutinin, neuraminidase and nucleoprotein. J Gen Virol. 1999;80:2559-64. (Pubitemid 29458615)
61. Mazanec MB, Kaetzel CS, Lamm ME, Fletcher D, Nedrud JG. Intracellular neutralization of virus by immunoglobulin A antibodies. Proc Natl Acad Sci USA. 1992;89:6901-5.
62. Mazanec MB, Coudret CL, Fletcher DR. Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J Virol. 1995;69:1339-43.
63. Yan H, Lamm ME, Björling E, Huang YT. Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model. J Virol. 2002;76:10972-9. (Pubitemid 35176214)
64. Huang YT, Wright A, Gao X, Kulick L, Yan H, Lamm ME. Intraepithelial cell neutralization of HIV-1 replication by IgA. J Immunol. 2005;174:4828-35.
65. Wright A, Yan H, Lamm ME, Huang YT. Immunoglobulin A antibodies against internal HIV-1 proteins neutralize HIV-1 replication inside epithelial cells. Virology. 2006;356:165-70. (Pubitemid 44750044)
66. Safety study of recombinant M2e influenza-A vaccine in healthy adults (FLU-A). http://clinicaltrials.gov/ct2/show/NCT00819013.
67. Acambis reports positive data from trials of universal influenza A vaccine. FierceBiotech. http://www.fiercebiotech.com/press-releases/acambis- reports-positive-data-trials-universal-influenza-vaccine#ixzz13KPQYAE2.
68. Talbot HK, Rock MT, Johnson C, Tussey L, Kavita U, Shanker A, Shaw AR, Taylor DN. Immunopotentiation of trivalent influenza vaccine when given with VAX102, a recombinant influenza M2e vaccine fused to the TLR5 ligand flagellin. PLoS One. 2010;5:e14442.
69. Safety and immunogenicity of VAX102 universal influenza vaccine when given in the same arm with the standard influenza vaccine in healthy adults. http://clinicaltrials.gov/ct2/show/NCT00921973?term=VAX102&rank=2.
70. Atsmon J, Kate-Ilovitz E, Shaikevich D, Singer Y, Volokhov I, Haim KY, Ben-Yedidia T. Safety and immunogenicity of mul-timeric-001 - a novel universal influenza vaccine. J Clin Immunol. 2012;32(3):595-603.
71. Turley CB, Rupp RE, Johnson C, Taylor DN, Wolfson J, Tussey L, Kavita U, Stanberry L, Shaw A. Safety and immunogenicity of a recombinant M2e-flagellin influenza vaccine (STF2.4xM2e) in healthy adults. Vaccine. 2011;29:5145-52.
72. Ben-Yedidia T. Multimeric-001: BiondVax's universal flu vaccine. Interview by Duc Le. Expert Rev Vaccines. 2010;9:241-2.
73. A double-dose safety study of an influenza vaccine (Multimeric-001) injected to elderly volunteers. http://www.clinicaltrials.gov/ct2/show/ NCT01010737?term=Multimeric-001&rank=1.
74. Further investigation of an intramuscular influenza vaccine (Multimeric-001). http://www.clinicaltrials.gov/ct2/show/NCT01146119?term= Multimeric-001&rank=4.
75. A double-dose safety study of an influenza vaccine (Multimeric-001). http://clinicaltrials.gov/ct2/show/NCT00877448?term=Multimeric-001&rank=3.
76. Pipeline: flu vaccine. http://www.dynavax.com/flu.html.
77. Janssen R, Ervin J, Martin T. Clinical evaluation of N8295, a universal influenza A vaccine containing M2e and NP antigens conjugated to an oligonucleotide immunostimulatory sequence. http://www.who.int/vaccine-research/ diseases/influenza/18-feb-2011-Robert-Janssen-ws.pdf.
78. Antrobus RD, Berthoud TK, Mullarkey CE, Hoschler K, Coughlan L, Zambon M, Hill AV, Gilbert SC. Coadministration of Seasonal influenza vaccine and MVA-NP+M1 Simultaneously achieves potent humoral and cell-mediated responses. Mol Ther. 2013.
79. Antrobus RD, Lillie PJ, Berthoud TK, Spencer AJ, McLaren JE, Ladell K, Lambe T, Milicic A, Price DA, Hill AV, Gilbert SC. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years. PLoS One. 2012;7:e48322.
80. A study to assess the safety and efficacy of a new influenza candidate vaccine MVA-NP?M1 in healthy adults. http://clinicaltrialsfeeds.org/clinical- trials/show/NCT00993083.
81. Study Of VGX-3400X, H5N1 avian influenza virus DNA plasmid + electroporation in healthy adults. http://www.clinicaltrials.gov/ct2/show/ NCT01142362?term=VGX-3400&rank=1.
82. VGX-3400: Phase I data. http://www.biocentury.com/weekinreview/ clinicalresults/2011-07-25/vgx-3400-phase-i-data-314011.
83. Comparative safety and immunogenicity of 1.0 lg intramuscular (i.m.) and 2.0 lg subcutaneous (s.c.) dosing with VAX102 (M2eflagellin) universal influenza vaccine in healthy adults. http://www.clinicaltrials.gov/ct2/show/NCT00921947? term=STF2.4xM2e&rank=2.
84. A study of a bivalent influenza peptide conjugate vaccine in healthy adults. http://www.clinicaltrials.gov/ct2/show/NCT00851266?term=influenza ?M2&rank=3.
85. A Phase 1, double-blinded study to evaluate the safety, tolerability, and immunogenicity of pandemic influenza plasmid DNA vaccines. http://www. clinicaltrials.gov/ct2/show/NCT00709800?term=influenza+M2&rank=10.