Research ArticleIMMUNOLOGY

Flavivirus serocomplex cross-reactive immunity is protective by activating heterologous memory CD4 T cells

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Science Advances  04 Jul 2018:
Vol. 4, no. 7, eaar4297
DOI: 10.1126/sciadv.aar4297
  • Fig. 1 Overlapping geographic distribution and polyprotein similarity of flaviviruses from various serocomplexes.

    Maps showing the current distributions of (A) DENV, (B) JEV, and (C) YFV serocomplexes reveal geographic regions where multiple flaviviruses cocirculate. (D) Phylogenic tree showing the genetic distances between the strains used for this study for DENV, JEV, and YFV, as well as representative strains of WNV, ZIKV, and Spondweni virus. The scale bar indicates the genetic distances in substitutions per amino acid.

  • Fig. 2 Cross-reactive and cross-protective flavivirus immunity.

    End-point titers of specific versus cross-reactive serum immunoglobulin G (IgG) against (A) DENV1, (B) YFV, or (C) JEV at each time point were measured by enzyme-linked immunosorbent assay (ELISA) for mice infected with DENV1, YFV, or JEV. Blood was collected 7, 14, and 21 days after infection. A comparison of specific versus cross-reactive end-point titers for mice infected with (D) DENV1, (E) JEV, or (F) YFV is presented for serum obtained 21 days after challenge. The avidity of serum antibodies toward each virus after challenge with (G) DENV1, (H) JEV, or (I) YFV is presented. Neutralization of virus by serum antibodies from mice infected with DENV1, YFV, and JEV or injected with saline was measured by plaque reduction neutralization test (PRNT) against (J) JEV, (K) YFV, or (L) DENV1. Results are presented as the percentage of neutralization compared to untreated control (virus alone) starting at a dilution of 1:10, with fourfold serial dilutions. For all viruses, only specific sera generated in mice inoculated with the same virus and not sera obtained after challenge with a related flavivirus were capable of virus neutralization. (M) Splenocytes were obtained from mice 5 weeks after infection and were used in proliferation assays. The results are given as the percentage of proliferation over splenocytes from the saline control mice. For all viruses, splenocyte proliferation was observed in response to homologous antigen, and for some viruses, splenocytes also proliferated in response to antigens from a heterologous virus. For (A) to (M), infections were performed by intraperitoneally injecting 1 × 106 plaque-forming units (PFU). (N) Serum and T cells were purified 4 to 5 weeks after infection from naïve and DENV1, JEV, or YFV post-immune mice (pooled from five mice per group). These products were transferred to naïve recipient mice (n = 5) before challenging all mice with DENV1. Alternatively, mice (n = 5) were given a secondary infection with DENV1 28 days after the primary challenge with DENV1, JEV, YFV, or saline. All secondary challenges were performed by subcutaneous injection with 1 × 105 PFU of DENV1. DENV1 was quantified in draining LNs after 24 hours by real-time reverse transcription polymerase chain reaction (RT-PCR). Results are expressed as a percentage relative to the primary DENV1 infection control (saline; followed by DENV1 infection). Viral clearance was enhanced during a homologous secondary DENV1 challenge after serum transfer, secondary infection, or T cell transfer. DENV1 was significantly reduced in JEV post-immune mice, while transfer of JEV post-immune serum enhanced DENV1 infection in LNs. Previous YFV immunity did not influence DENV1 viral load. For all panels, n = 5, *P < 0.05, and **P < 0.01. Cross-reactive low-avidity antibodies and T cells are generated by flavivirus infection; however, JEV, but not YFV, cross-reactive immunity enhances protection during secondary heterologous DENV1 challenge. ns, not significant.

  • Fig. 3 Serocomplex cross-reactive immunity boosts early serum neutralization after infection.

    The DENV1-neutralizing capacity of serum antibodies raised in mice infected with (A) DENV1 or YFV or (B) DENV1 or JEV (or saline) was measured 70 days after primary challenge. Mice that were injected with (C) DENV1, YFV, or saline or (D) DENV1, JEV, or saline by intraperitoneal injection were reinfected 70 days after with DENV1 via intraperitoneal injection. (C and D) Neutralizing antibodies (day 75) were boosted by a secondary challenge with DENV1 in DENV1- and JEV-immune animals but not in YFV-immune animals. (E) The percentage neutralization at the highest dilutions from (A) to (D) was compared by analysis of variance (ANOVA) among all groups, for serum isolated after both primary challenge with DENV1, YFV, JEV, or PBS and secondary challenge with DENV1 (75 days). (F) Avidity of antibodies against DENV1 was measured in serum isolated 38 days after primary infection (10 days after secondary infection). Antibody avidity was significantly increased in mice experiencing a homologous secondary challenge of DENV1- and JEV-immune mice experiencing a heterologous challenge with DENV1. n = 5 per group. (G) DENV1 infection levels were measured in LNs 5 days following secondary DENV1 challenge by RT-PCR. n = 4 per group. *P < 0.05, **P < 0.01. Cross-reactive preexisting immunity to JEV enhances the neutralization and avidity of anti-DENV1 antibodies and coincides with reduced viral burden in vivo.

  • Fig. 4 Priming of DC activation and TMEM cell recall by serocomplex cross-reactive immunity.

