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9-Misc: Field-testing a DNA canine melanoma vaccine in the USA



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TITLE:  Field-Testing a DNA Canine Melanoma Vaccine
SOURCE: The Institute of Science in Society, UK, by Joe Cummins
        http://www.i- sis.org.uk/FTADNACMC.php
DATE:   13 Dec 2005

------------------ archive:  http://www.genet-info.org/ ------------------


Field-Testing a DNA Canine Melanoma Vaccine

A proposal that uses "confidential business information" to conceal the
most critical aspects with regard to safety while dismissing genuine
safety concerns. Prof. Joe Cummins

This report has been submitted to US Department of Agriculture-Animal and
Plant Inspection Service on behalf of the Independent Science Panel

The United States Department of Agriculture-Animal and Plant Health
Inspection Service (APHIS) is considering granting authorization to ship
an unlicensed DNA canine melanoma vaccine for field-testing, as requested
by Merial, Inc., Athens, Georgia.

The company wants to conduct clinical studies that will provide efficacy
and safety data in dogs administered this vaccine. Efficacy will be
measured by the sparing effect of the vaccine in dogs diagnosed with
melanoma; and the safety of the vaccine will be evaluated in all animals
participating in the studies. The Assessment for Field Testing Canine
Melanoma Vaccine, DNA 11/15/2005 is open for public comment before 15
December 2005 at:
http://www.regulations.gov/fdmspublic- bld61/component/main

The environmental assessment dealt with the novel features of DNA
vaccines. But with large sections of the assessment blacked out as
"confidential business information" (CBI), full evaluation of the
assessment is impossible; and this is not in the public interest.
Nevertheless, the use of DNA vaccines to treat canine melanoma has been
discussed in the scientific literature.

DNA vaccines are normally delivered by intramuscular injection or a
biolistic device, or orally administered. The vaccines are normally
bacterial plasmids into which are spliced a promoter active in mammals,
such as the cytomegalovirus promoter, driving the coding sequence for an
antigen. The plasmid is taken up by the mammalian cells and reaches the
nucleus of some of those cells. There it is transcribed into RNA, which
is translocated to the cytoplasm and translated into antigen protein. The
bacterial plasmid sequences are rich in CpG sequences which act as
adjuvant to enhance the immune response. The DNA vaccines induce the full
spectrum of immune responses including antibodies, T helper cells and
cytotoxic T lymphocytes [1]; but concerns have been expressed over the
induction of autoimmunity and anti-DNA antibodies, which were observed in
rabbits immunized with plasmids bearing a HIV reverse transcriptase gene [2].

A phase one clinical trial of a DNA vaccine using a plasmid modified with
two peptides from human tyrosinase - an enzyme on the path to melanin
formation that is greatly elevated in melanoma cancer cells - was
undertaken on human subjects with stage IV melanoma, in which the
melanoma has spread from its site of origin. Plasmid DNA was injected
into the groin lymph nodes; and 16 of 24 patients survived for 12 months [3].

Metastatic (spreading) canine malignant melanoma 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 resulted
in anti- tyrosinase antibodies [4]. Dogs with advanced malignant melanoma
survived for more than a year when inoculated with a plasmid containing a
gene for a peptide from human tyrosinase. The trial supported the use of
the vaccine in both dogs and humans with advanced melanoma [5].

The report "Nucleic Acid-Mediated (Genetic) Vaccines Risk Analysis for
Melanoma DNA Vaccine (Product Code 9240.D0, Unlicensed)" [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). The only information on the plasmid 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 integration of the vaccine DNA,
the proponents and APHIS argued that the chance of integration was low
based on studies of antigen integration from the malaria parasite [7] or
influenza virus or HIV virus [8]. But 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 depend on
illegitimate recombination. It is surprising that APHIS and the proponent
failed to mention this 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 E. coli plasmids, and is certainly active in dogs and
cats [9].

The problem of auto-immunity and anti-DNA antibodies was dealt with in a
cursory manner; and so was the handling and escape of plasmid bearing
bacteria, with no data provided to support conclusions. The dissemination
of the vaccine plasmid in the environment was also considered in the
absence of experimental data. The conclusion that the plasmid ingested by
animals would be of no consequence was similarly based on no experimental
data, as was the dismissal of horizontal gene transfer.

The report claims that there is little or no chance of problems arising
from accidental spills of solutions containing the plasmid, because the
plasmid is not infectious and is unstable in the environment. Again, no
data were supplied to support that conclusion, which would appear at odds
with what we now know about the stability of DNA in all environment. 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. But no data were provided to support that conclusion; and 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, and hence unavailable to any member of the public 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; there are
at least 87 species of naturally transformable bacteria in the soil alone
[10].

In conclusion, the proposal for a field trial of a DNA vaccine to treat
canine melanoma suffers from serious defects, chief among which, using
CBI to conceal the most critical aspects of the proposal with regard to
safety while dismissing genuine safety concerns with no empirical
evidence. This proposal must be rejected and given no further
consideration unless and until those defects are made good.


References
1 Kowalczyk 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, 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, 98,144-54.
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,
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, 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 lnjection in mice. Intervirology 2000, 43(4-6), 258-72.
9 Krieg A. CpG Motifs in bacterial DNA and their immune effect. Ann Rev.
Immunol. 2002, 20, 709- 60.
10 de Vries J, Meier P and Wackernagel W. Microbial horizontal gene
transfer and the DNA release from transgenic crop plants. Plant and Soil
2004, 266, 91-104.




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