Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms

McCraw, Dustin M.; Gallagher, John R.; Harris, Audray K.

doi:10.1128/cvi.00085-16

Cited by 15 publications

(24 citation statements)

References 60 publications

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“…5A). After adjusting the pH to 5.25 for 10 min, BHA aggregates into rosettes commonly observed in solubilized fusion glycoproteins due to the formation of the stable postfusion helical bundle with tightly clustered fusion peptides (21)(22)(23).…”

Section: Resultsmentioning

confidence: 99%

Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments

Williams

Gui

Hom

et al. 2018

J Virol

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The neutralizing antibody (nAb) response against the influenza virus's hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency on inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor-binding pocket at HA's distal end, while FI6v3 binds primarily to the HA2 fusion subunit towards the base of the stalk. Surprisingly, HC19 inhibited HA's ability to induce lipid mixing by preventing structural rearrangement of HA under fusion activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding, but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to crosslink separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that inter-spike crosslinking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared down Fab-soluble HA structures.The influenza virus's hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of Ab-mediated neutralization largely relies on high resolution characterization of antigen binding fragments (Fab) in complex with soluble, isolated antigen constructs by cryo-EM single particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well-characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occurs by multiple coexisting mechanisms and is largely dependent on the specific epitope that is targeted and is highly dependent on the bivalent nature of IgG molecules.

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Section: Resultsmentioning

confidence: 99%

Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments

Williams

Gui

Hom

et al. 2018

J Virol

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“…The rHAs present in Flublok are extracted from insect cells with a detergent. When the detergent is later removed, the hydrophobic regions of the trimerization domain cluster together again and form rosettes (9,34,35). As a consequence, epitopes on the stalk might be partially hidden (compared to soluble HA trimers) but are comparable to subunit vaccines where rosettes will form as well.…”

Section: Discussionmentioning

confidence: 99%

Monoclonal Antibody Responses after Recombinant Hemagglutinin Vaccine versus Subunit Inactivated Influenza Virus Vaccine: a Comparative Study

Henry

Palm

Utset

et al. 2019

J Virol

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Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok) and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (MAbs) and 38 Flucelvax-induced MAbs for avidity, cross-reactivity, and any selectivity toward the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of MAbs targeting epitopes near the receptor-binding domain on HA head (hemagglutinin inhibition-positive MAbs) than Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive MAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive antibody-secreting cells, showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head. IMPORTANCE There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax, that use alternate production strategies can induce protective influenza-specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.

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“…Prediction software has been developed for the purpose of recognizing both conformational and linear BCR epitopes, indicating that receptors do preferentially interact with epitopes displaying certain quantifiable properties (i.e., patterns in sequence) (108). However, the utility of these predictions when considering PBV design is limited due to small and inconsistent datasets, difficulties predicting antigen 3D structure, and the structural heterogeneity exhibited by some PBVs (108)(109)(110)(111). The last consideration, epitope size, consists of constraints that are somewhat vague.…”

Section: Mechanisms Behind Bcr Recognition Of Pbvmentioning

confidence: 99%

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

et al. 2020

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Today, vaccinologists have come to understand that the hallmark of any protective immune response is the antigen. However, it is not the whole antigen that dictates the immune response, but rather the various parts comprising the whole that are capable of influencing immunogenicity. Protein-based antigens hold particular importance within this structural approach to understanding immunity because, though different molecules can serve as antigens, only proteins are capable of inducing both cellular and humoral immunity. This fact, coupled with the versatility and customizability of proteins when considering vaccine design applications, makes protein-based vaccines (PBVs) one of today's most promising technologies for artificially inducing immunity. In this review, we follow the development of PBV technologies through time and discuss the antigen-specific receptors that are most critical to any immune response: pattern recognition receptors, B cell receptors, and T cell receptors. Knowledge of these receptors and their ligands has become exceptionally valuable in the field of vaccinology, where today it is possible to make drastic modifications to PBV structure, from primary to quaternary, in order to promote recognition of target epitopes, potentiate vaccine immunogenicity, and prevent antigen-associated complications. Additionally, these modifications have made it possible to control immune responses by modulating stability and targeting PBV to key immune cells. Consequently, careful consideration should be given to protein structure when designing PBVs in the future in order to potentiate PBV efficacy.

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Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms

Cited by 15 publications

References 60 publications

Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments

Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments

Monoclonal Antibody Responses after Recombinant Hemagglutinin Vaccine versus Subunit Inactivated Influenza Virus Vaccine: a Comparative Study

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

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