BackgroundPRO 140 is a humanized monoclonal antibody targeting CCR5 with potent antiviral activity in patients with CCR5-tropic HIV-1 infection. In phase 2b studies, we evaluated the long-term efficacy, safety, and tolerability of PRO 140 monotherapy in maintaining viral suppression for over 24 months in patients who were stable on combination antiretroviral therapy on entry into the trials.Methods and ResultsForty-one adult patients, infected exclusively with CCR5-tropic HIV-1 with viral loads <50 copies/mL, were switched from daily oral combination ART regimens to weekly PRO 140 monotherapy for 12 weeks.Participants who completed 12 weeks of treatment without experiencing virologic rebound were allowed to self-administer PRO 140 as a 350 mg subcutaneous injection weekly, for up to an additional 160 weeks. Participants were monitored bi-weekly for one year, and every four weeks thereafter for virologic rebound.PRO 140 provided virologic suppression in 23/41 (56.1%) participants for 12 weeks and was well tolerated. Ten (10) participants are currently ongoing, of which nine participants have completed more than two years of monotherapy treatment (47–129 weeks). Participants experiencing virologic rebound achieved full viral suppression upon re-initiation of oral combination ART regimen. Anti-PRO 140 antibodies were not detected in any patient, and no drug-related major adverse events or treatment discontinuations were reported.ConclusionsPRO 140 has a potential to address an unmet need for a long-acting, single-agent, maintenance regimen for HIV infection in selected patients. Studies are underway to determine host and/or virologic factors that may predict treatment success on PRO 140 monotherapy. Moreover, it has sufficient potency for a prolonged period of monotherapy that it would be an excellent component of a multi long-acting drug combination.

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection.

The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

Green assay.Permissiveness of these cells for HCV RNA replication and virus production was assessed by transfection of the cells with a luciferase reporter virus genome.The frequency of all SNPs was investigated in vivo by direct sequencing of DNA from 120 healthy controls.Results: Two out of six SNPs in the coding region of human CypA were associated with lower efficiency of HCV RNA replication and virus production in vitro while all other SNPs had no influence on HCV RNA replication.The two SNPs, that reduce HCV RNA replication, are located near the active site of cyclophilin A and also near the binding site of cyclosporine A and alisporivir.However, both of these SNPs were very rare in our Caucasian population (allele frequency <1%).Further work investigating a confirmatory cohort and cohorts of other ethnic origins is ongoing.Conclusion: These results highlight the important role of CypA for HCV and indicate that SNPs in human cyclophilin A may play an important role in vitro and also in vivo.

BACKGROUND:PRO 140 is a humanized CCR5 monoclonal antibody that has demonstrated potent antiviral activity when it is administered intravenously to adults infected with CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1). This study is the first to evaluate subcutaneous administration. METHODS:A randomized, double-blind, placebo-controlled study was conducted among 44 subjects with HIV-1 RNA levels of >5000 copies/mL, CD4(+) cell counts of >300 cells/microL, no receipt of antiretroviral therapy for >or=12 weeks, and only R5 HIV-1 detectable. Subjects received placebo, 162 mg of PRO 140, or 324 mg of PRO 140 weekly for 3 weeks or 324 mg of PRO 140 every other week for 2 doses by means of subcutaneous infusion. Subjects were monitored for 58 days for safety, antiviral effects, and PRO 140 serum concentrations. RESULTS:Subcutaneous PRO 140 demonstrated potent and prolonged antiretroviral activity. Mean log(10) reductions in HIV-1 RNA level were 0.23, 0.99 (P=.009), 1.37 (P<.001), and 1.65 (P<.001) for the placebo, 162 mg weekly, 324 mg biweekly, and 324 mg weekly dose groups, respectively. Viral loads remained suppressed between successive doses. Treatment was generally well tolerated. CONCLUSIONS:This trial demonstrates proof of concept for a monoclonal antibody administered subcutaneously in HIV-1 infected individuals. Subcutaneous PRO 140 offers the potential for significant dose-dependent HIV-1 RNA suppression and infrequent patient self-administration. TRIAL REGISTRATION:ClinicalTrials.gov identifier: NCT00642707 .

ABSTRACT The anti-CCR5 antibody PRO 140 has shown potent and prolonged antiretroviral activity in subjects infected with CCR5-tropic (R5) HIV-1. Prior studies have examined single intravenous doses ranging up to 5 mg/kg of body weight or up to three subcutaneous doses ranging up to 324 mg. Here we report the results of a randomized, double-blind, placebo-controlled trial that examined the antiviral activity, tolerability, and pharmacokinetics of single 5-mg/kg and 10-mg/kg intravenous infusions of PRO 140 in 31 treated subjects. Eligibility criteria included HIV-1 RNA levels of >5,000 copies/ml, CD4 + cell counts of >300/μl, no antiretroviral therapy for ≥12 weeks, and detection of only R5 HIV-1 in the original Trofile assay. Following poststudy testing with an enhanced-sensitivity Trofile assay, one subject treated with 10 mg/kg was reclassified as having dual/mixed-tropic virus at screening, and the data for that subject were censored from efficacy analyses. The mean maximum reduction of the HIV-1 RNA level from the baseline level was 1.8 log 10 units for both the 5-mg/kg and 10-mg/kg doses ( P < 0.0001 relative to placebo). Viral loads reached their nadir at day 12 posttreatment and remained significantly ( P < 0.01) reduced through day 29 for both PRO 140 dose groups. Treatment was generally well tolerated, with no dose-limiting toxicity being observed. Peak serum concentrations and overall exposures increased proportionally with dose. In summary, single 5-mg/kg and 10-mg/kg doses of PRO 140 exhibited potent, long-lived antiviral activity and were generally well tolerated. The findings further delineate the safety and antiviral properties of this novel, long-acting antiretroviral agent.

