Archive for October, 2010

HPV vaccines: good, but out of reach for most

28 October, 2010

Human papillomavirus and cervical cancer - copyright Russell Kightley Media

The fact that genital Human papillomaviruses (HPVs) cause cervical cancer in women, as well as a variety of other growths and lesions in both men and women, is not in dispute.  The fact that cervical cancer is a major and growing scourge of women in developing countries is also non-contentious: of the more than 500 000 cases and 300 000 deaths due to the disease every year, more than 80% occur in the developing world.  This is largely because, unlike their counterparts in the developed world, poor Third World women either do not get screened using the relatively simple cytological detection method known as the Papanicolau (Pap) smear, or do not get treated thereafter.  Thus, cervical cancer really is a disease of poverty, given that most deaths occur due to a lack of simple procedures being provided in clinics.

The best method of prevention of an infectious disease is almost always a vaccine: HPV vaccines have been around a while now, and have proved to be both safe and efficacious – both primary requirements of a vaccine.  Both Merck and GlaxoSmithKline’s vaccines – the yeast-produced Gardasil and insect cell-produced Cervarix respectively – are virus-like particles (VLPs) composed of the major HPV coat protein L1 only; Cervarix contains particles of the high-risk HPV types (or species) 16 and 18 and Gardasil contains VLPs derived from HPVs 16 and 18 as well as the genital wart-causing 6 and 11.

The vaccines are both “blockbusters” – that is, they both have sales of over US$1 billion – are are possibly the best-researched human vaccines ever made.  They are also possibly among the most expensive: Gardasil went on sale in the USA at $120 per dose – and a full treatment consists of 3 doses, for a total cost per person treated of $360; Cervarix retails at around the same price.

This is so far beyond the budget of most people in most countries as to be akin to their expectation of winning a lottery – and of the order of 1000x as expensive as possibly the most widely distributed vaccine in the world, which is Bacillus Calmette-Guerin (BCG), the Mycobacterium tuberculosis vaccine.

It is a sad fact of life that the whole WHO Expanded Programme on Immunisation – EPI – six vaccine bundle of polio, measles, neonatal tetanus, diphtheria, pertussis (whooping cough) and tuberculosis vaccines “… costs no more than US$1 … (at UNICEF-discounted prices), and another US$14 for programme costs (laboratories, transport, the cold chain, personnel and research) to fully immunize a child”.  It is also a sad fact that the new generation of vaccines – exemplified by the yeast-made recombinant hepatitis B virus (HBV) subunit vaccine – are expensive even when discounted after patents have expired: thus, HBV vaccine launched at US$150 for three doses in 1986, and came down to around $10 now.  It is included in EPI bundles in some countries because of even greater discounting (down to ~$1); however, its cost is generally greater than the rest of the bundle combined.

So what should happen with HPV vaccines?  How are they going to get to the people who need them most, at the price they can afford – which is nothing?  The simple answer is that governments and international agencies must buy them, as is presently the case with the EPI package – and that they must be very heavily discounted, to allow this.

In fact, at the recent Papillomavirus Conference in July in Montreal (which we should write up in more detail elsewhere), I heard that the Mexican government has managed to secure  HPV vaccine at US$27/dose – or 25% of the regular price – for a campaign they are mounting in some regions to supply vaccine for free.  So it is possible – however, even this price is far too high, as it represents about the per capita per annum public health expenditure in the poorest countries who probably need it most.

It raises my blood pressure, therefore, when I read that in several highly-developed western countries there are a number of controversies (see also here) around HPV vaccination: yet again, on the heels of the measles and MMR (measles-mumps-rubella viruses) vaccines-cause-autism idiocy, people who can afford vaccines are among the most stupid when it comes to having them.

