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ABSTRACT: The Eastern woodchuck (Marmota monax) is a useful experimental model for evaluating antiviral therapy against chronic HBV infection. In the present study, an immunogenic complex (IGC) composed of immune sera containing PreS/S heterologous antibodies (anti-HBs) and serum-derived WHV particles containing 10(7) WHV-DNA copies/50 µl was developed. The IGC was administered to WHV-negative woodchucks and natural chronic WHV carriers, with the final aim of evaluating the outcome of WHV infection in both groups. A control group of three animals, infected experimentally with viral particles only, was also evaluated. Following IGC administration, two WHV-negative woodchucks exhibited persistent infection, with WHV-DNA levels 3-6 logs lower than the WHV-DNA levels of the controls that developed persistent infection. WHeAg seroconversion to anti-WHe was observed in these two woodchucks and in two control woodchucks which developed self-limited infection. In two of the four chronic carriers, the WHV-DNA level decreased significantly (by 4-6 logs) following IGC administration, with no rebound in viral load during follow-up. WHeAg seroconversion to anti-WHe was observed also in these animals. Analyses of the sequences derived from envelope proteins confirmed that IGC did not induce the emergence of resistant viral variants. The results of this study indicate that the IGC could be useful for breaking the tolerance in hepadnaviral infection and for boosting the host's innate and adoptive immune response.
Journal of Medical Virology 01/2011; 83(1):178-86. · 2.82 Impact Factor
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ABSTRACT: Studies focused on the understanding of the molecular mechanisms involved in recovery or progression to chronicity of HBV may take advantage of natural and experimental models that mimic its properties. This is also of relevance for associated diseases such as cirrhosis and hepatocellulocarcinoma. The eastern woodchuck (Marmota monax) infected by the hepadnavirus woodchuck Hepatitis B virus (WHV) has been applied as a predictive model to support development of new HBV vaccines, antivirals, immunotherapies and combination therapies. This report summarizes studies carried out by our and other groups, with the application of this model in natural and experimental infections. Using standardized viral inocula in neonate and adult animals and newly established assays, the presence of the specific patterns of markers of acute, chronic and resolved infections and their relationships in the different virus-host interactions have been shown. B and T cell responses and T(H)1 cytokine expression have been shown to play a crucial role in the outcome of infection. The availability of the WHV/Marmota monax model and specific standardized assays may allow evaluation of new formulations of multimodal therapeutic strategies based on antiviral chemotherapy and immunomodulation. These may also include specifically targeted immunocomplexes. Such therapies could constitute new frontiers for the treatment of HBV chronic disease.
Expert Opinion on Drug Discovery 12/2010; 5(12):1153-62. · 2.12 Impact Factor
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Retrovirology. 01/2010;
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ABSTRACT: HCV is a ssRNA virus belonging to the Flaviviruses and is found worldwide worldwide in humans. Following primary infection, persistent infection develops in more than 85% of cases, which in up to 30% of cases, may progress to liver disease, cirrhosis and hepatocellular carcinoma. The virus presents a high degree of genetic variability owing to the combination of a lack of proofreading by the RNA-dependent RNA polymerase and a high level of viral replication. This genetic variability allows the classification of genotypes, subtypes, isolates and quasispecies to which epidemiological and pathogenetic significance may be associated. The features and biological implications of HCV variability and of quasispecies dynamics in infection transmission, mechanisms of chronicity and resistance to antiviral therapy are discussed.
Future Microbiology 05/2009; 4(3):359-73. · 3.82 Impact Factor
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ABSTRACT: Present efforts of HBV vaccine research are aimed at defining targeted antigen compositions and adjuvancy systems for earlier and broader immune responses and optimization of immunotherapeutic approaches. We have demonstrated the applicability of the WHV/Marmota monax model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use. Protective activity was evaluated following the administrations of HBV CHO-PreS/S and adjuvanted S/Core vaccines. The administration of a complex constituted by HBV derived woodchuck PreS/S antibodies coupled with WHV particles was able to induce inhibition of viral replication. Future studies on treatment of HBV chronic infection should be addressed to the evaluation of therapies combined with antivirals, vaccines and immunomodulatory compounds.
