Article Review: PreClinical and Clinical Safety Studies on DNA Vaccines
Pre-Clinical and Clinical Safety Studies on DNA Vaccines
Johanna A.C. Schalk, Frits R. Mooi, Guy A.M. Berbers, Leon A.G.J.M. Van Aerts, Hans Ovelgonne and Tjeerd G. Kimman
Human Vaccines 2:2 45-53, March/April 2006; Landes Bioscience
The topic under review is the safety issues on the use of DNA vaccines. There are a number of studies cited by the authors. Findings from these were used in organizing the paper based on the major safety issues (genetic, immune-mediated and environmental) surrounding the preclinical and clinical use of DNA vaccines both in animal models and humans.
GENERAL SAFETY
The use of DNA vaccines in mice, rabbits and rats administered either intravenously, intramuscularly or intradermally were well tolerated by the subjects regardless of the doses applied. This was based on the experiments done by Parker et al (1999); Hanke et al (2002) and Tuomela et al (2005.) Based on the reports of McGergor et al. (1998), Moorthy et al. (2003), Mwau et al (2004) and Rottinghaus et al. (2003), all DNA vaccines used so far in humans are well tolerated with no local or systemic serious adverse effects.
GENETIC ISSUES
The authors raised three major genetic concerns on the use of DNA vaccines.
First, is the possible long-term persistence of the DNA vaccines which might facilitate the integration of plasmid DNA into the host’s genome and could cause long term skewing of the immune system influencing future immunizations and infections. Although current studies reported that the amount of plasmid DNA dissipates from the introduction site few minutes shortly after injection (Parker et al., 1999; Hoblwerg et al., 2001; Manam et al., 2000; Kim et al., 2003; and Bureau et al., 2004), it was also found out that DNA vaccines could be stably expressed for about 19 months (Wolf et al., 1992) and could even persist up to two years (Armengol et al., 2004). However in both cases, no integration was detected and the amount discovered was low, but at significant level.
Second, is the probable integration of plasmid DNA into the host’s genome. Integration could lead to activation of oncogenes, inactivation of tumor suppressor genes or to vertical transmission when integrated into the chromosomal DNA of germ line cells. Nevertheless, current studies show that the risk of mutation to plasmid integration is negligible as demonstrated by the experiments of Nichols et al. (1995) and Ledwith et al. (2000). Several studies also support the claim that various plasmid DNA vaccines did not demonstrate any integration event in rat (Kang et al., 2003; Yang et al., 2003), in fish (Kanellos et al., 1999) and in mice (Martin et.al, 1999 and Manam et.al, 2000). As to the manner of delivery, it was found out that even if the use of biojector increases the plasmid uptake of host cells as compared with needle injection, it did not still result in a detectable increase in integration frequency, likewise, in the use of aluminum phosphate adjuvant (Manam et al,2000).
However, in the study of Wang et al. (2004), they found out that subjecting the cell to electroporation results in increased plasmid frequency. That’s why integration events even at a very low frequency should not be neglected. It is for this reason that suicide vectors are now being developed to induce the apoptotic cell death of transfected cells (Xiao et al., 2004; Kohno et al.,1998; Chattergon et al, 2000; Sasaki et al.,2001; Kim et al.,2004) and alleviate the concerns of potential integration and cell transformation.
Third, is the issue on germ line integration and vertical transmission. The study of Parker et al. (1999) and Manam et al. (2000) revealed that plasmid DNA can be detected in gonads shortly after upon injection. However, its integration into chromosomal germ line DNA has not yet been observed so far. This raises some concerns because when plasmid is transmitted to the gonads, there is a possibility that germ line chromosomal integration and transmission could occur. However, it was reported that this will just be very minimal.
IMMUNE MEDIATED ISSUES
Four concerns related to immunity on the use of DNA vaccines were drawn by the reviewers.
