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Heterosubtypic antibodies to influenza A virus have limited activity against cell-bound virus but are not impaired by strain-specific serum antibodies - PubMed

Heterosubtypic antibodies to influenza A virus have limited activity against cell-bound virus but are not impaired by strain-specific serum antibodies

Arkadiusz Wyrzucki et al. J Virol. 2015 Mar.

Abstract

The majority of influenza virus-specific antibodies elicited by vaccination or natural infection are effective only against the eliciting or closely related viruses. Rare stem-specific heterosubtypic monoclonal antibodies (hMAbs) can neutralize multiple strains and subtypes by preventing hemagglutinin (HA)-mediated fusion of the viral membrane with the endosomal membrane. The epitopes recognized by these hMAbs are therefore considered promising targets for the development of pan-influenza virus vaccines. Here, we report the isolation of a novel human HA stem-reactive monoclonal antibody, hMAb 1.12, with exceptionally broad neutralizing activity encompassing viruses from 15 distinct HA subtypes. Using MAb 1.12 and two other monoclonal antibodies, we demonstrate that neutralization by hMAbs is virtually irreversible but becomes severely impaired following virus attachment to cells. In contrast, no interference by human anti-influenza virus serum antibodies was found, indicating that apically binding antibodies do not impair access to the membrane-proximal heterosubtypic epitopes. Our findings therefore encourage development of new vaccine concepts aiming at the induction of stem-specific heterosubtypic antibodies, as we provide support for their effectiveness in individuals previously exposed to influenza virus.

Importance: The influenza A virus hemagglutinin (HA) can easily accommodate changes in its antigenic structures to escape preexisting immunity. This variability restricts the breadth and long-term efficacy of influenza vaccines. Only a few heterosubtypic antibodies (hMAbs), i.e., antibodies that can neutralize more than one subtype of influenza A virus, have been identified. The molecular interactions between these heterosubtypic antibodies and hemagglutinin are well characterized, yet little is known about the functional properties of these antibodies. Using a new, extraordinarily broad hMAb, we show that virus neutralization by hMAbs is virtually irreversible and that efficient neutralization is possible only if stem-specific hMAbs bind to HA before the virus attaches to the cell surface. No interference between strain-specific human serum immunoglobulin and hMAbs was found, indicating that preexisting humoral immunity to influenza virus does not limit the efficacy of stem-reactive heterosubtypic antibodies. This knowledge supports the development of a pan-influenza virus vaccine.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Figures

FIG 1
FIG 1

Specificity of hMAb 1.12. (a) Half-maximal binding concentrations (EC50s) of hMAb 1.12 to recombinantly expressed HA proteins from 9 subtypes. The data represent the means of EC50s obtained in two independent experiments. (b) Half-maximal neutralizing concentrations (IC50s) of MAb 1.12 to a panel of 19 viruses from 15 subtypes. The HIV-1 gp120-specific MAb b12 was used as a negative control in both experiments (αHIV). A representative of at least 2 independent, consistent experiments performed in triplicate is shown. Avian viruses are depicted with open symbols, human isolates with solid symbols, and bat isolates with asterisk-like symbols. Isolates with zoonotic potential are designated with mixed symbols. Isolates from phylogenetic group 1 are depicted in blue and those of phylogenetic group 2 in red. A dashed line is used to indicate the detection limit of the assay. (c) The epitope recognized by hMAb 1.12 was roughly evaluated in a binding-competition ELISA using HA stem-reactive hMAb c179. ELISA plates coated with purified HA from A/Puerto Rico/8/1934(H1N1) were incubated with titrated amounts of hMAb 1.12, washed, and later incubated with a fixed concentration (1 μg/ml) of the murine hMAb c179. Binding of both antibodies was then detected using species-specific secondary antibodies. (d) Phylogenetic tree for all 18 HA subtypes. At total of 1,339 arbitrarily chosen recent nonidentical HA amino acid sequences (from the year 2000 to the present for frequent isolates; from 1985 to the present for rare isolates) were aligned using Muscle 3.8 (34). The tree was built using “neighbor” from the Phylip 3.69 software package (

http://evolution.genetics.washington.edu/phylip.html

) and illustrated as a rooted tree in FigTree 1.4 (

http://tree.bio.ed.ac.uk/software/figtree

). Phylogenetic group 1 is indicated in blue and group 2 in red.

FIG 2
FIG 2

Protection of mice and reversibility of neutralization. (a) Passive immunization of mice. Animals were injected intraperitoneally with the indicated dose of hMAb 1.12 in PBS or with PBS alone (control group) 24 h prior to intranasal infection with a lethal dose of A/Puerto Rico/8/34(H1N1) or mouse-adapted A/Hong Kong/1/1968(H3N2). Mice that dropped below 80 or 85% of their initial body weight were scored as dead and euthanized. For A/Puerto Rico/8/1934(H1N1), pooled data from two separate experiments are shown, each performed with 5 C57BL/6 females per group. One experiment was performed with 15% and one with 20% weight loss as the abortion criteria. For mouse-adapted A/Hong Kong/1/1968(H3N2), only the depicted experiment with a 20% weight loss abortion criterion was performed. (b) Reversibility of neutralization. Three HA stem-reactive antibodies were incubated at a concentration of 10 μg/ml with A/Puerto Rico/8/1934(H1N1), A/California/07/2009(H1N1), A/Brisbane/59/2007(H1N1), A/Brisbane/10/2007(H3N2), or A/Chicken/Vietnam/C58/2004(H5N3) virus (amounts corresponding to an MOI of ∼ 30) before the MAb-virus mixture was captured on magnetic beads. The beads were then processed so that no-dissociation and long-term-dissociation conditions were applied. In the last step, the residual infectivity of each sample was measured on MDCK cells. The HIV-1 gp120-specific MAb b12 was used as a negative control (αHIV). Uninfected and infected cells were included as positive and negative controls, respectively. Representatives of at least 2 consistent experiments (performed in duplicate) are shown. The error bars indicate standard deviations.

FIG 3
FIG 3

Neutralization of virus attached to cell surfaces. A/Puerto Rico/8/1934(H1N1) or A/California/7/2009(H1N1) virus was preadsorbed to MDCK cells at 4°C to avoid virus internalization. The attached viruses were then incubated with titrated amounts of HA stem-reactive (3.1, 1.12, and FI6) or hemagglutination-inhibiting (30D1 and H36-4) MAbs, and residual infectivity was detected. As a control, viruses were mock incubated in cell-free medium before titrated amounts of the MAbs were added. The HIV-1 gp120-specific MAb b12 (αHIV) was used as a nonneutralizing control. Gray areas highlight the differences between the neutralization curves, and numbers indicate the fold difference in the corresponding IC50s. Representatives of at least 2 independent, consistent experiments (performed in duplicate) are shown.

FIG 4
FIG 4

Competition with human sera. A/Puerto Rico/8/1934(H1N1) virus was first incubated with sera from the indicated donors for 1 h before the indicated hMAbs were added at a concentration of 10 μg/ml. Residual infectivity of the sample was evaluated and compared to the infectivity of samples without preincubation with human serum. The low serum concentration corresponds to the dilution giving saturated signals in ELISA while having only minor neutralizing activity against the virus. The high serum concentration is three times higher than the low concentration. Representatives of 2 independent, consistent experiments (performed in triplicate) are shown. The error bars indicate standard deviations.

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