Cells
BHK-21 cells (ATCC: CCL-10) and derived stable cell lines were cultured in BHK-21 Glasgow MEM (Gibco BRL, UK) supplemented with 5% FCS, 10% tryptose phosphate broth, 2 mM glutamine, 20 mM HEPES, 100 µg/ml streptomycin and 100 IU/ml penicillin (BHK complete medium). A20 lymphoma cells (ATCC® TIB-208) were cultured in RPMI medium supplemented with 10% FCS, glutamine and streptomycin/penicillin as previously described for BHK cells. Hybridoma cells were obtained by fusion of A20 cells with P3X63Ag8.653 (ATCC® CRL-1580™) as described15.
Plasmid constructs
Plasmid ncSFV-pac2A, which contains the P718T and R649H mutations in the nsp2 subunit of the SFV replicase and the pac gene downstream of the subgenomic promoter (sgPr) followed by the sequence coding for the foot and mouth disease virus 2A autoprotease (2A), was previously described by our group13,14 and used to generate ncSFV-pac plasmids expressing A20 mAb. For this purpose we first purifed total RNA from A20 lymphoma cells and synthesized cDNA using random primers. Since the sequence of the A20 mAb was already known7, we designed specific oligonucleotides to amplify the HC and LC sequences (Supplementary Table 1) from the obtained cDNA. We generated DNA fragments containing both the HC and LC chains of the antibody by PCR and subcloned them into the Xma I site of the ncSFV-pac2A plasmid (Fig. 1). PCR fragments were obtained as follows: (i) For ncSFV-pacA20sgPr, a crossover PCR was designed to insert an sgPr sequence between both chains. The first PCR was performed with oligonucleotides 1 + 2 (Supplementary Table 1) and the second PCR with oligonucleotides 3 + 4. Fragments obtained from both PCRs were purified, mixed in equal amounts and used as template for a crossover PCR using oligonucleotides 1 + 4, obtaining a 2191 bp fragment with the LC followed by sgPr and HC. This fragment was subcloned into pGEM-T, sequenced, digested with Xma I and subcloned into ncSFV-pac, using the same restriction site. (ii) For ncSFV-pac-A20HLC a similar strategy was followed using primers 5 + 6 and 7 + 8 to generate two independent fragments and primers 5 + 8 for the crossover PCR, generating a 2196 bp fragment with the HC followed by sgPr and LC. (iii) For ncSFV-pac-A20-2A a similar strategy was used using primers 5 + 9 and 10 + 8 to generate two independent fragments and primers 5 + 8 for the crossover PCR, generating a 2193 bp fragment with the HC followed by 2A sequence and LC. (iv) Finally, to generate ncSFV-pac-A20ires, we first used a similar strategy using primers 1 + 11 and primers 12 + 13 to generate two independent DNA fragments and primers 1 + 13 (in this case using as template a plasmid containing the IRES sequence16 for crossover PCR, obtaining a construct with the LC fused to the internal ribosome entry site (IRES) from encephalomyocarditis virus, which was cloned into pGEM-T (pLC-IRES). Another PCR was performed with primers 4 + 14 to generate a fragment with the HC sequence that was subcloned into pLC-IRES generating pLC-IRES-HC. From this intermediate plasmid a 2826 pb fragment with the LC followed by the IRES sequence and HC was extracted with Xma I and sucbloned into ncSFV-pac2A. Plasmid pSFV-S2-9-pac, referred here as ncSFV-pac was used to generate control cell lines14.
Schematic representation of vectors expressing murine follicular lymphoma-derived A20 mAb. Noncytopathic vectors (ncSFV-pac) contain mutations P718T and R649H in the nsp2 subunit of the replicase (Rep), indicated by two Xs, and express the pac gene downstream of the viral subgenomic promoter (sg Pr). The sequence of the small FMDV 2A autoprotease (2A) was used to fuse the pac gene to genes coding for the heavy (HC) or light chain (LC) of A20 mAb. A second sg Pr, the 2A sequence, or the internal ribosome entry site (IRES) from encephalomyocarditis virus were used to express separately HC and LC as indicated in the diagrams.
Selection of cell lines with ncSFVpac vectors
SFV-derived plasmids were linearized by digestion with SpeI and transcribed in the presence of cap analog (New England Biolabs, Ipswich, MA) using SP6 polymerase (New England Biolabs)17. Fifty µg of in vitro transcribed RNA were electroporated into 5 × 106 BHK-21 cells by electroporation as described previously13. After transfection, cells were allowed to recover for 24–28 h before addition of puromycin at 5 µg/ml (Sigma, St. Louis, MO). To select for puromycin resistant cells, medium was replaced every 2–3 days with fresh puromycin containing medium. Upon selection, cells were always passaged in the presence of puromycin at the indicated concentration.
