LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with Shingrix™ in VZV-primed mice

Abstract

Latent varicella-zoster virus (VZV) may be reactivated to cause herpes zoster, which affects one in three people during their lifetime. The currently available subunit vaccine Shingrix™ is superior to the attenuated vaccine Zostavax® in terms of both safety and efficacy, but the supply of its key adjuvant component QS21 is limited. With ionizable lipid nanoparticles (LNPs) that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers, and oligodeoxynucleotides containing CpG motifs (CpG ODNs) approved by the FDA for a subunit hepatitis B vaccine as immunostimulators, we developed a LNP vaccine encapsulating VZV-glycoprotein E (gE) and CpG ODN, and compared its immunogenicity with Shingrix™ in C57BL/6J mice. The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix™ as determined by enzyme-linked immunosorbent assay (ELISA). Most importantly, the LNP vaccine induced comparable levels of cell-mediated immunity (CMI) that plays decisive roles in the efficacy of zoster vaccines to Shingrix™ in a VZV-primed mouse model that was adopted for preclinical studies of Shingrix™. Number of IL-2 and IFN-γ secreting splenocytes and proportion of T helper 1 (Th1) cytokine-expressing CD4⁺ T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix™ boosted mice. All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities, indicating the potential of LNP-CpG-adjuvanted VZV-gE as a more cost-effective zoster vaccine.

Keywords: AS01B; Adjuvant; Cell-mediated immunity (CMI); CpG oligodeoxynucleotide (CpG ODN); Humoral immunity; Lipid nanoparticle (LNP); Subunit vaccine; Varicella zoster virus (VZV).

Fig. 1

Characterization of lipid nanoparticle (LNP) vaccines. A Diagram of LNP vaccines. Encapsulation efficiency of varicella-zoster virus glycoprotein E (gE) (B) and oligodeoxynucleotide containing CpG motifs (CpG ODN) (C). D The average diameter of LNP vaccine. E LNP vaccine polydispersity index (PDI) showing particle uniformity. Tests were repeated three times, and the data are shown as the mean ​± ​SD (standard deviation).

Fig. 2

Immunization schedule and gE-specific IgG titers. A Immunization schedules for different mice groups. Mice were randomly divided into 7 groups (N ​= ​6) and immunized with PBS or Oka strain with 4000 ​PFU, then boosted two times with four weeks interval. Mice were sacrificed two weeks after the last immunization and serum or splenocytes were collected for analysis. B gE-specific IgG titers induced by different immune strategies were detected by ELISA. Points represent individual mice. The data were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test, comparing the mean of each group with the mean of every other group. ∗, P ​< ​0.05.

Fig. 3

LNP vaccines induced cytokine responses at levels comparable to Shingrix™ in immunized mice. IL-2 (A) and IFN-γ (B) secreted by splenocytes upon stimulation with 10 ​μg/mL gE were detected by ELISA. C Representative images of IL-2-producing splenocytes in the enzyme linked immunospot (ELISPOT) assay, and numbers of IL-2-producing splenocytes (D) were calculated from the ELISPOT assay after 20 ​μg/mL gE stimulation. E Representative images of IFN-γ-producing splenocytes and numbers of IFN-γ-producing splenocytes (F) were calculated from the ELISPOT assay after 20 ​μg/mL gE stimulation. N ​= ​6, each dot represents an independent mouse. PBS controls were splenocytes from PBS immunized mice stimulated with gE and positive controls were cells stimulated with 12-myristate 13-acetate (PMA) ​+ ​ionomycin. Data were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test, comparing the mean of each group with the mean of every other group. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001.

Fig. 4

LNP vaccines induced T helper 1 (Th1) cytokine-expressing CD4⁺ T cells at levels comparable to Shingrix™ in immunized mice as detected by flow cytometry assay. A Pseudocolor images displaying representative results near the average value for gated IL-2/IFN-γ-expressing CD4⁺ T cells. B Proportion of IL-2-producing CD4⁺ T cells among splenocytes after 10 ​μg/mL gE stimulation. C Proportion of IFN-γ-producing CD4⁺ T cells among splenocytes after 10 ​μg/mL gE stimulation. N ​= ​6, points represent individual mice. PBS controls were splenocytes from PBS immunized mice stimulated with gE and positive controls were these cells stimulated with PMA⁺ ​ionomycin. Data were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test, comparing the mean of each group with the mean of every other group. ∗, P ​< ​0.05; ∗∗∗∗, P ​< ​0.0001.

Fig. 5

LNP vaccines induced CD4⁺ memory T cells at levels comparable to Shingrix™ in immunized mice as detected by flow cytometry assay. A Pseudocolor images displaying representative results near the average value for gated CD44⁺/CD62L⁺ cells. B Proportion of effector memory T cells. C Proportion of central memory T cells. N ​= ​6, points represent individual mice. Data were compared using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test, comparing the mean of each group with the mean of every other group.