A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.

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COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host’s cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

Abstract:In this study, we examined the expression of hepatitis C virus (HCV) components in hepatocytes and peripheral blood mononuclear cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA. The samples obtained from 25 randomly selected patients (13 of 25 patients showed no histological evidences of chronic hepatitis and had normal serum ALAT and GGTP levels) were studied by in situ Hybridization and Immunofluorescence assays. The findings show that HCV-RNA of both positive and negative polarity was carried in the hepatocytes of more than 80% of cases. The proportion of cells expressing the negative strand (mean ± SD, 10.25% ± 5.56%) was lower than those expressing positive strand (mean ± SD, 19.88% ± 9.19%) (p=0.0076; Student's t test). In addition, reaction products suggestive of HCV core, E1 and E2 antigens within hepatocytes were also observed. Both hybridization and immunofluorescence signals were localized in the cytoplasm suggesting that this is the place of active HCV replication. On the other hand, the HCV-RNA of positive polarity was detected in PBMC from 16 out of 17 samples analyzed (94%) while the HCV-RNA of negative polarity was detected in 82% of samples investigated. Positive hybridization signals were localized in the cytoplasm of PBMC. Interestingly, 12 out of 13 patients with clinical and histological resolution of hepatitis, contain HCV-RNA in either liver or PBMC. These results provide further evidences that the intermediate replicative form of the HCV genome can persist in hepatocytes and PBMC after apparently complete resolution of chronic hepatitis C.

Understanding the mechanism of Hepatitis C Virus (HCV) pathogenesis is an important part of HCV research. In this study, the presence of apoptosis in HCV-infected liver and Peripheral Blood Mononuclear Cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA was investigated. The samples obtained from 21 patients were studied by in situ Hybridization (ISH), Immunofluorescence, TUNEL reaction and caspase 3 activation assays. The findings show that both DNA fragmentation by TUNEL assay and activation of caspase 3 were detected in hepatocytes from patients histologically confirmed as bearing chronic hepatitis or with abnormal ALAT or GGTP as well as in patients with no histological evidences of chronic hepatitis and normalization of transaminases. Apoptotic cells were also detected in PBMC samples by the TUNEL assay. ISH analysis of liver biopsies and PBMC samples showed both positive and negative strands of the HCV genome localized in some cells showing nuclear characteristics of apoptosis such as chromatin margination, condensation and fragmentation. These typical morphological changes of apoptotic cell death were also observed in some hepatocytes showing reaction products suggestive of HCcAg. Data suggest that under certain conditions HCV induces apoptosis in the absence of liver injury. Induction of apoptosis in HCV-infected cells may interfere with viral replication, which may lead to undetectable levels of HCV-RNA in serum