Emilio D'Ugo

Hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-l-lysine: Synthesis of the carrier and pharmacological properties of the conjugate

BACKGROUND/AIMS The hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine (L-Poly(Lys)) must have a high solubility in order to be injected in a small volume compatible with the intramuscular route. In this paper the molecular weights of Poly(Lys) which allowed the synthesis of conjugates with the properties of high solubility and limited loss by the kidney were determined and a procedure for obtaining Poly(Lys) preparations with the required range of polymerization has been described. METHODS Conjugates were prepared using Poly(Lys) of different molecular weights obtained by the procedure described here or purchased from a commercial source. Their solubility and renal loss in mice was determined. RESULTS Poly(Lys) with molecular weights ranging from 45,000 and 65,000 Da guarantees high solubility and low renal elimination of the conjugates. Conjugate preparations with these properties, intramuscularly administered to woodchuck hepatitis virus-infected woodchucks for 37 days at a daily dose of 5.8 mg/kg exerted a strong antiviral activity. These preparations were devoid of acute toxicity in rat and caused no toxic effects when injected intramuscularly daily for 28 days at a dose ten times higher than that active in woodchucks. CONCLUSIONS The results support the possibility of a clinical use of L-Poly(Lys) to obtain liver targeting of adenine arabinoside monophosphate for the treatment of chronic hepatitis B virus infection.

New perspectives for hepatitis B vaccines and immunization.

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.

Detection of Human Enteric Viruses in Freshwater from European Countries.

The transmission of water-borne pathogens typically occurs by a faecal–oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.

Effect of an immunogenic complex containing WHV viral particles and non‐neutralizing anti‐HBs antibodies on the outcome of WHV infection in woodchucks

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 107 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 hosts innate and adoptive immune response. J. Med. Virol. 83:178–186, 2011.

The woodchuck hepatitis B virus infection model for the evaluation of HBV therapies and vaccine therapies

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 TH1 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.

Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on Mycobacterium spp

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected—24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012–2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

Sequence and phylogenetic analysis of the VP1 gene in two cell culture-adapted HAV strains from a unique pathogenic isolate

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.

[406] RAPID INSURGENCE OF A VIRAL RESISTANT MUTANT IN WHV CHRONICALLY INFECTED WOODCHUCKS TREATED WITH LAMIVUDINE AND A PRE-S/S CHO-DERIVED HEPATITIS B VIRUS VACCINE

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.