Archive for the ‘biotechnology’ Category

Happy centenary, phages!

17 February, 2015

Here am I, writing a not-so-brief history of the the discovery of viruses, and I miss The Centenary of the Phage!  How did THAT happen?!

Seriously: it took an email from Virologica Sinica alerting me to their commemorative issue, to jolt me into a better state of historical awareness.

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I wrote elsewhere:

Eaters of Bacteria: The Phages

Two independent investigations led to the important discovery of viruses that infect bacteria: in 1915, Frederick Twort in the UK accidentally found a filterable agent that caused the bacteria he was growing to lyse, or burst open.  While he was not sure whether or not it was a virus, Félix d’Hérelle in Paris published in 1917 that he had discovered a virus that lysed a bacterial agent he was culturing that causeddysentery, or diarrhoea.  He named the virus “bacteriophage”, or eater of bacteria, derived from the Greek term “phagein”, meaning to eat.

The discovery of bacteriophages was a landmark in the history of virology, as it meant that for the first time it was relatively easy to work with viruses: many kinds of bacteria could be grown in solid or liquid culture quite easily, and the life cycle of the viruses could be studied in detail.”

"Twort" by Obituary Notices of Fellows of the Royal Society, Vol. 7, No. 20. (Nov., 1951), pp. 504-517.. Licensed under Public Domain via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Twort.jpg#mediaviewer/File:Twort.jpg

“Twort” by Obituary Notices of Fellows of the Royal Society, Vol. 7, No. 20. (Nov., 1951), pp. 504-517.. Licensed under Public Domain via Wikimedia Commons – http://commons.wikimedia.org/wiki/File:Twort.jpg#mediaviewer/File:Twort.jpg

And so it has come to be: the study of phages helped to establish virology as a science, in the era before tissue culture and accurate assay of animal viruses; the birth of molecular biology was pretty much due to the famous Phage Group - and phages turn out to be possibly the most abundant form of life in the known galaxy.

Moreover, the wheel of phage therapy espoused by Félix d’Hérelle has turned full circle, with formerly-scorned Soviet-era institutes now suddenly courted by biotech companies: the Virologica Sinica issue has a an editorial review on the subject, and there is another review on the history of the Eliava Institute in Tbilisi, Georgia, complete with a picture of d’Hérelle there in the 1930s.

So, congratulations Frederick Twort, on the centenary of your discovery.  Your “ultramicroscopic viruses” have gone from strength to strength; your name is remembered – albeit shamefully late – and we really should think of how to put phages more into the public eye.

Figuratively and literally, possibly B-)

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PS: I discover to my delight that there is an entire site devoted to The Year of Phage, which has some amazing art as well as an entire book available for download.  Get yours NOW!

A Short History of the Discovery of Viruses – Part 3

29 January, 2015

The Phage Group and the birth of molecular biology

Some of the more fundamental discoveries in modern biology were facilitated either by the study of viruses, or by use of viruses as tools for exploring host cell mechanisms.  The foundations for this work were laid by Felix d’Hérelle and others, working after 1917 with bacterial viruses in cultured bacteria.  Indeed, Macfarlane Burnet’s first important work was in 1929, showing by use of plaque counting that a single bacterial cell infected with a single phage produced 20 – 100 progeny some 20 minutes following infection.  The fact that phages adsorbed irreversibly to their hosts as part of the infection process was shown by AP Krueger and M Schlesinger in 1930 – 1931.  Schlesinger later showed between 1934 and 1936 that the bacteriophage he worked with consisted of approximately equal amounts of protein and DNA, the first proof that viruses might be nucleoprotein in nature.

However, it took until 1939 for the former physicist Max Delbrück, working with the biologist Emory Ellis at Caltech, to elucidate the growth cycle of a sewage-isolated Escherichia coli bacteriophage in a now-classic paper simply entitled “The Growth of Bacteriophage”.  This used the simple technique of counting plaques in a bacterial lawn in a Petri dish, following infection of a standard bacterial inoculum with a dilution series of a phage preparation.

