BACKGROUND
equine melanoma, a tumor of pigment producing cells, is the most common skin tumor in aging grey horses with a prevalence of up to 95%  <cit> .
so far, conventional therapies such as surgical excision  <cit> , cryosurgery  <cit> , radiotherapy  <cit> , or chemotherapy with cisplatin  <cit>  or cimetidine  <cit>  have not been curative in advanced cases.
obviously, there is need for innovative approaches to treat equine melanoma lesions of later stages.
xenogenic dna vaccination against the melanoma differentiation antigens glycoprotein   <dig>  <cit>  and tyrosinase   <cit>  have been shown to overcome auto-tolerance and to elicit an immune response in mice, dogs, and humans  <cit>  and a clinical antitumoral effect in mice and dogs  <cit> .
in clinically healthy horses, specific antibodies at relatively low levels and a variable cellular immune response were elicited upon vaccination with a plasmid encoding the human tyrosinase   <cit> .
there are no reports about the immunogenicity of human gp <dig>  to horses, and clinical study results on the anti-melanoma effect of dna-encoded xenogenic tyrosinase and gp <dig> in grey horses have not been published to date.
cytokines such as interleukin   <dig> and il <dig> have been applied to increase cellular immunity and reduce angiogenesis in neoplasms  <cit>  and were shown to have synergistic antitumoral effects  <cit> .
to date, all immunological melanoma treatment efforts in horses were of limited effect.
a local effect was achieved with dna encoding human  <cit>  or either eqil <dig> or eqil <dig>  <cit> .
thus, combination of these interleukins and addition of either hgp <dig> or htyr, all encoded by dna vectors, seemed to be a promising approach.
the aim of the present clinical study was to evaluate whether or not treatment with eqil <dig> in combination with eqil <dig> each encoded by dna vectors has a local and systemic anti-tumoral effect on naturally occurring melanoma in grey horses, and whether or not this effect is augmented by dna vaccination against the xenogenic melanoma differentiation antigens hgp <dig> and htyr, respectively.
methods
patients
twenty-seven horses with one or more melanoma lesions and unaffected general condition were included in the study.
informed consent was obtained from all animal owners.
horses were not treated with any medication at least two weeks prior to immunisation.
pre-trial evaluation included a physical examination, hematology, and blood biochemistry profile.
age, breed, gender, degree of greying and number of melanoma lesions were documented .
the diagnosis of melanoma was confirmed by examination of fine needle aspirates by board certified pathologists in  <dig> of  <dig> horses.
in the remaining horses, diagnosis was made clinically with regard to typical localisation and appearance of the lesions, as the owners did not agree to aspiration biopsy.
patients were treated in the clinic for horses of the university of veterinary medicine hannover, foundation, from november  <dig> to july  <dig> in accordance with the ethical guidelines of the law of animal welfare approved by the “lower saxony state office for consumer protection and food safety, laves” approval no.
08/1522).table  <dig>
patient demographics and group assignment
treatment
horse id
age
breed
sex
color
number of melanoma
fna
localisation
legend:
horses were assigned to three treatment groups by a stratified biphasic model to achieve equal distribution of age  and number of melanoma lesions .
production of midge-th <dig> vectors
minimalistic immunogenically defined gene expression  vectors with a small peptide  attached were produced by mologen ag  as previously described  <cit>  from plasmids coding for hgp  <dig> , htyr , eqil <dig> and eqil <dig> .
oligonucleotides coding for the il-1beta receptor antagonist protein   were ligated to the il <dig> gene .
preparation of midge-th1/saint- <dig> complexes
midge-th1/saint- <dig>  methyl-pyridinium-chloride, synvolux therapeutics, groningen, the netherlands) complexes were prepared as described by endmann et al.
<cit> .
midge-th1/saint- <dig> complexes were formed at a ratio of 1 mg dna dissolved in pbs to  <dig>  μmol saint- <dig>  the pbs concentration in the final mixture was 1× pbs.
treatment
three groups of  <dig> grey horses each were treated on days  <dig>   <dig> and  <dig> with midge-th <dig> vectors coding for eqil <dig> and ilrap-eqil <dig> alone or in combination with hgp100midge-th <dig> or htyrmidge-th <dig> .
throughout the study period the test items were blinded by a color code and unblinded upon completion of data analysis only.table  <dig>
treatment substances administered to horses of the three treatment groups on days  <dig>   <dig> and 78
vaccine component
dose per injection
group eqil12/18
group hgp100
group htyr
for each treatment,  <dig>  ml  were injected intradermally  peritumorally around one pre-selected, well-measureable and easily-accessible melanoma .
injection was performed using a 25 g cannula and a 1 ml syringe.
another  <dig>  ml  were administered intramuscularly  into the semimembranosus muscle using a 22 g cannula and a 1 ml syringe.
the identical injection sites were used for the first, second and third immunisation.
clinical evaluation for determination of response to treatment
in each horse the locally treated melanoma and up to eight additional melanoma lesions  were monitored prior to each injection and on day  <dig> using calipers and ultrasound .
the ultrasonographic measurements were performed independently by two examiners.
all measurements were performed in triplicates.
the length , width , and depth  were documented and the tumor volume  calculated .
the relative volumes were calculated in reference to the volume on day one , which was defined as  <dig> %.
for all recorded non–locally treated melanoma lesions of each horse, a mean value per time point was calculated to statistically evaluate the effect on these tumors, i.e.
the systemic effect of the treatment.
furthermore, the change of tumor volume was calculated by subtraction of the relative volume on day  <dig>  from the relative volume on day  <dig> , with a negative value implying tumor reduction and a positive value standing for tumor growth.
these measurements and calculations were used in previous studies investigating the effect of gene therapies against equine melanoma  <cit>  and were found to be appropriate for this mostly slow growing tumor and for comparability with aforementioned studies.
