Posts Tagged ‘rotavirus’

PBVAB 5 Part 2

28 June, 2013

Session: Vaccines 1.

This session produced some of the most interesting talks of the conference, so I will go into some detail in describing them.

Charlie Arntzen (ASU, Tempe, AZ) gave a typically excellent presentation on their latest work on norovirus vaccine formulation for stability and oral delivery – using lyophilized aloe gel-derived nanoparticles.

Norovirus outbreaks are tracked by a CDC lab continuously; every 2 years or so new strains circulate, meaning vaccines will have to keep up.  Ligocyte makes VLPs in insect cells currently; however, plant expression has been shown to be able to respond quickly to strain changes, via Icon vectors used at KBP, with the possibility of very quickly making a lot of product.  Downstream formulation has been a problem, however, as the processing throws away lots of antigen protein downstream.

Ligocyte use MPLA and chitosan (an irritant) for nasal immunisation: this has 50% efficacy.  The FDA does not like adjuvants for nasal dosing – so they went for no adjuvant, and chose the nasal route as one gets a more uniform response for 5x less Ag than with oral administration.  The formulation is basically of VLP preparations with lyophilized and milled pectin content from aloe gel: the uniformly-sized nanoparticles absorb water, and stick to each other and to the nasal mucosa for 3 hrs+.

Charlie commented that “This is the one time I recommend putting white powder in your nose!”.

They have tried mixing VLPs of different virus types – and found that with 50 ug of each, you get same immune response as to one.  Apparently this virus is unusual as you can do virus challenge experiments quite easily: these cost $15K/patient, which is a bargain.  The group is looking at annual or biannual dosing for maximum protection, and is also formulating VLPs for oral vaccination.  Interestingly, the aloe gel also works for intramuscular vaccination – possibly as a result of a depot effect?

 

Yuri Gleba (Nomad / Icon Genetics, Halle, Germany) was supposed to speak on “Technology progress in PMP (=plant-made pharma) research” – but basically said “Transient technologies are the future!”, and then went on to demonstrate it.  He noted that KBP can process 1.2 tonnes biomass/day for agroinfiltrating plants, using a robot from a car factory and a converted industrial autoclave – and consequently have to grow plants in trays for infiltration. Nomad had therefore started investigating how spraying Agrobacterium onto plants might work – with a biosafe Agrobacterium as a prime requirement.  They also took the bold approach of doing transient agronomic trait engineering – for traits such as flowering control, drought tolerance, yield suites, cellulases and anti-microbials – and sold the idea to Bayer Crop Science.

Their technique uses an engineered Agrobacterium that is 100-1000x more efficient at gene delivery than standard strains, with surfactants that allow easy penetration of the leaf tissues.  In combination with the use of replicating vectors that spread cell-to-cell, they could get 100% of standard infiltration yields, by a far easier and much more scalable technique.  They found that spraying worked for most dicots and even for maize, albeit inefficiently, and that they could repeatedly dose plants for the same trait with no apparent harm.  Transient expression of cry1ab and cry2ab Bt toxins delivery worked well, as did delivery of the Cold Shock protein from B subtilis, which also works for drought tolerance.

Their technique does away with need for seed – they can do somatic trait addition / subtraction, they can use the technology outdoors, and there is no trait transmission and so no escape, as the genes do not get into seed.  It means they can produce proteins in  plants at commodity agricultural prices – which considerably broadens the scope of “biofarming” in terms of what products can viably be made!!

One good example was cellulases for bioethanol production: one needs 1-3% w/w relative to cellulose mass, meaning production must be high volume and cheap.  Yuri noted it was possible to store biomass as silage or possibly by vacuum-packing at room temperature for months and that the silage process also eliminated Agrobacterium.  He mused that it might be possible to make a sauerkraut-type oral formulation for recombinant protein delivery…B-)

As for antimicrobials in plants, he said organic crops have more microbes than standard, eg: the recent fatal infections caused by E coli in bean sprouts in Europe – and that a solution would be to make eg colicin E1 in the plants, to kill the bacteria in situ.  One can apply for GRAS status which is MUCH faster than for other routes of approval.  They were currently doing this for phage lysins, bacteriocins, and thaumatin, among others.  Yuri said transient expression tech was like flash drives vs old-style PCs: a versatile set of tools vs a one-trick pony.  He also mused that the technology could lead to reinvention of the old ideal of use of biofarming in undeveloped communities – presumably for low-cost remedies as well as for therapeutics, etc.

