ZIKV NS1–specific cellular immune responses were assessed by IFN-γ ELISpot assay. The 114-peptide array spanning the entire NS1 of the PRVABC59 strain of ZIKV was obtained through BEI Resources, the National Institute of Allergy and Infectious Diseases, and the National Institutes of Health: peptide array, ZIKV, and PRVABC59 (nonstructural protein 1, NR-50534). Individual peptides are 13- or 15-mers with 12–amino acid overlap, and detailed information on their length and sequence is provided by BEI Resources (www.beiresources.org/Catalog/BEIPeptideArrays/NR-50534.aspx).

The peptides were divided into four pools, each containing 27 to 29 individual peptides. Mouse IFN-γ ELISpot assay was performed on red blood cell–depleted splenocytes from immunized mice, which were stimulated with different NS1 peptide pools for 36 hours at 37°C, essentially as we described previously (43, 68). Briefly, multiscreen-IP HTS plates (Merck Millipore) were coated with anti-mouse IFN-γ (clone AN18, Mabtech), and secreted IFN-γ was detected with anti-mouse IFN-γ biotin (clone R4-6A2, Mabtech) followed by streptavidin–alkaline phosphatase (AP) (Sigma-Aldrich) and SigmaFast BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitroblue tetrazolium; Sigma-Aldrich).

The number of NS1-specific IL-2– and IFN-γ-producing cells in splenocytes from vaccinated mice was analyzed using FluoroSpot kit (Mabtech) according to the manufacturer’s instructions. In brief, splenocytes were stimulated with 4 μg of either NS1 peptide pool 3, pool 4, CTL, or TH pools. Samples were incubated for 36 hours at 37°C, after which spots were developed following the manufacturer’s instructions.

Developed spots were counted automatically by use of an ELISpot reader (Autoimmun Diagnostika GmbH, Germany), and the number of spots for unstimulated splenocytes (<50) was subtracted from the number of spots for the peptide pool–stimulated splenocytes to generate the number of specific spot-forming units per 106 cells. Data are presented as mean ± SEM.

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