FIGURE 2 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
| Amino acid sequences of VHH amplified from the transformants of VHH-E. coli library. (A)-Sequences of 15 randomly selected clones were aligned and regions of framework and CDRs are marked. Two cysteine residues (C21 and C97) that can mediate the formation of a disulfide bond are marked with asterisks. The similarity of residues within the framework region 1-3 and high variation of residues in CDR regions: 1-3 is noticeable in the consensus. (B)-A distance matrix plot generated on the sequences from (A). Number of amino acid residues differing in clone comparison is mentioned in each box. Various shades of gray correspond to the difference in residues in which white represents maximum difference and black represents no difference.
Source publication
Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A ³³ to K ⁶⁹ ) and the first coiled-coil dom...
Contexts in source publication
Context 1
... of the hBMECs (D3 cell line, Merck/Millipore), and extraction of the membrane proteins was performed as described in our previous publication . Four hundred micrograms of hBMEC proteins were separated on SDS-PAGE and transblotted on a nitrocellulose membrane (Supplementary Figure 2A). The membrane was cut vertically to obtain strips of 0.3 cm and they were subjected to western blot with 5 µg of rec-NadA as described previously ( Mertinková et al., 2020). ...
Context 2
... membrane was cut vertically to obtain strips of 0.3 cm and they were subjected to western blot with 5 µg of rec-NadA as described previously ( Mertinková et al., 2020). The specific interaction of rec-NadA with ∼15 kDa receptor of hBMECs was reported (Supplementary Figure 2B). In the present study, VHH targeting NadA-gd A33−K69 and NadAcc L121−K157 were tested either individually or in combination to block the interaction of rec-NadA with ∼15 kDa hBMEC proteins (possible receptor). ...
Context 3
... from fifteen randomly picked clones from the VHH-E. coli library were sequenced and their amino-acid sequences were aligned to define CDR and framework regions (Figure 2A). A distance matrix was plotted based on amino-acid sequences ( Figure 2B). ...
Context 4
... library were sequenced and their amino-acid sequences were aligned to define CDR and framework regions (Figure 2A). A distance matrix was plotted based on amino-acid sequences ( Figure 2B). The matrix showed that 3 clones (clone numbers 2, 6, and 7) carry similar VHH sequence. ...
Context 5
... clone numbers 10 and 11 had similar VHH sequence. All other clones were having a unique VHH sequence (Figure 2). VHH-E. ...
Context 6
... our recent study it was shown that rec-NadA interacts with hBMEC, mediated through ∼15 kDa receptor [(Mertinková et al., 2020), Supplementary Figures 2A,B]. One of the aims of the present study was to block this interaction using VHH. ...
Context 7
... the experimental blocking of the adhesion of NadA to hBMECs, the minimum concentration of rec-NadA required to detect visible interaction to the endothelial receptor was assessed. In the western blot targeting ∼15 kDa receptor, it was observed that a minimum of 125 ng of rec-NadA was necessary to detect the interaction (Supplementary Figure 2C). ...
Context 8
... sequence typically has two cysteine residues one in framework region 1 and another in framework region 3 (Figure 2A; Supplementary Figure 6) (Mitchell and Colwell, 2018). The formation of disulfide bonds between these two cysteine residues is essential to maintain the proper folding and functionality of the nanobodies ( de Marco, 2015). ...
Similar publications
Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene...
Background
The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti- mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents....
By inhibiting intracellular KRAS and STAT3, SBT-100 suppresses the growth of human tumors in vivo, decreases VEGF and PD-L1 expression, inhibits IL-6 function in cancers, suppresses IL-17, GM-CSF, IFN-gamma, IL-1-alpha, and downregulates Th17 cells.
Purpose of Review
Two VHH (camelid heavy chain variable region) antibodies are being used to treat...
Citations
... The bacterial strain was thoroughly characterised before the development of the animal model [14][15][16][17]. ...
Background:
Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds.
Method:
We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain-heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood-brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised.
Results:
The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group).
Conclusion:
We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.
... In a recent study, Nbs VHHG9 and VHHF3 targeting Neisseria adhesin A (NadA), which can antagonize the interaction between recombinant NadA and cell receptors, were developed. The preincubation of Neisseria meningitidis with VHHF3 and VHHG9 could significantly reduce the adhesion of neisseria meningitidis to human microvascular endothelial cells in situ and prevent it from crossing a BBB model (human BMECs) in vitro, providing a new treatment idea (127). Rabies is also an infectious disease in which the CNS is mainly affected. ...