To a question on what was the shelf-life of recombinant Agro he replied that there was already field use of live bacteria to combat pathogenic strains; that one can take a Petri dish and dilute in 100l water and spray, and then keep the suspension for two days…it was a very robust bug!  An interesting regulatory point that came up was that the USDA thinks a plant is a GMO even if it is transiently sprayed.

 

Andres Wigdorovitz (INTA, Buenos Aires, Argentina) spoke on their experience of a decade’s worth of work on plant-made veterinary vaccines.  He opened by noting that he has a major problem of getting money from companies in Argentina – partly die to what a “product” is defined as, because what happens is that a “researcher has an egg, whereas the company wants a butterfly”.

They made the decision to work in platforms – to make diagnostic kits initially, which teaches one how to make recombinant proteins.  They use baculovirus/insect cells and plants – in the form of transgenic alfalfa or transplastomic tobacco – to make the same proteins for comparison purposes, and as products, depending on which was more suitable.  While they had had considerable experience with FMDV vaccines made in plants, which had been protective, their current work focused on making novel vaccines and products.  An example was camellid-derived VHH nanobodies – and the fact that fusing the E2 protein of Bovine viral diarrhoea virus (BVDV) with a anti-E2 VHH gave a better alfalfa-produced immunogen for something that was already protective.  Their experimental vaccine was better than the commercial vaccine from the Ab response – and they could get total protection with 3 ug vaccine, and even better efficacy if they made an E2-HLA fusion.  He believed they will have a commercial vaccine in less than 2 yrs as they were engaged in getting regulatory approval now.

In other work, a FMDV VP1 peptide-GUS fusion expressed 10x better in transplastomic tobacco than in transgenic alfalfa.  A rotavirus VP8* fusion protein was also 10x increased in chloroplasts, and dry leaves preserved the protein very well.  They were also making VHH nanobodies against human rotavirus as vaccine coverage of local strains was not good – and VHH against the conserved VP6 could penetrate the outer capsid and bind and neutralize infectivity whereas larger proteins did not work.  They got 3% TSP expression in tobacco chloroplasts.  They were also making VHH to other rotavirus proteins, and to human noro- and influenza viruses.  All in all, it was a very heartening demonstration of a good business model, and that developing countries too can lead the field in some respects.

 

The remainder of the session was taken up with two talks from our group: these were given by Drs Ann Meyers and Inga Hitzeroth, on the parrot-infecting Beak and feather disease virus CP-elastin fusion protein production, and Human rotavirus CP and VLP production in N benthamiana via agroinfiltration, respectively.

The BFDV work has just been published with MSc student Lucian Duvenage as first author – from PubMed, then:

J Virol Methods. 2013 Jul;191(1):55-62. doi: 10.1016/j.jviromet.2013.03.028. Epub 2013 Apr 9.
Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides.
Duvenage L, Hitzeroth II, Meyers AE, Rybicki EP.
Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7700, South Africa.

Abstract

Psittacine beak and feather disease, caused by beak and feather disease virus (BFDV), is a threat to endangered psittacine species. There is currently no vaccine against BFDV, which necessitates the development of safe and affordable vaccine candidates. A subunit vaccine based on BFDV capsid protein (CP), the major antigenic determinant, expressed in the inexpensive and highly scalable plant expression system could satisfy these requirements. Full-length CP and a truncated CP (ΔN40 CP) were transiently expressed in tobacco (Nicotiana benthamiana) as fusions to elastin-like polypeptide (ELP). These two proteins were fused to ELPs of different lengths in order to increase expression levels and to provide a simple means of purification. The ELP fusion proteins were purified by inverse transition cycling (ITC) and it was found that a membrane filtration-based ITC method improved the recovery of ΔN40 CP-ELP51 fusion protein relative to a centrifugation-based method.

Essentially, Lucian managed in some very elegant work to show that BFDV CP fused to a 51-mer ELP allowed production and subsequent simple purification of quite high yields of fusion protein.  It remains to be seen how immunogenic or protective this is – however, it is a breakthrough, as expression of the CP alone has been VERY problematic, in everything from insect cells through E coli, to plants.

Inga spoke on our recent MSc student David Mutepfa’s work on expression in plants of the CPs of a South African rotavirus that is not well matched to current live attenuated vaccines.  The short story is that he succeeded very well indeed in expressing three of the four proteins.  From a recent publication from me and Nunzia Scotti on plant-made VLPs, then:

Current studies in the Rybicki laboratory have focused on expression of capsid proteins of a local isolate of human rotavirus (G9 P[6]) that is not well matched to available commercial vaccines.  Expression of VP2, VP4 and VP6 in N. benthamiana was targeted via co-agroinfiltration to the cytosol, endoplasmic reticulum, apoplast and chloroplast. Electron microscopy showed that co-expressed VP2/6 and VP2/6/4 produced virus-like particles in the cytosol, with yields as high as 1.1 g/kg of plant material, for batches of 100 g.

