A Pan-H1 Anti-Hemagglutinin Monoclonal Antibody with Potent Broad-Spectrum Efficacy<i>In Vivo</i>

Tan, Gene S.; Krammer, Florian; Eggink, Dirk; Kongchanagul, Alita; Moran, Thomas M.; Palese, Peter

doi:10.1128/jvi.00469-12

Cited by 156 publications

(209 citation statements)

References 36 publications

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“…The broadly neutralizing mAbs 6F12 (pan-H1) (7) and 9H10 (pan-H3) (8) that recognize the HA stalk have been described previously. The mAbs PY102, 6F12, XY102, and 9H10 were harvested from hybridomas and purified as described previously (7). The FLAG-specific mAb is a commercially available antibody (635691; Clontech).…”

Section: Methodsmentioning

confidence: 99%

“…However, because the globular head of the HA is antigenically more variable, antibodies that target the head and inhibit hemagglutination are limited in their capacity to neutralize divergent strains. In contrast, the stalk region is much more conserved, and antibodies elicited against conserved stalk epitopes have been shown to be broadly protective (6)(7)(8)(9)(10)(11)(12)(13).…”

mentioning

confidence: 99%

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Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Leon

Mullarkey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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108

107

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Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab′) 2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells. hemagglutinin | influenza virus | antibody-dependent cell-mediated immunity | broadly reactive antibodies | Fcγ receptor

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Leon

Mullarkey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

108

107

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“…Using these plasmids we were able to quickly construct baculoviral expression vectors for production of recombinant HA and NA in insect cells. This expression system is well established in our laboratory and has proven pivotal to many of our ongoing research projects [1][2][3][4][5][6][7][8] . Insect cells are able to correctly fold and post-translationally modify complex proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Their use as molecular baits allows to specifically sort for plasmablasts or other types of B-cells that can then be used to isolate (therapeutic) monoclonal antibodies (mAbs). Recombinant HA and NA molecules are further used in the characterization of these mAbs 8 . Other examples include study the pH stability or receptor specificity of HAs expressed by different virus isolates, or quantitatively assessing specific NA enzymatic activity or resistance to inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Margine

Palese

Krammer

2013

JoVE

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146

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The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins -the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins. Video LinkThe video component of this article can be found at

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“…Recently, broadly neutralizing antibodies against this domain of the HA have been isolated (16)(17)(18)(19)(20)(21)(22), suggesting that a vaccine based on the induction of such antibodies would protect from infection with divergent strains within a subtype and also against strains from other subtypes that have similar stalk structures. It is of note that these antibodies are HI negative and that their mechanism of neutralization is likely to be different from the mechanism through which antibodies against the globular head domain work (16,(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

Margine

Krammer

Hai

et al. 2013

J Virol

186

161

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f Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.

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A Pan-H1 Anti-Hemagglutinin Monoclonal Antibody with Potent Broad-Spectrum EfficacyIn Vivo

Cited by 156 publications

References 36 publications

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

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