So: thank you, anyone who clicked in, and regular visitors. You make it worthwhile!!
The WordPress.com stats helper monkeys prepared a 2012 annual report for this blog.
Here’s an excerpt:
4,329 films were submitted to the 2012 Cannes Film Festival. This blog had 33,000 views in 2012. If each view were a film, this blog would power 8 Film Festivals
Update, June 11th, 2018
We had a High Church Anglican funeral for Keith in St Margaret’s Church in Fishhoek, Cape Town, , with his Very Reverend son-in-law officiating. Poems were read and prayers were said – and it is the only funeral I have ever been to where poxviruses made it into the remembrances, and one of the featured photographs was of Keith signing the declaration of eradication of smallpox. We had a restrainedly joyous time remembering Keith with family, with stories never heard by most.
Anna-Lise Williamson brought back video of two tributes to Keith from the Poxvirus Conference in Taipei, when Geoff Smith and Grant McFadden remembered him. I attach a Dropbox link to the slightly enhanced version.
Update, May 31st 2018
I am sad to have to tell everyone that Professor Keith Dumbell died a few days ago, at the age of 95, of natural causes. His funeral is set for 6th June, in Cape Town. I would be happy to pass on messages from anyone who knew him.
It was wonderful, though, to have been able to celebrate his 95th birthday recently, in the same manner and with many of the same people who celebrated his 90th. Thanks to Niki Douglass – again – and old friends and family. I have video of both occasions, and of informal talks Keith gave in Medical Virology, that I will be compiling as a video tribute in the near future.
Go well, Keith!
Ed Rybicki
May 31st 2018
**********************************************************************
I have written here previously on how our extended Virology community in Cape Town was regularly getting together to listen to Keith Dumbell talk on his early days in virology – and now we are rapidly approaching his 90th birthday, and we would like to get as many people as possible who know him / know of him, to send birthday wishes and greetings.
Preferably as a sound or video file, although text is obviously also great. I will be incorporating it all into a video which we will give him – and may also put up for download, but will also send to his friends. Get recording…Mike Mackett, this means you – and Upton, and Bert Jacobs…
A technical development that was to greatly advance the study of viruses was begun in 1923, but only reached fruition by the 1930s: this was the ultracentrifuge, invented and developed first by Theodor (“The”) Svedberg in Sweden as a purely analytical tool, and later by JW Beams and EG Pickels in the USA as an analytical and preparative tool. The ultracentrifuge revolutionised first, the physical analysis of proteins in solution, and second, the purification of proteins, viruses and cell components, by allowing centrifugation at speeds high enough to allow pelleting of subcellular fractions.
Analytical centrifugation and calculation of molecular weights of particles gave some of the first firm evidence that certain proteins, and virus particles, were large, regular objects. Indeed, it came to be taken as a given that one of the fundamental properties of a virus particle was its sedimentation coefficient, measured in svedbergs (a unit of 10-13 seconds, shown as S20,W). This is also how ribosomes of pro- and eukaryotes came to be named: these are known as 70S (prokaryote) and 80S ribosomes, respectively, based on their different sedimentation rates.
In 1931, Robert Shope in the USA managed to recreate swine influenza by intranasal administration of filtered secretions from infected pigs. Moreover, he showed that the classic severe disease required co-inoculation with a bacterium – Haemophilus influenza suis – originally thought to be the only agent. He also pointed out the similarities between the swine disease and the Spanish Flu, where most patients died of secondary infections. However, he also suggested that the virus survived seasonally in a cycle involving the pig, lungworms, and the earthworm, which is now known to be completely wrong.
This notwithstanding, he found that people who had survived infection during the 1918 pandemic had antibodies protecting them against the swine flu virus, while people born after 1920 did not, which showed that the 1918 human and swine flu viruses were very similar if not identical. This was a very relevant discovery for what happened much later, in the 2009 influenza pandemic, when the same virus apparently came back into the human population from pigs after circulating in them continuously since 1918.
Shope went on in 1932 to discover, with Peyton Rous, what was first called the Shope papillomavirus and later Cottontail rabbit papillomavirus: this causes benign cancers in the form of long hornlike growths on the head and face of the animal. This may explain the sightings in the US Southwest of the near-mythical “jackalope”.
Influenza viruses in pigs
Patrick Laidlaw and William Dunkin, working in the UK at the National Institute for Medical Research (NIMR), had by 1929 successfully characterised the agent of canine distemper – a relative of measles, mumps and distemper morbilliviruses – as a virus, proved it infected dogs and ferrets, and in 1931 got a vaccine into production that protected dogs. This was made from chemically inactivated filtered tissue extract from infected animals. Their work built on and completely eclipsed earlier findings, such as those of Henri Carré in France in 1905, who first claimed to have shown it was a filterable agent, and Vittorio Puntoni, who first made a vaccine in Italy from virus-infected brain tissue inactivated with formalin in 1923.
Continuing from Laidlaw and Dunkin’s work in the same institute, Christopher Andrewes, Laidlaw and W Smith reported in 1933 that they had isolated a virus from humans infected with influenza from an epidemic then raging. They had done this by infecting ferrets with filtered extracts from infected humans – after the fortuitous observation that ferrets could apparently catch influenza from infected investigators! The “ferret model” was very valuable – see here for modern use of ferrets – as strains of influenza virus could be clinically distinguished from one another.
