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).

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).

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).

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.

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.

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