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.

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.

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.

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.

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.

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.

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.

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.

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.

Critical roles of the WASP N-terminal domain and Btk in LPS-induced inflammatory response in macrophages.

While Wiskott-Aldrich syndrome protein (WASP) plays critical roles in TCR signaling as an adaptor molecule, how it transduces innate immune signals remains to be elucidated. To investigate the roles of WASP in innate immune cells, we established bone marrow-derived macrophage (BMDM) cell lines from WASP15 transgenic (Tg) mice overexpressing the WASP N-terminal region (exons 1-5). Upon LPS stimulation, WASP15 Tg BMDM cell lines produce lower levels of inflammatory cytokines, such as TNF-α, IL-6, and IL-12p40 than the wild-type BMDM cell line. In addition, the production of nitric oxide by WASP15 Tg BMDM cells in response to LPS and IFN-γ was significantly impaired. Furthermore, we uncovered that the WASP N-terminal domain associates with the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk). Overexpression of the WASP N-terminal domain diminishes the extent of tyrosine phosphorylation of endogenous WASP in WASP15 Tg BMDM cells, possibly by interfering with the specific binding between endogenous WASP and Btk during LPS signaling. These observations strongly suggest that the interaction between WASP N-terminal domain and Btk plays important roles in the LPS signaling cascade in innate immunity.

Enhancement of antibody responses to native G protein-coupled receptors using E. coli GroEL as a molecular adjuvant in DNA immunization.

Antibody-based drug research involves the preparation of polyclonal and monoclonal antibodies, especially those that are reactive with native G protein-coupled receptors (GPCRs) on the cell membrane. Here, we report that DNA immunization of mice with a plasmid that encodes endothelin A receptor (ETAR) fused to Escherichia coli (E. coli) GroEL at its C-terminus (ETAR-GroEL) induced very strong and specific antibody responses to native ETAR. Co-injection of plasmids that expressed ETAR and GroEL (ETAR+GroEL) induced significantly lower antibody responses compared with the ETAR-GroEL plasmid. Monoclonal antibodies that are prepared by using GroEL as a molecular adjuvant could be used in immunoassays, such as flow cytometry, western blotting, and immunoprecipitation, to detect both exogenous and endogenous ETAR. The adjuvant activity of GroEL might involve inflammatory cytokine mediators via Toll-like receptor 4 in addition to the anticipated carrier effect. DNA immunization using GroEL might become a standard method for producing antibodies that are useful for the functional analysis of GPCRs.

Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells.

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

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.

Passive immune-prophylaxis against influenza virus infection by the expression of neutralizing anti-hemagglutinin monoclonal antibodies from plasmids.

The genetic delivery of therapeutic monoclonal antibodies (mAbs) by in vivo production may offer a new solution to the current problems in the mAb therapy for microbial diseases. Herein, plasmids encoding the neutralizing mAb against hemagglutinin (HA) of A/PR/8/34 influenza virus (IFV) were electro-transferred into mouse muscle and the relationship between serum recombinant anti-HA mAb (rHA mAb) levels and the prophylactic efficacy against lethal IFV infection were analyzed. Pretreatment of the muscle with hyaluronidase before electroporation and gene transfer into 3 muscles resulted in a marked enhancement of the mAb expression. After single gene transfer, peak serum concentrations were reached around 20 days after the gene transfer following sustained expression of >10 µg/ml of rHA mAbs. This level of rHA mAb expression was sufficient to protect all mice against a lethal IFV infection. Furthermore, a significant rHA mAb expression level sufficient to protect the host against lethal IFV infection was maintained for at least 130 days. Passive immune-prophylaxis with gene transfer using the plasmid encoding neutralizing mAbs may therefore provide effective protection against viral infections, including IFV.

Generation of a panel of monoclonal antibodies against atypical chemokine receptor CCX-CKR by DNA immunization.

INTRODUCTION: Chemokines are regulated by a family of 'atypical' chemokine receptors, D6, DARC and CCX-CKR, each of which efficiently internalizes its cognate chemokine ligands. Development of monoclonal antibodies (MAbs) that would recognize CCX-CKR on the cell surface will be helpful to identify primary CCX-CKR-expressing cell types and analyze the fate of CCX-CKR after ligand binding to the receptor. METHODS: We generated IgG MAbs recognizing the cell-surface CCX-CKR by DNA immunization using a molecular adjuvant, and analyzed the epitope recognized by the MAbs. Then, the reactivities of the MAbs with CCX-CKR-transfected cells, and also hepatocytes and hepatic tumor lines were evaluated. Finally, we also tested the ligand-like activities of the MAbs, namely, induction of internalization of CCX-CKR by the MAbs. RESULTS: A panel of MAbs reacting with CCX-CKR expressed on the cell surface was prepared. The panel was a small one, consisting of only ten MAbs, but was rich in terms of diversity of the Ig isotypes and of the epitopes. Epitope analyses revealed that all the 10 MAbs recognized at least three different, although very close, peptide structures of the N-terminal domain. Three MAbs, namely, 2F11, 13E11 and 14F10, were selected to represent the panel. All of the MAbs were applicable for flow cytometry and immunoflurescent assays and immunoprecipitation. The reactivity of the 2F11 MAb was also confirmed by western blotting. Endogenous expression of CCX-CKR on human hepatocytes and hepatic tumor cell lines was demonstrated using the 13E11 MAb. Interestingly, binding of the 13E11 MAb with B300-19 cells expressing CCX-CKR resulted in induction of CCX-CKR internalization. DISCUSSION: This panel of MAbs may be expected to prove valuable for further study of the functions of this silent chemokine receptor, including those related to the homeostasis of lymphoid cells, and to the growth and metastasis of hepatic cancer.

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.