Figure 4.

NC0321 mAb expression by rAAV and rSIV.F/HN vectors. (A) Schematic of a codon-optimised, single open-reading frame, human IgG mAb cDNA. Regions encoded include IgG heavy and kappa light chain variable and constant regions, with each proceeded by a human growth hormone signal sequence (hGH SS) and joined by a Furin/2A (F2A) protein cleavage site. (B) The SARS-CoV-2 receptor binding domain (RBD) protein binding activity of the anti-SARS-CoV-2 mAbs NC0321 and CR3022 single open reading frame protein expressed from an rSIV.F/HN vector) was examined by ELISA. Binding activity of OD at 450 nm is proportional to the amount of antibody bound to the SARS-CoV-2 RBD protein. (C) The neutralising activity of anti-SARS-CoV-2 mAb NC0321 (single open reading frame protein expressed from an rSIV.F/HN vector) to block S-LV infection (multiplicity of infection 1) of an hACE2-293T cell line was examined. In (B and C) a single open reading frame anti-influenza mAb T1-3B acted as an isotype negative control; and CR3022 an anti-SARS-CoV-2 neutralising mAb, that can bind but not neutralise SARS-CoV-2 was used as a comparator.⁴⁰ (D) Serum human IgG levels in mice were determined at day 7, 14 and 28 after transduction with the indicated doses of NC0321 expressing vector using ELISA (ANOVA, Dunnett’s multiple comparison against the unlabelled treatment group; *** and **** represent <0.001 and <0.0001, respectively). The levels of human IgG observed in naïve animals is indicated by the dotted line. (E) BALF of mice from (D) was collected at day 28 post-transduction with NC0321 expressing vector, and human IgG levels measured using ELISA; IgG levels in epithelial lining fluid (ELF) were computed by comparison of urea levels in BALF and serum (Kruskal Wallis, Dunn’s multiple comparison against the unlabelled treatment group; ns, **,*** and **** represent p>0.05, <0.01, <0.001 and <0.0001, respectively). Each dot represents an individual mouse and data are presented as endpoint titres (mean±SD).