Influenza virus and eggs: large-scale culture
Frank Macfarlane Burnet from Australia visited the NIMR in the early 1930s, and learned a number of techniques he used to great effect later on. Principal among these was the technique of embryonated egg culture of viruses – which he took back to Melbourne, and applied to the infectious laryngotracheitis virus of chickens in 1936. This is a herpesvirus, first cultivated by JR Beach in the USA in 1932: Burnet used it to demonstrate that it was possible to do “pock assays” on chorioallantoic membranes that were very similar to the plaque assays done for bacteriophages, with which he was also very familiar. Also in 1936, Burnet started a series of experiments on culturing human influenza virus in eggs: he quickly showed that it was possible to do pock assays for influenza virus, and that
“It can probably be claimed that, excluding the bacteriophages, egg passage influenza virus can be titrated with greater accuracy than any other virus.”
Max Theiler and colleagues in the USA took advantage of the new method of egg culture to adapt the French strain of yellow fever virus (YFV) he had grown in mouse brains to being grown in chick embryos, and showed that he could attenuate the already weakened strain even further – but it remained “neurovirulent”, as it caused encephalitis or brain inflammation in monkeys. He then adapted the first YFV characterised – the Asibi strain, from Ghana in 1927 – to being grown in minced chicken embryos lacking a spinal cord and brain, and showed in 1937 that after more than 89 passages, the virus was no longer “neurotrophic”, and did not cause encephalitis. The new 17D strain of YFV was successfully tested in clinical trials in Brazil in 1938 under the auspices of the Rockefeller Foundation, which has supported YFV work since the 1920s. The strain remains in use today, and is still made in eggs.
Given that the nature of viruses had prompted people to think of them as “chemical matter”, researchers had attempted from early days to isolate, purify and characterise the infectious agents. An early achievement was the purification of a poxvirus in 1922 by FO MacCallum and EH Oppenheimer.
Much early work was done with bacteriophages and plant viruses, as these were far easier to purify or extract at the concentrations required for analysis, than animal or especially human viruses.
CG Vinson and AM Petre, working with the infectious agent causing mosaic disease in tobacco – tobacco mosaic virus, or TMV – showed in 1931 that they could precipitate the virus from suspension as if it were an enzyme, and that infectivity of the precipitated preparation was preserved. Indeed, in their words:
“…it is probable that the virus which we have investigated reacted as a chemical substance”.
Viruses in Crystal
An important set of discoveries started in 1935, when Wendell Stanley in the USA published the first proof that TMV could be crystallised, at the time the most stringent way of purifying molecules. He also reported that the “protein crystals” were contaminated with small amounts of phosphorus. An important finding too, using physical techniques including ultracentrifugation and later, electron microscopy, was that the TMV “protein” had a very high molecular weight, and was in fact composed of large, regular particles. This was a very significant discovery, as it indicated that some viruses at least really were very simple infectious agents indeed.
TMV particle: 95% protein, 5% RNA
However, his conclusion that TMV was composed only of protein was soon challenged, when Norman Pirie and Frederick Bawden working in the UK showed in 1937 that ribonucleic acid (RNA) – which consists of ribose sugar molecules linked by phosphate groups – could be isolated consistently from crystallised TMV as well as from a number of other plant viruses, which accounted for the phosphorus “contamination”. This resulted in the realisation that TMV and other plant virus particles – now known to be virions – were in fact nucleoproteins, or protein associated with nucleic acid.
Stanley received a share of the Nobel Prize in Chemistry in 1946 for his work on TMV: it is instructive to read his acceptance speech from the time to realise what the state of the science that was becoming virology was at the time. He wrote:
“Since the original discovery of this infectious, disease-producing agent, known as tobacco mosaic virus, well over three hundred different viruses capable of causing disease in man, animals and plants have been discovered. Among the virus-induced diseases of man are smallpox, yellow fever, dengue fever, poliomyelitis, certain types of encephalitis, measles, mumps, influenza, virus pneumonia and the common cold. Virus diseases of animals include hog cholera, cattle plague, foot-and-mouth disease of cattle, swamp fever of horses, equine encephalitis, rabies, fowl pox, Newcastle disease of chickens, fowl paralysis, and certain benign as well as malignant tumors of rabbits and mice. Plant virus diseases include tobacco mosaic, peach yellows, aster yellows, potato yellow dwarf, alfalfa mosaic, curly top of sugar beets, tomato spotted wilt, tomato bushy stunt, corn mosaic, cucumber mosaic, and sugar cane yellow stripe. Bacteriophages, which are agents capable of causing the lysis of bacteria, are now regarded as viruses”.
Two of the most interesting things about the article, however, are the electron micrographs of virus particles – Stanley had one of the first electron micrsoscopes available at the time – and the table of sizes of viruses, proteins and cells that had been determined by then by techniques such as ultracentrifugation and filtration: TMV was known to be rodlike, 15 x 280 nm; vaccinia was 210 x 260 nm; poliomyelitis was 25 nm; phages like T2 were known to have a head-and-tail structure.
