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Pathogenesis and Immunity

Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses

Hongquan Wan, Jin Gao, Kemin Xu, Hongjun Chen, Laura K. Couzens, Katie H. Rivers, Judy D. Easterbrook, Kevin Yang, Lei Zhong, Mohsen Rajabi, Jianqiang Ye, Ishrat Sultana, Xiu-Feng Wan, Xiufan Liu, Daniel R. Perez, Jeffery K. Taubenberger, Maryna C. Eichelberger
Hongquan Wan
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Jin Gao
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Kemin Xu
bDepartment of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA
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Hongjun Chen
bDepartment of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA
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Laura K. Couzens
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Katie H. Rivers
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Judy D. Easterbrook
cViral Pathogenesis and Evolution Section, Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Kevin Yang
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Lei Zhong
dAnimal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
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Mohsen Rajabi
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Jianqiang Ye
eDepartment of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA
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Ishrat Sultana
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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Xiu-Feng Wan
eDepartment of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA
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Xiufan Liu
dAnimal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
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Daniel R. Perez
bDepartment of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA
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Jeffery K. Taubenberger
cViral Pathogenesis and Evolution Section, Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Maryna C. Eichelberger
aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
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DOI: 10.1128/JVI.01203-13
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  • Fig 1
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    Fig 1

    Reactivity of cross-reactive group B MAbs to N1 and other NA subtypes in IFAs. MDCK cells were infected with BR/07 (human H1N1) (A), CA/09 (human H1N1) (B), A/Chongqing/150/2007 (human H1N1) (C), A/Guangdong/51/2008 (human H1N1) (D), A/duck/Eastern China/1/2008 (avian H6N1) (E), A/duck/Eastern China/103/03 (Y103, avian H1N1) (F), A/duck/Eastern China/233/03 (Y233, avian H3N1) (G), A/turkey/Ontario/6188/68 (avian H8N4) (F), and other viruses as listed in Table 1. IFAs were performed with all group B MAbs, and the same reactivity patterns were observed. Shown are images generated with group B MAb 1H5. The remaining viruses tested (listed in Table 1) were negative for staining with group B MAbs, and therefore images are not shown.

  • Fig 2
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    Fig 2

    Reduction of plaque size by cross-reactive group B MAbs. MDCK cells growing in six-well plates were inoculated with cloned, wt BR/07, attenuated VN/04 (ΔVN/04), or reassortant H6N1CA/09 virus and overlaid with agar supplemented with 1 μg/ml (the top three rows) or 10 μg/ml (the bottom row) of MAbs. Shown are images of plaques formed by these viruses in the presence of strain-specific group A MAb 3A2 and cross-reactive group B MAbs 1H5 and 3H10. The virus control shows plaques formed in the absence of MAb.

  • Fig 3
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    Fig 3

    Effect of single amino acid mutations on MAb binding to N1. Binding of group A MAb 3A2 (A), group A MAb 4G2 (B), and group C MAb 1C7 (C) to mutant N1s was examined by cell-based ELISAs using HEK293 cells transfected with a panel of plasmids carrying each of 14 different single amino acid mutations. The signal generated with each MAb (1 μg/ml) was normalized to that of mouse hyperimmune serum and was expressed as relative binding.

  • Fig 4
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    Fig 4

    Location of 12 critical residues in N1 epitopes on an N1 dimer (Protein Data Bank code 3TI6). The image in the top panel was generated with PyMol software (Delano Scientific). Residues recognized by strain-specific group A MAbs and cross-reactive group B and E MAbs are highlighted in magenta, green, and yellow, respectively; residue 309 recognized by group F MAb 4C4 is at the bottom of the NA region and is shown in blue. Note that residues 339, 341, and 397 are shared by epitopes recognized by two or three groups of MAbs (Table 4 and Fig. 3). The bottom panel shows an alignment of the 12 residues in N1 viruses described in the present study.

  • Fig 5
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    Fig 5

    Prophylactic efficacy of cross-reactive group B MAbs in mice against H1N1 and H5N1 challenge. (A to I) DBA/2 mice (9/group) and (J to L) BALB/c mice (15/group) were treated with group A MAb 3A2 and group B MAb 1H5 or 3H10 (15 mg/kg) intraperitoneally 12 h before virus challenge. Mice treated with PBS served as a control. DBA/2 mice were challenged intranasally with 10 MLD50 of seasonal H1N1 BR/07 (A to C), 09pdm H1N1 CA/09-X179A (D to F), or attenuated H5N1 ΔVN/04 virus (G to I). BALB/c mice were challenged with 20 MLD50 of wt VN/04 virus. Mortality (A, D, G, and J) and body weight (B, E, H, and K) were monitored daily for 14 days, and on day 3 postchallenge, animals (4/group for DBA/2 mice and 5/group for BALB/c mice) were euthanized, and lung viral titers (C, F, I, and L) determined in MDCK cells.

