Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes

Abstract

The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca(2+) dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-alpha-induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8(+) T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

Figure 1.

iDCs and mDCs are productively infected by YF17D. (A) Intracellular FACS analysis of NS1 expression (indicated by the gate shown) in iDCs and mDCs infected with 2 or 20 PFU/cell 48 h after infection. (B) Intracellular staining of NS4 expression (indicated by the gate shown) in iDCs infected with 2 PFU/cell. (C) Virus production by iDC and mDC cultures measured by plaque assay on SW13. (D) Virus production by T cell– and monocyte-enriched PBMC fractions compared with iDCs. Values in C and D represent means ± SD.

Figure 2.

YF17D Infection of DCs is Ca²⁺ dependent. (A) YF17D infection of iDCs at 2 PFU/cell was monitored by intracellular FACS for YF NS4 24 h after infection. DCs were treated as indicated with 10 mM EDTA or EGTA. 6 mM Ca²⁺ or Mg²⁺ was added as indicated. The percentage of NS4-positive cells is indicated. (B) Intracellular YF NS4 or surface influenza hemagglutinin staining after 24 or 8 h, respectively. Infection was done with or without pretreatment with EDTA as indicated. (C) Bar graph represents the percentage of iDCs staining positive for YF17D NS4 24 h after infection. Samples preincubated with 10 μg/ml DC-SIGN or αvβ3 blocking antibodies or 100 μg/ml RGD peptides are indicated. Values represent means ± SD.

Figure 3.

YF17D infection does not induce or inhibit DC maturation. (A) iDCs infected with 10 PFU/cell were analyzed for CD83 surface expression. CD83 expression of the YF17D-infected population (compared with mock infected) was analyzed 36 h after infection. (B) iDCs were infected with 10 PFU/cell and exposed to TNF-α/PGE-2 maturation cocktail. CD83 expression of the YF-infected population (compared with mock infected) was analyzed after 36 h.

Figure 4.

Maturation protects iDCs from YF cytopathogenicity. (A) Caspase activation is measured using CaspaTag–FITC in SW13, iDCs, or iDCs + TNF-α, exposed to the indicated PFU of YF17D virus at 48 h. Tables indicate the percentage of cells that were CaspaTag positive. (B) YF17D- infected iDCs or iDCs + TNF-α were identified by intracellular staining with NS4. CD83 staining was used to distinguish iDCs versus mDCs. Caspase activation of the indicated cell populations is indicated by histograms, and the percentage that is CaspaTag positive is indicated.

Figure 5.

YF17D infection of DCs allows for processing and presentation of endogenous and model T cell epitopes. (A) DCs infected with influenza or YF17D were used to stimulate purified CD8⁺ T cells from YF17D-immunized donor 1 or a naive donor. Stimulation was measured using IFN-γ ELISPOT, and data are shown as spot-forming cells/10⁶ cells. (B) Schematic showing the insertion site of the M1 epitope at the 2B-3 junction. (C) DCs infected with influenza, YF17D, YF17D-M1, or pulsed with 10 μM M1 peptide were used to stimulate CD8⁺ T cells from a naive donor. T cell stimulation was measured by IFN-γ ELISPOT, as mentioned in Materials and methods. Values represent means ± SD.