Evaluation of the Immunological Efficacy of an LNP-mRNA Vaccine Prepared from Varicella Zoster Virus Glycoprotein gE with a Double-Mutated Carboxyl Terminus in Different Untranslated Regions in Mice

Abstract

Cell-mediated immunity (CMI) plays a key role in the effectiveness of varicella zoster virus (VZV) vaccines, and mRNA vaccines have an innate advantage in inducing CMI. Glycoprotein E (gE) has been used widely as an antigen for VZV vaccines, and carboxyl-terminal mutations of gE are associated with VZV titer and infectivity. In addition, the untranslated regions (UTRs) of mRNA affect the stability and half-life of mRNA in the cell and are crucial for protein expression and antigenic translational efficiency. In this study, three UTRs were designed and connected to the nucleic acid sequence of gE-M, which is double mutated in the extracellular region of gE. Then, mRNA with different nucleic acids was encapsulated in lipid nanoparticles (LNPs), forming three LNP-mRNA VZV vaccines, named gE-M-Z, gE-M-M, and gE-M-P. The immune response elicited by these vaccines in mice was evaluated at intervals of 4 weeks, and the mice were sacrificed 2 weeks after the final immunization. In the results, the gE-M-P group, which retains the nucleic acid sequence of gE-M and is connected to Pfizer/BioNTech's BNT162b2 UTRs, induced the strongest humoral immune response and CMI. Because CMI is crucial for protection against VZV and for the design of VZV vaccines, this study provides a feasible strategy for improving the effectiveness and economy of future VZV vaccines.

Keywords: CMI; UTR; VZV; mRNA vaccine.

Figure 1

Diagram and sequence of LNP-mRNA VZV vaccines synthesized in this study. (A) Schematic diagram of the components of the synthesis of the nucleic acid sequences of mRNA. Red boxes indicate the altered parts in each group. (B–D) Sequence differences in the 5′-UTR, 3′-UTR, and poly (D) tail between groups, respectively. ORF: open reading frame; UTR: untranslated regions.

Figure 2

Characterization of LNP mRNA vaccines. The group labels were gE-M-Z and gE-M-M gE-M-P. (A) Diameters tested by size analyzer; (B) polydispersity index of LNPs; (C) mRNA encapsulation efficiency; (D) loaded mRNA detected with 1% denatured agarose gel electrophoresis.

Figure 3

gE-specific IgG titers detected by enzyme-linked immunosorbent assay (ELISA). The mean value of each group was shown on the top of every bar. IgG titers were compared using one-way analysis of variance (ANOVA) followed by Kruskal–Wallis test, with the Shingrix group as a control. Each point represents an individual mouse, ** p < 0.01.

Figure 4

The secretion levels of IL-2 and IFN-γ in splenocytes were assayed by ELISA and ELISPOT. (A,B) The levels of IL-2 produced by splenocytes after gE stimulation; (C,D) the levels of IFN-γ produced by splenocytes after gE stimulation; (E) pictures of individual spots. Means were compared using one-way ANOVA followed by Dunnett’s multiple comparisons test, with the Shingrix group as a control. Points represent individual mice, * p < 0.05, p > 0.99, not significant.

Figure 5

Flow cytometry assay for gE-specific IL-2- and IFN-γ-producing T-cells. (A,B) Proportion of IL-2- and IFN-γ- producing CD4⁺ T-cells among splenocytes after stimulation with gE. (C,D) Proportion of IL-2- and IFN-γ- producing CD8⁺ T-cells among splenocytes after stimulation with gE. Data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test, with the Shingrix group as a control. Not significant (ns) means p > 0.99.