Long COVID After Bamlanivimab Treatment.

BACKGROUND: Prospective evaluations of long COVID in outpatients with coronavirus disease 2019 (COVID-19) are lacking. We aimed to determine the frequency and predictors of long COVID after treatment with the monoclonal antibody bamlanivimab in ACTIV-2/A5401. METHODS: Data were analyzed from participants who received bamlanivimab 700 mg in ACTIV-2 from October 2020 to February 2021. Long COVID was defined as the presence of self-assessed COVID symptoms at week 24. Self-assessed return to pre-COVID health was also examined. Associations were assessed by regression models. RESULTS: Among 506 participants, median age was 51 years. Half were female, 5% Black/African American, and 36% Hispanic/Latino. At 24 weeks, 18% reported long COVID and 15% had not returned to pre-COVID health. Smoking (adjusted risk ratio [aRR], 2.41 [95% confidence interval {CI}, 1.34- 4.32]), female sex (aRR, 1.91 [95% CI, 1.28-2.85]), non-Hispanic ethnicity (aRR, 1.92 [95% CI, 1.19-3.13]), and presence of symptoms 22-28 days posttreatment (aRR, 2.70 [95% CI, 1.63-4.46]) were associated with long COVID, but nasal severe acute respiratory syndrome coronavirus 2 RNA was not. CONCLUSIONS: Long COVID occurred despite early, effective monoclonal antibody therapy and was associated with smoking, female sex, and non-Hispanic ethnicity, but not viral burden. The strong association between symptoms 22-28 days after treatment and long COVID suggests that processes of long COVID start early and may need early intervention. CLINICAL TRIALS REGISTRATION: NCT04518410.

Preventing Infectious Complications of Immunomodulation in COVID-19 in Foreign-Born Patients.

Immunomodulating therapies for COVID-19 may carry risks of reactivating latent infections in foreign-born people. We conducted a rapid review of infection-related complications of immunomodulatory therapies for COVID-19. We convened a committee of specialists to formulate a screening and management strategy for latent infections in our setting. Dexamethasone, used in severe COVID-19, is associated with reactivation of latent tuberculosis, hepatitis B, and dissemination/hyperinfection of Strongyloides species and should prompt screening and/ or empiric treatment in appropriate epidemiologic contexts. Other immunomodulators used in COVID-19 may also increase risk, including interleukin-6 receptor antagonist (e.g., tocilizumab) and kinase inhibitors. People with specific risk factors should also be screened for HIV, Chagas disease, and endemic mycoses. Racial and ethnic minorities in North America, including foreign-born persons, who receive immunomodulating agents for COVID-19 may be at risk for reactivation of latent infections. We develop a screening and management pathway for such patients.

SARS-CoV-2 viral load is associated with increased disease severity and mortality.

The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). Here, we quantify SARS-CoV-2 viral load from participants with a diverse range of COVID-19 disease severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. We detected SARS-CoV-2 plasma RNA in 27% of hospitalized participants, and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, we report that a higher prevalence of detectable SARS-CoV-2 plasma viral load is associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, are associated with increased risk of mortality. Our data show that SARS-CoV-2 viral loads may aid in the risk stratification of patients with COVID-19, and therefore its role in disease pathogenesis should be further explored.

On the Road to a HIV Cure: Moving Beyond Berlin and London.

The Berlin patient, a famous example for human immunodeficiency virus (HIV) cure, had received a bone marrow transplantation with an HIV resistance mutation. The authors describe his case and others that had shown HIV control, like the Mississippi baby who was started on antiretroviral therapy very early after birth, and posttreatment controllers, like the VISCONTI cohort. Moreover, the authors outline various strategies, oftentimes informed by these individuals, that have been tried in vitro, in animal models, or in human trials, to deplete the latent reservoir, which is considered the basis of HIV persistence and the obstacle to cure.

