Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation.

Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFκB promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

Anti-WASP intrabodies inhibit inflammatory responses induced by Toll-like receptors 3, 7, and 9, in macrophages.

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we showed that the WASP N-terminal domain interacted with the SH3 domain of Bruton's tyrosine kinase (Btk), and that the complex formed by WASP and Btk was important for TLR2 and TLR4 signaling in macrophages. Several other studies have shown that Btk played important roles in modulating innate immune responses through TLRs in immune cells. Here, we evaluated the significance of the interaction between WASP and Btk in TLR3, TLR7, and TLR9 signaling. We established bone marrow-derived macrophage cell lines from transgenic (Tg) mice that expressed intracellular antibodies (intrabodies) that specifically targeted the WASP N-terminal domain. One intrabody comprised the single-chain variable fragment and the other comprised the light-chain variable region single domain of an anti-WASP N-terminal monoclonal antibody. Both intrabodies inhibited the specific interaction between WASP and Btk, which impaired the expression of TNF-α, IL-6, and IL-1ß in response to TLR3, TLR7, or TLR9 stimulation. Furthermore, the intrabodies inhibited the phosphorylation of both nuclear factor (NF)-κB and WASP in response to TLR3, TLR7, or TLR9 stimulation, in the Tg bone marrow-derived macrophages. These results suggested that WASP plays important roles in TLR3, TLR7, and TLR9 signaling by associating with Btk in macrophages.

Single-chain variable fragment intrabody impairs LPS-induced inflammatory responses by interfering with the interaction between the WASP N-terminal domain and Btk in macrophages.

Wiskott-Aldrich syndrome protein (WASP) plays important roles in both acquired and innate immune responses. We recently uncovered that the WASP N-terminal domain specifically binds the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk) in macrophages. Over-expression of the WASP N-terminal domain impairs LPS-induced inflammatory responses. To evaluate the significance of this interaction in LPS signaling, we established bone marrow-derived macrophage (BMDM) cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) intrabody. Anti-WASP scFv specifically bound endogenous WASP and inhibited its specific binding to the SH3 domain of Btk in the Tg BMDMs. Tyrosine phosphorylation in WASP was inhibited after LPS stimulation. As a result, TNF-α, IL-6, and IL-1ß gene transcription and NF-κB phosphorylation were impaired. These observations strongly suggest that the phosphorylation of WASP by Btk plays a pivotal role in transducing the LPS signaling pathway in macrophages.

Conjugation of cell-penetrating peptides leads to identification of anti-HIV peptides from matrix proteins.

Compounds which inhibit the HIV-1 replication cycle have been found amongst fragment peptides derived from an HIV-1 matrix (MA) protein. Overlapping peptide libraries covering the whole sequence of MA were designed and constructed with the addition of an octa-arginyl group to increase their cell membrane permeability. Imaging experiments with fluorescent-labeled peptides demonstrated these peptides with an octa-arginyl group can penetrate cell membranes. The fusion of an octa-arginyl group was proven to be an efficient way to find active peptides in cells such as HIV-inhibitory peptides.

Structural modulation study of inhibitory compounds for ribonuclease H activity of human immunodeficiency virus type 1 reverse transcriptase.

Reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) has two enzymatic functions. One of the functions is ribonuclease (RNase) H activity concerning the digestion of only RNA of RNA/DNA hybrid. The RNase H activity is an attractive target for a new class of anti-HIV drugs because no approved inhibitor is available now. In our previous studies, an agent bearing 5-nitro-furan-2-carboxylic acid ester core was found from chemical screening and dozens of the derivatives were synthesized to improve compound potency. In this work, some parts of the chemical structure were modulated to deepen our understanding of the structure-activity relationship of the analogous compounds. Several derivatives having nitro-furan-phenyl-ester skeleton were shown to be potent RNase H inhibitors. Attaching methoxy-carbonyl and methoxy groups to the phenyl ring increased the inhibitory potency. No significant cytotoxicity was observed for these active derivatives. In contrast, the derivatives having nitro-furan-benzyl-ester skeleton showed modest inhibitory activities regardless of attaching diverse kinds of functional groups to the benzyl ring. Both the modulation of the 5-nitro-furan-2-carboxylic moiety and the conversion of the ester linkage resulted in a drastic decrease in inhibitory potency. These findings are informative for designing potent inhibitors of RNase H enzymatic activity of HIV-1.

