James Netterwald, PhD, MT (ASCP)
DNA vaccine manufacturers look to take over the market aimed at protecting against viral pathogens.
Vaccines have always been difficult to create. Ever since the polio outbreak that followed a mishap in manufacturing of the polio vaccine in 1955, vaccine makers have had to be extremely careful in how they create vaccines. And there have been many different ways of making vaccines, most of which follow the classical design of using an inactivated viral particle of some sort. That is, until recently. Now, drug companies have found ways to create anti-viral vaccines that contain only a part of the virus, either one or more viral proteins or, more recently, viral DNA.
One researcher who is using basic DNA technology to create recombinant viral vaccines is Gregory Spies, PhD, staff scientist at the Fred Hutchinson Cancer Research Center, Seattle. Using this technology, Spies constructs plasmid vectors for making recombinant viruses. “We are looking at various genetic vectors and the ways to increase the immunogenicity of those vectors.” The problem with working with viral genomes is that most are too large to clone into a single plasmid. The solution used by workers in the field is to clone the viral gene they want to express into a relatively small shuttle plasmid which is easy to manipulate using standard DNA technology. Then, in the case of the vector system Spies is working with, the shuttle plasmid is recombined using Cre/Lox recombination in a test tube with the rest of viral genome to regenerate the complete virus genome. Cre is an enzyme that recognizes and joins LoxP sites engineered into the DNA in both the shuttle plasmid and the much larger plasmid containing the balance of the adenoviral genome. He uses this method to create an adenoviral vector for an experimental vaccine.
The vaccine regimen consists of sequential injections of plasmid DNA encoding proteins of human immunodeficiency virus type 1 (HIV) , followed a month or more later by a second immunization with replication-deficient adenovirus encoding the same proteins . Similar vaccines are now in phase II clinical trials, with data to be made available in 18 months. Enrolled in these efficacy trials are people who are high risk for HIV infection. “The idea is to statistically determine whether this vaccine would provide some form of protection to HIV infection,” says Spies.
Another strategy that is currently being used at the Fred Hutchinson Cancer Research Center to produce viral vaccines is to just transfect cells infected with wild-type virus with a shuttle plasmid containing the transgene of interest. For virus vectors like vaccinia virus whose genome is too large to fit in a plasmid vector, the idea is that homologous recombination will occur between the transgene containing shuttle plasmid and the homologous site in the viral genome; viral particles in which this event has occurred are later selected and used for vaccination.
DNA-based viral vaccine technology has attracted a lot of attention lately, including that of the National Institutes of Health (NIH), Bethesda, Md. “There is an
Of course, the efficacy of all vaccines must be validated before they can be marketed. And there are a number of ways to test vaccine efficacy, says Diana Noah, PhD, research virologist at the Southern Research Institute, Birmingham, Ala. Two major ways used at the institute are to use the hemagglutination inhibition assay (HIA) and the microneutralization assay (MNA), both of which are standard workhorses for vaccine validation. Amazingly, the methods both work by the same principle. “The idea is that if a vaccinated individual has built up an antibody, then that antibody in the person’s serum is combined with a specific amount of virus in the lab,” she says. If antibody binds to the virus, then it will either prevent the virus from a) agglutinating red blood cells (HIA) or b) infecting a subsequent cell type in culture (MNA).Although HIA has been around since the 1940s, there really is not a standardized way of performing the assay, and this makes comparing data from one lab to another nearly impossible. “The biggest problem that has come up for the international community as a whole is the fact that there is not an international standard to understand the parameters of these assays and getting everyone in-line with the same guidelines and the same results,” says Noah. By having this standard, Noah hopes that when a vaccine is made, the testing performed from site to site would produce the same information, regardless of who performed the testing.
