Pierre-Olivier Lavoie

Human metapneumovirus (HMPV) and human respiratory virus (HRSV) are two leading causes of acute respiratory tract infection in young children. While there is no licensed drug against HMPV, the monoclonal antibody (mAb) Palivizumab is approved against HRSV for prophylaxis use only. Novel therapeutics against both viruses are therefore needed. Here, we describe the identification of human mAbs targeting these viruses by using flow cytometry-based cell sorting. One hundred and two antibodies were initially identified from flow cytometry-based cell sorting as binding to the fusion protein from HRSV, HMPV or both. Of those, 95 were successfully produced in plants, purified and characterized for binding activity by ELISA and neutralization assays as well as by inhibition of virus replication in mice. Twenty-two highly reactive mAbs targeting either HRSV or HMPV were isolated. Of these, three mAbs inhibited replication in vivo of a single virus while one mAb could reduce both HRSV and HMPV titers in the lung. Overall, this study identifies several human mAbs with virus-specific therapeutic potential and a unique mAb with inhibitory activities against both HRSV and HMPV.

Partial neutralization of the Golgi lumen pH by ectopic expression of influenza virus M2 proton channel stabilizes acid-labile and protease-susceptible recombinant proteins in the plant cell secretory pathway. Here, we assessed the impact of M2 channel expression on the proteome of Nicotiana benthamiana leaf tissue infiltrated with the bacterial gene vector Agrobacterium tumefaciens , keeping in mind the key role of pH homeostasis on secreted protein processing and the involvement of protein secretion processes in plant cells upon microbial challenge. The proteomes of leaves agroinfiltrated with an empty vector or with an M2 channel-encoding vector were compared with the proteome of non-infiltrated leaves using a iTRAQ quantitative proteomics procedure. Leaves infiltrated with the empty vector had a low soluble protein content compared to non-infiltrated leaves, associated with a strong decrease of photosynthesis-associated proteins (including Rubisco) and a parallel increase of stress-related secreted proteins (including pathogenesis-related proteins, protease inhibitors and molecular chaperones). M2 expression partly compromised these alterations of the proteome to restore original soluble protein and Rubisco contents, associated with higher levels of translation-associated (ribosomal) proteins and reduced levels of stress-related proteins in the apoplast. Proteome changes in M2-expressing leaves were determined both transcriptionally and post-transcriptionally, to alter the steady-state levels of proteins not only along the secretory pathway but also in other cellular compartments including the chloroplast, the cytoplasm, the nucleus and the mitochondrion. These data illustrate the cell-wide influence of Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana plants responding to microbial challenge. They underline in practice the relevance of carefully considering the eventual off-target effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.