  • Fig 6
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    Fig 6

    Group B MAbs protect mice against H1N1 but not H3N2 virus challenge. DBA/2 mice (5/group) were treated with group B MAb 3H10 (15 mg/kg) intraperitoneally 12 h before challenge with 10 MLD50 of 09pdm H1N1 CA/09-X179A or H3N2 A/Hong Kong/1/1968-X31 (HK/68-X31) virus. Mice treated with PBS and challenged with HK/68-X31 served as a control. Mortality (A) and body weight (B) were monitored daily for 14 days.

  • Fig 7
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    Fig 7

    Dose-dependent prophylactic efficacy of group B MAbs against BR/07 (H1N1). DBA/2 mice (9/group) were treated with group B MAb 3H10 (15, 5, 2, 0.5, and 0.1 mg/kg) intraperitoneally 12 h before challenge with 10 MLD50 of BR/07. Mice treated with the 09pdm H1N1 NA-specific MAb CD6 (Table 3) at 15 mg/kg served as a control. Mortality (A) and weight (B) were monitored daily for 14 days. (C) lung viral titers were measured for 4 mice per group on days 3 and 6 postchallenge.

Tables

  • Figures
  • Table 1

    Wild-type influenza A viruses used in the present study

    Virus name (abbreviation)aSubtype
    A/Texas/36/1991 (TX/91)H1N1
    A/New Caledonia/20/1999 (NC/99)H1N1
    A/Solomon Islands/3/2006 (SI/06)H1N1
    A/Brisbane/59/2007 (BR/07)H1N1
    A/Chongqing/150/2007H1N1
    A/Guangdong/51/2008H1N1
    A/Bethesda/NIH50/2009 (NIH/09)H1N1
    A/California/07/2009 (CA/09)H1N1
    A/duck/Eastern China/103/03 (Y103)H1N1
    A/duck/Eastern China/233/03 (Y233)H3N1
    A/Vietnam/1203/2004 (VN/04)H5N1
    A/duck/Eastern China/1/2008H6N1
    A/turkey/Ontario/6188/68H8N4
    A/duck/Eastern China/01/05H8N4
    A/duck/eastern China/008/2008H5N5
    A/duck/eastern China/031/2009H5N5
    A/shearwater/Australia/1/72H6N5
    A/duck/Yangzhou/013/2008H6N5
    A/duck/Ukraine/1/63H3N8
    A/duck/Eastern China/18/05H3N8
    A/duck/Jiangsu/K1203/2010H5N8
    A/duck/Eastern China/163/02H6N8
    A/duck/Eastern China/36/02H3N2
    A/chicken/Shanghai/14/2001H9N2
    A/duck/China/397/03H10N3
    A/duck/China/376/04H4N6
    A/chicken/Germany/n/49H10N7
    A/duck/Memphis/546/74H11N9
    • ↵a Viruses in bold were tested in IFAs.

  • Table 2

    Reactivities of N1 MAbs with the NA of H1N1 and H5N1 viruses

    MAb groupMAbIsotypeReactivity with N1 of the indicated virusa
    BR/07NIH/09TX/91NC/99SI/06BM/18CA/09VN/04
    A1C4IgG2b++−−−−−−
    2D12IgG1++−−−−−−
    3A2IgG2a++−−−−−−
    4D6IgG2b++−−−−−−
    4G2IgG1++−−−−−−
    B1H5IgG2b++++++++
    2D9IgG2b++++++++
    2G6IgG2b++++++++
    3H10IgG2b++++++++
    4E9IgG2b++++++++
    C1C7IgG2a++−+±−−−
    2C3IgG2b++±++−−−
    2F4IgG2a++±++−−−
    3C2IgG1+++++−−−
    3G1IgG3+±±±±−−−
    D2B4IgG2a++±+++−−
    2B5IgG2a+++++±−−
    3C6IgG2a++++++−−
    3H4IgG2b++±+++−−
    E1C9IgG2a++++++−±
    1H8IgG2b++++++−±
    2D4IgG2b++++++−+
    3H3IgG2b++++++−+
    4B12IgG2b++++++−+
    F4C4IgG2a++−++++−
    • ↵a Values were determined in ELISAs with H1N1 viruses A/Brisbane/59/2007 (BR/07), A/Texas/36/1991 (TX/91), A/New Caledonia/20/1999 (NC/99), A/Solomon Islands/3/2006 (SI/06), and A/California/07/2009 (CA/09)-X179A and the NA of A/Bethesda/NIH50/2009 (NIH/09), A/Brevig Mission/1/1918 (BM/18), and A/Vietnam/1203/2004 (VN/04) expressed on HEK293 cells. +, OD490 of >0.3; −, OD490 of <0.15; ±, OD490 value between 0.15 and 0.30.

