Immunogenicity of novel vB_EcoS_NBD2 bacteriophage-originated nanotubes as a carrier for peptide-based vaccines.

Non-infectious virus-like nanoparticles mimic native virus structures and can be modified by inserting foreign protein fragments, making them immunogenic tools for antigen presentation. This study investigated, for the first time, the immunogenicity of long and flexible polytubes formed by yeast-expressed tail tube protein gp39 of bacteriophage vB_EcoS_NBD2 and evaluated their ability to elicit an immune response against the inserted protein fragments. Protein gp39-based polytubes induced humoral immune response in mice, even without the use of adjuvant. Bioinformatics analysis guided the selection of protein fragments from Acinetobacter baumannii for insertion into the C-terminus of gp39. Chimeric polytubes, displaying 28-amino acid long OmpA protein fragment, induced IgG response against OmpA protein fragment in immunized mice. These polytubes demonstrated their effectiveness both as antigen carrier and an adjuvant, when the OmpA fragments were either displayed on chimeric polytubes or used alongside with the unmodified polytubes. Our findings expand the potential applications of long and flexible polytubes, contributing to the development of novel antigen carriers with improved immunogenicity and antigen presentation capabilities.

Electrochemical biosensor for the evaluation of monoclonal antibodies targeting the N protein of SARS-CoV-2 virus.

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein.

Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.

Immunogenic Properties and Antigenic Similarity of Virus-like Particles Derived from Human Polyomaviruses.

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system.

Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages.

Viral antigens can activate phagocytes, inducing inflammation, but the mechanisms are barely explored. The aim of this study is to investigate how viral oligomeric proteins of different structures induce inflammatory response in macrophages. Human THP-1 cell line was used to prepare macrophages that were treated with filamentous nucleocapsid-like particles (NLPs) of paramyxoviruses and spherical virus-like particles (VLPs) of human polyomaviruses. The effects of viral proteins on cell viability, pro-inflammatory cytokines' production, and NLRP3 inflammasome activation were investigated. Filamentous NLPs did not induce inflammation while spherical VLPs mediated inflammatory response followed by NLRP3 inflammasome activation. Inhibitors of cathepsins and K+ efflux decreased IL-1ß release and cell death, indicating a complex inflammasome activation process. A similar activation pattern was observed in primary human macrophages. Single-cell RNAseq analysis of THP-1 cells revealed several cell activation states different in inflammation-related genes. This study provides new insights into the interaction of viral proteins with immune cells and suggests that structural properties of oligomeric proteins may define cell activation pathways.

vB_EcoS_NBD2 bacteriophage-originated polytubes as a carrier for the presentation of foreign sequences.

Virus-based nanoparticles constitute a promising platform for the creation of efficient vaccines and nanomaterials. Previously we demonstrated, that the recombinant tail tube protein gp39 of vB_EcoS_NBD2 bacteriophage self-assembles into extremely long (from 0.1 to >3.95 µm), flexible, and stable polytubes when produced in Saccharomyces cerevisiae. To develop a tubular platform for multivalent display of foreign antigens, yeast-derived recombinant tail tube protein gp39 was chosen as a scaffold. The carboxy-terminal fusions of gp39 with various antigens up to 238 amino acids in length resulted in different synthesis efficiency and self-assembly capacity. Recombinant gp39 fused with green fluorescent protein (eGFP) comprising 238 amino acid residues was capable to self-assemble into short fluorescent polytubes with retained eGFP functional activity. By demonstrating the display of active foreign antigens on the exterior surface of polytubes, these structures may provide a promising tool for diverse applications in nanotechnology.

Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018.

Intensive horizontal gene transfer may generate diversity and heterogeneity within the genus Gardnerella. Restriction-modification (R-M) systems and CRISPR-Cas are the principal defense tools against foreign DNA in bacteria. Nearly half of the tested Gardnerella spp. isolates harbored the CRISPR-Cas system. Several putative R-M systems of Gardnerella spp. strains were identified in the REBASE database. However, there was no experimental evidence for restriction endonuclease (REase) activity in the isolates. We showed that G. vaginalis strain ATCC 14018 contains the REase R.Gva14018I, which recognizes GGCC and most probably generates blunt ends on cleavage. Bioinformatics evidence and the activity of recombinant methyltransferase M.Gva14018I in vivo indicate that ATCC 14018 possesses a HaeIII-like R-M system. The truncated R.Gva14018I-4 lacking the C-terminal region was expressed in Escherichia coli and displayed wild-type REase specificity. Polyclonal antibodies against R.Gva14018I-4 detected the wild-type REase in the cell lysate of ATCC 14018. The cofactor requirements for activity and bioinformatics analysis indicated that R.Gva14018I belongs to the PD-(D/E)XK family of REases. The REase-like activity was observed in 5 of 31 tested Gardnerella spp. strains, although none of these matched the DNA digestion pattern of R.Gva14018I.

Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity.

BACKGROUND: Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker. METHODS: The recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model. RESULTS: The novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in Escherichia coli. The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70-140% of the SCF monomer activity (p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% (p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% (p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF.

