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. 2001 Nov;75(21):10208-18.
doi: 10.1128/JVI.75.21.10208-10218.2001.

Hemagglutinin 1-specific immunoglobulin G and Fab molecules mediate postattachment neutralization of influenza A virus by inhibition of an early fusion event

M J Edwards  1 , N J Dimmock
Affiliations

Hemagglutinin 1-specific immunoglobulin G and Fab molecules mediate postattachment neutralization of influenza A virus by inhibition of an early fusion event

M J Edwards et al. J Virol. 2001 Nov.

Abstract

In standard neutralization (STAN), virus and antibody are reacted together before inoculation of target cells, and inhibition of almost any of the processes concerned in the early interaction of virus and cell, including inhibition of virus attachment to cell receptors, can be the cause of neutralization by a particular monoclonal antibody (MAb). To simplify the interpretation of antibody action, we carried out a study of postattachment neutralization (PAN), where virus is allowed to attach to target cells before neutralizing antibody is introduced. We used influenza virus A/PR/8/34 (H1N1) and monoclonal immunoglobulin G (IgG) molecules and their Fabs specific to antigenic sites Sb (tip), Ca2 (loop), and Cb (hinge) of the hemagglutinin 1 (HA1) protein. All IgGs and Fabs gave PAN, although with reduced efficiency compared with STAN. Thus, bivalent binding of antibody was not essential for PAN. By definition, none of these MAbs gave PAN by inhibiting virus attachment, and they did not elute attached virus from the target cell or inhibit endocytosis of virus. However, virus-cell fusion, as demonstrated by R18 fluorescence dequenching or hemolysis of red blood cells, was inhibited in direct proportion to neutralization and in a dose-dependent manner and was thus likely to be responsible for the observed neutralization. However, to get PAN, it was necessary to inhibit the activation of the prefusion intermediate, the earliest known form on the fusion pathway that is created when virus is incubated at pH 5 and 4 degrees C. PAN antibodies may act by binding HA trimers in contact with the cell and/or trimers in the immediate vicinity of the virus-cell contact point and so inhibit the recruitment of additional receptor-HA complexes.

