Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues.

BACKGROUND: Macrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies. METHODS: To elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection. RESULTS: NOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 + and CD8 + T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H2O2 generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages. CONCLUSION: In light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.

TLR3/TRIF pathway confers protection against herpes simplex encephalitis through NK cell activation mediated by a loop of type I IFN and IL-15 from epithelial and dendritic cells.

Autosomal recessive (AR) and dominant (AD) deficiencies of TLR3 and TRIF are believed to be crucial genetic causes of herpes simplex encephalitis (HSE), which is a fatal disease causing focal or global cerebral dysfunction following infection with herpes simplex virus type 1 (HSV-1). However, few studies have been conducted on the immunopathological networks of HSE in the context of TLR3 and TRIF defects at the cellular and molecular levels. In this work, we deciphered the crosstalk between type I IFN (IFN-I)-producing epithelial layer and IL-15-producing dendritic cells (DC) to activate NK cells for the protective role of TLR3/TRIF pathway in HSE progression after vaginal HSV-1 infection. TLR3- and TRIF-ablated mice showed enhanced susceptibility to HSE progression, along with high HSV-1 burden in vaginal tract, lymphoid tissues and CNS. The increased HSV-1 burden in TLR3- and TRIF-ablated mice did not correlate with increased infiltration of Ly-6C+ monocytes, but it was closely associated with impaired NK cell activation in vaginal tract. Furthermore, using delicate ex vivo experiments and bone marrow transplantation, TRIF deficiency in tissue-resident cells, such as epithelial cells in vaginal tract, was found to cause impaired NK cell activation by means of low IFN-I production, whereas IFN-I receptor in DC was required for NK cell activation via IL-15 production in response to IFN-I produced from epithelial layer. These results provide new information about IFN-I- and IL-15-mediated crosstalk between epithelial cells and DC at the primary infection site, which suppresses HSE progression in a TLR3- and TRIF-dependent manner.

The Interplay between Host Immunity and Respiratory Viral Infection in Asthma Exacerbation.

Asthma is one of the most common and chronic diseases characterized by multidimensional immune responses along with poor prognosis and severity. The heterogeneous nature of asthma may be attributed to a complex interplay between risk factors (either intrinsic or extrinsic) and specific pathogens such as respiratory viruses, and even bacteria. The intrinsic risk factors are highly correlated with asthma exacerbation in host, which may be mediated via genetic polymorphisms, enhanced airway epithelial lysis, apoptosis, and exaggerated viral replication in infected cells, resulting in reduced innate immune response and concomitant reduction of interferon (types I, II, and III) synthesis. The canonical features of allergic asthma include strong Th2-related inflammation, sensitivity to non-steroidal anti-inflammatory drugs (NSAIDs), eosinophilia, enhanced levels of Th2 cytokines, goblet cell hyperplasia, airway hyper-responsiveness, and airway remodeling. However, the NSAID-resistant non-Th2 asthma shows a characteristic neutrophilic influx, Th1/Th17 or even mixed (Th17-Th2) immune response and concurrent cytokine streams. Moreover, inhaled corticosteroid-resistant asthma may be associated with multifactorial innate and adaptive responses. In this review, we will discuss the findings of various in vivo and ex vivo models to establish the critical heterogenic asthmatic etiologies, host-pathogen relationships, humoral and cell-mediated immune responses, and subsequent mechanisms underlying asthma exacerbation triggered by respiratory viral infections.

Dual TLR2/9 Recognition of Herpes Simplex Virus Infection Is Required for Recruitment and Activation of Monocytes and NK Cells and Restriction of Viral Dissemination to the Central Nervous System.

