Single dose of chimeric dengue-2/Zika vaccine candidate protects mice and non-human primates against Zika virus

Abstract

The development of a safe and effective Zika virus (ZIKV) vaccine has become a global health priority since the widespread epidemic in 2015-2016. Based on previous experience in using the well-characterized and clinically proven dengue virus serotype-2 (DENV-2) PDK-53 vaccine backbone for live-attenuated chimeric flavivirus vaccine development, we developed chimeric DENV-2/ZIKV vaccine candidates optimized for growth and genetic stability in Vero cells. These vaccine candidates retain all previously characterized attenuation phenotypes of the PDK-53 vaccine virus, including attenuation of neurovirulence for 1-day-old CD-1 mice, absence of virulence in interferon receptor-deficient mice, and lack of transmissibility in the main mosquito vectors. A single DENV-2/ZIKV dose provides protection against ZIKV challenge in mice and rhesus macaques. Overall, these data indicate that the ZIKV live-attenuated vaccine candidates are safe, immunogenic and effective at preventing ZIKV infection in multiple animal models, warranting continued development.

Fig. 1. Genomic organization and modification of chimeric D2/ZK viruses.

a Genomic map of the D2/ZK viruses with prM-E of ZIKV SPH in the genomic background of either DENV-2 16681 (D2/ZK-P, parental) or DENV-2 PDK-53 (D2/ZK-V, vaccine). Blue triangles denote the 3 primary PDK-53 attenuation loci, 5-NCR c57t, NS1-G53D and NS3-E250V. b Genetic substitutions and plaque images of several D2/ZK-V viruses. Engineered mutations and a representative image of plaque morphology in Vero cells are indicated for each virus.

Fig. 2. Growth kinetics and genetic stability of D2/ZK LAV candidates in Vero cells.

a Growth kinetics of D2/ZK viruses: geometric mean titers (GMT) and error bars (standard deviation, SD) of each virus group. Sample size (n) shown in the symbol legend indicates the number of independent culture flasks used for each virus. Dashed line represents the limit of detection (LOD) of the plaque assay. b Whole genome NGS of D2/ZK-V4, V5 and V5-Pr viruses at passage 1 (green), 5 (blue) and 10 (red) of 2 independent lineages (Flask A and B). SNVs with frequency ≥1% are displayed across the virus genome. SNVs at DENV-2 PDK-53 attenuation loci are highlighted in red font with an asterisk*. Nucleotide positions labeled with black font indicate SNVs ≥10% frequency (above dashed line), bold font indicates SNVs resulting in AA change. Vertical line between P5 and P10 indicates SNVs found in both passages of the lineage.

Fig. 3. In vitro attenuation markers of vaccine candidates.

a Virus plaque diameter in Vero cells. Box and whiskers plot shows the range of plaque sizes observed for each virus (n = 12 independent plaques for wt ZIKV; n = 20 plaques for other viruses). The whiskers represent the maximum and minimum diameters, the box is bound by the 25th and 75th percentiles with the median indicated by a solid line and the mean indicated with a cross. A two-tailed, unpaired t test was used to determine statistical differences between P and V viruses. ****p < 0.0001, ns not significant (p = 0.1988). b Temperature sensitivity of viruses in LLC-MK2 cells. Peak geometric mean titers (GMTs) with standard deviation (SD) from a growth kinetics study (Supplementary Fig. 1) are shown for each virus. The log₁₀ differences of peak GMTs between cultures at 37 °C and 39 °C are indicated as +(1–2.5 log₁₀), ++(2.5–4 log₁₀) or +++(>4 log₁₀). Dashed line represents the limit of detection (LOD) of the plaque assay. Black dots show data points from 2 independent cultures (n = 2). c Growth kinetics of viruses in C6/36 cells: GMTs and SD of each virus group over the time course of the experiment. Number (n) of independent cultures conducted is indicated in the symbol legend for each virus. Dashed line represents the LOD of the plaque assay.