Their principal finding was that viruses multiply inside cells in one step, and not by division and exponential growth like cells.. This was determined using the so-called “one-step growth curve”, which allowed the accurate determination of the titres of viruses released from bacteria that had been synchronously infected.  This allowed calculation of not only the time of multiplication of the virus, but also the “burst size” from individual bacteria, or the number of viruses produced in one round of multiplication.  This was a fundamental discovery, and allowed the rapid progression of the field of bacterial and phage genetics

One important facet of this work was that it showed that infection could be caused by single phages: the power of the plaque assay meant that even dilutions of phage preparations that contained only a single particle could produce a detectable plaque.

The Phage Group was started in the 1940s after Delbrück and Salvador Luria – also famous for inventing the Luria broth used to this day to grow bacteria -  met at a conference.  They soon began to collaborate, and in 1943 published the famous Luria–Delbrück experiment or Fluctuation Test: this showed that resistance to phage infection in bacteria could arise spontaneously and without selection pressure.

Also in 1943, they added Alfred Hershey to the group.  An important early result of their joint work was the proof that co-infection of one bacterium with two different bacteriophages could lead to genetic recombination, or mixing of the phage genomes.  Hershey and his assistant Martha Chase subsequently went on in 1952 to perform the legendary Hershey-Chase experiment in order to prove whether or not DNA was the genetic material of the phage: this purportedly used a new high speed Waring blender Hershey had purchased for his wife, but which never made it to her.  They grew up preparations of the E coli bacteriophage T2 separately in the presence of the radioisotopes 35S and 32P, to label the protein and nucleic acid components of the phage respectively.  Their most exciting result was achieved by allowing adsorption of phages to bacteria in liquid suspension for different times, then shearing off adsorbed phage particles from the bacteria using the blender.  Pelleting the bacteria by centrifugation and assaying radioactivity allowed them to determine that over 75% of the 35S – incorporated into cysteine and methionine amino acids – remained in the liquid, or outside the bacteria, whereas over 75% of the 32P – incorporated into the phage DNA - was found inside the bacteria.  Subsequent production of phage from the bacteria showed that DNA was probably the genetic material, and that protein was not involved in phage heredity.

Aside from their ground-breaking discoveries, the main influence of the Phage Group was felt via their establishment of the yearly summer phage course at Cold Spring Harbor Laboratory. From 1945 through to the 1960s, Delbrück and colleagues taught the fundamentals of bacteriophage biology and experimentation to generations of biologists, which helped to instill a culture of rigorous mathematical and analytical techniques in attendees – many of whom went on to help establish the emerging field of molecular biology.

Indeed, not only did Delbrück, Luria and Hershey receive the 1969 Nobel Prize for Physiology or Medicine for their work on bacteriophages, but Luria’s first graduate student James Watson was also awarded the prize in 1962 for his work with Francis Crick on elucidating the structure of DNA.  It is a not particularly well known fact that Watson honed his analytical skills for 3-D reconstructions from X-ray data of DNA with data from TMV, which he helped to show had helical virions.

Animal cell culture

Possibly the most important development for the study of animal viruses since their discovery was the growing of poliovirus in cell culture: this was reported in 1949 by John Enders, Thomas Weller and Frederick Robbins from the USA, and was rewarded with a joint Nobel Prize to them in 1954.  They did this around the same time as David Bodian and Isabel Morgan identified three distinct types of poliovirus.

While both bacterial and plant viruses could be both grown and assayed in “culture” – bacterial cells for phages, and plants for viruses like TMV – it was very difficult to grow and work with animal viruses, and especially to assay them, or measure their concentration.  While the pock assay done on egg membranes for influenza virus was very useful, it was not applicable to many viruses.  Indeed, people working with animal and human viruses were envious of the advantages enjoyed by their colleagues working with bacteriophages and plant viruses, because their assay systems were far more generally useful, even if local lesion assays on leaves for plant virus were limited compared to the precision obtainable for bacteriophages using pure cultures of bacterial cells on Petri dishes.  Titration or assay of poliovirus, for example, required the injection of virus preparations into the brains of monkeys, or later, in the case of the Lansing or Type II poliovirus strain, into brains of mice.