clinical evaluation of safety of treatment
horses were hospitalized for 3 days after each injection.
safety and tolerability of the treatment were evaluated by clinical examination, hematologic and blood biochemistry profile: a general clinical examination was performed before injection, on each day of hospitalisation and on day  <dig>  a hematologic examination was realized before injection, on the 3rd day after injection and on day  <dig>  a biochemistry profile was accomplished before each injection and on day  <dig>  the injection sites were monitored for signs of local inflammation and depigmentation.
other pigmented skin areas  were observed for signs of depigmentation.
measurement of the immune response induced by vaccination
to measure specific serum antibodies against hgp <dig> and htyr a cell based, flow cytometric antibody assay was performed.
therefore, sera from the patients were obtained before each injection and on day  <dig>  the sera were incubated with hgp <dig> and htyr plasmid transfected hek  <dig> cells.
evaluation was performed by flow cytometry after staining bounded antibodies with a secondary antibody .
at any time post-vaccination a humoral response was considered positive when fluorescence increased ≥  <dig> standard deviations over the mean value at baseline and had an absolute value >  <dig>  %.
the establishment of an assay to detect a potential t-cell response induced by the vaccine was not followed up after transfection rates of primary autologous dermal cells, derived from skin of patients were inconsistent and low.
thus expression of antigens was insufficient to induce t cell activation after co-culture of transfected cells and peripheral blood mononuclear cells .
in vitro expression of transgenes eqil <dig>  eqil <dig>  htyr and hgp <dig> on mrna level
chinese hamster ovary -k <dig> cells  were cultured in ham’s f <dig>  medium at 37 °c in 5% co <dig>  prior to transfection the culture medium was removed, cells were washed once with pbs, then detached with trypsin/edta and  <dig> e +  <dig> cells per well suspended in 500 μl transfection medium .
100 μl of each dna/saint- <dig> complex were prepared as follows: midge-th <dig> vectors were mixed with previously vortexed saint- <dig>  at a ratio of 5 μl saint- <dig> per μg dna and filled up to 100 μl with hbs.
complexes were allowed to form for 5 min.
cells were incubated with complexes containing a) midge-th <dig> vectors encoding eqil <dig> and eqil <dig> , b) midge-th <dig> vectors encoding eqil <dig> , eqil <dig>  and htyr , c) midge-th <dig> vectors encoding eqil <dig> , eqil <dig>  and hgp <dig>  and d) midge-th <dig> vectors encoding egfp  as positive control for the transfection method and cho-k <dig> expression efficiency .
salmon sperm dna  served as negative control item.
the dna saint- <dig> complexes were added to the cells, followed by a brief centrifugation step.
after  <dig>  hours of incubation, 1 ml of complete ham’s f <dig> was added and cells incubated for 24 hours at 37 °c in 5% co <dig>  cells were harvested and detached as described above, centrifuged and pellets stored on ice until rna was extracted using the nucleospin rna ii kit  as described in provider’s instructions.
mrna specific reverse transcription quantitative pcr  was performed with 100 ng mrna per reaction using the taqman® rna-to-ct 1-step kit  according to manufacturer’s instructions.
primers and probes  had specific sequences to generate and detect cdna of eqil12-p <dig> , eqil12-p <dig> , eqil <dig> , hgp <dig>  and htyr .
samples were measured in technical triplicates.
statistical analysis
an analysis of variance  for repeated measurements was performed to identify statistically significant influences on the relative tumor volume.
parameters included in the model were the individual horse, examination day , treatment group , locally treated versus non-locally treated melanoma lesions and examination method .
after starting with the full model, non-significant variables and correlations were eliminated stepwise.
for post-hoc testing a tukey-kramer multiple-comparison test was performed to compare the relative volumes on the different examination days.
an anova for repeated measurements was performed to analyse changes in body temperature over time.
a one-way anova was used to evaluate differences in body temperature between the groups.
a p–value <  <dig>  was considered significant for all statistical tests applied.
statistical analyses were performed using the statistical software jmp  <dig>   and ncss .
RESULTS
safety of treatment
based on the clinical examinations of the horses the vaccine was safe and well tolerated.
the only consistent abnormal finding was a significant increase in body temperature on the day after injection .
no difference in body temperature between the groups  was observed.
hematology and blood biochemistry profile revealed no abnormalities at any time point.
at the intradermal injection sites , horses showed mild subcutaneous swelling , reddening , exudation  and mild ulceration .
these signs of acute inflammation occurred within the first three days after injection and resolved until the next treatment except in horse  <dig> were they persisted for 21 days after the first injection.
twentysix of  <dig> horses developed local dermal depigmentation restricted to the intradermal injection sites, only.
the depigmentation was first observed after 22 days in  <dig> horses and after 81 days in  <dig> more horses.
there were no signs of depigmentation observed in the monitored pigmented regions or melanoma lesions.
no differences of local reactions were noted between treatment groups.
no local reactions were observed at the sites of intramuscular injections.figure  <dig> daily rectal temperatures  measured in  <dig> horses before each injection  and on three consecutive days.
there was a significant transient increase in the temperature on the first day after injection .
the center horizontal line of the box plot marks the median of the sample.
the edges of the box mark the first and third quartiles.
the dotted lines define the 75th percentile plus  <dig>  times the interquartile range  and the 25th percentile minus  <dig>  times iqr.
the singular dots represent the outside values.
