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[SANET-MG] field testing a vaccine for dog melanoma cancer

November 21, 2005

Prof. Joe Cummins

Field Testing a DNA Canine Melanoma Cancer Vaccine

The United States Department of Agriculture-Animal and Plant Health Inspection Service (APHIS) is considering granting authorization to ship an unlicensed Canine Melanoma Vaccine, DNA, for field testing. Merial, Inc., Athens, Georgia, has requestedauthorization to conduct clinical studies that will provide efficacy and safety data in dogsadministered this vaccine. The efficacy trial will measure the sparing effect of thevaccine on dogs diagnosed with melanoma. The safety of the vaccine will be evaluatedin animals participating in the studies. The public may comment on the environmental assessment APHIS-2005-0104-0001 Availability of an Environmental Assessment for Field Testing Canine Melanoma Vaccine, DNA 11/15/2005 public comments can be submitted on or before December 15, 2005. at the web address URL: http://www.regulations.gov/fdmspublic-bld61/component/main

The environmental assessment dealt with novel features of DNA vaccines. However, large sections of the assessment were blacked out as “confidential business information” (CBI) rendering full evaluation of the assessment impossible. Frankly, such lavish use of CBI seems to be a feckless way to avoid criticism of the project other than the comments made from bureaucrats who have access to the full information. Nevertheless, the use of DNA vaccines to treat canine melanoma has been discussed in the literature of science and those studies will be discussed below.

DNA vaccines are proving effective, normally they are delivered by intramuscular injection or use of a biolistic device, oral DNA vaccines are effective in some cases. The vaccines are normally bacterial plasmids into which are encoded a mammalian promoter such as the cytomegalovirus promoter driving the protein sequence for an antigen. The plasmids are taken up by the mammalian cells and reaches the nucleus of some of those cells. There it is transcribed , translocated to the cytoplasm, then translated into antigen protein. The bacterial plasmid sequences are rich in CpG sequences which act as aduvants which enhance the immune response. The DNA vaccines induce the full spectrum of immune responses including antibodies, T helper cells and cytotoxic T lymphocytes(1). Concerns have been expressed about the induction of autoimmunity and induction of anti-DNA antibodies, and ant-DNA antibodies were observed in rabbits immunized with plasmids bearing a HIV reverse trascriptase gene (2). However, DNA antibodies and autoimmunity have not proven top be major complications of DNA vaccination.

A phase one clinical trial of a DNA vaccine using a plasmid modified with two peptides from human tyrosinase (tyrosinase is an enzyme that is on the path to melanin formation which is greatly elevated in melanoma cancer cells) was undertaken on human subjects with stage IV melanoma (Patients with stage IV, or metastatic, melanoma have cancer that has spread from its site of origin to distant lymph nodes and/or distant sites.). Plasmid DNA was injected into groin lymph nodes. Survival of treated patients was surprisingly long , with 16 of 24 patients surviving for after 12 months(3).

Canine malignant melanoma , metastatic form of the disease is common and resistant to chemotherapy. A clinical study of dogs with malignant melanoma involved treatment with plasmids containing peptides from human or mouse tyrosinase. The study showed that the inoculations were safe and led formation of anti-tyrosinase antibodies (4). Dogs with advanced malignant melanoma survived for an extended period when inoculated with a plasmid containing a gene for a peptide from human tyrosinase, survival extended more than a year . The trial supported use of the vaccine with dogs and humans with advanced melanoma alike (5).

The report “Nucleic Acid-Mediated (Genetic) Vaccines Risk Analysis for Melanoma DNA Vaccine (Product Code 9240.D0, Unlicensed)” (reference 6) indicated that the DNA vaccine was derived from a bacterial plasmid but all of the pertinent information about the antigen sequence and antibiotic selection markers was blacked out presumably deemed confidential business information (CBI) in an astonishingly ill informed manner. The only information about the plasmid allowed (not blacked out) was that it was an E coli plasmid.

Among the issues considered in the review was the chance that the vaccine antigen would recombine with genes in the dog chromosomes causing mutations. No effort was made to measure gene integration form the vaccine antigen , the proponents and APHIS argued that the chance of integration was low based on studies of antigen integration from the malaria parasite (7) or from influenza virus or HIV virus (8). It is worth pointing out that the dog melanoma vaccines have all been based on genes present in the mammalian genomes with high levels of DNA homology allowing legitimate recombination at a much higher frequency than the antigen genes from parasites or viruses that have little or no homology with the mammalian genome and must recombine using illegitimate recombination, It is surprising that APHIS and the proponent failed to mention that important point.

The proponent and APHIS argue that immuno-modulator sequences such as the CpG motif are not known to be present in something blacked out related to the plasmid vaccine DNA. This point is clearly in error for the CpG motif is present in the E. coli plasmids and that is certainly active in dogs and cats (9).

