DNA vaccines in which the expressed protein is an immunogen have many benefits, as Amit Vasavada, director of process development engineering at Vical in the US, explains
Last year saw the first commercial approvals of plasmid DNA (pDNA) vaccines - one designed to protect farm-raised salmon from Infectious Hematopoietic Necrosis virus and the other to protect horses from West Nile virus. The vaccines have received considerable attention due to the simplicity of the technology, in comparison with traditional vaccine production methods. The technology involves the injection of engineered pDNA rather than proteins into humans or animals (see figure 1).
Plasmid DNA is a closed-loop double-stranded, non-chromosomal DNA that occurs naturally in bacteria. Bacterial pDNA carry genes for their own replication but often include genes essential for bacterial growth in diverse environments.
Using rDNA technology, gene coding for therapeutic proteins has been engineered into these plasmids, which replicate and express these genes to make proteins in bacteria.
For pDNA technology, genes critical only for replication in bacteria and those for protein expression in a target host cell are engineered. This ensures that therapeutic or antigenic proteins are produced only after pDNA injection into the host cells (see figure 2).
Typically for vaccine products, the gene of interest is "codon-optimised'in order to yield better expression, to eliminate elements potentially toxic either to the bacteria or the host, and to eliminate unnecessary portions of the antigen.
Production of recombinant proteins involves sizeable factories with multiple, large fermentors in which the microbes or mammalian cells are grown. Introduction of pDNA into living cells in higher animals causes those cells to produce, or "express," the encoded proteins, effectively allowing living cells in the body to be used as the protein factories.
But because, with this method, the biopharmaceutical product is the instructions for making proteins rather than the proteins themselves, production can be accomplished in much smaller facilities and at much lower cost.
Applications of pDNA currently under development include:
- vaccines for infectious diseases or cancer, in which the encoded protein is an immunogen;
- cancer immunotherapeutics, in which the encoded protein is an immune system stimulant;
- cardiovascular therapies, in which the encoded protein is an angiogenic growth factor.
A cell that has taken up the pDNA containing the expression cassettes of interest uses its normal machinery to read the genes and express the encoded proteins (see figure 3).
Depending on the nature of the antigen, how it is formulated, and how it is presented to the immune system, the result can be a humoral response via the B-cell pathway resulting in antibodies to the antigen, or a cellular response via the T-cell pathway which ultimately produces cytotoxic lymphocytes that are ready to kill the specific invader represented by the antigen. Either way, the presented antigen serves as a way to prepare the immune system to immediately fight the real disease at some time in the future.
Vical is developing a cytomegalovirus (CMV) vaccine product, currently in Phase 2 clinical trials, to combat the complications of CMV disease in hematopoietic cell transplants (HCTs). This vaccine consists of two codon-optimised components for mammalian expression. One is a truncated and secreted form of glycoprotein B (gB) and the other is a mutagenised form of pp65 that lacks a kinase domain.
The product design seeks to produce both antibody and cellular immune responses to protect HCT recipients from activation of pre-existing CMV disease in the subject or introduction of new CMV disease from the donor.
In another application, Vical's pandemic influenza programme is based on conserved proteins encoding hemagglutinin (HA), nucleocapsid protein (NP) and matrix 2 ion channel (M2) protein. The goal is to produce a vaccine that provides high-level protection against severe disease and mortality for any pandemic influenza strain and to reduce disease spread in the general population.
Selection of these antigens derives from attempting to circumvent the high mutation rate found in influenza. This is particularly important because in the event of a pandemic influenza crisis, the entire egg-based vaccine industry would not be able to produce enough vaccine before the pandemic had run its course. By using a pDNA vaccine based on conserved portions of the viral genome, vaccine could be stockpiled in advance, and could significantly reduce the morbidity and mortality associated with a pandemic.
rapid production
Plasmid DNA is typically produced using methods common to other biologically-derived products. Production is initiated with a homogenous preparation of the selected host E. coli cells, which are transformed with pDNA containing the expression cassette with the genes of interest. These are then grown up in media, combined with the cryoprotectant glycerol and stored frozen in a stock of vials called a Master Cell Bank (MCB).
