Maxi prep

The cDNA sequence encoding the ectodomain of desired HAs, or the variable domains from the heavy and light chains of the antibodies, were codon optimised for Chinese hamster (Cricetulus griseus) expression before synthesis as Geneblocks (Integrated DNA Technologies) together with the necessary 5′ and 3′ overlaps for subsequent InFusion cloning (ClonTech) into in-house generated mammalian expression vectors. These vectors contained either no trimerisation domain, the foldon trimerisation domain or the molecular clamp domain (derived from HIV-1 glycoprotein 41 heptad repeat (HR) regions, HR1 (aa540–576) and HR2 (aa619–656)) for the HA constructs, or the constant regions of human or mouse IgG1 heavy or light chain for the antibodies. The HA stem constructs, which were based on previously designed antigens³⁷ were cloned into a vector containing a monomeric Fc domain^{82 (link)}. After successful InFusion cloning, plasmid DNA was prepared by Midi- or Maxi-prep (Promega) and sequence confirmed at the Australian Genome Research Facility (AGRF).

Infectious CHIKV particles were produced by transfecting HEK-293T cells with the pACNR-CHIKV plasmid. The single-round infectious particles and virus-like-particles (VLPs) were produced in HEK-293T cells (producer cells) by either co-transfecting them with the pACNR-Rep and pVax-Help plasmids or just pVax-Help, respectively. The cells were seeded in a 6-well dish at a cell density of 250,000 cells/well. Twenty-four hours later, the cells were transfected with lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) reagent. The viral particles and VLPs were collected at 24 and 48 h post-transfection (h.p.t.), centrifuged at 1000 RFC for 10 min, and frozen at −80 °C. The expression of mKate2 and cell morphology were analyzed by fluorescence microscopy Lionheart FX Automated Microscope (BioTek Instruments, Winooski, VT, USA) using a Texas RED and DAPI filters, as well as a high-contrast phase mode. All the DNAs used for transfection were purified with the kit MaxiPrep (Promega Corporation, Fitchburg, WI, USA).