RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins

Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-β bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

Boron-Rich Boron Carbide Nanoparticles as a Carrier in Boron Neutron Capture Therapy: Their Influence on Tumor and Immune Phagocytic Cells

The aim of the work was to study the interaction between boron-rich boron carbide nanoparticles and selected tumor and immune phagocytic cells. Experiments were performed to investigate the feasibility of the application of boron carbide nanoparticles as a boron carrier in boron neutron capture therapy. Boron carbide powder was prepared by the direct reaction between boron and soot using the transport of reagents through the gas phase. The powder was ground, and a population of nanoparticles with an average particle size about 80 nm was selected by centrifugation. The aqueous suspension of the nanoparticles was functionalized with human immunoglobulins or FITC-labeled human immunoglobulins and was then added to the MC38 murine colon carcinoma and to the RAW 264.7 cell line of mouse macrophages. Flow cytometry analysis was used to determine interactions between the functionalized boron carbide nanoparticles and respective cells. It was shown that B4C–IgG nanoconjugates may bind to phagocytic cells to be internalized by them, at least partially, whereas such nanoconjugates can only slightly interact with molecules on the cancer cells’ surface.

Staphylococcal Protein A Induces Leukocyte Necrosis by Complexing with Human Immunoglobulins

Staphylococcus aureus is one of the largest health care threats faced by humankind, with a reported mortality rate within the United States greater than that of HIV/AIDS, tuberculosis, and viral hepatitis combined. One of the defining features of S. aureus as a human pathogen is its ability to evade and impair the human immune response through expression of staphylococcal protein A.

Framework for Characterizing Longitudinal Antibody Response in Children After Plasmodium falciparum Infection

Human Plasmodium infection produces a robust adaptive immune response. Time courses for 104 children followed for 42 days after initiation of Plasmodium falciparum chemotherapy were assayed for antibody levels to the five isotypes of human immunoglobulins (Ig) and 4 subclasses of IgG for 32 P. falciparum antigens encompassing all 4 parasite stages of human infection. IgD and IgE against these antigens were undetectable at 1:100 serum concentration, but other Ig isotypes and IgG subclasses were consistently observed against all antigens. Five quantitative parameters were developed to directly compare Ig response among isotypes and antigens: Cmax, maximum antibody level; ΔC, difference between Cmax and the antibody level at Day 0; tmax, time in days to reach Cmax; t1/2, Ig signal half-life in days; tneg, estimated number of days until complete loss of Ig signal. Classical Ig patterns for a bloodborne pathogen were seen with IgM showing early tmax and IgG production highest among Ig isotypes. However, some unexpected trends were observed such as IgA showing a biphasic pattern for many antigens. Variability among these dynamics of Ig acquisition and loss was noted for different P. falciparum antigens and able to be compared both quantitatively and statistically. This parametrization methodology allows direct comparison of Ig isotypes produced against various Plasmodium antigens following malaria infection, and the same methodology could be applied to other longitudinal serologic studies from P. falciparum or different pathogens. Specifically for P. falciparum seroepidemiological studies, reliable and quantitative estimates regarding the IgG dynamics in human populations can better optimize modeling efforts for serological outputs.

The study of interaction of different forms of human recombinant anti-mullerian hormone with a chimeric analogue of the AMH type II

The homodimeric glycoprotein, anti-mullerian hormone (AMH), described over 70 years ago by A. Jost, is the least studied member of the transforming growth factor beta superfamily. Despite the antitumor activity of AMH discovered at the end of the last century, the creation of effective drugs based on AMH is hindered primarily by the lack of information on the mechanism of various AMH forms interaction with a specific type II receptor (MISRII). Previously, we have shown that not only the full-length activated hormone but also its C-terminal fragment (C-rAMH) could bind to MISRII. In this work, using the surface plasmon resonance technique, we compared the interaction of three forms of recombinant AMH (rAMH) with the MISRII analogue — the chimeric protein MISRII-Fc containing AMH type II receptor and a Fc-fragment of the human IgG1 heavy chain. Comparison of the binding of MISRII-Fc, immobilized on a chip with group specificity for human immunoglobulins, to C-rAMH, to intact rAMH (pro-rAMH), and to rAMH containing one uncleaved monomer (hc-rAMH), showed that the KD of the complexes increased: 1.7 nM, 88 nM and 110 nM, respectively. Thus, we have shown that C-terminal fragment of AMH has the maximum affinity for the recombinant MISRII analogue, which indicates the prospects for the development of drugs based on this hormone derivative.