The technological advances that led to the breakthrough were incremental, and in fact had occurred over a period of over sixty years: Wilhelm Roux is credited with creating the first “tissue culture” with animal cells, by maintaining extracts of chicken embryos in warmed saline in 1885.  Other early workers had used minced-up chick embryos as far back as the early 1900s; roller-tube cultures had been in use for some time for studying viruses; a number of human and other tissues had been used to culture viruses.  Part of the development was, however, the increased ease of making the necessary reagents, such as ultrafiltered bovine serum, and a greater understanding of the requirements of cells for successful growth in culture.  Another major enabling factor was the post-Second World War availability of antibiotics, which meant contaminating microorganisms could be killed in culture – which had been impossible previously.

Enders, Weller and Robbins started with a suspended cell culture of human embryo skin and muscle tissue – a technique first described in 1928 – with the idea of studying varicella zoster herpesvirus.  However, in a case of chance favouring the prepared mind(s), the proximity of these tissue cultures and the Lansing strain of poliovirus in the same lab led to them using this instead, as part of an effort to determine whether all polioviruses exclusively multiplied in human nervous tissue.

Their cultures were started by inoculation with a suspension of infected mouse brains, and re-inoculation of mice with tissue culture fluids demonstrated that the virus was multiplying.  Injection of fluid into monkey brains after three passages of tissue culture resulted in typical symptoms of paralysis.  Later, Types I and III poliovirus were also successfully cultured – and suspended cell cultures of intestine, liver, kidney, adrenals, brain, heart, spleen, lung and brain derived from human embryos were also found to support growth of various polioviruses.

Adaptation of the culture technique to roller-tubes allowed higher yields of virus – and the possibility of direct observation of the effects of virus multiplication on large sheets of cells, rather than in clumps and pieces of tissue from suspension cultures.  These effects were termed “cytopathogenic” (now generally cytopathic) for the direct damage and morphological changes to cells that could be seen and measured, and roller-tubes made it far easier and quicker to do this by simple staining of cultures with various reagents such as haemotoxylin and eosin.

The technique of looking at cells for cytopathic effects (also abbreviated as CPE) quickly found application in assays of infectivity – and therefore of concentration – of poliovirus preparations.  It was also possible to do neutralisation assays with immune human sera.  There was also the observation that passaging the Lansing strain through cell suspensions reduced its virulence in mice, and similar passage of Type I poliovirus significantly reduced virulence in rhesus macaques.  These developments together were part of the advances that led to the development of live poliovirus vaccines soon afterwards.

These observations also quickly found application with a wide variety of other human and animal viruses, which triggered an explosion in these fields that led to them rapidly overtaking plant and bacterial virology in terms of understanding how the viruses replicated, and developing assays and vaccines for them.  Indeed, the poliovirus work was rapidly followed in the same lab by the isolation of herpes zoster and herpes simplex viruses; the agent of measles was characterised by Thomas Peebles and Enders via tissue culture by 1954; adenoviruses were discovered in 1953 by Wallace Rowe and Robert Huebner and shown to be associated with acute respiratory disease soon afterwards, by Maurice Hilleman and others.

Click here for Part 1: Introduction

and here for Part 2: Egg Culture and EM

Copyright Edward P Rybicki and Russell Kightley, February 2015, except where otherwise noted.

Ebola virus mutating, scientists say

29 January, 2015

Scientists at the Institut Pasteur in France who are tracking the Ebola outbreak in Guinea say the virus has mutated.

Source: www.bbc.com

I would be surprised it there weren’t evidence by now of adaptation to humans: never in any previous outbreak of EHD [Ebola haemorrhagic disease] has the person-person chain of transmission been sustained for so long, meaning never before has there been the opportunity for human-specific adaptations to become established.