“Antibody titer is the end point of vaccine efficacy in the US and Europe and the standard is a four-fold rise in titer between a pre-vaccination and post-vaccination sample from the same patient,” she says. Of course, tests other than HAI and MNA are necessary to determine vaccine efficacy, and efficacy trials in which an antigenic challenge is necessary must be performed in animal models because challenging an unvaccinated human would be unethical.
intramural effort here at NIH that is working to advance a very interesting combination vaccine that is currently in small clinical trials and will soon be launched into larger clinical trials,” says Margaret Johnston, PhD, director of the vaccine research program, division of AIDS, at the National Institute of Allergy and Infectious Diseases, Bethesda, Md. The vaccine she is referring to here is a combination of plasmid DNA molecules that code for protein antigens from HIV. This recombinant viral vaccine is now in phase II clinical trials here in the United States, the Caribbean, South America, and in several countries in Africa. Three doses of the plasmid DNA mixture are administered intramuscularly, after which there is an immunological boost with recombinant, replication-incompetent adenovirus vector that also codes for HIV proteins.
“The idea is to give [the vaccine] to people who are otherwise healthy before they become exposed to [HIV]. They mount an immune response to those HIV proteins made by the vaccine. And these immune responses hopefully enable them to prevent the infection completely,” says Johnston. Even if vaccination does not completely prevent infection, it may allow a recipient’s immune system to control viral replication and consequently lead a longer disease-free and treatment-free life. In fact, during proof-of-concept trials conducted in Simian Virus-infected non-human primates, the vaccine did not prevent infection, but did enable infected animals to control viral replication better than a placebo and allowed them to live longer. “This [DNA + adenovirus vaccine] is what we refer to as a non-classical preventive vaccine because a classical vaccine would completely eliminate the virus from the body.”
Some advantages of DNA viral vaccines over non-DNA viral vaccines include:
- Safe and well-tolerated
- Better immunological boosting after first dose
- No integration of viral DNA into host genome.
Despite these advantages, a major criticism of DNA-based viral vaccines is that the expression of viral immunogens from recombinant vectors is typically low. And this low expression decreases the immunogenicity of the vaccine.”I think the question of immunogenicity of the vectors is one that needs to be answered before we see where things are going with DNA-based viral vaccines,” says
Spies. “It might be that we have to design attenuated viruses, such as an HIV that is modified in some way, so that it does not cause disease but still delivers the antigen load—that might be a better vaccine than the recombinant.”
Several companies are actively pursuing DNA vaccines against infectious diseases. In fact, to make DNA vaccines more immunogenic, Vical Inc., San Diego,
has developed a novel proprietary adjuvant known as Vaxfectin. A lipid combination including a novel proprietary cationic lipid, Vaxfectin has two primary purposes:
- Protection of plasmids from nucleases allowing for high transfection efficiency
- Attraction of pro-inflammatory, antigen-presenting cells which subsequently stimulate the immune response.Vijay Samant, president and chief executive officer of Vical, says of one recent success of Vaxfectin, “We recently took the seasonal influenza vaccine—a protein subunit-based vaccine—and mixed it with our proprietary adjuvant Vaxfectin and showed that Vaxfectin can enhance immune response by approximately 10- to 60-fold therefore reducing the [required] vaccine dose by a factor of at least 10. And this bodes well for the problem with the pandemic influenza vaccine, which requires a 90-?g dose—six times the normal dose—and barely produces 40 to 50% immunogenicity.” Vical is now working with the US government to test the effectiveness of Vaxfectin as an adjuvant for the H5 influenza vaccine.According to Samant, the plasmid DNA found in DNA-based vaccines is also relatively easy to manufacture, because of the following characteristics:
- They are produced via simple Escherichia coli fermentation
- Require only two purification steps
- Require minimal sterile manipulation
- Do not require human cell bioreactors, thus eliminating concerns about potential cross-contamination
- The same manufacturing procedure is used to produce all vaccines, regardless of type of pathogen.In summary, DNA-based viral vaccines seem to breeze through the drug discovery and development process and are slowly taking over the viral vaccine market.
This article was published in Drug Discovery & Development magazine: Vol. 10, No. 6, June, 2007, pp. 24-27.
Filed Under: Drug Discovery