Safety and efficacy of meplazumab in healthy volunteers and COVID-19 patients: a randomized phase 1 and an exploratory phase 2 trial.

Recent evidence suggests that CD147 serves as a novel receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Blocking CD147 via anti-CD147 antibody could suppress the in vitro SARS-CoV-2 replication. Meplazumab is a humanized anti-CD147 IgG2 monoclonal antibody, which may effectively prevent SARS-CoV-2 infection in coronavirus disease 2019 (COVID-19) patients. Here, we conducted a randomized, double-blinded, placebo-controlled phase 1 trial to evaluate the safety, tolerability, and pharmacokinetics of meplazumab in healthy subjects, and an open-labeled, concurrent controlled add-on exploratory phase 2 study to determine the efficacy in COVID-19 patients. In phase 1 study, 59 subjects were enrolled and assigned to eight cohorts, and no serious treatment-emergent adverse event (TEAE) or TEAE grade ≥3 was observed. The serum and peripheral blood Cmax and area under the curve showed non-linear pharmacokinetic characteristics. No obvious relation between the incidence or titer of positive anti-drug antibody and dosage was observed in each cohort. The biodistribution study indicated that meplazumab reached lung tissue and maintained >14 days stable with the lung tissue/cardiac blood-pool ratio ranging from 0.41 to 0.32. In the exploratory phase 2 study, 17 COVID-19 patients were enrolled, and 11 hospitalized patients were involved as concurrent control. The meplazumab treatment significantly improved the discharged (P = 0.005) and case severity (P = 0.021), and reduced the time to virus negative (P = 0.045) in comparison to the control group. These results show a sound safety and tolerance of meplazumab in healthy volunteers and suggest that meplazumab could accelerate the recovery of patients from COVID-19 pneumonia with a favorable safety profile.

Modulation of Tim-3 Expression by Antigen-Dependent and -Independent Factors on T Cells from Patients with Chronic Hepatitis B Virus Infection.

T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) was up-regulated on viral specific T cells and contributed to T cells exhaustion during chronic hepatitis B virus (HBV) infection. However, modulation of Tim-3 expression was still not fully elucidated. To evaluate the potential viral and inflammatory factors involved in the inductor of Tim-3 expression on T cells, 76 patients with chronic HBV infection (including 40 chronic hepatitis B [CHB] and 36 asymptomatic HBV carriers [AsC]) and 40 of normal controls (NCs) were enrolled in this study. Tim-3 expressions on CD4+ and CD8+ T cells were assessed in response to HBV-encoding antigens, HBV peptide pools, and common γ-chain (γc) cytokines stimulation by flow cytometry. HBV peptides and anti-CD3/CD28 directly induced Tim-3 expression on T cells. γc cytokines also drive Tim-3 up-regulations on both CD4+ and CD8+ T cells in patients with chronic HBV infection. However, γc cytokines did not enhance the Tim-3 inductions by either anti-CD3/CD28 or HBV peptides stimulation. Furthermore, γc cytokines-mediated Tim-3 induction could not be abrogated by γc cytokine receptor-neutralizing antibodies. The current results suggested that elevation of Tim-3 expression on T cells could be regulated by both antigen-dependent and -independent manner in patients with chronic HBV infection. The role of γc cytokines in modulation of inhibitory pathway might be evaluated as immunotherapies in humans.

Notch Signaling Contributes to Liver Inflammation by Regulation of Interleukin-22-Producing Cells in Hepatitis B Virus Infection.

The mechanism of hepatitis B virus (HBV) induced liver inflammation is not fully elucidated. Notch signaling augmented interleukin (IL)-22 secretion in CD4+ T cells, and Notch-IL-22 axis fine-tuned inflammatory response. We previously demonstrated a proinflammatory role of IL-22 in HBV infection. Thus, in this study, we analyzed the role of Notch in development of IL-22-producing cells in HBV infection by inhibition of Notch signaling using γ-secretase inhibitor DAPT in both hydrodynamic induced HBV-infected mouse model and in peripheral blood cells isolated from patients with HBV infection. mRNA expressions of Notch1 and Notch2 were significantly increased in livers and CD4+ T cells upon HBV infection. Inhibition of Notch signaling in vivo leaded to the reduction in NKp46+ innate lymphoid cells 22 (ILC22) and lymphoid tissue inducer 4 (LTi4) cells in the liver. This process was accompanied by downregulating the expressions of IL-22 and related proinflammatory cytokines and chemokines in the liver, as well as blocking the recruitment of antigen-non-specific inflammatory cells into the liver and subsequent liver injury, but did not affect HBV antigens production and IL-22 secretion in the serum. Furthermore, IL-22 production in HBV non-specific cultured CD4+ T cells, but not HBV-specific CD4+ T cells, was reduced in response to in vitro inhibition of Notch signaling. In conclusion, Notch siganling appears to be an important mediator of the liver inflammation by modulating hepatic ILC22. The potential proinflammatory effect of Notch-mediated ILC22 may be significant for the development of new therapeutic approaches for treatment of hepatitis B.