The facts, as opposed to the hype, are these:

  • the vaccines were proven to be safe in extended clinical trials
  • they were proven to be efficacious in preventing infection and development of precancerous lesions and genital warts – in men as well as in women

Inflammatory stories about deaths due to HPV vaccines are just that – stories.  A recent publication from India, where the government suspended a vaccine study due to deaths of girls involved in the trial, puts things into perspective:

“The causes of death had been scrutinized by the State Government and reported to ICMR and Drugs Controller General of India; all were satisfied that no death was vaccine-related [ my emphasis]. We understand that there is an unusually high frequency of death among girls in this community, which is what deserves immediate enquiry and remedial interventions….
The death of a 14-year old British girl shortly after receiving HPV Vaccine,evoked considerable media attention across the world. The necropsy studies showed that she had malignant tumor affecting her heart and lungs…. The vaccine was not her cause of death.”

There is also considerable silliness surrounding the vaccination of girls – and, hopefully, boys! – against what is very largely a sexually transmitted virus.

Do people have the same problem with HBV?

Or – is it possible?? – they don’t know that it is also frequently a sexually-transmitted disease, among adults at least?

In any case, the kinds of prudishness-by-proxy that result in non-vaccination against HPV or HBV are simple foolishness.

And I would be happy to tell anyone so.

Meantime, we want to make HPV vaccines in plants. Any sponsors??

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They DO get everywhere, don’t they?

21 October, 2010

Euglena cells in pondwater. Image copyright Russell Kightley, http://www.rkm.com.au

Thanks to AJ Cann’s MicrobiologyBytes, and The Scientist:

Decoding the Genome of Chlorella Microalgae, a Promising Genus for Biofuel Production

ScienceDaily (Oct. 13, 2010)
“…the analysis of the Chlorella genome has also revealed numerous genes governing the synthesis of flagellar proteins, which suggests that this species could have a sexual cycle that has gone unnoticed until now. Last but not least, the ability of Chlorella algae to synthesize chitin could have been inherited from a virus (itself endowed with chitinase activity) having secured exclusive use of its host against other viruses incapable of piercing through its protective shell. This “monopoly” scenario illustrates a new mode of co-evolution between viruses and their hosts.”

Gotta love ’em – because maybe we and many other things couldn’t be here without ’em.  This builds on previous evidence that retroviruses probably helped in the evolution of placental mammals, that much of the planet’s oxygen may be due to viruses, and that viruses often aid hosts in developing resistance against them.

However, the parent paper is always preferable to a commentary – and I am indebted to Guillaume Blanc – the corresponding author – for a copy of the paper; our otherwise reliable library service fell down on access to the Plant Cell!  This allows me to quote the following (bold text my emphases):

The Chlorella variabilis NC64A Genome Reveals Adaptation to Photosymbiosis, Coevolution with Viruses, and Cryptic Sex

Guillaume Blanc et al., Plant Cell Advance Online Publication
Published on September 17, 2010; 10.1105/tpc.110.076406

With 233 predicted enzymes involved in carbohydrate metabolism, NC64A appears much better equipped for synthesizing and modifying polysaccharides than the other sequenced chlorophytes that have between 92 (O. tauri) and 168 (C. reinhardtii) of such predicted enzymes…. However, we did not find homologs of the Arabidopsis proteins involved in the synthesis of cellulose (cellulose synthase CesA) or hemicellulose (hemicellulose syn- thase CLS), the major components of the primary cell wall of land plants. Instead, experimental evidence suggests that the cell wall of Chlorella species, including NC64A, contain glucosamine polymers such as chitin and chitosan….

Chitin is a natural component of fungal cell walls and of the exoskeleton of arthropods but is not normally present in green algae. The origin of chitin and its derivatives in the Chlorella genus has long been an enigma. Except for the plant-type chitinase gene, which is found in land plants (but not in chlorophytes apart from Chlorella), the four gene classes involved in forming and remodeling chitin cell walls (i.e., chitin synthase, chitin deacetylase, chitinase, and chitosanase) are absent in all the other fully sequenced Viridiplantae species. By contrast, homologs for each of these families exist in genomes of Chlorella viruses. The viral genes are presumably involved in degradation of the Chlorella cell wall (chitinase and chitosanase)… and production of chitinous fibers on the external surface of virus-infected cells (chitin synthase and chitin deacetylase) …. Phylogenetic analysis suggests that the Chlorella ancestor exchanged the bacterial-type chitinase and chitin-deacetylase genes with the chloroviruses.

