Vaccines, Vol. 11, Pages 53: Increase in the Immune Response in Balb/c Mice after the Co-Administration of a Vector-Based COVID-19 Vaccine with Cytosine Phosphoguanine Oligodeoxynucleotide

Conceptualization, E.O.O. and A.K.M.; formal analysis, D.A.C.; investigation, D.A.C.; resources, J.K. and J.W.K.; data curation, D.A.C. and E.O.O.; writing—original draft preparation, D.A.C., E.O.O. and A.K.M.; writing—review and editing, D.A.C., E.O.O. and A.K.M.; supervision, E.O.O. and A.K.M.; funding acquisition, D.A.C. All authors have read and agreed to the published version of the manuscript.

Figure 1. The IgG antibody immune response to SARS-CoV-2 at 14 days after immunization. (A) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 4 × 109 VP of vaccine plus the CpG ODNS group showed statistical significance (p ≤ 0.001), 2-fold increase in IgG against 4 × 109 VP of vaccine. (B) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 8 × 109 VP of vaccine plus the CpG ODNs group. There was a 1.5-fold increase for the CPG-supplemented against 8 × 109 VP of the vaccine-only group. A 95% CI was considered for all tests. (**** p < 0.0001).

Figure 1. The IgG antibody immune response to SARS-CoV-2 at 14 days after immunization. (A) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 4 × 109 VP of vaccine plus the CpG ODNS group showed statistical significance (p ≤ 0.001), 2-fold increase in IgG against 4 × 109 VP of vaccine. (B) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 8 × 109 VP of vaccine plus the CpG ODNs group. There was a 1.5-fold increase for the CPG-supplemented against 8 × 109 VP of the vaccine-only group. A 95% CI was considered for all tests. (**** p < 0.0001).

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Figure 2. The IgG antibody immune response to SARS-CoV-2 at 28 days after immunization. (A) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 4 × 109 VP of vaccine plus the CpG ODNS group showed statistical significance (p ≤ 0.001) with a 5.6-fold increase in IgG against 4 × 109 VP of vaccine. (B) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 8 × 109 VP of vaccine plus the CpG ODNs group. There was a 1.2-fold increase against 8 × 109 VP of the vaccine group. A 95% CI was considered for all tests. A 95% CI was considered for all tests. (**** p < 0.0001).

Figure 2. The IgG antibody immune response to SARS-CoV-2 at 28 days after immunization. (A) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 4 × 109 VP of vaccine plus the CpG ODNS group showed statistical significance (p ≤ 0.001) with a 5.6-fold increase in IgG against 4 × 109 VP of vaccine. (B) The results of SARS-CoV-2 spike protein-specific IgG antibodies in the plasma of 8 × 109 VP of vaccine plus the CpG ODNs group. There was a 1.2-fold increase against 8 × 109 VP of the vaccine group. A 95% CI was considered for all tests. A 95% CI was considered for all tests. (**** p < 0.0001).

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Figure 3. IgG antibody response to SARS-CoV-2 using ELISA assays on days 14 and 28. The IgG response was significantly stronger in the 4 × 109 VP of vaccine supplemented by CpG ODNs mice, compared to the CpG ODNs and 8 × 109 VP of vaccinated mice (p < 0.06).

Figure 3. IgG antibody response to SARS-CoV-2 using ELISA assays on days 14 and 28. The IgG response was significantly stronger in the 4 × 109 VP of vaccine supplemented by CpG ODNs mice, compared to the CpG ODNs and 8 × 109 VP of vaccinated mice (p < 0.06).

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Figure 4. mRNA expression of TNF (A) and INF-γ (B). VACC 40 and VACC 80: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine concentration, respectively, VACC 40 +CpG and VACC 80 + CpG: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine plus CpG ODNs, CpG: group treated with CpG ODNs, and PBS: group treated with PBS.

Figure 4. mRNA expression of TNF (A) and INF-γ (B). VACC 40 and VACC 80: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine concentration, respectively, VACC 40 +CpG and VACC 80 + CpG: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine plus CpG ODNs, CpG: group treated with CpG ODNs, and PBS: group treated with PBS.

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Figure 5. Results of biochemical analysis. VACC 40 and VACC 80: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine concentration, respectively, VACC 40 +CpG and VACC 80 + CpG: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine plus CpG ODNs, CpG: group treated with CpG ODNs and PBS: group treated with PBS.

