Fig. 2 In vitro characterization of ZIKV SAM constructs. (A) Potency of the ZIKV SAM constructs. The RNA was in vitro transcribed, and 0.1 μg of the RNA from each vaccine construct was electroporated into BHK cells. Cells were collected 24 hours later and stained with an anti-dsRNA antibody. The upper and lower panels show the percentage and MFI of dsRNA-positive cells determined using flow cytometry, respectively. Error bars represent the SD of technical triplicates. Shown are the representatives of at least two experiments. (B) ZIKV SAM RNA expression of prM-E protein. BHK cells were electroporated with 4.0 μg of the indicated SAM RNA. Twenty-four hours later, cells were collected and lysates were analyzed by immunoblotting using antibody 4G2 specific to flavivirus E protein or to β-tubulin. Shown are the representatives of at least two experiments.
Fig. 3 ZIKV SAM (CNE) vaccines are immunogenic and protective in mice. (A) BALB/c mice were immunized with ZIKV SAM (CNE) vaccines or VRC5283 DNA vaccine at the indicated doses at days 0 and 21 and subsequently challenged with 100 FFU of ZIKV at day 49. (B and C) Neutralizing antibody titers were determined by RVP neutralization assay at days 0, 14, and 35. Horizontal lines and error bars represent the mean log10 reciprocal EC50 (half-maximal neutralization of infection) dilution ± SD of 10 mice per group, respectively. The dotted line represents the limit of confidence (LOC), a reciprocal titer of 60. Any replicates below LOC were assigned a value of 30 (0.5 LOC). Significant difference by two-way analysis of variance (ANOVA) with Tukey’s multiple comparison posttest between vaccines at the same doses as indicated: *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001. (D) Viral loads were determined by qRT-PCR at day 3 after challenge. Horizontal line and error bars represent mean log10 FFU equivalent/ml ± SD for 10 mice per group, respectively. Dotted line represents the LOC. Any replicates below the LOC were assigned a value of 0.5 LOC. #P < 0.0001 between all other groups by one-way ANOVA with Tukey’s multiple comparison posttest.
Fig. 4 ZIKV SAM (CNE) vaccines are immunogenic and protective in NHPs. (A) Rhesus macaques (n = 8 per group) were immunized with the indicated vaccines at days 0 and 28 and subsequently challenged with 1000 FFU of ZIKV at day 56. (B) Neutralizing antibody activity was determined by ZIKV RVP assay at days 0, 28, and 56. Horizontal line and error bars represent the mean log10 reciprocal EC50 dilutions ± SD of eight animals per group, respectively. Statistical difference is shown between vaccines at each time point by two-way ANOVA with Tukey’s multiple comparison posttest. Dotted line represents the LOC. Any replicates below the limit of detection were assigned a value of 0.5 LOC. (C) Viral loads were determined by qRT-PCR at days 3, 4, 5, and 7 after challenge. Each animal is depicted as a single line. Dotted line represents the LOC. Any replicates below the LOC were assigned a value of 0.5 LOC. (D) Postchallenge anamnestic antibody response. Fold change in EC50-neutralizing antibody activity is shown relative to day 56. Each animal is depicted as a single line. Dotted line indicates a fold change of 4.
- Table 1 Description of the SAM vaccine candidates.
Name Description WT-prM-E Wild-type (WT) prM-E sequence
from ZIKV Natal strain (Brazil)
KU527068.1.CO-prM-E Identical to WT-prM-E, except that
the prM-E sequence was codon
optimized (CO).CO-prM-E.ESS.1 Identical to CO-prM-E, except that
the prM signal peptide “c
region” contained the
enhanced signal sequence
(ESS) PQAQA mutation.CO-prM-E.ESS.2 Identical to CO-prM-E, except that
the prM signal sequence was
replaced with the
immunoglobulin G signal
sequence.CO-C-prM-E.1 Identical to CO-prM-E, except that
it additionally contained the
codon-optimized capsid (C)
protein with the native C/prM
cleavage site.CO-C-prM-E.2 Identical to CO-C-prM-E.1, except
that the prM signal peptide “c
region” contained the
enhanced signal sequence
(ESS) PQAQA mutation.CO-C-prM-E.3 Identical to CO-C-prM-E.1, except
that a 2A sequence was
inserted in the native cleavage
site between C and prM.VRC5283 CO prM-E sequence from the ZIKV
French Polynesian H/PF/2013
strain, with the prM signal
peptide replaced with that of
JEV.VRC5288 Identical to VRC5283, except that
both the prM signal peptide
and the last 98 amino acids of E
protein were replaced with
those of JEV.
Supplementary Materials
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/32/eaba5068/DC1
Additional Files
Supplementary Materials
Development of a potent Zika virus vaccine using self-amplifying messenger RNA
Kate Luisi, Kaitlyn M. Morabito, Katherine E. Burgomaster, Mayuri Sharma, Wing-Pui Kong, Bryant M. Foreman, Sonal Patel, Brian Fisher, Maya A. Aleshnick, Jason Laliberte, Madison Wallace, Tracy J. Ruckwardt, David N. Gordon, Christine Linton, Nicole Ruggiero, Jessica L. Cohen, Russell Johnson, Kunal Aggarwal, Sung-Youl Ko, Eun Sung Yang, Rebecca S. Pelc, Kimberly A. Dowd, Derek O’Hagan, Jeffrey Ulmer, Sally Mossman, Anna Sambor, Edith Lepine, John R. Mascola, Theodore C. Pierson, Barney S. Graham, Dong Yu
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