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A Single-Chain H-2D^b Molecule Presenting an Influenza Virus Nucleoprotein Epitope Shows Enhanced Ability at Stimulating CD8⁺ T Cell Responses In Vivo ¹

Michael J. Palmowski^2,*, Mathew Parker^3,, Kaushik Choudhuri^4,, Christopher Chiu^5,, Margaret F. C. Callan, P. Anton van der Merwe, Vincenzo Cerundolo^* and Keith G. Gould^6,

^* Tumour Immunology Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; Department of Immunology, Wright-Fleming Institute, Imperial College London, London, United Kingdom; Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; and Department of Rheumatology, Division of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom

We have generated a construct encoding a single-chain H-2D^b mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse β₂-microglobulin and the D^b H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous β₂-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D^b NP SCT primed a CD8⁺ T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8⁺ cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8⁺ T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8⁺ T cell responses is required.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the U.K. Medical Research Council. Work carried out by M.P. and V.C. was funded by Cancer Research U.K (C399/A2291) and the Harry Mahon Cancer Research Trust.

² Current address: UCB Celltech, 216 Bath Road, Slough SL1 4EN, U.K.

³ Current address: TwistDx, Meditrina (Building 260), Babraham Research Campus, Cambridge CB22 3AT, U.K.

⁴ Current address: Skirball Institute of Biomolecular Medicine, Program in Molecular Pathogenesis, Second Floor, Dustin Lab, 540 1st Avenue, New York, NY 10016.

⁵ Current address: Department of Microbiology, Imperial Healthcare NHS Trust, St Mary’s Hospital, Norfolk Place, London W2 1PG, U.K.

⁶ Address correspondence and reprint requests to Dr. Keith G. Gould, Department of Immunology, Wright-Fleming Institute, Imperial College London, Norfolk Place, London W2 1PG, U.K. E-mail address: k.gould{at}imperial.ac.uk

⁷ Abbreviations used in this paper: SCT, single-chain trimer; β₂m, β₂-microglobulin; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; NP, nucleoprotein; VV, vaccinia virus.

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