Posts Tagged ‘VLP’

Recombinant Bluetongue virus vaccines – or some, anyway

1 May, 2014
VIRUS-rota-200

General model of reo-like viruses. Copyright Russell Kightley Media

I picked up yesterday – via @MicrobeTweets’ Twitter feed – on a very useful list of papers in a “Virtual Special Issue” of Elsevier’s recent coverage of vaccines – for “World Immunization Week”. Great stuff, I thought to myself, as I browsed the list – and downloaded at least those that were Open Access, or which I can get via our Libraries’ IP range.

“Even better!”, I thought, as I saw a review entitled “Recombinant vaccines against bluetongue virus?”  A meaty, well-sourced review, I thought; good reading for me and my students / coworkers, and good meat for upcoming Introductions for papers yet to be written.  Indeed, it promised the following:

“The multiple outbreaks of BTV in Mediterranean Europe in the last two decades and the incursion of BTV-8 in Northern Europe in 2008 has re-stimulated the interest to develop improved vaccination strategies against BTV. In particular, safer, cross-reactive, more efficacious vaccines with differential diagnostic capability have been pursued by multiple BTV research groups and vaccine manufacturers. A wide variety of recombinant BTV vaccine prototypes have been investigated, ranging from baculovirus-expressed sub-unit vaccines to the use of live viral vectors. This article gives a brief overview of all these modern approaches to develop vaccines against BTV including some recent unpublished data.”

So, I parked the conveniently Open Access-ible window away on the side of my desktop, to be got back to with every expectation of delight.

Until I read it, that is: well-sourced it may be; excellent in its coverage, it is NOT.  In fact, apart from a brief discursion on subunit vaccines – concentrating almost exclusively on baculovirus / insect cell-produced proteins – it is almost exclusively concerned with live viral vectors for bluetongue proteins, and of poxviruses in particular.  Now, this is all very well, if that is what they work on – but to dismiss one of the potentially most exciting developments in recent Bluetongue vaccinology like this:

“VLPs of BTV have been also produced in plants recently using the cowpea mosaic virus and their use in a vaccination study produced no clinical manifestations in sheep after homologous challenge, although viremia was no [sic] evaluated (Thuenemann et al., 2013).”

– boggles the mind somewhat.  Really?  That’s all they have, compared to the screed immediately before it on baculovirus-produced antigens?  They get the expression system wrong – it is an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana involving a Cowpea mosaic virus-derived enhanced translation vector – and neglect to mention that the VLPs produced are as good as anything produced in insect cells; will be FAR cheaper to produce, and WORKED AS WELL AS THE CONVENTIONAL ATTENUATED LIVE VIRUS VACCINE IN A CHALLENGE EXPERIMENT IN SHEEP.  True!

This is a big deal, folks, really: successful production of significant amounts of VLPs requiring simultaneous expression of 4 structural proteins of BTV-8 in plants AND their subsequent assembly, AND performing as well as the standard vaccine in an animal trial.  But no – not good enough for our review’s authors….

I must declare vested interests up front here: first, we work on plant-made recombinant Bluetongue vaccines; second, I and others in my group are co-authors of the paper whose lack of coverage I am aggrieved about.

But that’s not the point: what IS the point is that this review is a slipshod piece of work that damns our collective endeavour with faint praise, in community that might otherwise have been alerted to an alternative to the far-too-expensive-for-animal-use baculovirus expression technology.

Ah, well.  I suppose that’s what blogs are for B-)

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Virus-like particle and Nano-particle vaccines 2012: a conference report

30 January, 2013

Alta van Zyl, Virology Group, Molecular & Cell Biology Department, UCT

Introduction:

VLP flusm

Haemagglutinin-only Influenza A virus VLP. Courtesy of Russell Kightley Media

The new international conference on virus-like particles and nano-particles (VLPNPV) took place in Cannes, France at The Novotel Montfleury Hotel from the 28th to the 30th of November 2012.  The scope of the conference included virus-like particles (VLPs), the plant-based expression of VLP vaccines as well as expression and optimisation of VLPs.

Other topics included in the conference were:

  • VLP platform delivery systems
  • VLP vaccines
  • Nano-particles and nano-particulate vaccines

A multitude of topics were covered during the conference and many of the talks pertained to the immunogenicity of the VLPs and nano-particles and how they compared with the immunogenicity of DNA or subunit vaccines.

Talks were given by researchers from companies such as Medicago, Mucosis, Pevion Vaccines and Novavax. These talks gave a perspective on factors that need to be considered when commercialising VLP/nano-particle vaccines and to be GMP compliant.

