Lectin Blot using <i>Ricinus communis</i> agglutinin I.

<p>Cellular supernatants were harvested 96 hpi and purified 3D6 antibodies were separated on SDS-PAGE and binding of biotinylated <i>Riccinus communis</i> agglutinin I to antibody heavy chains was tested. The lectin only bound SweetBac expressed antibodies (Hi5Glyco, Tn38Glyco), because of terminal galactose present on N-glycans. CHO and Mimic insect cell expressed antibodies were used as controls.</p

Expression of 3D6 antibody in lepidopteran insect cells.

<p>(A) <i>Tnao</i>38 and Hi5 cells were infected in triplicate with recombinant baculovirus expressing 3D6 antibody. Samples were taken every 24 h and the amount of secreted antibody was measured by ELISA. (B) <i>Tnao</i>38 and Hi5 cells were again infected in triplicate with recombinant baculovirus (Tnao38, Hi5) and SweetBac virus (<i>Tnao</i>38 glyco, Hi5 glyco) expressing 3D6 antibody. Samples were taken 72 and 96 hours post infection (hpi) and the amount of secreted antibody was measured by ELISA. Significance of the results was confirmed by Student's t-test (<i>p</i>-value<0,05).</p

MALDI-TOF-MS spectra of 3D6 IgG₁ N-glycans.

<p>N-glycan structures of an IgG₁ antibody expressed in <i>Tnao</i>38 (A) and Hi5 (C) cells (96 hpi) mostly consist of single and double fucosylated tri-mannose structures with maximally one terminal <i>N</i>-acetylglucosamine. The spectra of the mammalianised cells (B, D) show a shift of the dominant structures towards higher m/z values. Fucosylated biantennary N-glycan structures carrying two <i>N</i>-acetylglucosamine residues with one or two terminal galactose residues were identified as dominant N-glycan on IgG₁ recombinantly expressed in <i>Tnao</i>38 cells using SweetBac (B). The N-glycan structures identified from IgG₁ expressed in Hi5 cells using SweetBac (D) resemble the ones found in SweetBac infected <i>Tnao</i>38 cells with an even higher dominance of terminal galactose. Oligomannosidic glycans (Man_5–6GlcNAc₂) are indicated by asterisks. Graphical representations of glycans are consistent with the nomenclature of the Consortium for Functional Glycomics.</p

Binding of insect cell expressed 3D6 antibodies to 3D6 epitope and human FcγRI.

<p><i>Sf</i>9 cells presenting the 3D6 epitope were incubated with purified antibodies and target binding was analysed by FACS. Antibodies expressed in Hi5 and <i>Tnao</i>38 cells using standard baculoviral vectors (Hi5, <i>Tnao</i>38) as well as SweetBac (Hi5 Glyco, <i>Tnao</i>38 Glyco) show a highly specific binding. The broadening of the peaks can be explained by different amounts of 3D6 epitope displayed on <i>Sf</i>9 cells (A). Binding of antibodies to human Fc gamma receptor I was measured by incubating 3D6 antibodies with U937 cells. FACS analysis showed that all antibodies bound FcγRI present on the cellular surface, but the binding of SweetBac expressed variants (Hi5 Glyco, <i>Tnao</i>38 Glyco) was significantly increased.</p

Additional file 5: Figure S4. of New insights into HCV replication in original cells from Aedes mosquitoes

HCV Infection protocols. (A) for human HepaRG hepatocytes (“H”) and (B) for insect cells, Ktmos1 (“K”, Ae Aegypti) and C6/36 (“C”, Ae Albopictus). The infection was performed using HCVsp, LAT isolate, genotype 3. D, day; − before infection; D0, day of infection; D4, D7, D14, D21, D28, days post-infection and medium change. P17, P18, passages 17 and 18. HepaRG®, HepaRG cells from KIT902 (Biopredic International). Over the time, HepaRG and Ktmos1 cells in monolayer became more and more differentiated. (TIFF 300 kb

Additional file 3: Figure S2. of New insights into HCV replication in original cells from Aedes mosquitoes

Fluorescence observation of adherent Ktmos1 cells. The Ktmos1 Aedes aegypti cells were grown on thin glass (0,17 mm), 2 chambers LabTek (Nunc). The cells were fixed after different periods of cultivation with 2% PFA for 20 min at 37 °C. After permeabilization by PBS containing 0,1% Triton X100 for 2 min, the nuclei were stained by Hoechst 33,258 (Sigma). Observation was performed on motorized inverted Olympus IE81 microscope using the DIC (Differential Interference Contrast) and the DAPI filter. The panel (A) shows a late metaphase stage of a dividing cell. The panel (B) shows Ktmos1 cells in monolayer. (TIFF 925 kb

Additional file 7: Figure S6. of New insights into HCV replication in original cells from Aedes mosquitoes

Absence of HCV RNA detection in HEK 293 cells. Cells were collected at days 0 (D0), 4 (D4), 21 (D21) and 28 (D28) p.i. The inoculum HCVsp (LAT isolate, genotype 3) was used as positive control. Non-infected (mock) cells (−) and HCV-infected (+) HEK 293 cells. (TIFF 1236 kb

Additional file 2: Figure S1. of New insights into HCV replication in original cells from Aedes mosquitoes

Global process of the Ktmos1 cell generation from eggs hatching to the final supracellular structures. (A) Macroscopic picture showing the eggs of Aedes aegypti collected from insectary. (B) Large hollow vesicles developing at the cut ends of the larvae fragments. (C) Microscopic examination of the adherent cells and molecular identification of cells and larvae extracts by PCR targeting rDNA ITS: the upper panel shows cells in monolayer; the lower panel indicates species-diagnosis PCR of cellular samples with hollow vesicles (lane 1), adherent cells (lane 2) and “Dome-like” structures (lane 3). HEK 293 cells are the negative control, ground larvae extracts of Aedes aegypti bora bora strain are the positive control. The approximate size of the amplified product is 550 pb. (D) Microscopic examination of the hollow vesicles as supracellular structures (D1 and D2). (TIFF 751 kb