Comparison between bacterial and mammalian IgG.

<p>(A). Non-reducing SDS-PAGE of 2 µg of pooled bacterial IgG eluate after protein L purification (EL) and mammalian cell culture-derived IgG (IgG) indicates that the majority of purified IgG was fully assembled IgG (2H2L) although partially assembled IgG (2H1L, 1H1L) are also present. (B). Direct binding ELISA of serially diluted bacterial (-▪-)- and mammalian cell culture (-•-)-derived IgG against Dengue serotype-2 virus and epsilon toxin showing similar binding curves. Binding of antibody was detected using anti-Human IgG Fc-HRPO as the secondary antibody.</p

Small scale optimization of bacterial IgG expression.

<p>(A-E) Left and middle panels: Non-reducing immunoblot of 7.5 µl clarified cell lysate from small scale overnight expression of bacterial IgG in shaking culture. All blots were probed with anti-Human Fc-HRPO and adjusted to ensure equal intensity. Arrows indicate fully-assembled IgG. Right panel: Direct binding ELISA indicating levels of functional IgG. Background binding signal was negligible for all cell lysate samples at the indicated dilution or neat. Error bars were calculated from average of three or four observations. Expressed antibodies were 4G2 (A), PA38 (B), PA64 (C), ET21 (D), ET149 (E). (F) Variation in wet cell mass for all five antibodies under different induction conditions. Mass is indicated in mg</p

Large scale purification of bacterial IgG.

<p>(A) Non-reducing coomassie gel of peak fractions from Protein A HPLC purification of cell lysate. Left panel: 4G2 fractions 5 to 11 (F5 to F11). Right panel ET149 fractions 10 to 16 (F10 to F16). 30 µl of each fraction was run on the gel. A sample of the wash (LW) was run and shows no contaminants present indicating the column was sufficiently washed to remove non-binding proteins. Fully assembled IgGs (2H2L) are indicated. (B). Reducing coomassie gels (4G2-F7, ET149-F12 & 13) and adjacent immunoblots (I) showing representative fractions from each Protein A elution (indicated with * on panel A). 30 µl of each fraction was loaded for coomassie and 3.75 µl for immunoblot. Blots were probed with both anti-IgG Fc and anti-Kappa chain polyclonals showing that majority of protein bands in the fraction are neither IgG heavy chain nor light chain. A separate reducing coomassie gel shows 2 µg of bacterial IgG pooled eluate (EL) after Protein L purification showing successful removal of the contaminating proteins and degraded heavy chain fragments. The equivalent amount of mammalian cell culture-derived IgG (IgG) was loaded for comparison. Individual heavy (HC) and light (LC) chains are indicated although the light chain for ET149 appears as two separate species.</p

Initial bacterial IgG expression and periplasmic extraction.

<p>(A and B) Non-reducing and reducing immunoblot of periplasmic extract from overnight expression of 4G2 in HB2151 (HB) and BL21(DE3) (BL) <i>E. coli</i> strains, respectively. All blots were probed with anti-Human Fc-HRPO.</p

Construction of tet promoter bacterial IgG expression plasmid.

<p>pASK-IBA2 plasmid using a <i>tet</i> promoter was used as the backbone expression vector. The appropriate restriction sites for cloning in of the light and variable heavy chains, leader sequences (OmpA, PelB) and the constant heavy chain sequence (CH) were added. Light (LC) and variable chains (VH) were cloned in as a complete construct together with the intercistronic ribosomal binding site (RBS) from the phage display vector or as separate constructs from the 4G2 mammalian IgG expression vector.</p

Site-directed mutagenesis of predicted epitope on prM-E protein.

<p>(A) To confirm the binding epitope of D29 Fab-IgG, residues within the P3 and P9 predicted sequences were mutated to generate mutants 1–6 (M1-6); M3/4, M3/5, M4/5 and M5/6 contain combinational-mutations as stated. (B) Reactivity of antibodies with the mutants was tested by Western blot analysis. Cleared lysate of DENV2-infected Vero cells (DV2), pCMV-prM-E- (prM-E) or mutants-transfected HEK 293 T cells (M1-5/6) were separated on 12% SDS-PAGE in non-reducing condition, followed by detection with h4G2, m2H2 and D29 Fab-IgG.</p

Localization of D29 Fab-IgG predicted epitopes in 3D crystal structures of DENV2 prM-E heterodimer (PDB 3C6E) and the binding specificity of peptide-phages.

