Antiviral Immunity Research Articles

Generating prophylactic immunity against arboviruses in vertebrates and invertebrates.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

The effect of probiotic Bifidobacterium lactis Bl-04 on innate antiviral responses invitro

Consumption of certain probiotic strains may be beneficial for reducing the risk of acute upper respiratory tract infections (URTIs), however, underlying immunological mechanisms are elusive. Bifidobacterium lactis Bl-04™ has been reported in humans to significantly reduce the risk of URTIs, affect the innate immunity in the nasal mucosa, and reduce nasal lavage virus titer after a rhinovirus (RV) challenge. To study the immunological mechanisms, we investigated the effect of Bl-04 on cytokine production and transcriptomes of human monocyte-derived macrophages (Mfs) and dendritic cells (DCs), and further on RV replication and cytokine production in MRC-5 fibroblasts. The results showed that Bl-04 modulates antiviral immune responses and potentiates cytokine production during viral challenge mimic in immune cells. However, effect of Bl-04 on RV replication and cytokine production in fibroblasts was negligible. Overall, the findings suggest that Bl-04 mildly stimulates antiviral immunity in Mfs and DCs, and potentially influences viral replication in fibroblasts that however warrants further investigations.

African swine fever virus A137R protein inhibits NF-κB activation via suppression of MyD88 signaling in PK15 and 3D4/21 cells in vitro

African swine fever (ASF) is an infectious disease with high mortality caused by African swine fever virus (ASFV), which poses a great threat to the global swine industry. ASFV has evolved multiple strategies to evade host antiviral innate immunity by perturbing inflammatory responses and interferon production. However, the molecular mechanisms underlying manipulation of inflammatory responses by ASFV proteins are not fully understood. Here, we report that A137R protein of ASFV is a key suppressor of host inflammatory responses. Ectopic expression of ASFV A137R in HEK293T cells significantly inhibited the activation of IL-8 and NF-κB promoters triggered by Sendai virus (SeV), influenza A virus (IAV), or vesicular stomatitis virus (VSV). Accordingly, forced A137R expression caused a significant decrease in the production of several inflammatory cytokines such as IL-8, IL-6 and TNF-α in the cells infected with SeV or IAV. Similar results were obtained from experiments using A137R overexpressing PK15 and 3D4/21 cells infected with SeV or VSV. Furthermore, we observed that A137R impaired the activation of MAPK and NF-κB signaling pathways, as enhanced expression of A137R significantly decreased the phosphorylation of JNK, p38 and p65 respectively upon viral infection (SeV or IAV) and IL-1β treatment. Mechanistically, we found that A137R interacted with MyD88, and dampened MyD88-mediated activation of MAPK and NF-κB signaling. Together, these findings uncover a critical role of A137R in restraining host inflammatory responses, and improve our understanding of complicated mechanisms whereby ASFV evades innate immunity.

Complete Rescue of HTLV-1p12KO Infectivity by Depletion of Monocytes Together with NK and CD8+ T Cells.

The transient depletion of monocytes alone prior to exposure of macaques to HTLV-1 enhances both HTLV-1WT (wild type) and HTLV-1p12KO (Orf-1 knockout) infectivity, but seroconversion to either virus is not sustained over time, suggesting a progressive decrease in virus expression. These results raise the hypotheses that either HTLV-1 persistence depends on a monocyte reservoir or monocyte depletion provides a transient immune evasion benefit. To test these hypotheses, we simultaneously depleted NK cells, CD8+ T cells, and monocytes (triple depletion) prior to exposure to HTLV-1WT or HTLV-1p12KO. Remarkably, triple depletion resulted in exacerbation of infection by both viruses and complete rescue of HTLV-1p12KO infectivity. Following triple depletion, we observed rapid and sustained seroconversion, high titers of antibodies against HTLV-1 p24Gag, and frequent detection of viral DNA in the blood and tissues of all animals when compared with depletion of only CD8+ and NK cells, or monocytes alone. The infection of macaques with HTLV-1WT or HTLV-1p12KO was associated with higher plasma levels of IL-10 after 21 weeks, while IL-6, IFN-γ, IL-18, and IL-1β were only elevated in animals infected with HTLV-1WT. The repeat depletion of monocytes, NK, and CD8+ cells seven months following the first exposure to HTLV-1 did not further exacerbate viral replication. These results underscore the contribution of monocytes in orchestrating anti-viral immunity. Indeed, the absence of orf-1 expression was fully compensated by the simultaneous depletion of CD8+ T cells, NK cells, and monocytes, underlining the primary role of orf-1 in hijacking host immunity.

Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L

RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin leading to the synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis.

Autophagy-enhancing ATG16L1 polymorphism is associated with improved clinical outcome and T-cell immunity in chronic HIV-1 infection

Chronic HIV-1 infection is characterized by T-cell dysregulation that is partly restored by antiretroviral therapy. Autophagy is a critical regulator of T-cell function. Here, we demonstrate a protective role for autophagy in HIV-1 disease pathogenesis. Targeted analysis of genetic variation in core autophagy gene ATG16L1 reveals the previously unidentified rs6861 polymorphism, which correlates functionally with enhanced autophagy and clinically with improved survival of untreated HIV-1-infected individuals. T-cells carrying ATG16L1 rs6861(TT) genotype display improved antiviral immunity, evidenced by increased proliferation, revamped immune responsiveness, and suppressed exhaustion/immunosenescence features. In-depth flow-cytometric and transcriptional profiling reveal T-helper-cell-signatures unique to rs6861(TT) individuals with enriched regulation of pro-inflammatory networks and skewing towards immunoregulatory phenotype. Therapeutic enhancement of autophagy recapitulates the rs6861(TT)-associated T-cell traits in non-carriers. These data underscore the in vivo relevance of autophagy for longer-lasting T-cell-mediated HIV-1 control, with implications towards development of host-directed antivirals targeting autophagy to restore immune function in chronic HIV-1 infection.

Fangchinoline induces antiviral response by suppressing STING degradation

The stimulator of interferon genes (STING), an integral adaptor protein in the DNA-sensing pathway, plays a pivotal role in the innate immune response against infections. Additionally, it presents a valuable therapeutic target for infectious diseases and cancer. We observed that fangchinoline (Fan), a bis-benzylisoquinoline alkaloid (BBA), effectively impedes the replication of vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), influenza A virus (H1N1), and herpes simplex virus-1 (HSV-1) in vitro. Fan treatment significantly reduced the viral load, attenuated tissue inflammation, and improved survival in a viral sepsis mouse model. Mechanistically, Fan activates the antiviral response in a STING-dependent manner, leading to increased expression of interferon (IFN) and interferon-stimulated genes (ISGs) for potent antiviral effects in vivo and in vitro. Notably, Fan interacts with STING, preventing its degradation and thereby extending the activation of IFN-based antiviral responses. Collectively, our findings highlight the potential of Fan, which elicits antiviral immunity by suppressing STING degradation, as a promising candidate for antiviral therapy.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Insights into the modulation of bacterial NADase activity by phage proteins

The Silent Information Regulator 2 (SIR2) protein is widely implicated in antiviral response by depleting the cellular metabolite NAD+. The defense-associated sirtuin 2 (DSR2) effector, a SIR2 domain-containing protein, protects bacteria from phage infection by depleting NAD+, while an anti-DSR2 protein (DSR anti-defense 1, DSAD1) is employed by some phages to evade this host defense. The NADase activity of DSR2 is unleashed by recognizing the phage tail tube protein (TTP). However, the activation and inhibition mechanisms of DSR2 are unclear. Here, we determine the cryo-EM structures of DSR2 in multiple states. DSR2 is arranged as a dimer of dimers, which is facilitated by the tetramerization of SIR2 domains. Moreover, the DSR2 assembly is essential for activating the NADase function. The activator TTP binding would trigger the opening of the catalytic pocket and the decoupling of the N-terminal SIR2 domain from the C-terminal domain (CTD) of DSR2. Importantly, we further show that the activation mechanism is conserved among other SIR2-dependent anti-phage systems. Interestingly, the inhibitor DSAD1 mimics TTP to trap DSR2, thus occupying the TTP-binding pocket and inhibiting the NADase function. Together, our results provide molecular insights into the regulatory mechanism of SIR2-dependent NAD+ depletion in antiviral immunity.

