Tatsuya Yamazaki

Sa15‐21, a monoclonal antibody against mouse Toll‐like receptor (TLR) 4, can protect mice from lipopolysaccharide (LPS)/D‐galactosamine‐induced acute lethal hepatitis. Herein, we investigated the molecular mechanisms underlying Sa15‐21‐mediated regulation of TLR4 signaling in macrophages. Results showed that Sa15‐21 enhanced the production of proinflammatory cytokines and attenuated the production of anti‐inflammatory cytokines in LPS‐stimulated macrophages. Western blotting analysis revealed that Sa15‐21 pretreatment had no effect on NF‐κB and MAPK signaling in LPS‐stimulated macrophages; however, Sa15‐21 treatment alone led to a weak and delayed activation of NF‐κB and MAPK signaling without any effect on proinflammatory cytokine production. By contrast, Sa15‐21 failed to induce the activation of interferon regulatory factor 3. Taken together, our results indicate that Sa15‐21 sensitizes macrophages to facilitate the inflammatory response via TLR signaling.

For its cell surface expression, radioprotective 105 (RP105) – an orphan Toll‐like receptor – must form a complex with a soluble glycoprotein called myeloid differentiation 1 (MD‐1). The number of RP105‐negative cells is significantly increased in patients with systemic lupus erythematosus (SLE); however, to elucidate the mechanism underlying this increase, how RP105 is expressed on the cell surface depending on MD‐1 should be investigated. We demonstrated that RP105 exhibits two forms depending on MD‐1 and its two N‐glycosylation sites, N96 and N156. Cell surface expression of RP105 decreased in the presence of mutant MD‐1 (N96Q/N156Q). Nonglycosylated MD‐1 decreased the de novo cell surface expression of RP105 but not pre‐expressed RP105. Thus, the N‐glycans of MD‐1 may represent targets for SLE therapy.

For epidemiological studies of infectious diseases, pathogen-specific antibody levels in an area give us essential and appropriate information. The antibodies against pathogens are usually detected in blood, the drawing of which inconveniences people. Collection of blood increases the risk of accidental infections through blood, and it is difficult to obtain the participation of the target populations, especially the younger generation. On the other hand, urine samples, which contain a high enough level of antibodies for ELISA, can be harmlessly and easily collected and therefore have been used for epidemiological studies for diseases. The antibody examination of urine has been used for the epidemiology of parasitic diseases with a high sensitivity and specificity of serum samples. In this paper, we reviewed antibody assays with urine for seven parasitic diseases that urine diagnostic methods have reported in the past, and these are important infections included in NTDs, caused, for example, by Leishmania donovani, Wuchereria bancrofti, Schistosoma japonicum, Paragonimus westermani, Echinococcus granulosus, Echinococcus multilocularis, Strongyloides stercoralis, and Opisthorchis viverrini. The easy and safe urine surveillance system might be an admirable tool for future epidemiological studies for infectious diseases.

Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigenviathe cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105in vitro. To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccineviaa simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

Influenza viruses cause annual epidemics and occasional pandemics. The high diversity of viral envelope proteins permits viruses to escape host immunity. Therefore, the development of a universal vaccine and broadly neutralizing antibodies (bnAbs) is essential for controlling various mutant viruses. Here, we review some potentially valuable bnAbs for influenza; one is a novel passive immunotherapy using a variable domain of heavy chain-only antibody (V_HH), and the other is polymeric immunoglobulin A (pIgA) induced by intranasal vaccination. Recently, it was reported that a tetravalent multidomain antibody (MDAb) was developed by genetic fusion of four V_HHs, which are bnAbs against the influenza A or B viruses. The transfer of a gene encoding the MDAb–Fc fusion protein provided cross-protection against both influenza A and B viruses in vivo. An intranasal universal influenza vaccine, which can induce neutralizing pIgAs in the upper respiratory tract, is currently undergoing clinical studies. A recent study has revealed that tetrameric IgAs formed in nasal mucosa are more broadly protective against influenza than the monomeric and dimeric forms. These broadly neutralizing antibodies have high potential to control the currently circulating influenza virus.

Bee venom (BV) induces skin inflammation, characterized by erythema, blisters, edemas, pain and itching. Although BV has been found to have an inhibitory effect on toll-like receptors (TLRs), we here show that BV enhances keratinocyte responses to polyinosinic-polycytidylic acid [poly(I:C)], a ligand for TLR3. Our results revealed that the enhanced TLR activity was primarily induced by secretory phospholipase A2 (sPLA2), a component of BV (BV-sPLA2). PLA2 mediates the hydrolysis of membrane phospholipids into lysophospholipids and free fatty acids. We demonstrated that BV-sPLA2 increased the intracellular uptake of poly(I:C), phosphorylation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), and poly(I:C)-mediated interleukin 8 production in human keratinocytes. We further showed that the enzymatic activity of BV-sPLA2 was essential for the increased uptake of poly(I:C). These findings suggest that BV-sPLA2 may induce a modification of the cell membrane structure, leading to enhanced poly(I:C) uptake in keratinocytes. BV-sPLA2 might be able to promote wound healing by enhancing TLR3 responses.

Hemagglutinin (HA) of influenza virus is a major target for vaccines. HA initiates the internalization of the virus into the host cell by binding to host sialic acid receptors; therefore, inhibition of HA can significantly prevent influenza virus infection. However, the high diversity of HA permits the influenza virus to escape from host immunity. Moreover, the vaccine efficacy is poor in some high-risk populations (e.g., elderly or immunocompromised patients). Passive immunization with anti-HA monoclonal antibodies (mAbs) is an attractive therapy; however, this method has high production costs and requires repeated inoculations. To address these issues, several methods for long-term expression of mAb against influenza virus have been developed. Here, we provide an overview of methods using plasmid and viral adeno-associated virus (AAV) vectors that have been modified for higher expression of neutralizing antibodies in the host. We also examine two methods of injection, electro-transfer and hydrodynamic injection. Our results show that antibody gene transfer is effective against influenza virus infection even in immunocompromised mice, and antibody expression was detected in the serum and upper respiratory tract. We also demonstrate this method to be effective following influenza virus infection. Finally, we discuss the perspective of passive immunization with antibody gene transfer for future clinical trials.

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection.

Recently, we reported a novel function for C4b-binding protein (C4BP) in inhibiting the toll-like receptor (TLR)1/2 response by interacting with TLR2. TLRs share a common structure; hence, we examined the effect of C4BP on activation of other TLRs-TLR4 and TLR3. The results of immunoprecipitation assays suggest that C4BP interacts with TLR4/MD-2 but not TLR3. C4BP inhibits TLR4/MD-2-mediated, but not TLR3-mediated, proinflammatory cytokine production and nuclear factor (NF)-κB signaling. C4BP-deficient mice show increased interleukin (IL)-6 production in response to the TLR4/MD-2 ligand. A competition assay revealed that C4BP prevents an interaction between TLR4/MD-2 and its ligand. These findings indicate that C4BP binds to cell surface TLRs and inhibits the TLR-TLR ligand interaction, thereby inhibiting TLR activation.