The importance of TLR2 and TLR9 in the recognition of infection with herpes simplex virus (HSV) and HSV-caused diseases has been described, but some discrepancies remain concerning the benefits of these responses. Moreover, the impact of TLR2/9 on innate and adaptive immune responses within relevant mucosal tissues has not been elucidated using natural mucosal infection model of HSV. Here, we demonstrate that dual TLR2/9 recognition is essential to provide resistance against mucosal infection with HSV via an intravaginal route. Dual TLR2/9 ablation resulted in the highly enhanced mortality with exacerbated symptoms of encephalitis compared with TLR2 or TLR9 deficiency alone, coinciding with highly increased viral load in central nervous system tissues. TLR2 appeared to play a minor role in providing resistance against mucosal infection with HSV, since TLR2-ablated mice showed higher survival rate compared with TLR9-ablated mice. Also, the high mortality in dual TLR2/9-ablated mice was closely associated with the reduction in early monocyte and NK cell infiltration in the vaginal tract (VT), which was likely to correlate with low expression of cytokines and CCR2 ligands (CCL2 and CCL7). More interestingly, our data revealed that dual TLR2/9 recognition of HSV infection plays an important role in the functional maturation of TNF-α and iNOS-producing dendritic cells (Tip-DCs) from monocytes as well as NK cell activation in VT. TLR2/9-dependent maturation of Tip-DCs from monocytes appeared to specifically present cognate Ag, which effectively provided functional effector CD4+ and CD8+ T cells specific for HSV Ag in VT and its draining lymph nodes. TLR2/9 expressed in monocytes was likely to directly facilitate Tip-DC-like features after HSV infection. Also, dual TLR2/9 recognition of HSV infection directly activated NK cells without the aid of dendritic cells through activation of p38 MAPK pathway. Taken together, these results indicate that dual TLR2/9 recognition plays a critical role in providing resistance against mucosal infection with HSV, which may involve a direct regulation of Tip-DCs and NK cells in VT. Therefore, our data provide a more detailed understanding of TLR2/9 role in conferring antiviral immunity within relevant mucosal tissues after mucosal infection with HSV.

CCL2, but not its receptor, is essential to restrict immune privileged central nervous system-invasion of Japanese encephalitis virus via regulating accumulation of CD11b(+) Ly-6C(hi) monocytes.

Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2-CCL2 axis plays an important role in trafficking CD11b(+) Ly-6C(hi) monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2- and CCL2-ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2-ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2- and CCL2-ablated mice was unlikely to be mediated by innate natural killer and adaptive T-cell responses. Furthermore, CCL2 produced by haematopoietic stem cell-derived leucocytes played a dominant role in CNS accumulation of Ly-6C(hi) monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.

Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

BACKGROUND: Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis. METHODS: Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed. RESULTS: Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c(+) dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c(+) DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment. CONCLUSIONS: Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade.

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I-dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I-dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

Anti-herpes Activity of Vinegar-processed Daphne genkwa Flos Via Enhancement of Natural Killer Cell Activity.

Herpes simplex virus (HSV) is a common causative agent of genital ulceration and can lead to subsequent neurological disease in some cases. Here, using a genital infection model, we tested the efficacy of vinegar-processed flos of Daphne genkwa (vp-genkwa) to modulate vaginal inflammation caused by HSV-1 infection. Our data revealed that treatment with optimal doses of vp-genkwa after, but not before, HSV-1 infection provided enhanced resistance against HSV-1 infection, as corroborated by reduced mortality and clinical signs. Consistent with these results, treatment with vp-genkwa after HSV-1 infection reduced viral replication in the vaginal tract. Furthermore, somewhat intriguingly, treatment of vp-genkwa after HSV-1 infection increased the frequency and absolute number of CD3(-)NK1.1(+)NKp46(+) natural killer (NK) cells producing interferon (IFN)-γ and granyzme B, which indicates that vp-genkwa treatment induces the activation of NK cells. Supportively, secreted IFN-γ was detected at an increased level in vaginal lavages of mice treated with vp-genkwa after HSV-1 infection. These results indicate that enhanced resistance to HSV-1 infection by treatment with vp-genkwa is associated with NK cell activation. Therefore, our data provide a valuable insight into the use of vp-genkwa to control clinical severity in HSV infection through NK cell activation.

Functional restoration of exhausted CD4(+) and CD8(+) T cells in chronic viral infection by vinegar-processed flos of Daphne genkwa.

T-cell exhaustion has become an important issue in chronic infection because exhausted antigen-specific T cells show impaired abilities to eradicate persistently infected pathogens and produce effector cytokines, such as IFN-γ and TNF-α. Thus, strategies to either restore endogenous exhausted T cell responses or provide functional T cells are needed for therapeutics of chronic infection. Despite promising developments using antibodies and cell immunotherapy, there have been no reported attempts to restore exhausted T cells using treatment with materials derived from natural resources. Here, using a mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found that vinegar-processed flowers (flos) of Daphne genkwa (vp-genkwa), which was composed mainly of four index components, restored exhausted CD4(+) and CD8(+) T cells significantly, as corroborated by evidence that vp-genkwa treatment enhanced functional LCMV-specific CD4(+) and CD8(+) T cells, both quantitatively and qualitatively. Furthermore, pretreatment with vp-genkwa prevented the generation of exhausted LCMV-specific CD8(+) T cells. Such restorations of exhausted LCMV-specific CD4(+) and CD8(+) T cells by vp-genkwa were closely associated with reduced viral burden in sera and tissues. More interestingly, vp-genkwa treatment induced down-regulation of negative molecules, such as PD-1 and Tim-3, in exhausted CD4(+) and CD8(+) T cells with more apparent down-regulation of Tim-3, suggesting that Tim-3 molecule may be a major target in restoring exhausted T cell responses. Collectively, these results provide valuable new insights into the use of vp-genkwa to develop a therapeutic strategy for chronic human diseases, such as hepatitis B and C virus, human immunodeficiency virus, and cancers.

