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Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library

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Abstract

Immunomodulation by the ectopic expression of intracellular antibodies (‚intrabodies’) has a great potential for interfering with physiological or pathological functions in vivo in a highly specific manner. One of the major obstacles of this technology is the inability of most antibodies to properly fold and function in the reducing environment of the cytoplasm, which prevents the formation of essential disulfide bonds. We wished to assess the intracellular performance of antibodies derived from a semi-synthetic single-chain variable fragment (scFv) phage display library (‚F8 library’) built on a thermodynamically stable single-framework scaffold. To this purpose, we chose to modulate the infection of a pandemic plant pathogen, the cucumber mosaic virus (CMV). After in vitro ‚biopanning’ on immobilized virions, two scFvs were biochemically characterized, showing high affinity toward the antigen. They were transiently expressed at high yields as soluble molecules in the cytoplasm of Nicotiana benthamiana plants. Subsequently, they were expressed in the cytoplasm of transgenic tomato plants. Challenge with high viral dose showed that both scFvs were able to elicit a phenotypic effect and led to the identification of a transgenic line fully resistant to infection. In these plants, the scFv binds the virus in the inoculated leaves preventing viral long distance movement. This work represents the first demonstration that the ‚F8 library’ can be directly screened in vitro to rapidly isolate antigen-specific scFvs that act as effective intrabodies in vivo. These antibodies represent powerful tools to interfere with several intracellular targets, modulating pathogen infectivity and/or cellular metabolism.

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Abbreviations

AMCV:

artichoke mottled crinkle virus

CDR:

complementarity-determining region

CMV:

cucumber mosaic virus

CP:

coat protein

ELISA:

enzyme-linked immunosorbent assay

PBS:

phosphate-buffered saline

PVX:

potato virus X

scFv:

single-chain variable fragment

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SPR:

surface plasmon resonance

TAS-ELISA:

triple antibody sandwich ELISA

VH:

immunoglobulin heavy chain variable domain

VL:

immunoglobulin light chain variable domain

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Authors and Affiliations

  1. ENEA, Ente per le Nuove Tecnologie, l’Energia e l’Ambiente, UTS BIOTEC, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400, I-00100, Roma, Italy

    Maria Elena Villani, Orsola Bitti, Eugenio Benvenuto & Rosella Franconi

  2. CNR, Consiglio Nazionale delle Ricerche, Istituto di Virologia Vegetale (IVV), Area di Ricerca Torino, Strada delle Cacce 73, 10135, Torino, Italy

    Piero Roggero

Authors
  1. Maria Elena Villani
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  2. Piero Roggero
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  3. Orsola Bitti
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  4. Eugenio Benvenuto
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  5. Rosella Franconi
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Corresponding author

Correspondence to Rosella Franconi.

Additional information

We dedicate this work to Piero Roggero who prematurely passed away on June 19th 2003.

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Villani, M.E., Roggero, P., Bitti, O. et al. Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library. Plant Mol Biol 58, 305–316 (2005). https://doi.org/10.1007/s11103-005-4091-0

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  • DOI: https://doi.org/10.1007/s11103-005-4091-0

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