Generation and efficacy assessment of a chimeric antigen E2-CD154 as a marker Classical Swine Fever Virus subunit vaccine produced in HEK 293 and CHO K1 mammalian cells

The E2 glycoprotein is the major antigen that induces neutralizing and protective antibodies in CSFV infected pigs, thus a marker vaccine based on this antigen appears to be the most promising alternative to induce a protective immune response against CSFV. However, the structural characteristics of this protein state the necessity to produce glycoprotein E2 in more complex expression systems such as mammalian cells. In this study, we use a lentivirus-based gene delivery system to establish a stable recombinant HEK 293 and CHO K1 cell line for the expression of E2 fused to porcine CD154 as immunostimulatory molecule. In a first experiment, E2his and E2-CD154 were compared in an immunization trial. The average antibody titers in E2his immunized pigs was in the range of 30-40% of blocking and the average antibody titers for E2-CD154 are above 40% at day 14, meaning that the chimeric antigen is able to raise antibodies at positive levels in a shorter time. Additionally, the blocking rate of E2his vaccinated group in ELISA ranged between 66-88% and in the E2-CD154- vaccinated groups ranged between 86-92%, one week after booster immunization. The NPLA antibody titers also increased greatly. Later on, the protective capacity of purified E2-CD154 glycoprotein was demonstrated in a challenge experiment in pigs using a biphasic immunization schedule with 25 and 50 μg. The immunized animals developed neutralizing antibodies that were protective when the animals were faced to a challenge with 105 LD50 of ‘‘Margarita’’ CSFV highly pathogenic strain. No clinical signs of the disease were detected in the vaccinated pigs. Unvaccinated pigs in the control group exhibited symptoms of CSF at 3–4 days after challenge and were euthanized from 7–9 days when the pigs became moribund. These results indicate that E2-CD154 produced in recombinant HEK 293 and CHOK1cell line is a high quality candidate for the development of a safe and effective CSFV subunit vaccine. In the next steps, pilot and production scale, E2-CD154 expression levels should be increased in 10 to 50 fold, arriving to a very attractive productive platform for an implementation of a commercial subunit vaccine against CSF

Production and characterization of a chimeric antigen, based on nucleocapsid of SARS-CoV-2 fused to the extracellular domain of human CD154 in HEK-293 cells as a vaccine candidate against COVID-19.

Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases

Original images for SDS-PAGE and Western Blot.

Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.</div

Dose-response study in mice.

Dose-response study in mice. (A) Overall immunization schedule. Mice were intramuscularly immunized with 5, 10 or 20 μg of the N-CD protein or PBS (placebo) using alum as an adjuvant on days 0 and 21. The experimental groups were composed by 10 animals each one. Blood draws were performed at 0 (pre-immune serum) and 35 after the first immunization. (B) N-specific IgG endpoint titers at 35 days were measured by ELISA using plates coated with SARS-CoV-2 N protein expressed in E. coli. Serum samples were serially diluted from 1:1000 to 1:2048000. The titer was defined as the highest dilution which presents an optical density that is twice the value of the corresponding pre-immune serum. The graphic shows mean ± standard deviation. Kruskal-Wallis test followed by Dunn’s multiple comparisons test were used for comparisons between IgG endpoint titers from different experimental groups. (*) p (DOCX)</p

Multiple sequence alignment of extracellular domain of CD154 from different species.

(A) Protein sequences of mouse, macaque, swine and human extracellular domain of CD154 were aligned using the bioinformatic tool Clustal Omega 2.1 (https:// www.ebi.ac.uk/Tools/msa/clustalo/). (B) Percent identity matrix of the aligned sequences. (*) Positions with a single, fully conserved residue. (:) Positions with conservation between amino acid groups of similar properties. (.) Positions with conservation between amino acid groups of weakly similar properties. (DOCX)</p

Epitope mapping of antibodies against SARS-CoV-2 N protein by ELISA.

The absorbance values obtained from the assessment of the sera from monkeys immunized with the N-CD protein or the placebo group, using a peptide from SARS-CoV-2-N protein or the complete protein as coating antigen, are showed. A positive result was considered when the mean absorbance values were higher than 0.18 (media plus three times the standard deviation of absorbance values of negative control). Positive control: Serum from convalescent COVID-19 patient. Negative control: Serum from non-infected patient. Blank control: wells were filled only with washing buffer.</p

Excel sheets with the raw data of binding of CD154 to CD40 by ELISA, binding of N-CD to sera from COVID-19 convalescent individuals, ELISA results of immunogenicity in mice at days 21 and 35 post first immunization, dose experiments in mice with 5, 10 and 20 μg of N-CD protein for immunization and ELISA results of immunogenicity in monkeys (days 21, 28, 35, 42, 63, 84, 127, 144, 174 post first immunization).

Excel sheets with the raw data of binding of CD154 to CD40 by ELISA, binding of N-CD to sera from COVID-19 convalescent individuals, ELISA results of immunogenicity in mice at days 21 and 35 post first immunization, dose experiments in mice with 5, 10 and 20 μg of N-CD protein for immunization and ELISA results of immunogenicity in monkeys (days 21, 28, 35, 42, 63, 84, 127, 144, 174 post first immunization).</p

Design and functionality of the N-CD expression cassette cloned into the lentiviral plasmid pl6twblast.

(A) Schematic diagram of the plasmid pl6twblast-CMV-N-CD (hCMV promoter/enhancer + Igk-chain leader sequence + N gene + 6-His tail + linker + extracellular domain of CD154 gene). (B) 750 μL of supernatant of HEK-293 cells transfected with pl6twblast-CMV-N-CD harvested after 72 hours and analyzed under reducing conditions in 12.5% SDS-PAGE. Western Blotting analysis was performed using a HRP-conjugated anti-SARS-CoV-2 N protein monoclonal antibody. Lane 1: SARS-CoV-2 N protein expressed in E. coli, lane 2: protein standard, lanes 3 to 6: total proteins from supernatant of HEK-293 cells transfected with the lentiviral plasmid pl6twblast-CMV-N-CD (clones 15, 17, 18 and 19, respectively), lane 7: total proteins from supernatant of HEK-293 cells transfected with the plasmid pDisplay-CMV-N-CD (positive control), lane 8: total proteins from supernatant of HEK-293 cells transfected without DNA (negative control). The red arrow points out the non-degraded N-CD protein.</p

Humoral immune responses induced by N-CD protein in monkeys.

(A) Overall immunization schedule. Monkeys were intramuscularly immunized with 50 μg of the N-CD protein or PBS (placebo) using alum as an adjuvant on days 0 and 21. Both experimental groups were composed of 3 animals. Blood draws were performed at 0 (pre-immune serum) and at 9 different times during almost 6 months. (B) N-specific IgG endpoint titers at 21, 28, 35, 42, 63, 84, 127, 144 and 174 days were measured by ELISA using plates coated with SARS-CoV-2 N protein expressed in E. coli. The graphic shows mean ± standard deviation. One way-ANOVA followed by Tukey test for multiple comparisons were used for comparison between N-specific IgG endpoint titers from serum samples collected at different times after the immunization with the N-CD protein or placebo. Different letters indicate significant differences (p < 0.01) between different timepoints.</p