A self-amplifying mRNA vaccine expressing PRV gD induces robust immunity against virulent mutants

Abstract

Pseudorabies virus (PRV) causes an acute febrile infectious disease of pigs. Since 2011, PRV variants have appeared and spread nationwide in China. mRNA vaccines present a safe alternative and can stimulate humoral and cellular immunity. We constructed a self-amplifying mRNA (saRNA) vaccine containing gD gene of a highly virulent PRV variant. In mice, the PRV saRNA gD vaccine induced high levels of neutralizing antibodies. In piglets, vaccination with the PRV saRNA gD vaccine at a dose of as little as 5 μg induced very strong humoral immunity and cellular immunity, providing full protection against challenge infection with very virulent PRV variants. Protection following vaccination with saRNA gD induced significantly stronger protection than attenuated live vaccines and inactivated vaccines. In addition, our results confirm that a PRV saRNA vaccine expressing only one antigen can induce excellent protection in weaned piglets. Hence, the PRV saRNA gD vaccine might be a very promising substitute for inactivated and live vaccines. Finally, we provide proof-of-concept for the development of self-amplifying mRNA vaccines for members of the Alphaherpesvirinae, including herpes simplex virus 1 and 2 as well as varicella zoster virus.

Fig. 1. Production and characteristics of PRV gD-based saRNA vaccines.

a Illustration of the saRNA constructs, which include VEEV non-structural proteins (nsp) and the Luc and full-length gD genes, respectively. b saRNA-Luc was transfected into HEK-293 cells, and twenty hours later, images were captured with an in vivo imaging system. c, d Codon-optimized gD with a HiBiT tag was transfected into ST cells, and PK-15 cells and fusion proteins were detected by luciferase activity. e, f Representative particle size and polymer dispersity indices of the mRNA-LNPs. The particle size and PDI of the mRNA-LNPs were tested via dynamic light scattering on an NS-90Z instrument (OMEC, China).

Fig. 2. Immunogenicity of gD mRNA vaccines in mice.

a Experimental design for mouse vaccination, the mice were immunized on 0 and 28dpi. Schematics created with BioRender.com. b Body weights of the mice after immunization were measured. c Serum-neutralizing antibodies were determined at 14 and 28 dpi. d, e Levels of IFN-α and IL-12 in the serum were measured at 14 and 28 dpi (data are presented as mean ± SD). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001). f Histopathological evaluation of tissues from mice vaccinated with 100 ng saRNA at 3dpi (Scale bars: 20 μm, magnification: 40×).

Fig. 3. Immune responses induced by the saRNA-gD vaccine in piglets.

a Experimental design for vaccination and challenge, piglets were immunized on d 0 and 28dpi and challenge-infected on 42 dpi. Schematics created with BioRender.com. b Serum-neutralizing antibodies against PRV were measured weekly after immunization. c, d The levels of IL-4 and IFN-γ in the serum were measured at 14 and 35 dpi (data are presented as mean ± SD (n = 3 per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 4. Protective effect of the saRNA-gD vaccine in piglets.

a Daily rectal temperatures of piglets post-challenge. b Clinical scores (0-10 scale) of piglets post-challenge (data are presented as mean ± SD (n = 3 per group). Statistical significance was determined by two-way ANOVA with Šidák’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). c Survival rate of piglets post-challenge. d Viral titers in nasal swabs of PRV-challenged piglets at 3dpc and 4dpc (data are presented as mean ± SD (n = 3 per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ns no significant. e Protection rate of piglets post-challenge.

Fig. 5. Lung pathology assessment in PRV-challenged piglets.

a Representative gross lesion image and H&E-stained lung sections. b Proportional distribution of lesion severity (0–3 scale) across groups. c Comparative pathology scores (0–12 scale) showing significant reduction in the 5 μg two-dose saRNA group versus controls (****p < 0.0001), scale bars: 200 μm, magnification: 400×. d Quantification of PRV DNA loads in tissue samples of piglets. Statistical analysis by one-way ANOVA with Tukey’s test or two-way ANOVA with Šidák’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns no significant.