Prophylactic and therapeutic modulation of innate and adaptive immunity against mucosal infection of herpes simplex virus.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are the most common cause of genital ulceration in humans worldwide. Typically, HSV-1 and 2 infections via mucosal route result in a lifelong latent infection after peripheral replication in mucosal tissues, thereby providing potential transmission to neighbor hosts in response to reactivation. To break the transmission cycle, immunoprophylactics and therapeutic strategies must be focused on prevention of infection or reduction of infectivity at mucosal sites. Currently, our understanding of the immune responses against mucosal infection of HSV remains intricate and involves a balance between innate signaling pathways and the adaptive immune responses. Numerous studies have demonstrated that HSV mucosal infection induces type I interferons (IFN) via recognition of Toll-like receptors (TLRs) and activates multiple immune cell populations, including NK cells, conventional dendritic cells (DCs), and plasmacytoid DCs. This innate immune response is required not only for the early control of viral replication at mucosal sites, but also for establishing adaptive immune responses against HSV antigens. Although the contribution of humoral immune response is controversial, CD4(+) Th1 T cells producing IFN-γ are believed to play an important role in eradicating virus from the hosts. In addition, the recent experimental successes of immunoprophylactic and therapeutic compounds that enhance resistance and/or reduce viral burden at mucosal sites have accumulated. This review focuses on attempts to modulate innate and adaptive immunity against HSV mucosal infection for the development of prophylactic and therapeutic strategies. Notably, cells involved in innate immune regulations appear to shape adaptive immune responses. Thus, we summarized the current evidence of various immune mediators in response to mucosal HSV infection, focusing on the importance of innate immune responses.

Protective effects of red ginseng extract against vaginal herpes simplex virus infection.

Numerous studies have suggested that Korean red ginseng (KRG) extract has various immune modulatory activities both in vivo and in vitro. In this study, we used a mouse model to examine the effects of orally administered KRG extract on immunity against herpes simplex virus (HSV). Balb/c mice were administered with 100, 200, and 400 mg/kg oral doses of KRG extract for 10 d and then vaginally infected with HSV. We found that KRG extract rendered recipients more resistant against HSV vaginal infection and further systemic infection, including decreased clinical severity, increased survival rate, and accelerated viral clearance. Such results appeared to be mediated by increased vaginal IFN-γ secretion. Moreover, increased mRNA expression of IFN-γ, granzyme B, and Fas-ligand was identified in the iliac lymph node and vaginal tracts of KRG extract treated groups (200 and 400 mg/kg). These results suggest that the activities of local natural killer cells were promoted by KRG extract consumption and that KRG may be an attractive immune stimulator for helping hosts overcome HSV infection.

Modulation of Humoral and Cell-Mediated Immunity Against Avian Influenza and Newcastle Disease Vaccines by Oral Administration of Salmonella enterica Serovar Typhimurium Expressing Chicken Interleukin-18.

Interleukin-18 (IL-18) has been known to induce interferon-γ (IFN-γ) production and promote Th1 immunity. Although mammalian IL-18 has been characterized in great detail, the properties and application of chicken IL-18 remain largely uninvestigated as of yet. In this study, we evaluated the immunomodulatory properties of Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 (chIL-18) on immune responses induced by avian influenza (AI) and Newcastle disease (ND) vaccines. After oral administration of S. enterica serovar Typhimurium expressing chIL-18, chickens were vaccinated intramuscularly with the recommended dose of either inactivated AI H9N2 vaccine or ND (B1 strain) vaccine. Chickens receiving a primary vaccination were boosted using the same protocol 7 days later. Humoral and cell-mediated immune responses were evaluated in terms of HI antibody titers and proliferation and mRNA expression of IFN-γ and IL-4 of peripheral blood mononuclear cells (PBMC) in response to specific antigen stimulation. According to our results, oral administration of S. enterica serovar Typhimurium expressing chIL-18 induced enhanced humoral and Th1-biased cell-mediated immunity against AI and ND vaccines, compared to that of chickens received S. enterica serovar Typhimurium harboring empty vector. Therefore, we conclude that our proposed vaccination regimen using inactivated AI and ND viruses along with oral administration of S. enterica serovar Typhimurium expressing chIL-18 may provide a novel approach in protecting chicken from currently circulating AI and ND virus strains.