Compelling presentations:

Developing plant-made virus-like particle vaccine products: An integrated platform from discovery to commercial scale

Marc-Andre D’Aoust, Nathalie Landry, Sonia Trepanier, Michele Dargis, Manon Couture and Louis-Philippe Vezina (Medicago, Quebec City, Quebec, Canada)

This talk was about a plant-made VLP against both pandemic and seasonal influenza- these vaccines are now in the clinical trial phase. What was especially interesting was the view from an industry point of view where expression had to be scaled up to produce large amounts of vaccine.  The Medicago platform can synthesize and clone approximately 100 gene constructs in two weeks, they can prepare 100 bacterial cultures per week and they have automated infiltration where 200 plant transformations can be performed per day and 150 VLP engineering approaches can be tested in one week.  For influenza Medicago tested 48 different infiltration approaches in one day for HA, NA, M1, M2 as well as P1 Gag and HGalT.  Medicago has been able to produce 10 million doses of HA VLPs in just one month.

See also: 

  • D’Aoust et al (2010) PBJ 8:  607-619 – The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza.
  • http://www.medicago.com

Development of RNA-free plant VLPs a source of novel therapeutics

George Lomonossoff (John Innes Centre, Norwich, UK)

This group made empty Cowpea Mosaic Virus (CPMV) VLPs that contained no RNA.  CPMV VLPs are versatile nanoparticles to which organic, inorganic and biological molecules can be bound.  The empty nature of the particle means that they can be used as carrier molecules for therapies; this could prove to be potentially useful as a cancer-treatment therapy.  The system is advantageous because of the lack of RNA which makes the particles non-infectious and no bio-containment is needed for the production of these VLPs.

Immunogenicity of VLPs: an immunological perspective

Martin Bachmann (University of Zurich, Zurich, Switzerland)

Background was given from immunological point of view about what makes VLPs so immunogenic. Three properties contribute to the immunological properties of VLPs (1) their size, (2) the repetitiveness of the particle capsid which provides multiple sites for antibody binding and (3) TLR ligands – the particle can be disassembled, the RNA removed and replaced with a TLR ligand to enhance immunogenicity. Also, the size of VLPs is optimal for drainage to the lymph nodes.

Immunogenicity optimization strategies for public-sector development of vaccines: the critical role of optimizing the antigen.

Martin Howell Friede (WHO, Geneva, Switzerland)

This talk was about looking at VLPs from the vaccine development view.  Monomeric antigens are not very immunogenic; therefore adjuvants were developed and came into use. For an efficient vaccine the antigen must be multimeric as antigen alone is insufficient to be immunogenic without adjuvant. Two factors have to be considered when producing a vaccine for FDA approval; (1) optimise the antigen before using an adjuvant, (2) use an adjuvant that has already been approved by the FDA. VLPs as vaccines provide the potential for immune-stimulation without the addition of adjuvant as the multimeric presentation of the antigen will enhance its immunogenicity.

Enhancing the immunogenicity of VLP vaccines

Richard W. Compans (Emory University, Atlanta, Georgia, USA)

This talk highlighted strategies which could be used to enhance the immunogenicity of VLPs.

  1. Look at alternate routes for vaccine delivery (intranasal, intramuscular, subcutaneous etc)
  2. Increase the breadth of immunity by enhancing responses to conserved antigens/epitopes
  3. Increase the amount of antigen incorporated into VLPs
  4. Incorporate the adjuvant into the VLPs as part of the structure

See also:

  • Ye et al (2011) PLoS One 6(5):  e14813
  • Wang et al (2008) J Virol

Innate and adaptive responses to plant-made VLP vaccines

Brian Ward (McGill University, Montreal, Quebec, Canada)

Brain Ward is also the medical officer at Medicago.  Humans rarely react to plant proteins/antigens. The plant glycans fucose/xylose at the N-terminal is an allergen and can cause anaphylaxis in humans. During trial experiments with influenza no individuals developed IgE responses to plant glycans, therefore plant produced vaccine is safe. The H1 VLP induced long lasting memory multifunctional T-cell responses in humans.

Impressions of the conference:

The conference was well organised with leaders in the field presenting their work. Interaction with the delegates aid in building crucial networking opportunities and work relationships. The international arena is packed with new technology development allowing us the opportunity to learn and improve our own understanding of various concepts.

This conference proved to be an invaluable learning experience and I thank the NRF for this opportunity and for providing me with the funding to attend this conference.  The exposure to conferences, especially those in the international arena, aid in the development of students and contribute to the quality of research that is conducted at UCT.

References:

1. VLPNPV website

(http://www.meetingsmanagement.co.uk/index.php?option=com_content&view=article&id=33&Itemid=83)

2.  Personal notes taken at the conference

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-)