As development proceeds, a manufacturer may take the decision to produce a Manufacturer's Working Cell Bank (MWCB) from the MCB. This produces a larger stock of homogenous source material that typically is sized to complete clinical trial production and initial commercial production. In this manner, an MCB can generate multiple MWCBs.
Production of pDNA is commonly performed in batch fermentation mode, but a high yielding fed-batch approach is also practiced. Since the pDNA is retained within the host cell during fermentation, recovery of the pDNA requires cell lysis to liberate the host cell contents. Typically, cells are lysed chemically although other methods, such as heat lysis, have been successfully introduced.
Following lysis, cellular debris is removed by centrifugation or filtration and the resulting pDNA-containing filtrate is then purified further using a combination of methods. The resulting drug substance is then further processed to produce a formulation optimised for a specific use.
scale-up
Plasmid DNA is unique in the biotech industry in that it has a "one size fits all" method of manufacture. Once a method is developed, it is typically applied consistently for subsequent processes. Drivers for this approach are such elements as cost of goods and intellectual property.
Typically, host cells are optimised for a given plasmid and there are variations in fermentation conditions but the fermentation equipment and purification process will essentially be unchanged for all plasmids.
This is quite different from other biopharmaceutical product manufacturing procedures, which generally require a product-specific production scheme - particularly important when considering an emergency situation such as a pandemic influenza crisis. Bacterial fermentation and purification capacity and expertise can be found on every continent.
drug formulation
Vical's initial work centred on the discovery that skeletal muscle could take up pDNA at fairly high levels, resulting in measurable gene expression without the use of adjuvants. Although this method is effective, we have found that in certain applications the use of specific adjuvants can be highly advantageous. Hence we have continued our work on the use of cationic lipids, which have been shown to enhance pDNA uptake and enhance humoral immune responses in vaccine applications.
We also use poloxamers - block copolymers that enhance T-cell immune responses. These differences in immune system response drove the use of a polox-amer formulation in our Phase 2 CMV vaccine, which is aimed at reducing the complications associated with CMV disease in hematopoietic cell transplant recipients. Conversely, we have chosen the patented use of the Vaxfectin cationic lipid mixture developed at Vical for our vaccine programme aimed at pandemic influenza.
improved stability
To anyone that has ever worked on DNA with polymerase chain reaction, it is quite apparent that the DNA molecule can withstand a lot of stress, particularly in the area of high temperature exposure, before it shows permanent degradation. Use of lyophilization (freeze-drying), typically a production rate-limiting step, is not needed to achieve acceptable stability. Stability for in excess of three years under refrigerated conditions has been demonstrated.
Although more work remains to be done, it is conceivable that a room temperature-stable pDNA product would be possible and this could have significant implications on cold-chain use in distribution, particularly for vaccine products in the developing world.
While the applications for pDNA technology are quite broad, infectious disease vaccines, especially those against emerging pathogens, represent a particularly promising field. Key attributes supporting such programmes include the ability to target specific features of a pathogen, to invoke a desired combination of humoral and cellular immune responses, to manufacture quickly and efficiently using standardised processes in geographically dispersed facilities, and potentially to ship and store at room temperature.
Vical is independently developing pDNA vaccines against CMV and pandemic flu and has completed Phase 1 testing of a vaccine against anthrax. The company is leveraging the technology through collaborations with the National Institutes of Health and corporate partners for the development of vaccines against HIV, Ebola, SARS, West Nile virus, and certain animal health applications.
The company has granted non-exclusive licenses for its DNA delivery technology to several leading academic research institutions, including Stanford University, Harvard University, Yale University, and the Massachusetts Institute of Technology. The academic licenses are intended to encourage widespread commercial use of the company's DNA delivery technologies in the development of new antibodies, vaccines, therapeutic proteins, and diagnostics.
With initial commercial approvals in 2005 of the salmon and equine pDNA vaccines, the technology has advanced from theory to reality. Further refinements and continued development are expected to translate into the initial approvals of human pDNA vaccines in the future. Recombinant DNA technology (rDNA) has introduced a number of new protein drugs over the past three decades to treat human and animal diseases.