Poly I:C also works as a mucosal adjuvant for the induction of humoral and cell-mediated immune reactions C. proliferation of CD3+CD4+ T cells (solid lines) or CD3+CD4? T cells (dotted lines) was assessed in CFSE dilution assays, incubated for 7 days. KLH-specific proliferation was measured as the proportion of CFSElow cells, gating on CD3-, CD4-double positive or CD3-positive, CD4-bad cells, respectively. Background proliferation in medium only was subtracted from proliferation of KLH-stimulated PBMCs. The five-digit figures are monkey designations.(0.74 MB TIF) ppat.1000373.s002.tif (721K) GUID:?BE55DD08-4115-40BC-92F4-1192EB9405D3 Figure S3: Correlation between antibody titers measured by ELISA and neutralization assays. Titers in serum samples collected 12 weeks after the 1st immunization with HPV16 capsomeres (10 g/animal) only or together with 2 mg of poly ICLC or CpG-C are demonstrated for the individual animals. Neutralization titers are given as reciprocal of the highest dilution used in this experiment yielding 50% neutralizing activity. Dedication of ELISA titers is definitely explained in Materials and Methods.(0.76 MB TIF) ppat.1000373.s003.tif (747K) GUID:?46FBC843-8770-4F14-A340-D5443D40BFF9 Figure S4: Veralipride DC numbers in draining lymph nodes 18 hours after immunization of KLH plus poly ICLC. Numbers of CD1a, CD83, and CD208 positive cells per unit area were determined by immunohistochemistry in lymph node sections before and 18 hours after immunization with KLH plus poly ICLC at 0.5 mg/kg body weight (packed symbols) or 0.1 mg/kg (open symbols).(0.37 MB TIF) ppat.1000373.s004.tif (366K) GUID:?D2A2EEFC-D186-47E7-A5C2-DBC01065F3A5 Table S1: Maximum proliferative responses (mean cpm of wells in triplicates) after immunization of rhesus macaques with KLH (200 g) alone or together with poly I:C or poly ICLC (0.5 mg/kg body weight).(0.02 MB DOC) ppat.1000373.s005.doc (24K) GUID:?62384DCC-1F37-4916-A0E3-BE573CBBC483 Table S2: Individual titers of L1-binding antibodies after immunization with HPV16 capsomeres (10 g) alone or together with poly ICLC or CpG-C (2 mg/animal).(0.04 MB DOC) ppat.1000373.s006.doc (37K) GUID:?FB528FB3-19C1-4B15-8E01-667D968B5FB2 Abstract Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as with the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is definitely identified by TLR3 and additional intracellular receptors. Poly ICLC is definitely a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is definitely less harmful but also less stable in vivo than poly I:C, and TLR3 is essential for its acknowledgement. To study the results of these compounds within the induction of protein-specific immune Veralipride responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human being papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses becoming observed with 2 mg/animal poly ICLC Veralipride (p?=?0.002) or 6 mg/animal poly I:C12U (p?=?0.001) when compared with immunization with KLH alone. Notably, poly ICLCbut not CpG-C given at the same dosealso helped to induce HPV16-specific Th1 immune reactions while both adjuvants supported the induction of strong anti-HPV16 L1 antibody reactions as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 Veralipride capsomeres alone did not develop considerable HPV16-specific immune responses. Injection of dsRNA led to improved numbers of cells generating the T cellCactivating chemokines CXCL9 and CXCL10 as recognized by in situ hybridization in draining lymph nodes 18 hours after injections, and to improved serum levels of CXCL10 (p?=?0.01). This was paralleled from the Veralipride reduced production of the homeostatic T cellCattracting chemokine CCL21. Therefore, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates. Author Summary Novel adjuvants that facilitate the induction of strong cellular immunity could be of help in the design of vaccine strategies to combat infections such as HIV or tuberculosis. Our immune cells possess archaic receptors realizing constructions of infectious pathogens, and the interaction ETO of these receptors with their ligands results in an activation of the immune system. Here we exploited synthetic forms of one of these ligands, i.e., dsRNA, to define an adjuvant for the induction of cellular immune responses in primates. We injected model and viral proteins together with three different forms of dsRNA subcutaneously (s.c.) in rhesus macaques, and all compounds served as adjuvants for the induction of cellular immunity without the incidence of major side effects. These adjuvant effects depended around the adjuvant dose and coincided with profound alterations in the chemokine production in the draining lymph nodes. dsRNA also helped to induce cellular and humoral immune responses against capsomeres of low immunogenicity derived from the human papillomavirus 16, the causative agent.