The discovery in the early 1990s that administration in mice of plasmid DNA encoding both viral and nonviral antigens induced antibody responses held out the prospect of achieving broad immunogenicity using DNA vaccines without the safety issues associated with a replicating pathogen.
Despite this promise, the efficacy of first-generation DNA vaccines against HIV, HPV, and hepatitis was compromised by low antibody titers and sporadic immune cell responses. In addition, DNA vaccines face broader challenges encountered by other classes of vaccines such as manufacturing, scale-up, and purification, as well as vaccine resistance.
A number of companies are developing processes and technical platforms designed to circumvent these issues, with particular emphasis on improving the efficiency of delivery and uptake of vaccines by target cells, referred to as transfection, which can be confounded by differences in tissue and cell type accessibility between individuals, in addition to other factors.
Many of these companies were present at last month’s DNA Vaccines conference in San Diego. The meeting was sponsored by International Society of DNA Vaccines and organized by BioConferences International, a Mary Ann Liebert company.
“Vaccines have saved more lives than any other invention in human history”, explained J. Joseph Kim, Ph.D., president and CEO of Inovio Pharmaceuticals. “Conventional vaccines have been successfully used against the lower hanging fruit in terms of disease, and more complex diseases such as cancer represent both opportunities and challenges for DNA vaccines."
According to Dr. Kim, one of the biggest hurdles for DNA vaccines has been inducing sufficiently high immune responses for effective vaccination. “There are two issues at stake,” he said. “The first is to make plasmids more ‘people-friendly’ to optimize their expression once in the cell, and the second is to optimize the rapid transfer of the DNA across the cell membrane to avoid degradation by endonucleases.
Inovio’s approach in this area includes informatically assisted optimization of the coding sequence to expand immunogenicity across multiple targets and boost expression levels of immunogens in target cells, and to improve the delivery of the plasmid DNA into host cells to minimize exposure to extracellular nucleases. The development of one of Inovio’s candidate high-grade cervical dysplasia vaccines, VGX-3100, encoding the HPV16 and HPV18 E6/E7 antigens, incorporated a combination of these features to increase vaccine antigen immune potency.
“The VGX-3100 vaccine was developed using our SynCon platform and includes highly efficient leader and Kozak sequences,” said Dr. Kim. “We also introduced an endoproteolytic cleavage site to improve protein folding and cytotoxic T lymphocyte processing.”
In addition, Inovio has developed an electroporation-based delivery system that, according to Dr. Kim, allows for more efficient and safe targeting of the antigenic sequence to the target cell. “A low voltage electrical field is applied at the site of vaccine injection, causing the cell membranes to transiently realign into a more porous state,” he explained, adding that electroporation is the most efficient and safe mode of delivery of nucleic acids, requiring no additional chemicals, preservatives, or adjuvants.
Looking to the future, “Electroporation is not a static technology,” continued Dr. Kim. “We are developing new devices, referred to as surface electroporators, that sit on the surface of the skin, and a piezo-electric-based system that doesn’t even require contact with the skin.”
Annie DeGroot, M.D., CEO and CSO of EpiVax, highlighted immune escape as an important issue in the optimizing the amount of information that DNA vaccines deliver. Immune escape is thought to arise when an infectious agent or, in the case of cancer, a tumor, acquires a genetic profile distinct from that toward which a DNA vaccine was initially targeted, allowing it to escape the immune pressure of the vaccine.
“In response to the problem of immune escape, our approach at EpiVax has been to combine multiple key antigens or specific epitopes within these antigens, in a single recombinant vaccine,” noted Dr. DeGroot.
“We use in silico methods to optimize a gene sequence for improved vaccine expression,” added Lenny Moise, Ph.D., director of vaccine research at the company. “They include codon optimization and gene analysis, including 5´ end secondary structure, cryptic splice sites, human genome homology, bacterial promoters, eukaryotic promoters, inverted repeats, palindromes, tandem repeats, and nucleosome positioning.”
Similar to Inovio, Epivax’ transfection method is to rely on electroporation to enhance uptake to improve vaccine immunogenicity. “There are about 30 Phase I and II clinical trials using electroporation of DNA vaccines, more than any other delivery technology. It represents the most promising DNA vaccine delivery method with a path to the clinic,” said Dr. Moise.
Range of Technologies
Aldevron was the first company to produce a DNA vaccine used outside an experimental setting, a West Nile Virus DNA vaccine that has been used to protect endangered species.
“There are many reasons why so few cells are transfected by DNA vaccines,” said Michael Chambers, president and CEO of Aldevron. “One potential hurdle related to DNA vaccine manufacturing includes residual E. coli impurities like colanic acid,” referring to an exopolysaccharide common to many enterobacteria.
“These can cause toxicity and overall negative effects that hurt transfection efficiency,” he pointed out. In addition to impurities in the vaccine formulation, plasmid methylation patterns can also play a role in low transfection and expression. Other hurdles include issues related to vector size, regional micro environments around the DNA injection site, and the inherent toxicology of most compaction/transfection reagents.
“Aldevron has developed or in-licensed advanced manufacturing and purification systems to increase in vivo transfection efficiencies,” said Chambers, citing the use of Nature Technology’s HyperGro plasmid fermentation technology that enables them to produce large amounts of ultrapure DNA vaccines.
“Aldevron is also working with companies like Sekris Biomedical to develop new host strains that enable scientists to fine-tune the methylation patterns of the plastids they use for immunization,” he added.
Finally, Aldevron’s genetic immunization and antibody service allows our clients to try multiple delivery technologies like Ichor’s TriGrid electroporation system and Pharmajet’s needle-free delivery system to find the optimal way to administer their vaccines,” he noted.