It could be demonstrated that miR-1 targeted E2F5 and HDAC4 to inhibit cell growth and promote cell differentiation.WST-1 proliferation assay and H 3 incorporation assay showed of significant decrease of proliferation rate in miR-1 transfected cells.Cell cycle analysis demonstrated that miR-1 was able to arrest cell cycle at G1 phase.The differentiation-related genes Albumin and PEPCK were induced after miR-1 transfection.Consistently, HDAC4 inhibitor TSA or siRNA could also enhance HBV replication.Conclusions: miR-1 could target multiple cellular genes expression to promote cell differentiation which was beneficial for HBV replication.This is the first report about the potential role of miRNAs in the pathogenesis of HBV infection.

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.

Inflammatory hepatocellular adenomas (IHCA) are benign liver tumours defined by the presence of inflammatory infiltrates and by an overexpression of inflammatory proteins.IHCA are usually developed in women and their occurrence is frequently associated with obesity and alcohol intake.Recently, we identified somatic mutations activating gp130 in 60% of IHCA (Rebouissou et al, Nature, in press), thus defining gp130 as a new oncogene in human tumors.The aim of this work was to evaluate frequency of gp130 mutations in a wide series of tumors.Method: A series of 400 well-characterised hepatocellular tumors was screened including hepatocellular adenomas, carcinomas, hepatocholangio-carcinoma, fibrolamellar carcinomas and intra-hepatic cholangiocarcinomas. 100 tumors from different organs were also collected.To search for mutations, gp130 exons were screened in all samples and function of 7 different mutants was analysed in HEP3B.Results: We identified a somatic mutation in 65% of the IHCA including 24 different small in-frame deletions or duplications.Spectrum of mutations shows that mutations clearly target the IL-6 binding site in gp130.We further demonstrated that the expression of the most frequent gp130 mutants in HEP3B cells activates STAT3 in absence of IL-6.We searched for possible interaction and cooperation of gp130 activation with other pathways altered in hepatocellular adenoma.HNF1a inactivation and gp130 activation were mutually exclusive in HCA (P < 10 -4 ).By contrast, in half of the b-catenin activating HCA, we identified a gp130-activating mutation.For two IHCA associated with a malignant transformation, both an activating gp130 and b-catenin mutations were found.Further, analysis of 220 hepatocellular carcinomas revealed rare gp130 alterations in all cases accompanied by b-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant transformation of hepatocytes.Conclusion: This is the first identification of gp130 somatic activating mutations in human tumors; this finding reinforces the role of inflammation in hepatocarcinogenesis and particularly in the malignant transformation of HCA.Moreover, gp130 mutations enable to refine the molecular classification of hepatocellular tumors in human.

BACKGROUND The current goal of human immunodeficiency virus type 1 (HIV-1) therapy is to maximally suppress viral replication. Securing this goal requires new drugs and treatment classes. The chemokine receptor CCR5 provides an entry portal for HIV-1, and PRO 140 is a humanized monoclonal antibody that binds to CCR5 and potently inhibits CCR5-tropic (R5) HIV-1 in vitro. METHODS A randomized, double-blind, placebo-controlled, dose-escalating study was conducted in 39 individuals with HIV-1 RNA levels or =5000 copies/mL, CD4(+) cell counts > or =250 cells/microL, no antiretroviral therapy for 3 months, and only R5 HIV-1 detectable. Cohorts were randomized 3:10 to receive placebo or doses of PRO 140 of 0.5, 2, or 5 mg/kg. Subjects were monitored for 58 days for safety, antiviral effects, and serum concentrations of PRO 140. RESULTS PRO 140 was generally well tolerated and demonstrated potent, rapid, prolonged, and dose-dependent antiviral activity. Mean reductions in HIV-1 RNA level of 0.58 log(10), 1.20 log(10) (P= .0002) and 1.83 log(10) (P= .0001) were observed for the 0.5-, 2-, and 5-mg/kg dose groups, respectively. Reductions in mean viral load of > or =10-fold were observed within 4 days and persisted for 2-3 weeks after treatment. CONCLUSIONS This trial established clear proof of concept for PRO 140 as a potent antiretroviral agent with extended activity after a single dose. TRIAL REGISTRATION ISRCTN Register: ISRCTN45537485 .