*** significant difference .
caliper and ultrasonographic measurements of tumor size
in total  <dig> melanoma lesions  were measured.
after stepwise elimination of insignificant variables and correlations the only variable influencing the relative volume was the examination day  and an individual effect of the horses.
post-hoc testing showed a significant decrease of the relative melanoma-volume over time.
mean relative volume of all measured melanoma decreased significantly to  <dig>  ±  <dig> % - equalling to  <dig> % of tumor volume reduction - as measured by caliper and  <dig>  ±  <dig> % ,  <dig>  ±  <dig> %  as measured by ultrasonography  .
the relative tumor volume ranged from  <dig> to 205%  and  <dig> to  <dig>  196%  at day  <dig> figure  <dig> relative tumor volumes of melanoma lesions during the observation period of  <dig> days.
a): relative volumes  of locally and non-locally treated melanoma lesions calculated by caliper and ultrasound measurements  on days  <dig>   <dig>   <dig> and  <dig>  relative volume of melanoma lesions decreased significantly from day  <dig> to  <dig> .
there were no statistically significant differences detected between the treatment groups, locally or non-locally treated melanoma lesions and measurement modality/ultrasound examiner.
dots, squares and triangles represent the median and the vertical lines the standard deviations.
b): relative volumes  of non-locally treated melanoma lesions calculated by caliper measurements on days  <dig>   <dig>   <dig> and  <dig>  relative volume of all non-locally treated melanoma lesions decreased significantly on day  <dig>  there were no statistically significant differences detected between the treatment groups.
2c): relative volumes  of locally treated melanoma lesions calculated by caliper measurements on days  <dig>   <dig>   <dig> and  <dig>  relative volume of all locally treated melanoma lesions decreased significantly on day  <dig>  there were no statistically significant differences detected between the treatment groups.
2d): relative volumes  of non-locally treated melanoma lesions calculated by ultrasonographic measurements on days  <dig>   <dig>   <dig> and  <dig>  relative volume of all non-locally treated melanoma lesions decreased significantly on day  <dig>  there were no statistically significant differences detected between the treatment groups.
2e): relative volumes  of locally treated melanoma lesions calculated by ultrasonographic measurements on days  <dig>   <dig>   <dig> and  <dig>  relative volume of all locally treated melanoma lesions decreased significantly on day  <dig>  there were no statistically significant differences detected between the treatment groups.
*** significant difference .
relative volume of melanoma lesions measured by calipers in individual horses on day  <dig>  and number of new lesions by day 120
treatment
horse id
% of reduction of the tumor volumes at day  <dig> in reference to the volume on day 1
number of new lesions
peritumoral treated melanoma lesion
distant melanoma lesions
-
-
-
-
measurement of the humoral response induced by vaccination
no specific antibodies against htyr or hgp <dig> were detected in equine sera collected on days  <dig>   <dig>   <dig> and  <dig> .
proof of function of dna vectors: in vitro expression
the expression of all encoded genes was proven on mrna level after transfection of cells in vitro.
midge-th <dig> vectors were used in mixtures at weight ratios equal to those administered to the patients and complexed with saint- <dig>  the amount of cdna generated from mrna was analysed in a rt-qpcr assay using a plasmid standard, allowing for detection and estimation of the mrna expression levels produced from individual transgenes.
eqil <dig>  eqil <dig>  htyr and hgp <dig> were shown to be expressed from all respective mixtures of midge-th <dig> vectors .table  <dig>
results of rt-qpcr to evaluate
in vitro
expression of transgenes from midge-th <dig> vectors encoding eqil <dig>  eqil <dig>  htyr and hgp100
midge-th1
estimated mrna / cdna copy numbers per transgene
vectors
eqil12-p35
eqil12-p40
eqil18
htyr
hgp100
lod : = <  <dig> copies/reaction.
discussion
in the present clinical study, three treatments with mixtures of midge-th <dig> eqil <dig> and ilrap-eqil <dig> alone or in combination with midge-th <dig> hgp <dig> or midge-th <dig> htyr, all complexed with the cationic transfection agent saint- <dig>  resulted in a moderate reduction of the relative volume of examined melanoma lesions .
the lack of differences in treatment effects between the three groups suggests that midge-th <dig> hgp <dig> or midge-th <dig> htyr did not augment the effect of the other components of the treatment.
in former studies on equine melanoma the placebo group showed progression or no change in size during the study period of 64 days  <cit> .
a placebo group was not included in this study because a positive anti-tumoral effect of treatment with interleukin- <dig> and − <dig> dna had been demonstrated in these earlier studies.
ethical considerations regarding withholding any potentially effective treatment from client-owned horses precluded inclusion of an untreated cohort.
however, it is reasonable to attribute the tumor size reduction observed in this study to the treatment, as no spontaneous regression of equine melanoma lesions has been reported up to date.
importantly, the size reduction was not only seen in locally treated but also in non-locally treated melanoma lesions indicating a systemic effect of the treatment.
comparing the development of volume of peritumorally treated and distant melanoma lesions, there was no statistically significant difference observed.
the systemic effect might be based on a primarily unspecific immune response that, in the presence of melanoma antigens, converted into an anti-melanoma specific immune response.
however, this is a hypothesis only since such aspecific immune response has not been detected in our assays.
the present study was the first to use a combination of dna vectors encoding eqil <dig> and eqil <dig> for the therapy of equine melanoma.
previously either eqil <dig> or eqil <dig> dna injected intratumorally induced some size reduction in equine melanoma lesions  <cit>  but systemic effects were not evaluated.
experimental studies in mice  <cit>  have proven synergistic effects of il <dig> and il <dig>  suppressing collateral  <cit>  or distant tumor growth  <cit> .
in the present study the cationic-amphiphilic transfection agent saint- <dig> was used to enhance expression in vivo.
saint- <dig> has been reported to strengthen the immune response against antigens encoded by midge-th <dig> vectors  <cit> .
while the complexed dna was very well tolerated in mice and rats  <cit> , in the horses of the study presented here it caused local inflammation at the site of intradermal injection.
this could be either explained by an inflammatory reaction triggered by the two expressed cytokines eqil <dig> and eqil <dig> upon transfection of dermal cells, or respectively and by a higher sensitivity of horses against dna complexed with saint- <dig>  in mice, expressed cytokines were found to mediate antitumoral effects by reduction of angiogenesis  <cit>  as well as improved cellular immunity represented by increased ifnγ production and enhanced cytotoxic t- and natural killer cell activities  <cit> .
in addition to the local reaction, horses developed fever on the day post injection, a systemic reaction to the treatment.
dow et al.
