Immunogenicity, Efficacy and Twelve-Month Storage Stability Studies of a Lyophilized Rabies mRNA Vaccine

Abstract

Background: Many new mRNA-based vaccine candidates in liquid mRNA-LNP formulations are under development; however, their stability limitations necessitate frozen storage, posing a significant challenge for long-term storage and transportation. Methods: In this study, an mRNA-LNP rabies vaccine, ABO1005, was prepared, freeze-dried and stored at 2-8 °C for 12-month storage stability evaluation. The immunogenicity, vaccine potency (the NIH method), and protective efficacy of ABO1005 were assessed in mice or dogs and compared to a commercialized inactivated vaccine. Results: Research conducted in mice indicated that the lyophilized vaccine exhibited comparable immunogenicity to its liquid form counterpart. Furthermore, the vaccine candidate elicited a robust humoral response lasting at least 175 days, and the specific antibody titers were not affected by the pre-administration of hyperimmune serum. In comparison to the commercialized inactivated vaccine (HDCV or PVRV), ABO1005 elicited significantly higher levels of humoral and cellular immunity. Vaccine potency testing (NIH) revealed that the potency of ABO1005 at 15 μg/dose was 8.85 IU/dose, which is substantially higher than the standard required for the lot release of rabies vaccines for current human use. In a post-exposure prophylaxis (PEP) study in Beagle dogs, the lyophilized vaccine provided 100% protection for dogs following a two-dose regimen (D0-D7), whereas commercially approved inactivated vaccine offered 83% protection. After storage at 2-8 °C for 12 months, no notable changes were observed in the particle size, encapsulation efficiency, and integrity of mRNA or in the immunogenicity of the lyophilized vaccine. Conclusions: This study successfully developed a formulation and process of freeze-drying for a rabies mRNA vaccine, paving the way for future lyophilized mRNA vaccine development.

Keywords: beagles; lipid nanoparticles; long-term stability; lyophilization; mRNA vaccine; rabies.

Figure 1

The design and characterization of ABO1005. (A) ABO1005 composes a 5′ Cap, a 5′ UTR, a coding sequence (CDS) of RABV-G derived from the PM strain, a 3′ UTR and a poly (A) tail. (B) The integrity of the mRNA. (C) The cryogenic electron microscopy (Cryo-EM) of ABO1005. (D) The percentage of trimer-form RABV-G-positive cells in transfected HEK293F cells. (E) The Western blot analysis of RABV-G expression in transfected HEK293T cells.

Figure 2

Humoral immune responses of ABO1005 in mice and dogs. (A) The experimental layout of the immunization regimen study. (B) VNA titers of the sera from mice (n = 10) in the regimen study. (C) Survival curves of the mice (n = 10) intracerebrally (I.C.) infected with the virulent CVS-24 strain (50 × LD₅₀) in the regimen study. (D) The experimental layout of the dosage titration study. (E) VNA titers of the sera from mice (n = 5~9) in the dosage titration study. (F) Survival curves of the mice in the dosage titration study (n = 10). (G) The experimental layout of the immunogenicity study in rural dogs. (H) VNA titers in sera of rural dogs (n = 5) post I.M. immunization with a single dose of 15 μg of ABO1005. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns, not significant.

Figure 3

Cellular immune responses of ABO1005 in mouse model. On day 21 post initial immunization, splenic cells from mice (n = 5) were analyzed. (A) Representative pseudo-color plots of cytokine-positive CD4⁺ T cells (A) and CD8⁺ T cells (B). Statistical percentages of cytokine-positive CD4⁺ T cells (C) and CD8⁺ T cells (D). Representative IFN-γ (E)- and IL-2 (F)-secreting cell spots from five splenic samples of each group. Statistical spot-forming units (SFU) of IFN-γ (G)- and IL-2 (H)-secreting cells. *, p ≤ 0.05; **, p ≤ 0.01.

Figure 4

ABO1005 induces proliferation of Tfh and GCB in mouse spleens. On day 21 post initial immunization, splenic cells from mice (n = 5) were analyzed. Representative pseudo-color plots (A,C) and statistical percentages (B,D) of Tfh and GCB cells. **, p ≤ 0.01.

Figure 5

Duration of humoral immunity and effect of pre-administration with rabies-specific immunoglobulin on immunogenicity of ABO1005. (A) Experimental layout of duration study. (B) Duration of humoral immunity in mice. (C) Correlation analysis between RABV-specific IgG titers and VNA titers (n = 137). (D) Experimental layout of study on effect of pre-administration with rabies specific immunoglobulin. Groups of mice (n = 8) were injected I.V. with 2 IU (VNA titers) of rabies hyperimmune serum diluted in PBS buffer or PBS alone, followed by I.M. immunization with two doses of ABO1005. One group (n = 8) of mice received only I.V. injection of rabies hyperimmune serum. (E) RABV-specific IgG titers in sera were measured using ELISA. ns, not significant.

Figure 6

Lyophilized ABO1005 protected Beagle dogs in PEP model. (A) The images of lyophilized vials containing ABO1005. (B) Spherical nanoparticles of lyophilized ABO1005. (C) The comparison of immunogenicity between liquid and lyophilized ABO1005 (0.12 μg per dose on days 0 and 14) in mice (n = 8). (D) The experimental layout of the PEP study. (E) Survival curves of dogs (n = 6). (F) VNA titers of the survived dogs (n = 5~6) on day 60 post initial immunization (challenge). ns, not significant.

Figure 7

The lyophilized ABO1005 vaccine exhibited long-term stability. (A) The size distribution, encapsulation efficiency and mRNA integrity of the cargo encapsulated in lyophilized ABO1005. (B) At storage after months 0 and 12, the immunogenicity changes in lyophilized ABO1005 were determined in mice. ns, not significant.