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER.

BACKGROUND: DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown. RESULTS: In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage. CONCLUSIONS: The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.

Structural and biochemical study on the inhibitory activity of derivatives of 5-nitro-furan-2-carboxylic acid for RNase H function of HIV-1 reverse transcriptase.

Rapid emergence of drug-resistant variants is one of the most serious problems in chemotherapy for HIV-1 infectious diseases. Inhibitors acting on a target not addressed by approved drugs are of great importance to suppress drug-resistant viruses. HIV-1 reverse transcriptase has two enzymatic functions, DNA polymerase and RNase H activities. The RNase H activity is an attractive target for a new class of antiviral drugs. On the basis of the hit chemicals found in our previous screening with 20,000 small molecular-weight compounds, we synthesized derivatives of 5-nitro-furan-2-carboxylic acid. Inhibition of RNase H enzymatic activity was measured in a biochemical assay with real-time monitoring of florescence emission from the digested RNA substrate. Several derivatives showed higher inhibitory activities that those of the hit chemicals. Modulation of the 5-nitro-furan-2-carboxylic moiety resulted in a drastic decrease in inhibitory potency. In contrast, many derivatives with modulation of other parts retained inhibitory activities to varying degrees. These findings suggest the binding mode of active derivatives, in which three oxygen atoms aligned in a straight form at the nitro-furan moiety are coordinated to two divalent metal ions located at RNase H reaction site. Hence, the nitro-furan-carboxylic moiety is one of the critical scaffolds for RNase H inhibition. Of note, the RNase H inhibitory potency of a derivative was improved by 18-fold compared with that of the original hit compound, and no significant cytotoxicity was observed for most of the derivatives showing inhibitory activity. Since there is still much room for modification of the compounds at the part opposite the nitro-furan moiety, further chemical conversion will lead to improvement of compound potency and specificity.

SOCS1 is an inducible host factor during HIV-1 infection and regulates the intracellular trafficking and stability of HIV-1 Gag.

Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus-host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders.

Induction of cross-protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia.

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection.

Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus.

The effectiveness in cynomolgus macaques of intranasal administration of an influenza A H5N1 pre-pandemic vaccine combined with synthetic double-stranded RNA (polyI/polyC12U) as an adjuvant was examined. The monkeys were immunized with the adjuvant-combined vaccine on weeks 0, 3, and 5, and challenged with the homologous virus 2 weeks after the third immunization. After the second immunization, the immunization induced vaccine-specific salivary IgA and serum IgG antibodies, as detected by ELISA. The serum IgG antibodies present 2 weeks after the third immunization not only had high neutralizing activity against the homologous virus, they also neutralized significantly heterologous influenza A H5N1 viruses. The vaccinated animals were protected completely from the challenge infection with the homologous virus. These results suggest that intranasal immunization with the Double stranded RNA-combined influenza A H5N1 vaccine induce mucosal IgA and serum IgG antibodies which could protect humans from homologous influenza A H5N1 viruses which have a pandemic potential.

Broadly Neutralizing Antibodies for Influenza: Passive Immunotherapy and Intranasal Vaccination.

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 (VHH), 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 VHHs, 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.

An anti-perfringolysin O monoclonal antibody cross-reactive with streptolysin O protects against streptococcal toxic shock syndrome.

OBJECTIVE: Streptococcus pyogenes (Group A Streptococcus; GAS) causes a variety of infections that include life-threatening, severe invasive GAS infections, such as streptococcal toxic shock syndrome (STSS), with > 30% mortality rate, despite effective antibiotics and treatment options. STSS clinical isolates highly express streptolysin O (SLO), a member of a large family of pore-forming toxins called cholesterol-dependent cytolysins (CDCs). SLO is an important toxic factor for GAS and may be an effective therapeutic target for the treatment of STSS. Our aim was to identify a monoclonal antibody (mAb) that reacts with SLO and has therapeutic potential for STSS treatment. RESULTS: We focused on mAbs that had originally been established as neutralizing reagents to perfringolysin O (PFO), another member of the CDC family, as some cross-reactivity with SLO had been reported. Here, we confirmed cross-reactivity of an anti-PFO mAb named HS1 with SLO. In vitro analysis revealed that HS1 mAb sufficiently prevented human neutrophils from being killed by STSS clinical isolates. Furthermore, prophylactic and therapeutic injection of HS1 mAb into C57BL/6 mice significantly improved the survival rate following lethal infection with an STSS clinical isolate. These results highlight the therapeutic potential of HS1 mAb for STSS treatment.

