A Chimeric Japanese Encephalitis Vaccine Protects against Lethal Yellow Fever Virus Infection without Inducing Neutralizing Antibodies

Abstract

Recent outbreaks of yellow fever virus (YFV) in West Africa and Brazil resulted in rapid depletion of global vaccine emergency stockpiles and raised concerns about being unprepared against future YFV epidemics. Here we report that a live attenuated virus similar to the Japanese encephalitis virus (JEV) vaccine JE-CVax/Imojev that consists of YFV-17D vaccine from which the structural (prM/E) genes have been replaced with those of the JEV SA14-14-2 vaccine strain confers full protection in mice against lethal YFV challenge. In contrast to the YFV-17D-mediated protection against YFV, this protection is not mediated by neutralizing antibodies but correlates with YFV-specific nonneutralizing antibodies and T cell responses against cell-associated YFV NS1 and other YFV nonstructural (NS) proteins. Our findings reveal the potential of YFV NS proteins to mediate protection and demonstrate that chimeric flavivirus vaccines, such as Imojev, could confer protection against two flaviviruses. This dual protection may have implications for the possible off-label use of JE-CVax in case of emergency and vaccine shortage during YFV outbreaks. In addition, populations in Asia that have been vaccinated with Imojev may already be protected against YFV should outbreaks ever occur on that continent, as several countries/regions in the Asia-Pacific are vulnerable to international spread of the YFV.IMPORTANCE Efficient and safe vaccines against yellow fever (e.g., YFV-17D) that provide long-lasting protection by rapidly inducing neutralizing antibody responses exist. However, the vaccine supply cannot cope with an increasing demand posed by urban outbreaks in recent years. Here we report that JE-CVax/Imojev, a YFV-17D-based chimeric Japanese encephalitis vaccine, also efficiently protects against YFV infection in mice. In case of shortage of the YFV vaccine during yellow fever outbreaks, (off-label) use of JE-CVax/Imojev may be considered. Moreover, wider use of JE-CVax/Imojev in Asia may lower the risk of the much-feared YFV spillover to the continent. More generally, chimeric vaccines that combine surface antigens and replication machineries of two distinct flaviviruses may be considered dual vaccines for the latter pathogen without induction of surface-specific antibodies. Following this rationale, novel flavivirus vaccines that do not hold a risk for antibody-dependent enhancement (ADE) of infection (inherent to current dengue vaccines and dengue vaccine candidates) could be designed.

Keywords: antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent enhancement; chimeric YFV-17D vaccine; chimeric flavivirus vaccine; cross-protection; dual protection; flavivirus; nonneutralizing antibodies; off-label use of vaccine; protective T cell responses.

Copyright © 2020 Mishra et al.

Figures

FIG 1

In vivo evaluation of JE-CVax-mediated dual protection against lethal JEV SA14-14-2 and YFV-17D challenge. (A to D) AG129 and C57BL/6 mice were first vaccinated via the i.p. route with either 104 PFU of JE-CVax (blue), 1/6th of a human dose of Ixiaro (green), or assay medium as a negative control (red). Animals vaccinated with Ixiaro were boosted with another 1/6th of a human dose of Ixiaro 14 dpv. In order to facilitate vaccine virus replication (28), wild-type C57BL/6 mice receiving JE-CVax vaccination were treated with MAR1-5A3 antibody. AG129 mice were i.p. challenged with 103 of PFU JEV SA14-14-2 at 28 dpv (A) or with 103 PFU of YFV-17D at 28 dpv (B) or at 0, 4, 7, 14, 21, and 28 dpv (C). C57BL/6 mice were i.c. challenged with 104 PFU of YFV-17D at 28 dpv (D). Animals were observed for 5 weeks after challenge and were euthanized when humane endpoints were reached. The data represent cumulative results of at least two independent experiments. Log rank (Mantel-Cox) survival analysis test was performed for statistical significance. **, P ≤ 0.01; ****, P ≤ 0.001 compared to the nonvaccinated group.