<cit>  showed that i.v.
injections of plasmid dna complexed with lipid-protamine with non-coding dna, resulted in local inflammation of the lung and other organs of mice.
thus, it is possible that in the present study fever and the local inflammation were in part induced by the complexes of dna and transfection agent.
in mice injection of saint- <dig> formulated dna was tolerated well.
neither local nor systemic signs of intolerance were observed  <cit> .
the use of saint- <dig> in pigs proved to enhance their humoral immune response to dna vaccination.
the study did not mention adverse side effects of the transfection reagent  <cit> .
however both, the fever as well as the signs of acute inflammation were transient and are therefore considered not to be a serious drawback regarding the safety of the vaccination.
as dow et al.
<cit>  showed also that lipoplexes with dna encoding il <dig> induced a more effective antitumor response than lipoplexes with non-coding plasmid dna in mice the effect of the dna-saint-18-complex might have potentially enhanced the antitumoral effect of the interleukins.
the tumor size reduction in the present study was moderate and – according to the response evaluation criteria in solid tumors   <cit>  or immune-related response criteria   <cit>  used in human medicine - would not be considered as tumor regression but as stable disease.
however, wolchok et al.
consider stable disease as a positive therapeutic outcome in cancer immunotherapy  <cit> .
dosages of  <dig>   <dig> and 1500 μg plasmid dna for hgp <dig> in humans  <cit>  and for htyr in dogs  <cit>  successfully induced detectable immune responses.
in the studies of lembcke et al.
<cit>  and phillips et al.
<cit>  a dosage of only 100 μg plasmid induced a measurable, though variable and relatively weak, immune response in nontumor-bearing healthy horses.
thus the doses used in our study, i.e.
500 μg per antigen encoding midge-th <dig> vector per application should have been appropriate to induce a detectable immune response.
moreover, midge-vectors are devoid of plasmid backbone dna and therefore have a smaller molecule size, so their effective transgene dose per microgram dna is much higher than that of conventional plasmids  <cit> .
tyrosinase and gp <dig> were found to be expressed in equine melanoma lesions  <cit> .
homology between equine and human proteins is comparable to the proteins of mice compared to human for gp <dig>  and canine tyrosinase as well as the mouse and human equivalent .
since xenogenic dna vaccination encoding these proteins in mice and dogs induced an antitumoral immune response  <cit>  it can be assumed that the proteins chosen and the difference in the amino acid sequence are adequate.
the vaccination interval used in the recent study is comparable to immunisation strategies for infectious diseases and also to a therapeutic vaccination protocol against melanoma in humans  <cit> .
however, vaccination against tumors generally elicits a weaker immunity than vaccination against infectious diseases  <cit> .
in other studies, in which an immunological response was successfully induced, more frequent application schemes for therapeutical antitumor vaccines  <cit>  or cytokine gene therapy were used  <cit> .
lembcke et al.
<cit>  vaccinated healthy horses four times with human tyrosinase dna every two weeks, resulting in the successful induction of specific antibodies and ifnγ producing cells.
they stated that the altered immunologic status of tumor-bearing patients  might make it more challenging to induce a specific immune response to tumor antigens than in healthy animals  <cit> .
no specific humoral response could be observed in the present study.
in future studies additional tests to evaluate the cellular immune response  <cit>  might give additional information about immunological mechanisms.
however, in contrast to studies by lembcke et al.
<cit>  and phillips et al.
<cit> , we treated melanoma-bearing horses which might have developed an immune tolerance to gp <dig> and tyrosinase previously.
to overcome this specific immune tolerance, higher dna doses encoding xenogenic antigens and/or a more condensed immunisation schedule than in the present study might be necessary to induce a detectable immune response.
CONCLUSIONS
in the present study, treatment of horses with midge-th <dig> vectors encoding eqil <dig> and ilrap-eqil <dig> alone or in combination with midge-th <dig> vectors encoding hgp <dig> or human htyr, all complexed with the transfection agent saint- <dig> resulted in a measurable systemic antitumoral effect on equine melanoma lesions.
to our knowledge this is the first report on a systemic antitumoral effect against equine melanoma upon treatment with dna vectors.
because no specific immune response was detected, it remains to be elucidated whether this systemic antitumoral effect was caused by interleukins expressed from the dna vectors, or an unspecific immune reaction to the combination of dna and transfection reagent.
in future studies, a stronger anti-tumoral effect as well as a detectable specific immune response may be induced by an increased vaccine dose and/or an improved dosing schedule.
additionally, more sensitive immune assays should be applied to characterize the immune response in detail.
additional files
additional file 1:
supplemental digital content  <dig>  contains supplementary text file  <dig>
additional file 2:
figure s <dig>  no specific antibodies were detected in serum samples from vaccinated horses on days  <dig>   <dig>   <dig> and  <dig>
abbreviations
anovaanalysis of variance
dnadeoxyribonucleic acid
cdnacomplementary deoxyribonucleic acid
chochinese hamster ovary
edtaethylenediaminetetraacetic acid
egfpenhanced green fluorescent protein
eqequine
facsfluorescence-activates cell sorting
fcsfetal calf serum
gpglycoprotein
hgphuman glycoprotein
htyrhuman tyrosinase
i.d.intradermally
i.m.intramuscularly
ilinterleukin
ilrapil-1beta receptor antagonist protein
irrcimmune-related response criteria
midgeminimalistic immunogenically defined gene expression
mrnamessenger ribonucleic acid
pbsphosphate buffered saline
recistresponse evaluation criteria in solid tumors
rnaribonucleic acid
rt-qpcrreverse transcription quantitative polymerase chain reaction
tyrtyrosinase
competing interests
christiane juhls, detlef oswald and anne endmann are employees of mologen ag but have no additional financial interests.
mologen ag owns a patent for midge-th <dig> vectors .
the other authors have no financial interest in the subject matter or materials discussed in this manuscript.
authors’ contributions
km, hjs, kf and jmvc designed the concept of the study and the manuscript.
km and jmvc conducted the experiments of the present study.
km and mh did the ultrasonographic measurements of the tumors.
cj, ae, and do designed the treatment constructs and determined the application details.
md performed the statistical analysis.
all authors contributed to the critical revision and finalization of the manuscript.