Figure 5. Results of biochemical analysis. VACC 40 and VACC 80: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine concentration, respectively, VACC 40 +CpG and VACC 80 + CpG: groups treated with 4 × 109 VP and 8 × 109 VP of vaccine plus CpG ODNs, CpG: group treated with CpG ODNs and PBS: group treated with PBS.

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Table 1. Types of cytosine phosphoguanine oligodeoxynucleotides (CpG ODNs) used in the study.

Table 1. Types of cytosine phosphoguanine oligodeoxynucleotides (CpG ODNs) used in the study.

CpG ODNsSequences1828 9 (22 mer)5′-T*CCATGACGTTC*TACTGACGT*T- 3′18281-1 (22 mer)5′-T*C*C*A*T*G*A*C*G*T*T*C*T*A*C*T*G*A*C* G*T*T- 3′18281-2 (23 mer)5′-T*GA*CTGT*GAACGTTCGGATGAT*T-3′

Table 2. Quantitative Real-Time PCR Thermal Profile.

Table 2. Quantitative Real-Time PCR Thermal Profile.

StepTimeTemperatureCyclesPre-Denaturation10 Minutes95 °C1Denaturation15 Seconds95 °C45Annealing/Extension1 Minute60 °C

Table 3. Primers and expected Amplicon sizes for TNF and INF-γ toxicity analysis.

Table 3. Primers and expected Amplicon sizes for TNF and INF-γ toxicity analysis.

CytokineForward Primer
5′--3′Reverse Primer
5′--3′NCBISize of
AmpliconReferenceTumor necrosis factor (TNF)CTCCAGGCGGTGCCTATGTGAAGAGCGTGGTGGCCCNM_013693.376 bp[18]Interferon Gamma (IFN-γ)CTGATTTCAACTTCTTTGGCTTATCCGCTACATCTGAATGANM_008337.4136 bp[18]HPRT1 (Hypoxanthine guanine phosphoribosyl transferase 1)TCCTCCTCAGACCGCTTTTCCTGGTTCATCATCGCTAATCNM_013556.290 bp[17]

Table 4. Reliability of the vaccine concentrations among BALB/c mice at day 14.

Table 4. Reliability of the vaccine concentrations among BALB/c mice at day 14.

Compound/Molecule DoseOptical Density (OD) Signals GeneratesCoefficient of Variation % Mouse 1Mouse 2Mouse 3 Vac 400.2390.1940.23411.1Vac 800.3830.3290.29113.8Vac 40/CPG0.7210.7230.7290.6Vac 80/CPG0.6430.6630.6471.6CPG0.010.010.0115.7PBS0.030.030.033.4

Table 5. Reliability of the vaccine concentrations among BALB/c mice at day 28.

Table 5. Reliability of the vaccine concentrations among BALB/c mice at day 28.

Compound/Molecule DoseOptical Density (OD) Signals GeneratesCoefficient of Variation % Mouse 1Mouse 2Mouse 3 Vac 400.3290.3230.27210.2Vac 800.68350.7240.57911.3Vac 40/CPG2.30952.4722.3223.8Vac 80/CPG1.2511.2051.21452.0CPG0.01750.01450.01510.3PBS0.0250.0270.020513.8

Table 6. Results from Hematological Profiling.

Table 6. Results from Hematological Profiling.

ParametersUnityVACC 40VACC 80VACC 40 + CPGVACC 80 + CPGCPGPBS **LEUKOGRAMWBC*103/ul3.69.39.76.14.95.7NEUTROPHILS%15.79.010.310.76.09.0LYMPHOCYTES%73.784.784.084.386.785.7MONOCYTES%9.35.04.75.76.06.3EOSINOPHILS%1.31.31.01.31.31.7BASOPHILS%0.00.00.00.00.00.0ERYTHROGRAMRBC*106/Ul8.49.08.78.97.97.8HBg/dl16.115.815.815.513.714.7HCT%40.243.242.541.837.137.3MCVFl47.942.748.946.636.748.1MCHPg19.117.618.117.317.319.0MCHCg/dl39.936.837.137.136.939.3PLATELETS COUNTPLATELETS103/ul130.71003.01140.71172.0896.0895.7

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