Vandalia Research has developed the Triathlon, a system and process for performing the polymerase chain reaction (PCR) at the multiple-liter scale. This technology can produce fragments of DNA in a range of 50-5000bp for a variety of applications, including DNA vaccines. Our current goal is to produce a rapid response capability DNA vaccine process to use as a prophylactic for millions of people with very short turnaround times.  As has been well-documented, currently licensed vaccines have a number of limitations. They are often dependent on eggs for production, and the manufacturing cycle is long (at least six months). Attenuated vaccines could revert back to virulence. Cell culture-derived vaccines are also expensive, requiring high levels of sterility and skilled workers.  In 2004, Chiron identified bacterial contamination in some batches of the influenza vaccine, resulting in shortages.

DNA Vaccine Technology

The efficacy of DNA vaccines has been demonstrated against infectious diseases caused by viruses, bacteria, and parasites. They have been widely used in preclinical animal models and entered clinical trial testing in humans against HIV, malaria, tuberculosis, influenza, hepatitis B and C and CMV, and also have been used as potential immunotherapeutics against several cancers.DNA vaccines induce both humoral and cytotoxic T-cell responses. No dangerous infectious agents are involved, and DNA vaccines are very stable compared to existing vaccines. DNA vaccines are non-living, non-replicating, and non-spreading. The induction of immunity by DNA was even shown in newborns that have high levels of maternal antibodies that usually neutralize conventional vaccines. A single vaccine can be formulated against several diseases by encoding multiple immunogenic epitopes. One of the major advantages is that DNA vaccines can be quickly developed once the gene has been identified. This factor can be critical for controlling dangerous emerging pathogens (e.g. SARS or pandemic influenza) or bioterrorist attacks.

Most DNA vaccines today are plasmid constructs and have a number of limitations.About 75% of cytosine-phosphate-guanine (CpG) dinucleotides in mammalian DNA are methylated at cytosine residues, but are mostly non-methylated in bacteria. (Bird, 1985, Nature). The non-methylated CpG motifs in bacterial and plasmid DNA stimulate immune response in mammals and may lead to auto-immune diseases (Krieg, 2002, Ann. Rev. Immunol.).  Methylation of these motifs or the reduction of their number in plasmid DNA can reduce such immune responses. Plasmid DNA vaccines can potentially spread environmentally by shedding or consumption of vaccinated animals. There is a concern that integration of plasmid DNA into the host genome can cause insertional mutagenesis which can be especially dangerous if insertion occurs in or near an oncogene. One of the major safety concerns is the insertion of the antibiotic resistance genes present in all conventional plasmids into humans or animals. Plasmids are usually propagated in bacteria, therefore bacterial remnants such as lipopolysaccharides or toxins can be inadvertently present in the vaccines causing undesirable side effects.

Linear Expression Cassettes

An alternative to the circular DNA plasmid is a linear expression cassette, or LEC. These constructs are easily produced using the polymerase chain reaction, or PCR. Linear DNA molecules, being smaller in size and more flexible may more easily pass through nucleopores. (Salman, 2001, Proc Natl Acad Sci). One of the problems of plasmids is low and transient transgene expression, which is attributed mostly to the large size of plasmid DNA. When electroporation was used as a delivery method, PCR fragments showed better expression compared to the plasmids carrying the same gene. (Hirata, 2007, J. Pharmaceut Sci.). Linear DNA made by PCR products can be designed to lack any sequence not required for gene expression in the eukaryotic cells. The linear topology avoids coiling and yields a conformationally homogenous final product. It is also easier to control the immunostimulatory (CpG) sequence motifs in the vaccine. One known problem for linear DNA vaccines is that they can be a target to exonuclease activity, but proper protection via the use of phosphorothioate-modified primers (Johansson, 2002, Vaccine) or ligation of hairpin oligonucleotides to the end on linear molecule(Schakowski, 2001, Mol. Ther.) can greatly increase DNA stability. Linear DNA vaccines have also been utilized against feline immunodeficiency virus and leishmaniasis and induced strong antibody response in mice.

