Posts Tagged ‘vaccinia’

A Short History of the Discovery of Viruses – Part 2

7 February, 2012

The Ultracentrifuge, Eggs and Flu

The ultracentrifuge

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.

The Official Discovery of Influenza Virus

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

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.

Influenza and Ferrets: the Early Days

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.

Eggs and Flu and Yellow Fever

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.

Virus purification and the physicochemical era

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.

Seeing is Believing: the Electron Microscope

First Electron Microscope with Resolving Power Higher than that of a Light Microscope. Ernst Ruska, Berlin 1933 Wikipedia CC BY-SA 3.0,

First Electron Microscope with Resolving Power Higher than that of a Light Microscope. Ernst Ruska, Berlin 1933
Wikipedia CC BY-SA 3.0,

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

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.

Monkeypox vaccine?? We don’t need no monkeypox vaccine….

22 December, 2011

An in-press article in Vaccine that was tweeted by MicrobeTweets (well worth signing up to, BTW) has the intriguing title “Whither monkeypox vaccination?”

Now, some background to this: monkeypox virus is a rather nasty relative of smallpox (family Poxviridae; subfamily Chordopoxvirinae, genus Orthopoxvirus), meaning it is a large dsDNA virus (170-250 kb) with a complex structure.  The virus is endemic in remote forest areas in central Africa – principally in the Democratic Republic of the Congo – and naturally infects a number of animal species, including giant pouched rats (Cricetomys sp.), dormice (Graphiurus sp.) and African squirrels (Heliosciurus, Funisciurus), as well as laboratory monkeys, which is how it was isolated and got its name.

Monkeypox gets transmitted to humans by contact with infected animals: this includes by simple handling, as well as by exposure to meat and blood of butchered animals.  It causes a disease in humans that is very similar in appearance to smallpox, with a case fatality rate of 1-10%, but is apparently far less easily transmitted person-to-person.  It caused only sporadic and limited outbreaks in Africa and was of limited interest until an outbreak in the USA in 2003, which was linked to young prairie dogs kept in a pet store in close proximity to an infected Gambian pouched rat (Cricetomys gambianus) recently imported from West Africa. Seventy-three people were reportedly infected, among whom there were no fatalities.  The CDC recommends vaccination of people exposed to human or suspected animal cases with smallpox vaccine, as this protects animals from experimental lethal monkeypox challenge.

The Vaccine paper makes the point that the potential for monkeypox virus (MPX) to fill the disease niche recently vacated by smallpox was evaluated in the 1970s – and discounted, largely because human-to-human spread was inefficient enough for outbreaks not be self-sustaining – thus, although smallpox vaccine protected against MPX, the WHO thought there was insufficient justification to continue vaccination.

Now, however, the incidence of the virus in humans

“…appears to have markedly increased. In addition to diminished vaccine-induced orthopoxvirus immunity, there have been profound social and demographic changes that have increased human MPX exposures and the likelihood of severe disease. Recurrent civil war and subsequent economic decline have forced rural residents to flee deep into the rain forests for extended periods of time, disrupted traditional village life and increased dependence on hunting for sustenance, thus increasing exposure to animal reservoirs of MPX.”

So, in other words, people are getting a whole lot more exposure to sick animals.  Increasingly, by eating them.  The paper goes on to say:

“Although orthopoxviruses are relatively genetically stable MPX has diverged into two clades with different levels of virulence. As incidence rises, each new MPX infection provides an opportunity for viral evolution or adaptation that may result in a more virulent or contagious variant capable of sustained person-to-person transmission. These new circumstances merit a re-evaluation of the need for immunizing against MPX”.

So – that should be relatively simple, surely?  I mean, South Africa alone has millions of doses of smallpox vaccine safely frozen away from the 1970s?  Not so fast….

“However, in an era where the threat of smallpox is not imminent and there are conditions such as AIDS, tissue transplantation, and therapies for cancer and autoimmunity that cause immunodeficiency, the adverse events associated with live vaccinia are no longer considered acceptable for the general population.”

The paper goes on to mention how all sorts of supposedly safe new smallpox vaccines have been deposited into biodefence stockpiles, based on animal testing.

And there it is again – that word “biodefence”, in the context of human vaccines – implying that there is a “biothreat” to counter.  Specifically, in this case, the spectre of weaponised smallpox.

The authors go on to make reasonable statements about surveilling for monkeypox in central Africa, and vaccinating people at risk, and say that treatment options should also be investigated given that clinical diagnosis is relatively easy.

They also close with this:

“If immunization studies in developing countries are contemplated to support the licensure of orthopoxvirus vaccines for industrialized countries or for military purposes, then provisions from those countries or organizations should be secured to distribute successful products in endemic regions where the products were tested.” [my emphases]

I should hope so.  I should really, really hope so – because then one country’s biodefence interests could end up benefitting quite a few others, who are the ones who really need the product.  Now, while you’re busy with that, what about vaccines for Rift Valley fever, Crimean-Congo haemorrhagic fever and Chikungunya – which are actually far more serious a problem, in a much bigger geographical area?


HIV Vaccines From Bangkok – 1

14 September, 2011

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:

  • one needed a protective Ab response;
  • that IgA or IgG at mucosal surfaces may prevent transmission;
  • strong cellular responses will help control viral replication;
  • there is a good chance that we will get herd immunity;
  • the vaccine composition may have to change envelope component with time and or region;
  • regular boosters will probably be necessary;
  • public health may require universal vaccination of adolescents rather than only of high risk groups.

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….

A recycled virus to protect against TB?

25 August, 2011

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:

  1. the virus replicates in chicken cells and in chicken eggs, meaning it can be cultivated at large scale
  2. it does not replicate in primate, and in fact not in most mammal, cells

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!


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