Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice

Abstract

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 μg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

Fig. 1. Immunization schedule of ChulaCov19 in BALB/c mice.

a Experiment 1: mice were immunized twice intramuscularly (IM) with a 3-week interval with various dosages of ChulaCov19 at 0.2, 1, 10 and 30 µg. Experiment 2: heterologous prime-boost study, mice were primed with 1/10 of the approved human dosage of CoronaVac or AZD1222 and boosted 4 weeks later with 5 µg of ChulaCov19. Homologous prime/boost of each vaccine (CoronaVac, AZD1222, or ChulaCov19) were included as control groups. Experiment 3: antibody durability and effect of 3^rd dose of ChulaCov19 study, mice were immunized twice with 3 weeks interval with 5 µg of ChulaCov19 (1/10 of human dose used in clinical trial) then boosted again at week 20. Bleeding was performed at 2 weeks following each dose (and at week 18 for Experiment 3). Splenocytes were collected at 2 weeks after the second dose (Experiment 1 & 2). n = 5 per group for Experiment 1, 2 and 3. b Challenge study in K18-hACE2 transgenic mice, n = 6 in vaccinated groups and n = 5 in control (PBS-receiving) group. Animals were immunized IM with 1 µg or 10 µg of ChulaCov19 at weeks 0 and 3. Sera were collected at weeks 0, 2, 3, 4 + 6 days, and 5 + 6 days for NAb measurements. At week 5, mice were challenged intranasally with 2×10⁴ pfu of WT SARS-CoV-2. Tissues were collected at week 5 + 6 days for assessment of viral RNA. Figures were created with BioRender.com.

Fig. 2. Protein expression analysis at 24 h after mRNA transfection in VERO E6 cells.

a Intracellular S protein expression examined by immunofluorescent assay employing anti-RBD, -S1, -S2 or PCS as primary antibody, the nuclei were counter stained with DAPI (blue). FITC-tagged 2^nd Abs (green) were used for detection of RBD, S1, and S2 while AlexaFluor647-tagged 2^nd Ab (red) was used following PCS staining. b hACE-2 binding assay (merged): culture supernatant collected from ChulaCov19 transfected cells incubated with HEK293T- hACE-2 cells. S protein on HEK293T-hACE-2 cell surface was stained with the same antibodies used in 2a. c S protein expression in cell culture supernatant analyzed by western blot using anti-RBD, -S1, -S2 or PCS as primary antibody. Recombinant S protein with abolished S1/S2 cleavage site was used as positive control in HEK293T-hACE-2 binding assay (right panel of 2b) and western blot (right lane of each panel in 2c). S0 was used to depict unprocessed S protein. Experiments were repeated two times independently with similar results.

Fig. 3. S-specific IgG responses in ChulaCov19 immunized mice.

a Kinetics of total IgG at 2 weeks after receiving 1 or 2 doses of 0.2, 1, 10, and 30 µg of ChulaCov19. b S-specific IgG2a/IgG1 ratio measured at 2 weeks after the 2^nd dose. Note; the IgG2a/IgG1 ratio of 10 µg and 30 µg immunized mice were not analyzed due to limited volume of serum samples. The mid-point titers were determined in duplicate assays from 5 mice in each group. Bars (a) or horizontal lines (b) represent the geometric mean (GMT) for each group while error bars indicate the 95% confident interval. Each dot represents an individual animal. Statistical analysis significance was determined by two-sided Mann–Whitney test. Differences were considered significant at p < 0.05 with exact p-values shown. p < 0.05 and p < 0.01 are indicated by * and **, respectively. Source data are provided as a Source Data file.

Fig. 4. NAb responses in immunized mice.

Experiment 1: (a) Live-virus microneutralization (micro-VNT50) titers against WT (Wuhan-Hu1) live-virus at two weeks after receiving each vaccine dose. b Pseudovirus neutralization test (psVNT50) titers at two weeks after the second dose againt WT (Wuhan-Hu1), Delta (B.1.617.2), Omciron (BA.1, and BA.4/5) variants. Experiment 2: c micro-VNT50 titers against WT (Wuhan-Hu1), Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) live-virus at two weeks after receiving various homologous or heterologous prime/boost regimens. d psVNT50 NAb titer results at two weeks after the second dose in various prime/boost regimens againt Omicron BA.1 and BA.4/5 subvariants. Experiment 3: e psVNT50 NAb against WT (Wuhan-Hu1), Delta (B.1.617.2), and Omicron (BA.1 and BA.4/5) variants for NAb durability and effect of 3^rd dose of ChulaCov19 studies. The titers were determined in duplicate assays from 5 mice in each group. Note; 4 mice in 10 µg group were analyzed for psVNT50 against BA.4/5 due to the limited volume of serum samples. Each dot represents an individual animal. Bars represent the GMTs and 95% CI for each group. Statistical significance was determined by two-sided Mann–Whitney tests. Differences were considered significant at p < 0.05 with exact p-values shown. p < 0.05 and p < 0.01 are indicated by * and **, respectively. Source data are provided as a Source Data file.

Fig. 5. Induction of S-specific IFN-γ positive T cells in BALB/c mice.

a mice were immunized with various doses of ChulaCov19 analyzed at 2 weeks after the second dose. b heterologous prime/boost study; mice were primed with CoronaVac or AZD1222 vaccine and boosted with ChulaCov19 (5 µg). Homologouse prime/boost results of each vaccine were included. S-specific IFN-γ positive T cells were determined in duplicate assays from 5 mice in each group. Bars represent the mean ± SD of S-specific IFN-γ positive T cells after stimulated with overlapping peptide pools spanning the SARS-CoV-2 S1 (pooled #1-5) and S2 (pooled #6-10). Statistical significance was determined by two-sided Mann–Whitney test. Differences were considered significant at p < 0.05 with exact p-values shown. p < 0.05 and p < 0.01 are indicated by * and **, respectively. SD; standard deviation. Source data are provided as a Source Data file.

Fig. 6. Immune response and protective efficacy results in the challenge study.

a Kinetic response of micro-VNT50 titer after ChulaCov19 immunization and after challenge. The titers were determined in duplicate assays from control (n = 5) or vaccinated groups (n = 6), respectively. Data are presented as GMT of micro-VNT50 titer with 95% confident interval. Statistical analysis was performed to compare the GMT of micro-VNT50 between 1 and 10 μg dosed mice at each time point. b Body-weight values with SD are presented as a percentage of initial body weight before challenge (Day 0) through Day 6 post-challenge. In the control group, 3 out of 5 mice reached euthanasia criteria on Day 5 hence only 2 mice were analyzed for body weight on Day 6 after challenge. c SARS-CoV-2 viral RNA copies with SD detected by RT-qPCR in serum and homogenized tissues of challenged animals analyzed at euthanasia date (Day 6). Each dot represents an individual animal. Note: tissues from 3/5 animals in control group were collected at day 5. RNA copies were calculated as genomic equivalent/mg of tissue. Statistical significance was determined by two-sided Mann–Whitney test. p < 0.05 and p < 0.01 are indicated by * and **, respectively. SD; standard deviation. Source data are provided as a Source Data file.