Research ArticleIMMUNOLOGY

CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine

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Science Advances  04 Nov 2020:
Vol. 6, no. 45, eabb2154
DOI: 10.1126/sciadv.abb2154
  • Fig. 1 Generation of ZIKV vaccines using SMARRT.

    (A) Schematic of the ZIKV prM-E and ZIKV NS3 vaccines. All structural genes were removed from the Venezuelan equine encephalitis virus (strain TC-83) while retaining the four nonstructural proteins (nsP1 to nsP4) that encode the replicase. prM-E or NS3 genes from ZIKV strain SPH2015 replaced the structural genes downstream of the viral 26S promoter. BHK-21 cells were electroporated with water (Mock), an irrelevant RNA (bridging control), or RNA encoding either ZIKV prM-E or NS3 and then analyzed 20 hours after electroporation. UTR, untranslated region. (B) Western blot analysis of whole-cell lysates probed with anti-ZIKV E or NS3 Abs. MW, molecular weight. (C) Quantification of launch efficiency by intracellular staining of BHK-21 cells with an anti-dsRNA (J2) Ab. Data are presented as the mean ± SD of triplicates from one experiment, representative of two independent experiments. The nonparametric Mann-Whitney test was used to compare Mock versus each group; ****P < 0.0001.

  • Fig. 2 Immunogenicity of NS3 and prM-E vaccines in immunocompetent C57BL/6 mice.

    (A) C57BL/6 wild-type mice were intramuscularly injected with 10 μg of NS3 (n = 10) and prM-E (n = 11) vaccines or with saline (n = 4) and boosted in the same manner on day 28. (B and C) On day 49, splenocytes were stimulated with pooled ZIKV prM-E– or NS3-derived peptides. IFNγ-producing CD8+ T cells (B) and CD4+ T cells (C) were quantified as SFCs per 106 of splenocytes. (D) U937-DC-SIGN cell-based flow cytometric assay of ZIKV neutralizing activity (NT50) of sera collected on days 27 and 49. (E and F) Sera from ZIKV NS3- and prM-E–vaccinated C57BL/6 wild-type mice were collected on day 49, and 1 to 10 μl were intraperitoneally injected into 5- to 6-week-old AG129 mice (n = 6). Mice (n = 6) were injected with 15 μg of anti–DENV-prM Ab (2H2) or PBS as positive and negative controls, respectively. One day later, all AG129 mice were intravenously infected with 105 focus-forming units (FFU) of DENV2 S221. (F) Survival of mice treated as in (E). Data are presented as the mean ± SEM pooled from two independent experiments. The nonparametric Kruskal-Wallis and Mann-Whitney tests were used to compare three groups (D) and two groups (B and C), respectively. The Gehan-Breslow-Wilcoxon test was used to compare survival data.

  • Fig. 3 Induction of anti-ZIKV immune response by NS3 and prM-E vaccines in HLA-B*0702 transgenic mice.

    (A) Experimental protocol. (B) HLA-B*0702 Ifnar1−/− mice were intramuscularly injected with 10 μg (n = 4) or 1 μg (n = 5) of NS3 vaccine or with saline (n = 5) and boosted in the same manner on day 28. On day 49, splenocytes were stimulated with pooled ZIKV NS3 peptides and IFNγ-producing SFCs were evaluated by ELISpot. This experiment was performed once. (C and D) HLA-B*0702 Ifnar1−/− mice were intramuscularly injected with 10 μg (n = 6 and n = 11 for days 35 and 49) of NS3 or prM-E (n = 6 and n = 9 for days 35 and 49) vaccines or with saline (n = 5 and n = 10 for days 35 and 49) and boosted on day 28. (C) Peripheral blood mononuclear cells (day 35) and (D) splenocytes (day 49) were stimulated with pooled ZIKV NS3 or prM-E peptides. Cells producing IFNγ, IFNγ and TNF, or IFNγ, TNF, and IL-2 were quantified by flow cytometry. (E) U937-DC-SIGN cell-based flow cytometric assay of ZIKV neutralizing activity (NT50) of mouse sera collected on day 49. Data are presented as the mean or mean ± SEM from two independent experiments. The nonparametric Kruskal-Wallis and Mann-Whitney tests were used to compare three groups (B and E) and two groups (C and D), respectively.

  • Fig. 4 Control of ZIKV infection in HLA-B*0702 mice by NS3 and prM-E vaccines.

    (A) HLA-B*0702 Ifnar1−/− mice were intramuscularly injected with 10 μg of NS3 or prM-E vaccines or with saline and boosted on day 28. (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of ZIKV RNA levels in the serum, brain, spleen, and liver of vaccinated mice with NS3 or prM-E (n = 11) or saline (n = 12) on day 52. (C) Number of CD3+, CD8+, CD44highCD8+ T cells and (D) CD44highCD8+ T cells producing IFNγ, IFNγ/TNF, or IFNγ/TNF/IL-2 or (E) granzyme B were reported. (F) Neutralizing activity (NT50) against ZIKV SD001 on days 49 (pre-challenge) and 52 (post-challenge). On day 49, vaccinated mice (NS3, n = 6; prM-E, n = 6; saline, n = 5) were lethally challenged retro-orbitally and survival (G) and weight loss (H) were monitored. (I) HLA-B*0702 Ifnar1−/− mice were intramuscularly immunized with 20 μg of NS3, prM-E (n = 6), or NS3 + prM-E (n = 5) vaccines or with saline (n = 6) as represented. (J) qRT-PCR analysis of ZIKV RNA levels in the serum, brain, and liver after infection (day 73). Data are presented as the mean or mean ± SEM from two independent experiments. The nonparametric Kruskal-Wallis and Mann-Whitney tests were used to compare more than two groups (B, C, F, and J) or two groups (D and E), and the Gehan-Breslow-Wilcoxon test was used to compare survival data (G).