…with a picture to prove VLPs are made:

rota pic

 

Rotavirus VP2/6/4 co-expression in N benthamiana: protein ex- tract partially purified by sucrose gradient centrifugation, particles captured onto electron microscope grids with mouse-anti VP6 antibody. Bar = 200 nm

Trends in Intussusception Hospitalizations Among US Infants Before and After Implementation of the Rotavirus Vaccination Program, 2000–2009

27 May, 2012

See on Scoop.itVirology News

“A small increase in intussusception rates was seen among infants aged 8–11 weeks, to whom most first doses of rotavirus vaccine were given, but no sustained population-level change in overall intussusception hospitalizations rates in US infants was observed after implementation of the US rotavirus vaccination program. Although an association between intussusception and rotavirus vaccination cannot be established by this ecologic analysis alone, even if the low risk with the first dose exists, it is outweighed by the well-documented benefits of vaccination of US infants”

This is a big deal- a very important, big deal: human rotavirus kills more than 500 000 people a year (mainly very little), and rotavirus vaccines have been bedevilled with the suspicion that they cause telescoping of the intestine, or intussusception.  Which can be fatal, and is not something you want happening to your healthy baby.

However, and however: I have taught my students for years to be aware of relative risks when talking about vaccines, and there is absolutely no doubt that even the Wyeth vaccine could have been considered “safe” in a developing country environment, where the threat of death due to diarrhoea and dehyderation caused by rotavirus, would have been far greater than any threat from the vaccine.

I thank Rusdsell Kightley Media for the rotavirus graphic

See on jid.oxfordjournals.org

Scoop.it: Virology News

11 February, 2012

This is just to announce that I will be regularly posting “Virology News” updates on a new Scoop.it site I have just set up – as well as occasionally updating another Scoop.it site – “Virology and Bioinformatics from Virology.ca” – which is curated by Chris Upton, of Univ Victoria in Canada.

Even more ways to get your daily viral fix…B-)

Virology Africa 2011: viruses at the V&A Waterfront 2

19 December, 2011

We thank Russell Kightley for permission to use the images

Marshall Bloom (Rocky Mountain Laboratories, NIAID) opened the plenary session on Thursday the 1st of December, with a talk on probing the pathogen-vector-host interface of tickborne flaviruses.   Although thoroughly infected with a rhinovirus, he held our attention most ably while reminding us that while many flaviviruses are tick borne, the hard and soft body ticks that vector them are very phylogenetically different – as different as they are from spiders – meaning that if similar flaviruses replicated in them, these viruses may have much wider host range than we know.

He pointed out that while about 95% of the virus life cycle takes place in a tick, transmission to a vertebrate means suddenly adapting to a very different host.  Infection in ticks is persistent, as befits their vector role – but vertebrate infection generally is not.  It was interesting, as a sometime plant virologist, to hear that they look for dsRNA as a marker for replication, and do Ab staining for it: the technique was invented with plant viruses, and very few other virologists seem to appreciate that dsRNA can be quite easily isolated and detected.

They compared Vero and tick cells for virus replication, and saw significant differences: while tick cells could go out to 60+ days and look fine, Vero cells were severely affected at much shorter times post infection.  There was also 100-fold less virus in tick cells, and prominent tubular structures in old infected tick cells.  He noted that ticks evade host defences quite efficiently: eg they suppress host clotting during feeding, and there is huge gene activation in the tick during feeding.  In another study to envy, they are doing array work on ticks to see what is regulated and how.

 Linda Dixon (Institute for Animal Health, Pirbright, UK) recounted her lab’s work on African swine fever (ASFV), a poxvirus-like large DNA virus.  The virus is endemic to much of Africa, and keeps escaping – and there is no effective  vaccine to prevent spread, so regulation is by slaughter.  There are 3 types of isolate, with the most highly pathogenic causing up to 10% fatality and a haemorrhagic syndrome.  She described how in 2007 the virus had spread from Africa to Georgia, then in 2009 to southern Russia and all way to the far north, in wild boar.

There are more than 50 proteins in the dsDNA-containing virion; two infectious forms similar to the poxviruses with multilayer membranes and capsid layers can form, and neutralising Ab play no part in protection as a result.  They studied the interaction of viruses with cells and the immune system, and compared the genomes of pathogenic and non-pathogenic strains, in order to understand how to develop an effective vaccine.

