Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

Abstract

Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines.

Fig. 1. Expression of SARS-CoV-2 RBD protein in cells transfected with formulated RBD-mRNAs.

a Description of the different nanocarriers used for the encapsulation of RBD-mRNA. b Detection of RBD expression in cellular pellets and supernatants from 293T cells transfected with the different formulated RBD-mRNAs for 6 h by western-blotting analysis using a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (upper panels). Ponceau staining (lower panels) was used as loading control. All blots derive from the same experiment and were processed in parallel. c RBD expression and viability of human monocyte-derived dendritic cells (hMDDCs) from a healthy donor at 6 and 24 h after transfection with the different nanocarriers containing the RBD-mRNA. Mean with standard error of the mean (SEM) is represented.

Fig. 2. Humoral immune responses induced in C57BL/6 mice by different nanocarriers containing RBD-mRNA.

a Immunization schedule. Female C57BL/6 mice (n = 5) were immunized with two doses of 40 µg of the different formulations containing RBD-mRNA by intramuscular (i.m.) route as indicated. b SARS-CoV-2 RBD-specific IgG binding antibodies. Anti-RBD IgG titers were determined in individual sera obtained at 20 days post-prime (d20) or 21 days post-boost (d42) by ELISA. An unpaired nonparametric Mann–Whitney test of transformed data was used. ***p < 0.001. c SARS-CoV-2 neutralizing antibody responses. NT₅₀ titers were determined in individual sera harvested at d20 and d42 using a live virus microneutralization assay (MAD6 strain, containing D614G mutation). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. ***p < 0.001. d Neutralizing antibody responses induced against SARS-CoV-2 variants. NT₅₀ titers were evaluated in individual serum samples harvested at d42 by a live virus microneutralization assay using the SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants. An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. **p < 0.01; ***p < 0.001. Serum samples from mice similarly vaccinated with two doses of 5 μg of BNT162b2 vaccine (mRNA vaccine from Pfizer-BioNTech) were used as a reference value (BNT162b2 reference). Red dashed line represents the lower limit of detection of the assay. Mean with standard error of the mean (SEM) is represented.

Fig. 3. SARS-CoV-2-specific humoral responses elicited in transgenic K18-hACE2 mice by mLNP-RBD or LNP-1-RBD formulations before virus challenge.

a Immunization schedule. K18-hACE2 transgenic mice (n = 6) were immunized with two doses of 40 µg of mLNP-RBD or LNP-1-RBD formulations by i.m. route as indicated. At day 47 mice were challenged intranasally (i.n.) with 1 × 10⁵ PFU of SARS-CoV-2 (MAD6 isolate, containing D614G mutation). b SARS-CoV-2 S- and RBD-specific IgG binding antibodies. Anti-S and anti-RBD IgG titers were determined in individual sera obtained at 20 days post-prime (d20) or 21 days post-boost (d42) by ELISA. An unpaired nonparametric Mann-Whitney test of transformed data was used. *p < 0.05; **p < 0.005; ***p < 0.001. c SARS-CoV-2 neutralizing antibody responses. NT₅₀ titers were determined in individual sera harvested at d20 and d42 using a live virus microneutralization assay (MAD6 strain, containing D614G mutation). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. **p < 0.005. d Neutralizing antibody responses induced against SARS-CoV-2 variants. NT₅₀ titers were evaluated in individual sera collected at d42 by a live virus microneutralization assay using the SARS-CoV-2 Delta (B.1.617.2), Omicron (B.1.1.529), BQ1.1 and XBB1.5 variants. An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. *p < 0.05; **p < 0.005; ***p < 0.001. Serum samples from mice similarly vaccinated with two doses of 5 μg of BNT162b2 vaccine (mRNA vaccine from Pfizer-BioNTech) were used as a reference value (BNT162b2 reference). Red dashed line represents the lower limit of detection of the assay. Mean with standard error of the mean (SEM) is represented.

Fig. 4. Homologous prime/boost administration of mLNP-RBD or LNP-1-RBD fully protects transgenic K18-hACE2 mice from morbidity and mortality against SARS-CoV-2 infection.

Individual mice were daily monitored for changes of body weight (a) and mortality (b) for 14 days. Mice that lost more than 25% of the initial body weight were sacrificed. c Genomic (RdRp) and subgenomic (N) SARS-CoV-2 RNAs detected by RT-qPCR in lungs from individual mice at 14 days (groups 1 and 2) or 7 days (group 3) after SARS-CoV-2 challenge. Mean RNA copy numbers (copies/μl) with standard error of the mean (SEM) from duplicates of each lung sample is represented. Relative values are referred to uninfected mice (group 4). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. ***p < 0.001. d SARS-CoV-2 infectious virus in lung or nasal turbinates. Mean PFU (PFU/gram of lung tissue or PFU/mL of nasal turbinates) with SEM from triplicates of each sample is represented. An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. **p < 0.005; ***p < 0.001.

Fig. 5. Lung pathology in vaccinated and challenged transgenic K18-hACE2 mice.

a Lung inflammation scores observed in lung samples from vaccinated and challenged K18-hACE2 transgenic mice, and euthanized at day 7 (group 3) or day 14 post-challenge (groups 1, 2 and 4). Mean with SEM of cumulative histopathological lesion scores is indicated. An unpaired nonparametric Mann-Whitney test was performed.*p < 0.05; **p < 0.005. b Representative lung histopathological sections (H&E staining) from K18-hACE2 transgenic mice included in each of the experimental groups (scale bar: 200 µm). The severity and extent of inflammatory lung lesions observed in the immunized and challenged mice included in group 1 were similar to those described in the mice included in the non-immunized and challenged control group (group 3). Lesions included the presence of mild to moderate diffuse thickening of the alveolar septa, occasional small multifocal alveolar mononuclear cell infiltrates (black arrows) or mild multifocal perivascular and peribronchiolar mononuclear infiltrates (black arrowheads). Mice included in group 2 showed the lowest inflammatory scores. These animals showed only some lung areas with mild thickening of the alveolar septa (blue arrows) together with occasional small focal perivascular mononuclear infiltrates, showing an appearance highly similar to that observed in the PBS-treated non-challenged mice (group 4).

Fig. 6. mLNP-RBD and LNP-1-RBD formulations differentially regulate proinflammatory cytokine and chemokine profiles in lung from vaccinated and challenged transgenic K18-hACE2 mice.

Proinflammatory cytokines and chemokines were detected by RT-qPCR in lungs from individual mice at 14 days (groups 1 and 2) or 7 days (group 3) after SARS-CoV-2 challenge. Mean RNA levels (in A.U.) with SEM from duplicates of each lung sample is represented; relative values are referred to uninfected mice (group 4). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. *p < 0.05; **p < 0.005; ***p < 0.001.