Fig. 4. Mosquito infection, dissemination, and transmissibility potential of vaccine candidate viruses.

a, b Ae. aegypti (Poza Rica 2012), c, d Ae. albopictus (Lake Charles 1987), e, f Ae. albopictus (ELG 2018). a, c, e Infection, dissemination, and transmission rates. The number of positive bodies out of the total number of mosquitoes is indicated above the blue bar (infection). The number of mosquitoes with positive legs among the mosquitoes with virus-positive bodies is indicated above the red bar (dissemination). The number of mosquitoes with positive saliva among mosquitoes with positive legs is indicated above the green bar (transmission). NA indicates not analyzed, as no sample was tested due to previous negative infection or dissemination of the virus. A 0% rate was drawn at 1% for illustration purposes. b, d, f Virus titers in mosquito bodies (blue), legs (red) and saliva (green). GMT and SD are based on positive samples for each group. The dashed lines represent the LOD for each sample type. All tested samples, including negative ones (assigned as and graphed at 0) were included for statistical comparison between wt and vaccine groups. A two-tailed, unpaired t test was used to determine statistical differences. ***p = 0.0005, ****p < 0.0001. The sample size for each group used to derive statistics is indicated by the denominator above the bars in a, c and e.

Fig. 5. Chimeric viruses are non-neurovirulent in neonatal CD-1 mice.

a Kaplan–Meyer survival curves of 1-day-old CD-1 mice inoculated i.c. with 10⁴ pfu of virus. b Group mean weight ±SD post virus challenge. Inset figure shows a close-up of the graph between 6 and 10 days post challenge. The number (n) of mice in each virus group is indicated in the symbol legend of figure a.

Fig. 6. Protective efficacy and immunogenicity of single- or double-dose of D2/ZK-P5 and -V5 virus in AG129 mice.

Separated groups of mice were immunized once (day 0) or twice (day 0 and 42), and challenged with 10⁴ pfu of wt ZIKV on day 70. a NAb titers of immunized mice at 40 and 68 days after primary immunization. b Viremia after wt ZIKV challenge. c Weight changes after ZIKV challenge. d Kaplan–Meyer survival curves after lethal ZIKV challenge. Horizontal bars (in a and b) represent the group GMT, error bars represent SD. Negative samples are plotted at the half value of the assay LOD and have been included in determination of GMTs and statistical analysis (one-way ANOVA with Tukey’s test to correct multiple comparisons). ****p < 0.0001, Not significant: p > 0.05 (exact p values provided in graph). Number (n) of mice used in each group is indicated in the symbol legend.

Fig. 7. Single dose of D2/ZK LAV candidates protect AG129 mice from lethal ZIKV challenge.

D2/ZK-V candidates were tested at 2 single-dose levels, 10³ and 10⁴ pfu. Their parental chimeric control viruses, D2/ZK-P, were tested at 10⁴ pfu dose only. The naïve group received PBS. All mice were challenged with 10⁴ pfu of wt ZIKV on day 70. a NAb titers of mice at 40–42 days and 68–70 days after the single immunization. b Viremia after wt ZIKV challenge. All immunized animals were protected from viremia. c Weight changes after ZIKV challenge. d Kaplan–Meyer survival curves after wt ZIKV challenge. Horizontal bars (in a and b) represent the group GMT, error bars represent standard deviation. Negative samples are plotted at the half value of the assay LOD and have been included in determination of GMTs and statistical comparisons (one-way ANOVA with Tukey’s test to correct multiple comparisons). *p = 0.186, ****p < 0.0001. Number (n) of mice used in each group is indicated in the symbol legend. With exception of the D2/ZKV-V5-Pr group showing significantly lower NAb titers than its P counterpart group on day 40–42, no significant differences were observed between groups that received D2/ZK-V and -P viruses.

Fig. 8. D2/ZK-V4 and D2/ZK-V5 vaccination protects rhesus macaques following ZIKV challenge.

Animals were immunized on day 0 (D2/ZK-V4 and -V5) and day 91 (D2/ZK-V5 group only) before challenge with 10⁴ pfu of wt ZIKV on day 186. a Vaccine vRNAmia after immunization. b NAb kinetics (by R-mFRNT) after vaccination and wt ZIKV challenge. Black arrow indicates day of boost (D2/ZK-V5 group only) and red arrow indicates day of challenge. c ZIKV vRNAmia after wt ZIKV challenge. Negative samples were plotted at the half value of the assay LOQ or LOD. Dashed lines indicate assay limit of quantitation (LOQ, 3.3 log₁₀ copies/mL; in a and c) or LOD (1.0 log₁₀ EC₅₀, in b). Graphs in a and c show the mean of qRT-PCR results from samples tested in triplicate. Graph in b represents mean and SD of 2 (n = 2) independent titration experiments (samples were titrated in triplicate in each experiment).