Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2.

NF-κB essential modulator (NEMO) is a key regulatory protein that functions during NF-κB- and interferon-mediated signaling in response to extracellular stimuli and pathogen infections. Tight regulation of NEMO is essential for host innate immune responses and for maintenance of homeostasis. Here, we report that the E3 ligase MARCH2 is a novel negative regulator of NEMO-mediated signaling upon bacterial or viral infection. MARCH2 interacted directly with NEMO during the late phase of infection and catalyzed K-48-linked ubiquitination of Lys326 on NEMO, which resulted in its degradation. Deletion of MARCH2 resulted in marked resistance to bacterial/viral infection, along with increased innate immune responses both in vitro and in vivo. In addition, MARCH2-/- mice were more susceptible to LPS challenge due to massive production of cytokines. Taken together, these findings provide new insight into the molecular regulation of NEMO and suggest an important role for MARCH2 in homeostatic control of innate immune responses.

Fas-associated factor 1 mediates NADPH oxidase-induced reactive oxygen species production and proinflammatory responses in macrophages against Listeria infection.

Fas-associated factor 1 is a death-promoting protein that induces apoptosis by interacting with the Fas receptor. Until now, FAF1 was reported to interact potentially with diverse proteins and to function as a negative and/or positive regulator of several cellular possesses. However, the role of FAF1 in defense against bacterial infection remains unclear. Here, we show that FAF1 plays a pivotal role in activating NADPH oxidase in macrophages during Listeria monocytogenes infection. Upon infection by L. monocytogenes, FAF1 interacts with p67phox (an activator of the NADPH oxidase complex), thereby facilitating its stabilization and increasing the activity of NADPH oxidase. Consequently, knockdown or ectopic expression of FAF1 had a marked effect on production of ROS, proinflammatory cytokines, and antibacterial activity, in macrophages upon stimulation of TLR2 or after infection with L. monocytogenes. Consistent with this, FAF1gt/gt mice, which are knocked down in FAF1, showed weaker inflammatory responses than wild-type mice; these weaker responses led to increased replication of L. monocytogenes. Collectively, these findings suggest that FAF1 positively regulates NADPH oxidase-mediated ROS production and antibacterial defenses.

Neuroprotective Effect and Antioxidant Potency of Fermented Cultured Wild Ginseng Root Extracts of Panax ginseng C.A. Meyer in Mice.

Wild ginseng has better pharmacological effects than cultivated ginseng. However, its industrialization is limited by the inability to grow wild ginseng on a large scale. Herein, we demonstrate how to optimize ginseng production through cultivation, and how to enhance the concentrations of specific ginsenosides through fermentation. In the study, we also evaluated the ability of fermented cultured wild ginseng root extract (HLJG0701-ß) to inhibit acetylcholinesterase (AChE), as well as its neuroprotective effects and antioxidant activity. In invitro tests, HLJG0701-ß inhibited AChE activity and exerted neuroprotective and antioxidant effects (showing increased catalyst activity but decreased reactive oxygen species concentration). In invivo tests, after HLJG0701-ß was orally administered at doses of 0, 125, 250, and 500 mg/kg in an animal model of memory impairment, behavioral evaluation (Morris water maze test and Y-maze task test) was performed. The levels of AChE, acetylcholine (ACh), blood catalase (CAT), and malondialdehyde (MDA) in brain tissues were measured. The results showed that HLJG0701-ß produced the best results at a dose of 250 mg/kg or more. The neuroprotective mechanism of HLJG0701-ß was determined to involve the inhibition of AChE activity and a decrease in oxidative stress. In summary, both invitro and invivo tests confirmed that HJG0701-ß administration can lead to memory improvement.

HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I.

RIG-I is a key cytosolic sensor that detects RNA viruses through its C-terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While posttranslational modification has been demonstrated to regulate RIG-I signaling activity, its significance for the sensing of viral RNAs remains unclear. Here, we first show that the RIG-I C-terminal region undergoes deacetylation to regulate its viral RNA-sensing activity and that the HDAC6-mediated deacetylation of RIG-I is critical for viral RNA detection. HDAC6 transiently bound to RIG-I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG-I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild-type mice. These findings underscore the critical role of HDAC6 in the modulation of the RIG-I-mediated antiviral sensing pathway.

Released Tryptophanyl-tRNA Synthetase Stimulates Innate Immune Responses against Viral Infection.