Evaluation the frequencies of HLA alleles in moderate and severe COVID-19 patients in Iran: A molecular HLA typing study

BackgroundSevere acute respiratory syndrome coronavirus 2 was first reported in December 2019 and it has spread globally ever since. The HLA system is crucial in directing anti-viral immunity and recent studies are investigating the possible involvement of the HLA genes on the severity of immune inflammation in different phases of COVID-19. MethodsIn this cross-sectional study, peripheral blood-extracted genomic DNAs of 109 COVID-19 patients and 70 healthy controls were genotyped for different alleles of HLA-A, HLA-B, and HLA-DRB1 loci using sequence-specific primer PCR method. ResultsThe results indicated that frequencies of HLA-DRB1*11:01 and HLA-DRB1*04:03 were significantly higher in severe patients rather than moderates (p: <0.001 and 0.004, respectively). Also, it was observed that HLA-DRB1*04:01 was more frequent in moderate patients and healthy controls (p:0.002). In addition, HLA-B*07:35, and HLA-DRB1*07:01 showed higher frequencies in patients compared with controls (p: 0.031 and 0.003 respectively). Inversely, due to the higher frequencies of HLA-B*51:01 (p:0.027), HLA-DRB1*11:05 (p:0.003), HLA-DRB1*13:05 (p:0.022), and HLA-DRB1*14:01 (p:0.006) in healthy individuals rather than patients, they may be associated with COVID-19 resistance. ConclusionThe results show that, based on the population differences, the type of alleles related to the severity of COVID-19 is different, which should be clarified by designing large-scale studies in order to develop HLA-based treatments and vaccines.

Grass carp (Ctenopharyngodon idella) SIRT3 enhances MAVS-mediated antiviral innate immunity in response to GCRV infection

Grass carp (Ctenopharyngodon idella) is a widely cultivated freshwater fish in the world, which occupies a leading position in China's aquaculture industry. However, the outbreak of hemorrhagic disease resulted from grass carp reovirus (GCRV) infection causes tremendous loss in grass carp production. In this study, we cloned the cDNA of grass carp SIRT3 (gc-SIRT3) and found that it is highly conserved across species and abundantly expressed in grass carp liver. Upon GCRV infection, the expression of gc-SIRT3 is dynamically changed. In addition, grass carp SIRT3 positively regulates the expression of antiviral genes depending on its enzymatic activity. Mechanistically, gc-SIRT3 interacts with gc-MAVS to enhance the MAVS-mediated antiviral response. Furthermore, SIRT3 inhibition in zebrafish or grass carp larvae with the specific inhibitor, 3-TYP, attenuates the antiviral ability against GCRV infection. Our findings may provide clues for understanding the function of SIRT3 in antiviral innate immunity.

Insights into the mechanism of action of pterostilbene against influenza A virus-induced acute lung injury

BackgroundSevere respiratory system illness caused by influenza A virus infection is associated with excessive inflammation and abnormal apoptosis in alveolar epithelial cells (AEC). However, there are limited therapeutic options for influenza-associated lung inflammation and apoptosis. Pterostilbene (PTE, trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol that has been reported to limit influenza A virus infection by promoting antiviral innate immunity, but has not been studied for its protective effects on virus-associated inflammation and injury in AEC. PurposeOur study aimed to investigate the protective effects and underlying mechanisms of PTE in modulating inflammation and apoptosis in AEC, as well as its effects on macrophage polarization during influenza virus infection. Study design and methodsA murine model of influenza A virus-mediated acute lung injury was established by intranasal inoculation with 5LD50 of mouse-adapted H1N1 viruses. Hematoxylin and eosin staining, immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, Luminex and flow cytometry were performed. ResultsPTE effectively mitigated lung histopathological changes and injury induced by H1N1 viruses in vivo. These beneficial effects of PTE were attributed to the suppression of inflammation and apoptosis in AEC, as well as the modulation of M1 macrophage polarization. Mechanistic investigations revealed that PTE activated the phosphorylated AMP-activated protein kinase alpha (P-AMPKα)/sirtui1 (Sirt1)/PPARγ coactivator 1-alpha (PGC1α) signal axis, leading to the inhibition of nuclear factor kappa-B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling induced by H1N1 viruses, thereby attenuating inflammation and apoptosis in AEC. PTE also forced activation of the P-AMPKα/Sirt1/PGC1α signal axis in RAW264.7 cells, counteracting the activation of phosphorylated signal transducer and activator of transcription 1 (P-STAT1) induced by H1N1 viruses and the augment of P-STAT1 activation in RAW264.7 cells with interferon-gamma (IFN-γ) pretreatment before viral infection, thereby reducing H1N1 virus-mediated M1 macrophage polarization as well as the enhancement of macrophages into M1 phenotypes elicited by IFN-γ pretreatment. Additionally, the promotion of the transition of macrophages towards the M2 phenotype by PTE was also related to activation of the P-AMPKα/Sirt1/PGC1α signal axis. Moreover, co-culturing non-infected AEC with H1N1 virus-infected RAW264.7 cells in the presence of PTE inhibited apoptosis and tight junction disruption, which was attributed to the suppression of pro-inflammatory mediators and pro-apoptotic factors in an AMPKα-dependent manner. ConclusionIn conclusion, our findings suggest that PTE may serve as a promising novel therapeutic option for treating influenza-associated lung injury. Its ability to suppress inflammation and apoptosis in AEC, modulate macrophage polarization, and preserve alveolar epithelial cell integrity highlights its potential as a therapeutic agent in influenza diseases.

