Following the 2009 outbreak of the H1N1 pandemic flu and the numerous delays in producing vaccines against the virus, the US Department of Health and Human Services (HHS) recognized the need to invest in new vaccine technologies that can ensure national preparedness for a pandemic influenza or other diseases. Many other countries are focused on developing similar capabilities. There is also a significant requirement for therapeutic vaccines to treat existing and prevent further chronic viral infections. As a result, there is an urgent need to develop alternative vaccine production methods that can either generate large quantities of vaccines in a much shorter time and/or produce more broadly effective vaccines than is possible using traditional egg-based technology. Examples include cell-culture, synthetic DNA, chimeric antigen, and recombinant protein nanoparticle vaccine technologies.
Egg-vaccine technology limitations
Traditional vaccine manufacture begins with a “seed” virus identified and provided by the Centers for Disease Control and Prevention. This virus is introduced into fertilized chicken eggs. It then reproduces and builds up in the white (allantoic fluid) of the egg, which is collected and purified. It takes two eggs to generate enough vaccine for one dose, and thus large numbers of eggs must be produced in advance, which is always a challenge. The limited availability of eggs prevents the rapid production of vaccines, which is a major concern in a pandemic situation. In fact, it takes many months to organize egg supplies, incubate the virus, and obtain produced vaccine that can be delivered, according to Novartis.
Another difficulty with this approach is the fact the seed virus is not always accurately replicated, and as a result, the virus in the vaccine may not be the same as the infective strain, which thus provides reduced immunity. Extensive DNA and protein analysis is required to help avoid this problem, and further testing is conducted to ensure that no pathogens from the eggs are transferred to the vaccine. All of this testing extends the production time and increases the manufacturing cost.
Cell-culture technology
Cell-culture technology enables the use of raw materials that are readily available and not threatened by pandemic events, as well as closed-system bioreactors that reduce the required biosafety level for the manufacturing space. In addition, it is possible to provide rapid response to potential pandemic influenza threats while fulfilling the need for seasonal influenza vaccines, according to Novartis. The company received the 2013 Facility of the Year Award issued by the International Society for Pharmaceutical Engineering (ISPE) for its flu cell-culture technology, which it has implemented at a new facility in Holly Springs, North Carolina.
In cell-based flu culture, immortalized canine kidney cells are typically employed; these cells can be stored in advance until needed and then rapidly amplified, enabling the production of large quantities of vaccine in a much shorter period of time than is possible using fertilized eggs. Cell-culture technology also enables more robust virus production, and therefore, the virus in the vaccine is more consistent and more closely resembles the seed strain, which leads to increased efficacy. In addition, the size of the bioreactors required to produce large numbers of doses is much smaller than the space required to produce a similar number of doses using eggs. Furthermore, the use of bioreactors ensures a closed, sterile, controlled environment, and thus the risk of potential impurities is reduced. As a result, Novartis’ Flucelvax vaccine does not contain any preservatives, such as thimerosal, or any antibiotics. Finally, unlike vaccines produced in fertilized eggs, cell-culture-derived vaccines can be administered to patients who are allergic to eggs.
Development of Novartis’ technology and construction of the plant were funded in part by the HHS Office of the Assistant Secretary for Preparedness and Response and the Biomedical Advanced Research and Development Authority. The Holly Springs facility has the capability to produce seasonal flu cell-culture vaccine, pre-pandemic vaccine, and 150 million doses of pandemic vaccine within six months of an influenza pandemic declaration, according to Novartis. A fill-finish set up for the production of both flu and non-flu products has also been installed in the facility. The company received FDA approval for Flucelvax, the first cell-culture vaccine in the US designed to protect against seasonal influenza in individuals 18 years of age and older, in November 2012 and made its first shipments in August 2013.
