Preserved efficacy of lyophilized SARS-CoV-2 mRNA vaccine incorporating novel ionizable lipids after one year at 25 °C

Abstract

mRNA vaccines have shown great efficacy against SARS-CoV-2, yet challenges remain in optimizing vaccine components to achieve enhanced immune response and vaccine stability. In this study, we developed CPVax-CoV, a new lyophilized mRNA vaccine that features novel thiolactone-based ionizable lipids and newly designed untranslated regions (UTRs) for enhanced expression. Incorporation of these optimized components into our vaccine candidate CPVax-CoV significantly improved immune responses in mice compared to commercially available mRNA vaccines. Moreover, lyophilized CPVax-CoV has proven to be thermostable, maintaining its biological activity for up to one year at 4 °C and 25 °C after lyophilization, overcoming the cold-chain limitations of current mRNA vaccines. This vaccine demonstrates protective efficacy against ancestral SARS-CoV-2 and the Omicron XBB variant, offering a scalable solution for global distribution and pandemic preparedness. These findings underscore the potential of this platform for future next-generation mRNA vaccine development.

Fig. 1. Immunity induced by SARS-CoV-2 mRNA vaccine candidates formulated with novel ionizable lipids.

a Immunization scheme and sample collection schedule. BALB/c mice (n = 5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 3 (prior to boost) and 6, and specific antibody levels were determined in serum. T-cell response was evaluated in splenocytes 3 weeks post-boost. b Reciprocal endpoint titers of antigen-specific IgG antibodies in serum samples determined by ELISA using RBD recombinant protein from SARS-CoV-2 wild-type variant. c IFN-γ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pool (d, e) IFN-γ and IL-4 quantification in supernatants of splenocytes after overnight stimulation with SARS-CoV-2 Spike peptide pool determined by ELISA. Graphs are represented as mean ± SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001 as determined by two-way OVA (b) and one-way ANOVA (c, d) with Tukey post-test.

Fig. 2. Influence of mRNA sequence optimization on vaccine-elicited immune response.

a Immunization scheme and sample collection schedule. Mice (n = 5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 3 (prior to boost) and 6, and specific antibody levels were determined in serum. T-cell response was evaluated in splenocytes 3 weeks post-boost. b Reciprocal endpoint titers of antigen-specific IgG antibodies in serum samples determined by ELISA using RBD recombinant protein from wild-type variant. c Neutralization titers in serum samples were determined by neutralization assay using Spike-pseudotyped lentivirus and infection in HEK293T-ACE2-TMPRSS2 cells. NT50 titers refer to the dilution of a serum sample at which 50% of the pseudovirus infection is inhibited. d IFN-γ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pool. e IFN-γ quantification in supernatants of splenocytes after overnight stimulation with SARS-CoV-2 Spike peptide pool determined by ELISA. f T-lymphocyte CD4 and CD8 cell frequencies in spleen analyzed by flow cytometry. g Tfh and GC B cells analysis. Mice (n = 5) were immunized intramuscularly with 5 µg of mRNA/animal. Inguinal lymph nodes were extracted on day 7. h Tfh (CD45R^-, CD4⁺, CD44hi, CXCR5⁺, PD-1⁺) and GC B cell (CD19⁺, CD95⁺, GL7⁺) frequencies were determined by flow cytometry analysis. Graphs are represented as mean ± SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001 as determined by two-way ANOVA (a) and one-way ANOVA (c–h) with Tukey post-test.

Fig. 3. CPVax-CoV vaccine candidate biodistribution and preliminary safety evaluation.

BALB/c mice (male n = 3, female n = 3) were immunized intramuscularly with 5 µg of mRNA/animal encapsulated in Indium-111 labeled LNPs. a In vivo SPECT/CT representative images of coronal and axial sections acquired at different time points post-immunization for up to one week. The lower image shows the signal progression in the presacral lymph node (red arrow) with an adjusted threshold. b Signal quantification at the injection site at different points post-vaccination using standardized uptake value (SUV) units. c Signal quantification in the presacral lymph node at different points post-vaccination. d Ex vivo signal quantification at day 7 post-immunization, represented as injected dose per gram normalized to brain signal. e Serum biochemical analysis of hepatic enzymes 24 h post-immunization. Graphs are represented as mean ± SD. ns non-significant as determined by two-way ANOVA with Tukey post-test (e).

Fig. 4. Vaccine-induced protection against mouse-adapted SARS-CoV-2 infection.

a Immunization scheme and sample collection schedule of the mouse-adapted infection model. BALB/c mice were immunized intramuscularly at day 0 (prime) and 21 (boost) with 1 µg of mRNA/animal. At week 7, mice were challenged with 1 × 10⁴ TCID50 of the mouse-adapted strain MA20. At days 2 and 4 post-challenge, lung viral load and cytokine profile were evaluated (n = 5). Clinical signs and survival were monitored during 25 days after challenge in an independent experimental group of each condition (n = 10). b Body weight monitored over 25 days after challenge, calculated as a percentage of the initial weight (pre-challenge). c Survival rate over 25 days after challenge. d Lung viral load determined by virus titration assay in VERO E6 cells at day 2 and 4 post-infection. e Cytokine and chemokine profile in lung homogenates collected at day 2 and 4 post-challenge and analyzed by Luminex assay. Graphs are represented as mean ± SD. ns non-significant; ^**p < 0.01; ^****p < 0.0001 as determined by two-way ANOVA with Tukey post-test.

