Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate.

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.

A competitive ELISA for the quantitative determination of the novel anti-HIV drug candidate CIGB-210 in biological fluids.

The synthetic peptide CIGB-210 is a promising anti-HIV drug candidate shown to inhibit HIV replication in MT4 cells at the nanomolar range by triggering the rearrangement of vimentin intermediate filaments. Sensitive and specific analytical methods are required for pharmacological studies of CIBG-210 in animals. In this study, we describe the development of a competitive ELISA for the quantitative determination of CIGB-210 using an anti-CIGB-210 hyperimmune serum. After optimization of all the steps, the assay exhibited a dynamic range from 11.87 to 0.0095 µg/mL. The intra-assay coefficient of variation (CV) was lower than or close to 5% for all the six concentrations of the calibrator, and the inter-assay CV was below 10% in five out of the six concentrations tested. No interference of either murine or human plasma was observed. The analyte was stable in plasma after five freeze-thaw cycles, while the hyperimmune serum maintained its binding capacity after 10 freeze-thaw cycles. Furthermore, the ELISA was able to detect the two main metabolites of CIGB-210, although with a tenfold decrease in sensitivity. Our results demonstrate the utility and feasibility of this analytical method for pharmacological experiments in animals as humans.

Identification of Vimentin as a Potential Therapeutic Target against HIV Infection.

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.

Non-induced leukocyte extract reduces HIV replication and TNF secretion.

According to UNAIDS, the global HIV/AIDS epidemic increased to 40 million the number of people living with the virus around the world. Dialyzable leukocyte extract obtained by our group is a low molecular weight dialyzable material from peripheral human leukocytes previously in vitro induced with Sendai virus (DLE-ind), and more recently, from non-induced leukocytes (DLE n/i). Previous results have shown the ability of DLE-ind to inhibit HIV in vitro replication in MT4 cell; to reduce TNFalpha secretion, and to delay in vivo progression to AIDS in early stage of HIV infection. In this work we present evidences that DLE n/i also inhibits HIV in vitro replication and reduces TNFalpha secretion in human whole blood like DLE obtained from induced leukocytes. Taking together these results show that both properties of DLE, HIV in vitro inhibition and TNF production modulation, are not dependent on in vitro Sendai virus induction of leukocytes.