Archive for April, 2008

Influenza vaccines from plants??

22 April, 2008

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.

A plant-produced influenza subunit vaccine protects ferrets against virus challenge
Influenza and Other Respiratory Viruses 2008 2: 33–40

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….

Oxygen from viruses??

7 April, 2008

I thank my colleague Suhail Rafudeen for alerting me to this:

 “Some Of Our Oxygen Is Produced By Viruses Infecting Micro-organisms In The Oceans

ScienceDaily (Apr. 6, 2008) – Some of the oxygen we breathe today is being produced because of viruses infecting micro-organisms in the world’s oceans, scientists heard April 2, 2008 at the Society for General Microbiology’s 162nd meeting.

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.

“In major parts of the oceans, the micro-organisms responsible for providing oxygen and locking away carbon dioxide are actually single celled bacteria called cyanobacteria,” says Professor Nicholas Mann of the University of Warwick. “These organisms, which are so important for making our planet inhabitable, are attacked and infected by a range of different types of viruses.”

The researchers have identified the genetic codes of these viruses using molecular techniques and discovered that some of them are responsible for providing the genetic material that codes for key components of photosynthesis machinery.

“It is beginning to become to clear to us that at least a proportion of the oxygen we breathe is a by-product of the bacteria suffering from a virus infection,” says Professor Mann. “Instead of being viewed solely as evolutionary bad guys, causing diseases, viruses appear to be of central importance in the planetary process. In fact they may be essential to our survival.”

Viruses may also help to spread useful genes for photosynthesis from one strain of bacteria to another.

Adapted from materials provided by Society for General Microbiology, via EurekAlert!, a service of AAAS”

Fascinating concept: viruses as an essential link in the circle of life?!  Not so far-fetched, though: just because we know them largely because of their propensity to cause, and our fascination with, diseases that affect us and our livestock and crops…doesn’t mean that is all there is.

Viruses have been around as long as any other form of life, and it would be strange indeed if some form(s) of commensalism and/or symbiosis had not evolved.

…and see here for some fascinating speculations on the possible involvement of viruses with the origin of eukaryotes.

So there IS light at the end of the tunnel

1 April, 2008

After the shock of the second failure of an HIV-1 vaccine in Phase III trials recently – detailed to some extent here – we were surely due some relief.

And it is here: William Borkowsky and team have just published in AIDS and Human Retroviruses a paper which describes what amounts to successful “autologous immunisation” of a paediatric HIV-infected cohort by a series of progressively longer treatment interruptions, or drug holidays. 

The children, who ranged in age from 4 to 19, were all on HAART or highly active anti-retroviral drug therapy, and all had initially undetectable viral loads.  The subjects in the experimental arm of the trial were given a series of drug holidays of progressively increasing length over up to 17 cycles of treatment in some cases.  In the words of the authors:

“Increased HIV-specific immune responses and decreased HIV RNA were seen in those children who have had >10 cycles of antiretroviral discontinuations of increasing durations acting as autologous virus vaccinations. Other studies may have failed due to an insufficient number of exposures to HIV; most of the studies had fewer than six drug interruptions.”

This is a quite momentous finding: given that it is known that increased CD8+ T-cell responses to Gag proteins of HIV are correlated with decreased viral load in infected patients, this means that many times-repeated exposures of immunocompetent people to live virus seems to successfully elicit suitable immunity and reduce viral load, just as a vaccine could be wished to do.

But in all the vaccine trials, and in previous treatment interruption trials, no more than 4 vaccinations or 6 drug interruptions were performed – which may mean, given the lack of persistence of T-cell as compared to antibody responses, that simply too few treatments have been given in the past.

So is the solution to dose people considerably more often in prophylactic vaccine trials aimed at protecting against HIV infection? 

And possibly with subunit vaccines (such as our recent offering…B-) or killed whole-virus vaccines instead of “genetic vaccines” such as the DNA and virus-vectored HIV gene vaccines which have been so popular up to now?

We need to explore these possibilities – and to explore them soon.  There is a lot riding on the outcome….