First Electron Microscope with Resolving Power Higher than that of a Light Microscope. Ernst Ruska, Berlin 1933
Wikipedia CC BY-SA 3.0, https://www.flickr.com/photos/93452909@N00/176059674
The development of the electron microscope, in Germany in the 1930s, represented a revolution in the investigation of virus structures: while virions of viruses like variola and vaccinia could just about be seen by light microscopy – and had been, as early as 1887 by John Buist and others – most viruses were far too small to be visualised in this way.
While Ernst Ruska received a Nobel Prize in 1986 for developing the electron microscope, it was his brother Helmut who first imaged virus particles – using beams of electrons deflected off virus particles coated in heavy metal atoms. From 1938 through the early 1940s, using his “supermicroscope”, he imaged virions of poxviruses, TMV, varicella-zoster herpesvirus, and bacteriophages, and showed that they were all particulate – that is, they consisted of regular and sometimes complex particles, and were often very different from one another. He even proposed in 1943 a system of viral classification on the basis of their perceived structure.
While electron microscopy was also used medically to some extent thereafter – for example, in differentiating smallpox from chickenpox by imaging particles of variola virus and varicella-zoster virus, respectively, derived from patients’ vesicles – its use was limited by the expense and cumbersome nature of sample preparation. For example, the micrographs in Stanley’s 1946 paper were all done with samples “…prepared with gold by the shadow-casting technique”.
The use of the cumbersome technique of metal shadow-casting, and the highly inconvenient nature of electron microscopy as a routine tool all changed from 1959 onwards, when Sydney Brenner and Robert Horne published “A negative staining method for high resolution electron microscopy of viruses”. This method involves the use of viruses in liquid samples deposited on carbon-coated metal grids, and then stained with heavy-metal salts such as phosphotungstic acid (PTA) or uranyl acetate.
This simple technique revolutionised the field of electron microscopy, and within just a few years much information was acquired about the architecture of virus particles. Not only were the overall shapes of particles revealed, but also the details of the symmetrical arrangement of their components. Some beautiful examples can be seen here, at the Cold Spring Harbor site.
Depiction of the effects of using a heavy metal salt solution to negatively stain particles on a carbon film. The stain (dark) pools around the particles (light). Human rotavirus particles, stained from below (left) and by immersion (right). Images copyright LM Stannard
Click here for Part 1: Filters and Discovery
here for Part 3: Phages, Cell Culture and Polio
and here for Part 4: RNA Genomes and Modern Virology
Copyright Edward P Rybicki and Russell Kightley, February 2015, except where otherwise noted.
I have updated the blog on virus origins quite considerably – new pictures, more detail, more speculation!
Pathways on information flow for RNA viruses
Given that I am presently at the HIV Vaccine 2011 Conference here in Bangkok, I thought (belatedly) that I might blog on the proceedings, given Dorian McIlroy’s previous excellent example on CROI in recent months.
Reclining Buddha, Bangkok
Yesterday morning a Crown Princess of the Kingdom of Thailand was opening the first proper session of the oral proceedings: I was not there, as I needed breakfast after handling an email overload and didn’t feel like wearing a suit, so I missed an important performance by a Thai orchestra. Close call, that…!
We were there on Monday night, though, when a lineup of dignitaries presented in an opening plenary session. First up was Pratap Singhasivanon, the Conference chair from Thailand. He introduced for the ignorant the long history and impressive list of Thailand’s achievements in the world of HIV vaccinology and prevention. It was sobering to hear that 40% of injecting drug users and 33% of men who have sex with men (MSM) were HIV+, despite that history.
Josè Esparza, acting head of the HIV Vaccine Enterprise, came next. He was of the opinion that this is the Golden Age of HIV vaccines – an age of unprecedented successes and great promise, and that an HIV vaccine to end the pandemic is within reach. He told us that UNAIDS says that behaviour modification and testing is bringing down infection rates worldwide, which is another encouraging development. He thought that we Need increased and sustained financial support for the vaccine effort, however, including for a greater number of trials with short timelines so as to better test a wide range of possible vaccines.
Stanley Plotkin of Univ Pennsylvania is a luminary of the vaccine world, having helped as an industry insider to develop rubella and pentavalent rotavirus vaccines, among others: his job was to tell us how the success of other vaccines could inform the development of HIV vaccines. He said he had thought of saying “There are no lessons!” and sitting back down, but on reflection he had better not.
What he did share was that he thought that antibody response is king, but that it must be functional. A second lesson was that Ab at mucosal surfaces can give sterilising immunity. As an example, injected inactivated poliovirus vaccine (IPV) does not prevent shedding virus in gut while the live oral OPV does as it is much better at eliciting mucosal imm – but interestingly, at the pharynx both work. A lesson from human papillomavirus vaccination was that while low Ab concentration did not prevent binding of the virus to the first receptor, it did prevent binding to the second – so entry of the virus into susceptible cells was prevented. Another lesson from polio was that high challenge dose can overwhelm immunity, and that IPV was a lot less good at protecting against high challenge doses. It was important that one could still get protection from disease in the presence of infection: for example, Rotateq rotavirus vaccine prevents disease very well, but vaccinees often get infected.
Ab- and cell-mediated immunity can also synergise: with smallpox it was found that both B and T cells are necessary for survival from vaccination, but on secondary exposure to infection in vaccinees, only Ab was necessary to prevent infection.