Vaccine 03/2009; 27(25-26):3271-5. · 3.77 Impact Factor
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ABSTRACT: Several seroepidemiological population-based surveys carried out in Italy have shown a high prevalence of hepatitis C virus (HCV) infection. Camporeale (CP), a small Sicilian town with a 10.4% prevalence of HCV mostly genotype 1b, probably represents a specific context, since intravenous drug addiction, and sexual promiscuity are almost absent. In order to reconstruct the pattern of introduction and diffusion of HCV in this ecological niche, the NS5 genomic region of 72 HCV genotype 1 isolates (39 from CP and 33 collected throughout Sicily) was amplified and sequenced. Sequences were aligned and analyzed by BioEdit, PAUP and BEAST, and their molecular evolution compared. Thirty-eight HCV genotype 1b isolates from CP were associated in a monophyletic "transmission cluster." By applying Monte Carlo Markov simulation, it was calculated that HCV was introduced between the end of the 1940s and the beginning of the 1950s. The phylogenetic distance between the CP cluster and other Sicilian isolates confirmed its uniqueness and the local diffusion from a common ancestor. The data obtained from classic phylogenetic analysis, combined with the application of the Bayesian analysis to the study of the coalescence of phylogenetic trees, have shown that, in CP, few HCV native strains have been transmitted in a limited length of time probably through iatrogenic routes, and then have not spread further.
Journal of Medical Virology 11/2008; 80(10):1723-31. · 2.82 Impact Factor
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ABSTRACT: In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6P cy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the evolution of the SHIV-89.6P cy243 both in control and vaccinated infected monkeys, the sequence of the human immunodeficiency virus (HIV)-1 Tat protein and the C2-V3 Env region of the proviral-DNA-derived clones were analyzed in both control and vaccinated but unprotected animals. We also performed analysis of the T cell epitope using a predictive epitope model taking into consideration the phylogeny of the variants. Our results suggest that even though the viral evolution observed in both groups of monkeys was directed toward variations in the major histocompatibility complex (MHC)-I epitopes, in the control animals it was associated with mutational escape of such epitopes. On the contrary, it is possible that viral evolution in the vaccinated monkeys was linked to mutations that arose to keep high the viral fitness. In the vaccinated animals the reduction of epitope variability, obtained prompting the immune system by vaccination and inducing a specific immunological response against virus, was able to reduce the emergence of escape mutants. Thus the intervention of host's selective forces in driving CTL escape mutants and in modulating viral fitness appeared to be different in the two groups of monkeys. We concluded that in the vaccinated unprotected animals, vaccination with the Tat protein induced a broad antiviral response, as demonstrated by the reduced ability to develop escape mutants, which is known to help in the control of viral replication.
Virus Genes 03/2008; 36(1):105-15. · 1.85 Impact Factor
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ABSTRACT: In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven
monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6Pcy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the
evolution of the SHIV-89.6Pcy243 both in control and vaccinated infected monkeys, the sequence of the human immunodeficiency virus (HIV)-1 Tat protein and
the C2-V3 Env region of the proviral-DNA-derived clones were analyzed in both control and vaccinated but unprotected animals.
We also performed analysis of the T cell epitope using a predictive epitope model taking into consideration the phylogeny
of the variants. Our results suggest that even though the viral evolution observed in both groups of monkeys was directed
toward variations in the major histocompatibility complex (MHC)-I epitopes, in the control animals it was associated with
mutational escape of such epitopes. On the contrary, it is possible that viral evolution in the vaccinated monkeys was linked
to mutations that arose to keep high the viral fitness. In the vaccinated animals the reduction of epitope variability, obtained
prompting the immune system by vaccination and inducing a specific immunological response against virus, was able to reduce
the emergence of escape mutants. Thus the intervention of host’s selective forces in driving CTL escape mutants and in modulating
viral fitness appeared to be different in the two groups of monkeys. We concluded that in the vaccinated unprotected animals,
vaccination with the Tat protein induced a broad antiviral response, as demonstrated by the reduced ability to develop escape
mutants, which is known to help in the control of viral replication.