The article points out that on consequence of mutation may be that the virus becomes less virulent, leading to a greater incidence of asymptomatic infection – of which there is already evidence from previous outbreaks, and which has been implicated in the lessening incidence of transmission because of increasing herd immunity.

However, this same property might lead to increased transmission to the non-exposed, because of a lack of signs that contacts with the infected person(s) should be avoided – and for a disease as lethal as EHD, even a reduced mortality rate still means you should avoid it at all costs.

The idea of developing a modified live measles virus vaccine as an Ebola virus vaccine vector, which is what the Institut Pasteur is apparently doing, seems to be a very good one.  Measles is still a major potential problem in that part of the world, necessitating regular infant immunisations, and coupling anti-measles with an anti-Ebola vaccine in those countries is probably very good use of both a proven vaccine and existing EPI infrastructure.

 

See on Scoop.itVirology News

More Surprises in the Development of an HIV Vaccine

14 November, 2014

More Surprises in the Development of an HIV Vaccine

In the current issue of Frontiers in Immunology, Jean-Marie Andrieu and collaborators, report results from non-human primate experiments designed to explore a new vaccine concept aimed at inducing tolerance to the simian immunodeficiency virus (SIV) (1). This approach, which is significantly different from other vaccine concepts tested to date, resulted in a surprisingly high level of protection. If the results are confirmed and extended to the human immunodeficiency virus (HIV), this approach may represent a game changing strategy, which should be welcomed by a field that has been marred by mostly disappointing results.

 

HIV Graphic from Russell Kightley Media

 

Source: journal.frontiersin.org

This is a commentary by two well-respected friends of mine on a very surprising result published by the Andrieu group recently, which seems to have been ignored by the mainstream HIV vaccine world.

This is not surprising, in that Andrieu is an outsider in this field – he is a cancer researcher – but is typical of the disappointing tendency in science to ignore contributions from outside the various "Golden Circles" that exist for various specialties.

Something that should elicit interest, though, is that this group has shown that a previously obscure 

"…population of non-cytolytic MHCIb/E-restricted CD8+ T regulatory cells [that] suppressed the activation of SIV positive CD4+ T-lymphocytes".

This is interesting because Louis Picker’s groups’ recent findings, announced at the recent HIVR4P conference in Cape Town, highlighted the involvement of MHC-E proteins in what amounted to a cure of SIV infection in macaques by a modified Rhesus cytomegalovirus (RhCMV) HIV vaccine vector (see here: http://www.iavireport.org/Blog/archive/2013/09/13/cmv-based-vaccine-can-clear-siv-infection-in-macaques.aspx). 

I tweeted at the time:

"Universal MHC-E-restricted CD8+ T cells – break all the rules for epitope recognition"

Could this be a link between the two mechanisms – both from way outside the orthodoxy, I will point out?

It will be interesting to see.

See on Scoop.itVirology News

Ethical dilemma for Ebola drug trials

13 November, 2014

Public-health officials split on use of control groups in tests of experimental treatments.

With clinical trials of experimental Ebola treatments set to begin in December, public-health officials face a major ethical quandary: should some participants be placed in a control group that receives only standard symptomatic treatment, despite a mortality rate of around 70% for Ebola in West Africa?

Two groups planning trials in Guinea and Liberia are diverging on this point, and key decisions for both are likely to come this week. US researchers meet on 11 November at the National Institutes of Health (NIH) in Bethesda, Maryland, to discuss US-government sponsored trials. A separate group is gathering at the World Health Organization (WHO) in Geneva, Switzerland, on 11 and 12 November to confer on both the US effort and trials organized by the WHO with help from African and European researchers and funded by the Wellcome Trust and the European Union.

Source: www.nature.com

I have to say – faced with a deadly disease, I think it is UNethical to have control / placebo arms of any trial.

Seriously: what about comparing ZMapp and immune serum, for example, with historical records of previous standard of care outcomes rather than directly?