An important lesson for HIV was that several diseases required vaccine boosters in later life to maintain protection: with diphtheria, immunity in vaccinees declined dramatically while in those naturally infected it did not. Pertussis too needed boosters in children, and several more in ones lifetime to maintain functional immunity.
It was also important to revaccinate where pathogens changed significantly through time and with place – eg rotavirus was much more varied in Africa than elsewhere, as is HIV-1, and strains changed with time in one place, as do HIV and influenza viruses.
An important societal lesson was that vaccination of adolescents and high risk groups may not be accepted: Eg HPV vaccine coverage in the USA in adolescents was only 27% for all 3 doses, despite a very intensive campaign promoting the vaccine. HBV vaccination in high risk adults was also only at 50% and incidence only decreased when adolescents were vaccinated.
Herd immunity was also essential for public health success: eg pneumococcal vaccination of children protected old people indirectly as they were no longer exposed to the live pathogen in familial or sociatal settings.
His conclusions for HIV vaccines were that:
Sanjay Gurunathan of Sanofi Pasteur gave an industry view of how to move forward from the partially successful Thai RV144 vaccine trial, also reported here in Viroblogy. He observed that the traditional vaccine development model has large volume purchase in developed countries as the main driver, with industry doing R and D and clinical trials and the public sector doing purchase and delivery, with a trickle down to developing countries over time. He thought that HIV needs novel technology, and needs parallel development for 1st and developing worlds – with partnerships being of paramount importance together with guaranteed volume and price to some extent.
He noted that we must realise that for HIV vaccines failure will preceed success in an iterative process, that successes may be population-specific, that we may need multicomponent regimens, that we need to address developing country infrastructure – and that no company, NGO or even country can do it alone.
In this vein, he described a new partnership which was extending RV144 – this was P5, or the Poxvirus Protein Public Private Partnership, of the US NIAID, Gates Foundation, the HIV vaccine Trials Network, the US Military, Sanofi Pasteur and Novartis. This had in mind a broad poxvirus based protein boost regimen to further exploit the surprising success of the regimen in RV 144.
An important result from RV144 was that it was most efficacious at 12 months (60% efficacy) but that protection had dropped >30% by two years, indicating that boosting may significantly and positively impact level and durability of protection.
P5 want to increase efficacy to at least 50%, which would give a big impact for regional epidemics. There is historical precedent for this with cholera and meningococcal vaccines, neither of which is very good but which do impact public health. Their strategy will use a common regimen of poxvirus prime and a recombinant HIV gp120 boost, and will test MSM in Thailand and heterosexuals in South Africa. They planned to use MF59 or similar adjuvant to increase immune responses, unlike the earlier trial. Another new development was that they planned parallel development and clinical tracks, with a research arm in S Africa on NYVAC vaccinia plus protein and adjuvant and a DNA-poxvirus-protein combination.
An interesting evening – with promises of a major announcement to come the following day….
News from the University of Cape Town site:
“UCT is taking part in the Phase IIb proof-of-concept efficacy trial of a candidate tuberculosis vaccine, a study that will involve people living with the human immunodeficiency virus (HIV).
Researchers from the Institute of Infectious Disease and Molecular Medicine will screen and test patients living in Khayelitsha, using the vaccine known as MVA85A. The patients are HIV positive but have not been infected with TB.
This is the first proof-of-concept efficacy trial in people infected with HIV using MVA85A, which is being developed by the Oxford-Emergent Tuberculosis Consortium (OETC), a joint venture between the University of Oxford and Emergent BioSolutions, and Aeras, a non-profit partnership focusing on TB vaccine regimens.
The MVA85A vaccine candidate is intended to boost the response of immune-essential T-cells already stimulated by the Bacille Calmette-Guerin (BCG) vaccine, also used against tuberculosis.”
So – fantastic, and it involves the alma mater, but what does it have to do with viruses?? Note the throwaway “…using MVA85A…”: while this could be an adjuvant, or some kind of carrier, it is in fact a live virus. Modified Vaccinia Ankara, in fact, meaning it is a variant of the tried-and-true smallpox virus vaccines that have been with us since Edward Jenner did his thing on the 14th of May, 1796. Poxviruses, and especially vaccinia and fowlpox viruses, can also be genetically engineered to express foreign proteins, because they have large genomes and can tolerate even quite large insertions without it affecting the virus much. There is a useful recent paper on the subject in PLoS One; inevitably, Wikipedia has an article on it too. Not a very good one, however!
It does have this, though:
“Modified Vaccinia Ankara (MVA) virus, is a highly attenuated strain of vaccinia virus that was developed towards the end of the campaign for the eradication of smallpox by Professor Anton Mayr in Germany. Produced by hundreds of passages of vaccinia virus in chicken cells, MVA has lost about 10% of the vaccinia genome and with it the ability to replicate efficiently in primate cells”.
So, two important features:
It does, however, undergo a significant portion of the life cycle in mammalian cells – only virion maturation does not occur. This means that genes inserted into the MVA virus genome with appropriate poxvirus promoters may be expressed in cells containing virus particles even if the virus does not multiply. The other Wikipedia page mentioning it – the Vaccinia page – has this:
“Modified vaccinia Ankara: a highly attenuated (not virulent) strain created by passaging vaccinia virus several hundred times in chicken embryo fibroblasts. Unlike some other vaccinia strains it does not make immunodeficient mice sick and therefore may be safer to use in humans who have weaker immune systems due to being very young, very old, having HIV/AIDS, etc.”
