Comparing immunogenicity and protective efficacy of the yellow fever 17D vaccine in mice

Abstract

The live-attenuated yellow fever 17D (YF17D) vaccine is one of the most efficacious human vaccines and also employed as a vector for novel vaccines. However, in the lack of appropriate immunocompetent small animal models, mechanistic insight in YF17D-induced protective immunity remains limited. To better understand YF17D vaccination and to identify a suitable mouse model, we evaluated the immunogenicity and protective efficacy of YF17D in five complementary mouse models, i.e. wild-type (WT) BALB/c, C57BL/6, IFN-α/β receptor (IFNAR^-/-) deficient mice, and in WT mice in which type I IFN signalling was temporally ablated by an IFNAR blocking (MAR-1) antibody. Alike in IFNAR^-/- mice, YF17D induced in either WT mice strong humoral immune responses dominated by IgG2a/c isotype (Th1 type) antibodies, yet only when IFNAR was blocked. Vigorous cellular immunity characterized by CD4⁺ T-cells producing IFN-γ and TNF-α were mounted in MAR-1 treated C57BL/6 and in IFNAR^-/- mice. Surprisingly, vaccine-induced protection was largely mouse model dependent. Full protection against lethal intracranial challenge and a massive reduction of virus loads was conferred already by a minimal dose of 2 PFU YF17D in BALB/c and IFNAR^-/- mice, but not in C57BL/6 mice. Correlation analysis of infection outcome with pre-challenge immunological markers indicates that YFV-specific IgG might suffice for protection, even in the absence of detectable levels of neutralizing antibodies. Finally, we propose that, in addition to IFNAR^-/- mice, C57BL/6 mice with temporally blocked IFN-α/β receptors represent a promising immunocompetent mouse model for the study of YF17D-induced immunity and evaluation of YF17D-derived vaccines.

Keywords: Yellow fever 17D; correlate of protection; live-attenuated vaccine; mouse models; type I IFN response.

Figure 1.

YFV-specific humoral responses in different mouse models. YFV-specific nAbs (A, B) and total IgG (C, D) levels at day 7 and 21 from mice vaccinated with 2 × 10⁴ PFU of YF17D (n ≥ 9 from two to three independent experiments). Boxes and horizontal bars denote the IQR and medians, respectively; whisker end points are equal to the maximum and minimum values. Statistical significance was determined using two-way ANOVA analysis between MAR1-treated and non-treated compartments (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns = not significant). Dotted lines denote the lower limit of detection (LLOD) of the assay. Ratio of IgG2a or IgG2c over IgG1 (determined for minipools of three mice each at day 28) plotted and compared to theorigical limit between Th1 and Th2 responses (E). Data are median ± IQR. Statistical significance between groups was calculated by the nonparametric two-tailed Mann-Whitney U-test (ns  =  not significant, P  >  0.05).

Figure 2.

YFV-specific cellular responses in different mouse models. YFV-specific T cell responses were measured by IFN-γ ELISPOT and flow cytometry with intracellular cytokine straining of splenocytes harvested from mice immunized with 2 × 10⁴ PFU of YF17D at day 28 post-vaccination. (A) Spot counts for IFN-γ-producing cells per 10⁶ splenocytes after 48 h stimulation with YF17D total antigen (n ≥ 9 from two independent experiments). Percentage of IFN-γ (B), TNF-α (C) producing CD4⁺, and IFN-γ (D), TNF-α (E), GzmB (F) producing CD8⁺ T cells after overnight stimulation with YF17D total antigen (n ≥ 3 from single experiment). All values normalized by subtracting spots/percentage of positive cells in corresponding unstimulated control samples. Data are median ± IQR. Statistical significance between groups was calculated by the nonparametric two-tailed Mann-Whitney U-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns =  not significant, P > 0.05).

Figure 3.

Mice were vaccinated with 2 PFU or 2 × 10⁴ PFU of YF17D in presence or absence of MAR1 antibody, whereas IFNAR^-/- mice were vaccinated without MAR1-treatment. Mock group received culture medium alone (n  =  10 from two independent experiments). Three weeks post-vaccination, animals were challenged i.c. with 3 × 10³ PFU YF17D. Pre-challenge YFV-specific IgG (A, B) and nAbs (C, D) levels in mice. Boxes and horizontal bars denote the IQR and medians, respectively; whisker end points are equal to the maximum and minimum values. Statistical significance between groups was calculated by the nonparametric two-tailed Mann-Whitney U-test. (*P < 0.05; **P < 0.01; ***, P < 0.001; ****P < 0.0001, ns = not significant, P > 0.05). Protective efficacy of YF17D against lethal intracranial challenge in different mouse modes. Animals were monitored daily for survival for the next four weeks (E-F-G). Viral loads in the mouse brains at euthanasia. Brains were harvested from both sick mice at their euthanasia and mice that survived challenge until four weeks post-challenge (n ≥ 7 from two independent experiments, mice died in four days post-challenge were excluded from experiments). RT-qPCR was performed on brain homogenates to determine viral RNA copies in the brain (H-I-J). Data are median ± IQR. Statistical significance between groups was calculated by the nonparametric two-tailed Mann-Whitney U-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns = not significant, P > 0.05). Dotted lines denote the lower limit of detection (LLOD) of the assay.

Figure 4.

Correlation among vaccination dose (2 and 2 × 10⁴ PFU), IgG, nAbs, viral loads, and survival in different mouse models. Correlation analysis was performed with data (dose, IgG, nAbs, viral loads, and survival), obtained from the experiment shown in Figure 3. Spearman correlation was used to measure the strength of association among dose, IgG, nAbs, and viral loads. Point biserial correlation was used to measure the association between survival and dose, IgG, nAbs, or viral loads. All coefficients are represented in the same heatmap (Correlation matrix and P-values were shown in Table S4A-S4B).