HCV induces transforming growth factor ß1 through activation of endoplasmic reticulum stress and the unfolded protein response.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-ß1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-ß1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-ß1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-ß1 expression. ROS, ER stress, NFκB, and TGF-ß1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-ß1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-ß1 activation. We conclude that HCV activates NFκB and TGF-ß1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-ß1.

The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing.

BACKGROUND &/AIMS: The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. METHODS: We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. RESULTS: We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. CONCLUSIONS: The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Entry inhibitors and future treatment of hepatitis C.

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Furthermore, HCV-induced liver disease is the leading indication for liver transplantation. The recent introduction of direct-acting antivirals (DAAs) has revolutionized HCV treatment by making possible the cure of the majority of patients. However, their efficacy and safety in difficult-to-treat patients such as patients receiving immunosuppression, those with advanced liver disease, co-morbidity and HIV/HCV-co-infection remain to be determined. Furthermore, prevention of liver graft infection remains a pressing issue. HCV entry inhibitors target the very first step of the HCV life cycle and efficiently inhibit cell-cell transmission - a key prerequisite for viral spread. Because of their unique mechanism of action on cell-cell transmission they may provide a promising and simple perspective for prevention of liver graft infection. A high genetic barrier to resistance and complementary mechanism of action compared to DAAs makes entry inhibitors attractive as a new strategy for treatment of multi-resistant or difficult-to-treat patients. Clinical studies are needed to determine the future role of entry inhibitors in the arsenal of antivirals to combat HCV infection. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication."

Kinetic differences in the induction of interferon stimulated genes by interferon-α and interleukin 28B are altered by infection with hepatitis C virus.

UNLABELLED: Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 hours of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV-infected cells the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B. CONCLUSION: The kinetics of gene induction are fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes, suggesting distinct roles of these cytokines within the immune response. Furthermore, the observed differences are substantially altered by infection with HCV.

The AMPK-related kinase SNARK regulates hepatitis C virus replication and pathogenesis through enhancement of TGF-ß signaling.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis. METHODS: The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-ß signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression. RESULTS: Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-ß signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-ß signaling. CONCLUSIONS: Thus reciprocal regulation between HCV and SNARK promotes TGF-ß signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs.

A genetic screen identifies interferon-α effector genes required to suppress hepatitis C virus replication.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach. METHODS: We performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further. RESULTS: The screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, ß 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, ß isoform; peroxisomal proliferator-activated receptor-γ-DBD-interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα-mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this screen (92%) were not transcriptionally stimulated by IFNα; these genes represent a heretofore unknown class of non-IFN-stimulated gene IEGs. CONCLUSIONS: We performed a whole-genome loss-of-function screen to identify genes that mediate the effects of IFNα against human pathogenic viruses. We found that IFNα restricts HCV via actions of general and specific IEGs.

A functional genomic screen reveals novel host genes that mediate interferon-alpha's effects against hepatitis C virus.

BACKGROUND & AIMS: The precise mechanisms by which IFN exerts its antiviral effect against HCV have not yet been elucidated. We sought to identify host genes that mediate the antiviral effect of IFN-α by conducting a whole-genome siRNA library screen. METHODS: High throughput screening was performed using an HCV genotype 1b replicon, pRep-Feo. Those pools with replicate robust Z scores ≥2.0 entered secondary validation in full-length OR6 replicon cells. Huh7.5.1 cells infected with JFH1 were then used to validate the rescue efficacy of selected genes for HCV replication under IFN-α treatment. RESULTS: We identified and confirmed 93 human genes involved in the IFN-α anti-HCV effect using a whole-genome siRNA library. Gene ontology analysis revealed that mRNA processing (23 genes, p=2.756e-22), translation initiation (nine genes, p=2.42e-6), and IFN signaling (five genes, p=1.00e-3) were the most enriched functional groups. Nine genes were components of U4/U6.U5 tri-snRNP. We confirmed that silencing squamous cell carcinoma antigen recognized by T cells (SART1), a specific factor of tri-snRNP, abrogates IFN-α's suppressive effects against HCV in both replicon cells and JFH1 infectious cells. We further found that SART1 was not IFN-α inducible, and its anti-HCV effector in the JFH1 infectious model was through regulation of interferon stimulated genes (ISGs) with or without IFN-α. CONCLUSIONS: We identified 93 genes that mediate the anti-HCV effect of IFN-α through genome-wide siRNA screening; 23 and nine genes were involved in mRNA processing and translation initiation, respectively. These findings reveal an unexpected role for mRNA processing in generation of the antiviral state, and suggest a new avenue for therapeutic development in HCV.