First, is the possible induction of auto-immunity which is a general concern. In the experiment on knockout mice (normal and with lupus), it was confirmed that DNA vaccines can really induce the formation of anti-DNA antibodies, however, no evidence was found that they induce systemic auto-immune diseases. In another four separate experiments, it was found out that no anti-DNA antibodies at all were observed after DNA vaccination of mice, rat, rabbits, fish and non-human primates. However Donnely et al. (1997), Gurunthan et al. (2000)and Kreig et al. (2002) raised the immunostimulatory activity issue of the unmethylated CpG motifs in plasmid backbone because these can lead to the formation of anti-DNA antibodies which might accelerate the development of auto-immune diseases. It was also raised that introduction of other immunomodulatory molecules, the attachment of peptides to the DNA vectors and destruction of the injected muscle cells as a result of DNA vaccination may induce auto-immunity. Nevertheless, the authors believe that it is unlikely that DNA vaccines would pose any greater risk than conventional vaccines.
Second concern is on the induction of immunological tolerance particularly among infants and children. Given their immature immune system, there is a fear that vaccination may develop tolerance rather than immunity. This was confirmed by the studies of Ichino et al. (1999) and Mor et al. (1996) who found out that vaccination of 2-6 month old mice resulted in immunity but those younger than 7 days old did not develop an antibody response and remained unresponsive when revaccinated as adults. This tolerance was not induced by early vaccination which was antigen specific.
The third concern is about the altered immune responsiveness to other vaccines and infections. Plasmids that encode cytokines affect the immune capacity of the host cell. The coadministration of these immunostimulatory molecules can result in their release in the circulation causing undesirable systemic effects like enhanced susceptibility to infections and mortality rates that may range from substantial to high. However, in the experiment of Ishi et al. (1999), they found out that some plasmids that encode specific protein boosted immunity against a coadministered vaccine without unwanted side effects.
The fourth issue is on the toxicity and immunotoxicity of the DNA vaccines. Taylor et al. (2003) discovered that vaccination against TB in mice caused pulmonary necrosis. They manifested pyrogenic responses suggestive of a lung damage. This is not the case however in some other animals where plasmid DNA vaccines provided protection against lethal type. For example, immunization of mice with DNA plasmid, protected the mice against lethal challenges with tetanus toxin.
ENVIRONMENTAL ISSUES
Several studies reported that DNA vaccines can be spread in the environment by shedding or by consumption of vaccinated animals. They can also recombine with viruses, bacteria or parasites outside the vaccinated host after shedding or inside the vaccinated host and consequently generate the spread of genetically modified organism. And they can also spread via integration in the genome of germ line cells.
At first this is really a matter of great concern. We do not want more stress in our environment. However the study of Comerota et al. (2002), the only paper so far that addresses the environmental issues surrounding the use of DNA vaccines revealed that once shed in the environment, DNA will be degraded. This was proven by an experiment in humans that after intramuscular administration of a naked plasmid DNA, none was detectable in the urine.
Chances on recombination events particularly in resident microorganisms and subsequent shedding and long term transmission are extremely low especially when DNA vaccines are introduced intramuscularly. This is because DNA vaccines do not replicate in mammalian cells in contrast to live vaccines.
Thus release of plasmid DNA into the environment is not likely to have any direct impact on other organisms. Much more, the consumption of vaccinated animals may not pose greater risk as this would just be likened to the consumption of natural DNA.
REFLECTIONS
The use of DNA vaccines has been found so far to be safe in preclinical and clinical trials and holds a lot of promise for the improved well-being of humanity, particularly those in the third world countries.
The risks according to various studies are minimal, but there is no need to be complacent. I am so much concerned of the genetic effects of using DNA vaccines particularly their potential to turn on the oncogenes and shutting off the tumor suppressor genes. If this will not be addressed properly, we are going to create problem than solution. The development of suicide vectors must be given attention and focus as this would reduce the fears and worries of the possible integration of the plasmid to the patient’s genome. The process must be perfected in order to ensure the safety of the vaccine users.
The world has erstwhile bet on the live and weakened vaccines and has saved lives and I think to take another risk to something that is in the form of a molecule, handy, freezer free and which has the potential to create long term immunity is more than worth it.
Who knows one day…
… DNA vaccines against infectious diseases such as AIDS, rabies, malaria will be made available in the market already;
….scientists would be able to isolate plasmid DNA with multiple genes that could provide immunity against many diseases in one booster;
Then, this world will surely appreciate more what science and technology is all about!
