Anti-Bacterial Effect of CpG-DNA Involves Enhancement of the Complement Systems.

CpG-DNA activates the host immune system to resist bacterial infections. In this study, we examined the protective effect of CpG-DNA in mice against Escherichia coli (E. coli) K1 infection. Administration of CpG-DNA increased the survival of mice after E. coli K1 infection, which reduces the numbers of bacteria in the organs. Pre-injection of mice with CpG-DNA before E. coli K1 infection increased the levels of the complement C3 but not C3a and C3b. The survival of the mice after E. coli K1 infection was significantly decreased when the mice were pre-injected with the cobra venom factor (CVF) removing the complement compared to the non-CVF-treated mice group. It suggests that the complement has protective roles against E. coli K1 infection. In addition, the survival of complement-depleted mice was increased by CpG-DNA pre-administration before E. coli K1 infection. Therefore, we suggest that CpG-DNA enhances the anti-bacterial activity of the immune system by augmenting the levels of complement systems after E. coli K1 infection and triggering other factors as well. Further studies are required to investigate the functional roles of the CpG-DNA-induced complement regulation and other factors against urgent bacterial infection.

Prophylactic effect of a peptide vaccine targeting TM4SF5 against colon cancer in a mouse model.

Expression of transmembrane 4 superfamily member 5 protein (TM4SF5) was implicated in hepatocellular carcinoma (HCC) and colon cancer. Previously, we have shown that immunization with TM4SF5 peptide-CpG-DNA-liposome complex induces production of TM4SF5-specific antibodies and protects mice from HCC progression in an allograft model. Here, we confirmed expression of TM4SF5 in the mouse colon cancer cell line CT-26 and found that anti-TM4SF5 antibody inhibits growth of CT-26 cells. We then immunized mice with TM4SF5 peptide-CpG-DNA-liposome complex and transplanted CT-26 cells to investigate the vaccination effects. Robust production of TM4SF5-specific antibodies was induced by challenge with CT-26 cells and the tumor growth was significantly suppressed in the immunized mice. The peptide vaccine targeting TM4SF5 consequently showed a prophylactic effect against colon cancer development in a mouse model. These results suggest that the peptide vaccine can be potentially applied in humans to treat colon cancer.

CpG-DNA induces bacteria-reactive IgM enhancing phagocytic activity against Staphylococcus aureus infection.

CpG-DNA triggers the proliferation and differentiation of B cells which results in the increased production of antibodies. The presence of bacteria-reactive IgM in normal serum was reported; however, the relevance of CpG-DNA with the production of bacteria-reactive IgM has not been investigated. Here, we proved the function of CpG-DNA for the production of bacteria-reactive IgM. CpG-DNA administration led to increased production of bacteria-reactive IgM both in the peritoneal fluid and serum through TLR9 signaling pathway. When we stimulated B cells with CpG-DNA, production of bacteria-reactive IgM was reproduced in vitro. We established a bacteria-reactive monoclonal IgM antibody using CpG-DNA stimulated-peritoneal B cells. The monoclonal IgM antibody enhanced the phagocytic activity of RAW 264.7 cells against S. aureus MW2 infection. Therefore, we suggest that CpG-DNA enhances the antibacterial activity of the immune system by triggering the production of bacteria-reactive IgM. We also suggest the possible application of the antibodies for the treatment of antibiotics-resistant bacterial infections. [BMB Reports 2019; 52(11): 635-640].

Peritoneal Cells Mediate Immune Responses and Cross-Protection Against Influenza A Virus.

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.

Production of an anti-TM4SF5 monoclonal antibody and its application in the detection of TM4SF5 as a possible marker of a poor prognosis in colorectal cancer.

The cell surface transmembrane 4 superfamily member 5 protein (TM4SF5) has been implicated in various human cancers. Immunization with a peptide vaccine targeting human TM4SF5 has been shown to exert prophylactic and therapeutic effects against the development of hepatocellular carcinoma and colon cancer in mouse models. In this study, we developed a novel monoclonal antibody (mEC2­CF) targeting a cyclic epitope of TM4SF5 and evaluated its reactivity to TM4SF5 in colorectal cancer (CRC) cells and cancer tissues. The isotype of mEC2­CF was IgG2a and the antibody specifically recognized the cyclic peptide, based on ELISA. The antibody recognized recombinant TM4SF5 overexpressed in 293F cells, irrespective of N­glycosidase F treatment. The antibody was internalized into the cytosol after binding to the surface of TM4SF5­expressing CRC cells, suggesting that this antibody may be useful in therapeutics. In addition, we evaluated TM4SF5 expression in the tissues of patients with CRC patients to determine its prognostic significance. TM4SF5 expression was assessed by immunohistochemistry using mEC2­CF and tissue microarray blocks of 204 primary CRC samples. The overall rate of TM4SF5 overexpression in the samples (immunohistochemical score >4) was 27.0% (55 of 204). The increased expression of TM4SF5 was significantly associated with a shorter survival rate (P=0.0014) and a worse disease­free survival (P=0.0483) of patients with CRC. No association was observed between TM4SF5 expression and clinicopathological characteristics, apart from tumor depth of invasion (P=0.027). These results suggest that our novel antibody can be used to detect endogenous and recombinant TM4SF5, and that TM4SF5 may be a possible marker for the poor prognosis of patients with CRC.

