Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine

Abstract

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans¹. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 μg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

Fig. 1. Vaccine design and study schema.

a, Designs of the CVnCoV and CV2CoV mRNA vaccine candidates. Both vaccines are based on CureVac’s RNActive platform and encode SARS-CoV-2 spike protein with di-proline mutations. The vaccines differ in their unique non-coding regions, as shown. b, Non-human primate vaccine study schema. Cynomolgus macaques were immunized intramuscularly (i.m.) on day (D) 0 with CVnCoV (n = 6) or CV2CoV (n = 6) mRNA vaccine or were designated as sham (n = 6). The animals were boosted at week 4 and were challenged at week (W) 8. Samples were collected weekly after immunization and on days 0, 1, 2, 4, 7 and 10 after challenge for immunological and virological assays. PP, K986P and V987P mutations; HSL, histone stem–loop.

Fig. 2. CV2CoV elicits high levels of binding and neutralizing antibody responses in macaques.

Animals (n = 6 per group) were vaccinated twice with 12 µg of CVnCoV or CV2CoV on day 0 and on day 28 or remained untreated as negative controls (sham). a, b, Titres of RBD-binding antibodies (a) and pseudovirus neutralizing antibodies (NAb) against the ancestral SARS-CoV-2 strain (b) were evaluated at different time points after the first (weeks 0, 1, 2 and 4) and second (weeks 5, 6 and 8) vaccinations. c, d, Sera collected on day 42 (week 6) were analysed for pseudovirus (c) and live-virus (d) neutralizing antibody titres against virus with the D614G mutation and the B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. e, Sera collected from non-human primates immunized with 12 µg of CVnCoV or 30 µg of BNT162b2 on day 35 (week 5) after boosting were analysed for pseudovirus neutralizing antibody titres against the ancestral WA/2020 (WT) strain. Each dot represents an individual animal and bars depict the median; the dashed line shows the limit of detection. Source data

Fig. 3. Protective efficacy of CV2CoV.

Negative-control animals (sham) and animals (n = 6 per group) vaccinated on day 0 and day 28 with 12 µg of CVnCoV or CV2CoV were challenged with 1.0 × 10⁵ TCID₅₀ of SARS-CoV-2 (USA-WA1/2020) via the intranasal and intratracheal routes. a, b, BAL (a) and nasal swab (b) samples collected on days 1, 2, 4, 7 and 10 after challenge were analysed for levels of replicating virus by RT–PCR specific for sgRNA. Thin black lines represent individual animals and thick red lines depict the median; the dashed line shows the limit of detection. Source data

Fig. 4. Titres of binding and neutralizing antibodies elicited following CVnCoV and CV2CoV vaccination (n = 6 per group) correlate with protection against SARS-CoV-2.

a, b, Summary of peak viral loads following SARS-CoV-2 challenge in BAL and nasal swab (NS) samples. Animals were challenged with 1.0 × 10⁵ TCID₅₀ of SARS-CoV-2 derived from strain USA-WA1/2020 (NR-52281, BEI Resources). c–f, Antibody correlates of protection for binding antibodies (c, d) and neutralizing antibodies (e, f). Statistical analysis was performed using the two-tailed non-parametric Mann–Whitney test, and correlation was analysed by two-sided Spearman rank-correlation test. The bars indicate median values. Source data

Extended Data Fig. 1. Innate cytokine induction following mRNA immunization (6/group).

Sera isolated 24h post first injection were analyzed for a panel of 19 cytokines associated with viral infection using a U-PLEX Viral Combo kit from Meso Scale Discovery. Changes in cytokine levels above the detection limits were detectable for 9 cytokines. Each dot represents an individual animal, bars depict the median and the dotted line shows limit of detection. Statistical analysis was performed using two-tailed nonparametric Mann-Whitney test. Source data

Extended Data Fig. 2. Neutralizing antibody titers against variants.

Animals (6/group) were vaccinated twice with 12µg of CVnCoV or CV2CoV on d0 and d28 or remained untreated as negative controls (sham). Sera isolated on d42 (week 6) were analyzed for pseudovirus neutralizing antibody titers against C.37 (Lambda), B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. Each dot represents an individual animal, bars depict the median and the dotted line shows limit of detection. Source data

Extended Data Fig. 3. Memory B and T cell immune responses day 42 following immunization.

