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, 8 (1), 16509

Purified Inactivated Zika Vaccine Candidates Afford Protection Against Lethal Challenge in Mice

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Purified Inactivated Zika Vaccine Candidates Afford Protection Against Lethal Challenge in Mice

Whitney R Baldwin et al. Sci Rep.

Abstract

In response to the 2016 global public health emergency of international concern announced by the World Health Organization surrounding Zika virus (ZIKV) outbreaks, we developed a purified inactivated Zika virus vaccine (PIZV) candidate from ZIKV strain PRVABC59, isolated during the outbreak in 2015. The virus isolate was plaque purified, creating six sub-isolated virus stocks, two of which were selected to generate PIZV candidates for preclinical immunogenicity and efficacy evaluation in mice. The alum-adjuvanted PIZV candidates were highly immunogenic in both CD-1 and AG129 mice after a 2-dose immunization. Further, AG129 mice receiving 2 doses of PIZV formulated with alum were fully protected against lethal ZIKV challenge and mouse immune sera elicited by the PIZV candidates were capable of neutralizing ZIKVs of both African and Asian genetic lineages in vitro. Additionally, passive immunization of naïve mice with ZIKV-immune serum showed strong positive correlation between neutralizing ZIKV antibody (NAb) titers and protection against lethal challenge. This study supported advancement of the PIZV candidate toward clinical development.

Conflict of interest statement

J.A.L., H.K.P., H.K.D., K.J.B., S.S., and H.J.D. are employees of Takeda and own stocks and options. W.R.B. and H.A.G. are contract researchers on assignment with Takeda Vaccines working in C.Y-H.H.’s laboratory. C.Y-H.H.’s laboratory received research support from Takeda under a CRADA. Takeda Vaccines, Inc. and Centers for Disease Control and Prevention (J.A.L., H.A.G., W.R.B., H.J.D., and C.Y-H.H) have filed a patent application (pending, numbers: 62581500, 62592995) describing specific aspects of this manuscript. The other authors declare no competing financial or non-financial interests.

Figures

Figure 1
Figure 1
Characterization and preparation of PIZV candidates. (a) Plaque phenotype of ZIKV PRVABC59 P6 sub-isolates, compared to ZIKV PRVABC59 P1 stock. (b) Growth kinetics of ZIKV PRVABC59 P6 sub-isolates. (c) Flowchart of PIZV preparation. (d) Left panel: total protein on SDS-PAGE. Right panel: Immunoblot analysis detecting ZIKV E. Lane 1: recombinant ZIKV sE; lane 2 and 4: P7 virus supernatant; lane 3 and 5: PIZV made from P7 virus. (e) Representative electron micrograph of PIZV-e at 110,000X magnification.
Figure 2
Figure 2
PIZV immunogenicity in CD1 mice. (a) Test groups and schedule of dosing. (b) Serum NAb titers of immunized CD-1 mice as determined by RVP neutralization assay. Solid lines represent the geometric mean titer (GMT) of EC50 for each group. The limit of detection (1.9 log10) is represented by a dashed line. Samples with a titer below the detection limit were included in the GMT calculation. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test: **** indicates p < 0.0001.
Figure 3
Figure 3
PIZV immunogenicity and efficacy in AG129 mice. (a) Test groups and schedule of dosing. (b) Serum NAb titers as determined by R-mFRNT of immunized AG129 mice. Solid lines represent the group GMT of the reciprocal serum dilution required for at least 50% reduction of viral foci. The limit of detection (1.3 log10) is represented by a dashed line. Animals with no detectable titer (<1.3) were assigned a titer of 1.0 for visualization and GMT calculation. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test: ****indicates p < 0.0001. (c) Infectious virus titers of individual mouse serum samples two days post-challenge, shown as pfu/mL. Solid lines represent the GMT of a group. Error bars represent standard deviation. The limit of detection (2.0 log10) is represented by a dashed line. Animals with no detectable titer (<2.0) were assigned a titer of 1.0 for visualization and GMT calculation. (d) Kaplan-Meier survival curve of test groups post-challenge.
Figure 4
Figure 4
Passive immunization of ZIKV immune sera in AG129 mice. (a) Test groups. (b) Circulating serum NAb levels measured by R-mFRNT, prior to challenge. Solid lines with error bars represent the GMT with standard deviation. The limit of detection (1.3 log10) is represented by a dashed line. Animals with no detectable titer (<1.3) were assigned a titer of 1.0 for visualization and GMT calculation. (c) Infectious viremia titers of individual mice post-challenge. Solid lines with error bars represent the GMT with standard deviation. The limit of detection (2.0 log10) is represented by a dashed line. Animals with no detectable titer (<2.0) were assigned a titer of 1.0 for visualization and GMT calculation. (d) Kaplan-Meier survival curve of test groups post-challenge. (e) Correlation analysis of circulating serum NAb titers at the time of challenge and viremia after challenge. Data used for this analysis were from 3 mice/group based on the group peak viremia day. Horizontal dashed line represents the limit of detection of the plaque assay, and vertical dashed line represents the limit of detection of the R-mFRNT assay. A Spearman rank correlation test was used to determine a rho and p value. (f) Comparison of circulating serum NAb titers following passive transfer in protected and unprotected test groups at 28 days post challenge. Geometric mean is indicated by a solid bar. The **** symbol indicates a p value < 0.0001 using a Wilcoxon rank test. (g) Fitted logistic regression model for survival as a function of circulating NAb titers following passive transfer. An inverse prediction of 70% is indicated by a horizontal line. Dashed lines indicate a 95% confidence interval.

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