Background Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics. Methods and Findings To address this need, we generated whole-genome–fragment phage display libraries (GFPDL) expressing fragments of 15–350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformation-dependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 na？ve, H1N1 or H3N2 seropositive). Conclusion This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools.
References
[1]
Subbarao K, Joseph T (2007) Scientific barriers to developing vaccines against avian influenza viruses. Nat Rev Immunol 7: 267–278.
[2]
Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y (2008) Human infection with highly pathogenic H5N1 influenza virus. Lancet 371: 1464–1475.
[3]
Simmons CP, Bernasconi NL, Suguitan AL, Mills K, Ward JM, et al. (2007) Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. PLoS Med 4: e178. doi:10.1371/journal.pmed.0040178.
[4]
Kashyap AK, Steel J, Oner AF, Dillon MA, Swale RE, et al. (2008) Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies. Proc Natl Acad Sci U S A 105: 5986–5991.
[5]
Luke TC, Kilbane EM, Jackson JL, Hoffman SL (2006) Meta-analysis: convalescent blood products for Spanish influenza pneumonia: a future H5N1 treatment? Ann Intern Med 145: 599–609.
[6]
Zhou B, Zhong N, Guan Y (2007) Treatment with convalescent plasma for influenza A (H5N1) infection. N Engl J Med 357: 1450–1451.
[7]
Bui HH, Peters B, Assarsson E, Mbawuike I, Sette A (2007) Ab and T cell epitopes of influenza A virus, knowledge and opportunities. Proc Natl Acad Sci U S A 104: 246–251.
[8]
de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, et al. (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12: 1203–1207.
[9]
Khurana S, Needham J, Park S, Mathieson B, Busch MP, et al. (2006) Novel approach for differential diagnosis of HIV infections in the face of vaccine-generated antibodies: utility for detection of diverse HIV-1 subtypes. J Acquir Immune Defic Syndr 43: 304–312.
[10]
Stephenson I, Wood JM, Nicholson KG, Charlett A, Zambon MC (2004) Detection of anti-H5 responses in human sera by HI using horse erythrocytes following MF59-adjuvanted influenza A/Duck/Singapore/97 vaccine. Virus Res 103: 91–95.
[11]
Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, et al. (1999) Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol 37: 937–943.
[12]
Stephenson I, Das RG, Wood JM, Katz JM (2007) Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine 25: 4056–4063.
[13]
de Jong MD, Tran TT, Truong HK, Vo MH, Smith GJ, et al. (2005) Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med 353: 2667–2672.
[14]
Khurana S, Kennedy M, King LR, Golding H (2005) Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity. J Virol 79: 6791–6800.
[15]
Wiley DC, Wilson IA, Skehel JJ (1981) Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289: 373–378.
[16]
Smith CA, Barnett BC, Thomas DB, Temoltzin-Palacios F (1991) Structural assignment of novel and immunodominant antigenic sites in the neutralizing antibody response of CBA/Ca mice to influenza hemagglutinin. J Exp Med 173: 953–959.
[17]
Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, et al. (2006) The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 443: 45–49.
[18]
Gerhard W, Mozdzanowska K, Zharikova D (2006) Prospects for universal influenza virus vaccine. Emerg Infect Dis 12: 569–574.
[19]
Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P (2007) A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS Pathog 3: 1414–1421. doi:10.1371/journal.ppat.0030141.
[20]
Khurana S, Needham J, Mathieson B, Rodriguez-Chavez IR, Catanzaro AT, et al. (2006) Human immunodeficiency virus (HIV) vaccine trials: a novel assay for differential diagnosis of HIV infections in the face of vaccine-generated antibodies. J Virol 80: 2092–2099.
[21]
Bizebard T, Gigant B, Rigolet P, Rasmussen B, Diat O, et al. (1995) Structure of influenza virus haemagglutinin complexed with a neutralizing antibody. Nature 376: 92–94.
[22]
Wilson IA, Skehel JJ, Wiley DC (1981) Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289: 366–373.
[23]
Green N, Alexander H, Olson A, Alexander S, Shinnick TM, et al. (1982) Immunogenic structure of the influenza virus hemagglutinin. Cell 28: 477–487.
[24]
Kaverin NV, Rudneva IA, Govorkova EA, Timofeeva TA, Shilov AA, et al. (2007) Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies. J Virol 81: 12911–12917.
[25]
Kaverin NV, Rudneva IA, Ilyushina NA, Varich NL, Lipatov AS, et al. (2002) Structure of antigenic sites on the haemagglutinin molecule of H5 avian influenza virus and phenotypic variation of escape mutants. J Gen Virol 83: 2497–2505.
[26]
Smith GJ, Naipospos TS, Nguyen TD, de Jong MD, Vijaykrishna D, et al. (2006) Evolution and adaptation of H5N1 influenza virus in avian and human hosts in Indonesia and Vietnam. Virology 350: 258–268.
[27]
Throsby M, van den Brink E, Jongeneelen M, Poon LL, Alard P, et al. (2008) Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS ONE 3: e3942. doi:10.1371/journal.pone.0003942.
[28]
Vareckova E, Mucha V, Kostolansky F, Gubareva LV, Klimov A (2008) HA2-specific monoclonal antibodies as tools for differential recognition of influenza A virus antigenic subtypes. Virus Res 132: 181–186.
[29]
Sandbulte MR, Jimenez GS, Boon AC, Smith LR, Treanor JJ, et al. (2007) Cross-reactive neuraminidase antibodies afford partial protection against H5N1 in mice and are present in unexposed humans. PLoS Med 4: e59. doi:10.1371/journal.pmed.0040059.
[30]
Treanor JJ, Tierney EL, Zebedee SL, Lamb RA, Murphy BR (1990) Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J Virol 64: 1375–1377.
[31]
Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, et al. (1999) A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med 5: 1157–1163.
[32]
Fan J, Liang X, Horton MS, Perry HC, Citron MP, et al. (2004) Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine 22: 2993–3003.
[33]
Tompkins SM, Zhao ZS, Lo CY, Misplon JA, Liu T, et al. (2007) Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg Infect Dis 13: 426–435.
[34]
Fu TM, Freed DC, Horton MS, Fan J, Citron MP, et al. (2008) Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus. Virology 385: 218–226.
[35]
Black RA, Rota PA, Gorodkova N, Klenk HD, Kendal AP (1993) Antibody response to the M2 protein of influenza A virus expressed in insect cells. J Gen Virol 74 (Pt 1): 143–146.
[36]
Zamarin D, Ortigoza MB, Palese P (2006) Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. J Virol 80: 7976–7983.