Nanobodies are antibody fragments derived from camelids, naturally endowed with properties like low molecular weight, high affinity and low immunogenicity, which contribute to their effective use as research tools, but also as diagnostic and therapeutic agents in a wide range of diseases, including brain diseases. Also, with the success of Caplacizumab, the first approved nanobody drug which was established as a first-in-class medication to treat acquired thrombotic thrombocytopenic purpura, nanobody-based therapy has received increasing attention. In the current review, we first briefly introduce the characterization and manufacturing of nanobodies. Then, we discuss the issue of crossing of the brain-blood-barrier (BBB) by nanobodies, making use of natural methods of BBB penetration, including passive diffusion, active efflux carriers (ATP-binding cassette transporters), carrier-mediated influx via solute carriers and transcytosis (including receptor-mediated transport, and adsorptive mediated transport) as well as various physical and chemical methods or even more complicated methods such as genetic methods via viral vectors to deliver nanobodies to the brain. Next, we give an extensive overview of research, diagnostic and therapeutic applications of nanobodies in brain-related diseases, with emphasis on Alzheimer’s disease, Parkinson’s disease, and brain tumors. Thanks to the advance of nanobody engineering and modification technologies, nanobodies can be linked to toxins or conjugated with radionuclides, photosensitizers and nanoparticles, according to different requirements. Finally, we provide several perspectives that may facilitate future studies and whereby the versatile nanobodies offer promising perspectives for advancing our knowledge about brain disorders, as well as hopefully yielding diagnostic and therapeutic solutions.
Phage antibody display technology has revolutionized the field of bacterial immunodetection. This technology allows the expression of an antibody fused to the coat protein of a filamentous bacteriophage. The use of phage display makes it possible to obtain high-affinity antibodies by passing the stage of animal immunization, reducing the time for obtaining stable antibody-producing clones from several months to several weeks, significantly reducing the cost of the process. These advantages make phage antibodies an important tool for bacterial detection. The paper presents a brief description of the technological methods for obtaining phage antibodies to microbial cells. The possibilities and prospects for using phage antibodies as a selective agent in analytical systems, including biosensors, are discussed.
An infection caused by bacteria is one of the main factors that poses a threat to human health. A recent report from the World Health Organization (WHO) has highlighted that bacteria that cause blood infections have become increasingly drug-resistant. Therefore, it is crucial to research and develop new techniques for detecting and treating these infections. Since their discovery, nanobodies have exhibited numerous outstanding biological properties. They are easy to express, modify, and have high stability, robust permeability and low immunogenicity, all of which indicate their potential as a substitute. Nanobodies have been utilized in a variety of studies on viruses and cancer. This article primarily focuses on nanobodies and introduces their characteristics and application in the diagnosis and treatment of bacterial infections.
Antibody phage display, aimed at preparing antibodies to defined antigens, is a useful replacement for hybridoma technology. The phage system replaces all work stages that follow animal immunization with simple procedures for manipulating DNA and bacteria. It enables the time needed to generate stable antibody-producing clones to be shortened considerably, making the process noticeably cheaper. Antibodies prepared by phage display undergo several affinity selection steps and can be used as selective receptors in biosensors. This article briefly describes the techniques used in the making of phage antibodies to various antigens. The possibilities and prospects are discussed of using phage antibodies as selective agents in analytical systems, including biosensors.
Neisseria meningitidis is a gram-negative diplococcus and a transient commensal of the human nasopharynx. It shares and competes for this niche with a number of other Neisseria species including N. lactamica, N. cinerea and N. mucosa. Unlike these other members of the genus, N. meningitidis may become invasive, crossing the epithelium of the nasopharynx and entering the bloodstream, where it rapidly proliferates causing a syndrome known as Invasive Meningococcal Disease (IMD). IMD progresses rapidly to cause septic shock and meningitis and is often fatal despite aggressive antibiotic therapy. While many of the ways in which meningococci survive in the host environment have been well studied, recent insights into the interactions between N. meningitidis and the epithelial, serum, and endothelial environments have expanded our understanding of how IMD develops. This review seeks to incorporate recent work into the established model of pathogenesis. In particular, we focus on the competition that N. meningitidis faces in the nasopharynx from other Neisseria species, and how the genetic diversity of the meningococcus contributes to the wide range of inflammatory and pathogenic potentials observed among different lineages.
West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV’s envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIIIL297–S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1G299–K307 and DIII-2V371–R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1G299–K307 and DIII-2V371–R388 on a dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the WNV–like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC50) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAbA10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAbA10 targeting DIII-2V371–R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV–induced neuropathogenesis.
Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeutics. The common mammalian antibody immunoglobulin G (IgG) is a complex glycosylated macromolecule, composed of two heavy chains and two light chains held together by multiple disulfide bonds. For this reason, IgG and related antibody fragments are usually produced through secretion from mammalian cell lines, such as Chinese Hamster Ovary cells. However, there is growing interest in production of antibodies in prokaryotic systems due to the potential for rapid and cheap production in a highly genetically manipulable system. Research on oxidative protein folding in prokaryotes has enabled engineering of Escherichia coli strains capable of producing IgG and other disulfide bonded proteins in the cytoplasm, known as SHuffle. In this protocol, we provide a review of research on prokaryotic antibody production, guidelines on cloning of antibody expression constructs, conditions for an initial expression and purification experiment, and parameters which may be optimized for increased purification yields. Last, we discuss the limitations of prokaryotic antibody production, and highlight potential future avenues for research on antibody expression and folding.
Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.
