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DNA vaccines and replicons
DNA vaccines are based on bacterial plasmids - small rings of DNA found in bacterial cells into which other genes can be inserted. As bacteria can be grown rapidly, reliably, cheaply and in large quantities, it is simple to generate large numbers of genetically engineered plasmids. These plasmids may include, for example, selected genes from a virus. Once injected into an animal, the animal's own cells then effectively make the vaccine.
DNA vaccines are relatively stable at ordinary room temperatures, which greatly reduces problems of transport and storage. However, although they work very well in mice, it has been more difficult to get DNA vaccines working in primates, including humans. It appears that fine details, such as how much vaccine to give, where and when, are critical in eliciting immune responses.
The major problem with DNA vaccines is how to deliver the large quantities needed to bring about a strong immune response. The doses tested in mice, if scaled up for people based on body weight, would be very costly to make and require numerous injections. There is a practical limit of around 5mg per injection when a DNA vaccine is given intramuscularly. A 'real-life' HIV vaccine might require a mixture of DNA constructs for different HIV proteins or several strains. If each of these were to require multiple painful injections, the prospect becomes unacceptable.
Furthermore, there are safety considerations inherent in the design of DNA vaccines. In an extreme case, if the DNA encoded all of the genetic material of a virus like HIV, this could be a mechanism for infecting an organism with the virus. It is therefore important to ensure that the genes chosen are not sufficient to give rise to a self-replicating infection.
A further theoretical concern has been that the DNA might become integrated in the DNA of a cell in a way that switches on a cancer-causing gene. Although there has been no evidence that this has happened in animal studies, some researchers have opted to inject the vaccines into skin cells rather than muscle, as they have a shorter lifespan. It also appears that injecting into the skin may give stronger immune responses. However, the method of delivery of DNA into the skin has been unsuccessful in eliciting an immune response in macaque studies.
Scientists working with Apollon (now part of Wyeth-Lederle), were the first to report that their company's HIV-DNA vaccine had generated strong immune responses that appeared to protect chimpanzees against HIV. Subsequently, a research team from Chiron Corporation has achieved HIV-specific cytotoxic T cell responses using DNA vaccines and replicons (see below).
Some of the strongest evidence of vaccine-induced immune control in monkeys has now been obtained using DNA vaccines in combination with other approaches. Sadly, however, it does not seem likely that this can be translated easily into vaccines for human use.
Current research
It has been reported that a single mutation in HIV is sufficient to undermine the protection of an HIV vaccine that had apparently controlled HIV infection in a monkey for six months. Eight monkeys were given a DNA vaccine which was designed to elicit cell-killing immune responses to specific regions of gag. After they had developed responses, the animals were exposed to a highly aggressive form of SHIV. Although infection occurred in all the monkeys, virus levels remained low or became undetectable. However, one monkey subsequently suffered a shift in its virus population, became sick and died within six months of initial infection. The shift was enough to ensure that the main killer T-cell population produced in response to the vaccine was no longer able to control infection, and other T-cell responses were unable to make up for this (Barouch 2002).
This finding has raised fears that current vaccines which focus on orchestrating killer T-cell responses to control HIV after infection has occurred may not be enough, faced with HIV’s phenomenal mutation rate.
Some experts believe that vaccines which contain a small number of epitopes may be more prone to viral breakthroughs of the kind described above. There is evidence from studies of people with HIV to show that the number of different epitopes recognised by their killer T-cells correlates with survival. Researchers aiming at vaccines to stimulate cellular immunity are currently agreed that it is likely to need more than gag genes to get a broad enough range of response to be useful.
Several other groups, including researchers from the Pasteur Institute, have demonstrated that DNA vaccines can generate strong HIV-specific immune responses. However, these immune responses have not protected against infection in animal studies (Habel 2000; Okuda 2000). Nevertheless, a team from the Pasteur Institute did find that a DNA-plus-protein vaccine provided some protection against SHIV infection (Habel 2000).
