Biosensors for the Determination of SARS-CoV-2 Virus and Diagnosis of COVID-19 Infection

Monitoring and tracking infection is required in order to reduce the spread of the coronavirus disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, the development and deployment of quick, accurate, and sensitive diagnostic methods are necessary. The determination of the SARS-CoV-2 virus is performed by biosensing devices, which vary according to detection methods and the biomarkers which are inducing/providing an analytical signal. RNA hybridisation, antigen-antibody affinity interaction, and a variety of other biological reactions are commonly used to generate analytical signals that can be precisely detected using electrochemical, electrochemiluminescence, optical, and other methodologies and transducers. Electrochemical biosensors, in particular, correspond to the current trend of bioanalytical process acceleration and simplification. Immunosensors are based on the determination of antigen-antibody interaction, which on some occasions can be determined in a label-free mode with sufficient sensitivity.

Immunotherapy for Refractory Autoimmune Encephalitis

Autoimmune encephalitis (AE) is an immune-mediated disease involving the central nervous system, usually caused by antigen-antibody reactions. With the advent of autoantibody-associated diseases, AE has become a hot research frontier in neuroimmunology. The first-line conventional treatments of autoimmune encephalitis consist of steroids, intravenous immunoglobulin (IVIG), plasma exchange (PLEX), and second-line therapy includes rituximab. Despite considerable research and expanding clinical experience, current treatments are still ineffective for a significant number of patients. Although there is no clear consensus, clinical trial evidence limited, and the level of evidence for some of the drugs based on single reports, third-line therapy is a viable alternative for refractory encephalitis patients. With the current rapid research progress, a breakthrough in the treatment of AE is critical. This article aims to review the third-line therapy for refractory AE

Hepatitis B Immunoglobulin Inhibits The Secretion of HBV Via Antigen-Antibody Precipitation in The Multivesicular Body

Although the main action of human hepatitis B immunoglobulin (HBIG) of neutralizing the hepatitis B virus surface antigen (HBsAg) in the serum is known, HBIG is known to be localized in the cell. However, the effect of intracellularly located HBIG is not well elucidated due to the low purity of conventional plasma-derived HBIG (cHBIG). We attempted to clarify the mechanism of action of internalized HBIG using recombinant HBIG (lenvervimab). We used HBsAg cell lines, non-HBsAg cell lines and human HBsAg-producing hepatocytes. Autophagosome lysis pathway related proteins and Rab5, calnexin, giantin, and Rab7 were used to localize HBsAg and anti-HBs-IgG in the cytoplasm with Western blot and confocal microscopy.Intracellular anti-HBs-IgG (lenvervimab and cHBIG) transported by Fc receptor-mediated endocytosis increased the autophagosomes, but there was no change in autolysis. HBsAg and anti-HBs-IgG precipitated in the cytoplasm and co-localized in the multivesicular body. HBsAg secretion in the culture medium was decreased after lenvervimab. Simultaneously, the amount of cellular HBsAg increased in the cell lines but decreased in the human hepatocytes. Furthermore, intracellular lenvervimab was not easily washed out only in the HBsAg cell lines.Lenvervimab decreases the secretion of HBsAg, and HBsAg-antibody precipitation in the multivesicular body might play an important role.

Identifying protein sites contributing to vaccine escape via statistical comparisons of short-term molecular dynamics simulations

The identification of viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a molecular dynamic (MD) based approach that scales well to a desktop computer with a high-end modern graphics processor and enables the user to identify protein sites that are prone to vaccine escape in a viral antigen. We first implemented our MD pipeline to employ site-wise calculation of Kullback-Leibler divergence in atom fluctuation over replicate sets of short-term MD production runs to compare influenza hemagglutinin’s rapid motions in the presence and absence of three well-known neutralizing antibodies. Using this simple comparative method applied to motions of viral proteins, we successfully identified in silico all previously empirically confirmed sites of escape in hemagglutinin, predetermined via selection experiments and neutralization assays. After this validation of our computational approach, we identified potential hot spot residues in the receptor binding domain (RBD) of the SARS-CoV-2 virus in the presence of COVOX-222 and S2H97 antibodies. We identified sites in the antigen-antibody interface with strong dampening of fluctuation that may indicate potential antibody escape due to single mutations. Many of these sites were found to match known sites of mutations in SARS-CoV-2 variants of concern. The determination of single sites with large effect on antigen-antibody binding interfaces is crucial to discriminating neutral variants from potential escape variants. In summary, we provide a cheap, fast, and accurate in silico method to identify and quantify potential hot spots of functional evolution in antibody binding footprints.

