Today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. Compared to conventional antiviral drugs, monoclonal antibodies (mAbs) used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. Despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. The current high cost of mAbs' production, storage and administration (by injection only) and the consequent obstacles to development are outweighed by mAbs' clinical advantages. These are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses.

Background: The multidrug transporter P‐glycoprotein (P‐gp) is expressed in HIV‐1 target cells, in a range of pharmacological barriers and in AIDS‐associated tumours. P‐gp substrates include HIV‐1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug‐resistant (MDR) cells.Objectives: The aim of this study was to investigate the effect on human CD4 T‐lymphoblastoid CEM^rev cells of saquinavir and other PIs in terms of P‐gp expression and to characterize the functional and biochemical patterns of PI‐induced P‐gp molecules.Methods: CEM^rev cells no longer expressing detectable amounts of P‐gp were cultured for a prolonged period in the presence of 10 μg/mL saquinavir (CEM^saq10) and tested for P‐gp expression and function. Subsequently, CEM^saq10 cells were transferred into medium containing 15 μg/mL saquinavir (CEM^saq15) and cultured for several months. These cell lines were continuously monitored for P‐gp expression, function and immunochemical patterns. A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P‐gp expression in CEM^rev cells.Results: Compared with the drug‐diluent control, the exposure of CEM^rev cells to 10μg/mL saquinavir induced, in a consistent fraction of cells (45–50%), de novo expression of functioning P‐gp molecules. The transfer of CEM^saq10 cells to 15μg/mL saquinavir was associated with a dramatic increase in P‐gp expression and function (85–90% of CEM^saq15 cells expressed P‐gp and effluxed P‐gp dye substrates). These saquinavir‐induced P‐gp molecules included 75‐kDa molecules as well as the classical 170‐kDa form of P‐gp, suggesting induction of a particular isoform of P‐gp termed mini‐P‐glycoprotein. Conversely, ritonavir and indinavir induced transient P‐gp expression in a small percentage of the CEM^rev cells.Conclusions: Treatment of human CD4 T‐lymphoblastoid CEM^rev cells with saquinavir caused over‐expression of functioning P‐gp molecules. This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P‐gp were observed in CEM^saq10 and CEM^saq15 cells during prolonged in vitro culturing, even in drug‐free conditions.

Background: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration.Methods: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, byin vitromutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized.Results: The scFv MA39 antibody was affinity-maturated byin vitromutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells.Conclusion: The binding affinity of the scFv E8 is in a range for efficient,in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

Many recent studies have reported the onset of a robust antibody response to SARS-CoV-2 infection and highlighted produced antibodies’ specific qualitative and quantitative aspects, relevant for developing antibody-based diagnostic and therapeutic options. In this review, firstly we will report main information acquired so far regarding the humoral response to COVID-19; we will concentrate, in particular, upon the observed levels and the kinetics, the specificity spectrum and the neutralizing potential of antibodies produced in infected patients. We will then discuss the implication of humoral response’s characteristics in the development and correct use of serologic tests, as well as the efficacy and safety of convalescent plasma therapy and of neutralizing monoclonal antibodies for treating infected patients and preventing new infections. An update of the list of newly isolated specific neutralizing antibodies and suggestions for vaccine evaluation and development will be also provided.

Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/β) response, and represents a suitable target for the development of strategies to control EBOV infection. Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells. Monoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-β-induced RIG-I signaling cascade mediated by EBOV VP35. Five antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections.

Background: Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods: The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results: Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion: The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.