 equine melanoma, a tumor of pigment producing cells, is the most common skin tumor in aging grey horses with a prevalence of up to 95%. so far, conventional therapies such as surgical excision, cryosurgery, radiotherapy, or chemotherapy with cisplatin or cimetidine have not been curative in advanced cases. obviously, there is need for innovative approaches to treat equine melanoma lesions of later stages. xenogenic dna vaccination against the melanoma differentiation antigens glycoprotein and tyrosinase have been shown to overcome auto-tolerance and to elicit an immune response in mice, dogs, and humans and a clinical antitumoral effect in mice and dogs. in clinically healthy horses, specific antibodies at relatively low levels and a variable cellular immune response were elicited upon vaccination with a plasmid encoding the human tyrosinase. there are no reports about the immunogenicity of human gp to horses, and clinical study results on the anti-melanoma effect of dna-encoded xenogenic tyrosinase and gp in grey horses have not been published to date. cytokines such as interleukin and il have been applied to increase cellular immunity and reduce angiogenesis in neoplasms and were shown to have synergistic antitumoral effects. to date, all immunological melanoma treatment efforts in horses were of limited effect. a local effect was achieved with dna encoding human or either eqil or eqil. thus, combination of these interleukins and addition of either hgp or htyr, all encoded by dna vectors, seemed to be a promising approach. the aim of the present clinical study was to evaluate whether or not treatment with eqil in combination with eqil each encoded by dna vectors has a local and systemic anti-tumoral effect on naturally occurring melanoma in grey horses, and whether or not this effect is augmented by dna vaccination against the xenogenic melanoma differentiation antigens hgp and htyr, respectively. methods patients twenty-seven horses with one or more melanoma lesions and unaffected general condition were included in the study. informed consent was obtained from all animal owners. horses were not treated with any medication at least two weeks prior to immunisation. pre-trial evaluation included a physical examination, hematology, and blood biochemistry profile. age, breed, gender, degree of greying and number of melanoma lesions were documented. the diagnosis of melanoma was confirmed by examination of fine needle aspirates by board certified pathologists in of horses. in the remaining horses, diagnosis was made clinically with regard to typical localisation and appearance of the lesions, as the owners did not agree to aspiration biopsy. patients were treated in the clinic for horses of the university of veterinary medicine hannover, foundation, from november to july in accordance with the ethical guidelines of the law of animal welfare approved by the “lower saxony state office for consumer protection and food safety, laves” approval no. 08/1522).table patient demographics and group assignment treatment horse id age breed sex color number of melanoma fna localisation legend: horses were assigned to three treatment groups by a stratified biphasic model to achieve equal distribution of age and number of melanoma lesions. production of midge-th vectors minimalistic immunogenically defined gene expression vectors with a small peptide attached were produced by mologen ag as previously described from plasmids coding for hgp, htyr, eqil and eqil. oligonucleotides coding for the il-1beta receptor antagonist protein were ligated to the il gene. preparation of midge-th1/saint- complexes midge-th1/saint- methyl-pyridinium-chloride, synvolux therapeutics, groningen, the netherlands) complexes were prepared as described by endmann et al.. midge-th1/saint- complexes were formed at a ratio of 1 mg dna dissolved in pbs to  μmol saint- the pbs concentration in the final mixture was 1× pbs. treatment three groups of grey horses each were treated on days and with midge-th vectors coding for eqil and ilrap-eqil alone or in combination with hgp100midge-th or htyrmidge-th. throughout the study period the test items were blinded by a color code and unblinded upon completion of data analysis only.table treatment substances administered to horses of the three treatment groups on days and 78 vaccine component dose per injection group eqil12/18 group hgp100 group htyr for each treatment,  ml were injected intradermally peritumorally around one pre-selected, well-measureable and easily-accessible melanoma. injection was performed using a 25 g cannula and a 1 ml syringe. another  ml were administered intramuscularly into the semimembranosus muscle using a 22 g cannula and a 1 ml syringe. the identical injection sites were used for the first, second and third immunisation. clinical evaluation for determination of response to treatment in each horse the locally treated melanoma and up to eight additional melanoma lesions were monitored prior to each injection and on day using calipers and ultrasound. the ultrasonographic measurements were performed independently by two examiners. all measurements were performed in triplicates. the length, width, and depth were documented and the tumor volume calculated. the relative volumes were calculated in reference to the volume on day one, which was defined as %. for all recorded non–locally treated melanoma lesions of each horse, a mean value per time point was calculated to statistically evaluate the effect on these tumors, i.e. the systemic effect of the treatment. furthermore, the change of tumor volume was calculated by subtraction of the relative volume on day from the relative volume on day, with a negative value implying tumor reduction and a positive value standing for tumor growth. these measurements and calculations were used in previous studies investigating the effect of gene therapies against equine melanoma and were found to be appropriate for this mostly slow growing tumor and for comparability with aforementioned studies. clinical evaluation of safety of treatment horses were hospitalized for 3 days after each injection. safety and tolerability of the treatment were evaluated by clinical examination, hematologic and blood biochemistry profile: a general clinical examination was performed before injection, on each day of hospitalisation and on day a hematologic examination was realized before injection, on the 3rd day after injection and on day a biochemistry profile was accomplished before each injection and on day the injection sites were monitored for signs of local inflammation and depigmentation. other pigmented skin areas were observed for signs of depigmentation. measurement of the immune response induced by vaccination to measure specific serum antibodies against hgp and htyr a cell based, flow cytometric antibody assay was performed. therefore, sera from the patients were obtained before each injection and on day the sera were incubated with hgp and htyr plasmid transfected hek cells. evaluation was performed by flow cytometry after staining bounded antibodies with a secondary antibody. at any time post-vaccination a humoral response was considered positive when fluorescence increased ≥  standard deviations over the mean value at baseline and had an absolute value >  %. the establishment of an assay to detect a potential t-cell response induced by the vaccine was not followed up after transfection rates of primary autologous dermal cells, derived from skin of patients were inconsistent and low. thus expression of antigens was insufficient to induce t cell activation after co-culture of transfected cells and peripheral blood mononuclear cells. in vitro expression of transgenes eqil eqil htyr and hgp on mrna level chinese hamster ovary -k cells were cultured in ham’s f medium at 37 °c in 5% co prior to transfection the culture medium was removed, cells were washed once with pbs, then detached with trypsin/edta and e +  cells per well suspended in 500 μl transfection medium. 100 μl of each dna/saint- complex were prepared as follows: midge-th vectors were mixed with previously vortexed saint- at a ratio of 5 μl saint- per μg dna and filled up to 100 μl with hbs. complexes were allowed to form for 5 min. cells were incubated with complexes containing a) midge-th vectors encoding eqil and eqil, b) midge-th vectors encoding eqil, eqil and htyr, c) midge-th vectors encoding eqil, eqil and hgp and d) midge-th vectors encoding egfp as positive control for the transfection method and cho-k expression efficiency. salmon sperm dna served as negative control item. the dna saint- complexes were added to the cells, followed by a brief centrifugation step. after  hours of incubation, 1 ml of complete ham’s f was added and cells incubated for 24 hours at 37 °c in 5% co cells were harvested and detached as described above, centrifuged and pellets stored on ice until rna was extracted using the nucleospin rna ii kit as described in provider’s instructions. mrna specific reverse transcription quantitative pcr was performed with 100 ng mrna per reaction using the taqman® rna-to-ct 1-step kit according to manufacturer’s instructions. primers and probes had specific sequences to generate and detect cdna of eqil12-p, eqil12-p, eqil, hgp and htyr. samples were measured in technical triplicates. statistical analysis an analysis of variance for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. parameters included in the model were the individual horse, examination day, treatment group, locally treated versus non-locally treated melanoma lesions and examination method. after starting with the full model, non-significant variables and correlations were eliminated stepwise. for post-hoc testing a tukey-kramer multiple-comparison test was performed to compare the relative volumes on the different examination days. an anova for repeated measurements was performed to analyse changes in body temperature over time. a one-way anova was used to evaluate differences in body temperature between the groups. a p–value <  was considered significant for all statistical tests applied. statistical analyses were performed using the statistical software jmp and ncss.  safety of treatment based on the clinical examinations of the horses the vaccine was safe and well tolerated. the only consistent abnormal finding was a significant increase in body temperature on the day after injection. no difference in body temperature between the groups was observed. hematology and blood biochemistry profile revealed no abnormalities at any time point. at the intradermal injection sites, horses showed mild subcutaneous swelling, reddening, exudation and mild ulceration. these signs of acute inflammation occurred within the first three days after injection and resolved until the next treatment except in horse were they persisted for 21 days after the first injection. twentysix of horses developed local dermal depigmentation restricted to the intradermal injection sites, only. the depigmentation was first observed after 22 days in horses and after 81 days in more horses. there were no signs of depigmentation observed in the monitored pigmented regions or melanoma lesions. no differences of local reactions were noted between treatment groups. no local reactions were observed at the sites of intramuscular injections.figure daily rectal temperatures measured in horses before each injection and on three consecutive days. there was a significant transient increase in the temperature on the first day after injection. the center horizontal line of the box plot marks the median of the sample. the edges of the box mark the first and third quartiles. the dotted lines define the 75th percentile plus times the interquartile range and the 25th percentile minus times iqr. the singular dots represent the outside values. *** significant difference. caliper and ultrasonographic measurements of tumor size in total melanoma lesions were measured. after stepwise elimination of insignificant variables and correlations the only variable influencing the relative volume was the examination day and an individual effect of the horses. post-hoc testing showed a significant decrease of the relative melanoma-volume over time. mean relative volume of all measured melanoma decreased significantly to  ±  % - equalling to % of tumor volume reduction - as measured by caliper and  ±  %,  ±  % as measured by ultrasonography. the relative tumor volume ranged from to 205% and to 196% at day figure relative tumor volumes of melanoma lesions during the observation period of days. a): relative volumes of locally and non-locally treated melanoma lesions calculated by caliper and ultrasound measurements on days and relative volume of melanoma lesions decreased significantly from day to. there were no statistically significant differences detected between the treatment groups, locally or non-locally treated melanoma lesions and measurement modality/ultrasound examiner. dots, squares and triangles represent the median and the vertical lines the standard deviations. b): relative volumes of non-locally treated melanoma lesions calculated by caliper measurements on days and relative volume of all non-locally treated melanoma lesions decreased significantly on day there were no statistically significant differences detected between the treatment groups. 2c): relative volumes of locally treated melanoma lesions calculated by caliper measurements on days and relative volume of all locally treated melanoma lesions decreased significantly on day there were no statistically significant differences detected between the treatment groups. 2d): relative volumes of non-locally treated melanoma lesions calculated by ultrasonographic measurements on days and relative volume of all non-locally treated melanoma lesions decreased significantly on day there were no statistically significant differences detected between the treatment groups. 2e): relative volumes of locally treated melanoma lesions calculated by ultrasonographic measurements on days and relative volume of all locally treated melanoma lesions decreased significantly on day there were no statistically significant differences detected between the treatment groups. *** significant difference. relative volume of melanoma lesions measured by calipers in individual horses on day and number of new lesions by day 120 treatment horse id % of reduction of the tumor volumes at day in reference to the volume on day 1 number of new lesions peritumoral treated melanoma lesion distant melanoma lesions - - - - measurement of the humoral response induced by vaccination no specific antibodies against htyr or hgp were detected in equine sera collected on days and. proof of function of dna vectors: in vitro expression the expression of all encoded genes was proven on mrna level after transfection of cells in vitro. midge-th vectors were used in mixtures at weight ratios equal to those administered to the patients and complexed with saint- the amount of cdna generated from mrna was analysed in a rt-qpcr assay using a plasmid standard, allowing for detection and estimation of the mrna expression levels produced from individual transgenes. eqil eqil htyr and hgp were shown to be expressed from all respective mixtures of midge-th vectors.table results of rt-qpcr to evaluate in vitro expression of transgenes from midge-th vectors encoding eqil eqil htyr and hgp100 midge-th1 estimated mrna / cdna copy numbers per transgene vectors eqil12-p35 eqil12-p40 eqil18 htyr hgp100 lod: = <  copies/reaction. discussion in the present clinical study, three treatments with mixtures of midge-th eqil and ilrap-eqil alone or in combination with midge-th hgp or midge-th htyr, all complexed with the cationic transfection agent saint- resulted in a moderate reduction of the relative volume of examined melanoma lesions. the lack of differences in treatment effects between the three groups suggests that midge-th hgp or midge-th htyr did not augment the effect of the other components of the treatment. in former studies on equine melanoma the placebo group showed progression or no change in size during the study period of 64 days. a placebo group was not included in this study because a positive anti-tumoral effect of treatment with interleukin- and − dna had been demonstrated in these earlier studies. ethical considerations regarding withholding any potentially effective treatment from client-owned horses precluded inclusion of an untreated cohort. however, it is reasonable to attribute the tumor size reduction observed in this study to the treatment, as no spontaneous regression of equine melanoma lesions has been reported up to date. importantly, the size reduction was not only seen in locally treated but also in non-locally treated melanoma lesions indicating a systemic effect of the treatment. comparing the development of volume of peritumorally treated and distant melanoma lesions, there was no statistically significant difference observed. the systemic effect might be based on a primarily unspecific immune response that, in the presence of melanoma antigens, converted into an anti-melanoma specific immune response. however, this is a hypothesis only since such aspecific immune response has not been detected in our assays. the present study was the first to use a combination of dna vectors encoding eqil and eqil for the therapy of equine melanoma. previously either eqil or eqil dna injected intratumorally induced some size reduction in equine melanoma lesions but systemic effects were not evaluated. experimental studies in mice have proven synergistic effects of il and il suppressing collateral or distant tumor growth. in the present study the cationic-amphiphilic transfection agent saint- was used to enhance expression in vivo. saint- has been reported to strengthen the immune response against antigens encoded by midge-th vectors. while the complexed dna was very well tolerated in mice and rats, in the horses of the study presented here it caused local inflammation at the site of intradermal injection. this could be either explained by an inflammatory reaction triggered by the two expressed cytokines eqil and eqil upon transfection of dermal cells, or respectively and by a higher sensitivity of horses against dna complexed with saint- in mice, expressed cytokines were found to mediate antitumoral effects by reduction of angiogenesis as well as improved cellular immunity represented by increased ifnγ production and enhanced cytotoxic t- and natural killer cell activities. in addition to the local reaction, horses developed fever on the day post injection, a systemic reaction to the treatment. dow et al. showed that i.v. injections of plasmid dna complexed with lipid-protamine with non-coding dna, resulted in local inflammation of the lung and other organs of mice. thus, it is possible that in the present study fever and the local inflammation were in part induced by the complexes of dna and transfection agent. in mice injection of saint- formulated dna was tolerated well. neither local nor systemic signs of intolerance were observed. the use of saint- in pigs proved to enhance their humoral immune response to dna vaccination. the study did not mention adverse side effects of the transfection reagent. however both, the fever as well as the signs of acute inflammation were transient and are therefore considered not to be a serious drawback regarding the safety of the vaccination. as dow et al. showed also that lipoplexes with dna encoding il induced a more effective antitumor response than lipoplexes with non-coding plasmid dna in mice the effect of the dna-saint-18-complex might have potentially enhanced the antitumoral effect of the interleukins. the tumor size reduction in the present study was moderate and – according to the response evaluation criteria in solid tumors or immune-related response criteria used in human medicine - would not be considered as tumor regression but as stable disease. however, wolchok et al. consider stable disease as a positive therapeutic outcome in cancer immunotherapy. dosages of and 1500 μg plasmid dna for hgp in humans and for htyr in dogs successfully induced detectable immune responses. in the studies of lembcke et al. and phillips et al. a dosage of only 100 μg plasmid induced a measurable, though variable and relatively weak, immune response in nontumor-bearing healthy horses. thus the doses used in our study, i.e. 500 μg per antigen encoding midge-th vector per application should have been appropriate to induce a detectable immune response. moreover, midge-vectors are devoid of plasmid backbone dna and therefore have a smaller molecule size, so their effective transgene dose per microgram dna is much higher than that of conventional plasmids. tyrosinase and gp were found to be expressed in equine melanoma lesions. homology between equine and human proteins is comparable to the proteins of mice compared to human for gp and canine tyrosinase as well as the mouse and human equivalent. since xenogenic dna vaccination encoding these proteins in mice and dogs induced an antitumoral immune response it can be assumed that the proteins chosen and the difference in the amino acid sequence are adequate. the vaccination interval used in the recent study is comparable to immunisation strategies for infectious diseases and also to a therapeutic vaccination protocol against melanoma in humans. however, vaccination against tumors generally elicits a weaker immunity than vaccination against infectious diseases. in other studies, in which an immunological response was successfully induced, more frequent application schemes for therapeutical antitumor vaccines or cytokine gene therapy were used. lembcke et al. vaccinated healthy horses four times with human tyrosinase dna every two weeks, resulting in the successful induction of specific antibodies and ifnγ producing cells. they stated that the altered immunologic status of tumor-bearing patients might make it more challenging to induce a specific immune response to tumor antigens than in healthy animals. no specific humoral response could be observed in the present study. in future studies additional tests to evaluate the cellular immune response might give additional information about immunological mechanisms. however, in contrast to studies by lembcke et al. and phillips et al., we treated melanoma-bearing horses which might have developed an immune tolerance to gp and tyrosinase previously. to overcome this specific immune tolerance, higher dna doses encoding xenogenic antigens and/or a more condensed immunisation schedule than in the present study might be necessary to induce a detectable immune response.  