tetano
Editor, Senior Moderator
MethodsX. 2016 Dec 15;4:11-24. doi: 10.1016/j.mex.2016.12.001. eCollection 2017.
[h=1]Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays.[/h] Ferrara F[SUP]1[/SUP], Temperton N[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
[h=4]KEYWORDS:[/h] Chimeric haemagglutinin; Chimeric influenza HA pseudotype production; Influenza pseudotypes; Virus neutralization
PMID: 28070500 DOI: 10.1016/j.mex.2016.12.001
[PubMed - in process]
[h=1]Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays.[/h] Ferrara F[SUP]1[/SUP], Temperton N[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
[h=4]KEYWORDS:[/h] Chimeric haemagglutinin; Chimeric influenza HA pseudotype production; Influenza pseudotypes; Virus neutralization
PMID: 28070500 DOI: 10.1016/j.mex.2016.12.001
[PubMed - in process]