• Authors: Du Y. et al.
  • Year: 2022
  • Journal: Thorax
  • Applications: in vitro / DNA / PEIpro
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T


rSIV.F/HN: recombinant Simian Immunodeficiency Virus (SIV) pseudotyped with the Fusion (F) and Haemagglutinin-Neuraminidase (HN) surface glycoproteins from Sendai virus. S-LV: a SARS-CoV-2 pseudovirus created from a recombinant HIV1 lentiviral vector pseudotyped with the D614G derivative of the SARS-CoV-2 Spike (S) protein The production of rAAV, rSIV.F/HN and S-LV vectors was performed by cotransfection of HEK-293T cells with genome and packaging plasmids using (rAAV) Polyethylenimine (PEI, PolySciences) or (rSIV.F/HN and SLV) PEIpro® (Polyplus). All rSIV vectors were pseudotyped with the F and HN proteins of Sendai virus as described previously. The S-LV (rHIV.Spike.G614+∆19aa.CMV) vector was pseudotyped with the SARS-CoV-2 Spike protein Wuhan sequence (GenBank accession: 43740568) modified by inclusion of a D614G mutation and removal of the 19 C-terminal amino acids. Vectors were purified by (rAAV) discontinuous Iodixanol gradient ultracentrifugation 7 or (rSIV.F/HN, S-LV) anion exchange chromatography and tangential flow filtration. Physical titre (genome copies/mL: GC/mL) of rAAV vectors was determined by quantitative polymerase chain reaction (qPCR) analysis with primers and a probe against WPRE 8. Functional titre of rSIV.F/HN vectors (transducing units/mL: TU/mL) was determined using the same primer/probes on DNA extracted following transduction of HEK-293F cells with dilutions of vector preparations Physical titre (ng p24) of S-LV particles was determined using a p24 immunoassay


Background: The COVID-19 pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines. Aims: We hypothesised that passive immunity engineered by the in vivo expression of anti-SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored-immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age. Methods: We developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs. Conclusions: A single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV-2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV-2 vaccines. The in vivo expression of multiple anti-SARS-CoV-2 mAbs could enhance protection and prevent rapid mutational escape.