Glutamine and leucine provide enhanced protective immunity against mucosal infection with herpes simplex virus type 1.

Besides their role as building blocks of protein, there are growing evidences that some amino acids have roles in regulating key metabolic pathways that are necessary for maintenance, growth, reproduction, and immunity. Here, we evaluated the modulatory functions of several amino acids in protective immunity against mucosal infection of herpes simplex virus type 1 (HSV-1). We found that glutamine (Gln) and leucine (Leu) showed enhanced protective immunity to HSV-1 mucosal infection when two administration of Gln and single administration of Leu per day, but not when administered in combinations. Ameliorated clinical signs of HSV-1 challenged mice by the intraperitoneal administration of Gln and Leu were closely associated with viral burden and IFN-γ production in the vaginal tract at 2 and 4 days post-infection. In addition, the enhanced production of vaginal IFN-γ appeared to be caused by NK and HSV-1 antigen-specific Th1-type CD4+ T cells recruited into vaginal tract of mice treated with Gln and Leu, which indicates that IFN-γ, produced by NK and Th1-type CD4+ T cells, may be critical to control the outcome of diseases caused by HSV-1 mucosal infection. Collectively, our results indicate that intraperitoneal administration of Gln and Leu following HSV-1 mucosal infection could provide beneficial effects for the modulation of protective immunity, but dosage and frequency of administration should be carefully considered, because higher frequency and overdose of Gln and Leu, or their combined treatment, showed detrimental effects to protective immunity.

Enhancement of Th1-biased protective immunity against avian influenza H9N2 virus via oral co-administration of attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 along with an inactivated vaccine.

BACKGROUND: Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) as natural immunomodulators. RESULTS: Oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens. CONCLUSIONS: Our results indicate the value of combined administration of chIFN-α and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2.

Co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interleukin-18 and interferon-α provides enhanced Th1-biased protective immunity against inactivated vaccine of pseudorabies virus.

The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration. In light of the current situation, we evaluated the immunomodulatory functions of the combined administration of swine interleukin-18 (swIL-18) and interferon-α (swIFN-α) against an inactivated PrV vaccine using attenuated Salmonella enterica serovar Typhimurium as a cytokine delivery system. Co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α produced enhanced Th1-biased humoral and cellular immune responses against the inactivated PrV vaccine, when compared to single administration of S. enterica serovar Typhimurium expressing either swIL-18 or swIFN-α. Also, enhanced immune responses in co-administered piglets occurred rapidly after virulent PrV challenge, and piglets that received co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α displayed a greater alleviation of clinical severity following the virulent PrV challenge, as determined by clinical scores and cumulative daily weight gain. Furthermore, this enhancement was confirmed by reduced nasal shedding of PrV following viral challenge. Therefore, these results suggest that oral co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α provide enhanced Th1-biased immunity against inactivated PrV vaccine to alleviate clinical signs caused by PrV challenge.

Oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 enhances the alleviation of clinical signs caused by respiratory infection with avian influenza virus H9N2.

The combined use of cytokines has shown synergistic and/or additive effects in controlling several viral infections of livestock animals. However, little is known concerning the practical use of chicken cytokine combinations to control avian diseases. Here, we investigated the antiviral efficacy of oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) using attenuated Salmonella enterica serovar Typhimurium in chickens infected with avian influenza virus (AIV) H9N2. Our results demonstrate that oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 produced a greater alleviation of clinical signs caused by respiratory infection with AIV H9N2 in chickens, when compared to administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18 alone. Mortality, clinical symptom severity, and feed and water intake were used to access treatment effectiveness. This enhancement of antiviral immunity was further confirmed by evidence of reduced rectal shedding and decreased replication of AIV H9N2 in several different tissues of challenged chickens including trachea, lung, cecal tonsil, and brain. Furthermore, oral co-administration of chIFN-α and chIL-18 more efficiently modulated the immune responses of chickens against AIV H9N2 by enhancing both humoral and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results suggest that the combined administration of two chicken cytokines, chIFN-α and chIL-18, using attenuated S. enterica serovar Typhimurium as an oral carrier, provides an effective means for controlling respiratory disease caused by AIV H9N2 infection.

