A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.

NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

The tumor suppressor PTEN has a critical role in antiviral innate immunity.

The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3.

Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.

MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.

In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.

Non-transcriptional IRF7 interacts with NF-κB to inhibit viral inflammation.

Interferon (IFN) regulatory factors (IRF) are key transcription factors in cellular antiviral responses. IRF7, a virus-inducible IRF, expressed primarily in myeloid cells, is required for transcriptional induction of interferon α and antiviral genes. IRF7 is activated by virus-induced phosphorylation in the cytoplasm, leading to its translocation to the nucleus for transcriptional activity. Here, we revealed a nontranscriptional activity of IRF7 contributing to its antiviral functions. IRF7 interacted with the pro-inflammatory transcription factor NF-κB-p65 and inhibited the induction of inflammatory target genes. Using knockdown, knockout, and overexpression strategies, we demonstrated that IRF7 inhibited NF-κB-dependent inflammatory target genes, induced by virus infection or toll-like receptor stimulation. A mutant IRF7, defective in transcriptional activity, interacted with NF-κB-p65 and suppressed NF-κB-induced gene expression. A single-action IRF7 mutant, active in anti-inflammatory function, but defective in transcriptional activity, efficiently suppressed Sendai virus and murine hepatitis virus replication. We, therefore, uncovered an anti-inflammatory function for IRF7, independent of transcriptional activity, contributing to the antiviral response of IRF7.

IRF3 inhibits nuclear translocation of NF-κB to prevent viral inflammation.

Interferon (IFN) regulatory factor 3 (IRF3) is a transcription factor activated by phosphorylation in the cytoplasm of a virus-infected cell; by translocating to the nucleus, it induces transcription of IFN-ß and other antiviral genes. We have previously reported IRF3 can also be activated, as a proapoptotic factor, by its linear polyubiquitination mediated by the RIG-I pathway. Both transcriptional and apoptotic functions of IRF3 contribute to its antiviral effect. Here, we report a nontranscriptional function of IRF3, namely, the repression of IRF3-mediated NF-κB activity (RIKA), which attenuated viral activation of NF-κB and the resultant inflammatory gene induction. In Irf3-/- mice, consequently, Sendai virus infection caused enhanced inflammation in the lungs. Mechanistically, RIKA was mediated by the direct binding of IRF3 to the p65 subunit of NF-κB in the cytoplasm, which prevented its nuclear import. A mutant IRF3 defective in both the transcriptional and the apoptotic activities was active in RIKA and inhibited virus replication. Our results demonstrated IRF3 deployed a three-pronged attack on virus replication and the accompanying inflammation.

Intranasal Sendai virus-based SARS-CoV-2 vaccine using a mouse model.

The coronavirus disease 2019 (COVID-19) epidemic remains worldwide. The usefulness of the intranasal vaccine and boost immunization against severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) has recently received much attention. We developed an intranasal SARS-CoV-2 vaccine by loading the receptor binding domain of the S protein (S-RBD) of SARS-CoV-2 as an antigen into an F-deficient Sendai virus vector. After the S-RBD-Fd antigen with trimer formation ability was intranasally administered to mice, S-RBD-specific IgM, IgG, IgA, and neutralizing antibody titers were increased in serum or bronchoalveolar lavage fluid for 12 weeks. Furthermore, in mice that received a booster dose at week 8, a marked increase in neutralizing antibodies in the serum and bronchoalveolar lavage fluid was observed at the final evaluation at week 12, which neutralized the pseudotyped lentivirus expressing the SARS-CoV-2 spike protein, indicating the usefulness of the Sendai virus-based SARS-CoV-2 intranasal vaccine.

ADAR1 Biology Can Hinder Effective Antiviral RNA Interference.

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.

Age-Dependent Reduction in Asthmatic Pathology through Reprogramming of Postviral Inflammatory Responses.

Asthma is a chronic disease of childhood, but for unknown reasons, disease activity sometimes subsides as children mature. In this study, we present clinical and animal model evidence suggesting that the age dependency of childhood asthma stems from an evolving host response to respiratory viral infection. Using clinical data, we show that societal suppression of respiratory virus transmission during coronavirus disease 2019 lockdown disrupted the traditional age gradient in pediatric asthma exacerbations, connecting the phenomenon of asthma remission to virus exposure. In mice, we show that asthmatic lung pathology triggered by Sendai virus (SeV) or influenza A virus is highly age-sensitive: robust in juvenile mice (4-6 wk old) but attenuated in mature mice (>3 mo old). Interestingly, allergen induction of the same asthmatic traits was less dependent on chronological age than viruses. Age-specific responses to SeV included a juvenile bias toward type 2 airway inflammation that emerged early in infection, whereas mature mice exhibited a more restricted bronchiolar distribution of infection that produced a distinct type 2 low inflammatory cytokine profile. In the basal state, aging produced changes to lung leukocyte burden, including the number and transcriptional landscape of alveolar macrophages (AMs). Importantly, depleting AMs in mature mice restored post-SeV pathology to juvenile levels. Thus, aging influences chronic outcomes of respiratory viral infection through regulation of the AM compartment and type 2 inflammatory responses to viruses. Our data provide insight into how asthma remission might develop in children.

