I will be blogging in a LOT more detail soon concerning my group’s attendance of the 5th Plant-Based Vaccines, Antibodies and now also Biologics meeting in Verona less than two weeks ago – but one presentation so caught my eye that I thought I would feature it as a preview of my overall report.
It did not hurt that the presenter, one Diego Orzaez, who describes himself in an email as that “…guy from Valencia who showed those pictures of the virus mosaic”, is also a Twitter and Scoop.it Virology News fan, which will always endear someone to me…no, seriously, I think the potential of the technology is huge – so let me get right to it!
A paper describing the basics of the phenomenon Diego and colleagues utilised in order to potentially get expression of multiple monoclonal Abs in a single leaf, is described in the following paper from April 2013:
Plant Molecular Biology
April 2013, Volume 81, Issue 6, pp 553-564A coat-independent superinfection exclusion rapidly imposed in Nicotiana benthamiana cells by tobacco mosaic virus is not prevented by depletion of the movement protein
José Manuel Julve, Antoni Gandía, Asun Fernández-del-Carmen, Alejandro Sarrion-Perdigones, Bas Castelijns, Antonio Granell, Diego Orzaez
New evidence is emerging which indicates that population variants in plant virus infections are not uniformly distributed along the plant, but structured in a mosaic-like pattern due to limitation to the superinfection imposed by resident viral clones. The mechanisms that prevent the infection of a challenge virus into a previously infected cell, a phenomenon known as superinfection exclusion (SE) or Homologous Interference, are only partially understood. By taking advantage of a deconstructed tobacco mosaic virus (TMV) system, where the capsid protein (CP) gene is replaced by fluorescent proteins, an exclusion mechanism independent of CP was unveiled. Time-course superinfection experiments provided insights into SE dynamics. Initial infection levels affecting less than 10 % of cells led to full immunization in only 48 h, and measurable immunization levels were detected as early as 6 h post-primary infection. Depletion of a functional movement protein (MP) was also seen to slow down, but not to prevent, the SE mechanism. These observations suggest a CP-independent mechanism based on competition for a host-limiting factor, which operates at very low virus concentration. The possible involvement of host factors in SE has interesting implications as it would enable the host to influence the process.
Basically, what this means is that agroinfiltrating a leaf of N benthamiana with a mixture of Agrobacterium tumefaciens clones of deconstructed TMV constructs expressing different fluoresecent proteins, results in “clonal” mosaics of leaf cells, with each individual “tile” expressing a single construct. This is extremely well shown below, in a screen capture from a MP4 file illustrating progression of the tiling effect from initial stages following agroinfiltration, to several days later.
Expression mosaic in N benthamiana leaf infiltrated with different TMV-based vector constructs
This is just a mindblowingly visual proof of superinfection exclusion – and is a phenomenon which could be harnessed for doing things like expressing an Agrobacterium library of an antibody variable region repertoire.
Which would effectively allow a single leaf, or preferably a collection of plants, to express the equivalent of a polyclonal serum, rather than a single monoclonal antibody – something that is pretty much impossible in any other expression system.
Molecular farming is SO cool…B-)
And thanks, Diego!
Tags: deconstructed vector, MAbs, mosaic
ViroBlogy 
27 June, 2013 at 07:33 |
The super-infection exclusion hypothesis is based on the idea of homologous interference, which is the ability of an established infection with one virus to interfere with secondary viral infection. Specific examples of this phenomenon have been documented in cell culture not only with flaviviruses (Randolph and Hardy 1988 , Sundin and Beaty 1988 , Burivong et al. 2004 , Pepin et al. 2008 ), but also with other arboviruses of the genera Alphavirus (Karpf et al. 1997 ), Orbivirus (Ramig et al. 1989 ), and Vesiculovirus (Legault et al. 1977 , Whitaker-Dowling et al. 1983 ). Such observations have led to the proposition that super-infection exclusion may be a generalized phenomenon that occurs broadly across the genus Flavivirus (Farfan-Ale et al. 2009 , Kim et al. 2009 ), but evidence from the laboratory has been equivocal. For example, Vero cells infected with St. Louis encephalitis virus can be super-infected with two related flaviviruses, Japanese encephalitis virus and Yellow fever virus (Randolph and Hardy 1988 ), although not with the same strain of St. Louis encephalitis virus. On the whole, laboratory studies seem to suggest that superinfection exclusion can occur between closely related viruses but that more distantly related viruses do not generally interfere with each other. Our results demonstrating natural coinfection of individual mosquitoes and positive statistical association between CxFV and WNV suggest that the homologous interference concept may not apply to highly divergent flaviviruses such as the insect-specific flaviviruses and their vertebrate pathogen relatives, and that superinfection exclusion may be of limited relevance in a field setting.
21 August, 2013 at 11:21 |
[…] Orzaez (IPMCP-CSIC, Valencia, Spain) spoke next, on the same technology I have previously described (with beautiful pictures from Diego) here: that is, the enabling of tools for multigene engineering of plants – and specifically in this […]