A hothouse for drug r&d

Growing vaccines in fertilised chicken eggs is expensive and time-consuming and involves educated guesswork regarding the strain of virus to be vaccinated against. In contrast, plants can be used to produce large quantities of vaccines on a much shorter timescale, and because the virus-like particles (VLPs) produced lack the core genetic material of the virus, they are non-infectious and unable to reproduce

Medicago operates a 14,000 ft2 production facility in Quebec City, which includes a 10,000ft2 biosafety level 2 greenhouse

Vaccines are traditionally grown in fertilised chicken eggs in a process that takes several months. Now researchers are looking at the plant world as an alternative process route. Andy Sheldon, ceo of Medicago, explains why.

The first avian flu virus to infect humans arose in Hong Kong in 1997; hundreds have since become sick with the virus, and 60% of those who have fallen ill have died. Meanwhile, it took about eight months to deliver the first meaningful doses of H1N1 vaccines to fight the swine flu epidemic that swept the US in 2009, infecting millions of Americans. What was the nature of the delay?

Traditionally, vaccines are grown in egg culture, a process that is time-consuming and expensive. In the case of influenza vaccines – which must be produced every year to adapt to the changing strains of the virus – researchers must guess which strains of influenza to include in the vaccine based on the best information available about what to expect for the autumn and winter flu season.

The production of vaccine starts months ahead of time, so that a sufficient amount of vaccine is available for distribution prior to the start of flu season. If a novel or alternate strain emerges, it is usually too late to incorporate that strain in the vaccine, and as a result many individuals may fall victim to the new strain.

Québec-based Medicago believes a better solution is available. Instead of employing chicken eggs to produce vaccines on a scale of months, plants – such as the tobacco relative Nicotiana benthamiana – can be made to grow large quantities of vaccines on a timescale of as little as three weeks.

Plants – such as the tobacco relative Nicotiana benthamiana – can be made to grow large quantities of vaccines on a timescale of as little as three weeks

Manufacturing plant-based vaccines is a process involving several steps. At Medicago, it begins with synthesising the DNA sequence from the major surface protein of the latest flu strain, the haemagglutinin: the ‘H’ in a flu strain name. The mature tobacco leaves are then dipped into a solution containing Agrobacterium, an organism that easily transfers DNA between itself and the plants. The organism is engineered to then move the ‘H’ DNA sequence into the plant cells without transferring its own genetic material and compromising the purity of entities known as Virus Like Particles (VLPs), which are key to the process and are explained in more detail later.

The tobacco leaves stay in the solution for two minutes under vacuum to remove air, soaking up the bacteria particles in between the plant cells when the vacuum is broken, the bacteria pushing the virus DNA coding for the flu protein into the cells for replication. The plants are then incubated in a greenhouse for about six days, producing substantial amounts of VLPs in the tobacco leaves.

At the time of writing, more than 100 recombinant proteins, including vaccine antigens and antibodies, have been expressed in different plant tissues, including those of Medicago.

VLPs represent an emerging vaccine technology for developing effective and long-lasting protection. They consist either of protein or lipid shells studded with short strands of the proteins specific to whatever disease the vaccine is intended to address. VLPs are made to look like a virus, allowing them to be recognised readily by the body’s immune system; however, they lack the core genetic material, making them non-infectious and unable to replicate.

In contrast to traditional influenza vaccines that are manufactured with an inactivated virus, VLP-based vaccines do not require an actual sample of the virus ­– they simply require the genetic sequence of the virus or bacterium.

VLP-based vaccines have also been shown to provide protection against different strains of a virus other than those for which the vaccine was formulated

A separate advantage of VLPs is that they appear to activate more effectively key aspects of the immune response to achieve potent immune stimulation and to provide immunological memory. VLP-based vaccines have also been shown to provide protection against different strains of a virus other than those for which the vaccine was formulated. As mentioned earlier, with many different strains of influenza viruses to address, the previous year’s vaccines produced in eggs frequently cannot be used. VLP technology enables the development of a vaccine that precisely matches the particular influenza strain in circulation in a timely fashion, and has the potential to offer strong cross-protection against other subtypes of a virus.

Following incubation in a greenhouse for about six days, the tobacco plant leaves produce substantial amounts of VLPs

Charles Arntzen, currently co-director of the Center for Infectious Diseases and Vaccinology, The Biodesign Institute and Florence Ely Nelson Presidential Chair at the Arizona State University, is a pioneer in research on vaccines derived from plants. It was his research group that proved HBsAg – the surface antigen of the Hepatitis-B virus – derived from plants was identical both antigenically and physically to HBsAg particles produced using human serum and recombinant yeast. This was evidence that transgenic plants could be used to produce vaccines.

Similarly, researcher T. A. Haq and his group showed that E.coli LT-B (heat-labile enterotoxin) made in tobacco and potato plants were identical physically and functionally to E.coli-produced protein. The first expression of vaccines in plants was carried out in 1990 by Curtiss and co-workers with the expression of the SpaA surface protein antigen of S.mutans in tobacco.

