Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

Persistent hepatitis B virus and HIV coinfections in dually humanized mice engrafted with human liver and immune system.

Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including human immunodeficiency virus type (HIV) and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV, which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV, which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection, and preclinical testing of novel immunotherapeutics.

Upregulation of the Long Noncoding RNA HULC by Hepatitis C Virus and Its Regulation of Viral Replication.

BACKGROUND: Many long noncoding RNAs (lncRNAs) have important roles in biological processes. The lncRNA HULC was found to be upregulated in human hepatoma tissues. HULC is thought to be involved in multiple steps of hepatoma development and progression; however, the relationship between HULC and hepatitis C virus (HCV) infection, which is a leading cause of hepatoma, remains unclear. METHODS: We examined the effect of HCV replication on HULC expression and the underlying mechanism using cell culture systems. Subsequently, we tested the effect of HULC suppression and overexpression on HCV replication. Finally, we examined the impact of HCV eradication on HULC expression using human liver tissue and blood samples. RESULTS: HCV replication increased HULC expression in cell cultures. A promoter assay showed that an HCV nonstructural protein, NS5A, increased HULC transcription. HULC suppression inhibited HCV replication; conversely, its overexpression enhanced HCV replication. These effects on HCV replication seemed to occur by the modification of HCV translation. Measurements from human liver and blood samples showed that HCV eradication significantly reduced HULC levels in the liver and blood. CONCLUSIONS: HCV infection increases HULC expression in vitro and in vivo. HULC modulates HCV replication through an HCV internal ribosome entry site-directed translation step.

Unexpected Replication Boost by Simeprevir for Simeprevir-Resistant Variants in Genotype 1a Hepatitis C Virus.

Simeprevir is a novel NS3/4A protease inhibitor (PI) of hepatitis C virus (HCV). The baseline polymorphism NS3-Q80K is frequently observed in genotype (GT) 1a HCV and often associated with treatment failure in simeprevir-containing regimens. We aimed to elucidate mechanisms of treatment failure due to NS3-Q80K. We included a Q80R mutation in our study and generated a series of Huh-7.5 cell lines, each of which harbored either wild-type GT 1a strain H77S.3 or the Q80K or Q80R variant. The cells were cultured with increasing concentrations of simeprevir, and NS3 domain sequences were determined. The mutations identified by sequence analyses were subsequently introduced into H77S.3. The sensitivity of each mutant to the NS3/4A PIs simeprevir, asunaprevir, grazoprevir, and paritaprevir was analyzed. We introduced the mutations into GT 1b strain N.2 and compared the sensitivity to simeprevir with that of GT 1a strain H77S.3. While simeprevir treatment selected mutations at residue D168, such as D168A/V in the wild-type virus, an additional mutation at residue R155, R155K, was selected in Q80K/R variants at simeprevir concentrations of <2.5 µM. Sensitivity analyses showed that simeprevir concentrations of <1 µM significantly boosted the replication of Q80K/R R155K variants. Interestingly, this boost was not observed with the other NS3/4A PIs or in Q80R R155Q/G/T/W variants or GT 1b isolates. The boosted replication of the Q80K+R155K variant by simeprevir could be related to treatment failure in simeprevir-containing antiviral treatments in GT 1a HCV-infected patients with the NS3-Q80K polymorphism. This result provides new insight into how resistance-associated variants can cause treatment failure.

Siglec-H-/- Plasmacytoid Dendritic Cells Protects Against Acute Liver Injury by Suppressing IFN-γ/Th1 Response and Promoting IL-21+ CD4 T Cells.

BACKGROUND & AIMS: Siglec-H is a receptor specifically expressed in mouse plasmacytoid dendritic cells (pDCs), which functions as a negative regulator of interferon-α production and plays a critical role in pDC maturation to become antigen-presenting cells. The function of pDCs in autoimmune and inflammatory diseases has been reported. However, the effect of Siglec-H expression in pDCs in liver inflammation and diseases remains unclear. METHODS: Using the model of concanavalin A-induced acute liver injury (ALI), we investigated the Siglec-H/pDCs axis during ALI in BDCA2 transgenic mice and Siglec-H-/- mice. Anti-BDCA2 antibody, anti-interleukin (IL)-21R antibody, and Stat3 inhibitor were used to specifically deplete pDCs, block IL21 receptor, and inhibit Stat3 signaling, respectively. Splenocytes and purified naive CD4 T cells and bone marrow FLT3L-derived pDCs were cocultured and stimulated with phorbol myristate acetate/ionomycin and CD3/CD28 beads, respectively. RESULTS: Data showed that specific depletion of pDCs aggravated concanavalin A-induced ALI. Remarkably, alanine aminotransferase, hyaluronic acid, and proinflammatory cytokines IL6 and tumor necrosis factor-α levels were lower in the blood and liver of Siglec-H knockout mice. This was associated with attenuation of both interferon-γ/Th1 response and Stat1 signaling in the liver of Siglec-H knockout mice while intrahepatic IL21 and Stat3 signaling pathways were upregulated. Blocking IL21R or Stat3 signaling in Siglec-H knockout mice restored concanavalin A-induced ALI. Finally, we observed that the Siglec-H-null pDCs exhibited immature and immunosuppressive phenotypes (CCR9LowCD40Low), resulting in reduction of CD4 T-cell activation and promotion of IL21+CD4 T cells in the liver. CONCLUSIONS: During T-cell-mediated ALI, Siglec-H-null pDCs enhance immune tolerance and promote IL21+CD4 T cells in the liver. Targeting Siglec-H/pDC axis may provide a novel approach to modulate liver inflammation and disease.