Synthetic DNA vaccines
One of the biggest drawbacks of traditional vaccines is that for diseases that change rapidly and evolve into many different strains, the genetic makeup of the antigen introduced to the body by the vaccine may be different than what it encounters in an actual pathogen in the future, according to J. Joseph Kim, president and CEO of Inovio Pharmaceuticals. “Since the immune system isn’t looking for the mutated version of the antigen, it may not be able to prevent or fight an infection,” he explains.
To address this problem, Inovio has developed synthetic DNA vaccines based on the genetic codes of viruses. “We can consciously manipulate and engineer protein sequences that are flexible and can recognize multiple viruses in a particular subfamily, which provides more comprehensive protection and is a more universal approach to the development of influenza and other vaccines,” Kim states.
The synthetic DNA is encoded with instructions that enable cells in the body to produce only the targeted antigen relating to the pathogen or cancer of interest and cannot replicate or cause the disease. “This approach results in the body creating an immunogen capable of inducing strong, multi-faceted immune responses similar to actual pathogens,” Kim observes. As a result, the vaccines generate very strong T-cell immune responses, and particularly T-cells that are able to kill the targeted infected or diseased cells.
In animal models and early clinical studies, Inovio has shown that its H1N1 vaccine provides protection against all strains of the H1N1 virus identified over the last 90 years (since the 1918 Spanish Flu). “We believe the synthetic DNA vaccines are a paradigm-changing technology,” says Kim.
There are other advantages to the technology. The synthetic DNA vaccines are produced in pure water and do not contain any live virus, parts of a virus, adjuvants, or preservatives and are thermostable (i.e., no cold-chain requirements). The DNA plasmids are manufactured using conventional fermentation technology (i.e., no eggs) that is scalable, is engineered for maximum expression in the host cells, and can be rapidly produced in large quantities in a pandemic situation. Delivery is achieved through injection followed by in vivo electroporation, which involves application of a brief low-voltage electric field (three pulses of 0.05 s) that causes the cell membrane to open and allow entry of the DNA plasmid.
In addition to its H1N1 vaccine, Inovio has a therapeutic vaccine for cervical cancer in Phase II trials. It currently has a partnership with vaccine manufacturing company VGXI for clinical trial quantities, and will be selecting a contract manufacturer for the production of Phase III and commercial quantities later in 2014. Inovio is also participating in a National Institutes of Health malaria vaccine initiative focused on the development of novel technologies and is developing new candidate DNA vaccines for various cancers and other diseases, particularly prostate cancer and hepatitis B, which are being pursued in conjunction with Roche.
Mimicking natural antigen presentation pathways
The Chimigen vaccine technology developed by Akshaya Bio is based on chimeric antigens that bind to specific receptors on antigen-presenting cells, particularly dendritic cells (Fcγ and Lectin receptors), and mimic natural human antigen presentation pathways to generate antigen-specific, balanced, cellular (for clearing virus-infected and cancer cells) and humoral (antibody-mediated) immune responses, according to president and CSO Rajan George. As a result, the vaccines generate broad immune responses, “re-educate the immune system,” and “break tolerance” to chronic, infections and cancer. “Because a Chimigen vaccine has the characteristics of both an antigen and antibody in a single entity (i.e., it is a fusion protein), the technology is versatile and highly adaptable to disease-specific multiple molecular antigens and can be used to develop both prophylactic vaccines and immunotherapeutic agents,” he notes. The level of each type of response depends on the type of antigens used in the vaccine.
George adds that the incorporation of a xenotypic antibody fragment makes the entire molecule “foreign” and thus more immunogenic. Production of the vaccines in insect cells is also beneficial, because they impart non-mammalian glycosylation, thus increasing the immunogenicity and enabling the vaccines to be effective at low doses, according to George. Furthermore, adjuvant-related adverse reactions are not an issue with this technology, and cellular responses are promoted because no adjuvants are used in the vaccine formulations.
