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. 2010 Apr 15;184(8):4423-30.
doi: 10.4049/jimmunol.0903955. Epub 2010 Mar 8.

Single-chain HLA-A2 MHC trimers that incorporate an immundominant peptide elicit protective T cell immunity against lethal West Nile virus infection

Sojung Kim  1 , Lijin Li, Curtis P McMurtrey, William H Hildebrand, Jon A Weidanz, William E Gillanders, Michael S Diamond, Ted H Hansen
Affiliations

Single-chain HLA-A2 MHC trimers that incorporate an immundominant peptide elicit protective T cell immunity against lethal West Nile virus infection

Sojung Kim et al. J Immunol. .

Abstract

The generation of a robust CD8(+) T cell response is an ongoing challenge for the development of DNA vaccines. One problem encountered with classical DNA plasmid immunization is that peptides produced are noncovalently and transiently associated with MHC class I molecules and thus may not durably stimulate CD8(+) T cell responses. To address this and enhance the expression and presentation of the antigenic peptide/MHC complexes, we generated single-chain trimers (SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, beta(2)-microglobulin, and H chain connected by flexible linkers. In this study, we test whether the preassembled nature of the SCT makes them effective for eliciting protective CD8(+) T cell responses against pathogens. A DNA plasmid was constructed encoding an SCT incorporating the human MHC class I molecule HLA-A2 and the immunodominant peptide SVG9 derived from the envelope protein of West Nile virus (WNV). HLA-A2 transgenic mice vaccinated with the DNA encoding the SVG9/HLA-A2 SCT generated a robust epitope-specific CD8(+) T cell response and showed enhanced survival rate and lower viral burden in the brain after lethal WNV challenge. Inclusion of a CD4(+) Th cell epitope within the SCT did not increase the frequency of SVG9-specific CD8(+) T cells, but did enhance protection against WNV challenge. Overall, these findings demonstrate that the SCT platform can induce protective CD8(+) T cell responses against lethal virus infection and may be paired with immunogens that elicit robust neutralizing Ab responses to generate vaccines that optimally activate all facets of adaptive immunity.