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Figures

FIG. 1
FIG. 1
Model of an HA1 monomer of PR8 influenza virus showing the positions of the antigenic sites and epitopes of antibodies used in this study (adapted from references and 56).
FIG. 2
FIG. 2
Comparison of the percent PAN and STAN of PR8 virus by plaque assay in MDCK cell monolayers, as a function of IgG or Fab concentration (M). For PAN, virus was allowed to attach to a prechilled monolayer at 4°C and washed, and then antibody was added at 4°C for 120 min or 37°C for 60 min. For STAN, virus-antibody mixtures were incubated together at 20°C and inoculated onto monolayers at 37°C for 60 min. Monolayers were washed to remove virus and antibody before being overlaid with agar. (a and d) H36 IgG (a) and Fab (d); (b and e) H37 IgG (b) and Fab (e); (c and f) H9 IgG (c) and Fab (f). ⧫, PAN at 4°C; ▴, PAN at 37°C; ■, STAN at 37°C. Virus controls gave approximately 50 PFU/plate. Data are the mean of three experiments, and the bars represent the standard error of the mean. Curves were generated by Prism Graphpad software. R was >0.97 for all curves. The value of P1 shows the significance of the difference between the two curves of 4°C PAN and 37°C STAN, and that of P2 shows the same for 37°C PAN and 37°C STAN. These were calculated using an unpaired t test. P < 0.05 is considered significant.
FIG. 3
FIG. 3
Analysis of the kinetics of PAN of PR8 virus by H36 IgG and Fab at 4 and 37°C in MDCK cell monolayers, as a function of antibody concentration (M). PAN is expressed as a percentage. (a) IgG (55 nM); (b) Fab (100 nM). Data were generated from plaque assays and are the mean of two experiments. Bars represent the standard error of the mean. Curves were generated as in Fig. 2. ▴, 4°C; ■, 37°C.
FIG. 4
FIG. 4
Analysis of the relationship between PAN and internalization of PR8 virus by MDCK cell monolayers as a function of antibody concentration (M). PAN and internalization were assayed in parallel in the same batch of monolayers in 96-well trays, both by ELISA, and are expressed as a percentage. (a) H36 IgG; (b) Fab. ▴, PAN. Columns represent internalization. The internalization virus control gave values of 1.2 to 1.4 OD units, and the PAN virus control gave a value of 1.1 OD units. Data are the mean of three experiments, and the bars represent the standard error of the mean. Curves were generated as in Fig. 2. R > 0.97 for all curves.
FIG. 5
FIG. 5
Analysis of the relationship between PAN and virus-cell fusion of R18-labeled PR8 virus as a function of antibody concentration (M). PAN and fusion are expressed as a percentage. All assays were carried out at the same time in the same batch of 3-cm-diameter monolayers. PAN was measured by ELISA, and fusion was measured by fluorescence dequenching of R18. (a and b) H36 IgG (a) and Fab (b); (c) H9 Fab. ▴, PAN; ■, fusion inhibition. Virus controls gave values of 1.2 to 1.4 OD units. All data are the mean of three experiments, and the bars represent the standard error of the mean. Curves were generated as in Fig. 2. R > 0.97 for all curves. P values were calculated using an unpaired t test, where P < 0.05 is considered significant.
FIG. 6
FIG. 6
Inhibition of hemolysis of RBCs by preattached PR8 virus by antibody. Hemolysis is presented as a percentage. Virus was incubated with cells at 4°C for 30 min, and antibody was added for 1 h at 37°C. Incubation was then continued at pH 5 and 37°C for 45 min to allow hemolysis to occur. (a and b) H36 IgG (a) and Fab (b); (c) H9 Fab. Cells were pelleted, and released hemoglobin was determined spectrophotometrically at 520 nm. Data are the mean of four experiments, and the bars represent the standard error of the mean.
FIG. 7
FIG. 7
H9, H36, H37, and Y8 IgGs bound to virus that had been treated at pH 5. Virus was captured by HA-specific IgA and treated at pH 5 or 7.5 at 37°C as shown. The pH was then adjusted to 7.5, and the virus was incubated with various IgGs. MAb Y8 is specific for a low-pH-induced epitope in the Sa site of HA1. Binding of antibodies was determined by ELISA. PBS controls show the level of nonspecific binding of IgGs. Data are the mean of four experiments, and the bars represent the standard error of the mean.
FIG. 8
FIG. 8
Hemolysis of chicken RBCs by the prefusion intermediate of PR8 and its inhibition by neutralizing amounts of H36 IgG and Fab. Virus (10,000 HAU/ml) was mixed with H36 IgG (67 nM) or H36 Fab (200 nM). These concentrations of antibody gave 90% PAN of the virus used here in MDCK cells. Virus was attached to RBCs at 4°C for 30 min at pH 7.5 and then treated at pH 5 and 4°C for 30 min to activate the prefusion intermediate or at pH 7.5. Virus in column 7 was treated at pH 5 and 37°C before being mixed with RBCs. PBS or H36 IgG or Fab was then added at 37°C for 60 min, and mixtures were incubated for 45 min at pH 5 and 37°C for hemolysis to occur. Released hemoglobin was determined spectrophotometrically at 520 nm. Data are the mean of three experiments, and the bars represent the standard error of the mean. Numbers in parentheses refer to each experimental procedure.
FIG. 9
FIG. 9
PAN amounts of H36 Fab fail to inhibit the appearance of low pH-induced conformational changes in the HA1 of PR8 virus bound to fixed MDCK cells monolayers. Virus (200 HAU) was allowed to attach to cells at 4°C and then incubated with 200 nM H36 Fab at 4 or 37°C as indicated. The Fab gave >99% PAN of the virus used here in MDCK cells. After washing, virus-cell complexes were incubated at pH 5 or 7.5 and at 4 or 37°C for 30 min as shown and then probed with IgGs (as shown) specific for the pH-sensitive epitopes of the HA. Binding of antibodies was determined by ELISA. Data are the mean of three experiments, and the bars represent the standard error of the mean. Numbers in parentheses refer to each experimental procedure.
FIG. 10
FIG. 10
H36 Fab fails to inhibit the acquisition of protease sensitivity of PR8 HA at pH 5. Virus (100,000 HAU) was incubated with 2.5 μM H36 Fab at 37°C for 1 h. The Fab gave 90% STAN of the virus used here in MDCK cells. The mixture was then held at pH 5 or 7.5 at 37°C for 30 min as shown. After the pH was restored to 7.5, all mixtures were reacted with proteinase K (1 μg/ml) at 37°C for 30 min to determine if conformational changes to the HA had taken place. Lanes 3 and 5 contain Fab and no virus. Virus not incubated with protease was indistinguishable from virus in lane 2 (data not shown). Proteins were analyzed by PAGE under reducing conditions and stained with Coomassie blue.
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References

    1. Arendrup M, Sönnerborg A, Svennerholm B, Åkerblom L, Nielsen C, Clausen H, Olofsson S, Nielsen J O, Hansen J-E S. Neutralizing antibody response during human immunodeficiency virus type 1 infection: type and group specificity and viral escape. J Gen Virol. 1993;74:855–863. - PubMed
    1. Armstrong S J, Dimmock N J. Neutralization of influenza virus by low concentrations of HA-specific polymeric IgA inhibits viral fusion activity but activation of the ribonucleoprotein is also inhibited. J Virol. 1992;66:3823–3832. - PMC - PubMed
    1. Armstrong S J, Dimmock N J. Varying temperature-dependence of post-attachment neutralization of human immunodeficiency virus type 1 by monoclonal antibodies to gp120: identification of a very early fusion-independent event as a neutralization target. J Gen Virol. 1996;77:1397–1402. - PubMed
    1. Armstrong S J, McInerney T L, McLain L, Wahren B, Hinkula J, Levi M, Dimmock N J. Two neutralizing anti-V3 monoclonal antibodies act by affecting different functions of human immunodeficiency virus type 1. J Gen Virol. 1996;77:2931–2941. - PubMed
    1. Bentz J. Minimal aggregate size and minimal fusion unit for the first fusion pore of influenza hemagglutinin-mediated membrane fusion. Biophys J. 2000;78:227–245. - PMC - PubMed

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