Johanna A.C. Schalk, Frits R. Mooi, Guy A.M. Berbers, Leon A.G.J.M. Van Aerts, Hans Ovelgonne and Tjeerd G. Kimman
Human Vaccines 2:2 45-53, March/April 2006; Landes Bioscience
The topic under review is the safety issues on the use of DNA vaccines. There are a number of studies cited by the authors. Findings from these were used in organizing the paper based on the major safety issues (genetic, immune-mediated and environmental) surrounding the preclinical and clinical use of DNA vaccines both in animal models and humans.
GENERAL SAFETY
The use of DNA vaccines in mice, rabbits and rats administered either intravenously, intramuscularly or intradermally were well tolerated by the subjects regardless of the doses applied. This was based on the experiments done by Parker et al (1999); Hanke et al (2002) and Tuomela et al (2005.) Based on the reports of McGergor et al. (1998), Moorthy et al. (2003), Mwau et al (2004) and Rottinghaus et al. (2003), all DNA vaccines used so far in humans are well tolerated with no local or systemic serious adverse effects.
GENETIC ISSUES
The authors raised three major genetic concerns on the use of DNA vaccines.
First, is the possible long-term persistence of the DNA vaccines which might facilitate the integration of plasmid DNA into the host’s genome and could cause long term skewing of the immune system influencing future immunizations and infections. Although current studies reported that the amount of plasmid DNA dissipates from the introduction site few minutes shortly after injection (Parker et al., 1999; Hoblwerg et al., 2001; Manam et al., 2000; Kim et al., 2003; and Bureau et al., 2004), it was also found out that DNA vaccines could be stably expressed for about 19 months (Wolf et al., 1992) and could even persist up to two years (Armengol et al., 2004). However in both cases, no integration was detected and the amount discovered was low, but at significant level.
Second, is the probable integration of plasmid DNA into the host’s genome. Integration could lead to activation of oncogenes, inactivation of tumor suppressor genes or to vertical transmission when integrated into the chromosomal DNA of germ line cells. Nevertheless, current studies show that the risk of mutation to plasmid integration is negligible as demonstrated by the experiments of Nichols et al. (1995) and Ledwith et al. (2000). Several studies also support the claim that various plasmid DNA vaccines did not demonstrate any integration event in rat (Kang et al., 2003; Yang et al., 2003), in fish (Kanellos et al., 1999) and in mice (Martin et.al, 1999 and Manam et.al, 2000). As to the manner of delivery, it was found out that even if the use of biojector increases the plasmid uptake of host cells as compared with needle injection, it did not still result in a detectable increase in integration frequency, likewise, in the use of aluminum phosphate adjuvant (Manam et al,2000).
However, in the study of Wang et al. (2004), they found out that subjecting the cell to electroporation results in increased plasmid frequency. That’s why integration events even at a very low frequency should not be neglected. It is for this reason that suicide vectors are now being developed to induce the apoptotic cell death of transfected cells (Xiao et al., 2004; Kohno et al.,1998; Chattergon et al, 2000; Sasaki et al.,2001; Kim et al.,2004) and alleviate the concerns of potential integration and cell transformation.
Third, is the issue on germ line integration and vertical transmission. The study of Parker et al. (1999) and Manam et al. (2000) revealed that plasmid DNA can be detected in gonads shortly after upon injection. However, its integration into chromosomal germ line DNA has not yet been observed so far. This raises some concerns because when plasmid is transmitted to the gonads, there is a possibility that germ line chromosomal integration and transmission could occur. However, it was reported that this will just be very minimal.
IMMUNE MEDIATED ISSUES
Four concerns related to immunity on the use of DNA vaccines were drawn by the reviewers.