FIG 2

Serological analysis of serum of JE-CVax-vaccinated and YFV-17D- or ZIKV-MR766-challenged animals. (A) Detection of nAbs against JEV and YFV. CPE neutralization tests (CPENT) for JE-CVax (circles) and YFV-17D (squares) were performed on sera day 0 prior to vaccination (preimmune, red), day 28 after vaccination (blue), and after challenge (study endpoint, orange) for samples of JE-CVax-vaccinated AG129 mice, of JE-CVax-vaccinated mice after subsequent YFV-17D-challenge (n = 34), of mice hyperimmunized with JE-CVax (n = 13; first bleed 2 weeks post last booster immunization, blue), and of mice vaccinated with Ixiaro (green). Limit of detection (LOD) for virus neutralization was log10 20 (1.3). Data are presented as log10 CPENT50 (mean ± SD). The data presented are from ≥3 independent experiments. Statistical significance was determined using one-way ANOVA. ****, P ≤ 0.0001 for mean log10 CPENT50 titers against JEV or YFV compared to mean log10 CPENT50 titers before JE-CVax vaccination and before YFV-17D-challenge, respectively. (B) Quantitation of anti-YFV NS1 binding antibodies by direct ELISA. Serum from naive, nonvaccinated mice (red) or mice that had been vaccinated with 103 to 105 of PFU JE-CVax (blue) or that had been infected with 104 PFU of YFV-17D (orange) or with 105 PFU of ZIKV-MR766 (pink) were collected either 28 days postimmunization or when euthanized at the humane endpoint (n ≥ 5). The data are means of two independent analyses. Statistical significance was determined using one-way ANOVA. **, P ≤ 0.01 compared to YFV-17D. (C) Binding of serum antibodies to NS1-expressing cells. HEK 293 cells were transfected with a plasmid expressing YFV-17D NS1 as a transcriptional fusion to GFP (top) or infected with the YFV-17D-mCherry reporter virus (bottom). Either 48 h after transfection or 72 h after infection, cells were stained with the anti-YFV NS1-specific MAb 1A5 (left), with serum from mice that were vaccinated with JE-CVax (center), or with serum from naive, nonvaccinated mice (right). Graphs show flow cytometric analysis of GFP or mCherry fluorescence and visualization of anti-YFV NS1 antibody binding using a PE-Cy7-conjugated goat anti-mouse IgG secondary antibody. The fraction of NS1-positive cells (GFP or mCherry) stained by MAb 1A5 or serum of JE-CVax-immunized mice (anti-mouse IgG) is given as a percentage in the upper right quadrant. Data from one representative experiment out of four independent experiments are shown.

FIG 3

Role of antibody-dependent cell-mediated cytotoxicity (ADCC) conferred by JE-CVax hyperimmune serum in the protection against YFV. JE-CVax hyperimmune serum was tested for its ability to mediate ADCC activity compared to serum of nonvaccinated mice (normal serum) at 3:1 (A) and 10:1 (B) effector (E)-to-target (T) cell ratios. Experiments were conducted twice, each in triplicate, and data are presented as means ± SEMs for fold changes compared to control (CC) (i.e., mean reporter signal plus three SDs from E:T in the absence of hyperimmune serum). Values from noninfected target cells incubated with E in the presence of either hyperimmune serum or normal serum at highest antibody concentrations (dilution 1:9) are indicated as Control-9. Statistical significance was determined using two-way ANOVA. * and ****, P ≤ 0.05 and 0.0001 compared to normal serum.

FIG 4

Detection of protective T cell responses directed against YFV. (A to C) ELISpot assay data showing TNF-α (A) IFN-γ (B and C) and production by splenocytes of AG129 mice (n = 5; A and B) or C57BL/6 (n = 10; C) at 18 and 4 weeks, respectively, after vaccination with 104 PFU of JE-CVax, following 16 h ex vivo restimulation with either an MHC class I-restricted peptide derived from YFV-17D NS3 (32) or the lysate of YFV-17D- or JEV SA14-14-2-infected Vero E6 cells. Stimulation using lysate of noninfected Vero E6 cells served as a negative control. The data are derived from two independent experiments. Spot counts were normalized by subtraction of the number of spots in corresponding wells stimulated with uninfected Vero E6 cell lysate. (D) Cytokine expression profile of YFV-specific T cells. Shown are IFN-γ and TNF-α production profiles of YFV-specific CD4+ and CD8+ T cells from JE-CVax-vaccinated AG129 and C57BL/6 mice 18 and 4 weeks, respectively, postvaccination, as determined by intracellular cytokine staining. Mouse splenocytes were stimulated 16 h ex vivo with either an MHC class I-restricted NS3 peptide, cell lysate of YFV-17D-infected Vero E6 cells, or lysate of uninfected Vero E6 cells. The data are derived from two independent experiments and normalized by subtraction of number of cytokine-secreting T cells in corresponding samples in which uninfected Vero E6 cell lysate was used as recall antigen. (E) T cell-mediated in vivo protection against YFV. Loss of protection resulting from antibody-mediated T cell depletion (30, 52) suggests a direct functional involvement of CD4+ and CD8+ T cells in JE-CVax-mediated immunity against YFV in C57BL/6 mice (n ≥ 7) that had been vaccinated with 104 PFU of JE-CVax and subsequently challenged intracranially with 104 PFU of YFV-17D. Depletions were performed by administration of 0.5 mg of anti-mouse CD4 and/or anti-mouse CD8 antibodies i.p. on day −2 and day 0 each prior to YFV challenge. Log rank (Mantel-Cox) survival analysis test was performed for statistical significance. * and **, P ≤ 0.05 and 0.01 compared to vaccinated group (n = 5); +, P ≤ 0.05 compared to CD4-depleted group (n = 8).