The biggest differences were large deletions in non-virulent isolates, including genes coding for  proteins responsible for binding to RBC, and various immune evasion multicopy genes.  They planned to target regions to delete to make an attenuated virus for vaccine.  They had found non-essential genes involved in immune evasion, and ones that lower virulence, and had been systematically cutting them out.  She noted that pigs can be protected if they survive natural infection and if vaccinated with TC-attenuated virus, and can be protected by passive transfer of Abs from immune pigs – which indicated that an effective live vaccine was very possible.

Subunit vaccines were being investigated, and they had found partial protection with baculovirus-expressed proteins.  They were doing genome-wide screens for protective Ag, and were pooling Ags expressed from predicted ORFs in immunization trials – up to 47 Ags without reduction in specific  T cell responses.

Discovery One

My former labmate Dion du Plessis (Onderstepoort Veterinary Institute, OVI) made a welcome return to Cape Town, with a talk entitled “2011: A Phage Odyssey”.  He explained the title by noting the distinct resemblance of P1 coliphage to the Discovery One spacecraft dreamed up by Arthur C Clarke and Stanley Kubrick – and then went on to exuberantly and idiosyncratically recount a brief history of bacteriophages and their use in biotechnology since their discovery.  A revelation from his talk was that the first discovery of phages was probably described by a gentleman named Hankin, in 1896 in Annales de l’Institut Pasteur: he

The 1896 paper from Annales de l'Institut Pasteur

showed that river water downstream of cholera-infested towns on the Jumma river in India contained no viable Cholera vibrio – and that this was a reliable property of the water.  We were also introduced to the concept of turtles as undertakers in the Ganges….

He took us through the achievements of the Phage Group of Max Delbruck and others – where science was apparently fun, but also resulted in the establishment of modern molecular biology – through to the use of phages as exquisitely sensitive indicators immunochemistry studies in the 1960s.

All too soon we got to the modern uses of phages, with 3 types of gene library – random peptide, fragmented gene, and antibody V regions – being used to make recombinant phage tail proteins to be used for “panning” and enrichment purposes, in order to select either specific antibodies or antigens.  Dion manages a research programme at OVI aimed at developing a new generation of veterinary vaccines – and has for some years now been making significant progress in generating reagents from a chicken IgY single-chain Fv phage display library.

Carolyn Williamson (IIDMM, UCT) gave us an update on CTL epitopes associated with control of HIV-1 subtype C infections.  She said that it was now known that genome-wide association studies (GWAS) gives you certain HLAs which are associated with low viral load, and others with high – meaning that to some extent at least, control of infection was down to genetic luck.  She noted that they and others had shown that CTL escape was quick: this generally happened in less than 5 weeks in acute phase infections.

They had looked for evidence of a fitness cost of CTL escape – and shown that it exists.  She noted that this meant that even if one has “bad” HLA genes, if one was infected with a virus with fitness cost mutations from another, that one could still control infection.

It had been shown that “controllers” mainly have viruses with attenuating mutations, or have escapes in the p24 region – and it was a possible vaccine strategy to include these mutated epitopes in vaccines to help people with infections control their infections.

An interesting topic she broached was that of dual infections – there was the possibility of modelling if infection with two different viruses results in increased Ab neutralisation breadth, and if one would get different results if infections were staggered, possibly with increased nAb evolution if isolates were divergent.  She noted it was possible to track recombination events with dual virus infections too.

It was interesting that, as far as Ab responses went, there were independent responses to 2 variants and one could get a boost in Ab titres to the superinfecting virus, but not a boost to Abs reacting with the originally-infecting virus

Carolyn was of the opinion that HIV vaccines needed to include CTL epitopes where escape is associated with fitness cost.  She also reiterated that superinfection indicated that one can boost novel responses, which I take to mean that therapeutic applications are possible.

Ulrich Desselberger (University of Cambridge) is a long-time expert on rotaviruses and the vaccines against them, and it was a pleasure to finally hear him speak – and that he was mentoring young people in South Africa.  He said that more than a third of children admitted to hospital worldwide were because of rotavirus infections, meaning that the viruses were still a major cause of death and morbidity – and they were ubiquitous.

He reviewed the molecular biology and replication cycle of rotaviruses in order to illustrate where they could be targeted for prevention of infection or therapy, and noted that drugs that interfere with lipid droplet homeostasis interfere with rotavirus replication because 2 viral proteins associated proteins of lipid droplets.

