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. 2017 Nov 7;7:14769. doi: 10.1038/s41598-017-15039-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Construction, Western blot, and immunogenicity of MVA-ZIKV-NS1 vaccine in CD-1/ICR mice following single or prime-boost immunization. (a) ZIKV NS1 gene (Suriname 2015 isolate Z1106033) was inserted into the MVA restructured and modified deletion III. This insertion site has been identified to support high expression and insert stability. P_mH5, modified H5 promoter. Numbers are coordinates in the MVA genome. MVA-ZIKV-NS1 is replication competent in avian cells (producing both MVA and inserted transgenes) but is replication deficient in mammalian cells producing mainly inserted transgenes (e.g. NS1) but not infectious MVA viruses. Image of the ZIKV NS1 dimer is from RCSB PDB (www.Rcsb.org) of PDB ID 5IY3⁴¹ (b) Western blot, DF1 chicken fibroblasts cells were infected with either wild type MVA (wt MVA) or MVA-ZIKV-NS1 (MVA-NS1) at multiplicity of infection (MOI) of 0.1. The expression of full-length NS1 was confirmed using anti-ZIKV-NS1-protein mouse monoclonal antibody (IgG1). Recombinant ZIKV NS1 protein (Rec. NS1) served as a positive control. A loading control lane (Ctrl lane) served as another negative control. Like other flaviviral NS1 proteins, ZIKV NS1 obtained from infected cell lysates (LYS) migrates as a doublet (intracellular NS1 (non-glycosylated, lower band) and cell-surface NS1 (glycosylated, upper band)²⁰. Only fully glycosylated NS1 was found in the supernatants (SUP). (c) Single dose group, endpoint dilution geometric mean titer (GMT) determined by ELISA using sera from mice obtained at 2 and 4 weeks post-immunization (vac) and 3 weeks post-i.c. challenge (Ch) with 10⁵ PFU of MR766. (d) Prime-boost group, endpoint dilution GMT determined by ELISA using sera from mice obtained at 2 and 4 weeks (post-prime) and at 4 weeks (post boost) immunization and 3 weeks post i.c. challenge with 10⁵ PFU of MR766.