Stefania Catone

Surveillance of suspected poliomyelitis in Albania, 1980–1995: suggestion of increased risk of vaccine associated poliomyelitis

Surveillance of suspected poliomyelitis cases was conducted in Albania from 1980 through 1995. A total of 93 cases were reported, 11 of which were clinically defined as poliomyelitis cases according to WHO criteria. Poliovirus was isolated from six subjects who were defined as contact vaccine-associated cases. Characterization of isolates by both antigenic and molecular methods showed that, in all cases, the disease was associated with type 2 or 3 polioviruses of vaccine origin with retromutations known to be associated with loss of Sabin attenuated phenotype. Infection occurred despite the fact that all patients had records of previous immunization with oral polio vaccine (OPV), suggesting a failure of vaccination. Four of the five patients from which poliovirus could not be isolated were classified as possible recipient vaccine-associated poliomyelitis on the basis of serology data (presence of antibodies against all three polioviruses) and the temporal association between the latest dose of vaccine received and onset of paralysis. Virological investigation on healthy contacts of the poliomyelitic patients yielded the isolation of a further 12 Sabin-like polio revertant viruses, mostly type 2 and 3. A detailed study of the non-polio acute flaccid paralysis (AFP) cases and their healthy contacts revealed the presence of several enteroviruses, namely Echo, coxsackie and, in three cases type 2 or 3 Sabin-like polioviruses. Overall, these data suggest the absence of circulation of wild-type poliovirus in Albania from 1980 to 1995, before the recent outbreak of poliomyelitis in 1996, and emphasize the need for active surveillance of AFP and laboratory characterization of virus isolates to monitor vaccination efficacy.

T-cell-mediated protective efficacy of a systemic vaccine approach in cynomolgus monkeys after SIV mucosal challenge.

Abstract:  The immunogenicity and the protective efficacy of a new polyvalent triple vector (DNA/SFV/MVA) based vaccine against mucosal challenge with pathogenic SIVmac251 were investigated. Cynomolgus monkeys (Macaca fascicularis) were primed intradermally with DNA, boosted twice subcutaneously with recombinant Semliki Forest virus (rSFV) and finally intramuscularly with recombinant Modified Vaccinia Virus Ankara strain (rMVA). Both DNA and recombinant viral vectors expressed SIV proteins (Gag, Pol, Tat, Rev, Nef and Env). The vaccinated monkeys developed T helper proliferative responses to viral antigens after the second immunization while interferon (IFN)‐γ enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT) specific responses appeared only after the last boost with rMVA. Upon intrarectal challenge with pathogenic SIVmac251, three of four vaccinated monkeys were either fully protected or exhibited a dramatic reduction of virus replication up to undetectable level. A major contribution to this protective effect appeared to be the anamnestic T‐cell IFN‐γ ELISPOT responses to vaccine antigens (Gag, Rev, Tat, Nef) that mirrored the viral clearance. These results underline the efficacy of a multiprotein approach in combination with a triple vector system of antigen delivery.

Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation

BackgroundHepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.MethodsWe used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.ResultsWe found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.ConclusionsHCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIVmacJ5 on the kinetics of SIV replication in cynomolgus monkeys

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIVmac251/32H/J5) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA‐J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIVmac251. All control monkeys, previously inoculated with non‐recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post‐challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA‐J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell‐ and plasma‐associated viral load.

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.

Kinetics of WHV-HDV replication in acute fatal course of woodchuck hepatitis

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Isolation and biological characterization of 3(2H)-isoflavene-resistant and -dependent poliovirus type 2 Sabin mutants.

Poliovirus type 2 Sabin mutants were selected for drug resistance and dependence by plating on HeLa cell monolayers in the presence of 3(2H)-isoflavene, a compound related to dichloroflavan, which prevents the shut-off of host translation and poliovirus RNA and protein synthesis. The drug-resistant mutants grew equally well in the presence and in the absence of the drug, while the drug-dependent mutants only grew in the presence of the compound. One dependent and one resistant mutant were characterized biologically in more detail. The resistant mutant did not exhibit thermolability. The mild thermolability exhibited by the dependent mutant was not affected by the addition of 3(2H)-isoflavene, indicating that the substance does not bind the poliovirus type 2 Sabin capsid. The translation of viral proteins and the shut-off of host protein translation during cell infection were not inhibited in either mutant. In the absence of the drug, the cleavage of the precursor VPO, a step in virus protein processing, was affected in the dependent mutant. The dependence of the mutant on the drug was due to the inability of 75S empty particles to reach maturation: our results strongly suggest that this phenomenon is strictly dependent on the reduction of RNA synthesis, confirming the existence of a dynamic equilibrium between RNA production and genome encapsidation during the poliovirus replication cycle.

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.

Generation of a human immunodeficiency virus type 1 chronically infected monkey B cell line expressing low levels of endogenous TRIM5α

Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5α has been shown to block human immunodeficiency virus type 1 (HIV‐1) infection in several types of Old World monkey cells. Here we report a novel HIV‐1 chronically infected monkey B cell line, F6/HIV‐1, characterized by very low levels of TRIM5α expression that allows HIV‐1 to overcome the restriction. Virus produced by F6/HIV‐1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV‐1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV‐1 is the first monkey cell line chronically infected by HIV‐1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV‐1 pathogenesis. J. Cell. Physiol. 221: 760–765, 2009.