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Figures

FIGURE 1
FIGURE 1
Identification of an immunodominant peptide in HLA-A*0201 transgenic mice. HHDII mice were infected with 102 PFU of WNV, and 7 d later spleen cells were harvested. A, Cells were stimulated in vitro with peptides for 4 h in the presence of Golgi-blocking agent. Then cells were stained for CD8 and intracellular IFN-γ. The percentages of IFN-γ+ of CD8+ T cells after each peptide stimulation (x-axis) from infected (•) or naive (Δ) mice are shown. B, Cells were stimulated in vitro with SVG9 peptide for 7 d and stained with anti-CD8 Ab and SVG9/HLA-A*0201 or control tetramers. Cells were gated on propidium iodide-negative events. Numbers indicate the percentage of cells in each quadrant of total cells. *p < 0.05 (unpaired t test).
FIGURE 2
FIGURE 2
Epitope expression on WNV-infected cells. A, Primary DCs derived from HLA-A*0201–positive PBMCs were incubated with SVG9 (dotted line) or SLF9 (solid line) peptide for 4 h and stained with TCRm Ab, RL14C, or RL15A. Numbers indicate mean fluorescence intensity (MFI) of each histogram. Shaded histogram indicates unpulsed cells. B, Primary DCs were infected with WNV at a multiplicity of infection of 1. Forty-eight hours later, cells were stained with anti-WNV E24 (solid line) or isotype control mAb (dotted line). Shaded histogram indicates uninfected cells. C, Uninfected (left panels) or infected (right panels) DCs were stained with RL14C (upper panels) or RL15A (lower panels). Numbers indicate the percentages of TCRm-positive cells among total cells. D, A [51Cr]-release assay was performed in the presence of TCRm Ab. HLA-A*0201–expressing HeLa (HelaA2) cells were used as targets. SVG9-specific bulk CTLs were incubated with 1 µM SVG9-pulsed HeLaA2 cells for 4.5 h in the presence of RL14C or RL15A. All data are representative of at least two independent experiments with similar results.
FIGURE 3
FIGURE 3
Cytotoxic activity of the identified epitope-specific T cells in vitro and in vivo. A and B, A [51Cr]-release assay was performed with SVG9-specific CTLs as effectors. A, HeLaA2 (HLA-A2–transduced HeLa) targets were incubated with CTLs for 4.5 h in the presence of 1 or 10 µMof SVG9 peptide. B, HeLa or HeLaA2 cells infected with WNV-KUN were used as targets. C, In vivo cytotoxic assay. For targets, PHK26-labeled naive HHDII splenocytes were pulsed with SVG9 or irrelevant peptide and labeled with 1 µM or 50 nM of CFSE, respectively. Differentially labeled targets were mixed at 1:1 ratio and transferred i.v. into a HHDII mouse at day 7 postinfection. Six hours later, spleen cells from the recipients were analyzed. Histograms were gated on PKH26+ events. The percentage of CFSEhi or CFSElo population of PKH26+ cells is indicated with MFI in parentheses. All data are representative of at least two independent experiments with similar results. D, Donor cells were obtained from HHDII mice that had been infected with 102 PFU of WNV. Cells were restimulated in vitro for 7 d with 0.1 µM of SVG9 peptide. CD8+ T cells were then isolated by negative selection and transferred into HHDII mice at day 1 postinfection with 102 PFU of WNV (2 × 106 CD8+ T cells per mouse). Survival was monitored over 28 d. The results were combined from two independent experiments (n = 10–11 per group). **p < 0.0005 (log-rank test).
FIGURE 4
FIGURE 4
Generation of SVG9/HLA-A*0201 SCTs and specific CD8+ T cell responses. A, Diagram of the SCT DNA encoding SVG9 or mammoglobin A (mamA) peptide/chimeric HLA-A*0201 complex. B, Cell surface expression of SVG9 SCT. HeLa cells were transfected with SVG9 SCT DNA and stained with anti–HLA-A2 Ab (BB7.2, dotted line), RL15A (solid line), or isotype control Ab (shaded histogram). Numbers indicate MFI of each histogram. C, Lysis of target cells expressing SVG9 SCT. SVG9 SCT-expressing HeLa cells were used as targets in a [51Cr]-release assay with SVG9-specific CTLs. For controls, HeLaA2 cells or HeLaA2 cells pulsed with SVG9 peptide were also used as targets. D and E, Generation of specific CD8+ T cell responses in vivo. HHDII mice were immunized with SVG9 or control SCT DNA three times at 3 d intervals. At 5 d after the last immunization, splenocytes were harvested. D, Splenocytes from control (left panel) or SVG9 SCT-immunized mice (right panel) were stained with anti-CD8 Ab and SVG9/HLA-A*0201 tetramers. Numbers indicate the percentage of cells in each quadrant among total cells. Representative figures of the flow cytometry data. E, Splenocytes from each group were stimulated in vitro with SVG9 or irrelevant peptide overnight, and IFN-γ secretion was measured by ELISPOT. Error bars indicate SE of the experiment. The data presented are from one representative experiment of two performed independently with each group containing four to five mice. SS, signal sequence.
FIGURE 5
FIGURE 5
Protection against viral infection by SVG9 SCT DNA vaccination. A, HHDII mice were immunized with SVG9 or control SCT DNA with PADRE. At 5 d after the last immunization, mice were infected with 102 PFU of WNV. Mice were monitored over 60 d (n = 11–12 per group). *p < 0.05 (log-rank test). B, Diagram of a SVG9 SCT DNA construct with or without an insertion of PADRE sequence. C and D, HHDII mice were immunized with SVG9 SCT with or without PADRE or control SCT DNA. C, At 5 d after the last immunization, splenocytes were stimulated in vitro with 1 µM SVG9 or irrelevant peptide overnight. IFN-γ secretion was measured by ELISPOT. Error bars indicate SD with n = 5 mice per group. n.s., unpaired t test. D, At 5 d after the last immunization, mice were infected with 102 PFU of WNV and monitored over 30 d (n = 8– 9 per group). *p < 0.05 (unpaired t test) compared with SVG9 SCT without PADRE. SS, signal sequence.
FIGURE 6
FIGURE 6
Viral clearance and induction of non-sterilizing immunity by SCT DNA immunization. A and B, Brain tissues and serum were collected at day 8 after viral challenge of HHDII mice that had been immunized with SVG9 or control SCT DNA. A, Viral burdens in brain tissues were determined by a plaque assay. Naive mice were used as a negative control. B, The levels of anti-WNV IgG (left panel) and IgM (right panel) in the serum were measured by ELISA. n.s., unpaired t test. C–E, Spleen and serum samples were taken from SVG9 SCT DNA-vaccinated mice at day 65 post-WNV infection. Naive mice were used as a negative control. C, Splenocytes were stained with anti-CD8, anti-CD44 mAb, and SVG9/HLA-A*0201 tetramers. Mean percentage of tetramer-positive CD44hiCD8+ T cells of four mice in each group was shown. Error bars indicate SD. D, Splenocytes were stained for CD8 and intracellular IFN-γ after 4 h stimulation in vitro with SVG9 or irrelevant peptide in the presence of Golgi-blocking agent. E, WNV-specific IgG titers in the serum were measured by ELISA. Data were expressed as reciprocal log endpoint titers after regression analysis. *p < 0.05 (unpaired t test).
FIGURE 7
FIGURE 7
Long-term immunity provide by SVG9 SCT DNA vaccination. HHDII mice were vaccinated with SVG9 or control SCT DNA with PADRE as above and, at 45 d after the last immunization, challenged with 102 PFU of WNV. Mice were monitored over 25 d (n = 11–13 per group). *p < 0.05 (log-rank test).
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