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.].

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.

Rubicon Modulates Antiviral Type I Interferon (IFN) Signaling by Targeting IFN Regulatory Factor 3 Dimerization.

Rubicon is part of a Beclin-1-Vps34-containing autophagy complex. Rubicon induces antimicrobial responses upon Toll-like receptor (TLR) stimulation and functions as a feedback inhibitor to prevent unbalanced proinflammatory responses depending on dectin-1 signaling. However, the role played by Rubicon during antiviral immune responses, particularly the type I interferon (IFN) responses, remains largely unknown. Here, we report that Rubicon acts as a negative regulator for virus-triggered IFN signaling. Knockdown of Rubicon promoted type I interferon signaling and inhibited virus replication, while overexpression of Rubicon had the opposite effect. Rubicon specifically interacts with the interferon regulatory factor (IRF) association domain (IAD) of IRF3, and this interaction leads to inhibition of the dimerization of IRF3, which negatively regulates IFN-mediated antiviral response. Thus, our findings suggest the novel additional role of Rubicon as a negative regulator that inhibits the IFN signaling and cellular antiviral responses, providing a novel cellular mechanism of IRF3 inhibition.IMPORTANCE The type I IFN system is a critical innate immune response that protects organisms against virus infection. However, type I IFN signaling must be tightly regulated to avoid excessive production of IFNs. Hence, negative regulatory mechanisms for type I IFN signaling are important, and to date, several related molecules have been identified. Here, we show that Rubicon is a major negative regulator of type I IFN signaling, and unlike previous reports of cellular molecules that inhibit IRF3 activation via proteasomal degradation or dephosphorylation of IRF3, we show that Rubicon interacts with IRF3 and that ultimately this interaction leads to inhibition of the dimerization of IRF3. Thus, we identified a novel negative regulator of type I IFN signaling pathways and a novel cellular mechanism of IRF3 inhibition. The results of this study will increase our understanding of the role of negative-feedback mechanisms that regulate type I IFN signaling and maintain immune homeostasis.

Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

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.

Immune responses in pigs and cattle vaccinated with half-volume foot-and-mouth disease vaccine.

With the current commercial foot-and-mouth disease vaccine, inoculating twice increases the formation of denatured meat due to granuloma or residual adjuvant at the injection site in pigs, resulting in economic loss. Therefore, we investigated protective antibody levels after reducing the amount of adjuvant in the vaccine. Field applicability of the experimental vaccine, made with a new adjuvant ISA 201, was tested by vaccinating farm animals with half-volume doses (1 mL/animal) of commercial vaccine and monitoring their immunogenicity. Among pigs, the group that received a half-volume dose showed similar or higher titers of structural protein antibody and neutralizing antibody than those receiving the standard dose (2 mL). In pigs, the durable effects of antibody titer of the reduced vaccine volume did not diminish up to the time of slaughter. Among cattle, boosting with a second 1 mL vaccine increased virus neutralizing antibody for the protective effects. The boosting effects were more marked in cattle than in pigs. The immune responses differed between species with the effect of the half-volume vaccination being lower in cattle than in pigs. In conclusion, the immune response to the half-volume vaccine was similar to that from the standard volume vaccine in pigs, but not in cattle.

Inhibition of highly pathogenic avian influenza (HPAI) virus by a peptide derived from vFLIP through its direct destabilization of viruses.

The antiviral activities of synthesized Kα2-helix peptide, which was derived from the viral FLICE-like inhibitor protein (vFLIP) of Kaposi's sarcoma-associated herpesvirus (KSHV), against influenza A virus (IAV) were investigated in vitro and in vivo, and mechanisms of action were suggested. In addition to the robust autophagy activity of the Kα2-helix peptide, the present study showed that treatment with the Kα2 peptide fused with the TAT peptide significantly inhibited IAV replication and transmission. Moreover, TAT-Kα2 peptide protected the mice, that were challenged with lethal doses of highly pathogenic influenza A H5N1 or H1N1 viruses. Mechanistically, we found that TAT-Kα2 peptide destabilized the viral membranes, depending on their lipid composition of the viral envelop. In addition to IAV, the Kα2 peptide inhibited infections with enveloped viruses, such as Vesicular Stomatitis Virus (VSV) and Respiratory Syncytial Virus (RSV), without cytotoxicity. These results suggest that TAT-Kα2 peptide is a potential antiviral agent for controlling emerging or re-emerging enveloped viruses, particularly diverse subtypes of IAVs.

