Since B-cells with a mutation in TLR4 were unable to proliferate in the presence of rpHsp90, it could be considered that TLR4 is at least one of the BIBW2992 EGFR/HER2 inhibitor receptors involved in the interaction. Our results suggest that plant Hsp90s interact with the mutant TLR4 receptor, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. These data provide a new example of a non-pathogen-derived ligand of TLRs. An interesting observation is that the proliferation of the highly purified B cells is significantly lower than that obtained with the whole spleen cell population, suggesting that this proliferation could also be assisted by accessory cells. In fact, several reports demonstrated that some Hsps bind to the surface of professional antigen-presenting cells and are internalized spontaneously by receptor-mediated endocytosis demonstrating the existence of specific receptors for Hsp on professional APC . The main advantage of plant expression systems over other vaccine CP-690550 production systems is the reduced manufacturing cost . Over the past two decades, vaccine antigens expressed via the plant nuclear genome have elicited appropriate immunoglobulin responses and have conferred protection upon oral delivery . However, stably integrated nuclear transgenes typically yield relatively low levels of expression . In consequence, several strategies have been developed to increase the levels of recombinant proteins for plant production systems . Fusion of a foreign protein or peptide to a second recombinant protein that has been shown to be stably expressed in plants can act to stabilize the target protein or peptide . The fusion of antigen to the Vibrio cholerae toxin B subunit or the E. coli heat labile enterotoxin B subunit has contributed to the improvement of the humoral and cellular response due to adjuvant properties . An alternative to this is the use of Hsp90 as a safer adjuvant. It has been demonstrated that LiHsp83, a member of the Hsp90 family, is a good candidate to carry antigens and develop an adjuvant-free vaccine . Although future researches are necessary to understand the specific role of the pHsp90 as adjuvant, e.g. to evaluate whether these proteins are able to active macrophages and dendritic cells, the immunostimulatory properties of rAtHsp90 and rNbHsp90 observed here together with the high level of expression still under normal conditions, support the idea that these proteins could be excellent carriers to interesting vaccine antigens and peptides expressed in plants.