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication

ABSTRACT Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host’s antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-β promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection. Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4’,6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-β:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2

Prior infection with unrelated neurotropic virus exacerbates influenza disease and impairs lung T cell responses

Immunity to infectious diseases is predominantly studied by measuring immune responses towards a single pathogen, although co-infections are common. In-depth mechanisms on how co-infections impact anti-viral immunity are lacking, but are highly relevant to treatment and prevention. We established a mouse model of co-infection with unrelated viruses, influenza A (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems. SFV infection eight days before IAV infection results in prolonged IAV replication, elevated cytokine/chemokine levels and exacerbated lung pathology. This is associated with impaired lung IAV-specific CD8+ T cell responses, stemming from suboptimal CD8+ T cell activation and proliferation in draining lymph nodes, and dendritic cell paralysis. Prior SFV infection leads to increased blood brain barrier permeability and presence of IAV RNA in brain, associated with increased trafficking of IAV-specific CD8+ T cells and establishment of long-term tissue-resident memory. Relative to lung IAV-specific CD8+ T cells, brain memory IAV-specific CD8+ T cells have increased TCR repertoire diversity within immunodominant DbNP366+CD8+ and DbPA224+CD8+ responses, featuring suboptimal TCR clonotypes. Overall, our study demonstrates that infection with an unrelated neurotropic virus perturbs IAV-specific immune responses and exacerbates IAV disease. Our work provides key insights into therapy and vaccine regimens directed against unrelated pathogens.

A Variety of Mouse PYHIN Proteins Restrict Murine and Human Retroviruses.

PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.

Abstract 1190: NK cell subsets as potential correlates of antitumor immunity in HPV+ cancers

Abstract High-risk type human papillomaviruses (HPV) are associated with genital and oral cancers, and the incidence of head and neck squamous cell cancers is fast increasing in the USA and worldwide. Survival rates for patients with locally advanced disease are poor and variable after standard of care (SOC) chemoradiation treatment (CRT). Identifying the antitumor host immune mediators important for treatment response and designing strategies to promote them are essential for improving clinical outcome. Antitumor immunity comprises of adaptive and innate immune effectors. The immediate innate immune response by natural killer (NK) cells is a critical component for antitumor efficacy. The effector function of NK cells is modulated by various receptors via a wide array of activation or inhibitory signals. Among these receptors is HLA-DR, a recognized activation marker on CD8 T and NK cells. Literature studies refer to HLA-DR+ NK cells as pre-mNK. Preclinical data demonstrated therapeutic vaccine mediated induction of pre-mNK equivalent to contribute to tumor-free long-term survival. In our studies with clinical samples from HPV+ cancer patients, we’ve observed a dysfunctional phenotype of circulating pre-mNK when compared to healthy donors, demonstrated by flow cytometry methods. The correlates for dysfunction include increased PD-1 expression, a known inhibitory receptor, and reduced CD16, an NK receptor for Antibody Dependent Cellular Cytotoxicity (ADCC). Further, we observed circulating pre-mNK cells in HPV+ head and neck patients gain functionality post-treatment, as shown by significant increases in granzyme B and CD16, both known contributors for NK cytotoxicity. In cervical cancer patients, we had the opportunity to analyze the tumor microenvironment (TME). Interestingly, we found a significant increased frequency of cytotoxic pre-mNK cells, shown by granzyme B, in the TME that was not present in circulation. Overall, pre-mNK represent a uniquely activated immune subset for antiviral and antitumor immunity. Citation Format: Madison O'Hara, Jagannadha Sastry. NK cell subsets as potential correlates of antitumor immunity in HPV+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1190.

