**3. Results**

All the vaccine dilutions stimulated the TLR 4 but not the TLR2. The negative control (HA powder) did not stimulate any TLR. The SDS PAGE revealed two bands in the 95 kDa region which were previously demonstrated to be HSPs rich bands (fig. 3) The dot blots revealed that all the vaccine contained gp96 and HSP70 but the amount was different (fig.3 and 4).

Neither dog showed side effects after the injections, whether systemic or local. Two showed a decrease in nodule volume of less than 50% following the first month of injections and were rated MR. The other were rated SD. All dogs were rated PD about one month before they died.

No sign of infection such as fever was observed during the first month of vaccination. The Jagdterrier was diagnosed with an abscess of the collar by the fourth month, which was cured surgically. The lung radiographs did not reveal any lung metastases. The blood count did not show any anomaly in the white cell count up to the last month of survival.

0.2 ml of the previous solution was used to make electrophoretic control. The solution was centrifuged at 1000g 30 for seconds. The supernatant was discarded and the powder in the pellet washed with 0.1 ml of a 0.5 M NaCl solution. The solution was again centrifuged and the supernatant was used for a SDS-Page and for protein quantification using UV spectrometer. 10 μl of the solution was also used for dot blot with anti HSP70 and anti gp90 antibodies on a nitrocellulose membrane. The antibody labelling was evidenced using a westernbreeze™

The dogs were injected with 0.5 ml (about 40 μg of proteins) of the vaccine subcutaneously on day 0. The dose schema consisted of 0.5 ml/dose every week for one month, followed by one

Some HSPs are TLR (Toll like receptor) agonist and the TLR activation was checked using two cell lines secreting an embryonic phosphatase alkaline when the TLR 2 or 4 are stimulated. Two cell lines were used for TLR receptor stimulation. HEK-BlueTM-hTLR2 and HEK-BlueTMhTLR4 cells (invivogen –France) were grown in DMEM supplemented with 10% fetal calf serum and 0,5% normocin (invivogen-France) to avoid mycoplasma contamination. 20000 cells were introduced in wells of 96-well plate, 4 hours later 20 μl of different concentrations of the vaccine was added and incubated at 37°C for 20 hours. Then the medium was removed and replaced by 180 μl of resuspended QUANTI- blueTM (Invivogen) and incubated for 1 hour and the positivity evaluated. The negative control was the HA- powder without any protein immobilized on its surface. The minimal concentration turning blue the medium was noted. It was estimated that the vaccine was functional when the minimal active concentration was

All the vaccine dilutions stimulated the TLR 4 but not the TLR2. The negative control (HA powder) did not stimulate any TLR. The SDS PAGE revealed two bands in the 95 kDa region which were previously demonstrated to be HSPs rich bands (fig. 3) The dot blots revealed that

Neither dog showed side effects after the injections, whether systemic or local. Two showed a decrease in nodule volume of less than 50% following the first month of injections and were rated MR. The other were rated SD. All dogs were rated PD about one month before they died. No sign of infection such as fever was observed during the first month of vaccination. The Jagdterrier was diagnosed with an abscess of the collar by the fourth month, which was cured surgically. The lung radiographs did not reveal any lung metastases. The blood count did not

all the vaccine contained gp96 and HSP70 but the amount was different (fig.3 and 4).

show any anomaly in the white cell count up to the last month of survival.

(invitrogen) kit according to the manufacturer instructions.

**2.5. Vaccination protocol**

dose every month for four months.

506 Advances in Biomaterials Science and Biomedical Applications

less than the vaccine concentration.

**3. Results**

**2.6. activation of TLR2 and 4**

**Figure 3.** L1 was obtained after washing the powder with 0.3M NaCl, L2 was obtained after washing at 0.02M, L3 was obtained with 0.5M. At 0.02M almost all the contaminant proteins are eluted from the powder. At 0.3 M, the remain‐ ing proteins after the 0.02M elution are located in the 95 and 70 kDa zone. The 0.5 M column shows that after after 0.3 M there is almost no proteins remaining on the powder.

The poodle was euthanazised 11 months, and the Jagdterrier 6 months, after the disease was discovered. During their survival period, both dogs had a normal activity. The Jagdterrier was still hunting two weeks before to be euthanazied. The two other dogs were euthanazied at 155 and 173 days (table 2). The mean overall survival time is 210 days.


**Table 2.** Characteristics of the dogs treated by the vaccination protocol and overall survival time (OS) in days.

**Figure 4.** Dot blot after anti gp90 labelling

**Figure 5.** Dot blot after anti HSP70 labelling