He stated that there were lots of recent whole-genome sequences – we already there were many types, based on the 2 virion surface proteins; we  now know that other genes are also highly variable.  As far as correlates of immunity were concerned, VP7 & 4 were responsible for eliciting neutralising Ab.  Additionally, protective efficacy of VP6 due to elicitation of non-neutralising Ab had been shown in mice – but not in piglets, and not convincingly in humans.  Abs to VP2, and NSP2 and 4 were also partially protective in humans.  It was interesting that protection was not always correlated with high titre nAb responses.

He noted that in clinical disease primary infections partially protected against subsequent infections which are normally milder; subsequently no disease was seen even when infection occurred.  Cross-protection occurred at least partially after initial infection, and this got better after more exposure.  There was evidence one could get intracellular neutralisation by transcytosed Ab, and especially to VP6.  Ab in the gut lumen was a good indication of protection.

As far as the live modern vaccines were concerned, Merck’s Rotateq elicited type-specific nAb, with 9% of recipients shedding live virus.  GSK’s Rotarix gets elicits cross-reactive nAb and one gets 50% of recipients shedding virus.

While the vaccines seemed safe, he noted that where vaccines had been introduced, efficacy ranged from 90% in the USA and Europe, down to as low as 48% in Bangladesh, Malawi and SA, due to type mismatch, and that efficacy was correlated inversely with disease incidence and child mortality generally.  He mentioned that there had been much VLP work, but that none of the candidates was near licensure.

Johan Burger (Stellenbosch University) spoke on one of the more important non-human virus problems in our immediate environment – specifically, those affecting wine grape production in our local area.  He opened by stating that SA now produced 3.7% of the world’s wine, making grapes a nationally and especially locally important crop.  Leafroll disease was a major worldwide problem – as well as being the reason for the wonderful autumn reddening seen in grapevines, it also significantly limited production in affected vineyards.  His laboratory has done a lot of work in both characterising viruses in grapevine, and trying to engineer resistance to them.  Lately they were also investigating the use of engineered miRNAs as a response to and means of controlling, virus infection.

His group has for a couple of years been involved in “metaviromic” or high-throughput sequencing studies of grapevines, with some significant success in revealing unsuspected infections.  In this connection, he and Don Cowan pointed out that they had lots of data that they ignore – but which we should keep and study, as a resource for other studies not yet thought of.

As far as Johan’s work went, novel viruses kept popping up, including grapevine virus E (GVE), which hitherto had only been found in Japan.  They were presently looking at Shiraz disease, which was unique to SA, and was still not understood.  This was infectious, typified by a lack of lignification which led to rubbery vines, and kills plants in 5 years.  It also limits the production of the eponymous grapes – a crime when SA shirazes seem to be doing so well!

Veterinary Virology and Vaccines parallel session.

I again dodged the clinical / HIV session because of my personal biases, and was again treated to a smorgasbord of delight: everyone spoke well, and to time, and I was really gratified to see so many keen, smart young folk coming through in South African virology.  It was also very interesting to see highly topical subjects like Rift Valley fever and rare bunyavirus outbreaks being thoroughly covered, so I will concentrate on these.

P Jansen van Veeren (NICD, Johaanesburg) was again a speaker, this time representing his absent boss, Janusz Paweska.  He gave an account of the 2010 Rift Valley fever outbreak in SA, and epidemiological findings in humans – something of keen interest to me.  He said there had been some forecasting success for outbreaks in East Africa; however, there were long gaps between outbreaks, which were generally linked to abnormal rainfall and movement of mosquito and animal hosts.  RVFV isolates differed in pathogenicity but were structurally and serologically indistinguishable – because virulence was due to the NSs protein, and not a virion component.  He recounted how artificial flooding of a dambo in Kenya resulted in a population boom in the floodwater Aedes mosquitoes responsible for inititating an outbreak, and then of the Culex which maintained the epidemic.  He said there was a strong correlation between viral load and disease severity.

In terms of South African epidemiology, there had been smaller outbreaks from 2008 round the Kruger National Park (NE SA), then in the Northern Cape and KZN in 2009.  People had been infected from autopsy of animals, and handling butchered animal parts.  The 2010 outbreak started in the central Free State after an unusually wet period, and had then spread to all provinces except Limpopo and KZN.  In-house serological methods at the NICD were validated in-house too: these were HAI screening and IgM and IgG ELISAs and a virus neutralisation test.  They had got 1600+ samples of human serum, and confirmed 242 cases of disease and 26 deaths for 2010.