A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza.

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 antigen via the 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 RP105 in 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 vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells.

We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4(+) T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventional DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4(+) T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.

Production of antibodies against multipass membrane proteins expressed in human tumor cells using dendritic cell immunization.

Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, and seven transmembrane prostate specific G-protein coupled receptor (PSGR). Mice were immunized with these DCs and followed by repeated booster immunization with plasmids expressing each protein. The immunized mice produced significant amounts of antibodies against these proteins. Our results suggest that DC immunization is an effective method to produce antibodies reactive to extracellular regions of plasma membrane proteins with multiple-transmembrane domains, and may be useful to develop antibody mediated antitumor therapies.

DNA immunization using in vivo electroporation for generating monoclonal antibodies Against Mouse IL-9R.

Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins have been generated by immunization of animals with recombinant proteins produced in Escherichia coli (E. coli) or peptides derived from the protein. However, such antibodies often fail to recognize native proteins on cell surfaces because these antigens lack posttranslational modification and natural protein conformations. To circumvent such problems, we have developed a mouse immunization method, the DNA-immunization utilizing hyaluronidase and E. coli GroEL. Herein, we report an application of the original mouse immunization method in rats to generate anti-mouse IL-9R mAbs which could react with the native form of mouse IL-9R on cell surfaces. Thus, we suggest that the DNA-immunization method is feasible for generating monoclonal antibodies against cell surface proteins in rats.

Neutralizing Anti-Hemagglutinin Monoclonal Antibodies Induced by Gene-Based Transfer Have Prophylactic and Therapeutic Effects on Influenza Virus Infection.

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.

Combined therapy with gas gangrene antitoxin and recombinant human soluble thrombomodulin for Clostridium perfringens sepsis in a rat model.

Cases of Clostridium perfringens septicemia, such as liver abscess, often develop a rapidly progressive intravascular hemolysis and coagulation; the mortality rate with current standard care including antibiotics and surgery is high. Herein, we firstly investigated the effects of gas gangrene antitoxin (GGA) (antitoxin against C. perfringens) and recombinant human soluble thrombomodulin (rTM) on the hemolysis, coagulation status, inflammatory process, and mortality in α-toxin-treated rats. Male 11-week-old Sprague Dawley rats were randomly divided into five groups: control group, α-toxin group, GGA group, rTM group, and combined GGA and rTM (combination group). After α-toxin injection, mortality and platelet counts, and hemolysis were observed for 6 h. The fibrin/fibrinogen degradation products (FDP), and plasma high-mobility group box 1 (HMGB1) were also measured at 6 h. The combination group demonstrated 100% survival compared with 50% survival in the α-toxin group and demonstrated significantly improved hemolysis, platelet counts, and lactate levels compared with those in the α-toxin group (p < .01). The FDP and HMGB1 levels in the combination therapy group were significantly lower than those in the α-toxin group (p < .05). Combination therapy with GGA and rTM administration is applicable as adjunct therapy for fatal C. perfringens sepsis.

Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection.

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.

Intrinsic promoter activities of primary DNA sequences in the human genome.

In order to understand an overview of promoter activities intrinsic to primary DNA sequences in the human genome within a particular cell type, we carried out systematic quantitative luciferase assays of DNA fragments corresponding to putative promoters for 472 human genes which are expressed in HEK (human embryonic kidney epithelial) 293 cells. We observed the promoter activities of them were distributed in a bimodal manner; putative promoters belonging to the first group (with strong promoter activities) were designated as P1 and the latter (with weak promoter activities) as P2. The frequencies of the TATA-boxes, the CpG islands, and the overall G + C-contents were significantly different between these two populations, indicating there are two separate groups of promoters. Interestingly, similar analysis using 251 randomly isolated genomic DNA fragments showed that P2-type promoter occasionally occurs within the human genome. Furthermore, 35 DNA fragments corresponding to putative promoters of non-protein-coding transcripts (ncRNAs) shared similar features with the P2 in both promoter activities and sequence compositions. At least, a part of ncRNAs, which have been massively identified by full-length cDNA projects with no functional relevance inferred, may have originated from those sporadic promoter activities of primary DNA sequences inherent to the human genome.