DNA vaccines which trigger production of cytokines have also been tested. Incorporating the genes for cytokines into a DNA vaccine could make this approach affordable for a mass-produced vaccine, where direct injections of recombinant cytokines might not be. Experiments in monkeys show that this approach works surprisingly well. However, a Wyeth-Lederle DNA vaccine, using env/rev DNA, has stimulated only weak HIV-specific chemokine responses (Boyer 2000)
Given disappointing initial results with DNA-only vaccines, researchers are looking to combine them with other vaccines. A key line of research has been the vector vaccine. A DNA vector vaccine created using the common cold virus or adenovirus vector and parts of HIV’s genetic material has proved successful in maintaining viral load at undetectable levels for more than 500 days in monkey studies carried out by Merck (Shiver 2002).
Several DNA vaccines have been or are now in clinical trials with a view to using them in such combinations, notably those developed at Oxford University and by Merck, as described in Recombinant vectored vaccines . For details of research into DNA vaccines for therapeutic purposes in people with HIV, see DNA vaccines in Drugs used by people with HIV: Therapeutic vaccines.
Replicons
DNA vaccines are only one way of delivering genetic material into cells. In recent years, a number of research teams have created virus-like particles that contain unrelated genetic material, called 'replicons'. These are formed by using a carrier, or 'source' virus to take the genetic material of an unrelated virus into a cell. Replicons have the same physical properties as viruses, including the ability to enter cells of specific kinds, but they cannot reproduce themselves.
The major advantage of replicons over recombinant vectored viruses is that because they do not reproduce after entering the human cell, there is little or no immune response to the carrier virus. Thus, one replicon system could be used repeatedly in the same person, to deliver a series of different vaccines or gene therapies.
The potential choice of carrier viruses is vast. HIV itself is being developed as the basis for replicons, which are initially being designed as vehicles for genetic therapy in HIV disease.
When it comes to HIV vaccines, however, the three leading replicon systems are now based on alphaviruses called Venezuelan equine encephalitis (VEE) and Semliki forest virus (SFV), and an unrelated virus called adeno-associated virus (AAV).
VEE replicons use a vaccine strain developed by the United States military and now licensed to a company called AlphaVax based at the University of North Carolina. VEE replicons are reported to target lymphoid tissues and to be effective at inducing both cellular and antibody-mediated immune responses. Protection of monkeys against illness from a pathogenic strain of SIV has already been demonstrated (Olmsted 2000).
SFV is being studied by several groups of researchers and has been adopted by the EuroVacc consortium as one of several vectors into which they plan to insert the same target HIV gene sequences. This will enable a variety of different prime-boost combinations to be tested.
Adeno-associated viruses (AAVs) are unrelated to adenoviruses, although AAVs can only replicate in cells which are already infected with adenoviruses. Despite this, they are relatively common but have not themselves been associated with illness or pathology in humans. AAV has been adapted as a vector vaccine and tested in mice and macaques. High levels of antibodies and moderately high levels of cytotoxic T cells were produced (Xin 2001).
AAV is of particular interest because it seems to give rise to sustained expression of gene sequences included in the replicon system. This offers the prospect of a single injection instead of a prime-boost combination.
Papillomaviruses have also been developed as the basis for replicons which appear, from studies in mice, to offer the possibility of mucosal immunity to HIV antigens following oral immunisation (Zhang 2004). However, it remains to be seen if this can be translated into comparable effects in monkeys or people.
Bacterial delivery of DNA
As DNA vaccines are grown inside bacteria, it may be possible to use the bacteria themselves as the vaccine. Immune responses can be induced to DNA plasmids included in weakened vaccine strains of such bacteria as Salmonella and Shigella, which usually infect humans via the oral route and induce strong mucosal immune responses. A group of researchers at the Institute of Human Virology is now working to develop a number of vaccine candidates of this kind. One of these is the first vaccine to be based on a Nigerian strain.
If this strategy succeeds, it opens the way to an oral vaccine that could be produced very cheaply. However, there may still be an issue about the amount of DNA that can be delivered by this route, and how the immune response to it can be measured.