He noted that with winter rains there was a continuous outbreak in the Western Cape, and in 2011 the epidemic had started again in the Eastern and Western Cape Provinces, but has since tailed off.  Some 82% of human cases were people who occupationally handled dead animals, although there was some possibility of transmission by mosquitoes.

In human cases there was viraemia from 2-7 days, with IgM present transiently from 3 days at low level.  They had sequenced partial GP2 after PCR from 47 isolates, and showed some recombination occurring.  The 2010 isolates were very closely related to each other, and to a 2004 Namibian isolate.  There had been no isolation from mosquitoes yet.

Two talks on FMDV followed: Belinda Blignaut (OVI and Univ Pretoria) spoke on indirect assessment of vaccine matching by serology, and Rahana Dwarka (OVI) on a FMDV outbreak in KZN Province in 2011.  Belinda’s report detailed how 6 of 7 serotypes of FMDV occur in SA, with SAT-1 and -2 and O the most common – and that vaccines needed to be matched to emerging strains.  This was done by indirect vaccine matching tests such as serological r-value, determined by the ratio of the reciprocal serum titre to the heterologous virus against that to the homologous virus.  They had put 4 different viruses into cattle and got sera to test a range of 26 newly isolated viruses.  While they had not got sequence from the test panel viruses, indications were that topotype 3 viruses are antigenically more disparate and that a vaccine consisting of topotype 1 or 2 antigens may not be effective in the control of FMD.

In introducing Rahana’s talk, the chair (Livio Heath, OVI) mentioned that there had been 5 different major animal pathogens causing outbreaks in SA over the last 3 years – and that they had to produce reagents and validate tests for ASFV, classical swine fever (CSF) and FMDV, etc, with each outbreak.  Rahana described how they had neutralisation assays and blocking and competition ELISA for FMDV, as well as a big database of isolates from buffalo in KZN – so they were well-placed to type viruses found in cattle in the region.

C van Eeden (Univ Pretoria) had an intriguing account of their investigation of the occurrence of an orthobunyavirus causing neurological symptoms in horses and wildlife.  Horses seem to be particularly vulnerable to many of the viruses involved in such disease, and so are a useful sentinel species.  Shuni virus was first isolated from Culicoides midges and sheep and a child in Nigeria in the 1960s.  SA workers subsequently found it in some livestock and Culex mosquitoes and in horses.  The virus was shown to be a neurologic disease agent in horses and wildlife – then disappeared for some 30 years, much like Ebola.  There is apparently a new research unit at UP with a BSL3 lab, so they are well equipped to do tests with the virus.

Ms van Eeden noted that the incidence of encephalitic disease in humans and animal in SA is underreported, and the causes are mainly unknown – a revelation to me!  Horses are susceptible to many of the agents, and are useful sentinels – workers have identified flavi- and alphaviruses in some outbreaks, but many are not IDed.  They had done cell culture and EM on samples from an ataxic horse: they got a bunyavirus-like virus by EM, and did bunya-specific PCR, and got Shuni virus back.  Sequence relationships showed no linkage to type of animal or date, in subsequent samplings from horses, crocodiles,  a rhino and a warthog, and from blood, brain and spinal cord.  All positive wildlife were sampled in Limpopo Province; horses only from most other provinces.

She noted that latest cases were neurological, whereas previously these were mainly febrile.  The virus accounted for 10% all neurological cases, with a 50% fatality rate.  She noted further that vets often work without masks or gloves, and so had no protection from exposure in such cases….  There was no idea on what the vector was, but they would like to test mosquitoes, etc.  Ulrich Desselberger suggested  rodents may be a reservoir, but they don’t know if this is true.

Stephanie van Niekerk (Univ Pretoria) investigated alphaviruses as neurological disease agents in African wildlife.  The most common alphaviruses in SA are Sindbis and Middelburg viruses.  Old World alphaviruses are usually not too bad, and cause arthritic and febrile symptoms, while New World cause severe neurological diseases.  Sindbis was been found in SA outbreaks in 1974.  However, Stephanie noted that a severe neurological type had appeared since 2008 in horses.  Accordingly, they looked at unexplained cases in wildlife in the period 2009-2011: brain and spinal cord samples were investigated for all cases.  They found alphavirus in a number of rhinos, buffalo, warthog, crocodiles and jackal – and all except for one rhino were Middelburg virus.  They want to isolate viruses in cell culture, and increase the size of regions used for cDNA PCR.  Stephanie said the opinion was that the values of the animal involved justifies the development of vaccines.

 

Vaccines are safe: now use them!

