Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct 15;187(8):4268-79.
doi: 10.4049/jimmunol.1101970. Epub 2011 Sep 14.

Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

Daniela Weiskopf  1 , Lauren E Yauch, Michael A Angelo, Daisy V John, Jason A Greenbaum, John Sidney, Ravi V Kolla, Aruna D De Silva, Aravinda M de Silva, Howard Grey, Bjoern Peters, Sujan Shresta, Alessandro Sette
Affiliations

Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

Daniela Weiskopf et al. J Immunol. .

Abstract

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Identification of DENV-derived epitopes recognized by CD8+ T cells
DENV specific epitope identification was performed in four different HLA transgenic mouse strains (A) A*0201; (B) A *1101; (C) A*0101; (D) B*0702. For all strains tested, IFNγ ELISPOT was performed using spleenic T cells isolated from HLA transgenic IFN-α/βR−/− mice (black bars) and HLA transgenic IFN-α/βR+/+ mice (white bars). Mice were infected retro-orbitally with 1×1010 GE of DENV2. Seven days post-infection, CD8+ T cells were purified and tested against a panel of DENV2 predicted peptides. The data are expressed as mean number of SFC/106 CD8+ T cells of two independent experiments. Error bars represent SEM. Responses against peptides were considered positive if the stimulation index (SI) exceeded double the mean negative control wells (effector cells plus APCs without peptide) and net spots were above the threshold of 20 SFCs/106 CD8+ T cells in two independent experiments. Asterisks indicate peptides, which were able to elicit a significant IFNγ response in each individual experiment, according to the criteria described above.
Fig. 2
Fig. 2. Identification of DENV-derived epitopes recognized by CD4+ T cells
IFNγ ELISPOT was performed using CD4+ T cells isolated from DRB1*0101 transgenic IFN-α/βR−/− (black bars) and IFN-α/βR+/+ (white bars) mice. Mice were infected retro-orbitally with 1×1010 GE of DENV2. Seven days post-infection, CD4+ T cells were purified and tested against a panel of DENV2 predicted peptides. The data are expressed as mean number of SFC/106 CD4+ T cells of two independent experiments. Error bars represent SEM. Responses against peptides were considered positive if the stimulation index (SI) exceeded double the mean negative control wells (effector cells plus APCs without peptide) and net spots were above the threshold of 20 SFCs/106 CD4+ T cells in two individual experiments. Asterisks indicate peptides, which were able to elicit a significant IFNγ response, according to the criteria described above.
Fig. 3
Fig. 3. Determination of the optimal epitope
To determine the optimal epitope, HLA-transgenic IFN-α/βR−/− mice were infected with 1 × 1010 GE of DENV2 and spleens were harvested seven days post-infection. CD8+ T cells were purified and incubated for 24 hours with ascending concentrations of nested peptides. Panel A shows pairs of nested epitopes where the 10-mer as well as a nested 9-mer peptide was able to elicit an immune response. Panel B shows two B*0702 restricted epitopes where 8- and 9-mer peptides carrying alternative dominant B7 motifs were synthesized and tested for T cell recognition. The peptides, which were able to elicit stronger IFNγ responses at various concentrations, were then considered the optimal epitope.
Fig. 4
Fig. 4. MHC-restriction of identified epitopes
HLA A*0201 (A) and HLA A*1101 (B) transfected 721.221 cells as well as the non-transfected cell line as a control were used as antigen presenting cells in titration experiments to determine MHC restriction. Mice were infected retro-orbitally with 1×1010 GE of DENV2. Seven days post-infection purified CD8+ T cells from DENV2 infected HLA A*A0201 and HLA A*1101 transgenic IFN-α/βR−/− mice were incubated with ascending concentrations of peptides and tested for IFNγ production in an ELISPOT assay. Representative graphs of CD8+ T cell responses are shown, when incubated with HLA transfected cell lines (A and B; black lines) and non-tranfected cell lines (A and B, grey lines) are shown. The dotted line indicates the 25 net SFCs/106 cells threshold used to define positivity.
Fig. 5
Fig. 5. Antigenicity of identified epitopes in human donors
To validate the epitopes identified in the HLA-transgenic IFN-α/βR−/− mice, we tested their capacity to stimulate PBMC from human donors. Thus PBMC [2×106/ml] were stimulated in the presence of 1 μg/ml individual peptide for 7 days and then tested in an IFNγ ELISPOT assay. Figures A-E show IFNγ responses/106 PBMC after stimulation with A*0101, A*0201, A*1101, B*0702 and DRB1*0101 restricted peptides, respectively. Donors, seropositive for DENV, were grouped in HLA matched and non-HLA matched cohorts, as shown in panels 1 and 2 of each figure. All epitopes identified were also tested in DENV seronegative individuals. The average IFNγ responses elicited by PBMC from DENV seropositve non-HLA matched and DENV seronegative donors plus 3 times the standard deviation (SD) was set as a threshold for positivity, as indicated by the dashed line. Figure F shows the mean IFNγ response/106 T cells from HLA transgenic mice (black bars) and HLA matched donors (white bars) grouped by HLA restriction of the epitopes tested.
Fig. 6
Fig. 6. Protein location of identified epitopes
All identified epitopes were grouped according to the DENV protein they are derived from. Black bars show the total IFNγ response of all epitopes from a given protein. Numbers in parenthesis indicate the number of epitopes that have been detected for this protein.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kyle JL, Harris E. Global spread and persistence of dengue. Annu Rev Microbiol. 2008;62:71–92. - PubMed
    1. WHO. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva, Switzerland: 2009. - PubMed
    1. Burke D, Monath T. Flaviviruses. In: Knipe aPHD., editor. Field’s Virology. William and Wilkins; Philadelphia: 2001. pp. 1043–1126.
    1. Rothman AL. Cellular immunology of sequential dengue virus infection and its role in disease pathogenesis. Curr Top Microbiol Immunol. 2010;338:83–98. - PubMed
    1. Mathew A, Rothman AL. Understanding the contribution of cellular immunity to dengue disease pathogenesis. Immunol Rev. 2008;225:300–313. - PubMed

Publication types