    Mice were injected subcutaneously with JEV, YFV, or saline and then rechallenged with DENV1 on day 21. LNs were isolated 24 hours after infection (n = 6). (A) Activated DCs were enumerated by flow cytometry after staining for CD11c, CD80, and CD86, showing increased numbers of activated DCs in the LNs of JEV-immune, but not YFV-immune, mice compared to naïve controls. Representative histograms of costimulatory molecule expression are shown in fig. S2 (A and B). (B) Gating strategy to define T cell subsets and their activation after staining against CD3, CD4, CD8, CD44, CD69, and CD62L. (C) The graph represents the ratio of activated TN cells (CD62L+CD44CD69+) to activated TEM cells (CD62LloCD44+CD69+) for CD4+ and CD8+ T cells. Higher proportions of activated CD4+ and CD8+ TEM cells in JEV post-immune mice and CD8+ TEM cells in YFV post-immune mice were detected. Total CD4+ and CD8+ (D) Teff, (E) TCM, and (F) TEM cells in LNs are presented. Significantly increased LN TEM cells staining intracellularly for the cytokines (G) IFN-γ and (H) IL-2 were observed in JEV- and DENV1-immune animals. (I) Donor Th1.2+ T cells from DENV1, JEV, and YFV post-immune mice were transferred 5 weeks after primary challenge to recipient Thy1.1+ mice. Recipient mice were given a secondary DENV1 challenge, and after 5 days, draining LNs were isolated. Donor Tfh cells with a memory phenotype (Thy1.2+CD4+CD44+CD62−/loPD-1+CXCR5+BCL6+) were higher in the LNs of DENV1- and JEV-immune animals. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. When trending toward significance (P < 0.1), P values are shown on the graph. Gating strategies for (G) to (I) are presented in fig. S2 (D and E). Increased TEM activation and decreased TN and TCM activation are associated with early DENV1-immune responses in animals with JEV preexisting immunity, and donor cross-reactive TEM cells adopt a Tfh phenotype in LNs during a secondary DENV1 challenge.

  • Fig. 5 Identification and validation of flavivirus cross-reactive CD4 epitopes.

    Of the 17 regions of the DENV1 full-length polypeptide that were predicted MHC-II binders, we identified the homologous sequences in the JEV and YFV polypeptides. (A) The chart depicts the proportions of the DENV1 peptides for which the homologous regions of JEV and YFV either were not predicted to bind to MHC-II (DENV only) or were also predicted binders for JEV, YFV, or both (JEV and YFV). DENV1 peptides were selected for validation from those where homologous regions were predicted MHC-II binders for (B) JEV, (C) YFV, or (D) both JEV and YFV. For (B) and (C), DENV1 peptides are shown aligned to the corresponding regions of YFV, JEV, and ZIKV. Numbers indicate the amino acid position in the polyprotein, and its protein location is labeled blue. (E) Heat map representation of TMEM expansion after stimulation with synthetic peptide-pulsed APCs, relative to the no peptide control. The percentage of CD3+CD4+CD44+ of total T cells (CD3+) was calculated after analysis by flow cytometry. Raw data and bar graph representation are provided in fig. S3, and proliferation and cytokine production were validated in fig. S4.

  • Fig. 6 Confirmation of serocomplex cross-reactive T cell responses in flavivirus-immune humans.

    PRNT assays were used to determine immunity to DENV1, JEV, YFV, or ZIKV in (A) presumed naïve or (B to D) JEV-immunized individual donors. (E and F) Specific and cross-reactive IgG titers against each virus were measured by ELISA. Average titers for JEV-immune donors are shown in (E), and individual titers are shown in (F). N, not detected. (G) Avidity of antibodies for JEV was high in JEV-immune donors and lower for cross-reactive antibodies. PBMCs from (H) flavivirus-naïve or (I to K) JEV-immunized donors were incubated with either JEV, DENV1, ZIKV, YFV, or control antigens for 3 days, followed by flow cytometry to identify the CD4+ TEM population. Activation of TEM cells from JEV-immune donors to homologous and heterologous flaviviruses was observed based on up-regulated staining for HLA-DR. Percentages of HLA-DR+CD4+ TEM cells of total T cells are included on the histograms. The flow cytometry gating strategy is presented in fig. S5. The frequency of spot-forming cells (SFC) after stimulation with flavivirus-derived or control antigens was determined for (L) flavivirus-naïve and (M to O) JEV-immunized donors by IFN-γ ELISPOT.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/7/eaar4297/DC1

    Table S1. List of the 17 predicted peptides with DENV1 MHC-II binding capacity and their position on the DENV1 polyprotein.

    Fig. S1. Enhanced DENV1 clearance in LNs of DENV1- and JEV-immune mice.

    Fig. S2. Assessment of LN cellularity following secondary DENV1 challenge.

    Fig. S3. Relative TMEM expansion after stimulation with synthetic peptide-pulsed APCs.

    Fig. S4. Validation of TEM proliferation and IFN-γ production after stimulation with synthetic peptides.

    Fig. S5. Gating strategy to identify activated human CD4 TEM cells by flow cytometry.

  • Supplementary Materials

  • This PDF file includes:
    • Table S1. List of the 17 predicted peptides with DENV1 MHC-II binding capacity and their position on the DENV1 polyprotein.
    • Fig. S1. Enhanced DENV1 clearance in LNs of DENV1- and JEV-immune mice.
    • Fig. S2. Assessment of LN cellularity following secondary DENV1 challenge.
    • Fig. S3. Relative TMEM expansion after stimulation with synthetic peptide-pulsed APCs.
    • Fig. S4. Validation of TEM proliferation and IFN-γ production after stimulation with synthetic peptides.
    • Fig. S5. Gating strategy to identify activated human CD4 TEM cells by flow cytometry.

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