Topical resiquimod promotes priming of CTL to parenteral antigens.

We explored the topical use of resiquimod (R-848), a Toll-like receptor (TLR) 7/8 agonist, in gel formulation, to enhance cross-priming to subcutaneously administered protein antigen in a murine model. Resiquimod application at the time of subcutaneous administration of ovalbumin generated robust antigen-specific CTL as detected by tetramers, IFN-gamma ELISPOT assays and standard cytotoxicity assays. Induced CTL were capable of mediating antigen-specific killing in vivo as measured by in vivo cytotoxicity assays and an ability to protect against B16-OVA tumor challenge. Multiple serial applications of topical resiquimod increased the frequency of antigen-specific CTL when compared to single application. This enhanced frequency was noted despite a marked inhibition of adjuvant mediated pro-inflammatory cytokine release following repeated administration. Topical resiquimod is a potent adjuvant for locally administered subcutaneous vaccines, inducing clinically relevant CTL responses following single application at the time of subcutaneous vaccination.

Topical CpG enhances the response of murine malignant melanoma to dacarbazine.

Malignant melanoma is a potentially fatal skin cancer that is increasing in incidence. Standard chemoimmunotherapy consisting of dacarbazine (DTIC) given with IFN-alpha has had disappointing results. We describe a chemoimmunotherapy protocol for cutaneous melanoma that combines the administration of DTIC with the topical application of CpG oligodinucleotide (ODN). Subcutaneous B16 melanoma tumors in C57BL/6 mice were treated with intraperitoneal injections of DTIC followed by the topical application of CpG-ODN over the tumors. This therapeutic approach abrogated the growth of established tumors and significantly enhanced survival. Topical CpG application was more effective than intratumoral CpG. Cell depletion studies indicated that the antitumor effect was dependent on both CD4(+) and CD8(+) cells but not on natural killer (NK) cells. Tumor-specific cytotoxic T-lymphocyte activity was generated in treated animals and was highest in topically treated animals. Immunohistochemical analysis revealed that DTIC, but not CpG, enhanced tumor cell apoptosis. Further, topical CpG induced an expansion of a B220(+)CD8(+) subset of dendritic cells and a subset of NK1.1(+) CD11c(+) cells within the tumors. By enhancing both tumor cell death and local immune activation, DTIC/topical CpG chemoimmunotherapy induced an effective T-cell-dependent host-immune response against melanoma.

Topical TLR9 agonists induce more efficient cross-presentation of injected protein antigen than parenteral TLR9 agonists do.

Topical application of adjuvant to the skin promotes the generation of immune responses to co-administered peptide or protein antigen. We demonstrate that topical administration of CpG adjuvant (a TLR9 agonist) induces the cross-presentation of, and antigen-specific CTL induction to, locally injected soluble protein antigen. C57BL/6 mice were immunized by subcutaneous or intramuscular injection with ovalbumin (OVA) protein as model antigen. Application of CpG to the local skin induced more efficient cross-presentation of the injected antigen than co-injected adjuvant. Robust antigen-specific CTL responses were generated, as determined by antigen-specific CTL enumeration using tetramers, IFN-gamma ELISPOT analysis and cytotoxicity assays. Long-term memory CTL responses were induced. Topical administration of adjuvant induced Langerhans cell migration, local type 1 IFN-dependent myxovirus-resistance protein A expression and bystander dendritic cell (DC) activation. Soluble antigen-bearing DC within the skin draining lymph nodes were mainly CD11chiCD11bhilangerinloDEC205lo. Topical administration did not result in the splenomegaly or systemic cytokine induction (including TNF-alpha, IL-12, IFN-gamma and MCP-1) noted with parenteral administration. Topical TLR9 family agonists may be used to modulate the immune response to soluble protein vaccines administered by standard percutaneous route. Topical adjuvant administration increases efficacy of CTL induction and reduces toxicity when compared to parenteral adjuvant administration.

Chronic granulomatous disease with unusual clinical manifestation, outcome, and pattern of inheritance in an Iranian family.

CGD is a rare phagocytic disorder manifesting as recurrent, severe bacterial and fungal infections. We describe an Iranian family with eight children, of whom six, five males and one female were diagnosed with CGD resulting in diffuse pulmonary sterile granulomatous lesions. Three died despite multiple courses of antibiotic and antituberculosis medications while three are alive, to date they are asymptomatic but with imaging and pathologic findings of pulmonary granulomatosis, treated with steroids. The parents are healthy. Our report describes the clinical manifestations and outcome in this family. The inheritance pattern suggests an autosomal recessive pattern with high penetrance.

Antigen targeting to CD11b allows efficient presentation of CD4+ and CD8+ T cell epitopes and in vivo Th1-polarized T cell priming.

Bordetella pertussis adenylate cyclase (CyaA) is an invasive bacterial toxin that delivers its N-terminal catalytic domain into the cytosol of eukaryotic cells bearing the alpha(M)beta(2) integrin (CD11b/CD18), such as myeloid dendritic cells. This allows use of engineered CyaA for targeted delivery of CD8(+) T cell epitopes into the MHC class I pathway of APC and induction of robust and protective cytotoxic responses. In this study, we demonstrate that CyaA can efficiently codeliver both a CD8(+) T cell epitope (OVA(257-264)) and a CD4(+) T cell epitope (MalE(100-114)) into, respectively, the conventional cytosolic or endocytic routes of processing of murine bone marrow-derived dendritic cells. Upon CyaA delivery, a strong potentiation of the MalE(100-114) CD4(+) T cell epitope presentation is observed as compared with the MalE protein, which depends on CyaA interaction with its CD11b receptor and its subsequent clathrin-mediated endocytosis. In vivo, CyaA induces strong and specific Th1 CD4(+) and CD8(+) T cell responses against, respectively, the MalE(100-114) and OVA(257-264) epitopes. These results underscore the potency of CyaA for design of new vaccines.

Epicutaneous application of CpG oligodeoxynucleotides with peptide or protein antigen promotes the generation of CTL.

Immunostimulatory oligodeoxynucleotides (ODN) are effective adjuvants in the induction of humoral and cellular immune responses when administered parenterally with antigen. The skin has recently become a target organ for the design of non-invasive vaccine technologies. Using ovalbumin (OVA) as a model antigen, we demonstrate that the application of ODN sequences to tape-stripped skin promotes the induction of potent cytotoxic T lymphocyte (CTL) responses to co-administered peptide. Induction of peptide-specific CTL required the presence of CpG motifs within the ODN. CTL afforded tumor protection against a tumor expressing an immunodominant OVA CTL epitope. CTL could also be induced to whole protein administered onto the skin. Differential CpG sequence activity was noted with respect to the induction of CTL to epicutaneous protein with an ODN sequence containing a poly-G motif having an optimal effect. Peptide-specific CTL could be detected in the peripheral blood as early as 6 d after a single immunization. These results highlight the potential of the bare skin as a route for vaccine development and indicate an important role for immunostimulatory ODN as adjuvants to generate functional CTL with the help of the skin immune system.