Recruitment of phosphatase PP2A by RACK1 adaptor protein deactivates transcription factor IRF3 and limits type I interferon signaling.

The transcription factor IRF3 is a central regulator of type I interferon (IFN) signaling. The mechanisms underlying deactivation of IRF3 are poorly understood although many studies suggest that IRF3 activity is terminated through degradation after viral infection. Here we report that IRF3 is deactivated via dephosphorylation mediated by the serine and threonine phosphatase PP2A and its adaptor protein RACK1. The PP2A-RACK1 complex negatively regulated the IRF3 pathway after LPS or poly(I:C) stimulation or Sendai virus (SeV) infection. After challenge with LPS, poly(I:C), or low-titer SeV, activated IRF3 was dephosphorylated and returned to resting state without being degraded, although high-titer SeV infection triggered the degradation of IRF3. Furthermore, PP2A-deficient macrophages showed enhanced type I IFN signaling upon LPS, poly(I:C), and SeV challenge and protected mice from lethal vesicular stomatitis virus infection. Therefore, dephosphorylation of IRF3 is a deactivation mechanism that contributes to termination of IRF3-type I IFN signaling.

Atopic Neutrophils Prevent Postviral Airway Disease.

Respiratory syncytial virus (RSV) infection in infancy is associated with increased risk of asthma, except in those with allergic disease at the time of infection. Using house dust mite allergen, we examined the effect of pre-existing atopy on postviral airway disease using Sendai virus in mice, which models RSV infection in humans. Sendai virus drives postviral airway disease in nonatopic mice; however, pre-existing atopy protected against the development of airway disease. This protection depended upon neutrophils, as depletion of neutrophils at the time of infection restored the susceptibility of atopic mice to postviral airway disease. Associated with development of atopy was an increase in polymorphonuclear neutrophil-dendritic cell hybrid cells that develop in Th2 conditions and demonstrated increased viral uptake. Systemic inhibition of IL-4 reversed atopic protection against postviral airway disease, suggesting that increased virus uptake by neutrophils was IL-4 dependent. Finally, human neutrophils from atopic donors were able to reduce RSV infection of human airway epithelial cells in vitro, suggesting these findings could apply to the human. Collectively our data support the idea that pre-existing atopy derives a protective neutrophil response via potential interaction with IL-4, preventing development of postviral airway disease.

ZNF205 positively regulates RLR antiviral signaling by targeting RIG-I.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic viral RNA receptor. Upon viral infection, the protein recognizes and then recruits adapter protein mitochondrial antiviral signaling (MAVS) protein, initiating the production of interferons and proinflammatory cytokines to establish an antiviral state. In the present study, we identify zinc finger protein 205 (ZNF205) which associates with RIG-I and promotes the Sendai virus (SeV)-induced antiviral innate immune response. Overexpression of ZNF205 facilitates interferon-beta (IFN-ß) introduction, whereas ZNF205 deficiency restricts its introduction. Mechanistically, the C-terminal zinc finger domain of ZNF205 interacts with the N-terminal tandem caspase recruitment domains (CARDs) of RIG-I; this interaction markedly promotes K63 ubiquitin-linked polyubiquitination of RIG-I, which is crucial for RIG-I activation. Thus, our results demonstrate that ZNF205 is a positive regulator of the RIG-I-mediated innate antiviral immune signaling pathway.

Single-virus assay reveals membrane determinants and mechanistic features of Sendai virus binding.

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative-sense RNA virus in the Paramyxoviridae family and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein. Receptor binding induces a conformational change in HN, which allosterically triggers the viral fusion (F) protein to catalyze membrane fusion. While it is known that SeV binds to α2,3-linked sialic acid receptors, and there has been some study into the chemical requirements of those receptors, key mechanistic features of SeV binding remain unknown, in part because traditional approaches often convolve binding and fusion. Here, we develop and employ a fluorescence microscopy-based assay to observe SeV binding to supported lipid bilayers (SLBs) at the single-particle level, which easily disentangles binding from fusion. Using this assay, we investigate mechanistic questions of SeV binding. We identify chemical structural features of ganglioside receptors that influence viral binding and demonstrate that binding is cooperative with respect to receptor density. We measure the characteristic decay time of unbinding and provide evidence supporting a "rolling" mechanism of viral mobility following receptor binding. We also study the dependence of binding on target cholesterol concentration. Interestingly, we find that although SeV binding shows striking parallels in cooperative binding with a prior report of Influenza A virus, it does not demonstrate a similar sensitivity to cholesterol concentration and receptor nanocluster formation.

Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFκB p50 recruitment.

Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1-/- deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1-/- deletions eliminated G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a-GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1-/- deletions. G9a-GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a-GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a-GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents.

A phase I dose-escalation, safety/tolerability, and preliminary efficacy study of the intratumoral administration of GEN0101 in patients with advanced melanoma.

Despite recent advance in immunotherapy agents, safe new therapies that enhance the effects of immune checkpoint inhibitors are still required to develop. We previously demonstrated that hemagglutinating virus of Japan-envelope (HVJ-E) induced not only direct tumor cell death but also antitumor immunity through the activation of T and natural killer (NK) cells, thereafter, developed a manufacturing process of HVJ-E (GEN0101) for clinical use. We here performed a phase Ia clinical trial of intratumoral GEN0101 administration in six patients with stage IIIC or IV malignant melanoma. The primary aim was to evaluate the safety and tolerability of GEN0101, and the secondary aim was to examine the objective tumor response. Patients were separated into two groups (n = 3 each) and received a low dose of 30,000 and high dose of 60,000 mNAU of GEN0101. All patients completed a two-week follow-up evaluation without severe adverse events. The overall response rate was 33% (2 of 6), with 2 partial responses in the high-dose group and 2 with stable disease, and 2 with progressive disease in the low-dose group. Local complete or partial responses were observed in 11 of 18 (61%) target lesions. One patient demonstrated shrinkage of lung metastases after the treatment. The activity of NK cells and interferon-γ levels were increased in the circulation, indicating augmentation of antitumor immunity by GEN0101. This trial showed not only the safety and tolerability but also the significant antitumor effect of GEN0101, suggesting that GEN0101 might be a promising new drug for patients with advanced melanoma.

Structural Insight into the Interaction of Sendai Virus C Protein with Alix To Stimulate Viral Budding.

Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae, harbors an accessory protein, named C protein, which facilitates viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles by binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, small interfering RNA (siRNA)-mediated gene knockdown studies suggested that neither Alix nor C protein is related to SeV budding. In the present study, we determined the crystal structure of a complex comprising the C-terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å to investigate the role of the complex in SeV budding. The structure revealed that a novel consensus sequence, LXXW, which is conserved among Respirovirus C proteins, is important for Alix binding. SeV possessing a mutated C protein with reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h postinfection compared with nonmutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, for binding to Alix. All together, these results highlight the critical role of C protein in SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been studied extensively to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating viral reproduction, but its role in budding remains controversial. In the present study, the crystal structure of the C-terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutant C proteins with different binding affinities to Alix and showed that the interaction between C and Alix is vital for viral budding. These findings provide new insights into the development of new antiviral drugs against hPIV1.

Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition.

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA; however, its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data show that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal among NSVs, including Ebola virus, Lassa virus, and measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues for developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines.IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus, and Ebola virus. These viruses possess RNA genomes that are unreadable to the host, as they require specific viral RNA-dependent RNA polymerases in conjunction with other viral proteins, such as nucleoprotein, to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here, we demonstrate the balance that negative-sense RNA viruses must achieve both to replicate efficiently and to avoid induction of the host defenses.

CD8 T Cells Show Protection against Highly Pathogenic Simian Immunodeficiency Virus (SIV) after Vaccination with SIV Gene-Expressing BCG Prime and Vaccinia Virus/Sendai Virus Vector Boosts.

Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection.IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

The Kv1.3 ion channel acts as a host factor restricting viral entry.

Virus entry into cells is the initial stage of infection and involves multiple steps, and interfering viral entry represents potential antiviral approaches. Ion channels are pore-forming membrane proteins controlling cellular ion homeostasis and regulating many physiological processes, but their roles during viral infection have rarely been explored. Here, the functional Kv1.3 ion channel was found to be expressed in human hepatic cells and tissues. The Kv1.3 was then revealed to restrict HCV entry via inhibiting endosome acidification-mediated viral membrane fusion. The Kv1.3 was also demonstrated to inhibit DENV and ZIKV with an endosome acidification-dependent entry, but have no effect on SeV with a neutral pH penetration. A Kv1.3 antagonist PAP-1 treatment accelerated animal death in ZIKV-infected Ifnar1-/- mice. Moreover, Kv1.3-deletion was found to promote weight loss and reduce survival rate in ZIKV-infected Kv1.3-/- mice. Altogether, the Kv1.3 ion channel behaves as a host factor restricting viral entry. These findings broaden understanding about ion channel biology.