Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation.

The 21st century has already brought us a plethora of new threats related to viruses that emerge in humans after zoonotic transmission or drastically change their geographic distribution or prevalence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first spotted at the end of 2019 to rapidly spread in southwest Asia and later cause a global pandemic, which paralyzes the world since then. We have designed novel immunosensors targeting conserved protein sequences of the N protein of SARS-CoV-2 based on lab-produced and purified anti-SARS-CoV-2 nucleocapsid antibodies that are densely grafted onto various surfaces (diamond/gold/glassy carbon). Titration of antibodies shows very strong reactions up to 1:72 900 dilution. Next, we showed the mechanism of interactions of our immunoassay with nucleocapsid N protein revealing molecular recognition by impedimetric measurements supported by hybrid modeling results with both density functional theory and molecular dynamics methods. Biosensors allowed for a fast (in less than 10 min) detection of SARS-CoV-2 virus with a limit of detection from 0.227 ng/ml through 0.334 ng/ml to 0.362 ng/ml for glassy carbon, boron-doped diamond, and gold surfaces, respectively. For all tested surfaces, we obtained a wide linear range of concentrations from 4.4 ng/ml to 4.4 pg/ml. Furthermore, our sensor leads to a highly specific response to SARS-CoV-2 clinical samples versus other upper respiratory tract viruses such as influenza, respiratory syncytial virus, or Epstein-Barr virus. All clinical samples were tested simultaneously on biosensors and real-time polymerase chain reactions.

Chimeric virus-like particles presenting tumour-associated MUC1 epitope result in high titers of specific IgG antibodies in the presence of squalene oil-in-water adjuvant: towards safe cancer immunotherapy.

BACKGROUND: Immunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP-based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy. RESULTS: In this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen. CONCLUSIONS: The obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens.