The immunized mice were injected once with Alum (33.0?mg/kg mouse), Az (2.25?mg/kg mouse) or PolyI:C (6.65?mg/kg mouse). the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines. Adjuvants stimulate host immunity to administered vaccine antigens and, as a result, are clinically useful for prevention of infection by pathogenic bacteria and virus1,2,3,4. Specifically, adjuvants enable the use of smaller numbers and doses of vaccine injections by enhancing immune responses to vaccines. Despite recent progress made in their discovery5,6,7, only a small number of small molecule-based adjuvants have been approved for clinical use. Thus, a greater effort needs to be made for the development of efficacious small molecule adjuvants8,9. The Hsp70 protein family is known to play diverse roles in biological processes10,11. The two major members of this family, constitutive Hsc70 and inducible Hsp70, are composed of an N-terminal ATPase domain (or a nucleotide binding domain), which binds and catalyzes the hydrolysis of ATP to ADP, and a C-terminal substrate binding domain, which associates with peptide/protein substrates. The two domains are functionally coupled in such a way that hydrolysis of ATP by ATPase activity induces conformational changes in the adjacent substrate binding domain of the proteins. Alterations of the substrate binding domain lead to increases in binding affinities of substrates12. A representative function of the Hsp70 family is chaperone activity such as protein folding, suppression of aggregation of denatured proteins, removal of misfolded proteins and regulation of assembly/disassembly of protein complexes13,14,15,16. In addition, members of this protein family are also known to be involved in suppression of apoptotic cell death through multiple anti-apoptotic processes17,18,19,20,21. In particular, their suppression of cancer cell death leads to tumor cell survival and progression. Because of their pathological significance, small molecule-based inhibitors of these proteins have been exploited for use as potential therapeutic agents and/or chemical probes22,23,24. For example, apoptozole (Az, Fig. 1), which inhibits Hsp70 and Hsc70 activities by binding to ATPase domains15,20,21, and phenylethynesulfonamide (PES), which binds to the C-terminus of Hsp70 but not to Hsc7025, display anticancer activities. In addition, inhibitors of these proteins cause a reduction in the accumulation of misfolded tau and promote membrane trafficking of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis cells15,26. Open in a separate window Figure 1 Chemical structures of Az and DSG. Although extensive investigations of the chaperone and anti-apoptotic activities of members of the Hsp70 family have been performed, only a few studies focusing on Hsp70 associated immune responses have been reported27,28,29. For instance, Hsp70 JNJ-26481585 (Quisinostat) was found to block lipopolysaccharide (LPS)-induced generation of inflammatory cytokines by suppressing NF-B activation27. In addition, a reduced level of Hsp70 expression in cancer cells triggers specific immune responses, presumably by enhancing cell death injected daily five times from 0 to 4 days after antigen immunization. For the purpose of comparison, 15-deoxyspergualin (DSG, 12?mg/kg mouse, Fig. 1), which is known to have immunosuppressive activity31,32,33, was injected into mice administered with KLH under the same conditions as was Az. DSG binds to Hsc70 but not Hsp70 and it does not affect the substrate binding ability of Hsc70. Control groups were immunized with each protein antigen alone. Sera were collected at different times after antigen immunization, and production of total IgG, IgG1 and IgG2c antibodies was then determined by using an ELISA. The results of immunoassays show that the injection of Az leads to an enhancement in JNJ-26481585 (Quisinostat) production of total IgG and IgG1 antibodies specific to KLH or OVA compared to that of a control group untreated with Az (Fig. 2). Specifically, production of total IgG and IgG1 at 2C5 weeks after immunization increases the most when the concentrations of administered Az are JNJ-26481585 (Quisinostat) 2.25C3.75?mg/kg mouse. Az treatment also leads to an MIHC increase in the production of IgG2c antibody against KLH or OVA (Fig. 2). In marked contrast.