CpG-DNA exerts antibacterial effects by protecting immune cells and producing bacteria-reactive antibodies.

CpG-DNA activates various immune cells, contributing to the host defense against bacteria. Here, we examined the biological function of CpG-DNA in the production of bacteria-reactive antibodies. The administration of CpG-DNA increased survival in mice following infection with methicillin-resistant S. aureus and protected immune cell populations in the peritoneal cavity, bone marrow, and spleen. CpG-DNA injection likewise increased bacteria-reactive antibodies in the mouse peritoneal fluid and serum, which was dependent on TLR9. B cells isolated from the peritoneal cavity produced bacteria-reactive antibodies in vitro following CpG-DNA administration that enhanced the phagocytic activity of the peritoneal cells. The bacteria-reactive monoclonal antibody enhanced phagocytosis in vitro and protected mice after S. aureus infection. Therefore, we suggest that CpG-DNA enhances the antibacterial activity of the immune system by protecting immune cells and triggering the production of bacteria-reactive antibodies. Consequently, we believe that monoclonal antibodies could aid in the treatment of antibiotic-resistant bacterial infections.

Extracellular Release of CD11b by TLR9 Stimulation in Macrophages.

CpG-DNA upregulates the expression of pro-inflammatory cytokines, chemokines and cell surface markers. Investigators have shown that CD11b (integrin αM) regulates TLR-triggered inflammatory responses in the macrophages and dendritic cells. Therefore, we aimed to identify the effects of CpG-DNA on the expression of CD11b in macrophages. There was no significant change in surface expression of CD11b after CpG-DNA stimulation. However, CD11b was released into culture supernatants after stimulation with phosphorothioate-backbone modified CpG-DNA such as PS-ODN CpG-DNA 1826(S). In contrast, MB-ODN 4531 and non-CpG-DNA control (regardless of backbone type and liposome-encapsulation) failed to induce release of CD11b. Therefore, the context of the CpG-DNA sequence and phosphorothioate backbone modification may regulate the effects of CpG-DNA on CD11b release. Based on inhibitor studies, CD11b release is mediated by p38 MAP kinase activation, but not by the PI3K and NF-κB activation. CD11b release is mediated by lysosomal degradation and by vacuolar acidification in response to CpG-DNA stimulation. The amount of CD11b in the exosome precipitant was significantly increased by CpG-DNA stimulation in vivo and in vitro depending on TLR9. Our observations perhaps give more insight into understanding of the mechanisms involved in CpG-DNA-induced immunomodulation in the innate immunity.

Anti-metastatic effect of the TM4SF5-specific peptide vaccine and humanized monoclonal antibody on colon cancer in a mouse lung metastasis model.

Transmembrane 4 superfamily member 5 protein (TM4SF5) is a potential therapeutic target for hepatocellular carcinoma (HCC) and colon cancer. In a previous study, we demonstrated the prophylactic and therapeutic effects of a TM4SF5-specific peptide vaccine and monoclonal antibody in HCC and colon cancer in a mouse model. Here, we designed a cyclic peptide targeting TM4SF5. Cyclic peptide-specific antibodies were produced in mice after immunization with a complex of the peptide, CpG-DNA, and liposomes. Intravenous injection of the CT-26 mouse colon cancer cell line into mice induced tumors in the lung. Immunization with the peptide vaccine improved the survival rate and reduced the growth of lung tumors. We established a monoclonal antibody specific to the cyclic TM4SF5-based peptide and humanized the antibody sequence by complementarity determining region-grafting. The humanized antibody was reactive to the cyclic peptide and TM4SF5 protein. Treatment of CT-26 cells with the humanized antibody reduced cell motility in vitro. Furthermore, direct injection of the humanized anti-TM4SF5 antibody in vivo reduced growth of lung tumors in mouse metastasis model. Therefore, we conclude that the immunization with the cyclic peptide vaccine and injection of the TM4SF5-specifc humanized antibody have an anti-metastatic effect against colon cancer in mice. Importantly, the humanized antibody may serve as a starting platf.