Novel Utility of Leucine-Rich Alpha-2-Glycoprotein as a Biomarker in Ulcerative Colitis: A Predictor of Endoscopic Remission Independent of Symptoms.

Introduction: Leucine-rich alpha-2-glycoprotein (LRG) is a potential biomarker for disease activity and reflects mucosal healing in patients with ulcerative colitis (UC). However, only a few studies have described a detailed sensitivity analysis of LRG in predicting mucosal healing in patients. This study aimed to evaluate the association between LRG and the endoscopic activity of UC and its predictability for mucosal healing and explore the utility and clinical application of LRG. Methods: The diagnostic accuracy of biomarkers, including LRG, in predicting the endoscopic activity of UC was evaluated. All consecutive patients who underwent total colonoscopy between April 2021 and September 2022 were included. The Mayo endoscopic subscore (MES) was used for assessing endoscopic activity. Furthermore, endoscopic remission was defined as an MES of ≤1. Clinical activity was evaluated based on stool frequency and bloody stool. Receiver operating characteristic curve analysis and binary logistic regression were performed to assess the diagnostic accuracy of the biomarkers. We evaluated LRG trends and treatment response in patients with MES ≥2 who underwent induction therapy. Results: This study comprised 214 patients. The proportions of endoscopically and clinically active patients were 33.6% and 49.1%, respectively. LRG had an area under the curve (AUC) of 0.856, with a higher diagnostic accuracy than other biomarkers, such as C-reactive protein, leukocyte, neutrophil, platelet, and albumin. The cutoff value for LRG was 15.6 µg/mL (sensitivity, 72.2%; specificity, 86.6%). Using the MES, patients with higher scores had higher LRG levels than those with lower scores. The cutoff value, AUC, sensitivity, and specificity varied with a higher AUC for left-sided colitis and pancolitis than for proctitis. Logistic regression analysis showed that LRG was an independent predictor of endoscopic remission using multivariate analysis, even with the factor of clinical activity. The change ratio of LRG pre- and post-treatment was statistically significant in the higher LRG group. Conclusion: LRG reflected endoscopic activity independently, regardless of clinical symptoms. An LRG below the cutoff value could indicate a significantly low probability of endoscopic activity in asymptomatic patients, and follow-up endoscopy (not for cancer screening) may be unnecessary. Furthermore, a higher LRG level might be more useful as an indicator of treatment efficacy.

Type I interferons and TGF-ß cooperate to induce liver fibrosis during HIV-1 infection under antiretroviral therapy.

Liver diseases have become a major comorbidity health concern for people living with HIV-1 (PLWH) treated with combination antiretroviral therapy (cART). To investigate if HIV-1 infection and cART interact to lead to liver diseases, humanized mice reconstituted with progenitor cells from human fetal livers were infected with HIV-1 and treated with cART. We report here that chronic HIV-1 infection with cART induced hepatitis and liver fibrosis in humanized mice, associated with accumulation of M2-like macrophages (M2LMs), elevated TGF-ß, and IFN signaling in the liver. Interestingly, IFN-I and TGF-ß cooperatively activated human hepatic stellate cells (HepSCs) in vitro. Mechanistically, IFN-I enhanced TGF-ß-induced SMAD2/3 activation in HepSCs. Finally, blockade of IFN-I signaling reversed HIV/cART-induced liver diseases in humanized mice. Consistent with the findings in humanized mice with HIV-1 and cART, we detected elevated markers of liver injury, M2LMs, and of IFN signaling in blood specimens from PLWH compared with those of healthy individuals. These findings identify the IFN-I/M2LM/HepSC axis in HIV/cART-induced liver diseases and suggest that inhibiting IFN-I signaling or M2LM may provide a novel therapeutic strategy for treating HIV/cART-associated liver diseases in PLWH treated with antiretroviral therapy.

