ovalbumin and nonaarginine

Background We previously have shown that nona-arginine protein transduction domain (R9-PTD) induced efficient protein-antigen (Ag) transduction of dendritic cells (DCs) in vitro, resulting in the efficient induction of strong Ag-specific immune responses mediated by CD8+ and CD4+ T cells and in superior antitumour effects in vivo in cancer-bearing mice. Objectives The Ag-specific immune responses caused by intradermal (i.d.) injections of R9-PTD-containing protein Ags without DC preparation were investigated. We also investigated the antitumour effects by intratumoral (i.t.) injections of rR9-containing protein Ags. Methods Synthesized SIINFEKL peptide, or recombinant ovalbumin fusion proteins (rOVA, rR9-OVA), were directly injected into abdominal skin in naïve C57BL/6 mice. OVA-specific cytotoxic T lymphocyte (CTL) activity, serum IgG titre and cytokine profiles were investigated. Histopathological analyses were also performed. In a cancer vaccination model, EG.7 (OVA-cDNA transfectants thymoma) cells were inoculated intradermally in C57BL/6 mice, and the antitumour effects were evaluated by i.t. injections of rR9-OVA in a treatment setting. Results i.d. injections of rR9-OVA into naïve C57BL/6 mice elicited OVA-specific CTLs and produced IgG2-dominant immunoglobulin. The i.d. injections of rR9-OVA also induced inflammatory cell infiltrates containing neutrophils, monocytes and lymphocytes, as well as production of inflammatory cytokines such as interferon (IFN)-gamma, interleukin-2 and IFN-inducible protein 10, with presenting SIINFEKL epitopes on major histocompatibility complex (MHC) class I molecules at the injection area. i.t. injections of rR9-OVA into EG.7 tumour mass significantly suppressed tumour growth, and these effects were completely abrogated by the depletion of CD8+ T cells. These antitumour effects were superior to those elicited by i.t. injections of rR9-OVA-treated DCs. Conclusions i.d. injections of rR9-containing immunogenic Ag without adjuvants simultaneously induce dual immunological effects: the induction of Tc1- and Th1-dominant immune responses, and the induction of inflammatory and CTL-mediated immune responses at the injection area by expressing Ag epitopes on MHC class I molecules as targets. This simple vaccination approach with R9-PTD-containing fusion proteins might be useful as prophylactic immunotherapy for cancer or infectious diseases.

Topics: Animals; Antigen Presentation; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytokines; Female; Histocompatibility Antigens; Immunoglobulin G; Injections, Intradermal; Mice; Mice, Inbred BALB C; Oligopeptides; Ovalbumin; Recombinant Fusion Proteins; Skin; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Th1 Cells

Active immunotherapy is accomplished by two critical factors; (1) induction of strong antigen-specific immune responses, and (2) abundant expression of antigen-epitopes on target cells. Previously, we have shown that the nona-arginine protein-transduction domain (R9-PTD) induced efficient protein-antigen transduction to a variety of cell types in vitro and in vivo. We have also demonstrated that intradermal (i.d.) injections of R9-PTD-containing immunogenic foreign antigens (rR9-OVA) induced dual immunological effects: the induction of OVA-specific Tc1- and Th1-dominant immune responses, and the induction of CTL-mediated immune responses at the injection area by expressing OVA-epitopes.. We investigated and compared the antitumor effects by intratumoral (i.t.) injections of rR9-containing natural tumor-associated autoantigen (TAA) and other rR9-containing proteins, including rR9-OVA, into B16 melanoma.. Our results clearly demonstrate that multiple i.t. injections of rR9-OVA, but not rR9-containing TAA, elicited strong antitumor effects in B16-bearing mice, and resulted in complete tumor regression in some (50%) animals. These antitumor effects were abrogated by depletion of CD8(+) T cells, and SIINFEKL-specific CTL lysed rR9-OVA-treated B16 cells in vitro. I.t. injections with rR9-OVA altered the proportion of CD4(+) T cell subsets in B16-bearing mice. Interestingly, B16-tumor growth at the untreated site was also reduced following multiple injections of rR9-OVA at the other B16 tumor site. Finally, we confirmed that mice that had rejected B16 tumors by i.t. injections of rR9-OVA subsequently acquired CTL activities to Trp2-epitope/B16 cells.. Multiple i.t. injections of rR9-PTD-containing immunogenic foreign Ags elicit strong antitumor effects, and thereby may provide important clinical benefits to melanoma patients.

Topics: Analysis of Variance; Animals; Antigens; Antigens, Neoplasm; Female; Immunotherapy, Active; Injections, Intralesional; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Oligopeptides; Ovalbumin; Recombinant Proteins; T-Lymphocytes, Cytotoxic