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Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays

Ferrara, Francesca, Temperton, Nigel J. (2016) Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays. MethodsX, 4 . pp. 11-24. ISSN 2215-0161. (doi:10.1016/j.mex.2016.12.001) (KAR id:60125)


Recently chimeric influenza haemagglutinins (cHAs) have been generated as potential 'universal' vaccination antigens and as tools to identify HA stalk-directed antibodies via their use as antigens in ELISA, and virus or pseudotype-based neutralization assays. The original methods [1], [2] used for their generation require the amplification of regions of interest (head and stalk) using primers containing SapI sites and subsequent cloning into pDZ plasmid. This requires precise primer design, checking for the absence of SapI sites in the sequence of interest, and multi-segment ligation. As an alternative strategy we have developed and optimized a new protocol for assembling the cHA by exploiting Gibson Assembly. •This method also requires precise primer design, but it is rapid and methodologically simple to perform. We have evaluated that using this method it is possible to construct a cHA encoding DNA in less than a week.•Additional weeks are however necessary to optimize the production of pseudotyped lentiviral particles and to perform neutralization assays using them as surrogate antigens.•In comparison to the original protocols, we have also observed that performing parallel neutralization assays using pseudotypes harbouring the two parental HAs, permits effective delineation between stalk and head antibody responses in the samples tested.

Item Type: Article
DOI/Identification number: 10.1016/j.mex.2016.12.001
Subjects: Q Science > QR Microbiology > QR355 Virology
Divisions: Divisions > Division of Natural Sciences > Medway School of Pharmacy
Depositing User: Nigel Temperton
Date Deposited: 27 Jan 2017 13:21 UTC
Last Modified: 16 Feb 2021 13:42 UTC
Resource URI: (The current URI for this page, for reference purposes)

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