Establishment and Characterization of a New Cell Line Permissive for Hepatitis C Virus Infection.

Hepatitis C virus (HCV) cell culture systems have facilitated the development of efficient direct-acting antivirals against HCV. Huh-7.5, a subline of the human hepatoma cell line Huh-7, has been used widely to amplify HCV because HCV can efficiently replicate in these cells due to a defect in innate antiviral signalling. Recently, we established a novel cell line, KH, derived from human hepatocellular carcinoma, which showed atypical uptake of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) in a Gd-EOB-DTPA-enhanced magnetic resonance imaging study. KH cells expressed hepatocyte markers including microRNA-122 (miR-122) at a lower level than Huh-7.5 cells. We demonstrated that KH cells could support the entire life cycle of HCV; however, HCV replicated at a lower rate in KH cells compared to Huh-7.5 cells, and virus particles produced from KH cells seemed to have some disadvantages in viral assembly compared with those produced from Huh-7.5 cells. KH cells had more robust interferon-stimulated gene expression and induction upon HCV RNA transfection, interferon-α2b addition, and HCV infection than Huh-7.5 cells. Interestingly, both miR-122 supplementation and IRF3 knockout in KH cells boosted HCV replication to a similar level as in Huh-7.5 cells, suggesting that intact innate antiviral signalling and lower miR-122 expression limit HCV replication in KH cells. KH cells will enable a deeper understanding of the role of the innate immune response in persistent HCV infection.

Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

Sphingosine-1-phospate is a potent bioactive lipid metabolite that regulates cancer progression. Because sphingosine kinase 1 and sphingosine kinase 2 (SPHK 1/2) are both essential for sphingosine-1-phospate production, they could be a therapeutic target in various cancers. Peretinoin, an acyclic retinoid, inhibits post-therapeutic recurrence of hepatocellular carcinoma via unclear mechanisms. In this study, we assessed effects of peretinoin on SPHK expression and liver cancer development in vitro and in vivo. We examined effects of peretinoin on expression, enzymatic and promoter activity of SPHK1 in a human hepatoma cell line, Huh-7. We also investigated effects of SPHK1 on hepatocarcinogenesis induced by diethylnitrosamine using SPHK1 knockout mice. Peretinoin treatment of Huh-7 cells reduced mRNA levels, protein expression and enzymatic activity of SPHK1. Peretinoin reduced SPHK1 promoter activity; this effect of peretinoin was blocked by overexpression of Sp1, a transcription factor. Deletion of all Sp1 binding sites within the SPHK1 promoter region abolished SPHK1 promoter activity, suggesting that peretinoin reduced mRNA levels of SPHK1 via Sp1. Additionally, diethylnitrosamine-induced hepatoma was fewer and less frequent in SPHK1 knockout compared to wild-type mice. Our data showed crucial roles of SPHK1 in hepatocarcinogenesis and suggests that peretinoin prevents hepatocarcinogenesis by suppressing mRNA levels of SPHK1.

Efficient Suppression of Hepatitis C Virus Replication by Combination Treatment with miR-122 Antagonism and Direct-acting Antivirals in Cell Culture Systems.

Direct-acting antivirals (DAAs) against Hepatitis C virus (HCV) show effective antiviral activity with few side effects. However, the selection of DAA-resistance mutants is a growing problem that needs to be resolved. In contrast, miR-122 antagonism shows extensive antiviral effects among all HCV genotypes and a high barrier to drug resistance. In the present study, we evaluated three DAAs (simeprevir, daclatasvir, and sofosbuvir) in combination with anti-miR-122 treatment against HCV genotype 1a in cell cultures. We found that combination treatments with anti-miR-122 and a DAA had additive or synergistic antiviral effects. The EC50 values of simeprevir in simeprevir-resistant mutants were significantly decreased by combining simeprevir with anti-miR-122. A similar reduction in EC50 in daclatasvir-resistant mutants was achieved by combining daclatasvir with anti-miR-122. Combination treatment in HCV-replicating cells with DAA and anti-miR-122 sharply reduced HCV RNA amounts. Conversely, DAA single treatment with simeprevir or daclatasvir reduced HCV RNA levels initially, but the levels later rebounded. DAA-resistant mutants were less frequently observed in combination treatments than in DAA single treatments. In summary, the addition of miR-122 antagonism to DAA single treatments had additive or synergistic antiviral effects and helped to efficiently suppress HCV replication and the emergence of DAA-resistant mutants.

The acyclic retinoid Peretinoin inhibits hepatitis C virus replication and infectious virus release in vitro.

Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.