Oral administration of Salmonella enterica serovar Typhimurium expressing swine interleukin-18 induces Th1-biased protective immunity against inactivated vaccine of pseudorabies virus.

Enhancing and/or modulating innate and adaptive immunity by cytokines appears to be greatly useful to provide effective protective immunity against infectious diseases. However, an effective delivery system for mass administration in livestock industry is needed because of limitations such as cost, labor, time, and protein stability. Here the immunomodulatory functions of swine interleukine-18 (swIL-18), known as IFN-γ-inducing factor (IGIF), were evaluated in a vaccination model of pseudorabies virus (PrV) using attenuated Salmonella enterica serovar Typhimurium as the oral delivery system. The oral administration of S. enterica serovar Typhimurium expressing swIL-18 prior to vaccination with inactivated PrV vaccine induced enhanced levels of serum PrV-specific IgG and its IgG2 isotype, compared to administration of S. enterica serovar Typhimurium harboring the empty vector. Furthermore, S. enterica serovar Typhimurium expressing swIL-18 mounted Th1-biased cellular immune responses against PrV antigen, as evaluated by the production of IFN-γ and IL-4 from peripheral blood mononuclear cells of piglets. Subsequently, Th1-biased immunity induced by S. enterica serovar Typhimurium expressing swIL-18 showed rapid response and rendered piglets displayed more alleviated clinical signs following the virulent PrV challenge. Also, this alleviation of clinical signs was further confirmed by the reduction of nasal excretion of PrV after challenge. The present study demonstrates the extended use of immunomodulatory functions of swIL-18 orally delivered by attenuated S. enterica serovar Typhimurium.

Enhanced protection against infection with transmissible gastroenteritis virus in piglets by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-α and interleukin-18.

The enhanced effect of cytokine combinations has been assessed empirically, based on their immunobiological mechanisms. However, far less is known of the enhanced protection of practical cytokine combinations against viral infection in the livestock industry, due to cost and production issues associated with mass administration. This study demonstrates the enhanced protection of oral co-administration of swine interferon-α (swIFN-α) and interleukin-18 (swIL-18) against infection with transmissible gastroenteritis virus (TGEV) in piglets using attenuated Salmonella enterica serovar Typhimurium as carrier of cytokine proteins. A single oral co-administration of S. enterica serovar Typhimurium expressing swIFN-α and swIL-18 induced enhanced alleviation of the severity of diarrhea caused by TGEV infection, compared to piglets administered S. enterica serovar Typhimurium expressing swIFN-α or swIL-18 alone. This enhancement was further observed by the reduction of TGEV shedding and replication, and the expression of IFN-stimulated gene products in the intestinal tract. The results suggest that the combined administration of the swIFN-α and swIL-18 cytokines using attenuated S. enterica serovar Typhimurium as an oral carrier provides enhanced protection against intestinal tract infection with TGEV.

Oral administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α alleviates clinical signs caused by respiratory infection with avian influenza virus H9N2.

Low pathogenic avian influenza (LPAI) H9N2 has attracted considerable attention due to severe commercial losses in the poultry industry. Furthermore, avian influenza virus (AIV) H9N2-infected chickens can be a reservoir for viral transmission to mammals including pigs and humans, complicating control of viral mutants. Chicken interferon-alpha (chIFN-α) may be useful as an exogenous antiviral agent to control AIV H9N2 infection. However, a superior vehicle for administration of chIFN-α is needed because of challenges of protein stability, production cost, and labor associated with mass administration. Presently, oral administration of single dose of attenuated Salmonella enterica serovar Typhimurium expressing chIFN-α alleviated clinical signs and histopathological changes caused by respiratory infection with AIV H9N2 and reduced the excretion of virus in cloacal swab samples. Similarly, chickens administered S. enterica serovar Typhimurium expressing chIFN-α showed inhibited replication of AIV H9N2 in several different tissues including trachea, lung, cecal tonsil, and brain. Furthermore, immune responses specific for challenged AIV H9N2 were enhanced in chickens administered S. enterica serovar Typhimurium expressing chIFN-α, as determined by hemagglutination inhibition assay of sera, proliferation and IFN-γ and interleukin-4 expression by AIV H9N2 antigen-stimulated peripheral blood mononuclear cells and splenocytes. Therefore, oral administration of S. enterica serovar Typhimurium expressing chIFN-α can successfully control clinical signs caused by respiratory infection with AIV H9N2, which provides valuable insight into the use of attenuated Salmonella vaccine as an oral delivery system of chIFN-α to prevent the replication of AIV H9N2 in respiratory tract.

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus.

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.