Chimeric influenza haemagglutinins: Generation and use in pseudotype neutralization assays

Abstract

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.

Keywords: Chimeric haemagglutinin; Chimeric influenza HA pseudotype production; Influenza pseudotypes; Virus neutralization.

Graphical abstract

Fig. 1

Chimeric haemagglutinin and cloning strategy to generate it. A. Structure of influenza haemagglutinin polypeptide highlighting the cleavage site, the head, the stalk, Cys52 and Cys277 that need to be identified in the HA sequences to proceed with cHA cloning; B. Cloning strategy used to build cHA using Gibson assembly method: after primer design the HA head and the HA stalk with the plasmid backbone are amplified by PCR from two different plasmids encoding the parental HAs; following purification of fragments, a Gibson assembly reaction is set up to obtain a cHA.

Fig. 2

NEBuilder Assembly Tool preferences.

Fig. 3

Entry of the parental 2 HA (stalk donor) sequence.

Fig. 4

Preferences to be set for parental 2 HA (stalk donor) sequence.

Fig. 5

Entry of the parental 1 HA (head donor) sequence.

Fig. 6

Preferences to be set for parental 1 HA (head donor) sequence.

Fig. 7

Example of designed primers. Oligonucleotide sequences to be ordered are reported in the table with information about the regions that they anneal to, and the Annealing Temperature (3′Ta) that should be used in the PCR to amplify the stalk plus vector and the head fragments.

Fig. 8

Amplification of HA stalk plus vector and HA head. In the agarose gel, two bands corresponding to the amplified product of the HA stalk plus vector of phCMV1-A/South Carolina/1/1918 H1 and of the HA head of phCMV1-A/duck/Memphis/546/1974 H11 can be observed.

Fig. 9

Production of chimeric haemagglutinin pseudotypes.