Fig. 5. Vaccine-induced protection against SARS-CoV-2 infection in a humanized mouse model.

a Immunization scheme and challenge schedule of the K18 infection model. C57BL/6-hACE2 K18 mice (n = 5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. At week 7, mice were challenged with 1 × 10⁵ TCID50 of SARS-CoV-2 alpha strain. At day 3 post-challenge, lung viral load was evaluated. b Body weight monitoring after 4 days of infection was calculated as a percentage of the initial weight (pre-challenge). c Lung viral load determined by virus titration assay in VERO E6 cells at day 4 post-infection. d Cytokine and chemokine profile in lung homogenates collected at day 4 post-challenge and analyzed by Luminex assay. Graphs are represented as mean ± SD. ns non-significant; ^***p < 0.001; ^****p < 0.0001 as determined by two-way ANOVA (b) and one-way ANOVA (c) with Tukey post-test.

Fig. 6. Assessment of the CPVax-CoV platform’s ability to trigger immune responses against VOCs.

a Timeline of CPVax-CoV-XBB immunity evaluation. BALB/c mice (n = 5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. Blood samples were obtained at week 6, and serum neutralizing ability was determined. T-cell response was evaluated in splenocytes 3 weeks post-boost. b NT50 neutralization titers in serum of vaccinated mice determined at 3 weeks post-boost by Spike pseudovirus-neutralization assay using wild type and XBB.1.5 Spike variants. c IFNγ-secreting splenocytes quantification by ELISPOT after O.N. stimulation with SARS-CoV-2 Spike peptide pools of wild type and XBB.1.5 variants. d Immunization scheme and challenge schedule of the K18 infection model with the XBB strain. C57BL/6-hACE2 K18 mice (n = 5) were immunized intramuscularly at day 0 (prime) and 21(boost) with 1 µg of mRNA/animal. At week 7, mice were challenged with 5 × 10³ TCID50 of SARS-CoV-2 XBB.1.5 strain. At day 3 post-challenge, lung viral load was evaluated. e NT50 neutralization titers in the serum of mice vaccinated were determined at 3 weeks post-boost by Spike pseudovirus-neutralization assay using wild type and XBB.1.5 Spike variants. f Lung viral load determined by virus titration assay in VERO E6 cells at day 3 post-infection. Graphs are represented as mean ± SD. ns non-significant; ^*p < 0.05; ^**p < 0.01; as determined by two-way ANOVA (c), unpaired t-test (e), and one-way ANOVA (f) with Tukey post-test.

Fig. 7. Long-term stability assessment of lyophilized vaccine formulation.

a Timeline of the stability study. Lyophilized CPVax-CoV was stored at 4 °C and 25 °C, and standard liquid vaccines were stored at −80 °C as controls. Aliquots of each condition were reconstituted at the indicated time-points to characterize their physicochemical properties and to evaluate their in vivo activity. At 0, 3, 6, and 9 months, production of specific antibodies in serum was analyzed by ELISA after a single immunization of BALB/c mice with 1 µg of mRNA/animal. At 12 months, an efficacy study was conducted. After prime-boost immunization, animals were infected with mouse-adapted SARS-CoV-2, and neutralization, viral load and cytokines in lungs were evaluated. b Determination of diameter, PDI, Z potential, and encapsulation efficacy of liquid and lyophilized LNPs at the indicated time-points of storage. c Reciprocal endpoint titers of anti-RBD IgG antibodies in serum samples collected at day 21 post-immunization were determined by ELISA after the indicated time-points of storage. d Determination of diameter, PDI, Z potential, and encapsulation efficacy at different time-points after resuspension and storage at 4 °C of CPVax-CoV. e Reciprocal endpoint titers of anti-RBD IgG antibodies in serum samples collected at day 21 post-immunization were determined by ELISA at different time-points after resuspension and storage at 4 °C of CPVax-CoV. Graphs are represented as mean ± SD. ns non-significant; as determined by one-way ANOVA with Tukey post-test. wks weeks.

Fig. 8. Protective efficacy of lyophilized CPVax-CoV.

a–e Protection of one-year stored lyophilized CPVax-CoV at 4 °C and 25 °C in BALB/c mice infected with a viral dose of 1 × 10⁴ TCID50 of the MA20 strain. a Immunization scheme and challenge schedule of the MA20 infection model. b NT50 neutralization titers in the serum of vaccinated mice were determined at 3 weeks post-boost by Spike pseudovirus-neutralization assay. c Body weight monitoring during the 3 days after infection was calculated as a percentage of the initial weight (pre-challenge). d Lung viral load was determined by virus titration assay in VERO E6 cells at day 3 post-infection. e Cytokine and chemokine profile in lung homogenates collected at day 3 post-challenge and analyzed by Luminex assay. f Protective efficacy of lyophilized CPVax-CoV and CPVax-CoV-XBB in C57BL/6 humanized ACE2 model against 1 × 10⁵ or 5 × 10³ TCID50 of alpha or XBB infection, respectively. Lung viral load determined by virus titration assay in VERO E6 cells on day 3 post-infection. Graphs are represented as mean ± SD. ns non-significant; ^**p < 0.01; ^****p < 0.0001 as determined by one-way ANOVA (b, d, and f) and two-way ANOVA (c) with Tukey post-test.