Construction of stabilized and tagged foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine.

Antiviral Effects of Novel Herbal Medicine KIOM-C, on Diverse Viruses.

In order to identify new potential antiviral agents, recent studies have advocated thorough testing of herbal medicines or natural substances that are traditionally used to prevent viral infections. Antiviral activities and the mechanism of action of the total aqueous extract preparation of KIOM-C, a novel herbal medicine, against diverse types of viruses were investigated. In vitro antiviral activity against A/Puerto Rico/8/34 (H1N1) (PR8), vesicular stomatitis virus (VSV), and Newcastle disease virus (NDV) through the induction of type-I interferon related protein phosphorylation and up-regulation of pro-inflammatory cytokines in murine macrophage cells (RAW264.7) were determined. In vivo, KIOM-C-treated BALB/c mice showed higher survivability and lower lung viral titers when challenged with A/Aquatic bird/Korea/W81/2005 (H5N2), A/PR/8/34(H1N1), A/Aquatic bird/Korea/W44/2005(H7N3) or A/Chicken/Korea/116 /2004(H9N2) influenza subtypes in contrast with the non-treated group. The present study revealed that total aqueous extract preparation of KIOM-C stimulates an antiviral state in murine macrophage cells and in mice leading to inhibition of viral infection and protection against lethal challenges.

Genetic and immunologic relationships between vaccine and field strains for vaccine selection of type A foot-and-mouth disease virus circulating in East Asia.

Of the seven known serotypes of foot-and-mouth disease virus (FMDV), type A has the most diverse variations. Genetic variations also occur frequently at VP1, VP2, VP3, and VP4 because these proteins constitute the viral capsid. The structural proteins of FMDV, which are closely related to immunologic correlations, are the most easily analyzed because they have highly accessible information. In this study we analyzed the type A vaccine viruses by alignment of available sequences in order to find appropriate vaccine strains. The matching rate of ASIA topotype-specific sites (20 amino acids) located on the viral surface, which are mainly VP1 and VP2, was highly related to immunologic reactivity. Among the available vaccines analyzed in this study, we suggest that A Malaysia 97 could be used as a vaccine virus as it has the highest genetic similarity and immunologic aspects to field strains originating in East Asia.

Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) Induce broadly protective immune responses against divergent influenza subtypes.

The development of a universal influenza vaccine that provides broad cross protection against existing and unforeseen influenza viruses is a critical challenge. In this study, we constructed and expressed conserved sM2 and HA2 influenza antigens with cholera toxin subunit A1 (CTA1) on the surface of Lactobacillus casei (pgsA-CTA1sM2HA2/L. casei). Oral and nasal administrations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and their isotypes (IgG1 & IgG2a) as well as mucosal IgA. The mucosal administration of pgsA-CTA1sM2HA2/L. casei may also significantly increase the levels of sM2- or HA2-specific cell-mediated immunity because increased release of both IFN-γ and IL-4 was observed. The recombinant pgsA-CTA1sM2HA2/L. casei provided better protection of BALB/c mice against 10 times the 50% mouse lethal doses (MLD50) of homologous A/EM/Korea/W149/06(H5N1) or A/Aquatic bird/Korea/W81/2005 (H5N2) and heterologous A/Puerto Rico/8/34(H1N1), or A/Chicken/Korea/116/2004(H9N2) or A/Philippines/2/08(H3N2) viruses, compared with L. casei harboring sM2HA2 and also the protection was maintained up to seven months after administration. These results indicate that recombinant L. casei expressing the highly conserved sM2, HA2 of influenza and CTA1 as a mucosal adjuvant could be a potential mucosal vaccine candidate or tool to protect against divergent influenza viruses for human and animal.

Antigenic properties and virulence of foot-and-mouth disease virus rescued from full-length cDNA clone of serotype O, typical vaccine strain.