<p>(A) Clusters of conformational epitopes predicted by Pepitope server are displayed on the prM-E heterodimer crystal structure at neutral pH with their solvent-accessible surfaces highlighted. The prM is pink, EDI is red, EDII is yellow, EDIII is blue, FP is cyan. (B) The binding specificity of peptide-phages (P1-P9) to D29 Fab-IgG was tested in a direct ELISA format with 10 µg/ml of D29 Fab-IgG, h3H5, m2H2 and non-DENV specific human antibody (Hu) immobilized on a Maxisorb plate. Ability of peptide-phages to inhibit D29 Fab-IgG binding to DENV2 was investigated by (C) ELISA and (D) Western blot analysis. For ELISA, peptide-phages (10¹² pfu/ml) were incubated with D29 Fab-IgG for 1 hr at RT before application to immobilized DENV2 for 5 min at RT. Bound D29 Fab-IgG was detected with HRP-conjugated anti-Human IgG-Fc. The percentage of inhibition of D29 Fab-IgG binding by the peptide-phages shown is the average of three experiments. Error bars represent the standard errors of the mean (***p-value <0.005). For Western blot analysis, 0.5 µg/ml of D29 Fab-IgG was incubated with 8×10⁶ pfu of purified DENV2, 5% SM or 4×10¹¹ pfu of peptide-phage clones for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT.</p

Localization of P3 and P9 peptide sequence on 3D crystal structure of prM-E heterodimer (PDB 3C6E).

<p>P3 (Purple) and P9 (green) peptide sequences were aligned with the predicted clusters (Cluster 1 – Navy blue; Cluster 2 – Black) on the prM-E crystal structure. The path of peptide phage sequences was highlighted with the participating residues from the cluster and the peptide phage labeled. Matched residues were purple (P3) or green (P9) and numbered accordingly; mis-matched residues were grey. The respective proteins and domains were highlighted as above.</p

Competition Assays and Western Blots of D29 Fab-IgG and monoclonal anti-DENV antibodies with known epitope.

<p>(A) Serially diluted h3H5, h4G2 or m2H2 was incubated with immobilized DENV2 (2×10⁷ pfu/ml) for 1 hr at RT before addition of 2.5 µg/ml of D29 Fab-IgG for a further hour at RT. Bound D29 Fab-IgG was detected by HRP-conjugated anti-human IgG-Fc antibody. 2.5 µg/ml of HRP-conjugated m2H2 was used to compete against m2H2. Values displayed are the average of three independent experiments and error bars represent standard errors of the mean. For Western Blot analysis, DENV1-4 (D1-D4) infected Vero cell lysate and 0.5 µg of recombinant DENV2 E protein (ecto-domain) (rE) were separated on 12% SDS-PAGE in (B) non-reducing, (C) reducing conditions; followed by detection with 1 µg/ml of h3H5, h4G2, m2H2 and D29 Fab-IgG. (D) For competition Western blot analysis, 0.5 µg/ml of D29 Fab-IgG was incubated with 1 µg/ml of h4G2, h3H5, m2H2 for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT.</p

Immunoprecipitation of DENV2 proteins with anti-DENV antibodies.

<p>(A) For immunoprecipitation in native condition, DENV2-infected BHK cells were incubated with media supplemented with 250 µCi/ml ³⁵S-methionine before lysis. Viral proteins were precipitated with D29 Fab-IgG or h4G2. (B) For immunoprecipitation with 1.25% SDS, cleared lysate of DENV2-infected BHK cells were incubated with h3H5, h4G2, m2H2, D29 Fab-IgG or non-DENV specific human antibody (IgG) followed by analysis with silver staining and Western blotting. Precipitated prM and E proteins were verified by (anti-prM) HRP-conjugated m2H2 or (anti-E) h3H5 respectively. HC - antibody heavy chain; LC - antibody light chain.</p