30 August, 2011

At last, someone heavy has gone and nailed it down: The Scientist (http://the-scientist.com/2011/08/26/vaccines-are-safe/) reports that

“Vaccines are safe and not the cause of autism, according to a new report from the Institute of Medicine, the health arm of the National Academies.  The panel based its conclusions on the review of more than 1,000 studies on eight vaccines commonly given to children, including those for chickenpox, meningitis, tetanus, and measles, mumps, and rubella (MMR).”

One.  Thousand.  Studies.  At least!  There’s more:

“Some serious side effects were linked to vaccines, but occurred very rarely. Among them: those who receive the chicken pox vaccine could later come down with pneumonia or meningitis if their immune systems become compromised by diseases such as cancer, and the MMR vaccine occasionally sets off brain inflammation or seizures, Nature reports. Six of the eight vaccines can also cause allergic reactions. The more serious side effects most commonly occur in children who have underlying immune problems.”

As I taught my captive second- and third-years for close on thirty years, there’s a risk-benefit calculation to be done for every vaccine, and indeed, for every drug that you or yours may be exposed to.  If there’s no benefit – because there’s no risk – then do without.  If, on the other hand, your baby stands a reasonable chance of getting seriously ill, and maybe even suffering permanent damage from getting infected by a preventable dissease – vaccinate!

And that’s rotavirus I’m talking about, not even something really nasty like measles.  I did the same survey over several years, asking 70+ students if they would give kids a vaccine that had a POSSIBLE 1/40 000 chance of causing a possibly fatal intestinal complication (intussusseption, or telescoping of the bowel) in (a) an environment where no children died of rotavirus, (b) an environment like in many places in the developing world, where 1/100 children might die.  Nearly every single one, every time, would not use it in (a), but would have no hesitation in scenario (b).

Regulars on this blog will know what I think about not vaccinating against measles.  So now, folks: reach out to a relative or a friend who espouses this utter nonsense linking vaccines to autism – and smack them solidly.  Then get their kids vaccinated.

OK, maybe not – but remonstrate with them, point them towards the evidence, tell them just how nasty some of the preventable diseases can be, and that they should realise measles, polio or rotavirus are only someone else’s plane trip away.

Doctor, there’s a…pig virus in my vaccine??

6 June, 2010

rotavirus particle

I have for some time taught my third year students about how one must weigh relative risk vs. relative benefit when it comes to vaccination – with the Wyeth live rotavirus vaccine that was withdrawn in 2000 or so due to isolated incidents of intussusception (=telescoping of the bowel) as an object example.

Consider: the vaccine MAY have caused a couple of incidents (which granted, were serious) – but on the whole was protective, and well tolerated.  The publication referred to has this as the relative risk:

“…epidemiologic evidence supports a causal relationship, with a population attributable risk of ~1 per 10 000 (range of 1 in 5000 to 1 in 12 000) vaccine recipients.”

While this may be an unacceptable risk in a North American community – which is where it was tested, where children mostly just get sick from rotavirus infection – what about in a developing country, where the risk of an infant dying from rotavirus diarrhoea is far higher than 1 / 10 000?  Indeed, the same article says:

“Because perceptions of vaccine safety derive from the relative disease burdens of the illness prevented and adverse events induced, the acceptance of rare adverse events may vary substantially in different settings. [ my emphasis]”

Yes – like the vaccine may well have done a great deal of good, and very little harm, in a developing country setting where rehydration therapy is not the norm.  But it was pulled, and the world had to wait for Merck’s Rotateq pentavalent live vaccine and GSK’s Rotarix tetravalent live vaccine, YEARS later, and probably a lot of children died that may not have needed to.

I note that the Merck product has this as a warning, too:

“In post-marketing experience, intussusception (including death) and Kawasaki disease have been reported in infants who have received RotaTeq”.

So the vaccine has the same risk profile as Wyeth’s – yet it has been widely distributed, and is apparently highly effective – as is Rotarix.  In fact, in 2009 the WHO issued a recommendation “…that health authorities in all nations routinely vaccinate young children against rotavirus…”.

And then…news that must have made many a heart sink, in March 2010:

Pig virus contamination halts GSK Rotarix use

“GlaxoSmithKline’s oral rotavirus vaccine Rotarix has been temporarily shelved in the U.S. due to a pig-virus contamination. Researchers stumbled on DNA from porcine circovirus type 1–believed nonthreatening to humans–while using new molecular detection techniques. More work is being done to determine whether the whole virus or just DNA pieces are present.

Additional testing has confirmed presence of the matter in the cell bank and seed from which the vaccine is derived, in addition to the vaccine itself. So the vaccine has been contaminated since its early stages of development.”

