BackgroundThe screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers.ResultsWe present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells.ConclusionsOur overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

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Intraperitoneal inoculation with live influenza A virus confers protection against intranasal infections in mice and ferrets. However, the responses of peritoneal cells to influenza A virus have not been investigated. Here we show that intraperitoneal inoculation with A/WSN/1933 (H1N1) virus induced virus-reactive IgG production in the peritoneal cavity in mice. The infection resulted in substantial but transient B cell and macrophage depletion along with massive neutrophil infiltration, but virus growth was not detected. Influenza A viruses bound to α-2,6-linked sialic acids of B cells and macrophages and induced apoptotic death of peritoneal cavity cells. However, re-infection with A/WSN/1933 virus did not have adverse effects on immune cells most likely because of the neutralizing antibodies produced in response to the first exposure. Infection of BALB/c mice with A/WSN/1933 induced cross-protection against an otherwise lethal intraperitoneal dose of A/Hongkong/4801/2014 (H3N2) virus. This information suggests that immunological responses in the peritoneal cavity can induce effective defense against future virus infection. Considering the unexpected potent immunoregulatory activity of the peritoneal cells against influenza viruses, we suggest that comparative studies on various immune reactions after infection through different routes may contribute to better selection of vaccination routes in development of efficacious influenza vaccines.

Background: The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9). Results: We found that a variant of ASB9 that lacks the SOCS box (ASB9ΔSOCS) was naturally detected in human cell lines but not in peripheral blood mononuclear cells or normal hepatocytes. We also identified ubiquitous mitochondrial creatine kinase (uMtCK) as a new target of ASB9 in human embryonic kidney 293 (HEK293) cells. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of uMtCK in a SOCS boxindependent manner. The overexpression of ASB9, but not ASB9ΔSOCS, induces ubiquitination of uMtCK. ASB9 and ASB9ΔSOCS can interact and colocalise with uMtCK in the mitochondria. However, only expression of ASB9 induced abnormal mitochondrial structure and a decrease of mitochondrial membrane potential. Furthermore, the creatine kinase activities and cell growth were significantly reduced by ASB9 but not by ASB9ΔSOCS. Conclusions: ASB9 interacts with the creatine kinase system and negatively regulates cell growth. The differential expression and function of ASB9 and ASB9ΔSOCS may be a key factor in the growth of human cell lines and primary cells.

Although peptide vaccines have been actively studied in various animal models, their efficacy in treatment is limited. To improve the efficacy of peptide vaccines, we previously formulated an efficacious peptide vaccine without carriers using the natural phosphodiester bond CpG-DNA and a special liposome complex (Lipoplex(O)). Here, we show that immunization of mice with a complex consisting of peptide and Lipoplex(O) without carriers significantly induces peptide-specific IgG2a production in a CD4+ cells- and Th1 differentiation-dependent manner. The transmembrane 4 superfamily member 5 protein (TM4SF5) has gained attention as a target for hepatocellular carcinoma (HCC) therapy because it induces uncontrolled growth of human HCC cells via the loss of contact inhibition. Monoclonal antibodies specific to an epitope of human TM4SF5 (hTM4SF5R2-3) can recognize native mouse TM4SF5 and induce functional effects on mouse cancer cells. Pre-immunization with a complex of the hTM4SF5R2-3 epitope and Lipoplex(O) had prophylactic effects against tumor formation by HCC cells implanted in an mouse tumor model. Furthermore, therapeutic effects were revealed regarding the growth of HCC when the vaccine was injected into mice after tumor formation. These results suggest that our improved peptide vaccine technology provides a novel prophylaxis measure as well as therapy for HCC patients with TM4SF5-positive tumors.