Tryptophanyl-tRNA synthetase (WRS) is one of the aminoacyl-tRNA synthetases (ARSs) that possesses noncanonical functions. Full-length WRS is released during bacterial infection and primes the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to elicit innate immune responses. However, the role of WRS in viral infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early phase of viral infection and functions as an antiviral cytokine. Treatment of cells with recombinant WRS protein promotes the production of inflammatory cytokines and type I interferons (IFNs) and curtails virus replication in THP-1 and Raw264.7 cells but not in TLR4-/- or MD2-/- bone marrow-derived macrophages (BMDMs). Intravenous and intranasal administration of recombinant WRS protein induces an innate immune response and blocks viral replication in vivo These findings suggest that secreted full-length WRS has a noncanonical role in inducing innate immune responses to viral infection as well as to bacterial infection.IMPORTANCE ARSs are essential enzymes in translation that link specific amino acids to their cognate tRNAs. In higher eukaryotes, some ARSs possess additional, noncanonical functions in the regulation of cell metabolism. Here, we report a novel noncanonical function of WRS in antiviral defense. WRS is rapidly secreted in response to viral infection and primes the innate immune response by inducing the secretion of proinflammatory cytokines and type I IFNs, resulting in the inhibition of virus replication both in vitro and in vivo Thus, we consider WRS to be a member of the antiviral innate immune response. The results of this study enhance our understanding of host defense systems and provide additional information on the noncanonical functions of ARSs.

Correction: FAS-associated factor-1 positively regulates type I interferon response to RNA virus infection by targeting NLRX1.

[This corrects the article DOI: 10.1371/journal.ppat.1006398.].

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

FAS-associated factor-1 positively regulates type I interferon response to RNA virus infection by targeting NLRX1.

FAS-associated factor-1 (FAF1) is a component of the death-inducing signaling complex involved in Fas-mediated apoptosis. It regulates NF-κB activity, ubiquitination, and proteasomal degradation. Here, we found that FAF1 positively regulates the type I interferon pathway. FAF1gt/gt mice, which deficient in FAF1, and FAF1 knockdown immune cells were highly susceptible to RNA virus infection and showed low levels of inflammatory cytokines and type I interferon (IFN) production. FAF1 was bound competitively to NLRX1 and positively regulated type I IFN signaling by interfering with the interaction between NLRX1 and MAVS, thereby freeing MAVS to bind RIG-I, which switched on the MAVS-RIG-I-mediated antiviral signaling cascade. These results highlight a critical role of FAF1 in antiviral responses against RNA virus infection.

Inactivated enterovirus 71 with poly-γ-glutamic acid/Chitosan nano particles (PC NPs) induces high cellular and humoral immune responses in BALB/c mice.

Enterovirus 71 (EV71) is the major causative agent of hand-foot-and-mouth disease (HFMD) and many neurological manifestations. Recently, this virus has become a serious concern because of consecutive epidemics in the Asia-Pacific region. However, no effective vaccine for EV71 has been discovered except two EV71 vaccines which are being used in local communities of China. To develop a safe and efficient EV71 vaccine candidate, we generated inactivated EV71 and evaluated its efficacy with γ-PGA/Chitosan nanoparticles (PC NPs), which are safe, biodegradable and effective as an adjuvant. The subcutaneous administration of inactivated EV71 with PC NPs adjuvant induces higher levels of virus-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4). Additionally, inactivated EV71 with PC NPs adjuvant induces significantly higher virus neutralizing antibody responses compared to the virus only group, and resulted in a long lasting immunity without any noticeable side effects. Together, our findings demonstrate that PC NPs are safe and effective immunogenic adjuvants which may be promising candidates in the development of more efficacious EV71 vaccines.

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses.

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.

Programming of Influenza Vaccine Broadness and Persistence by Mucoadhesive Polymer-Based Adjuvant Systems.

The development of an anti-influenza vaccine with the potential for cross-protection against seasonal drift variants as well as occasionally emerging reassortant viruses is essential. In this study, we successfully generated a novel anti-influenza vaccine system combining conserved matrix protein 2 (sM2) and stalk domain of hemagglutinin (HA2) fusion protein (sM2HA2) and poly-γ-glutamic acid (γ-PGA)-based vaccine adjuvant systems that can act as a mucoadhesive delivery vehicle of sM2HA2 as well as a robust strategy for the incorporation of hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21. Intranasal coadministration of sM2HA2 and the combination adjuvant γ-PGA/MPL/QS21 (CA-PMQ) was able to induce a high degree of protective mucosal, systemic, and cell-mediated immune responses. The sM2HA2/CA-PMQ immunization was able to prevent disease symptoms, confering complete protection against lethal infection with divergent influenza subtypes (H5N1, H1N1, H5N2, H7N3, and H9N2) that lasted for at least 6 mo. Therefore, our data suggest that mucosal administration of sM2HA2 in combination with CA-PMQ could be a potent strategy for a broad cross-protective influenza vaccine, and CA-PMQ as a mucosal adjuvant could be used for effective mucosal vaccines.

Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016.

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).

Genetic characteristics of highly pathogenic H5N8 avian influenza viruses isolated from migratory wild birds in South Korea during 2014-2015.

The continuous worldwide spread of highly pathogenic avian influenza (HPAI) H5N8 viruses among wild birds and poultry is a potential threat to public health. In the present study, we investigated the genetic characteristics of recent H5N8 viruses continuously isolated from migratory birds over two winters (2013-2014 and 2014-2015) in South Korea. Genetic and phylogenetic analysis demonstrated that the 2014-2015 HPAI H5N8 viruses are closely related to the 2013-2014 viruses, including virulence markers; however, all eight gene segments of 2014-2015 H5N8 viruses clustered in different phylogenetic branches from 2013-2014 H5N8 viruses, except the A/Em/Korea/W492/2015 virus. The H5N8 viruses of Europe and North America belong to sublineages of the 2013-2014 Korean H5N8 viruses but differ from the 2014-2015 Korean H5N8 viruses. Further hemagglutination inhibition (HI) assay results showed that there were 2-to-4 fold differences in HI titer between 2013-2014 and 2014-2015 H5N8 viruses. Taken together, our results suggested that the 2014-2015 Korean H5N8 viruses were genetically and serologically different from those of 2013-2014 winter season H5N8 viruses, including those from Europe and North America.

Intranasal administration of poly-gamma glutamate induced antiviral activity and protective immune responses against H1N1 influenza A virus infection.

BACKGROUND: The global outbreak of a novel swine-origin strain of the 2009 H1N1 influenza A virus and the sudden, worldwide increase in oseltamivir-resistant H1N1 influenza A viruses highlight the urgent need for novel antiviral therapy. METHODS: Here, we investigated the antiviral efficacy of poly-gamma glutamate (γ-PGA), a safe and edible biomaterial that is naturally synthesized by Bacillus subtilis, against A/Puerto Rico/8/1934 (PR8) and A/California/04/2009 (CA04) H1N1 influenza A virus infections in C57BL/6 mice. RESULTS: Intranasal administration of γ-PGA for 5 days post-infection improved survival, increased production of antiviral cytokines including interferon-beta (IFN-ß) and interleukin-12 (IL-12), and enhanced activation of natural killer (NK) cells and influenza antigen-specific cytotoxic T lymphocytes (CTL) activity. CONCLUSIONS: These results suggest that γ-PGA protects mice against H1N1 influenza A virus by enhancing antiviral immune responses.

Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-ß activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses.

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA(225G) and NA(315N) substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA(225G) and NA(315N) as potential virulence markers in mammals.

The homologous tripartite viral RNA polymerase of A/swine/Korea/CT1204/2009(H1N2) influenza virus synergistically drives efficient replication and promotes respiratory droplet transmission in ferrets.

We previously reported that influenza A/swine/Korea/1204/2009(H1N2) virus was virulent and transmissible in ferrets in which the respiratory-droplet-transmissible virus (CT-Sw/1204) had acquired simultaneous hemagglutinin (HAD225G) and neuraminidase (NAS315N) mutations. Incorporating these mutations into the nonpathogenic A/swine/Korea/1130/2009(H1N2, Sw/1130) virus consequently altered pathogenicity and growth in animal models but could not establish efficient transmission or noticeable disease. We therefore exploited various reassortants of these two viruses to better understand and identify other viral factors responsible for pathogenicity, transmissibility, or both. We found that possession of the CT-Sw/1204 tripartite viral polymerase enhanced replicative ability and pathogenicity in mice more significantly than did expression of individual polymerase subunit proteins. In ferrets, homologous expression of viral RNA polymerase complex genes in the context of the mutant Sw/1130 carrying the HA225G and NA315N modifications induced optimal replication in the upper nasal and lower respiratory tracts and also promoted efficient aerosol transmission to respiratory droplet contact ferrets. These data show that the synergistic function of the tripartite polymerase gene complex of CT-Sw/1204 is critically important for virulence and transmission independent of the surface glycoproteins. Sequence comparison results reveal putative differences that are likely to be responsible for variation in disease. Our findings may help elucidate previously undefined viral factors that could expand the host range and disease severity induced by triple-reassortant swine viruses, including the A(H1N1)pdm09 virus, and therefore further justify the ongoing development of novel antiviral drugs targeting the viral polymerase complex subunits.

Early regulation of viral infection reduces inflammation and rescues mx-positive mice from lethal avian influenza infection.