And THAT’S why MVA as a TB vaccine vector in what amounts to a high-risk environment for HIV infection in South Africa: because the vaccine won’t cause complications in immune-suppressed individuals.
As I have previously discussed here, MVA has also been used as a vector for a component of the South African HIV-1 vaccine developed at UCT that is currently in Phase I clinical trial in SA and in the USA: there the MVA was engineered to express both a gp150 Env and a polygenic fusion protein GRTTN (Gag-RT-Tat-Nef), and was the boost component to a dual-component DNA vaccine expressing both singly.
It is encouraging that technology that has been touted for many years is finally seeing the mainstream: a large clinical trial combining immunogenicity with efficacy. Malaria antigens are also being delivered by MVA in clinical trials; HIV Env antigens were delivered using avian poxviruses in the only HIV vaccine efficacy trial that showed any positive effects at all – so the promise is finally being fulfilled.
A sword turned into a ploughshare. We need to see more of them!
While visiting the Australian National University in Canberra recently, I noticed in the lobby outside their Club dining room, a most interesting tapestry. Interesting, because it looked like a colourised electron micrograph (it depicts myxomavirus), and because it had a plaque beneath it commemorating their own Professor Frank Fenner. I was familiar with him because he authored an extremely useful book – Medical Virology – which I had used for educating myself and for teaching; I was also aware that he was an extremely eminent poxvirologist who had been active in the field for decades – and was still working despite having retired in 1979.
Variola virus, the agent of smallpox. Image courtesy Russell Kightley Media.
And then today I heard that he had died this week, at the age of 95.
Virology is still a young field, as I discover while trying to research its history for my sabbatical project: the concept dates only from 1898; only a couple of generations of scientists have been active in this field since it started – and Frank Fenner probably overlapped with nearly all of them. He was born in 1914, which meant he was in at the morning of virology as we know it, while many of the first practitioners were still around – and he stayed active until very recently, when the science had changed almost out of all recognition. He will be missed.
I am sharing this message that was sent out by the Director of the John Curtin School of Medical Research at ANU, where Fenner worked as an Emeritus Professor, as it is probably the best short account of his life.
“It is with great sadness that I communicate to you the passing of Professor Frank Fenner.
Frank John Fenner AC, CMG, MBE, FRS, FAA (born 21 December 1914, died 22 November 2010) was an Australian scientist with a distinguished career in the field of virology. His two greatest achievements are cited as overseeing the eradication of smallpox during his term as Chairman of the Global Commission for the Certification of Smallpox Eradication, and the control of Australia’s rabbit plague through the introduction of myxoma virus.
Professor Fenner was Director of the John Curtin School from 1967 to 1973. During this time he was also Chairman of the Global Commission for the Certification of Smallpox Eradication. In 1973 Professor Fenner was appointed to set up the new Centre for Resource and Environmental Studies at the Australian National University (ANU). He held the position of Director until 1979.
Professor Fenner has been elected a fellow of numerous faculties and academies, including Foundation Fellow of the Australian Academy of Science (1954), Fellow of the Royal Society (1958), and Foreign Associate of the United States National Academy of Sciences (1977). During his career Professor Fenner received many awards. Among these are the Britannica Australia Award for Medicine (1967), the Australia and New Zealand Association for the Advancement of Science Medal (1980), the World Health Organization Medal (1988), the Japan Prize (1988), the Senior Australian Achiever of the Year (1999), the Albert Einstein World Award for Science (2000), and the Prime Minister’s Science Prize (2002).
A man of decisive scientific action and strong opinions, Professor Fenner’s last interview with The Australian is extremely thought provoking and can be found here:
A summary of Frank’s remarkable career can be found here:
http://jcsmr.anuedu.au/about/Fenner/index.phphttp://jcsmr.anu.edu.au/about/Fenner/index.php
The last public recognition of Professor Frank Fenner’s accomplishments occurred here at JCSMR during the First International Meeting on Translational Medicine earlier this month: On 1 November 2010 Professor Fenner received a standing ovation by world leaders in academic medicine during the opening of the Conference and on 2 November 2010 he was recognized by the Conference as he and Sir Gus Nossal stood by their portraits, which hang side by side at the National Portrait Gallery. A picture of Professor Fenner at JCSMR taken on 1 November 2010 next to Gus Nossal is attached.
Further notices will be sent with information regarding Professor Fenner as plans to honor his accomplishments evolve.
With best regards,
Julio
Professor Julio Licinio
Director John Curtin School of Medical Research
The Australian National University
Canberra, ACT 2601, Australia ”
Thanks to Bertram Jacobs of ASU for sharing this with me.
Ed Rybicki
Cells stimulated by HIV vaccines Copyright Russell Kightley Media
It has taken a while for me to get to this, because I have been waiting for the fallout / comment storm to settle a bit, so that I could get a good clear objective view.
And that is…that the recent Thai trial showed hints of promise, but was largely a failure. At least it did no harm…!
First things first: Nature News’ Elie Dolgin had this to say on 24th September:
Vaccine protects against HIV virus [!!! sic – I had something to say about this, see Comments]
The largest HIV vaccine trial to date has shown moderate success at preventing infection by the virus.