Role of Nogo­A in the regulation of hepatocellular carcinoma SMMC­7721 cell apoptosis.

Nogo-A has been identified as an inhibitor of neurite outgrowth specific to the central nervous system. However, little is known about the role of Nogo-A in hepatocellular carcinoma (HCC), the most common primary malignant tumor with a high mortality rate. This study aimed to investigate the role of endogenous Nogo-A in human liver cancer cells. Reverse transcription polymerase chain reaction was used to detect the expression of Nogo-A in four liver cancer cell lines. A lentivirus vector was then constructed to mediate RNA interference (RNAi) targeting of Nogo­A (LV­Nogo-A­siRNA) and was confirmed to successfully suppress the expression of the Nogo-A gene in SMMC-7721 cells. Furthermore, Nogo-A was observed to be highly expressed in liver cancer cell lines. RNAi of Nogo-A using the LV­Nogo-A­siRNA construct significantly decreased Nogo-A protein expression and specifically inhibited the growth of SMMC-7721 cells. This growth inhibitory effect may be attributed to an increase in G2/M phase arrest and apoptosis in SMMC-7721 cells containing Nogo-A­siRNA. The results of this study demonstrate that Nogo-A may represent a novel therapeutic target for the treatment of liver cancer, in addition to its potent roles in neural systems.

Meta-analysis of the short-term effects of lamivudine treatment for severe chronic hepatitis B.

PURPOSE: To evaluate the short-term effect of lamivudine (LMV) treatment for severe chronic hepatitis B. METHOD: Patient data related to the safety and efficacy of using lamivudine (LMV) to treat hepatitis B virus (HBV)-induced liver failure or severe hepatitis were acquired from previous literature. These studies were retrieved from PubMed, Ovid, SpringerLink, Biosis Previews, Academic Search Premier, ProQuest Medical Library, Cochrane Library, China National Knowledge Infrastructure Full-text Database, VIP Chinese Scientific Journal Database, and Chinese Biomedicine. Relative risk and weighted mean difference were used to measure the effects. The major predictors observed included total bilirubin (TBIL), prothrombin activity (PTA), survival rate, and HBV-DNA negative change rate. Groups were further divided according to the clinical course and disease staging. RESULTS: A total of 242 studies were retrieved from the databases. At weeks 4, 8, and 12 of the treatment course, the survival rates and PTA of the test group were distinctively higher than those of the control group. However, TBIL concentrations in the test group were lower than the control group. The HBV-DNA negative change rate was distinctively higher throughout the 12 weeks of LMV treatment. For patients who started LMV treatment in the middle stage, the mortality rate of the test group was lower. For patients who started LMV treatment during the advanced stage, no significant difference was observed between the test and control groups. CONCLUSION: LMV decreased HBV-DNA levels in the serum, improved liver function in patients, and enhanced survival rate during the early and medium stages of severe chronic hepatitis B.

[Generation of six genotypes of infectious HCV pseudo-particles and detection of neutralizing antibodies in HCV patients].

OBJECTIVE: To generate hepatitis C virus pseudo-particles (HCVpp) containing the complete E1-E2 envelope glycoprotein, in order to establish a HCVpp database covering the six major genotypes of HCV (1b, 2a, 3b, 4, 5, and 6) and to develop a simple and effective method for detection of neutralizing antibodies in HCV patients. METHODS: HCVpp were generated for the six genotypes by co-transfecting 293T cells with a plasmid expressing the respective E1-E2 (p HR, CMVA 8.2 construct) and a MLV-GFP plasmid. Titration of each HCVpp was carried out by p24 ELISA. Infectivity of each HCVpp was assessed by mixing the harvested supernatant of producer cells with sera from HCV patients, adding the mixture to Huh-7 cells, and detecting the subsequent titers of neutralizing antibodies against HCVpp. RESULTS: All six types of HCVpp were able to infect Huh-7 cells in vitro. For healthy HCV carriers, only two genotypes of HCVpp (1b and 2a) produced neutralizing antibody titers more than 1:40. For cured HCV patients, only the 1b genotype produced neutralizing antibody titers more than 1:40. One patient showed titer of 1:200 for genotype 4. A healthy spouse of a chronic hepatitis C patient showed titers more than 1:40 for four genotypes of HCVpp (3a, 4, 5, 6). CONCLUSION: We generated six different genotypes of HCVpp successfully, established the in vitro neutralizing antibody detection method, and provided an effective model for screening antiviral drugs.

