Sub-epitopic dissection of HCV E1315-328HRMAWDMMMNWSPT sequence by similarity analysis.

Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1(315-328)HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315-328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH(2) and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host's proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.

Effect of HCV infection on THP-1 monocytoid cells.

Hepatitis C virus (HCV) is the main cause of hepatocellular carcinoma in industrialized countries. HCV-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution of chronic active hepatitis towards cirrhosis and hepatocellular carcinoma. The present work shows that THP-1 monocytoid cells are susceptible to HCV infection, of strain 1b, and that this strain can induce cellular modifications in this cell line. Infection of HCV was demonstrated by positivity for the E2 antigen within THP-1 cells and by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells from day 7 post-inoculation. Cell shape and membrane surface antigens varied upon viral infection, which is also capable of inducing oxygen radicals. In particular we underline the relevant intracellular accumulation of ferritin that paralleled an increase of cell surface expression of the transferrin receptor. Evaluation of cellular events upon HCV infection in THP-1 cells may represent a useful tool with which to identify alteration in monocytes metabolism and to study therapeutic approaches for such alterations.

HCV infective virions can be carried by human platelets.

It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV)-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests.

Cervico-vaginal dysplasia--papillomavirus-induced and HIV-1 infection: role of correlated markers for prognostic evaluation.

Sexually-transmitted diseases (STD) can facilitate the progression of HIV-1 infection. Among them, as we have previously demonstrated, cervico-vaginal dysplasia-papillomavirus (HPV)-induced, together with HSV-2 co-infection, seems to be correlated with a more evident immunodepression in HIV-positive women, compared with other sexually transmitted diseases. Here we have analysed some of the main correlated markers of HIV-1 infection progression: CD4 + T lymphocyte concentration, CD4 +/CD8 + T cells ratio, HIV-1 RNA loads and haemoglobin (Hb) concentration in 30 HIV-1 positive women co-infected with HPV, and suffering from cervico-vaginal dysplasia, in different stages. In particular, we noticed a positive correlation, evaluated by Spearman's test, between the degree of progression of dysplastic stages (CIN1 --> 3) until invasive carcinoma (IC) and HIV-1 RNA loads (C(s) = +0.78; p < 0.001), and in contrast, a negative correlation between the same stages of progression and respectively CD4 + T cell concentration (C(s) = -0.54; p = 0.01), ratio (C(s) = - 0.63; p = 0.002) and Hb concentration (C(s) = -0.85; p < 0.001). In conclusion, it is important to underline that low levels of Hb generally paralleled the degree of immunodepression. In fact CD4 + T cell levels and ratio positively correlated with Hb concentrations respectively, with C(s) = + 0.83; p < 0.001 and C(s) = + 0.90; p < 0.001. Finally, the most efficacious antiretroviral combined therapy (HAART = Highly Active Antiretroviral Therapy) can improve the above described laboratory parameters in HIV-1/HPV co-infected women and seems to prevent the progression of CIN1 to the following stages of the dysplastic disease.

Human herpesvirus 8 and associated diseases in a group of 67 human immunodeficiency virus-seropositive individuals.

Human herpesvirus 8 (HHV-8) is involved in the pathogenesis of Kaposi's sarcoma, of B-cells lymphomas, and of Castelman's disease. However, the role of this virus is not yet well known. To investigate the relationship between HHV-8 infection and diseases correlated with human immunodeficiency virus (HIV), we studied a cohort of 67 HIV-seropositive subjects, some of them coinfected with HHV-8. An indirect immunofluorescence test was employed to detect the antibodies against this virus. Positive cases were 31 (46.3%); among the 67 patients, 14 were weakly positive, or + (20.9%); 11 were significantly positive, or ++ (16.4%); and 6 were strongly positive, or (8.9%). These last six patients were the most affected by opportunistic infections, and all were affected by neoplastic pathologies. Moreover, the HHV-8 positive subjects showed hematologic and martial alterations more severe than those in the negative subjects. HHV-8 seroprevalence in HIV-seropositive patients of our cohort was higher (46.3%) than in normal population (0-10%). The presence of disseminated Kaposi's sarcoma and other neoplasms associated with high HIV-RNA levels in HHV-8-positive patients, and particularly in those with strong positivity, corroborates the hypothesis that the virus is correlated with the progression of HIV infection and with its related diseases, especially those that are neoplastic. Last, the severe alterations of iron metabolism found in the patients coinfected with HHV-8 and the negative effect of this virus on the lymphocytic populations can contribute to the unfavorable evolution of HIV infection and also might facilitate tumor development.

Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type-1.

To investigate whether transferrin receptor (CD71) expression is affected by acute HIV-1 infection, three different lymphoid cell lines (MT-4, SUPT-1, H9) were infected with HIV-1 and tested for surface CD71 expression after different incubation periods depending on cell survival after infection. We found that expression of surface CD71 was lower in cells infected with HIV-1 than in uninfected controls: the timing and extent of this down-modulation depended apparently on the different susceptibility of the cell lines to HIV-1 infection and cytopathogenicity. Citrate, a molecule capable of chelating iron, dose-dependently prevented down-modulation of surface CD71 in HIV-1 infected cells as well as viral cytopathic effects. We conclude that (i) expression of surface transferrin receptors is down-modulated by acute HIV-1 infection in T lymphoid cells, that (ii) this cell phenotypic modulation is associated with the cytopathic effects of the virus, and that (iii) these phenomena are modulated by iron chelation. These results support the view that iron metabolism may be an important area for interaction between HIV-1 and human cells.