Mechanism of a Decrease in Potency for the Recombinant Influenza A Virus Hemagglutinin H3 Antigen During Storage

Hickey, John M.; Holtz, Kathleen M.; Manikwar, Prakash; Joshi, Sangeeta B.; McPherson, Clifton E.; Buckland, Barry C.; Srivastava, Indresh K.; Middaugh, C. Russell; Volkin, David B.

doi:10.1002/jps.23848

Cited by 17 publications

(11 citation statements)

References 20 publications

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“…Indeed, for the H3 subtype HAs, the K58I mutant and other stalk region mutations have been well characterized for recognition by a wide panel of conformation-specific monoclonal antibodies that target several of the antigenic regions of HA; in nearly all cases, the mutations have no affect on the antigenic properties of mutant HAs (52)(53)(54). Acid-stable HAs could also prove useful for improving inactivated vaccines and those based on expressed recombinant proteins, as these would be less susceptible to HA degradation (69,70), and the expression and purification of bromelain-released HA ectodomains can prove problematic for HAs with elevated fusion pH phenotypes (53,71). Therefore, incorporation of stabilizing mutations such as K58I might prove useful to improve yield or shelf life of HAs whether such reagents were to be utilized for vaccine purposes, for generating analytical sera, or assessing immune responses to new vaccine candidates and is broadly applicable to a wide range of subtypes across the phylogenetic spectrum of influenza A viruses.…”

Section: Discussionmentioning

confidence: 99%

Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

Byrd-Leotis

Galloway

Agbogu

et al. 2015

J Virol

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Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles. IMPORTANCEThe influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components. N early every year seasonal influenza A viruses contribute to hundreds of thousands of human deaths worldwide and place an extraordinary burden on health care systems and the global economy. Of even greater concern is the unpredictable appearance of antigenically novel influenza A strains in humans, with the potential for causing devastating pandemics. A major obstacle for controlling influenza is the diversity of influenza virus A strains that circulate in the avian species, since this provides a vast genetic pool of vi...

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Section: Discussionmentioning

confidence: 99%

Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

Byrd-Leotis

Galloway

Agbogu

et al. 2015

J Virol

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“…Moreover, specific point mutations can be introduced to enhance features such as stability. For example, it has recently been reported that purified hemagglutinin protein, the protective antigen in influenza vaccines, appears unstable in the SRID potency assay due to cross‐linking of specific cysteine residues in the protein [20]. By mutating these cysteine residues to non‐thiol residues, it was possible to prevent cross‐linking and enhance stability [21].…”

Section: Bevs Platform Features: Advantages and Considerationsmentioning

confidence: 99%

The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors

Felberbaum¹

2015

Biotechnology Journal

208

141

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The baculovirus expression vector system (BEVS) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been approved - four for human use (Cervarix(®), Provenge(®), Glybera(®) and Flublok(®)) and five for veterinary use (Porcilis(®) Pesti, BAYOVAC CSF E2(®), Circumvent(®) PCV, Ingelvac CircoFLEX(®) and Porcilis(®) PCV). The BEVS platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. This combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize BEVS to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. In this review, we explore the BEVS platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. To underscore the growth in opportunities for BEVS-derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of Glybera(®) and Flublok(®), respectively. We anticipate that the utility of the platform will expand even further as new BEVS-derived products attain licensure. Finally, we touch on some of the areas where new BEVS-derived products are likely to emerge.

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“…One approach to preparing for a pandemic outbreak is to establish stockpiles of vaccines for high-risk pandemic threats. However, the constant antigenic drift of HA and the limited shelflives of conventional vaccines make them not conducive to stockpiling and millions of doses of influenza vaccines must still be produced annually to replace the expired vaccines (13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Designed Nanoparticles Elicit Cross-Reactive Antibody Responses To Conserved Influenza Virus Hemagglutinin Stem Epitopes

et al. 2019

Preprint

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Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that an alpha-helical fragment (helix-A) from the conserved stem region can be displayed on nanoparticles. The stem region of HA on these nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, H1nanoparticles protected mice from lethal challenge with H1N1 influenza virus. Importantly, antibodies elicited by these nanoparticles demonstrated homosubtypic and heterosubtypic crossreactivity. The helix-A stem nanoparticle design represents a novel approach to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens up opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes. SignificanceInfluenza virus is a public health issue that affects millions of people globally each year.Commercial influenza vaccines are based on the hemagglutinin (HA) surface glycoprotein, which can change antigenically every year, demanding the manufacture of newly matched vaccines annually. HA stem epitopes have a higher degree of conservation than HA molecules contained in conventional vaccine formulations and we demonstrate that we are able to design nanoparticles that display hundreds of HA stem fragments on nanoparticles. These designed nanoparticles are heat-stable, elicit antibodies to the HA stem, confer protection in mouse challenge models, and show cross-reactivity between HA subtypes. This technology provides promising opportunities to improve seasonal vaccines, develop pandemic preparedness vaccines, and facilitate the development of a universal influenza vaccine.

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Mechanism of a Decrease in Potency for the Recombinant Influenza A Virus Hemagglutinin H3 Antigen During Storage

Cited by 17 publications

References 20 publications

Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors

Designed Nanoparticles Elicit Cross-Reactive Antibody Responses To Conserved Influenza Virus Hemagglutinin Stem Epitopes

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