We cloned the full-length cDNA of O Manisa, the virus for vaccinating against foot-and-mouth disease. The antigenic properties of the virus recovered from the cDNA were similar to those of the parental virus. Pathogenesis did not appear in the pigs, dairy goats or suckling mice, but neutralizing antibodies were raised 5-6 days after the virus challenge. The utilization of O Manisa as a safe vaccine strain will increase if recombinant viruses can be manipulated by inserting or removing a marker gene for differential serology or replacing the protective gene from another serotype.

Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs and goats.

The immunity and protective capability produced by vaccines can vary remarkably according to the kinds of adjuvants being used. In the case of foot-and-mouth disease (FMD) vaccines in pigs, only oil-adjuvant vaccines have been used, and these tend to show lower immunity in pigs than in cattle. New adjuvants for these vaccines are therefore needed. We made different experimental FMD vaccines using new adjuvants (ISA 201, Carbigen, Emulsigen-D) and well-known adjuvants (ISA 206, aluminum hydroxide gel) and then conducted tests to compare the enhancement in pig immunity. More effective immune responses and protection against challenge were observed with the new adjuvants Emulsigen-D and ISA 201 compared to existing adjuvants. In the case of dairy goats, a mixture of Emulsigen-D and aluminum hydroxide gel produced rapid neutralizing antibody responses that were similar to results from tests conducted with pigs.

Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/c mice.

To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with (pgsA-CTA1-sM2/L. casei) or without (pgsA-sM2/L. casei) cholera toxin subunit A1 (CTA1) on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD50 of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/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 recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes.

The highly conserved HA2 protein of the influenza A virus induces a cross protective immune response.

Existing influenza vaccines protect mostly homologous subtypes and acted most effectively only when well matched to the circulating strain. Immunization with an updated vaccine is therefore necessary to maintain long-term protection and the development of a broadly protective influenza vaccine against the threat of pandemic outbreak. The highly conserved HA2 glyco-polypeptide (HA2 gp) is a promising new candidate for such an influenza vaccine. Helical domain and the fusion peptide (residues 15-137) of surface antigen from influenza A subtype A/EM/Korea/W149/06 (H5N1) was used to assess the potentiality of HA2 vaccination against multiple subtypes of the influenza viruses. The construct, named H5HA2 was expressed in Escherichia coli and allowed to refold from inclusion bodies. Purified proteins were used to investigate the immunogenicity of H5HA2 and its potential for cross protection. The immunization of mice with H5HA2 induced HA2 antibodies, HA2 specific T-cell responses, and protection against homologous A/EM/Korea/W149/06 (H5N1) influenza. Immunized mice were also protected from two distinct heterosubtypes of influenza: A/Puerto Rico/1/34(H1N1) and bird/Korea/w81/2005(H5N2). Results suggest that recombinant proteins based on the highly conserved residues within HA2 are candidates for the development of vaccines against pandemic outbreaks of emergent influenza variants.

Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate/chitosan nanoparticles induces protection against highly pathogenic influenza A virus.

Intranasal administration of recombinant influenza hemagglutinin (rHA) antigen or inactivated virus with nanoparticles (NPs) composed of poly-γ-glutamic acid (γ-PGA) and chitosan which are safe, natural materials, and able to target the mucosal membrane as a mucosal adjuvant, could induce a high degree of protective mucosal immunity in the respiratory tract. Intranasal immunization with mixture of rHA antigen or inactivated virus and γ-PGA/chitosan nanoparticles (PC NPs) induced not only a high anti-HA immunoglobulin A (IgA) response in lung and IgG response in serum, including anti-HA neutralizing antibodies, but also an influenza virus-specific cell-mediated immune response. Also, PC NPs could function as a potential mucosal adjuvant when it was compared with the well-known mucosal adjuvant, cholera toxin (CT). Intranasal administration of rHA antigen or inactivated virus with PC NPs protected mice against challenge with a lethal dose of the highly pathogenic influenza A H5N1 virus. These results suggested that application of PC NPs with a subunit antigen of influenza produced by prokaryotic expression system provides several solutions to the current problems of the influenza vaccines using inactivated influenza virus. Moreover, our finding about a sufficient function of PC NPs to elevate vaccine efficacy led us to consider that it can be useful in clinical applications as a potent mucosal adjuvant with safety.