Journal: Biotechnology Journal

Article Title: An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

doi: 10.1002/biot.70216

Figure Lengend Snippet: Construction, expression, and characterization of recombinant adenoviral vectors encoding the SARS‐CoV‐2 Spike protein. (A) Schematic representation of the adenoviral expression plasmid construction using Gateway LR recombination. The entry plasmid (pDONR223 SARS‐CoV‐2 S) containing attL1/attL2 recombination sites was recombined with the destination vector (pAd/CMV/V5‐DEST) containing attR1/attR2 sites using LR clonase enzyme mix. The resulting transfer plasmid (pAd5Spike) includes the full‐length Spike gene under a CMV promoter along with adenoviral vector backbone sequences. Simulation images were generated using SnapGene. (B) Agarose gel electrophoresis analysis of pAd5Spike plasmids. The left panel shows the simulated gel image, and the right panel displays actual gel electrophoresis of plasmids isolated from three independent bacterial colonies (lanes 1–3). MW: 1 kb Plus DNA Ladder (Thermo, SM0313). (C) Immunocytochemical analysis of SARS‐CoV‐2 Spike protein expression in HEK293T cells transfected with pAd5Spike. Cells transfected with pAd5Spike show strong brown immunostaining, indicating Spike expression, while control cells (transfected with PEI alone) show no signal. (D) CsCl density gradient ultracentrifugation of recombinant adenoviral particles. Two distinct bands are visible: the lower band represents genome‐containing Ad5Spike particles, and the upper band corresponds to empty viral capsids, indicating successful viral purification. (E) Immunofluorescence analysis of spike protein expression in HT1080 cells transduced with Ad5Spike at increasing multiplicities of infection (MOI: 10 2 –10 5 ). Cells were stained 72 h post‐transduction using an anti‐Spike antibody (green, Alexa Fluor 488). Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm.

Article Snippet: Codon‐optimized cDNAs encoding the full‐length SARS‐CoV‐2 Spike glycoprotein (3822 bp; Addgene #149329) or GFP were initially obtained in the Gateway entry vector pDONR223, flanked by attL1 and attL2 recombination sites.

Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Isolation, Transfection, Immunostaining, Control, Purification, Immunofluorescence, Transduction, Infection, Staining

Journal: Biotechnology Journal

Article Title: An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

doi: 10.1002/biot.70216

Figure Lengend Snippet: Humoral immune responses induced by Ad5Spike vaccination in BALB/c mice. Female BALB/c mice (6–8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with the Ad5Spike vaccine vector at doses of 10 8 , 10 10 , or 10 1 2 viral particles. Control groups received PBS or an AdGFP vector. (A) Serum anti‐Spike IgG antibody titers were quantified by ELISA at days 30 and 90 post‐immunization. Data are presented as mean ± SD, with each data point representing an individual mouse. (B) SARS‐CoV‐2 Wuhan‐1 variant–specific neutralizing antibody titers were assessed in sera collected at days 30 and 90 using a pseudovirus‐based neutralization assay. Titers are expressed as ID 50 values (reciprocal serum dilution). Each dot represents an individual animal; box plots indicate the interquartile range with whiskers representing the 95% confidence interval. Normality was assessed using the Shapiro–Wilk test. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test, with comparisons made against the AdGFP vector–immunized control group. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Codon‐optimized cDNAs encoding the full‐length SARS‐CoV‐2 Spike glycoprotein (3822 bp; Addgene #149329) or GFP were initially obtained in the Gateway entry vector pDONR223, flanked by attL1 and attL2 recombination sites.

Techniques: Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Variant Assay, Neutralization

Journal: Biotechnology Journal

Article Title: An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

doi: 10.1002/biot.70216

Figure Lengend Snippet: Construction, expression, and characterization of recombinant adenoviral vectors encoding the SARS‐CoV‐2 Spike protein. (A) Schematic representation of the adenoviral expression plasmid construction using Gateway LR recombination. The entry plasmid (pDONR223 SARS‐CoV‐2 S) containing attL1/attL2 recombination sites was recombined with the destination vector (pAd/CMV/V5‐DEST) containing attR1/attR2 sites using LR clonase enzyme mix. The resulting transfer plasmid (pAd5Spike) includes the full‐length Spike gene under a CMV promoter along with adenoviral vector backbone sequences. Simulation images were generated using SnapGene. (B) Agarose gel electrophoresis analysis of pAd5Spike plasmids. The left panel shows the simulated gel image, and the right panel displays actual gel electrophoresis of plasmids isolated from three independent bacterial colonies (lanes 1–3). MW: 1 kb Plus DNA Ladder (Thermo, SM0313). (C) Immunocytochemical analysis of SARS‐CoV‐2 Spike protein expression in HEK293T cells transfected with pAd5Spike. Cells transfected with pAd5Spike show strong brown immunostaining, indicating Spike expression, while control cells (transfected with PEI alone) show no signal. (D) CsCl density gradient ultracentrifugation of recombinant adenoviral particles. Two distinct bands are visible: the lower band represents genome‐containing Ad5Spike particles, and the upper band corresponds to empty viral capsids, indicating successful viral purification. (E) Immunofluorescence analysis of spike protein expression in HT1080 cells transduced with Ad5Spike at increasing multiplicities of infection (MOI: 10 2 –10 5 ). Cells were stained 72 h post‐transduction using an anti‐Spike antibody (green, Alexa Fluor 488). Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm.

Article Snippet: Neutralizing antibody responses were assessed using a pseudovirus neutralization assay based on third‐generation lentiviral particles pseudotyped with SARS‐CoV‐2 Spike (Wuhan‐1 sequence; Addgene, #155130), encoding an RFP reporter gene.

Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Isolation, Transfection, Immunostaining, Control, Purification, Immunofluorescence, Transduction, Infection, Staining

Journal: Biotechnology Journal

Article Title: An Ad5‐Based COVID‐19 Vaccine Encoding SARS‐CoV‐2 Spike Glycoprotein Induces Measurable Antibody and Cytokine Responses in Mice

doi: 10.1002/biot.70216

Figure Lengend Snippet: Humoral immune responses induced by Ad5Spike vaccination in BALB/c mice. Female BALB/c mice (6–8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with the Ad5Spike vaccine vector at doses of 10 8 , 10 10 , or 10 1 2 viral particles. Control groups received PBS or an AdGFP vector. (A) Serum anti‐Spike IgG antibody titers were quantified by ELISA at days 30 and 90 post‐immunization. Data are presented as mean ± SD, with each data point representing an individual mouse. (B) SARS‐CoV‐2 Wuhan‐1 variant–specific neutralizing antibody titers were assessed in sera collected at days 30 and 90 using a pseudovirus‐based neutralization assay. Titers are expressed as ID 50 values (reciprocal serum dilution). Each dot represents an individual animal; box plots indicate the interquartile range with whiskers representing the 95% confidence interval. Normality was assessed using the Shapiro–Wilk test. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test, with comparisons made against the AdGFP vector–immunized control group. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Neutralizing antibody responses were assessed using a pseudovirus neutralization assay based on third‐generation lentiviral particles pseudotyped with SARS‐CoV‐2 Spike (Wuhan‐1 sequence; Addgene, #155130), encoding an RFP reporter gene.

Techniques: Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Variant Assay, Neutralization