**Author details**

Although gp96 has been described as able to stimulate TLR4, it is not sure that these proteins are the only proteins responsible for TLRs stimulation in this case. There are contaminating proteins in various bands of the SDS which could interfere with the TLRs. The non stimulation

Hydroxyapatite has been used as an adjuvant for various infectious vaccines such as diphtheria and tetanus [21]. The Hydroxyapatite lattice is a hexagonal structure which allows numerous substitutions. Ions and small amino acids can thus be trapped in the HA lattice. Consequently, HA powder has been used to purify DNA, proteins or even viruses from biological solutions [22]. In this case, the surface properties of the particles allowed both the purification of HSPs

The HA-powder characteristics seems well suited for its role of cancer vaccine adjuvant. The granulometry range allows the phagocytosis by the APCs as it was demonstrated previously [14]. Furthermore, the grain boundaries in each particle is degraded by the cells making the particles fragmented and thus decreasing the real granulometry a few days after injection. It was also demonstrated that the phagocytosis of these particles by the APCs induced the synthesis of various cytokines and lymphokines necessary for the cross-priming of the CD8. The HA-adjuvant effect does not seem to be due to TLR activation as HA-particles alone do not trigger TLR2 and 4 activation. Other mineral adjuvants such as aluminum oxide have been demonstrated to activate the inflamasome. The inflamasome is a multi-protein complex involved in the production of mature IL-1β. The alumn-induced release of IL-1β in macro‐ phages is done under activation of the NLRP3 [23]. It is suggested from unpublished results that the adjuvancy effect of the HA-particles is also due to the inflamasome activation.

Tumor associated antigens show a very poor antigenicity. Thus the presence of an adjuvant like calcium phosphate particles is essential in order to increase the visibility of these antigens

This method was used in a short pilot study in humans and proved to be very safe, as only local effects (erythema) were reported in some patients (24). Although it was not the goal of the pilot study, some remission in the extent of the tumour was observed and constituted a good proof of concept. Gp96 has already been used in human medicine to treat a series of patients with indolent non Hodgkin's lymphoma [23]. The results are difficult to compare, as the patients could have been treated by chemo- or radiotherapy more than six weeks before the vaccination protocol. However, at three months most of the patients were rated SD, including those who were resistant to previous therapy. No patients suffered side effects.

The HA-particles are essential in this protocol both for their adsorption properties for proteins of interest and their adjuvant properties. They constitute a tool which allows to purify tumor

of the TLR2 indicates that the TLR4 is not activated by contaminating endotoxins.

and their use as a vector to APCs and as an adjuvant.

510 Advances in Biomaterials Science and Biomedical Applications

by the patient immune system.

**5. Conclusions**

associated antigens in a fast and reproducible way.

Michel Simonet1 , Nicole Rouquet2 and Patrick Frayssinet2\*

1 Veterinary hospital, Nuit Saint Georges, France

2 Urodelia, St Lys, France