Virus Genes 01/2008; 36(1):105-115. · 1.85 Impact Factor
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Stefano Dettori, Claudio Argentini,
Fabrizio Marcucci,
Enea Spada,
Paola Chionne,
Angela Candido,
Elisabetta Madonna,
Anna Rita Ciccaglione,
Elvira Bianco,
Emilio Iannitto,
Pellegrino Musto,
Vincenzo Liso,
Amalia De Renzo,
Livio Pagano,
Grazia Nieddu,
Alessandro Pulsoni,
Alfonso Mele,
Maria Rapicetta
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ABSTRACT: We compared the E2-HVR1 region in HCV-1b positive B-NHL cases from a multicenter study with sequences from studies related to lymphoproliferative disorders and B cell compartmentalisation. We found rare and unique mutations both in B-NHL isolates and in cases with lymphoproliferative disorders and lymphocyte infection. These rare mutations could have an important effect on HVR1 region and, as a consequence, on the binding of E2 on CD81, with a possible implication for both antigenic stimulation and HCV entry. In conclusion, the HCV predominants circulating in B-NHL cases seem to be associated with clonal selection of rare variants.
The new microbiologica: official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC) 08/2007; 30(3):265-70. · 1.00 Impact Factor
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Emilio D'Ugo,
Loreta A Kondili,
Andrea Canitano,
Stefania Catone,
Roberto Giuseppetti,
Bruno Gallinella,
Giampiero Palmieri,
Sara Orobello, Claudio Argentini,
Reinhard Glück,
Maria Rapicetta
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ABSTRACT: To determine whether the addition of a pre-S/S human vaccine increases the antiviral activity of lamivudine, four woodchucks were treated with a daily dose of 100 mg/kg lamivudine and four 50 microg doses of CHO-derived pre-S/S human vaccine. WHV DNA titres decreased up to two logarithms in three woodchucks. At week 4, in three of the animals, the sequence analysis showed a predominant strain containing a nucleotide change from A to T at position 1696 of domain B of the WHV DNA polymerase. Vaccination did not further suppress WHV DNA, despite anti-HBs production in three animals. The woodchuck remains a useful model for characterising the biology and kinetics of the emergence of drug-resistant variants and could be used for pre-clinical studies of combinations of new antiviral drugs.
Vaccine 07/2007; 25(26):4895-902. · 3.77 Impact Factor
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ABSTRACT: Acute viral hepatitis due to hepatitis A virus is a self-limited illness that infrequently has a severe clinical course.
We analyzed the virological characteristics of acute hepatitis A in a patient with a severe clinical presentation (peak total and conjugated bilirubin levels, 65.5 mg/dL and 40.1 mg/dL, respectively) and a course of disease that lasted 7 months.
Hepatitis A virus sequencing revealed coinfection with 2 subgenotypes of hepatitis A virus (Ia and Ib) as etiological factors of the illness.
Hepatitis A virus Ia and Ib coinfection may have accounted for the prolonged and severe course of illness.
Clinical Infectious Diseases 06/2007; 44(9):e73-7. · 9.15 Impact Factor
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ABSTRACT: We evaluated whether a non-adjuvanted vaccine derived from Chinese hamster ovary cells was capable of providing protection against woodchuck hepatitis virus (WHV). Three woodchucks were vaccinated with four 50-microg doses and challenged with a previously characterized virus isolate (WHV197). In all three animals, titre levels of antibodies against hepatitis B surface antigens (anti-HBs) exceeded 10 mIU/ml, peaking at 150 mIU/ml. Challenge resulted in productive acute infection in the two non-vaccinated woodchucks yet in none of the vaccinated woodchucks. In the vaccinated animals, there was evidence of abortive infection. The results demonstrate that a human vaccine is able to protect woodchucks from WHV infection.