in the present study, treatment of horses with midge-th vectors encoding eqil and ilrap-eqil alone or in combination with midge-th vectors encoding hgp or human htyr, all complexed with the transfection agent saint- resulted in a measurable systemic antitumoral effect on equine melanoma lesions. to our knowledge this is the first report on a systemic antitumoral effect against equine melanoma upon treatment with dna vectors. because no specific immune response was detected, it remains to be elucidated whether this systemic antitumoral effect was caused by interleukins expressed from the dna vectors, or an unspecific immune reaction to the combination of dna and transfection reagent. in future studies, a stronger anti-tumoral effect as well as a detectable specific immune response may be induced by an increased vaccine dose and/or an improved dosing schedule. additionally, more sensitive immune assays should be applied to characterize the immune response in detail. additional files additional file 1: supplemental digital content contains supplementary text file additional file 2: figure s no specific antibodies were detected in serum samples from vaccinated horses on days and abbreviations anovaanalysis of variance dnadeoxyribonucleic acid cdnacomplementary deoxyribonucleic acid chochinese hamster ovary edtaethylenediaminetetraacetic acid egfpenhanced green fluorescent protein eqequine facsfluorescence-activates cell sorting fcsfetal calf serum gpglycoprotein hgphuman glycoprotein htyrhuman tyrosinase i.d.intradermally i.m.intramuscularly ilinterleukin ilrapil-1beta receptor antagonist protein irrcimmune-related response criteria midgeminimalistic immunogenically defined gene expression mrnamessenger ribonucleic acid pbsphosphate buffered saline recistresponse evaluation criteria in solid tumors rnaribonucleic acid rt-qpcrreverse transcription quantitative polymerase chain reaction tyrtyrosinase competing interests christiane juhls, detlef oswald and anne endmann are employees of mologen ag but have no additional financial interests. mologen ag owns a patent for midge-th vectors. the other authors have no financial interest in the subject matter or materials discussed in this manuscript. authors’ contributions km, hjs, kf and jmvc designed the concept of the study and the manuscript. km and jmvc conducted the experiments of the present study. km and mh did the ultrasonographic measurements of the tumors. cj, ae, and do designed the treatment constructs and determined the application details. md performed the statistical analysis. all authors contributed to the critical revision and finalization of the manuscript. 
for post-hoc testing a tukey-kramer multiple-comparison test was performed to compare the relative volumes on the different examination days. affiliation to treatment group, local treatment and examination modality had no significant influence on the results. an analysis of variance for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. in all groups, the relative tumor volume decreased significantly to  ±  % by day. prior to each injection and on day the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. this is the first clinical report on a systemic effect against equine melanoma following treatment with dna vectors encoding eqil and eqil and formulated with a transfection reagent. neither a cellular nor a humoral immune response directed against htyr or hgp was detected. xenogenic dna vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. a one-way anova was used to evaluate differences in body temperature between the groups. an anova for repeated measurements was performed to analyse changes in body temperature over time. specific serum antibodies against hgp and htyr were measured using cell based flow-cytometric assays. in a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups and vaccinated on days and with dna vectors encoding for equine il- and il- alone or in combination with either human glycoprotein or human tyrosinase. addition of dna vectors encoding hgp respectively htyr did not potentiate this effect. equine melanoma has a high incidence in grey horses. a p–value <  was considered significant for all statistical tests applied. horses had an increased body temperature on the day after vaccination.
 equine melanoma has a high incidence in grey horses. xenogenic dna vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. in a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups and vaccinated on days and with dna vectors encoding for equine il- and il- alone or in combination with either human glycoprotein or human tyrosinase. horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. prior to each injection and on day the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. specific serum antibodies against hgp and htyr were measured using cell based flow-cytometric assays. an analysis of variance for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. for post-hoc testing a tukey-kramer multiple-comparison test was performed to compare the relative volumes on the different examination days. an anova for repeated measurements was performed to analyse changes in body temperature over time. a one-way anova was used to evaluate differences in body temperature between the groups. a p–value <  was considered significant for all statistical tests applied.  in all groups, the relative tumor volume decreased significantly to  ±  % by day. affiliation to treatment group, local treatment and examination modality had no significant influence on the results. neither a cellular nor a humoral immune response directed against htyr or hgp was detected. horses had an increased body temperature on the day after vaccination.  this is the first clinical report on a systemic effect against equine melanoma following treatment with dna vectors encoding eqil and eqil and formulated with a transfection reagent. addition of dna vectors encoding hgp respectively htyr did not potentiate this effect. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords horsemelanomainterleukinglycoprotein 100tyrosinasedna vaccineissue-copyright-statement© the author 2015 