Journal: Vaccines

Article Title: A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

doi: 10.3390/vaccines8020169

Figure Lengend Snippet: 2A protein sequences used in expressing polycistronic EBV glycoprotein transcript.

Article Snippet: Chinese hamster ovary (CHO); EBV-positive Burkitt lymphoma B cell (Raji); human embryonic kidney (HEK-293); a derivative of HEK-293 stably expressing EBNA1 protein for enhanced ability to produce recombinant proteins (HEK-293 6E); and anti-gp350 monoclonal antibody (mAb) 72A1 hybridoma producer (HB168) cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Sequencing

Journal: Vaccines

Article Title: A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

doi: 10.3390/vaccines8020169

Figure Lengend Snippet: Generation of a Chinese hamster ovary (CHO) cell line stably expressing five recombinant Epstein-Barr virus (EBV) glycoproteins. ( a ) Schematic of the chimeric glycoprotein construct sequence inserted into a modified pCAGGS vector for expression in CHO cells. The construct consists of the five EBV glycoproteins indicated, interspersed with unique 2A autocleavable linker sequences. The ectodomains of gp350, gB, and gH are fused to the transmembrane/cytoplasmic (TMC) domain of Newcastle disease virus-F (NDV-F) protein to facilitate incorporation onto the virus-like particle (VLP) surface. Amino acid numbers in each glycoprotein covered by the construct are shown; ( b ). Sequential enrichment of CHO cells expressing five EBV glycoproteins. CHO cells were co-transfected with pCAGGS-EBV-gp350-F-gB-F-gp42-gL-gH-F and pCI-Puro plasmids. Forty-eight h post-transfection, transfected cells were selected using 10 µg/mL puromycin selection media. Selected cells were stained with anti-gp350 mAb 72A1, followed by staining with AF488-conjugated secondary anti-mouse IgG, and sorted via FACS (first sorting). The collected cells were maintained in 10 µg/mL puromycin, and the sorting process was repeated twice (second and third sorting) until >90% cells were positive for gp350 expression; ( c ) Fluorescence-activated cell sorting (FACS) analysis of stable CHO cells expressing five EBV glycoproteins. To confirm stable expression of all glycoproteins in the gp350-positive CHO cells, cells were additionally stained with anti-gB mAb (CL55), anti-gp42 mAb (F-2-1), anti-gH/gL mAb (CL59), or anti-gL mAb (E1D1) followed by staining with AF488-conjugated secondary anti-mouse IgG and analyzed by FACS. The transfected cells were compared to unstained CHO (shown), CHO stained with primary antibody alone, CHO stained with secondary antibody alone, or CHO stained with isotype control.

Article Snippet: Chinese hamster ovary (CHO); EBV-positive Burkitt lymphoma B cell (Raji); human embryonic kidney (HEK-293); a derivative of HEK-293 stably expressing EBNA1 protein for enhanced ability to produce recombinant proteins (HEK-293 6E); and anti-gp350 monoclonal antibody (mAb) 72A1 hybridoma producer (HB168) cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Expressing, Recombinant, Virus, Construct, Sequencing, Modification, Plasmid Preparation, Transfection, Selection, Staining, Fluorescence, FACS, Control

Journal: Vaccines

Article Title: A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

doi: 10.3390/vaccines8020169

Figure Lengend Snippet: Production and characterization of EBV-like particles (EBV-LPs). ( a ) Generation of EBV-LPs in CHO cells stably expressing five EBV glycoproteins. CHO cells stably expressing the five glycoproteins were co-transfected with plasmids encoding NDV-M and NDV-NP proteins (amino acid numbers in each of the NDV proteins are shown) to induce production of EBV-LPs. After transfection, supernatants were collected between 24–120 h post-transfection and EBV-LPs were pelleted by ultracentrifugation and purified through a sucrose density gradient (EBV-LP layer shown by red arrow); ( b ) Immunoblot analysis of purified EBV-LPs. After lysis, purified EBV-LPs were resolved on a 4–12% polyacrylamide gel, transferred to a polyvinylidene fluoride membrane, and analyzed by immunoblot with anti-2A polyclonal, anti-NDV-NP polyclonal, anti-gp350 monoclonal (72A1), and anti-gH/gL polyclonal primary antibodies, as indicated. Untransfected CHO cells (CHO), CHO cells transfected with “empty” pCAGGS vector (CHO pCAGGS), CHO cells transfected with pCAGGS-NDV-NP vector alone (CHO NP), CHO cells transfected with pCAGGS-gH/gL-WT (CHO gH/gL), and stable CHO cells expressing EBV gp35-F-gB-F-gp42-gL-gH-F (CHO EBV 5in1) served as controls when indicated; ( c ) TEM analysis of purified EBV-LPs. Purified EBV virions and EBV-LPs were fixed in 4% paraformaldehyde and adsorbed to glow-discharged, carbon-coated, 200-mesh EM grids. Micrographs were collected using an FEI Tecnai 12 TEM and recorded with a Gatan 2 × 2 k CCD camera at a magnification of 21,000X and a defocus value of ∼1.5 μm.