PBMCs from negative control (sham), CVnCoV or CV2CoV vaccinated animals (6/group) isolated on d42 of the experiment were stained for (a) RBD and (b) Spike-specific activated memory B cells and analyzed by high-parameter flow cytometry. IFNγ responses to pooled spike peptides were analyzed via ELISPOT (c). Each dot represents an individual animal, bars depict the median and the dotted line shows limit of detection. Statistical analysis was performed using two-tailed nonparametric Mann-Whitney test. PBMC = peripheral blood mononuclear cell; SFC = spot forming cells. Source data

Extended Data Fig. 4. Binding and neutralizing antibody titers correlate with protection against SARS-CoV-2.

Summary of area under curve (AUC) viral load values following SARS-CoV-2 challenge in BAL and nasal swab samples (6/group) (a, b); antibody correlates of protection for binding antibodies (c, d) and neutralizing antibodies (e, f). Statistical analysis was performed using two-tailed nonparametric Mann-Whitney test. Correlations was analyzed by two-sided Spearman rank-correlation test. NAbs = neutralizing antibodies, BAL = bronchoalveolar lavage NS = nasal swab. Source data

Extended Data Fig. 5. Infectious virus titers after SARS-CoV-2 challenge (6/group).

Infectious virus titers of BAL and nasal swab samples collected 2 days post challenge were analyzed by TCID₅₀ assays. Each dot represents an individual animal, bars depict the median and the dotted line shows limit of detection. Statistical analysis was performed using two-tailed nonparametric Mann-Whitney test. Source data

Extended Data Fig. 6. Post-challenge binding and neutralizing antibody responses (6/group).

Negative control (sham) or animals vaccinated on d0 and d28 of the experiment with 12 µg of CVnCoV or CV2CoV as indicated were subjected to challenge infection using 1.0×10⁵ TCID₅₀ SARS-CoV-2 via intranasal (IN) and intratracheal (IT) routes. (a) Titers of RBD binding antibodies and (b) pseudovirus neutralizing antibodies against ancestral SARS-CoV-2 strain were evaluated before (week 8) and a week after challenge infection (week 9). Each dot represents an individual animal, bars depict the median and the dotted line shows limit of detection. Statistical analysis was performed using two-tailed nonparametric Mann-Whitney test. NAbs = neutralizing antibodies. Source data

Extended Data Fig. 7. CVnCoV and CV2CoV protect the lungs from pathological changes upon viral challenge (6/group).

Eight lung lobes (4 sections from right and left, caudal to cranial) were assessed and scored (1-4) for each of the following lesions: 1) Interstitial inflammation and septal thickening 2) Eosinophilic interstitial infiltrate 3) Neutrophilic interstitial infiltrate 4) Hyaline membranes 5) Interstitial fibrosis 6) Alveolar infiltrate, macrophage 7) Alveolar/Bronchoalveolar infiltrate, neutrophils 8) Syncytial cells 9) Type II pneumocyte hyperplasia 10) Broncholar infiltrate, macrophage 11) Broncholar infiltrate, neutrophils 12) BALT hyperplasia 13) Bronchiolar/peribronchiolar inflammation 14) Perivascular, mononuclear infiltrates 15) Vessels, endothelialitis. Each feature assessed was assigned a score of 0 = no significant findings; 1 = minimal; 2 = mild; 3 = moderate; 4 = marked/severe. (a) Cumulative scores per animal (b) Cumulative scores per lung lobe. Individual animals are represented by symbols. Representative histopathology from sham vaccinated (c-h), CnVCoV vaccinated (i, j), and Cv2CoV vaccinated (k, l) animals showing (c, d, inset) alveolar macrophage infiltrate, (e, f, inset) syncytial cells (arrowheads) and type II pneumocyte hyperplasia, inset (g, h, inset) bronchiolar epithelial necrosis with neutrophilic infiltrates (i) alveolar neutrophilic infiltrate and alveolar septal thickening (j) focal consolidation with inflammation composed of macrophages, neutrophils, and syncytial cells (k) focal pneumocyte hyperplasia, syncytial cells and inflammatory infiltrates (l) peribronchiolar inflammation. Statistical analysis was performed using two-tailed nonparametric Mann-Whitney test. Scale bars: 100 microns (c), 50 microns (e, g) 20 microns (i-l). BALT bronchus associated lymphoid tissue. Source data