  • Table 3

    Inhibition of enzyme activity by N1-specific MAbs

    MAb groupMAbIC50 for the indicated virus (ng/ml)a
    BR/07CA/09VN/04
    A3A210.4>>
    4G27.7>>
    B1H525.9>1,670
    2D968.1>2,160
    2G631.2>1,410
    3H1023.6>1,250
    4E927.0>2,420
    C1C785.7>>
    3C233.2>>
    D2B528.2>>
    3H463.4>>
    E1H814.7>>
    4B1250.9>8,599
    2D4196>>
    F4C41,184>>
    CD6b>44.0>
    Negative controlc>>>
    • ↵a IC50, median inhibition concentration measured by ELLA of the reassortant H6N1 viruses containing the NA of each indicated virus (see Materials and Methods). >, greater than 32,000 ng/ml.

    • ↵b CD6 is a MAb raised against 09pdm H1N1 NA in our laboratory using a protocol similar to that for generating BR/07 NA MAbs.

    • ↵c Negative control, mouse MAb against influenza A virus nucleoprotein (ViroStat, Portland, ME).

  • Table 4

    Amino acid changes in NA of mutant BR/07 viruses selected with N1 MAbs

    MAb groupMAb for selectionMutantaAmino acid changeb
    A3A23A2v1A250S
    4G24G2v1K249E
    4G2v2A343E
    B1H51H5v1T339A
    1H5v2T339I
    1H5v3T339N
    2D92D9v1T339I
    2D9v2T339P
    2G62G6v1T339A
    2G6v2T339I
    3H103H10v1N273D
    3H10v2V338 M
    3H10v3T339N
    3H10v4T339P
    4E94E9v1V338 M
    C1C71C7v1D341N
    3C23C2v1T339A
    3C2v2T339I
    D2B52B5v1T339N
    3H43H4v1T397N
    E1H81H8v1T397A
    2D42D4v1I396T
    2D4v2W456G
    F4C44C4v1N309S
    • ↵a MAb escape mutants of wt BR/07 were selected in eggs, followed by plaque purification.

    • ↵b Only a single mutation was detected in the NA of each escape variant.

  • Table 5

    Inhibition of NA activity of MAb escape mutants by selecting MAbs

    MAb groupMAbIC50 (ng/ml) of MAb escape mutant bearing the indicated mutationa
    wt BR/07bK249EA250SA343EN273DV338MT339AT339IT339NT339PD341NI396TT397AT397NW456GN309S
    A3A210.4>
    4G27.7>>
    B1H525.9>>>
    2G668.1>>
    2D931.2>>
    3H1023.6>>>>
    4E927.01,269
    C1C785.7>
    3C233.2>>
    D2B528.2>
    3H463.4>
    E1H814.7200
    2D4196>>
    F4C41184≫
    • ↵a IC50, median inhibition concentration measured with enzyme-linked lectin assay. Each mutant virus was tested only with the MAb used for its selection. >, no inhibition at 2 μg/ml, the highest MAb concentration tested; ≫, no inhibition at 16 μg/ml, the highest MAb concentration tested.

    • ↵b IC50 of wt BR/07 virus was included for comparison.

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Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses
Hongquan Wan, Jin Gao, Kemin Xu, Hongjun Chen, Laura K. Couzens, Katie H. Rivers, Judy D. Easterbrook, Kevin Yang, Lei Zhong, Mohsen Rajabi, Jianqiang Ye, Ishrat Sultana, Xiu-Feng Wan, Xiufan Liu, Daniel R. Perez, Jeffery K. Taubenberger, Maryna C. Eichelberger
Journal of Virology Jul 2013, 87 (16) 9290-9300; DOI: 10.1128/JVI.01203-13

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Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses
Hongquan Wan, Jin Gao, Kemin Xu, Hongjun Chen, Laura K. Couzens, Katie H. Rivers, Judy D. Easterbrook, Kevin Yang, Lei Zhong, Mohsen Rajabi, Jianqiang Ye, Ishrat Sultana, Xiu-Feng Wan, Xiufan Liu, Daniel R. Perez, Jeffery K. Taubenberger, Maryna C. Eichelberger
Journal of Virology Jul 2013, 87 (16) 9290-9300; DOI: 10.1128/JVI.01203-13
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