A large obstacle to using LECs in the past has been a scalable, inexpensive, and reproducible method of manufacturing. Vandalia Research’s technology for DNA production, the Triathlon system, scales the PCR to multiple liters, providing a solution to the aforementioned obstacle. Production of LECs by PCR is significantly easier and does not require highly skilled operators compared to bacterial fermenter operation. Linear DNA vaccines can be setup and produced much faster than both plasmid DNA vaccines and conventional vaccines, where the set-up can take up to several weeks or months. They can be quickly developed once the gene of interest has been identified and produced enzymatically using simple time saving and easily controlled single-step procedure at low cost.

According to http://www.pandemicflu.gov, in an epidemic event, between 16 and 24 million people will need to be vaccinated to provide critical services to the general population. Assuming a dosage of 0.2 mg of DNA per person for 20 million people, such vaccination would require 4 kg of DNA. The Triathlon technology is scalable and can provide this surge capacity within a few weeks. What is more important, the machines will be ready to produce linear DNA vaccines that are unknown now and will only be developed in the future. One cannot stockpile the vaccines to all the potential epidemic agents. Some of them will be absolutely new; the others might represent new viral or bacterial strains for which the existing vaccines will not perform efficiently. For these scenarios it is crucial to have the ability for a rapid de novo production.

Recent Data on LEC Vaccine

Inovio Biomedical, a collaborator of Vandalia Research, designed and tested DNA vaccine candidates against influenza A based on NP and M2 of influenza strain H1N1. DNA vaccines were constructed in the form of traditional plasmids or as fully synthetic LECs expressing the same antigens, produced by Vandalia Research. Inovio compared antigen expression, immunogenicity and efficacy of the two forms of vaccines delivered by intra-muscular (IM) electroporation (EP) or non invasive intradermal (ID) methods.

Immunogenicity of the LEC constructs was studied in mice. Balb/c mice in groups of five were immunized with plasmid or equal molar of LEC by IM EP or non invasive ID EP. Antibody responses to NP or M2e were studied by ELISA using serum. CTL responses against NP147 (TYQRTRALV) and HTL response against M2e (MSLLTEVETPIRNEWGCRCNDSSD) were studied by intracellular cytokine staining (ICS) using splenocytes.  When mice were immunized with 10 ug/mouse plasmid expressing NP or equal molar of LEC only once, two weeks after immunization, strong CTL responses against NP147 were induced by both form of antigen. The level induced by LEC was about one third of the level induced by plasmid. Total IgG against NP show the similar pattern. Non invasive intradermal EP is slightly better in generating both CTL and antibody responses compared to IM EP.   When mice were immunized with 10 ug/mouse of plasmid expressing NP or M2 or equal molar of LEC, five weeks after immunization, strong CTL responses were observed against NP147 or HTL response against M2e but the difference between plasmid and LEC forms became smaller or none. The responses generated by non invasive ID EP were lower than IM EP but significantly higher than ID without EP. 

The immune protection study was also performed using mice. Balb/c mice in groups of 7 were immunized and boosted with DNA expressing NP plus DNA expressing M2 by IM EP or non invasive ID EP. While all the naïve mice died, the immunized mice showed 100% survival following a lethal (100 x LD50) challenge of the heterologous and highly pathogenic H5N1 influenza virus (A/Vietnam/1203/04) and showed only minor weight loss.  These results demonstrate that PCR products manufactured in the Triathlon™ are effective in DNA vaccination protocols. 

In summary, linear DNA vaccines offer several advantages. There are no antibiotic resistance genes or unnecessary origins of replication. Because the constructs are generally 2-3x smaller than their plasmid DNA counterpart, there are fewer immunostimulatory CpG motifs. Finally, an ability to easily chemically-modify the DNA ends, and a cleaner, more reproducible product, make linear expression cassettes an exciting option for vaccination.

The hurdles to linear DNA vaccines have been scalability, speed, and cost. Vandalia Research has addressed these issues, with its Triathlon technology, and is now providing milligram and gram quantities of specific, high-quality dsDNA products by PCR. The company has produced DNA for applications in diagnostics, gene therapy, standards, and now, DNA vaccines.