HIV infection increases HCV-induced hepatocyte apoptosis.

BACKGROUND & AIMS: HCV related liver disease is one of the most important complications in persons with HIV, with accelerated fibrosis progression in coinfected persons compared to those with HCV alone. We hypothesized that HCV-HIV coinfection increases HCV related hepatocyte apoptosis and that HCV and HIV influence TRAIL signaling in hepatocytes. METHODS: We analyzed the effect of HIV in JFH1-infected Huh7.5.1 cells. Apoptosis was measured by Caspase-Glo 3/7 assay and Western blotting for cleaved PARP. TRAIL, TRAIL receptor 1 (DR4), and 2 (DR5) mRNA and protein levels were assessed by real-time PCR and Western blot, respectively. We also investigated activation of caspase pathways using caspase inhibitors and assessed expression of Bid and cytochrome C. RESULTS: We found increased caspase 3/7 activity and cleaved PARP in JFH1 HCV-infected Huh7.5.1 cells in the presence of heat-inactivated HIV, compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Both DR4 and DR5 mRNA and protein expression were increased in JFH1-infected cells in the presence of inactivated HIV compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Pancaspase, caspase-8, and caspase-9 inhibition blocked apoptosis induced by HCV, inactivated HIV, and HCV plus inactivated HIV. A caspase-9 inhibitor blocked apoptosis induced by HCV, HIV, and HCV-HIV comparably to pancaspase and caspase-8 inhibitors. HCV induced the activation of Bid cleavage and cytochrome C release. The addition of HIV substantially augmented this induction. CONCLUSIONS: Our findings indicate that hepatocyte apoptosis is increased in the presence of HCV and HIV compared to HCV or HIV alone, and that this increase is mediated by DR4 and DR5 up-regulation. These results provide an additional mechanism for the accelerated liver disease progression observed in HCV-HIV co-infection.

HIV and HCV cooperatively promote hepatic fibrogenesis via induction of reactive oxygen species and NFkappaB.

HIV/HCV coinfection leads to accelerated hepatic fibrosis progression, with higher rates of cirrhosis, liver failure, and liver death than does HCV mono-infection. However, the profibrogenic role of HIV on hepatocytes and hepatic stellate cells (HSC) has not been fully clarified. We hypothesized that HIV, HCV induce liver fibrosis through altered regulation of the production of extracellular matrix and matrix metalloproteinases. We examined the fibrogenesis- and fibrolysis-related gene activity in LX2 HSC and Huh7.5.1 cells in the presence of inactivated CXCR4 and CCR5 HIV, as well as HCV JFH1 virus. The role of reactive oxygen species (ROS) upon fibrosis gene expression was assessed using the ROS inhibitor. Fibrosis-related transcripts including procollagen α1(I) (CoL1A), TIMP1, and MMP3 mRNA were measured by qPCR. TIMP1 and MMP3 protein expression were assessed by ELISA. We found that inactivated CXCR4 HIV and CCR5 HIV increased CoL1A, and TIMP1 expression in both HSC and Huh7.5.1 cells; the addition of JFH1 HCV further increased CoL1A and TIMP1 expression. CXCR4 HIV and CCR5 HIV induced ROS production in HSC and Huh7.5.1 cells which was further enhanced by JFH1 HCV. The ROS inhibitor DPI abrogated HIV-and HCV-induced CoL1A and TIMP1 expression. HIV and HCV-induced CoL1A and TIMP1 expression were also blocked by NFκB siRNA. Our data provide further evidence that HIV and HCV independently regulate hepatic fibrosis progression through the generation of ROS; this regulation occurs in an NFκB-dependent fashion. Strategies to limit the viral induction of oxidative stress are warranted to inhibit fibrogenesis.