I know if I were an Ebola patient, and I saw someone else getting the experimental therapy and I didn’t, that I would have a few things to say.

It’s not as if these therapies have not been tested in primates, after all – in fact, both the ChAd3 and MVA-based vaccines and ZMapp have been thoroughly tested in macaques, as have the other therapeutics, with no adverse events there.

I say if people say clearly that they want an experimental intervention, that they should get one: after all, the first use of immune serum was not done in a clinical trial, but rather as a last-ditch let’s-see-if-this-works intervention – yet its use does not seem controversial?

See on Scoop.itVirology News

Virology Africa 2015: consider yourselves notified!

7 November, 2014

Dear ViroBlogy and Virology News followers:

Anna-Lise Williamson and I plan to have another in our irregular series of “Virology Africa” conferences in November-December 2015, in Cape Town.

As previously, the conference will run over 3 days or so, possibly with associated workshops, and while the venue is not decided, we would like to base it at least partially in the Victoria & Alfred Waterfront.

We also intend to cover the whole spectrum of virology, from human through animal to plant; clinical aspects and biotechnology.

We intend to make it as cheap as possible so that students can come. We will also not be inviting a slate of international speakers, as we have found that we always get quite an impressive slate without having to fund them fully.

It is also the intention to have a Plant Molecular Farming workshop – concentrating on plant-made vaccines – concurrently with the conference, in order to leverage existing bilateral travel grants with international partners. If anyone else has such grants that could be similarly leveraged, it would be greatly appreciated.

See you in Cape Town in 2015!

Ed + Anna-Lise

ZMapp in an HIV context

30 October, 2014

It was truly a pleasure to run into Kevin Whaley of Mapp BioPharmaceutical today, here at the HIVR4P inernational conferrence in Cape Town – so I made him come and have coffee with me and Anna-Lise, so we could chat about molecular farming.
Of course, it is the ZMapp plant-made therapeutic antibody that has set the molecular farming world alight, that was the main topic. Apparently Mapp is looking at a January 2015 date for a clinical trial in the affected West African countries, alongside the adenovirus and RSV-vectored vaccines. The plants for the production of the thousands of doses that will be needed – and recall, that’s a couple of grams per dose at 50 mg/kg – are already growing at Kentucky Bioprocessing in Louisville, so one imagines that a pile of work will be coming their way in the near future.
It’s also sobering to realise that even though plants ARE a more scalable and POTENTIALLY cheaper means of production of biologics, that therapeutic antibody production in particular, MAY be better suited right now to conventional technologies, such as CHO cell or even fungal production.
This is because large quantities of MAbs will be needed, and there is established capacity for production of hundreds of thousands of litres of cell culture right now, and yields and production costs have been driven right down to US$10 / gram for MAbs already, according to Kevin.
This partly answers a question I had during the HIVR4P sessions: if one is to use 20-50 mg/kg dosages for anti-HIV neutralising MAbs such as VRC01, how would it be remotely possible to make the amounts required for use in a developing country setting, where the patient can almost definitely NOT pay?
I still think there is a role for plants – but maybe this will be in the area of prophylactic use of MAbs, where much lower doses may be effective because there is not nearly as much virus to neutralise or inactivate.
And of course, Mapp is involved here too, with plant-made VRC01 in particular being incorporated into microbicides.
A great bunch of people, with really noble aims.

Rabies Vaccine Protects Nonhuman Primates against Deadly Ebola Virus

26 October, 2014

The research team is pursuing the inactivated rabies/Ebola vaccine for use in humans. The live vaccine is being developed for use in protecting wildlife at risk of Ebola virus infection in Africa, which could also serve to prevent transmission into the human population.

Source: www.niaid.nih.gov

I missed this one at the time – and it is an interesting piece of news.  Basically, the research team cloned the Ebola envelope glycoprotein GP1 into the extant rabies virus vaccine strain genome, and tested a live version, a replication-deficient version, and a killed whole virion version in macaques.

