In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays

https://doi.org/10.1016/j.ab.2010.12.022Get rights and content

Abstract

There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans. Such assays require reliable subtype-specific sources of soluble and authentically folded seroreactive hemagglutinin (HA), one of the integral membrane proteins that determine the serological subtype of influenza viruses. To this purpose, a bigenic pFastBacDual baculovirus transfer vector allowing efficient in vivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed. An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum (ER). Highly seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay (ELISA) matrices, thereby avoiding any purification steps. The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic in vivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase. The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenza A virus serodiagnosis.

Section snippets

Target-primed plasmid amplification

Target-primed plasmid amplification as described previously [10] was used throughout. Briefly, megaprimers for target-primed plasmid amplification were amplified using a DeepVent (New England Biolabs) proofreading polymerase and primers (Biomers) of 60 nt length (detailed primer information is available on request). Amplified megaprimers were column- or gel-purified (Illustra DNA purification kit, GE Heathcare; QiaEx Band extraction kit, Qiagen). Megaprimers were used in concentrations of at least

Construction of recombinant baculoviruses

Initial attempts to express full-length IAV HA in insect cells failed because the recombinant antigen was trapped in insoluble intracellular deposits from which the protein could not be solved under native conditions using Triton X-100, Na-desoxycholate, Igepal Ca-630, zwittergent, Chaps, or N-dodecyl β-d-maltoside detergents (data not shown).

Therefore, alternative strategies of secretory baculovirus-based expression of a soluble HA variant were adopted and several constructs were produced (

Discussion

We have produced an array of recombinant influenza A HA subtype H5 and H7 constructs for expression in insect cells. Secretable fusion proteins that were in vivo mono-biotinylated using BirA biotin ligase-expressing bigenic baculoviruses proved to be superior in handling ease and authentic antigenicity over conventionally tag-purified and chemically biotinylated forms as a diagnostic antigen. The HA constructs were designed for primary use in serodiagnostic applications by developing a system

Acknowledgments

This study was funded by the Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV), Germany (FSI project 2-1.2.1). We are grateful to J. Stech (Institute for Molecular Biology, Friedrich Loeffler Institute, Germany) for offering his advice concerning target-primed plasmid amplification and to S. Boudko (Shriners Hospital, Portland, OR, USA) for providing the fibritin–foldon sequence. We are also grateful to the technical staff of the National Reference Laboratory for Avian

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