Toll-like receptor 2 senses hepatitis C virus core protein but not infectious viral particles.

Toll-like receptors (TLRs) are pathogen recognition molecules activating the innate immune system. Cell surface expressed TLRs, such as TLR2 and TLR4, have been shown to play an important role in human host defenses against viruses through sensing of viral structural proteins. In this study, we aimed to elucidate whether TLR2 and TLR4 participate in inducing antiviral immunity against hepatitis C virus (HCV) by sensing viral structural proteins. We studied TLR2 and TLR4 activation by cell culture-derived infectious virions and serum-derived virions in comparison to purified recombinant HCV structural proteins and enveloped virus-like particles. Incubation of TLR2 or TLR4 transfected cell lines with recombinant core protein resulted in activation of TLR2-dependent signaling. In contrast, neither infectious virions nor enveloped HCV-like particles triggered TLR2 and TLR4 signaling. These findings suggest that monomeric HCV core protein but not intact infectious particles are sensed by TLR2. Impairment of interaction between TLR and the core in infectious viral particles may contribute to escape from innate antiviral immune responses.

Detection of IKKbeta-IKKgamma subcomplexes in monocytic cells and characterization of associated signaling.

The IkappaB kinase (IKK) complex is one major step in the regulation of the NF-kappaB/Rel system that is involved in inflammatory and immune responses as well as in proliferation and apoptosis. At present it is not clear whether besides the "classical" IKKalpha-IKKbeta-IKKgamma configuration additional complexes exist in vivo that solely contain IKKbeta and IKKgamma (without IKKalpha). In the current study we were able to demonstrate in monocytic cells that endogenous complexes, which only include IKKbeta as the kinase-active molecule do indeed exist in vivo and that these complexes contain IKKgamma as an additional component. Furthermore, we showed that these IKKbeta-IKKgamma complexes are involved in mainstream NF-kappaB activation cascades because they can be activated by tumor necrosis factor. In contrast, these subcomplexes appear not to participate in NIK-dependent pathways. As a next step we showed that exogenous IKKbeta-IKKgamma complexes can be formed in an intact cell by overexpression and that these artificial complexes fulfill the requirement for participation in regular signaling. Finally, in the absence of IKKalpha we found a retarded proteolysis of IkappaBalpha, but not of IkappaB in, which is associated with a reduced IKK activity. Differential pathways represented by various IKK subcomplexes may open attractive possibilities in treatment of inflammation or cancer allowing specific therapeutic intervention.

Involvement of NF-kappaB signalling in skin physiology and disease.

Transcription factors of the nuclear factor-kappaB (NF-kappaB)/Rel family play a crucial role in gene regulation during a variety of different cellular processes. This review focuses on the increasing knowledge of the role of NF-kappaB in skin physiology and pathology. Several studies demonstrate that NF-kappaB, or components of the system such as IkappaB kinase (IKK)-alpha, seem to be involved in epidermal development and differentiation. Furthermore, a dysregulation of NF-kappaB is suggested to play an important role in skin pathology, including proliferative disorders, e.g. psoriasis, inflammatory processes such as incontinentia pigmenti (IP), sunburn, Lyme disease, allergic contact dermatitis and autoimmune diseases, as well as also in skin carcinogenesis. However, although the knowledge concerning the role of NF-kappaB in the homeostasis of the skin is steadily increasing, many more questions need to be answered.