Their results are interesting enough – 100% protection against challenge for live, 50% for the other two – that they plan to follow up to see whether or not additional doses could improve protection in the two non-replicating versions, and to make a “multivalent filovirus vaccine”.

This can only be welcome news against the backdrop of the still-ongoing epidemic in West Africa – where two other vaccines (recombinant vesicular stomatitis and chimpanzee adenovirus) are probably going to be trialled next year. The rabies version at least is based on a very well characterised vaccine that already protects against an extremely deadly disease – it remains to be seen how well the other two do.

I forgot to mention that I found reference to this article on “The Zombie Research Society”‘s blog site: http://zombieresearchsociety.com/archives/25562. A very apt place if one considers the parallels that are already being drawn between Ebola and a “zombie virus”.

And because I like zombies B-)

See on Scoop.itVirology News

Norway to get world’s last dose of ZMapp – update

8 October, 2014

The Norwegian woman, infected by the Ebola in Sierra Leone and currently receiving treatment in Oslo, will get the last dose of the virus treatment medicine ZMapp

Source: m.thelocal.no

…and yet again, the emphasis is on how slow it is to make it – when the whole point of biofarming and transient expression is that it is supposed to be QUICK to make things, and easy to scale up production!!

What is the problem here?  KBP has facilities – or says it does – for large-scale production of proteins via transient expression in N benthamiana via rTMV or even BeYDV-based vectors. SO why has it been so difficult to make more ZMapp??

Why, in fact, are we told via other reports that the US government is considering getting Caliber to make it, or even to make the cocktail in CHO cells, because of capacity, when KBP has the equipment?

It can’t be supply of plants, surely: if they’d planted out a big greenhouse or two of N benth the moment ZMapp hit the news, they’d have enough to make many grams of ZMapp right now – given that it takes just a few days of incubation post-infiltraiton to make the protein.

Surely it’s not a protein purification thing – because THAT’S pretty quick too, once the plants have been mushed.

So what IS the bottleneck? cGMP requirement? Lack of certified protocols / equipment? Can someone tell us??  Otherwise, a posterchild for biofarming will end up being made by good old stainless steel cell culture technology, and our favourite way of doing things will have been found to be wanting.

NOTE ADDED 10th October:

Never let it be said I was unwilling to get schooled by a former colleague…Kenneth Palmer just told me what the problem is:

“You may not be aware that the human dose of Zmapp is 12 grams per patient, 3 infusions of 4 grams each.  Check the dose in recent Nature paper. If yield of one antibody is 100 mg per kg and you have to produce three antibodies for Zmapp… If you do the arithmetic you will see why the process is “slow””.

So…. Doing just that, you end up with 30 kg N benthamiana per gm of ZMapp as a best-case yield – meaning 360 kg PER PATIENT.

That’s a LOT of N benth – and tooling up for that sort of plant production takes time. Thanks, Kenneth!

I would be VERY interested in a cost breakdown of ZMapp vs CHO cell-produced MAbs – because producing at that sort of scale MUST be prohibitively expensive in stainless steel?

 

See on Scoop.itPlant Molecular Farming

Effect of Formaldehyde Inactivation on Poliovirus

23 September, 2014

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

 

Source: jvi.asm.org

People have been treating poliovirus with formaldehyde for over 60 years – and it’s only NOW that someone thought to study in detail what happens!

I love this stuff: analytical centrifugation could have been done any time in the last fifty years (and has been, in determining structural transitions) but the newer techniques such as flow cytometry and RT-PCR could not. Analytically determining now what was empirically observed to work when polio vaccines were first made, is a historically important vindication of pioneering work that has almost made the viruses go away.

Simple and obvious findings, essentially – it is obvious that methylene bridging between amino acids would affect structural transitions; so too that HCHO treatment would kill viral ssRNA – but it hadn’t been DONE properly previously.  Great stuff!

See on Scoop.itVirology and Bioinformatics from Virology.ca


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