  • Fig. 5 Effects of NS3 and prM-E vaccines on the phenotypes of ZIKV-infected pregnant mice and fetuses.

    (A) Experimental protocol. HLA-B*0702 Ifnar1−/− dams (all n = 12) were intramuscularly immunized with 10 μg of NS3 or prM-E vaccines or with saline and boosted in the same manner on day 28. Dams were then mated with BALB/c sires. On embryonic day 7.5 (E7.5), pregnant mice were infected retro-orbitally with 103 FFU of ZIKV SD001. Seven days later (E14.5), mice were sacrificed, and fetuses and maternal tissues were harvested for analysis. (B) Representative placenta and fetuses from dams in the NS3, prM-E, and saline groups at E14.5. Color-coded arrows indicate viable fetus (blue), growth restriction (green), and fetal resorption (red). (C and D) Fetal body weight (C) and size (D) on E14.5. (E and F) qRT-PCR analysis of ZIKV RNA levels in the placentas (E) and maternal serum and brain (F) on E14.5. Data were pooled from independent experiments and represent a total of 32 fetuses from 4 mothers for the NS3 group, 40 fetuses from 5 mothers for the prM-E group, and 25 fetuses from 4 mothers for the saline group. Data are presented as the mean ± SEM. The nonparametric Kruskal-Wallis test was used to compare three groups. (B) Photo credit: J. A. Regla-Nava, La Jolla Institute for Immunology, CA.

  • Fig. 6 Contribution of CD8T cells to NS3 vaccine protection against ZIKV infection.

    (A) HLA-B*0702 Ifnar1−/− mice were vaccinated with 10 μg of NS3 vaccine (n = 11) or saline (n = 12), boosted, and then intraperitoneally treated with CD8+ cell–depleting Ab (2.43) or isotype control Ab before ZIKV SD001 infection. (B) Serum, brain, liver, and spleen ZIKV RNA levels were analyzed by qRT-PCR (CD8- or isotype-treated mice). (C) HLA-B*0702 Ifnar1−/− mice were vaccinated as described for (A). On day 49, CD8+ T cells were isolated and transferred into naïve HLA-B*0702 Ifnar1−/− mice (1 × 107 cells). One day later, all mice were infected retro-orbitally with 103 FFU of ZIKV SD001. (D) ZIKV RNA levels in serum, brain, liver, and spleen were analyzed by qRT-PCR (NS3, n = 5; saline, n = 4). (E) HLA-B*0702 Ifnar1−/− mice were immunized as described for (A) (NS3, n = 12; saline, n = 12), and pooled sera from day 49 were passively transferred. (F) qRT-PCR analysis of ZIKV RNA in the serum, brain, liver, and spleen of mice transferred with serum from NS3-vaccinated mice (n = 6) or mice injected with saline (n = 5) 3 days after infection. Data are pooled from two independent experiments and are presented as the mean ± SEM. The nonparametric Kruskal-Wallis and Mann-Whitney tests were used to compare three groups and two groups, respectively.

  • Table 1 Summary of ZIKV-derived CD8+ and CD4+ T cell epitopes.

    Mouse modelSequence positionSequencePeptides per poolT cell specificity
    Wild-type C57BL/6NS31656–1664VVIKNGSYV2CD8
    NS31866–1874PSVRNGNEICD8
    NS31740–1754GLPVRYMTTAVNVTH1CD4
    PrM169–177ATMSYECPM3CD8
    E294–302IGVSNRDFVCD8
    E297–305SNRDFVEGMCD8
    E644–658PVGRLITANPVITES2CD4
    E346–360VRSYCYEASISDMASCD4
    HLA-B*0702 Ifnar1−/−NS3206–215APTRVVAAEM5CD8
    NS3427–436GPMPVTHASACD8
    NS3574–582KPRWMDARVCD8
    NS3405–413RVIDSRRCLCD8
    NS3309–317FPDSNSPIMCD8
    prM4–12LPSHSTRKL6CD8
    E38–45KPTVDIELCD8
    E170–178TPNSPRAEACD8
    E173–180SPRAEATLCD8
    E233–242TPHWNNKEALCD8
    E337–347GPCKVPAQMAVCD8

Supplementary Materials

  • Supplementary Materials

    CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine

    Annie Elong Ngono, Thasneem Syed, Anh-Viet Nguyen, Jose Angel Regla-Nava, Mercylia Susantono, Darina Spasova, Allison Aguilar, Melissa West, Jessica Sparks, Andrew Gonzalez, Emilie Branche, Jason L. DeHart, Jerel Boyd Vega, Priya Prakash Karmali, Padmanabh Chivukula, Kurt Kamrud, Parinaz Aliahmad, Nathaniel Wang, Sujan Shresta

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