Obtaining of the Stable Formulation of Reference Sample for Human IgG Antibodies to Tick-Borne Encephalitis

To ensure a long-term stability of the reference IgG antibodies to tick-borne encephalitis is an urgent task. The shelf life of immunoglobulin isolated from human immune blood plasma can be increased by freeze-drying an antibody solution stabilized with L-proline and glycine at a concentration of 12.5 g/L each and adjusting the pH value to 5.0± 0.5. The proposed method was used to obtain 3 batches of a reference sample with a shelf life from 30 to 48 months. During this time, the tested samples retained constant potency, fragmentation, absobance (400 nm) and dissolution time, which allows predicting its further stability during storage. The proposed method can also be helpful for obtaining stable antibody lyophilisates for use as standards in the analysis of the specific activity of human immunoglobulins against tick-borne encephalitis. reference standard, human tick-borne encephalitis immunoglobulin, L-proline, glycine, lyophilization, specific activity, production technology The work was carried out in accordance with the state assignment "Improvement of Quality Control Methods for Human Immunoglobulins".

Switching from Multiplex to Multimodal Colorimetric Lateral Flow Immunosensor

Multiplex lateral flow immunoassay (LFIA) is largely used for point-of-care testing to detect different pathogens or biomarkers in a single device. The increasing demand for multitargeting diagnostics requires multi-informative single tests. In this study, we demonstrated three strategies to upgrade standard multiplex LFIA to multimodal capacity. As a proof-of-concept, we applied the strategies to the differential diagnosis of Human Immunodeficiency Virus (HIV) infection, a widespread pathogen, for which conventional multiplex LFIA testing is well-established. In the new two-parameter LFIA (x2LFIA), we exploited color encoding, in which the binding of multiple targets occurs in one reactive band and the color of the probe reveals which one is present in the sample. By combining the sequential alignment of several reactive zones along the membrane of the LFIA strip and gold nanoparticles and gold nanostars for the differential visualization, in this demonstration, the x2LFIA can furnish information on HIV serotype and stage of infection in a single device. Three immunosensors were designed. The use of bioreagents as the capturing ligand anchored onto the membrane or as the detection ligand labelled with gold nanomaterials affected the performance of the x2LFIA. Higher detectability was achieved by the format involving the HIV-specific antigens as capturing agent and labelled secondary bioligands (anti-human immunoglobulins M and protein G) as the probes.

Rapid response platform to manufacture human immunoglobulins

A rapidly deployable drug product for use in the early stages of a disease outbreak could prevent further spread of the pathogen. Emergent BioSolutions is developing a rapid response platform (RRP) to manufacture human immunoglobulins (HIG) for use as a passive immunotherapy during public health emergencies. Passive immunotherapies derived from either convalescent plasma or purified immunoglobulins (Ig) have a long history of success in treating a range of viral, bacterial and toxin diseases, as they provide the immediate benefit of a protective response. Emergent's RRP is being developed to address the need for front-line therapy/prophylaxis with immediate benefit with the potential to overcome the current limitations of convalescent plasma. Emergent's proposed RRP begins with identification of plasma donors with measurable levels of pathogen-specific antibodies using a field deployable real-time donor screening assay. Plasma collected from eligible donors is treated using the Mirasol® Pathogen Reduction Technology, pooled, purified and concentrated into HIG using Emergent's rapid HIG manufacturing process to produce product formulated for intramuscular administration. Our rapid HIG process is chromatography based and each 30 L run aims to purify, formulate, and fill ∼65 doses (1 g of total IgG) with testing and release onsite for immediate use. Multiple innovations to streamline operations, including single-use technologies and minimal human interfaces, allow for the manufacture of HIG product in ∼30 hours. To house the process, Emergent has designed and constructed a working modular manufacturing unit (MMU) prototype. The MMU consists of a modified 53-foot shipping container designed for multiple modes of transport, to meet ISO 8 environment requirements with self-contained utilities, including HVAC, and can run off a diesel generator or local power. Product from multiple development runs within the MMU have been characterized and validation work is ongoing. Key messages A modular manufacturing unit is being developed with the potential to provide a local capability to produce health solutions for endemic diseases. A modular manufacturing unit is being developed with the potential to rapidly manufacture human immunoglobulins during public health emergencies and pandemics.