Analysis of protein expression
The amount of A20 mAb present in supernatants from cells transfected with ncSFV-pac-A20 vectors was quantified by a total mouse IgG ELISA kit (Mabtech, Sweden) using as standard A20 mAb purified from the A20 hybridoma cell line. This standard mAb was previously quantified by Bradford analysis. For Western blot experiments, supernatants from cells transfected with SFV vectors expressing A20 mAb were analyzed under reducing (with dithiothreitol, DTT) and non-reducing (without DTT) conditions in 8% or 12% polyacrylamide gels, respectively, incubating with a polyclonal goat antibody specific for mouse IgG conjugated with horseradish peroxidase (Sigma, St. Louis, MO). Cell lysates were also analyzed with rabbit polyclonal antisera specific for SFV nsp213 or for actin (Sigma). Proteins were visualized using the Western Lightning Chemiluminiscence Reagent Plus (Perkin Elmer Life Sciences, Waltham, MA).
Purification of A20 mAb
A cell line selected with ncSFV-pac-A20sgPr vector was grown to a final quantity of approximately 2 × 108 cells, corresponding to six consecutive passages. Cells were first incubated with 500 ml EX-CELL™ CD CHO medium without FBS (Sigma) during 36 h at 33 °C. This medium was collected and then cells were incubated again with 500 ml of new medium for 24 h at the same temperature. Culture media from the two incubations were pooled and centrifuged at 1000g for 5 min. Recombinant mAb was purified by Protein A Sepharose column (HiTrap Protein A HP, GE Healthcare Bio-Sciences, Pittsburgh, PA) at 4 °C and at a constant speed of 1 ml/min during approximately 16 h using an ÄKTA protein purification system (GE Healthcare Bio-Sciences). The mAb was eluted with 100 mM glycine (pH 3). The presence of the mAb was analyzed at each purification step by SDS-PAGE followed by Coomassie staining or Western blot as described earlier. The purified mAb was gel digested with trypsin and protein identification was obtained by analysis of the digests in a coupled liquid chromatography and tandem mass spectrometry (LC–MS/MS). A20 mAb produced by the hybridoma cell line had been previously purified using a similar procedure.
Analysis of A20 glycosylation
For glycosylation analysis, samples of purified mAb were treated with N-glycosidase (Calbiochem, San Diego, CA) according to the manufacturer’s instructions. Briefly, samples were heated at 100 °C for 5 min, cooled to room temperature, and incubated with glycosidase during 3 h at 37 °C in the presence of 0.75% Triton X-100. After treatment, samples were analyzed by Western blot as described earlier.
Vaccination studies
Purified A20 mAb was first conjugated to Keyhole Limpet Hemocyanin (KLH, Biosyn Corporation, Carlsbad, CA) as previously described15. Four-week-old Balb/c female mice (Envigo, Spain) were randomly distributed in three groups that were immunized with A20 mAb purified from hybridoma or ncSFV-pac-A20sgPr cells, or with PBS. In the first two cases, mice received four subcutaneous immunizations, given once a week, of 25 µg of purified mAb conjugated with the same amount of KLH in combination with 10,000 U of GM-CSF (BD Pharmingen) in a total volume of 50 µl. Control mice received the same volume of PBS. In addition, at 24 h, 48 h, and 72 h after each A20 mAb vaccination, mice received 10,000 U of GM-CSF diluted in 50 µl of PBS which were administered subcutaneously close to the site of the vaccine injection (Fig. 5A). All mice were retroorbitally bled before the first vaccination and 10 days after the last vaccine boost. Fifteen days after the last vaccine dose all mice were subcutaneously injected with 2 × 105 A20 lymphoma cells and tumor growth was determined at different time points by measuring two perpendicular diameters. Tumor volume was calculated with the formula: d2 × D/2 where d = smaller diameter; D = larger diameter. One mouse out of 18 and one mouse out of 22 were excluded from the A20-SFV and PBS groups in Fig. 5A, since they were detected as outlayers using Prism software (GraphPad Software, San Diego, CA). Animal studies were approved by the Universidad de Navarra ethical committee (study 063/14-E44/15) for animal experimentation under Spanish regulations. This study was carried out in compliance with the ARRIVE guidelines.
A20 specific ELISA
ELISA plates were coated with purified A20 mAb at a concentration of 10 µg/ml. After blocking with PBS containing 1% fat-free milk, plates were incubated with serial dilutions of mice sera (in PBS with 1% fat free milk). Finally, plates were incubated with horseradish peroxidase-conjugated anti mouse IgG1 (Pharmingen).
Statistical analyses
Graphpad Prism software (version 9.2.0) was used for statistical analysis. Survival of tumor-bearing animals is represented by Kaplan–Meier plots and was analyzed by log-rank test. To compare tumor size in experimental groups, the Kruskal–Wallis test, followed by the Dunn multiple-comparison test, was used for nonparametric data.