And as I have often said (well, mostly to myself, but also in MicrobiologyBytes) – “Profound Insight (No. 4): in order to understand viruses, we should all be working on seawater…. That is where the diversity is, after all; that is where the gene pool that gave rise to all viruses came from originally – and who knows what else is being cooked up down there?”.

Amen.  But let’s add ponds to that.

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Lassa, come home!

21 October, 2010

Lassa virus: image copyright Russell Kightley Media

Lassa fever is a nasty acute viral haemorrhagic fever (HF), caused by Lassa virus.  This is a member of the genus Arenavirus, family Arenaviridae, comprising a collection of 2-component ss(-)RNA enveloped viruses which also includes Lymphocytic choriomeningitis virus – a favourite model organism – and a host of South American HF viruses.  It is also a BSL-4 pathogen, or “hot virus” – one that needs to be worked with in a spacesuit environment, meaning it is pretty difficult to study in the lab.

Arenaviruses are interesting for molecular virologists because of they are one of several ssRNA(-)RNA viruses with “ambisense” genomes, meaning their genomic RNAs have stretches which can be directly read into protein by ribosomes, instead of having to be transcribed first.

The virus and the fever are endemic in the West African countries of Nigeria – from where it was first described in 1969 – Sierra Leone, Liberia, Guinea and the Central African Republic, but almost certainly occur more widely.  There are an estimated 300 000 cases a year, with 5 000 deaths attributed to the virus annually – again, probably an underestimate, as in epidemics mortality can go up to 50%.  The virus is vectored by what is probably the most common type of rodent in equatorial Africa, multimammate rats in the genus Mastomys, mainly via aerosolised faces and urine, which contain high concentrations of virions.  The rat can maintain infection as a persistent asymptomatic state.  It is also possible to spread the disease from person to person, via body fluids.

The CDC has this to say about Lassa fever:

In areas of Africa where the disease is endemic (that is, constantly present), Lassa fever is a significant cause of morbidity and mortality. While Lassa fever is mild or has no observable symptoms in about 80% of people infected with the virus, the remaining 20% have a severe multisystem disease. Lassa fever is also associated with occasional epidemics, during which the case-fatality rate can reach 50%.

While this may seem to be of mild interest only to the international community – after all, it is a seasonal disease limited to one part of Africa, and only 5 000 people die annually, compared to 400 000+ for influenza – it is and remains a nasty disease, with significant side effects, which include temporary or permanent deafness in those who recover – various degrees of deafness occur in up to one-third of cases – and spontaneous abortion of about 95% of third trimester foetuses in infected mothers, and a death rate of >80% in the women.  Moreover, while the term “limited to West Africa” may make it sound of local interest only, it is worth noting that that part of Africa is bigger than the whole of Western Europe – in fact, it’s the size of the whole of the USA – and is home to close to 200 million people.  Moreover, there is serious concern that the incidence of Lassa fever may be increasing, and that it is emerging from its endemic regions into newer pastures with changing regional weather patterns.  However, while fears of rampant spread via air travel do exist, like “Ebola Preston“, these are largely scare stories – which are admirably efficiently debunked here.

A tragic fact about Lassa fever is that it is treatable with drugs, if caught early: JB McCormick and others showed in 1986 that intravenous ribavirin given within 6 days of the onset of fever reduced mortality of patients with a serum aspartate aminotransferase level greater than or equal to 150 IU per litre at the time of hospital admission, from 55% to 5% – whereas patients whose treatment began seven or more days after the onset of fever had a case-fatality rate of 26 percent.  Moreover, oral ribavirin was also effective in patients at high risk of death.

So WHY isn’t ribavirin distributed widely and freely in West Africa for use in clinics??  Why, indeed…that doyen of the US biowarfare / hot virus community, CJ Peters, had this to say in an online book:

Both antiviral vaccines and drugs suffer from major development problems. They would require an expensive developmental effort that has never been able to attract industrial support based on disease activity in endemic areas, even when the U.S. Department of Defense has expressed an interest and provided an additional market.

In other words, no-one would manufacture it for a market that couldn’t pay for it in a sustainable way – another of the unacceptable faces of modern capitalism.