The experimental vaccine — a combination of two older shots that failed to work on their own — reduced the risk of someone contracting HIV by nearly a third. Scientists, however, are still scratching their heads as to how the double-shot approach blocks the virus….
The US$119 million study involved more than 16,000 HIV-negative men and women from Thailand aged 18–30. The trial was launched in October 2003, conducted by the Thai health ministry and sponsored by the US Army Surgeon General. It tested a two-shot infection-fighting strategy using drugs made by Sanofi-Pasteur of Lyon, France, and VaxGen of Brisbane, Australia. Over the course of 24 weeks, participants received four doses of a ‘primer’ vaccine — a disabled bird virus [canarypox – Ed] containing synthetic versions of three HIV genes [ALVAC, subtype B env, gag and pro – Ed] — and two doses of a ‘booster’, which consisted of a protein called gp120 [AIDSVAX subtypes B/E – Ed], a major component of HIV’s outer coat. [see here for link describing the components]. Clinicians tested for HIV infection every 6 months for 3 years….
Many HIV vaccine experts had previously criticized the approach as a waste of time because each of the vaccine components had a poor track record. The primer, called ALVAC, conferred little to no immune protection in multiple early-phase clinical trials, and the booster, called AIDSVAX, had flopped twice in high-profile, large-scale trials.
And here’s a thing: a high profile crew of scientists had, in 2004, written an open letter to Science magazine, stating in no uncertain terms that they thought the trial ought to be stopped. In their words:
“Concerns are expressed by a group of AIDS researchers about the U.S. government’s plans to conduct a phase III trial of a combination HIV-1 vaccine in Thailand despite the cancellation of a trial of a very similar combination vaccine in the U.S.A. last year. One of the vaccine components, recombinant monomeric gp120, has already been shown to be ineffective in phase III trials in Thailand and the United States; the other component, a recombinant canarypox vector, is also poorly immunogenic. The scientific rationale that has been offered for the new trial in Thailand is considered by the authors to be weak.”
And now we have Dan Barouch – not a signatory to the 2004 letter, I note – quoted by Dolgin as saying:
“I don’t think anybody knows why this worked the way it did,” says Dan Barouch, an immunologist at the Beth Israel Deaconess Medical Center in Boston, Massachusetts. “It’s the largest step forward that’s ever occurred in the HIV-vaccine field, but there’s a tremendous amount of more work that will need to be done.”
But exactly what is it that people are hailing as a breakthrough here? Dolgin again:
The two-pronged vaccine did not affect the amount of virus circulating in the blood of those who acquired HIV during the study. But it did show a protective effect — vaccinated individuals were 31% less likely to become infected. New infections occurred in 74 of the 8,198 people who received dummy shots, but only 51 of the 8,197 in the vaccine group [my emphasis – Ed], the researchers, led by Supachai Rerks-Ngarm of the Thai Ministry of Public Health’s Department of Disease Control, found.
Dorian McIlroy, a regular contributor to Viroblogy, had this to say on the 24th September in an email to me:
I just read the news story about the ALVAC/AIDSVAX trial results in Thailand. From the numbers on this press release:
http://www3.niaid.nih.gov/news/newsreleases/2009/ThaiVaxStudy.htm
The significance level is extremely slim. For example, if you go to this site
http://www.statpages.org/ctab2x2.html
and type in the numbers you will find that p=0.048 by Fisher’s exact test.
If one more person in the vaccine arm had been infected, or if one less person in the placebo arm had been infected, the difference between the groups would not have been significant. [my emphasis – Ed]
None of the experts (Wayne Koff, Frances Gotch, for example) interviewed in different news stories seems to have noticed just how borderline the “statistical significance” really is, and seem to have accepted the bottom-line 30% reduction figure.
Ah well, I just thought I had to tell someone….
Dorian
Lecturer in Microbiology and Cell Biology,
University of Nantes
Others have also picked up on this – which shows just how desperately slim the hope is. However, it does remain – although (pleasingly…B-) the pundits have been thrown into a state of confusion, as some strongly-held views have not been vindicated. Another Nature News article – from Erika Check Hayden, on October 1st – has this to say:
As the dust settles from last week’s surprising announcement that an HIV vaccine combination may protect some people from the virus, scientists are talking about what else the vaccine trial might tell them.
On 24 September, leaders of a US$119-million study of 16,000 people in Thailand reported that the combination of two shots had reduced the risk of HIV infection by one-third …. Now, the vaccine’s fate will depend on whether scientists can figure out its ‘correlate of protection’ — in other words, what caused it to partially protect some people from HIV. The key does not seem to be anything scientists had predicted, which has led to much head-scratching — and some unease.
“It’s a humbling thing, because for the first time we got a positive signal and it doesn’t jump out at us as being related to any classical parameters you would expect from a successful vaccine,” says Anthony Fauci, head of the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland, which supported the trial. “That tells us maybe we were not measuring the right thing.” [my emphasis – Ed]
Amen, brother Tony…a clearer proof of Clarke’s First Law I have yet to see.
So what ARE the things that fall out from this? First, I would suspect, is that the value of a heterologous prime-boost combination seems to have been shown, albeit weakly. Second, the use of a poxvirus vaccine in particular in combination with a protein may be a good thing to chase. I note here that the South Africa / US joint Phase I human trial currently underway with the SAAVI DNA / SAAVI MVA (=modified vaccinia virus Ankara, a poxvirus) was almost certainly considerably more immunogenic in non-humanprimates than either of the ALVAC / AIDSVAX vaccines, so the gleam of hope may soon get brighter.