Abstract 2470: Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy

Abstract Immunotherapy has had very limited success in pancreatic cancer, due to its low mutational burden and immunosuppressive microenvironment. Our approach consists in redirecting pre-existing antiviral immunity against pancreatic tumors by delivering viral antigens using the tumor penetrating peptide iRGD .This peptide, targets the tumor vasculature through αv integrins and neuropilin-1, delivering conjugated or co-administered cargo to tumors. Here we used mouse cytomegalovirus (CMV) as an infection model. CMV is a β-herpesvirus that induces a strong T-cell response in mice and humans, comprising &amp;gt;10% of all circulating CD4 and CD8 T cells mostly with effector-memory phenotype. Importantly, human CMV infects over 60% of the world’s population rendering it a suitable candidate for translation. Mice latently infected with CMV were orthotopically implanted with KPC pancreatic tumor cells and treated with systemic injections of vehicle or iRGD plus CMV class I and II binding peptides. Another cohort of age matched uninfected mice was used as a control. Tumor growth and mouse weight were monitored twice a week and CMV specific immune responses were measured in spleen and tumor by flow cytometry, using tetramer staining and CMV specific peptide recall. CMV infected mice receiving iRGD + CMV peptides responded to treatment as evidenced by delayed tumor growth associated with increased tumor necrosis and T cell infiltration. In addition, flow cytometry analysis of tumor and spleen showed a significant increase in CMV specific T cells that produced significant amounts of IFN-γ and TNF-α upon CMV peptide recall. Furthermore, tumor regulatory T cells (Treg) were significantly reduced and the CD8/T reg ratio was greatly increased. Survival studies showed a 68% increase in median survival in the treated and infected group. Combining this strategy with anti-PD1 and or anti-IL10R did not significantly improve the benefits of iRGD + CMV therapy alone. Other combinations with chemotherapy and TME remodeling agents are being tested.Taken together, this data shows we can deliver antigens to pancreatic tumors via iRGD and elicit an anti-tumor immune response that results in delayed tumor growth and has a survival benefit. This is a mutation agnostic approach with high translational value, since more than 60% of the population has previous CMV immunity and iRGD in combination with chemotherapy is already in phase II clinical trials for advanced pancreatic cancer. Citation Format: Remi Marrocco, Jay Patel, Siming Sun, Rithika Medari, Kevin Gulay, Eduardo Lucero-Meza, Evangeline Mose, Andrew Lowy, Chris Benedict, Tatiana Hurtado de Mendoza. Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2470.

Platycodin D facilitates antiviral immunity through inhibiting cytokine storm via targeting K63-linked TRAF6 ubiquitination.

Influenza virus infection is a worldwide challenge that causes heavy burdens on public health. The mortality rate of severe influenza patients is often associated with hyperactive immunological abnormalities characterized by hypercytokinemia. Due to the continuous mutations and the occurrence of drug-resistant influenza virus strains, the development of host-directed immunoregulatory drugs is urgently required. Platycodon grandiflorum is among the top 10 herbs of traditional Chinese medicine used to treat pulmonary diseases. As one of the major terpenoid saponins extracted from Platycodon grandiflorum, Platycodin D (PD) has been reported to play several roles, including anti-inflammation, analgesia, anti-cancer, hepatoprotection, and immunoregulation. However, the therapeutic roles of PD to treat influenza virus infection remains unknown. Here, we show that PD can protect the body weight loss in severely infected influenza mice, alleviate lung damage, and thus improve the survival rate. More specifically, PD protects flu mice via decreasing the immune cell infiltration into lungs and downregulating the overactivated inflammatory response. Western blot and immunofluorescence assays exhibited that PD could inhibit the activation of TAK1/IKK/NF-κB and MAPK pathways. Besides that, CETSA, SPR and immunoprecipitation assays indicated that PD binds with TRAF6 to decrease its K63 ubiquitination after R837 stimulation. Additionally, siRNA interference experiments exhibited that PD could inhibit the secretion of IL-1β and TNF-α in TRAF6-dependent manner. Altogether, our results suggested that PD is a promising drug candidate for treating influenza. Our study also offered a scientific explanation for the commonly used Platycodon grandiflorum in many anti-epidemic classic formulas. Due to its host-directed regulatory role, PD may serve as an adjuvant therapeutic drug in conjunction with other antiviral drugs to treat the flu.

USP46 promotes the interferon antiviral signaling in black carp by deubiquitinating TBK1

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN, PKR, and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates.