Archive for September, 2009

Influenza virus A H1N1 2009: gets to parts the other flu doesn’t reach

14 September, 2009

Flu virus life cycle. Copyright Russell Kightley Media

The September 2009 issue of Nature Biotechnology has a letter concerning the receptor specificity of AH1N1 2009 pandemic influenza virus – which accounts pretty well for why it CAN be pretty nasty, and for why it may get nastier yet.

Childs et al., in a letter entitled “Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray“, describe what amounts to a tour de force analysis of the receptor binding of a number of influenza viruses, which concludes with the statement that:

“The differences in receptor binding between the 2009 pandemic and seasonal H1N1 viruses may therefore account, at least in part, for the higher virus replication and greater pathology reported in the lungs of ferrets, mice and nonhuman primates infected with pandemic viruses, than observed with contemporary seasonal viruses.”

Which would help explain why some otherwise healthy young people are dying of the virus, while others are getting only mildly ill.  But we get ahead of ourselves: in January last year I wrote in MicrobiologyBytes about recpetor specificities of A-type influenza viruses, in the context of how H5N1 was less likely to mutate to easy human-to-human transmissibility than had origianlly been thought.

I wrote at the time:

According to a letter in the January 2008 issue of Nature Biotechnology, it is a characteristic structural topology, and not just the α2,6 linkage, that enables specific binding of HA to α2,6 sialylated glycans. The authors state:

…recognition of this topology may be critical for adaptation of HA to bind glycans in the upper respiratory tract of humans. An integrated biochemical, analytical and data mining approach demonstrates that HAs from the human-adapted H1N1 and H3N2 viruses, but not H5N1 (bird flu) viruses, specifically bind to long α2-6 sialylated glycans with this topology. This could explain why H5N1 viruses have not yet gained a foothold in the human population.

Apparently the critical shape in humans is umbrella-like, whereas the avian receptor is characteristically cone-like. Again from the paper:

The topology of α2-3 and α2-6 is governed by the glycosidic torsion angles of the trisaccharide motifs-Neu5Aca2-3Galb1-3/4GlcNAc and Neu5Aca2-6Galb1-4GlcNAc, respectively (Supplementary Fig. 3 online).

Ram Sasisekharan and colleagues showed that human-adapted viruses with mixed α2,3/α2,6 binding ability that bound the umbrella-type receptor were efficiently transmitted, whereas viruses with the same basic specificity that did not have HA binding specificity to “long” α2,6, were not.

The present paper reports the following investigation:

“We have compared directly, by carbohydrate microarray analysis, the receptor-binding characteristics of two isolates of the novel pandemic H1N1 virus, Cal/09 and A/Hamburg/5/2009 (Ham/09), with those of a seasonal human H1N1 virus, A/Memphis/14/96-M (Mem/96), as representative of a virus well adapted to humans [and a reassortant human H3N2 virus A/Aichi/2/68 x PR8 (X31)]. As the HA of the novel H1N1 pandemic virus originated from a virus similar to triple reassortant swine H1N1 viruses, we compared one such example, A/Iowa/1/2006 (Iowa/06), isolated from a human infection, and an older close relative of classical swine H1N1 viruses, A/New Jersey/76 (NJ/76), the human isolate that initiated the concern of a pandemic threat in 1976.”

This is a really comprehensive analysis – for such a short communication – which throws up a number of interesting points.  First, I was not aware it was possible to do “carbohydrate microarrays”!  Second, the paper shows quite conclusively that the swine-derived AH1N1 viruses have a significantly wider range of receptor specificities than a standard seasonal AH1N1 virus, and – but to a lesser extent – than the reassortant H3N2 virus X31.

Carbohydrate microarray analyses of the six viruses investigated.
From the following article (with permission from NBT):
Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray.
Robert A Childs, Angelina S Palma, Steve Wharton, Tatyana Matrosovich, Yan Liu, Wengang Chai, Maria A Campanero-Rhodes, Yibing Zhang, Markus Eickmann, Makoto Kiso, Alan Hay, Mikhail Matrosovich & Ten Feizi.
Nature Biotechnology 27, 797 – 799 (2009).


Numerical scores for the binding signals are shown as means of duplicate spots at 5 fmol per spot (with error bars). The microarrays consisted of eighty sialylated and six neutral lipid-linked oligosaccharide probes, printed on nitrocellulose-coated glass slides. These are listed in Supplementary Table 1 and arranged according to sialic acid linkage, oligosaccharide backbone chain length and sequence. The various types of terminal sialic acid linkage are indicated by the colored panels as defined at the bottom of the figure.

And what does all this mean, exactly?  The authors sum it up well:

These results indicate that no major change in receptor-binding specificity of the HA was required for the emergent pandemic virus to acquire human-like characteristics and become established in the human population. …

The broader specificity, namely, the ability to bind to 2-3- in addition to 2-6-linked receptors is also pertinent to the greater virulence of the pandemic virus than seasonal influenza viruses observed in animal models, and its capacity to cause severe and fatal disease in humans, despite the generally mild nature of most infections. Binding to 2-3-linked receptors is thought to be associated with the ability of influenza viruses to infect the lower respiratory tract where there is a greater proportion of 2-3- relative to 2-6-linked sialyl glycans, although long chain 2-3-linked sialyl (poly-N-acetyllactosamine) sequences are present in ciliated bronchial epithelial cells in humans where they are the receptors for another human pathogen, Mycoplasma pneumoniae.

So there you have it: the viruses can get deeper in to your lungs than the standard flu – which, if it happens, can make you seriously ill.

So what happens if it gets better at binding the 2,3-type receptors in humans?  Well, we’re only in the middle of the pandemic.  We may yet find out the hard way.

Plant therapy creeping in….

8 September, 2009

The August issue of Nature Biotechnology has a very interesting snippet of news – from two points of view. From a strict virology point of view, it is interesting that commercial production of a therapeutic enzyme in an industrial plant can be shut down because of infection of their mammalian cell line with a contaminating mammalian virus.

From the second point of view…well, our lab has a very strong interest in producing recombinant proteins (and especially candidate vaccine proteins) in plants – and here is a story showing just why plants may be a really good alternative means of production for pharmaceuticals.  First, the story:

Nature Biotechnology 27, 681 (2009) doi:10.1038/nbt0809-681a
Virus stalls Genzyme plant by Victor Bethencourt

Genzyme of Cambridge, Massachusetts, faces millions in lost revenue from its top-selling specialty drugs Cerezyme and Fabrazyme as result of a viral contamination at its Allston, Massachusetts plant. The company has announced that it will temporarily shut down the facility owing to a bioreactor contamination with Vesivirus 2117 [my emphasis – Ed], which does not cause human infections, but impairs growth of the biologics-producing Chinese hamster ovary (CHO) cells. It reportedly originated from tainted nutrient medium and belongs to the same strain that caused delays at the Allston site and its European biologics plant in Belgium last year. Genzyme anticipates supply constraints of Cerezyme (imiglucerase), a treatment for Gaucher disease, and Fabrazyme (agalsidase beta), used to treat Fabry disease, while the facility shuts down for 6 to 8 weeks to allow decontamination.

OK, really interesting, that: a vesivirus – genus Vesivirus, family Caliciviridae, nice link here for structure, and here for Genzyme’s press release – that is being transmitted around via cell culture media, between manufacturing plants.  One of the perils of using mammalian cells to make things…!

Vesivirus via kwout

The article goes on:

…With sales of $1.2 billion for Cerezyme and $494 million for Fabryzyme in 2008, analysts estimate the manufacturing crisis will result in $100–300 million in lost sales. The US Food and Drug Administration (FDA) has contacted rival manufacturers Shire of Basingstoke, UK, and Carmiel, Israel–based Protalix, who have enzyme replacement therapies for Gaucher disease in clinical trials, to file treatment protocols, which would allow physicians to use their drugs ahead of approval.

And of course, Protalix makes its glucocerebrocidase in cultured carrot cells, in disposable “bioreactor bags”….  In completely defined chemical media, with no risk of plant virus contamination – not that plant viruses can infect cultured plants cells, by any means short of being shot in on gold beads!  Their web site Press Room page had this to say as of 25th August:

Aug. 25, 2009 (Business Wire) — Protalix BioTherapeutics, Inc. (NYSE-Amex:PLX), announced today that it has received Fast Track Designation from the U.S. Food and Drug Administration (FDA) for prGCD, the Company’s proprietary plant-cell expressed recombinant form of glucocerebrosidase (GCD) for the treatment of Gaucher disease.

I wrote the following about Protalix after attending the Plant-Based Vaccines and Antibodies Conference in Verona in June this year (September issue of Expert Rev Vaccines, 8: 1151-1155, 2009):

“Einat Almon-Brill (Protalix Biotherapeutics, Israel) described their production of recombinant human glucocerebrosidase (rGCD) as a therapy for Gaucher disease, caused by a hereditary mitochondrial defect.  They used a contained disposable bioreactor system with suspension-cultured carrot or tobacco cells, and claimed there were no mammalian cell culture risk factors; they obtained uniform glycosylation, and the exposed mannose allowed rapid macrophage uptake.  The rGCD half-life was twice as long as commercial product, and had been trialled in Europe, Israel, South Africa, and North and South America.”

I wish I’d bought stock…or had the money to, or knew how to!  The time of plant-made pharmaceuticals – PMPs – is coming.

Be ready…B-)

At last, a podcast! Of sorts….

6 September, 2009

Given that the bandwidth here in South Africa is so sadly lacking, I have refrained from doing what my less byte-challenged colleagues elsewhere do with gay abandon: yes, MicrobiologyBytes and virology blog, I speak of you!

However: given that a local newspaper saw fit to ask me about flu and other vaccines, and put up a podcast, I shall link to it from here.   A little abridged – so you can’t tell what we are actually working on – but not too bad (my wife tells me).

From The Times website

Gene discrimination

3 September, 2009

In the latest online issue of Nature, there is an article entitled “Keeping genes out of terrorists’ hands“, by Erika Check Hayden.  Like an article a little while ago in Nature Biotechnology, it makes the apparently quite reasonable point that

“the way that the industry screens orders for hazardous toxins and genes, such as pieces of deadly viruses and bacteria…could be crucial for global biosecurity”.

Yes.  Well.  They would say that, wouldn’t they??  “They” being anyone in the developed world who has a paranoid fantasy about bearded extremists in caves (or crew-cut extremists in leafy suburbs) gleefully unwrapping their couriered DNA and brewing up a nice little necrotising poxvirus, or an airborne Ebola, or possibly an H5N1 variant that spreads human-to-human better than the present versions.

I wrote the following reply to the article:

While “all right-thinking people” – for which, read “those easily scared by the unrealistic prospect of mail-order killer bugs” may agree that some kind of limitations are required on what synthetic DNA is sent out, and to whom…there is a baby being thrown out with the bathwater here.

My laboratory has just, despite many previously successful orders from the same company, been denied permission (or told to obtain clearance from the relevant government, which amounts to the same thing) to have a coat protein gene synthesised for a bluetongue virus (BTV) strain now found all over western Europe. Because, apparently, BTV is on the “Australia Group”‘s prohibited list of biological agents – and South Africa is not a signatory to this group, which started out for arms control but has apparently ramified somewhat.

This is so ridiculous as to beggar belief: the viruses are endemic to Africa; the world’s expert on cDNA cloning of their genomes is in South Africa; why would anyone want to build a BTV from synthetic DNA when they could go out and sample a sheep for some REAL virus??

A closer look at the list throws up all sorts of interesting things. It is prohibited, for example, to order genes for H5N1 influenza – although curiously, not pandemic H1N1 – and dengue viruses. This rather puts a spoke in the wheels of anyone who might want to…oh, let’s say…MAKE A VACCINE to those agents, in any country not signatory to the agreement – where the viruses happen to be endemic!!

The ways of limiting spread of genes that are being proposed are first, unnecessary; second – discriminatory in the extreme.

And may just provide a good deal of business for firms operating in developing countries who otherwise would have been ignored because of quality issues. Imagine that: a lab in Pakistan, or South Africa, or Indonesia, using home-made genes to make a vaccine.

Because that is a LOT more likely than using them to make a pathogen.

I know of a passage written some years ago in a reputable science magazine which described how easy it would be to smuggle naturally-occurring foot and mouth disease virus worldwide – with no science involved whatever.  I have enough purified material of a particular plant virus in my cold room right now to kill all the wheat grown in my country – given some carborundum and a crop sprayer.

There are enough people on this planet infected with pandemic H1N1 who live in close enough proximity to birds infected with H5N1 to make coinfection of one or the other with both a certainty – the only uncertainty remaining being what will come of it.  For that matter, where DID the H1N1 come from?  Where did Lujo virus come from?

We DON’T NEED TO MAKE VIRUSES from mail-order DNA – and only Craig Venter et al. could even dream of making whole microbes.  There are more than enough nasty agents out there that are relatively easy to obtain, and do simple kitchen-based microbiology with, to keep entire cave complexes and Montana libertarian enclaves busy for years, without resorting to complicated molecular biology.

So DO let’s keep things in perspective, shall we??  And let reputable labs doing reputable work order the materials they need to work with.

…and the virus marches on….

1 September, 2009

From News24 this evening:

SA’s H1N1 deaths now 27

2009-09-01 17:03

Cape Town – The number of swine flu deaths in South Africa has risen to 27, and confirmed infections to 5 841, the National Institute for Communicable Diseases said on Tuesday.

“There is also ongoing and widespread community transmission,” it said in a statement.

Of the 27 fatal cases, 12 were pregnant women, five of whom had no identified underlying conditions.

The institute repeated its standard warning that people with depressed immunity, asthma, diabetes, or chronic lung, kidney and heart problems, or who were pregnant should seek early treatment with antivirals.

and on 26th August:

SA wants own H1N1 flu vaccine

2009-08-26 22:29

Cape Town – South Africa has no choice but to develop its own H1N1 flu vaccine, Health Minister Aaron Motsoaledi said on Wednesday, citing concerns treatment will not be available to poorer nations.

“South Africa has arrived at a situation where we have no option but to start developing our own vaccine capacity, not only for H1N1, but generally,” Motsoaledi told parliament.

“The disturbing feature about today’s world… has been expressed by the minister of health for Cambodia… who noted that the developed world, after producing the vaccine, may want to cover their own population first before thinking about the developing world,” Motsoaledi said.

Anyone remember reading that before, anywhere?  Watch this space….

Chimaeric plant virus stimulates influenza virus-specific CD8+ T-cell responses

1 September, 2009

Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice

 Chiara Lico, Camillo Mancini, Paola Italiani, Camilla Betti, Diana Boraschi, Eugenio Benvenuto, Selene Baschieri

 Vaccine (2009) 27: 5069 – 5076

 The authors used plant Potexvirus Potato virus X (PVX) to display the Db-restricted nonapeptide ASNENMETM of the nucleoprotein (NP) from influenza A virus (strain A/PR/8/34) to activate specific CD8+ T cells in mice. They paid great attention to the design of the NP-peptide to ensure optimum plant virus stability and antigen processing. The modified NP-peptide was fused to the N-terminal of the coat protein (CP) from PVX creating the pVXSma-NP construct that was subsequently inoculated into tobacco leaves. The resulting chimeric virus particles (NP-CVP) were stable and pure with a yield of approximately 1.1 mg NP-CVP / g fresh leaf tissue. Endotoxin tests were also performed to exclude their contribution to the immunoregulatory effects of the CVPs. Mice were inoculated with two different doses of NP-CVP (50 µg or 167 µg) with or without incomplete Freund’s adjuvant (IFA). The IFN-γ ELISPOT assays indicated that NP-CVPs activated the ASNENMETM-specific CD8+ response, especially the highest concentration of the NP-CVP without the adjuvant. Results also indicated that the CP of PVX contained T helper epitopes that contributed to the CD8+ T cell response. Thus, PVX is not only an epitope carrier but an adjuvant as well. This study illustrates the potential of implementing plant viruses displaying foreign epitopes to elicit T cell responses in vaccine development.

Contributed by Dr Elizabeth (Liezl) Mortimer