Vaccine 06/2005; 23(28):3649-56. · 3.77 Impact Factor
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ABSTRACT: The immunogenicity and the protection induced by an hepatitis delta virus (HDV) vaccine consisting of the small nucleoprotein (HDAg) (p24) and adjuvanted with MF59 or Freund's adjuvant (FA) were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) and challenged with hepatitis delta virus. Humoral and T-cell-mediated responses to HDAg were measured. Anti-HD antibodies appeared earlier in the FA/p24 animals. After challenge, all MF59/p24 vaccinated animals showed a response to HDAg-derived peptides, compared to two of the five FA/p24 animals and one of the control animals. Serum HDV-RNA peak values and persistence were considerably reduced in immunized animals, in comparison to controls. Furthermore, HDV-RNA was absent in autopsy liver tissues of 50% of the MF59/p24 animals, whereas high levels were present in all of the FA/p24 animals and controls. Histological liver analysis performed before and after challenge revealed the presence of acute hepatitis-like lesions only in the controls. Overall, the results suggest that the MF59/p24 vaccine better controls the infection in terms of viral replication and survival.
Vaccine 02/2004; 22(3-4):457-66. · 3.77 Impact Factor
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ABSTRACT: Evaluation of sequence evolution as well as structural defects and mutations of the human immunodeficiency virus-type 1 (HIV-1) nef gene in relation to disease progression in infected children.
We examined a large number of nef alleles sequentially derived from perinatally HIV-1-infected children with different rates of disease progression: six non-progressors (NPs), four rapid progressors (RPs), and three slow progressors (SPs).
Nef alleles (182 total) were isolated from patients' peripheral blood mononuclear cells (PBMCs), sequenced and analysed for their evolutionary pattern, frequency of mutations and occurrence of amino acid variations associated with different stages of disease.
The evolution rate of the nef gene apparently correlated with CD4+ decline in all progression groups. Evidence for rapid viral turnover and positive selection for changes were found only in two SPs and two RPs respectively. In NPs, a higher proportion of disrupted sequences and mutations at various functional motifs were observed. Furthermore, NP-derived Nef proteins were often changed at residues localized in the folded core domain at cytotoxic T lymphocytes (CTL) epitopes (E(105), K(106), E(110), Y(132), K(164), and R(200)), while other residues outside the core domain are more often changed in RPs (A(43)) and SPs (N(173) and Y(214)).
Our results suggest a link between nef gene functions and the progression rate in HIV-1-infected children. Moreover, non-progressor-associated variations in the core domain of Nef, together with the genetic analysis, suggest that nef gene evolution is shaped by an effective immune system in these patients.
AIDS 07/2003; 17(9):1291-301. · 6.24 Impact Factor
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ABSTRACT: The presence of Deleted Genomes has been shown in a number of viral models including Hepadnaviridae. The analysis of woodchuck hepatitis B virus (WHV) population after experimental infection of woodchuck 197 (W197) with WHV7-PI inoculum revealed the presence of two Deleted Genomes: DG600 lacking a 1330 bp region (Core/Polymerase/PreS1) and DG900 showing a deletion of 869 nts (Pol/PreS/S). These mutants were also present in WHV7-PI. The successive WHV experimental infections in adult animals were performed using W197-w7 inoculum containing DG600 and DG900. Infections were divided into three groups presenting different patterns of viral replication, different presence of markers, occurrence of variants and persistence of infection. The first group displayed 2-3 weeks viremic phase and WHV-DNA titres of 10-30 ng/ml; the second a longer viremic phase (8-9 weeks) and higher WHV-DNA titres (up to 78 ng/ml). In contrast, the third group exhibited lifetime presence of WHV-DNA and WHVeAg in serum and viral replication in liver. The Deleted Genomes were transmitted in the newly infected animals with the same genomic organization. DG600 was persistently found only in chronically infected woodchuck, whereas a different pattern of presence was described for DG900. The characterization of these classes of deleted mutants in woodchuck-WHV model raises new questions on the link between DGs and persistent infections.