Virtual immunology: An educational software to encourage antigen-antibody interaction learning

Immunology is a knowledge area of paramount importance in life sciences and health care professional education with diverse applications, as well as for a general public understanding of issues related to vaccination. However, many concepts are complex and difficult to be understood based only on conventional classes or static images. The use of tools, such as educational software, may enhance the learning of dynamic molecular phenomena that occur in our bodies. Virtual Immunology is a software that aims to facilitate the learning of certain complex immunology concepts. Herein, we present the "antigen-antibody interactions" module that was used and evaluated by 127 students and three teachers from medical schools from two universities, one public and one private, both in the state of Rio de Janeiro, Brazil. The pre-test/post-test research design was used to assess student learning in a randomized sample. To evaluate user perceptions concerning software quality, 14 statements were analyzed using a Likert scale. Results indicate suitable evaluations from both students and teachers concerning the "antigen-antibody module" as an auxiliary tool in immunology teaching. The software was well rated an educational resource since it allows dynamically viewing immunological phenomena. In addition, its ease of use and immunological process visualization were the best-evaluated parameters by the students, which recommended this software module as an auxiliary learning tool. The use of the evaluated software may motivate students and aid in the understanding of immunology-related concepts, becoming a complementary tool that may enhance the teaching-learning process.

Review on the Lymphatic Vessels in the Dental Pulp

Despite many studies, opinions on the lymphatic system of the teeth are still incompatible. Studies using light and electron microscopy and directly using methods such as a radioisotope (radionuclide) scan and interstitial fluid pressure measurement reported incomplete results. Immunohistochemistry (IHC) plays the main role in investigating presence of the lymphatic system in dental tissues. This method uses labeled antibodies against antigens typical of lymphatic vessels. The use of appropriate staining enables the detection of antigen-antibody reaction products using a light (optical), electron or fluorescence microscope. However, these studies do not show the system of vessels, their histologic structure under physiological conditions and inflammation as well as the lymphangiogenesis process in the dental pulp. Unfortunately, there is a lack of studies associating the presence of lymphatic vessels in the dental pulp with local lymphatic nodes or large vessels outside the tooth. In the scientific and research environment, the evaluation of the lymphatic system of the teeth is problematic because it is quite difficult to clearly distinguish lymphatic vessels from small blood vessels. Despite many indications of the presence of lymphatic vessels in the pulp chamber, this problem remains open and needs further research.

Analysis of Proteins by Immunoblotting

In immunoblotting (western blotting), proteins are first separated by SDS-PAGE and then transferred electrophoretically from the gel onto a support membrane that binds proteins tightly. After the unreacted binding sites of the membrane are blocked to suppress nonspecific adsorption of antibodies, the immobilized proteins are reacted with a specific polyclonal or monoclonal antibody. Antigen–antibody complexes are visualized using chromogenic, fluorescent, or chemiluminescent reactions. Immunoblotting protocols are reagent specific and, owing to the wide assortment of equipment, reagents, and antibodies available, highly diverse. Presented here is an example of a workable protocol for developing a blot using horseradish peroxidase (HRP)–conjugated secondary antibody and enhanced chemiluminescence (ECL). ECL is based on the emission of light during the HRP-catalyzed oxidation of luminal or other substrates. Emitted light is captured on film or by a CCD camera, for qualitative or semiquantitative analysis. Because ECL is so sensitive, it has become a popular detection method. This protocol can be modified for different membranes, antibodies, and detection systems. Optimal dilutions of the primary and secondary antibodies need to be determined empirically, but recommendations provided by the manufacturer are usually a good starting point.