Verónica L. Mathet

Detection of Hepatitis B Virus (HBV) Genotype E Carried—Even in the Presence of High Titers of Anti-HBs Antibodies—by an Argentinean Patient of African Descent Who Had Received Vaccination against HBV

ABSTRACT Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg.

Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.

Antiviral activity against the hepatitis C virus (HCV) of 1-indanone thiosemicarbazones and their inclusion complexes with hydroxypropyl-β-cyclodextrin

The hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis in humans. Approximately 5% of the infected people die from cirrhosis or hepatocellular carcinoma. The current standard therapy comprises a combination of pegylated-interferon alpha and ribavirin. Due to the relatively low effectiveness, the prohibitive costs and the extensive side effects of the treatment, an intense research for new direct-acting anti-HCV agents is taking place. Furthermore, NS3 protease inhibitors recently introduced into the market are not effective against all HCV subgenotypes. Thiosemicarbazones (TSCs) have shown antiviral activity against a wide range of DNA and RNA viruses. However, their extremely low aqueous solubility and high self-aggregation tendency often preclude their reliable biological evaluation in vitro. In this work, we investigated and compared for the first time the anti-HCV activity of two 1-indanone TSCs, namely 5,6-dimethoxy-1-indanone TSC and 5,6-dimethoxy-1-indanone N4-allyl TSC, and their inclusion complexes with hydroxypropyl-β-cyclodextrin (HPβ-CD) in Huh-7.5 cells containing the full-length and the subgenomic subgenotype 1b HCV replicon system. Studies of physical stability in culture medium showed that free TSCs precipitated rapidly and formed submicron aggregates. Conversely, TSC complexation with HPβ-CD led to more stable systems with minimal size growth and drug concentration loss. More importantly, both TSCs and their inclusion complexes displayed a potent suppression of the HCV replication in both cell lines with no cytotoxic effects. The mechanism likely involves the inhibition of non-structural proteins of the virus. In addition, findings suggested that the cyclodextrin released the drug to the culture medium over time. This platform could be exploited for the study of the drug toxicity and pharmacokinetics animal models.

Drug delivery systems and liver targeting for the improved pharmacotherapy of the hepatitis B virus (HBV) infection.

ABSTRACTIn spite of the progress made in vaccine and antiviral therapy development, hepatitis B virus (HBV) infection is still the most common cause of liver cirrhosis and hepatocellular carcinoma, with more than 400 million people chronically infected worldwide. Antiviral therapy with nucleos(t)ide analogues and/or immunomodulating peptides is the only option to control and prevent the progression of the disease in chronic hepatitis B (CHB)-infected patients. So far, the current antiviral monotherapy remains unsatisfactory because of the low efficacy and the development of drug resistance mutants. Moreover, viral rebound is frequently observed following therapy cessation, since covalent closed circular DNA (cccDNA) is not removed from hepatocytes by antiviral therapy. First, this review describes the current pharmacotherapy for the management of CHB and the new drug candidates being investigated. Then, the challenges in the development of drug delivery systems for the targeting of antiviral drugs to the liver parenchyma are discussed. Finally, perspectives in the design of a more efficient pharmacotherapy to eradicate the virus from the host are addressed.

Unusual Naturally Occurring Humoral and Cellular Mutated Epitopes of Hepatitis B Virus in a Chronically Infected Argentine Patient with Anti-HBs Antibodies

ABSTRACT Serum hepatitis B virus (HBV) DNA was extracted from a chronically infected patient with cocirculation of hepatitis B surface antigen (HBsAg) and anti-HBs antibodies. Direct PCR and clone-derived sequences of the S and overlapped P genes were obtained. DNA sequences and phylogenetic analysis ascribed this isolate to genotype A (serotype adw2). Five of six HBV DNA clones exhibited point mutations inside and outside the major hydrophilic region, while the sixth clone exhibited a genotype A “wild-type” amino acid sequence. Observed replacements included both humoral and/or cellular (major histocompatibility complex class I [MHC-I] and MHC-II) HBV mutated epitopes, such as S45A, P46H, L49H, C107R, T125A, M133K, I152F, P153T, T161S, G185E, A194T, G202R, and I213L. None of these mutants were individually present within a given clone. The I213L replacement was the only one observed in the five clones carrying nonsynonymous mutations in the S gene. Some of the amino acid substitutions are reportedly known to be responsible for the emergence of immune escape mutants. C107R replacement prevents disulfide bonding, thus disrupting the first loop of the HBsAg. Circulation of some of these mutants may represent a potential risk for the community, since neither current hepatitis B vaccines nor hyperimmune hepatitis B immune globulin are effectively prevent the liver disease thereto associated. Moreover, some of the recorded HBsAg variants may influence the accuracy of the results obtained with currently used diagnostic tests.

Naturally occurring hepatitis B virus (HBV) variants with primary resistance to antiviral therapy and S-mutants with potential primary resistance to adefovir in Argentina.