There is hope, however – people are working on vaccines, and there have been significant successes in primate models: in 2005, Geisbert et al. described a

“…replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease.”

Very promising, at first glance.  This is, however, a live virus vaccine – with all of the attendant problems of purification of whole virus, contamination, manufacture, cold chain – and cost….  Given the recent global experience with virus vaccines both live and dead – and recent rotavirus and papillomavirus vaccines would be excellent recent examples, with unit costs at over US$40 per shot  – this vaccine will not debut, if it does so at all, at a cost that is even remotely affordable in the target market in West Africa.

Unless the target market is in fact the US military – which, given the fact that the lead author’s address is given as “Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick”, can be considered quite likely.

Another more recent, and – to my biased mind at least – more promising candidate vaccine, is one described by Luis M Branco et al. in a brand-new Virology Journal article.  This one is also associated with the US military –  with 3 of 11 authors with addresses “@usarmy.mil” – but describes a virus-like particle vaccine candidate rather than a recombinant live virus.

Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever

Virology Journal 2010, 7:279 doi:10.1186/1743-422X-7-279

Published: 20 October 2010

Luis M Branco, Jessica N Grove, Frederick J Geske, Matt L Boisen, Ivana J Muncy, Susan A Magliato, Lee A Henderson, Randal J Schoepp, Kathleen A Cashman, Lisa E Hensley and Robert F Garry

Background

Lassa hemorrhagic fever (LHF) is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infection with intravenous Ribavirin, a nucleotide analogue drug, is possible and greatly efficacious if administered early in infection. However, this therapeutic platform has not been approved for use in LHF cases by regulatory agencies, and the efficacy of oral administration has not been demonstrated. Therefore, the development of a robust vaccine platform generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform.

Results

A mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though all host cell components remain largely unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles.

Conclusions

These results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.

So what they have done is to make non-infectious particles which strongly resemble native virions of Lassa virus, at high yield in a mammalian cell expression system, under low containment conditions – meaning it is safe for workers. The VLPs are highly and appropriately immunogenic, and appear to have significant potential as a Lassa virus vaccine.  This is very similar to previously reported work on Rift Valley fever VLPs made in insect cells, and HPAI and pandemic influenza HA-containing VLPs made in plants, in that VLPs are produced at good yield in an established expression system.

Except that they’re using mammalian cells, with all of the cost implications inherent in that.  And they’re using transfection of plasmids – not the world’s cheapest method of producing proteins.  And they didn’t show efficacy….

Ah, well, there’s still hope – and they could still go green…B-)

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Sendai don’t do it like they said

13 October, 2010

It is a sad fact of virological life that quite a lot of what we see, in the experiments we do, is artefactual: that is, the way we do experiments leads us to see results that do not necessarily reflect reality, but rather, the scenario we inadvertently selected for.

And it is electron microscopy that is at once our friend and our foe in this regard: over the last thirty years I have revised several aspects of my teaching on how virus particles interact with cells in particular, as what was once considered common knowledge has subsequently been proved to be false.  This is usually a consequence of having to use large numbers of virus particles – or high multiplicities of infection – and cultured cells, which may lead to rare events being selected for simply because they may be easier to detect.  An important example of this was the revelation that poliovirus (and presumably other picornaviruses) almost certainly enters cells via receptor-mediated endocytosis, rather than via some mysterious direct passage mechanism as is often depicted in textbooks (or here).

 


Paramyxovirus: image by Linda M Stannard

 

One of the long-time models for entry of enveloped viruses into animals has been Sendai paramyxovirus: this ss(-)RNA virus was supposed to fuse its membrane with that of the host cell, and uncoat via diffusion of its envelope glycoproteins into the host membrane, and deposit of virion internal components into the host cell cytoplasm.

Except, it turns out, that this is probably wrong: in a Journal of Virology Minireview published in July of 2010, Anne Haywood of the University of Rochester (NY, USA) describes how Sendai virions uncoat via a “connecting structure” that largely preserves the virion envelope.