Third: take heed of Arthur C Clarke before you go sticking your neck out making predictions about HIV vaccines…B-)
This was originally written as an Answer to a Question posted to Scientific American Online; however, as what they published was considerably shorter and simpler than what I wrote, I shall post the [now updated] original here.
The answer to this question is not simple, because, while viruses all share the characteristics of being obligate intracellular parasites which use host cell machinery to make their components which then self-assemble to make particles which contain their genomes, they most definitely do not have a single origin, and indeed their origins may be spread out over a considerable period of geological and evolutionary time.
Viruses infect all types of cellular organisms, from Bacteria through Archaea to Eukarya; from E. coli to mushrooms; from amoebae to human beings – and virus particles may even be the single most abundant and varied organisms on the planet, given their abundance in all the waters of all the seas of planet Earth. Given this diversity and abundance, and the propensity of viruses to swap and share successful modules between very different lineages and to pick up bits of genome from their hosts, it is very difficult to speculate sensibly on their deep origins – but I shall outline some of the probable evolutionary scenarios.
The graphic depicts a possible scenario for the evolution of viruses: “wild” genetic elements could have escaped, or even been the agents for transfer of genetic information between, both RNA-containing and DNA-containing “protocells”, to provide the precursors of retroelements and of RNA and DNA viruses. Later escapes from Bacteria, Archaea and their progeny Eukarya would complete the virus zoo.
It is generally accepted that many viruses have their origins as “escapees” from cells; rogue bits of nucleic acid that have taken the autonomy already characteristic of certain cellular genome components to a new level. Simple RNA viruses are a good example of these: their genetic structure is far too simple for them to be degenerate cells; indeed, many resemble renegade messenger RNAs in their simplicity.
RNA virus supergroups and RdRp and CP cassettes
What they have in common is a strategy which involves use of a virus-encoded RNA-dependent RNA polymerase (RdRp) or replicase to replicate RNA genomes – a process which does not occur in cells, although most eukaryotes so far investigated do have RdRp-like enzymes involved in regulation of gene expression and resistance to viruses. The surmise is that in some instances, an RdRp-encoding element could have became autonomous – or independent of DNA – by encoding its own replicase, and then acquired structural protein-encoding sequences by recombination, to become wholly autonomous and potentially infectious.
A useful example is the viruses sometimes referred to as the “Picornavirus-like” and “Sindbis virus-like” supergroups of ssRNA+ viruses, respectively. These two sets of viruses can be neatly divided into two groups according to their RdRp affinities, which determine how they replicate. However, they can also be divided according to their capsid protein affinities, which is where it is obvious that the phenomenon the late Rob Goldbach termed “cassette evolution” has occurred: some viruses that are relatively closely related in terms of RdRp and other non-structural protein sequences have completely different capsid proteins and particle morphologies, due to acquisition by the same RdRp module of different structural protein modules.
Given the very significant diversity in these sorts of viruses, it is quite possible that this has happened a number of times in the evolution of cellular organisms on this planet – and that some single-stranded RNA viruses like bacterial RNA viruses or bacteriophages and some plant viruses (like Tobacco mosaic virus, TMV) may be very ancient indeed.
However, other ssRNA viruses – such as the negative sense mononegaviruses, Order Mononegavirales, which includes the families Bornaviridae, Rhabdoviridae, Filoviridae and Paramyxoviridae, represented by Borna disease virus, rabies virus, Zaire Ebola virus, and measles and mumps viruses respectively – may be evolutionarily much younger. In this latter case, the viruses all have the same basic genome with genes in the same order and helical nucleocapsids within differently-shaped enveloped particles.
Their host ranges also indicate that they originated in insects: the ones with more than one phylum of host either infect vertebrates and insects or plants and insects, while some infect insects only, or only vertebrates – indicating an evolutionary origin in insects, and a subsequent evolutionary divergence in them and in their feeding targets.
HIV: a retrovirus
The ssRNA retroviruses – like HIV – are another good example of possible cell-derived viruses, as many of these have a very similar genetic structure to elements which appear to be integral parts of cell genomes – termed retrotransposons – and share the peculiar property of replicating their genomes via a pathway which goes from single-stranded RNA through double-stranded DNA (reverse transcription) and back again, and yet have become infectious. They can go full circle, incidentally, by permanently becoming part of the cell genome by insertion into germ-line cells – so that they are then inherited as “endogenous retroviruses“, which can be used as evolutionary markers for species divergence.
The Retroid Cycle
Indeed, there is a whole extended family of reverse-transcribing mobile genetic elements in organisms ranging from bacteria all the way through to plants, insects and vertebrates, indicating a very ancient evolutionary origin indeed – and which includes two completely different groups of double-standed DNA viruses, the vertebrate-infecting hepadnaviruses or hepatitis B virus-like group, and the plant-infecting badna- and caulimoviruses.
Metaviruses and pseudoviruses
These are two families of long terminal repeat-containing (LTR) retrotransposons, with different genetic organisations.