The major technical challenge that Akshaya Bio is currently tackling relates to the production of the vaccine. “In heterologous protein production, the quantity of protein produced depends on the type of antigen (intracellular/extracellular) and the size of the protein molecule,” George explains. To date the company has been able to produce ~5-7 mg/L for vaccines with a size of ~75 kilodaltons (KDa) to ~3-5 mg/L for vaccines with a size large than ~250 KDa. Due to the high immunogenicity of Chimigen vaccines, however, George notes that the lower production levels are still economical.
The company’s lead candidate is a therapeutic vaccine for the treatment of chronic hepatitis B virus (HBV) infections, for which there is currently no effective treatment. The Chimigen HBV vaccine is in late pre-clinical development, having achieved ex vivo proof of concept and is ready for out-licensing to a pharmaceutical/biotech partner for clinical development, according to George. The vaccine also has prophylactic application in non-responders to currently available vaccines. Akshaya also has a Chimigen vaccine for hepatitis C virus (HCV), for which there are some newer treatments that are effective, but a vaccine is desirable for preventing new infections. The Chimigen HCV vaccine is ready for clinical development and Akshaya is looking for partnership opportunities.
Additional earlier-stage pipeline products include products for various cancers, influenza, malaria, and HIV. The HIV vaccine, which is being tested in animals to evaluate immune responses, is being developed with support from a Government of Canada/Bill & Melinda Gates Foundation partnership and the National Resource Council Canada Industrial Research Assistance Program. The Chimigen malaria vaccine was initially developed using a Grand Challenge Exploration award from the Gates Foundation.
“The short-term goal is to establish proof of concept in humans for at least one of the Chimigen vaccines. In the long-term, we look forward to Akshaya’s products helping to alleviate suffering due to infectious diseases and cancer,” George remarks.
Flexibility and speed
Novavax’s recombinant protein nanoparticle vaccine technology combines the flexibility and speed of genetic engineering with the efficiency of single-use disposable technology to produce highly immunogenic nanoparticle vaccines, according to the company, which is developing vaccines against viral, bacterial, and parasitic diseases.
With the Novavax technology, the genetic code of a virus of interest can be used to produce, within a few weeks, a vaccine candidate that is designed to generate protective immunity for that specific virus. Two different types of immunogenic particles are used: virus-like particles (VLPs) and recombinant protein micelles. Novavax’ seasonal and pandemic influenza vaccines consist of VLPs or recombinant particles with matrix proteins that provide a structure onto which the surface proteins hemagglutinin and neuraminidase are incorporated. “VLP constructs resemble the virus they are designed to protect against; however, because they do not contain any RNA, they are not infectious, and thus are generally highly immunogenic,” states Novavax’s vice-president of Vaccine Development, Gale Smith.
The recombinant protein micelles are generally composed of a single target protein, engineered to assemble into stable nanoparticles that elicit an immunogenic response like the virus itself. For example, Novavax’s Respiratory Syncytial Virus (RSV) vaccine candidate is composed of recombinant micelles engineered as modified full-length fusion (F) proteins with the potential to induce protection against all strains of RSV, according to Smith.
The vaccine nanoparticles are produced in Sf9 fall army worm (Spodoptera frugiperda) ovary cells, which grow in perpetuity in special culture media. The cells produce recombinant proteins when infected with an insect virus (Baculovirus or BV) engineered to carry the foreign gene or genes of interest. “The Sf9/BV genetic engineering technology is now well established in the biopharmaceutical industry, and has for example been used for the production of a licensed human papilloma virus vaccine and a recently licensed influenza subunit vaccine,” Smith observes. In addition, Sf9/BV efficiently expresses large antigens and particles with proper folding, which promote superior immunogenicity and better functional immunity. Smith also notes that the adoption of single-use manufacturing technology accelerates process validation and analytical testing for Novavax’s vaccines and may allow for ultimate regulatory approval of the company’s vaccines derived from a common platform.
Novavax has also produced new vaccine candidates for the prevention of SARS and the newly emerged MERS-CoV virus, malaria, rabies, and other diseases where new or improved vaccines are needed.
About the Author
Cynthia A. Challener is a contributing editor to BioPharm International.