cho k1 parental cell line  (ATCC)

Journal: bioRxiv

Article Title: Single-component multilayered self-assembling protein nanoparticles presenting glycan-trimmed uncleaved prefusion optimized envelope trimers as HIV-1 vaccine candidates

doi: 10.1101/2022.07.01.498503

Figure Lengend Snippet: ( A ) Negative stain EM analysis to assess the structural integrity of SApNPs produced from 8 and 5 CHO-K1 cell clones that stably express E2p and I3-01v9 SApNPs, respectively. ( B )-( F ) Neutralization of purified IgG at week 11 from the mouse study. Groups of eight mice were immunized with BG505 Env immunogens mixed with AddaVax or aluminum phosphate adjuvants at weeks 0, 3, 6 and 9. Two glycan trimming regimens were tested: regimen 1 administers four doses of glycan-trimmed immunogens, whereas regimen 2 administers two doses of glycan-trimmed immunogens as prime and two doses of wildtype immunogens as boost. Immunization was repeated for some formulation/regimen groups to confirm the findings and are labeled in the summary. ( B )-( D ) Neutralization of week 11 IgG from mice immunized with ( B ) the BG505 UFO trimer and ( C ) UFO trimer-presenting SApNPs E2p and ( D ) I3-01v9 against autologous tier 2 clade A BG505.T332N, tier 1 clade B SF162, and MLV as a negative control. No IgG was available for one mouse in the UFO trimer/AP regimen 1 group and for two mice in the I3-01v9/AP regimen 1 (1 st repeat) group, as the mice died during the immunization study. Of note, IgG samples from the two groups that tested trimer/AP and four groups that tested E2p/AV and I3-01v9/AP in regimens 1 and 2 were selected for further analyses in (E) or (E-F) respectively, which are shown below. ( E ) Neutralization of week 11 IgG from select mice (more than 30% autologous neutralization) in the abovementioned six mouse groups against a BG505.T332N variant with the C3/V4 knockout mutation (I396R). IC 50 titers and neutralization curves are shown, with BG505.T332N data from (B-D) included for comparison. ( F ) Neutralization of week 11 IgG from the four abovementioned SApNP groups against a 12-virus global panel. IC 50 and IC 30 titers were calculated and are listed in two tables. For all the TZM-bl neutralization assays performed in (B)-(F), a starting IgG concentration of 300 µg/ml and a 3-times dilution series were used. Each IgG sample was run in duplicate in (B)-(D) except for some mice in the MLV assays and all mice in the 12-virus assays in (F). Of note, mice for which IgG samples were no longer available at the time of the MLV or 12-virus assays are marked ‘-‘ in the IC 50 /IC 30 titer tables. ( G )-( J ) Neutralization of purified IgG from the rabbit study. Four groups of rabbits (6 per group) were immunized to test the E2p/AV and I3-01v9/AP formulations for glycan trimming regimens 1 and 2, using a similar protocol to the mouse study. ( G ) Neutralization of week 0 rabbit IgG against BG505.T332N, SF162, and MLV. ( H ) Neutralization of week 11 rabbit IgG against BG505.T332N, SF162, and MLV. It should be noted that R5 IgG from the I3-01v9/AP regimen 2 group showed MLV neutralization and might contain non-specific antibodies. ( I ) Neutralization of select week 11 rabbit IgG (more than 30% autologous neutralization) against four BG505.T332N glycan hole mutant viruses. ( J ) Neutralization of select week 11 rabbit IgG (more than 30% autologous neutralization) against a 12-virus global panel. IC 50 and IC 30 titers were calculated and are listed in two tables with neutralization curves shown for each IgG sample against all 12 viruses. For all the TZM-bl neutralization assays performed in (G)-(J), a starting IgG concentration of 300 µg/ml and a 3-times dilution series were used. Each IgG sample was run in duplicate in (G-I) but in singlet in (J) due to the limited sample availability.

Article Snippet: Stable CHO cell lines expressing BG505 UFO-E2p-L4P and UFO-10GS-I3-01v9-L7P NPs were generated by the cationic lipid mediated transfection of a parental suspension culture adopted CHO K1 host cell line (ATCC no. CCL-61) using bacterial artificial chromosomes (BACs) as gene transfer vehicles ( ).

Techniques: Staining, Produced, Clone Assay, Stable Transfection, Neutralization, Purification, Glycoproteomics, Formulation, Labeling, Negative Control, Variant Assay, Knock-Out, Mutagenesis, Comparison, Virus, Concentration Assay