At the present time, a variety of research programmes are underway to develop and commercialise the technology of plant-based vaccines. For example, Medicago uses tobacco plants to express its VLPs, which are then purified to produce the vaccines. The company’s technology can deliver a vaccine for testing in less than a month following the identification and reception of genetic sequences from a pandemic strain. The company has already obtained positive Phase II data for a pandemic influenza vaccine and positive Phase I data for a seasonal flu vaccine. It has developed two proprietary technologies for the development of vaccines: its plant-based Proficia technology and its VLPs. Proficia is a vaccine and antibody production system based on transient protein expression in plant leaves.

Medicago's technology can deliver a vaccine for testing in less than a month following the identification and reception of genetic sequences from a pandemic strain

Having previously signed a US$21m (€16.5m) Technology Investment Agreement with the US Defense Advanced Research Projects Agency (DARPA) to demonstrate the scalable manufacturing of its plant-expressed VLP vaccines, Medicago has developed a vaccine facility covering more than 97,000ft2 in Research Triangle Park, North Carolina. This state-of-the-art facility is a large, cost-effective and scaled-up facility for VLP plant-based vaccine technology, ultimately for the delivery of current good manufacturing practice (cGMP) grade vaccines.

The company intends to demonstrate its capacity to produce 10 million doses/month of influenza vaccines, with the potential for further expansion in the future. This DARPA project is part of the Blue Angel influenza vaccine rapid response demonstration project, which seeks to identify novel techniques for producing large amounts of high-quality vaccine-grade protein in less than three months in response to emerging and novel biologic threats.

In addition to this new facility, the company operates a 14,000ft2 production facility located in the Parc Technologique du Québec Métropolitain in Québec City, Canada. This facility includes a 10,000ft2 biosafety level 2 greenhouse and a 3,000ft2 extraction and primary purification unit that is cGMP compliant. Medicago also rents approximately 13,000ft2 in Québec City, of which 2,500ft2 are dedicated to the final steps of purification under cGMP conditions, and the remaining is for storage and office space.

The company had a particularly busy year in 2011. In January, it announced the publication of its Phase I study results with its pandemic H5N1 influenza VLP candidate. This was the first time a scientific report on a clinical trial for a plant-based influenza VLP vaccine in humans was published in a peer-reviewed journal. The same month, the company was also selected to collaborate with the Infectious Disease Research Institute on a multimillion-dollar grant awarded to IDRI from the US Department of Defense. The purpose of the collaboration was designated as the proposed development of a single-dose H5N1 influenza vaccine that could be rapidly and widely administered in the case of avian pandemic flu outbreak.

In February, Medicago reported positive Phase II interim results for its avian flu pandemic vaccine, and also received regulatory clearance from the US Food and Drug Administration to initiate a Phase I clinical trial in the US for an H1N1 VLP vaccine candidate. Phase I clinical testing of its H1N1 VLP vaccine commenced in March. The following month, the company announced a research collaboration for the development of a non-influenza vaccine candidate with a top 10 global pharmaceutical company (the first stage of this collaboration was completed seven months later).

May saw the company announce a research collaboration for the development of a VLP vaccine candidate for the prevention of Ebola with the US Army Medical Research Institute of Infectious Diseases and in June the company reported positive US clinical trial results for its H1N1 seasonal influenza vaccine, as well as positive phase II final results for its avian flu pandemic vaccine. It commenced operations at its North Carolina vaccine facility in September.

innovation driven

The company’s programme of innovation has continued through 2012. In January, it signed a research agreement with Cellectis plant sciences, a subsidiary of Cellectis SA, the French specialist in genome engineering, to collaborate to improve therapeutic proteins expressed from tobacco leaves. Cellectis manufactures DNA scissors called nucleases that can cut precise DNA sequences, allowing for the creation of a wide range of specific tools to modify a target gene. Cellectis’s nucleases have the potential to enable the modification of protein glycosylation patterns in plants with unprecedented control and uniformity, allowing for increased efficacy of therapeutic products.

In addition, the company announced that it had successfully completed initial studies towards the development of a new VLP vaccine candidate for rabies. Results with the rabies VLP vaccine demonstrated that two doses of 1 or 4µg induced protective levels of neutralising antibodies in a mouse model.

It expects to move ahead with GMP process development and a GLP toxicology study in 2012, and following this, a Phase I clinical trial.

It has also announced plans to invest approximately C$4m (€3.1m) to enhance the capacity of its pilot production facility located in Québec City. This investment is to accelerate preclinical and clinical development timelines of future product candidates.

Eventually, due to the need for low-cost vaccines that can be rapidly produced, much of the world’s vaccine manufacturing capacity may be plant-based

The company has also recently established a strategic alliance with Mitsubishi Tanabe Pharma Corporation to develop and commercialise at least three new vaccines. Medicago is eligible to receive up to C$33m (€25.5m) in upfront and milestone payments for the first target, a rotavirus VLP vaccine candidate. Mitsubishi Tanabe will fund all r&d costs, as well as regulatory and commercialisation responsibilities. Royalty payments on future sales will also be due. Agreements for the additional two vaccines will be decided upon in the next three years.

Vaccines are currently a US$20bn per year industry, with many analysts projecting growth to $35bn over the next five years. However, only a few major pharma firms currently manufacture the majority of the world’s vaccine supply, which is one of the reasons that governments and philanthropies are sponsoring alternative manufacturing systems. Eventually, due to the need for low-cost vaccines that can be rapidly produced, much of the world’s vaccine manufacturing capacity may be plant-based.