The problem of auto-immunity and anti-DNA antibodies was dealt with in a cursory manner. The handling and escape of plasmid bearing bacteria was discussed in a cursory manner and no data was provided to support conclusions. The dissemination of the vaccine plasmid in the environment was also considered without experimental data. Conclusion that the plasmid ingested by animals would be of no consequence did not provide data support that conclusion. Horizontal gene transfer was discounted without supporting data.

The proposal by the proponent and APHIS claims that here is little or no chance of having problems related to accidental spills of solutions containing plasmid. Because the plasmid is not infectious and is unstable in the environment. However no data were supplied to support that conclusion. The report maintains that plasmid shed or released from test animals posed no concern because the levels of plasmid released by those animals would be low. Even though data was not provided to support that conclusion there was no indication that feces , urine or vomited materials would be handled in any special way to prevent dispersal of the plasmid in the environment. The antibiotic resistance markers associated with the plasmid were designated CBI , thus information on them is unavailable to any bystander exposed to the plasmid from surface or groundwater, in air associated with dust particles or in bacteria. Many bacteria are capable of taking up DNA molecules and integrating them into the bacterial chromosome , such as Acinobacter and a wide array of gram positive bacteria.

In conclusion, the proposal for a field trial of a DNA vaccine to treat canine had serious defects that make the project appear to be threatening rather than beneficial. Using CBI to hide the most elementary aspects of the proposal seemed to hide serious defects in the experiment as did the failure to provide supporting date to support far reaching conclusions. The proposal seems to cry out there must be collusion between APHIS and the vaccine promoters to provide fast and dirty remedies for important problems. The proponents should have provided a full and truthful report , without the feckless, even, silly CBI designations of fundamental information. Such proposals make it appear that bureaucrats and corporations connive to exclude the public from deliberations on biotechnology. This unfortunate turn of events has transpired because the public has allowed secretive field tests of dangerous genetic constructions which allows the proponents for such tests to evade being implicated in the injury from such tests. Now, bureaucrats serve private interests and participate in withholding fundamental information designated CBI . The practice prostitutes the primary scientific dictum that the results of experiments must be reported fully and truthfully.


1Kowalczyk,D. and Ertl,H. Immune response to DNA vaccines CMLS Cell. Mol. Life Sci. 1999 , 55, 751-70

2.Isaguliants MG, Iakimtchouk K, Petrakova NV, Yermalovich MA, Zuber AK, Kashuba VI, Belikov SV, Andersson S, Kochetkov SN, Klinman DM and Wahren B. Gene immunization may induce secondary antibodies reacting with DNA. Vaccine. 2004 Mar 29;22(11-12):1576-85.

3. Tagawa ST, Lee P, Snively J, Boswell W, Ounpraseuth S, Lee S, Hickingbottom B, Smith J, Johnson D and Weber JS. Phase I study of intranodal delivery of a plasmid DNA vaccine for patients with Stage IV melanoma Cancer. 2003 Jul 1;98(1):144-54.

4. Bergman PJ, Camps-Palau MA, McKnight JA, Leibman NF, Craft DM, Leung C, Liao J, Riviere I, Sadelain M, Hohenhaus AE, Gregor P, Houghton AN, Perales MA and Wolchok JD. Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center. Vaccine. 2005 Sep 23; [Epub ahead of print]

5. Bergman PJ, McKnight J, Novosad A, Charney S, Farrelly J, Craft D, Wulderk M, Jeffers Y, Sadelain M, Hohenhaus AE, Segal N, Gregor P, Engelhorn M, Riviere I, Houghton AN and Wolchok JD. Long-term survival of dogs with advanced malignant melanoma after DNA vaccination with xenogeneic human tyrosinase: a phase I trial. Clin Cancer Res. 2003 Apr;9(4):1284-90.

6. Merial, Inc. Environmental Assessment for Field Testing Canine Melanoma Vaccine, DNA 2005 Nucleic Acid-Mediated (Genetic) Vaccines Risk Analysis for Melanoma DNA Vaccine (Product Code 9240.D0, Unlicensed)” http://www.regulations.gov/fdmspublic-bld61/component/main

7. Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, Hobart P and Lew D. Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther. 1999 Mar 20;10(5):759-68.

8. Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Beare CM, Bagdon WJ and Nichols WW. Plasmid DNA Vaccines: Investigation of Integration into Host Cellular DNA following Intramuscular Injection in Mice Intervirology. 2000;43(4-6):258-72

9. Krieg A. CpG Motifs in bacterial DNA and their immune effect Annu, Rev. Immunol. 2002 ,20,709-60

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