Differing sensitivity of influenza A viruses to antiviral effects of the Myxovirus resistance (Mx) protein implies varying global gene expression profiles in the host. The role of Mx protein during lethal avian influenza (AI) virus infection was examined using Mx1-deficient C57BL/6 (B6-Mx1(-/-)) and congenic Mx1-expressing (B6-Mx1(+/+)) mice infected with a virulent, mouse-adapted avian H5N2 Ab/Korea/ma81/07 (Av/ma81) virus. After infection, B6-Mx1(+/+) mice were completely protected from lethal AI-induced mortality, and exhibited attenuated clinical disease and reduced viral titers and pathology in the lungs, compared with B6-Mx1(-/-) mice. Transcriptional profiling of lung tissues revealed that most of the genes up-regulated after infection are involved in activation of the immune response and host defense. Notably, more abundant and sustained expression of cytokine/chemokine genes was observed up to 3 dpi in B6-Mx1(-/-) mice, and this was associated with excessive induction of cytokines and chemokines. Consequently, massive infiltration of macrophages/monocytes and granulocytes into lung resulted in severe viral pneumonia and potentially contributed to decreased survival of B6-Mx1(-/-) mice. Taken together, our data show that dysregulated gene transcriptional activity corresponded to persistent induction of cytokine/chemokines and recruitment of cytokine-producing cells that promote inflammation in B6-Mx1(-/-) mouse lungs. Thus, we provide additional evidence of the interplay of genetic, molecular, and cellular correlates governed by the Mx1 protein that critically determine disease outcome during lethal AI virus infection.

Heterosubtypic protective immunity against widely divergent influenza subtypes induced by fusion protein 4sM2 in BALB/c mice.

BACKGROUND: Regular reformulation of currently available vaccines is necessary due to the unpredictable variability of influenza viruses. Therefore, vaccine based on a highly conserved antigen with capability of induction of effective immune responses could be a potential solution. Influenza matrix protein-2 (M2) is highly conserved across influenza subtypes and a promising candidate for a broadly protective influenza vaccine. For the enhancement of broad protection, four tandem copies of consensus M2 gene containing extracellular (ED) and cytoplasmic (CD) without the trans-membrane domain (TM) reconstituted from H1N1, H5N1 and H9N2 influenza viruses were linked and named as 4sM2. The construct was effectively expressed in Escherichia coli, purified and proteins were used to immunize BALB/c mice. Humoral and cell-mediated immune responses were investigated following administration. RESULTS: Mice were intramuscularly immunized with 4sM2 protein 2 times at 2 weeks interval. Two weeks after the last immunization, first humoral and cell mediated immune response specific to sM2 protein were evaluated and the mice were challenged with a lethal dose (10MLD50) of divergent subtypes A/EM/Korea/W149/06(H5N1), A/PR/8/34(H1N1), A/Aquatic bird/Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses. The efficacy of 4sM2 was evaluated by determining survival rates, body weights and residual lung viral titers. Our studies demonstrate that the survival of mice immunized with 4sM2 was significantly higher (80-100% survival) than that of unimmunized mice (0% survival). We also examined the long lasting protection against heterosubtype H5N2 virus and found that mice vaccinated with 4sM2 displayed 80% of protection even after 6 months of final vaccination. CONCLUSION: Taken together, these results suggest that prokaryotic expressed multimeric sM2 protein achieved cross protection against lethal infection of divergent influenza subtypes which are lasting for the long time.

Mucosal Administration of Lactobacillus casei Surface-Displayed HA1 Induces Protective Immune Responses against Avian Influenza A Virus in Mice.

Avian influenza is a serious threat to both public health and the poultry industry worldwide. This respiratory virus can be combated by eliciting robust immune responses at the site of infection through mucosal immunization. Recombinant probiotics, specifically lactic acid bacteria, are safe and effective carriers for mucosal vaccines. In this study, we engineered recombinant fusion protein by fusing the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) at the surface of Lactobacillus casei (pgsA-HA1/L. casei). Using subcellular fractionation and flow cytometry we confirmed the surface localization of this fusion protein. Mucosal administration of pgsA-HA1/L. casei in mice resulted in significant levels of HA1-specific serum IgG, mucosal IgA and neutralizing antibodies against the H5N2 virus. Additionally, pgsA-HA1/L. casei-induced systemic and local cell-mediated immune responses specific to HA1, as evidenced by an increased number of IFN-γ and IL-4 secreting cells in the spleens and higher levels of IL-4 in the local lymphocyte supernatants. Finally, mice inoculated with pgsA-HA1/L. casei were protected against a 10LD50 dose of the homologous mouse-adapted H5N2 virus. These results suggest that mucosal immunization with L. casei displaying HA1 on its surface could be a potential strategy for developing a mucosal vaccine against other H5 subtype viruses.