I should have known Alan Cann would find this one; it’s just too good to miss – so I am going to add to what he said, as a way of further exploring what they could/should have done, as a result of discussions in our Journal Club this morning.
Alan wrote:
Influenza vaccines from plants
Posted by ajcann on April 16, 2008
Our major defense against infection with influenza viruses is immunization of individuals with an annually updated vaccine that is currently produced in chicken eggs, with a global annual capacity of about 400 million doses, a scale of production insufficient to combat a pandemic. Furthermore, at least six months is required between the identification of new virus strains to be included in the vaccine formulation and the manufacture of bulk quantities. Uncertainties over the robustness of egg-based vaccine production are intensified even further by the emergence of H5N1 strains that are highly virulent to both chickens and eggs. There is a need to develop alternative vaccine production systems capable of rapid turnaround and high capacity. Recombinant subunit vaccines should circumvent some of the concerns regarding our current dependence on egg-based production.This paper reports on the production and evaluation of domains of influenza haemagglutinin (HA) and neuraminidase (NA) fused to the thermostable enzyme lichenase. All vaccine targets were produced using a plant-based transient expression system (Nicotiana). When tested in ferrets, vaccine candidates containing these engineered plant-produced influenza HA and NA antigens were highly immunogenic, and were protective against infection following challenge with homologous influenza virus. This plant-based production system offers safety and capacity advantages, which taken together with the protective efficacy data reported, demonstrates the promise of this approach for subunit influenza vaccine development.
There are a couple of interesting features of this paper, chief among them being the complete obscurity of the reasons why they use lichenase fusions, and what exactly their “launch vector” – which is what they use to express their proteins transiently – is. Because the reference they give is incorrect – it is to a journal they erroneously call “Influenza”, which is not listed by PubMed, and turns out to be Influenza and Other Respiratory Viruses in fact – and is unavailable at our institution. I am assuming, given the system uses a CaMV 35S promoter to drive RNA production, and they talk of “viral replication and target sequence expression from the [TMV] CP subgenomic mRNA promoter”, that the vector is a TMV-based replicon. I was alerted by colleagues at the Journal Club to the fact that the same group used the same system – pBID4 “launch vector”, fusions to lichenase – for production of a HPV E7 vaccine in plants. And referred to the same paper as this one does, for the vector and constructs. Aargh! I still don’t know why lichenase fusions are such a good idea!!
A hint is given in the E7 paper: they say that “…these LicKM fusion proteins alone are able to activate both innate and adaptive antigen-specific immune responses”. But they found in the paper under discussion here that alum was needed to get the best response…and they got the best yield AND immunogenicity out of their NA protein, which was expressed as a (presumably) soluble truncated native protein. So the reason is still obscure.
The purification section of this paper is also woefully inadequate: saying “…recombinant antigens were enriched by ammonium sulphate precipitation followed by immobilised metal affinity chromatography and anion exchange chromatography, with dialysis after each step, to at least 80% purity” is NOT a method! It is an anecdote, fit for a 1-minute talk maybe, but NOT for the Methods section of a paper. Naughty, naughty!
Another interesting thing is the complexity of the vaccine constructs – again, exactly the same type of constructs as made for HPV E7; assembly-line vaccine producers, these guys! These consist of the Gene of Choice (GoC) with a poly-His tag AND a KDEL (ER retention) tag at the C-terminus, AND the signal sequence of Nicotiana tabacum PR1a protein at their N-terminus. This means (a) proteins get into the ER lumen, (b) get retained in the ER, (c) can be purified by Ni or other metal affinity column. In addition to being fused to LickM. Granted, the PR1 signal sequence is lost and the His tags can be removed – but the proteins still have significant “other” constituents – which is rather frowned on in a vaccine intended for humans.
I am also interested that they did not do the standard thing with their plant-produced HA GD protein and test for haemagglutination / RBC binding: this was in any case superseded by the fact that the vaccines were protective and antisera elicited by them worked in HI [haemagglutination-inhibition] assays, but it has long been regarded as a necessary first step. I like these guys’ approach: forget the biochemistry; let’s see if it works!
All in all, a good paper despite our criticisms, which points up the very distinct possibility of being able to use plant production of influenza virus antigens for the rapid production of effective vaccines.
But I wish they’d included some more details….
About half the world’s oxygen is being produced by tiny photosynthesising creatures called phytoplankton in the major oceans. These organisms are also responsible for removing carbon dioxide from our atmosphere and locking it away in their bodies, which sink to the bottom of the ocean when they die, removing it forever and limiting global warming.
And it is here: William Borkowsky and team have just published in AIDS and Human Retroviruses 
ViroBlogy 