First, is the possible induction of auto-immunity which is a general concern. In the experiment on knockout mice (normal and with lupus), it was confirmed that DNA vaccines can really induce the formation of anti-DNA antibodies, however, no evidence was found that they induce systemic auto-immune diseases. In another four separate experiments, it was found out that no anti-DNA antibodies at all were observed after DNA vaccination of mice, rat, rabbits, fish and non-human primates. However Donnely et al. (1997), Gurunthan et al. (2000)and Kreig et al. (2002) raised the immunostimulatory activity issue of the unmethylated CpG motifs in plasmid backbone because these can lead to the formation of anti-DNA antibodies which might accelerate the development of auto-immune diseases. It was also raised that introduction of other immunomodulatory molecules, the attachment of peptides to the DNA vectors and destruction of the injected muscle cells as a result of DNA vaccination may induce auto-immunity. Nevertheless, the authors believe that it is unlikely that DNA vaccines would pose any greater risk than conventional vaccines.
Second concern is on the induction of immunological tolerance particularly among infants and children. Given their immature immune system, there is a fear that vaccination may develop tolerance rather than immunity. This was confirmed by the studies of Ichino et al. (1999) and Mor et al. (1996) who found out that vaccination of 2-6 month old mice resulted in immunity but those younger than 7 days old did not develop an antibody response and remained unresponsive when revaccinated as adults. This tolerance was not induced by early vaccination which was antigen specific.
The third concern is about the altered immune responsiveness to other vaccines and infections. Plasmids that encode cytokines affect the immune capacity of the host cell. The coadministration of these immunostimulatory molecules can result in their release in the circulation causing undesirable systemic effects like enhanced susceptibility to infections and mortality rates that may range from substantial to high. However, in the experiment of Ishi et al. (1999), they found out that some plasmids that encode specific protein boosted immunity against a coadministered vaccine without unwanted side effects.
The fourth issue is on the toxicity and immunotoxicity of the DNA vaccines. Taylor et al. (2003) discovered that vaccination against TB in mice caused pulmonary necrosis. They manifested pyrogenic responses suggestive of a lung damage. This is not the case however in some other animals where plasmid DNA vaccines provided protection against lethal type. For example, immunization of mice with DNA plasmid, protected the mice against lethal challenges with tetanus toxin.
ENVIRONMENTAL ISSUES
Several studies reported that DNA vaccines can be spread in the environment by shedding or by consumption of vaccinated animals. They can also recombine with viruses, bacteria or parasites outside the vaccinated host after shedding or inside the vaccinated host and consequently generate the spread of genetically modified organism. And they can also spread via integration in the genome of germ line cells.
At first this is really a matter of great concern. We do not want more stress in our environment. However the study of Comerota et al. (2002), the only paper so far that addresses the environmental issues surrounding the use of DNA vaccines revealed that once shed in the environment, DNA will be degraded. This was proven by an experiment in humans that after intramuscular administration of a naked plasmid DNA, none was detectable in the urine.
Chances on recombination events particularly in resident microorganisms and subsequent shedding and long term transmission are extremely low especially when DNA vaccines are introduced intramuscularly. This is because DNA vaccines do not replicate in mammalian cells in contrast to live vaccines.
Thus release of plasmid DNA into the environment is not likely to have any direct impact on other organisms. Much more, the consumption of vaccinated animals may not pose greater risk as this would just be likened to the consumption of natural DNA.
REFLECTIONS
The use of DNA vaccines has been found so far to be safe in preclinical and clinical trials and holds a lot of promise for the improved well-being of humanity, particularly those in the third world countries.
The risks according to various studies are minimal, but there is no need to be complacent. I am so much concerned of the genetic effects of using DNA vaccines particularly their potential to turn on the oncogenes and shutting off the tumor suppressor genes. If this will not be addressed properly, we are going to create problem than solution. The development of suicide vectors must be given attention and focus as this would reduce the fears and worries of the possible integration of the plasmid to the patient’s genome. The process must be perfected in order to ensure the safety of the vaccine users.
The world has erstwhile bet on the live and weakened vaccines and has saved lives and I think to take another risk to something that is in the form of a molecule, handy, freezer free and which has the potential to create long term immunity is more than worth it.
Who knows one day…
… DNA vaccines against infectious diseases such as AIDS, rabies, malaria will be made available in the market already;
….scientists would be able to isolate plasmid DNA with multiple genes that could provide immunity against many diseases in one booster;
Then, this world will surely appreciate more what science and technology is all about!
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