Membrane Uncoating of Intact Enveloped Viruses
Anne M. Haywood
JOURNAL OF VIROLOGY, Vol. 84, No. 21, Nov. 2010, p. 10946–10955
Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously “uncoated” with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.

Interesting: a whole model for entry of viruses into cells was predicated upon the interactions of a  laboratory-derived virus strain which produced damaged particles.

Haywood presents a new model for virus entry, based upon the observation that “early harvest” virions differ substantially form the “late harvest virions” previously used, in that “…the RNP is regularly folded parallel to the long axis of the virions…”, while  late-harvest particles “…have RNP strands that are randomly distributed in the virus rather than regularly arranged in relation to the membrane”.

She goes on to review a qualitatively very different alphavirus – Sindbis virus, an enveloped ss(+)RNA virus – for which similar things had been claimed, and shows that virus particles that have been gently treated also make a connector.  Moreover, she says that:

“…there is a structure that has no electron-dense material and is released from the cell. It is identified as viral by antibodies conjugated with gold beads. This release of an empty viral membrane has not been noted before, but the use of labeled antibodies meant such a structure would be revealed. If the envelope membrane disengages from the cell instead of merging with the host membrane, then not only would the cell not have viral proteins on its surface until the virus replicates but the released membrane pieces could serve as immunologic decoys.” [my emphasis]

Interestinger and interestinger…so enveloped viruses may have an entry mechanism which serves to hide them more effectively than we knew – by keeping their membranes intact, and possibly even using them as releasable decoys?

I note that in the case of HIV – possibly the best-studied single organism on the planet just recently – it has also recently been shown that virions probably enter cells via endosomal vesicles.

I hear the grinding sound of a shifting paradigm, folks: time for a relook at some other cherished models, possibly??

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Something rabid this way comes

5 October, 2010

Rabies virus: also known more officially as

The relevant ICTVdB (Intl Comm on Taxonomy of Viruses Database) page describes the viruses as follows:

Rabies virus virion

Morphology

Virions consist of an envelope and a nucleocapsid. Virus capsid is enveloped. Virions are bullet-shaped. Virions measure 45-100 nm in diameter; 100-430 nm in length. Surface projections are densely dispersed, distinctive spikes that cover the whole surface except for the quasi-planar end. Capsid/nucleocapsid is elongated with helical symmetry.

Nucleic Acid

The Mr of the genome constitutes 1-2% of the virion by weight. The genome is not segmented and contains a single molecule of linear, negative-sense, single-stranded RNA. The complete genome is 11900 nucleotides long, is fully sequenced.

A description of the replication of these viruses is given here.

There has been a fair bit of media fuss here in South Africa recently – and in Gauteng in particular – about a rabies outbreak, and the need to get pets and possibly dependants vaccinated against the virus.

The urgency of this campaign was underlined by the recently reported death of a child, scratched by a rabid puppy.

The literature available locally to inform prevention is a bit dated – 1997 – but it is comprehensive and well-researched.  This is a PDF document available here; more recent material can be found at the CDC site.

Important points to note about rabies are the following:

  • If untreated, it is effectively 100% fatal in both susceptible animals and in humans
  • There are effective vaccines for the prevention of infection – veterinarians and staff working with animals are routinely vaccinated – and
  • There is an effective therapy for people already bitten, which involves the injection of anti-rabies antibodies

News currently coming out of Gauteng Province reported in Business Day indicates that this outbreak is the first in that province in many years, and that over R30 million (~US$4 million) will be required to stamp it out – with the requirement that >70% of Gauteng’s estimated 1,4-million cats and dogs be vaccinated, otherwise the disease could become endemic.

While the disease has been known for centuries, and vaccines and therapy date back to the time of Louis Pasteur, it is alarming to realise that, in the words of the CDC Rabies Homepage,

“…Rabies in humans is 100% preventable through prompt appropriate medical care. Yet, more than 55,000 people, mostly in Africa and Asia, die from rabies every year – a rate of one person every ten minutes.”

A horrific disease to die of, and relatively easily preventable.  We just need more and cheaper vaccines and therapy.  Roll on the plants…!

Oh, and simple common sense, and widespread compliance….

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