Article Snippet: Chinese hamster ovary (CHO); EBV-positive Burkitt lymphoma B cell (Raji); human embryonic kidney (HEK-293); a derivative of HEK-293 stably expressing EBNA1 protein for enhanced ability to produce recombinant proteins (HEK-293 6E); and anti-gp350 monoclonal antibody (mAb) 72A1 hybridoma producer (HB168) cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Expressing, Transfection, Purification, Western Blot, Lysis, Membrane, Plasmid Preparation

Journal: Vaccines

Article Title: A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

doi: 10.3390/vaccines8020169

Figure Lengend Snippet: Antibody response in EBV-LP-immunized New Zealand white rabbits. ( a ) New Zealand white rabbit immunization and bleeding schedule schematic. Rabbits were immunized and bled as detailed in the Materials and Methods; ( b ) Biochemical characterization of recombinant EBV proteins used as ELISA target antigens. Coomassie stain (left) and immunoblot (right; performed using anti-6×His primary antibody) of soluble gp350 ectodomain, and recombinant EBV gB, gp42, and gH/gL proteins, which were used as target antigens in ELISA assay in panel C; ( c ) EBV-specific antibody responses in sera. IgG titers in immunized animals were measured by ELISA for each glycoprotein; proteins described in panel B were used as target antigens at 25 ng/well, and sera from immunized rabbits for each treatment group and timepoint were pooled, serially diluted, and used as primary antibody (1:100 dilution shown). Primary mouse mAbs anti-gp350 (72A1), anti-gB (CL55), anti-gp42 (F-2-1), anti-gL (E1D1), and anti-gH/gL (CL59) were used as positive controls where appropriate (not shown). Antibody binding was detected with HRP-labeled anti-rabbit IgG secondary antibody, and optical density (OD) was read at 405 nm with a spectrophotometer. ELISA assay was performed for each sample in quadruplicate, and results are expressed as mean ± standard deviations (SD). The assay was independently repeated two times with either individual animal serum or pooled sera.

Article Snippet: Chinese hamster ovary (CHO); EBV-positive Burkitt lymphoma B cell (Raji); human embryonic kidney (HEK-293); a derivative of HEK-293 stably expressing EBNA1 protein for enhanced ability to produce recombinant proteins (HEK-293 6E); and anti-gp350 monoclonal antibody (mAb) 72A1 hybridoma producer (HB168) cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Binding Assay, Labeling, Spectrophotometry

Journal: Vaccines

Article Title: A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

doi: 10.3390/vaccines8020169

Figure Lengend Snippet: In vitro neutralizing activity of purified IgGs from rabbits immunized with EBV-LPs. ( a ) Titration of purified Day 70 IgGs specific to EBV. Equal amounts of Day 70 sera from immunized rabbits from each treatment group (UV-EBV, EBV-LP, gp350, and TNE) were pooled, and total IgG antibodies were purified via protein A spin columns. Total IgGs were serially diluted (25, 12.5, 6.25, 3.125, and 1.56 µg/mL) and EBV-glycoprotein-specific IgG titers were determined in each dilution in quadruplicate by ELISA as described in C. Results are expressed as mean ± SD; ( b ) EBV-eGFP neutralization assay in Raji B cells and HEK-293 epithelial cells. Neutralization activity of purified Day 70 IgGs was determined by incubating known quantities of EBV-eGFP that result in 40–70% infectivity with serially diluted purified IgGs (50, 25, 12.5, 6.25, 3.125, and 1.56 µg/mL µg/mL) from all treatment groups for 1 h at 37 °C. The mixtures of purified IgGs and virus were then added to previously seeded cells and incubated for 2 h at 37 °C, after which the cells were thoroughly washed three times with 1×PBS and given complete media. Cells were collected after 48 h and infected cells (eGFP-positive) were quantified using FACS. Cells incubated with virus or media alone served as positive and negative controls for infection, respectively, and resulting infectivity was used to calculate % neutralization. Neutralizing anti-gp350 mAb 72A1 and non-neutralizing anti-gp350 mAb 2L10 served as positive and negative controls for neutralization, respectively. Results were normalized to the TNE group and are shown as mean ± SD.

Article Snippet: Chinese hamster ovary (CHO); EBV-positive Burkitt lymphoma B cell (Raji); human embryonic kidney (HEK-293); a derivative of HEK-293 stably expressing EBNA1 protein for enhanced ability to produce recombinant proteins (HEK-293 6E); and anti-gp350 monoclonal antibody (mAb) 72A1 hybridoma producer (HB168) cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: In Vitro, Activity Assay, Purification, Titration, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Virus, Incubation

Journal: Vaccines

Article Title: A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

doi: 10.3390/vaccines8020169

Figure Lengend Snippet: EBV neutralization IC 50 values for purified IgGs from gp350, UV-EBV and EBV-LP -treated rabbits, and for anti-gp350 mAbs 72A1 and 2L10.

Article Snippet: Chinese hamster ovary (CHO); EBV-positive Burkitt lymphoma B cell (Raji); human embryonic kidney (HEK-293); a derivative of HEK-293 stably expressing EBNA1 protein for enhanced ability to produce recombinant proteins (HEK-293 6E); and anti-gp350 monoclonal antibody (mAb) 72A1 hybridoma producer (HB168) cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Neutralization, Purification