Virus Genes 11/2002; 25(2):147-57. · 1.85 Impact Factor
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ABSTRACT: In the woodchuck hepatitis virus (WHV)/woodchuck model for hepatitis B virus-induced hepatocellular carcinoma, frequent activation of N-myconcogenes by WHV integration has been firmly established. N-myc2, the most frequently affected gene, was reported to be activated by WHV insertion either in the proximity of the gene or in a distant uncoding locus,win. We previously reported that a WHV integration cloned from a liver tumor was located in a chromosomal locus already described by others as the site of WHV integration in another hepatocellular carcinoma. On this basis, the locus, namedb3n, was defined as a recurrent site of WHV integration. A scaffold or matrix attachment region (S/MAR) element was subsequently shown to be located in this locus ∼1 kb from the WHV insertion sites. S/MARs are genetic elements involved both in structural and functional organization of chromosomal DNA and in stimulation of gene expression. In the present work, we investigated the possibility that an N-mycgene might be affected by integration inb3n. Analysis of a liver tumor harboring WHV integration in this locus showed N-myc2 overexpression. By restriction analysis, theb3nlocus was shown to be located downstream of N-myc2, so the known sites of viral insertion inb3nwere ∼11 kb downstream of the N-myc2 promoter. Although these data support that WHV insertion inb3nactivates N-myc2, the mechanisms previously described to be involved in N-myc2 activation do not appear to properly account for activation in this subset of WHV integrations. Available data suggest that activation of N-myc2 by WHV integration inb3nmight be mediated by the S/MAR located near the WHV insertion.
Virology 06/1999; · 3.35 Impact Factor
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ABSTRACT: The nucleotide sequences of the VP1 coding region of two newly characterized, cell culture-adapted hepatitis A virus (HAV) strains (RG-SB11 and RG-SB16) were analyzed and compared with homologous regions of previously characterized HAV strains of human or monkey origin, and at different levels of tissue-culture adaptation. In particular, HM175wt and its derivative strains and MBB, LCDC1, PA21, and AGM27 isolates were considered. RG-SB11 and RG-SB16 HAV strains were derived from a pathogenic isolate from an acutely infected patient, purified from stool, and subjected to different strategies of adaptation. Several nucleotide differences were observed, but high conservation was found in the predicted VP1 protein sequences, which confirms structural constraints for this region. Furthermore, comparative amino-acid sequence analysis of VP1 from all HAV isolates studied has shown, particularly for those from naturally infected monkeys, that differences are limited to the amino and carboxy-terminal part of the molecule. The results of phylogenetic analysis have confirmed the common origin of the RG-SB11 and RG-SB16 strains. The complete nucleotide sequences of the VP1 coding region of the RG-SB11/16, HM175 derivative strains and of other HAV strains has shown that branch-length evolution can give a measure of the evolution of HAV during adaptation processes.
Virus Genes 01/1995; 10(1):37-43. · 1.85 Impact Factor
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ABSTRACT: The woodchuck hepatitis B virus (WHV), the closest genetically related virus of HBV, and its natural host Marmota monax constitute a well recognized animal model. The application of this model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use, for lamivudine-CHO-PreS/S vaccine therapy and WHV particles coupled with HBV derived woodchuck PreS/S antibodies (IC complex) studies, as shown that the PreS/S-CHO vaccine is the first human vaccine able to elicit non sterilizing protection in the woodchuck model. The very early appearance and selection of the domain B FLLA motif resistant mutant not neutralized by the antibodies produced following vaccination, has confirmed that more potent antivirals and/or multiple targeted options with possible inclusion of immune complexes should be considered.
Procedia in Vaccinology 1(1):35-40.