Hepatitis B virus (HBV) variants may either emerge in patients with chronic hepatitis B (CHB) as a result of positive selection pressure exerted by their own immune response, or during therapy with nucleos(t)ide analogues (NAs). Naturally occurring HBV variants with primary antiviral resistance are rarely observed. The aim of this study was to retrospectively analyze the (eventual) circulation of HBV variants with natural resistance to NAs currently used as therapy for CHB in Argentina. This study reports 13 cases of CHB-infected patients with natural antiviral resistance to at least one NA. Five of them were also carriers of S-variants that might escape the humoral immune system recognition with potential resistance to adefovir. In addition to the already reported A2 HBV subgenotype association to NAs natural resistance, E and F genotypes association to such resistance is described for the first time. These findings suggest that sequence analysis of the HBV reverse transcriptase might be an essential tool before starting antiviral therapy, in order to choose the proper NAs for optimizing the therapeutic management of chronically infected patients. Moreover, the circulation and transmission of S-mutants with resistance to such antiviral drugs should be of public health concern as they may represent an additional risk for the community.

In Vitro Detection of Dissimilar Amounts of Hepatitis C Virus (HCV) Subtype-Specific RNA Genomes in Mixes Prepared from Sera of Persons Infected with a Single HCV Genotype

ABSTRACT The level of in vitro detection of viral genomes in mixes with two different hepatitis C virus (HCV) subtypes was investigated by artificially mixing previously measured subtype-specific HCV RNA genomes. The RNAs in these mixtures were reverse transcribed and then PCR amplified by using two sets of primers corresponding to the 5′ untranslated region and digested with endonucleases to analyze the restriction fragment length polymorphism patterns. This approach facilitated detection of a wider range of type-specific HCV genomes than originally described, beyond equimolar concentrations of contributing HCV subtypes. Moreover, by using computerized image analysis, this study also demonstrated that the true contribution of each virus type—and consequently of mixed infections—may be underestimated when only visual observation is carried out. These results may be useful for comparing data obtained from this and other currently used methodologies.

Single and multiple mutations in the human cytomegalovirus UL97 gene and their relationship to the enzymatic activity of UL97 kinase for ganciclovir phosphorylation

In this study we determined that the double mutant M460V/D605E in the UL97 gene of an HCMV isolate from an immunocompromised patient (MMT isolate) is related to resistance to ganciclovir (GCV) therapy. Our results suggest that the aspartic acid-to-glutamic acid substitution at codon 605 may be associated with a natural polymorphism of the UL97 gene, and not with positive selection pressure exerted by the antiviral drug. We also determined that GCV resistance due to the M460V mutation in the HCMV UL97 gene is not offset by a second mutation (D605E) at codon 605. Furthermore, we showed that when the two mutations related to GCV resistance were simultaneously detected in the same HCMV construct, virus-drug resistance might be enhanced in comparison to that of the single mutants studied separately. To our knowledge for the first time, seven of 12 amino acid changes (F102L, D118V, M330T, T400A, R507P and C511R and I533V) in the UL97 gene of an isolate are herein reported.

Hepatitis C Virus and GBV-C/Hepatitis G Virus in Argentine Patients with Porphyria Cutanea Tarda

To investigate hepatitis C virus (HCV) and GBV-C/hepatitis G virus (HGV) genotype prevalence among HCV-infected porphyria cutanea tarda (PCT) patients, 19 HCV-infected patients with associated PCT were studied. A control group of 53 age-matched HCV-infected patients without associated PCT was selected. Eighteen of the 19 serologically positive HCV-PCT patients showed HCV RNA in serum. Genotype 1b was the most prevalent among both HCV-PCT patients (72.2%; 13/18) and age-matched HCV controls (50.9%; 27/53). Such different genotypic prevalence failed to reach statistical significance (χ2 with Yates’ correction, p = 0.19). The single HCV-PCT patient without detectable HCV RNA was also infected with genogroup 3 GBV-C/HGV. This GBV-C/HGV RNA prevalence (5.3%) among HCV-PCT patients is not statistically different from that observed among Argentine blood donors (5.5%; 11/200). To our knowledge, these results show for the first time the molecular epidemiology of both HCV and GBV-C/HGV associated to PCT in America.

Successful interferon treatment in a patient chronically infected with hepatitis B virus carrying unusual S- (and P-) mutants in the presence of anti-HBs antibodies.

Background: Hepatitis B virus (HBV) immune escape mutants with point mutations within the S gene may arise during the natural course of HBV infection, due to a positive selection pressure exerted by the host immune response. Mutations within the immunodominant B and T cell epitopes of hepatitis B surface antigen (HBsAg) allow the resulting S‐mutants to propagate even in the presence of neutralizing anti‐HBs antibodies and the HBV‐specific T‐cell immune response.