[Peg-IFNa-2a/RBV antiviral efficacy in cirrhotic hepatitis C patients after splenectomy or partial splenic embolization].

To investigate the antiviral efficacy of combination therapy with pegylated-interferon alpha (peg-IFNa)-2a and ribavirin (RBV) in hepatitis C patients with liver cirrhosis after splenectomy or partial splenic embolization. Forty-nine hepatitis C patients with liver cirrhosis who were unable to use antiviral therapy because of hypersplenism were recruited for study and treated with splenectomy or partial splenic embolization. Three months later, a regimen of antiviral combination therapy was initiated with peg-IFNa-2a (once-weekly subcutaneous injection: 135 µg or 180 µg) and RBV (daily oral: 800 to 1200 mg), and was maintained for 48 weeks. The patients were followed up at treatment weeks 1, 2, 4, 6, 8, and 12. Thereafter, follow-up was conducted every four weeks. The patients were observed until 24 weeks after treatment discontinuation. Follow-up testing included liver function, blood chemistry, renal function, and HCV RNA level. Any adverse reactions were recorded. Liver cirrhosis patients complicated by hypersplenism can be treated effectively with peg-IFNa-2a/RBV combination antiviral therapy after splenectomy or partial splenic embolization. The antiviral-induced sustained viral response rates was 65.00% in cirrhotic/hypersplenic hepatitis C patients receiving splenectomy and 58.62% in those receiving partial splenic embolization. Hypersplenism patients with hepatitis C-related cirrhosis achieved a good antiviral therapeutic effect with peg-IFNa-2a/RBV combination therapy following splenectomy or partial splenic embolization. This sequence of treatment may help to decrease incidences of chronic hepatitis C-induced liver failure and liver cancer in these patients.

Therapeutic effect of RANTES-KDEL on inhibition of HIV-1 in CD34(+) human hematopoietic stem cells (hHSC).

Cell surface receptors, such as the CCR5 chemokine receptors, represent key determinants of the human immunodeficiency virus type 1 (HIV-1) entry into target cells. The CC-chemokine, RANTES (regulated upon activation, normal T-cell expressed and secreted), a ligand for CCR5, have been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL (ER-retention signal) fusion termed RANTES-KDEL and this construct was found to prevent effectively transport of newly synthesized CCR5 to the cell surface. Lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, an HIV-based lentiviral vector expressing RANTES-KDEL, pLenti6/V5-R-K, was constructed and then cotransfected with the ViraPower Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce a replication-incompetent lentivirus stock. The lentiviral stock was titrated using HeLa cells, and the expression of the gene of interest, RANTES, was detected by indirect immunofluorescence. Based on the above results, the lentiviral stock was transduced into CD34(+) human hematopoietic stem cells (hHSC) separated magnetically from the cord blood (the purity was 96.8% evaluated by flow cytometry). Finally, the levels of p24 in the cultures of pLenti6/V5-R-K-transduced CD34(+) hHSC were detected after infection by HIV-1 DP1 (a R5-tropic HIV-1 strain, which was isolated by the Centers for Disease Control and Prevention of China in Henan province in 2000 from a Chinese man who had asymptomatic HIV-1 infection with a history of blood transfusions). It was shown that pLenti6/V5-R-K transduction inhibited expression of the DP1 p24 antigen by 51%, 58% and 60% on the 4th, 7th and 10th day respectively (P<0.05).

[TLR7 protein expression induced by bacterial lipopolysaccharide in DC2.4 cells].

AIM: To investigate the role of bacterial Lipopolysaccharide (LPS) in inducing TLR7 protein expression in DC2.4 cells. METHODS: DC2.4 cells were cultured in RPMI1640 medium supplemented with 100 mL/L FBS. Optical microscope was used to observe the cells morphology before and after LPS induction. RT-PCR and Western blot were employed to analyze the expression of TLR7 at mRNA and protein lever, respectively. RESULTS: Without LPS induction, DC2.4 cells were small and shine with a few dendrites, didn't express TLR7 protein. However, DC2.4 cells become bigger and the dendrites of them become more robust with LPS induction. DC2.4 cells start to express TLR7 protein after 12 hours LPS induction, and there is no much difference on TLR7 protein expression after 12 h, 24 h, 48 h LPS stimulation. CONCLUSION: LPS can induce TLR7 protein expression in DC2.4 cells.

[Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].

OBJECTIVE: To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively. METHODS: The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. RESULTS: Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. CONCLUSION: The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.

A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo.

AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA). METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was transfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccines line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay. RESULTS: Constructive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constructively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus. CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.