The finding of the porcine circovirus type 1 (PCV-1) DNA in the vaccine was due to what seems to have been publication of an academic investigation in February 2010 of “Viral Nucleic Acids in Live-Attenuated Vaccines” by Eri L Delwart and team, mainly from Blood Systems Research Institute and University of California, San Francisco.  They used deep sequencing and microbial array technology to:

“…examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides.”

And they found:

“Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles [!!], while simian retrovirus (SRV) was present as genetically defective DNA.”

Whooooo…possibly live animal retroviruses in human vaccines??  But most importantly for our purposes:

Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids.”

I don’t know about you, but I’d be more worried about the retroviruses!  The authors concluded [my emphases in bold and red]:

“Given that live-attenuated viral vaccines are safe, effective, and relatively inexpensive, their use against human and animal pathogens should be encouraged. The application of high-throughput sequencing and microarrays provides effective means to interrogate current and future vaccines for genetic variants of the attenuated viruses and the presence of adventitious viruses. The wide range of sequences detectable by these methods (endogenous retroviruses, bacterial and other nucleic acids whose taxonomic origin cannot be determined, and adventitious viruses, such as PCV1) is an expected outcome of closer scrutiny to the nucleic acids present in vaccines and not necessarily a reflection of unsafe products. In view of the demonstrated benefit and safety of Rotarix, the >implications (if any) for current immunization policies of the detection of PCV1 DNA of unknown infectivity for humans need to be carefully considered.”

So they find all these bits of adventitious nucleic acids in a live human vaccine, then tell us it’s all right?  They go to say, however:

“As an added note, recent testing by GSK indicates that PCV1 was also present in the working cell bank and viral seed used for the generation of Rotarix used in the extensive clinical trials that demonstrated the safety and efficacy of this vaccine. These trials indicate a lack of detectable pathogenic effects from PCV1 DNA on vaccinees.”

So: a clinical trial in retrospect, then??  Interesting idea, that – it’s OK because they inadvertently tested it already and no obvious harm came of it!  Mind you, the same thing happened with SV40 in poliovirus vaccines over a lot longer period and on a much larger scale – and while the jury is still out on long-term effects, it appears as though there were none.

The first outcome of the finding, though, was that the FDA recommended in March that use of Rotarix be suspended, pending further investigations.

The same GSK press release reminds us that:

“Rotavirus is the leading cause of severe gastroenteritis among children below five years of age and it is estimated that more than half a million children die of rotavirus gastroenteritis each year – a child a minute [my bold – Ed]. It is predicted that rotavirus vaccination could prevent more than 2 million rotavirus deaths over the next decade. The continued availability of rotavirus vaccines around the world remains critical from a public health perspective to protect children from rotavirus disease. “

Cementing the risk/benefit argument very firmly and pre-emptively, then!

The next development was that Merck’s Rotateq, initially thought to be free of PCVs, was found to contain both PCV-1 AND PCV-2 DNA.  From their press release:

“In March 2010, an independent research team and the FDA tested for PCV DNA in rotavirus vaccines; at that time, PCV DNA was not detected in ROTATEQ by the assays that were used initially. Subsequently, Merck initiated PCV testing of ROTATEQ using highly sensitive assays. Merck’s testing detected low levels of DNA from PCV1 and PCV2 in ROTATEQ. Merck immediately shared these results with the FDA and other regulatory agencies.”

Alarming at first sight – but a variety of someones had done their relative risk calculations, and by mid-May, both vaccines had been cleared by the FDA – much to Merck and GSK stockholder relief, one imagines.

“The agency’s decision follows a May 7 recommendation from an FDA advisory panel, which said the PCV contamination didn’t appear to be harmful to humans and the vaccines’ benefits outweighed any “theoretical” risk the products might pose.

In announcing its decision, FDA said that both vaccines have strong safety records, including clinical trials of the vaccines in tens of thousands of patients, plus clinical experience with their administration in millions more. PCV isn’t known to cause illness in humans, whereas the rotavirus these vaccines ward off can cause severe illness and even death.”

All in all, what appears to be a sensible, logical decision, based on evidence – whether collected in retrospect or not – and common sense.  After all, as GSK points out in a press release,

“[PCV] is found in everyday meat products and is frequently eaten with no resulting disease or illness.”

Like plant viruses in vegetables, retroviruses in undercooked chicken and other meat, and a myriad other viruses and bacteria that live in, on, and with us – you really can’t keep away from everything.

But there’s still a good case to be made for killed vaccines….


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