Members of family Pseudoviridae, also known as Ty1/copia elements, have polygenic genomes of 5-9 kb ssRNA which encode a retrovirus-like Gag-type protein, and a polyprotein with protease (PR), integrase (IN) and reverse transcriptase / RNAse H (RT) domains, in that order. While some members also encode an env-like ORF, the 30-40 nm particles that are an essential replication intermediate have no envelope or Env protein. They are not infectious. Host species include yeasts, insects, plants and algae.
Metaviruses – family Metaviridae – are also known as Ty3-gypsy elements, and have ssRNA genomes of 4-10 kb in length. They replicate via particles 45-100 nm in diameter composed of Gag-type protein, and some species have envelopes and associated Env proteins. Gene order in the genomes is Gag-PR-RT-IN-(Env), as for retroviruses. One virus – Drosophila melanogaster Gypsy virus – is infectious; however, as for pseudoviruses, most are not. The genomes have been found in all lineages of eukaryotes so far studied in sufficient detail.
Both pseudovirus and metavirus genomes are clearly related to classic retroviruses; moreover, RT sequences point to metavirus RTs being most closely related to plant DNA pararetrovirus lineage of caulimoviruses. This gives rise to the speculation that pseudoviruses and metaviruses have a common and ancient ancestor – and that two different metavirus lineages gave rise to retroviruses and caulimoviruses respectively.
All of these cellular elements and viruses have in common a “reverse transcriptase” or RNA-dependent DNA polymerase, which may in fact be an evolutionary link back to the postulated “RNA world” at the dawn of evolutionary history, when the only extant genomes were composed of RNA, and probably double-stranded RNA. Thus, a part of what could be a very primitive machinery indeed has survived into very different nucleic acid lineages, some viral and many wholly cellular in nature, from bacteria through to higher eukaryotes.
The possibility that certain non-retro RNA viruses can actually insert bits of themselves by obscure mechanisms into host cell genomes – and afford them protection against future infection – complicates the issue rather, by reversing the canonical flow of genetic material. This may have been happening over aeons of evolutionary time, and to have involved hosts and viruses as diverse as plants (integrated poty– and geminivirus sequences), honeybees (integrated Israeli bee paralysis virus) – and the recent discovery of “…integrated filovirus-like elements in the genomes of bats, rodents, shrews, tenrecs and marsupials…” which, in the case of mammals, transcribed fragments “…homologous to a fragment of the filovirus genome whose expression is known to interfere with the assembly of Ebolavirus”.
There are also obvious similarities in mode of replication between a family of elements which include bacterial plasmids, bacterial single-strand DNA viruses, and viruses of eukaryotes which include geminiviruses and nanoviruses of plants, parvoviruses of insects and vertebrates, and circoviruses and anelloviruses of vertebrates.
Geminivirus particle
These agents all share a “rolling circle” DNA replication mechanism, with replication-associated proteins and DNA sequence motifs that appear similar enough to be evolutionarily related – and again demonstrate a continuum from the cell-associated and cell-dependent plasmids through to the completely autonomous agents such as relatively simple but ancient bacterial and eukaryote viruses.
Mimivirus particle, showing basic structure
However, there are a significant number of viruses with large DNA genomes for which an origin as cell-derived subcomponents is not as obvious. In fact, one of the largest viruses yet discovered – mimivirus, with a genome size of greater than 1 million base pairs of DNA – have genomes which are larger and more complex than those of obligately parasitic bacteria such as Mycoplasma genitalium (around 0.5 million), despite their sharing the life habits of tiny viruses like canine parvovirus (0.005 million, or 5000 bases).
Mimivirus has been joined, since its discovery in 2003, by Megavirus (2011; 1.2 Mbp) and now Pandoravirus (2013; 1.9 -2.5 Mbp).
The nucleocytoplasmic large DNA viruses or NCLDVs – including pox-, irido-, asfar-, phyco-, mimi-, mega- and pandoraviruses, among others – have been grouped as the proposed Order Megavirales, and it is proposed that they evolved, and started to diverge, before the evolutionary separation of eukaryotes into their present groupings.
It is a striking fact that the largest viral DNA genomes so far characterised seem to infect primitive eukaryotes such as amoebae and simple marine algae – and they and other large DNA viruses like pox- and herpesviruses seem to be related to cellular DNA sequences only at a level close to the base of the “tree of life”.
Variola virus, the agent of smallpox. Image courtesy Russell Kightley Media.
This indicates a very ancient origin or set of origins for these viruses, which may conceivably have been as obligately parasitic cellular lifeforms which then made the final adaptation to the “virus lifestyle”.
However, their actual origin could be in an even more complex interaction with early cellular lifeforms, given that viruses may well be responsible for very significant episodes of evolutionary change in cellular life, all the way from the origin of eukaryotes through to the much more recent evolution of placental mammals. In fact, there is informed speculation as to the possibility of viruses having significantly influenced the evolution of eukaryotes as a cognate group of organisms, including the possibility that a large DNA virus may have been the first cellular nucleus.
In summary, viruses are as much a concept as a unitary entity: all viruses have in common, given their polyphyletic origins, is a base-level strategy for replicating their genomes. Otherwise, their origins are possibly as varied as their genomes, and may remain forever obscure.
I am indebted to Russell Kightley for use of his excellent virus images.
Updated 12th August 2015
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