An mRNA-based vaccine candidate against SARS-CoV-2 elicits stable immuno-response with single dose

Abstract

D614G genotype of SARS-CoV-2 virus is highly infectious and responsible for almost all infection for 2nd wave. However, there are currently no reports with D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine with D614G variant and characterization in animal model. We have used special mRNA-architecture and formulation that provides suitable response of the product. The surface plasmon resonance (SPR) data with spike protein (S) revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice neutralized SARS-CoV-2-pseudoviruses in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, single-dose immunization protected mice-lungs from homotypic-pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The data suggested great promise for immediate translation of the technology to the clinic.

Keywords: COVID; Coronavirus; D614G; Immunization; LNP; Lipid nanoparticle; Vaccination.

Fig. 1

Target construction, amplification, IVT optimization, purification, and LNP formation. A representative data set from 3 independent experiments are shown. (a) Graphical representation of linear DNA construct for mRNA transcription, (b) IVT optimization where Lane 4 is the optimized condition, (c) DNA sequencing electropherogram data of D614G sequence in the target, (d) Identification of purified capped mRNA by SEC-HPLC, (e) size distribution of mRNA-LNP dose formulation.

Fig. 2

Local tolerance and CBC analysis. A representative data set from 2 independent experiments are shown, where n = 3 for each experiment. (a) check for sign of visible adverse reaction of administration before and after injection, (b) HE stained tissue from site of injection for erythema and edema, (c) WBC, white blood count, (d) RBC, red blood cell, (e) HGB, hemoglobin, (f) MCV, mean corpuscular volume, (g) MCH, mean corpuscular hemoglobin, (h) MCHC, mean corpuscular hemoglobin concentration, (i) HCT, hematocrit, (j) PLT, platelet, (k) ALT/SGPT, alanine transaminase, (l) AST/SGOT, aspartate aminotransferase, (m) BUN, blood urea nitrogen. Data were analyzed using one-way ANNOVA method, and found statistically non-significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Antibody titer and affinity analysis. A representative data set from 2 independent experiments are shown, where n = 4 for each experiment (unless mentioned otherwise). (a) antibody titer analysis from serum of different groups after 14 and 35 days of immunization (n = 6), data were compared by Mann-Whitney test, ****= p-value < 0.0001, ***= p-value < 0.001, **= p-value < 0.01 (b) ratio of IgG2a and IgG1 in treatment 2 and treatment 3 group, (c) ratio of IgG2a + IgG2b and IgG1 + IgG3 in treatment 2 and treatment 3 group, (d) serum antibody affinity analysis, (e) resin pull-down serum antibody affinity analysis.

Fig. 4

Cellular immune response analysis (cellular and secretory cytokine) in control and treatment group; unpaired T-test were performed between control and treatment groups; ***= p-value < 0.001, **= p-value < 0.01, *= p-value < 0.05. A representative data set from 2 independent experiments are shown, where control n = 3 and treatment n = 3 for each experiment (unless mentioned otherwise). (a) IFN-gamma expressing cell population percentage at Day 14, treatment n = 2 (b) IL-2 expressing cell population percentage at Day 14, (c) TNF-α expressing cell population percentage at Day 14, (d) IL-6 expressing cell population percentage at Day 14, (e) secretory IFN-gamma concentration at 6 and 18 h, (f) secretory IL-2 concentration at 6 and 18 h, (g) secretory IL-4 concentration at 6 and 18 h, (h) secretory IL-6 concentration at 6 and 18 h, (i) CD4 + cell population at Day 91, stimulated with S1 peptide pool, (j) CD4 + cell population at Day 91, stimulated with S2 peptide pool, (k) CD8 + cell population at Day 91, stimulated with S1 peptide pool, (l) CD8 + cell population at Day 91, stimulated with S2 peptide pool, (m) memory B cell population at Day 91, stimulated with S1 peptide pool.

Fig. 5

In vitro neutralization assay. A representative data set from 3 independent experiments are shown, where n = 4 for each experiment. (a) Image of Green fluorescence protein (GFP) expression after adeno-based SARS-CoV-2 pseudovirus neutralization assay from 2 to 4 sample dilution, (b) correlation between SARS-CoV-2 antibody from mice sera and intensity of GFP in different experimental group. For treatment group with the decrease of the antibody concentration, the intensity of GFP expression increased, which indicated the inhibition of SARS-Cov-2 pseudovirus into ACE2 overexpressed HEK293 cell (ACE2-HEK293 cell), (c) adeno-based SARS-CoV-2 pseudovirus neutralization percentage at different sample dilution, analyzed by real-time PCR, (d) HIV-1 based SARS-CoV-2 pseudovirus copy number analysis by real-time PCR; all the samples were compared by one-way ANNOVA method, ***= p-value < 0.001, **= p-value < 0.01, *= p-value < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

In vivo neutralization assay, lung section, * indicates trachea and arrow indicate infection. A representative data set from 2 independent experiments are shown, where n = 3 for each experiment. (a) the inhibition and neutralization of the SARS-CoV-2-S pseudo-type virus, data were analyzed using one-way ANNOVA method, ***= p-value < 0.001. (b) fluorescence image of lung section of control group mouse, (c) trans image of lung section of control group mouse, (d) overlay image of lung section of control group mouse, (e) fluorescence image of lung section of treatment group mouse, (f) trans image of lung section of treatment group mouse, (g) overlay image of lung section of treatment group mouse, intentional green color enhancement was done to observe any GFP intensity for panel g.