Soluble Aß oligomers and protofibrils induce NLRP3 inflammasome activation in microglia.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder causing memory loss, language problems and behavioural disturbances. AD is associated with the accumulation of fibrillar amyloid-ß (Aß) and the formation of neurofibrillary tau tangles. Fibrillar Aß itself represents a danger-associated molecular pattern, which is recognized by specific microglial receptors. One of the key players is formation of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome, whose activation has been demonstrated in AD patient brains and transgenic animal models of AD. Here, we investigated whether Aß oligomers or protofibrils that represent lower molecular aggregates prior to Aß deposition are able to activate the NLRP3 inflammasome and subsequent interleukin-1 beta (IL-1ß) release by microglia. In our study, we used Aß preparations of different sizes: small oligomers and protofibrils of which the structure was confirmed by atomic force microscopy. Primary microglial cells from C57BL/6 mice were treated with the respective Aß preparations and NLRP3 inflammasome activation, represented by caspase-1 cleavage, IL-1ß production, and apoptosis-associated speck-like protein containing a CARD speck formation was analysed. Both protofibrils and low molecular weight Aß aggregates induced a significant increase in IL-1ß release. Inflammasome activation was confirmed by apoptosis-associated speck-like protein containing a CARD speck formation and detection of active caspase-1. The NLRP3 inflammasome inhibitor MCC950 completely inhibited the Aß-induced immune response. Our results show that the NLRP3 inflammasome is activated not only by fibrillar Aß aggregates as reported before, but also by lower molecular weight Aß oligomers and protofibrils, highlighting the possibility that microglial activation by these Aß species may initiate innate immune responses in the central nervous system prior to the onset of Aß deposition. Cover Image for this issue: https://doi.org/10.1111/jnc.14773.

Activation of Macrophages by Oligomeric Proteins of Different Size and Origin.

Activation of macrophages is one of the key processes in generating the immune response against pathogens or misfolded/aggregated otherwise unharmful host's proteins. Antigens and their immune complexes (IC) may shape macrophage phenotype in various directions. Data on the impact of protein structure during inflammation are evident; however, some separate steps of this process involving changes in macrophage phenotype are not fully understood. Our aim was to investigate the phenotype of macrophages after activation with different oligomeric proteins and their IC. We have used amyloid beta (Aß 1-42) that plays a role in neurodegenerative inflammation as a model of host-associated protein and three oligomeric viral antigens as pathogen-associated proteins. Murine cell lines J774, BV-2, and macrophage primary cell culture were treated with oligomeric proteins and their IC. After 48 h, expression of surface markers F4/80, CD68, CD86, and CD206 and secreted cytokines IL-10, IL-12, IL-23, and TNF-α was analysed. Aß 1-42 oligomers stimulated expression of both inflammatory and anti-inflammatory molecules; however, fibrils induced less intense expression of markers investigated as compared to small and large oligomers. Two out of three viral oligomeric proteins induced the inflammatory response of macrophages. Data suggest that macrophage activation pattern depends on the origin, size, and structure of oligomeric proteins.

Investigation of markers of allergic sensitization and viral infections in children with allergy and asthma.

BACKGROUND: Allergic diseases are the most prevalent chronic diseases in the developed countries. It is believed that early allergic sensitization and respiratory viral infections play an important role in the development of allergic diseases and asthma. METHODS: The current study investigated the correlation between asthma, allergy, and various markers - allergen-specific IgE, IgG4 and IgA, ECP, IgM, and IgG antibodies against respiratory viruses hRSV and hPIV1-4 - in blood serum samples from 80 children (mean age 5.2 years) recruited from the Lithuanian birth cohort. Children were divided into three groups according to their diagnosis: asthma (n = 25), allergy without asthma (n = 14), and control group (n = 41). RESULTS: Based on retrospective data, airway infections and bronchitis by the age of two years were associated with asthma in later childhood. The presence of IgM and IgG antibodies against hRSV and hPIV1-4 at the age of five years were not associated with asthma and allergy: a high rate of persistent or past respiratory viral infections was revealed in all three groups. Among allergic children, increased levels of allergen-specific IgE and d1-specific IgG4 were determined. CONCLUSION: The current study provides new insights into the relationships between allergic sensitization and respiratory virus infections in children.

Construction, Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor.

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.

Generation of recombinant Schmallenberg virus nucleocapsid protein in yeast and development of virus-specific monoclonal antibodies.

Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

Antibodies bound to Aß oligomers potentiate the neurotoxicity of Aß by activating microglia.

Beta amyloid (Aß) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aß immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aß would prevent the neurotoxicity of Aß oligomers in primary neuronal-glial cultures. However, surprisingly,the antibodies dramatically increased the neurotoxicity of Aß. Antibodies bound to monomeric Aß fragments were non-toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody-antibody oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aß oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal-glial cultures via Fc-dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aß or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.

Immunogenic properties of amyloid beta oligomers.

BACKGROUND: The central molecule in the pathogenesis of Alzheimer's disease (AD) is believed to be a small-sized polypeptide - beta amyloid (Aß) which has an ability to assemble spontaneously into oligomers. Various studies concerning therapeutic and prophylactic approaches for AD are based on the immunotherapy using antibodies against Aß. It has been suggested that either active immunization with Aß or passive immunization with anti-Aß antibodies might help to prevent or reduce the symptoms of the disease. However, knowledge on the mechanisms of Aß-induced immune response is rather limited. Previous research on Aß1-42 oligomers in rat brain cultures showed that the neurotoxicity of these oligomers considerably depends on their size. In the current study, we evaluated the dependence of immunogenicity of Aß1-42 oligomers on the size of oligomeric particles and identified the immunodominant epitopes of the oligomers. RESULTS: Mice were immunized with various Aß1-42 oligomers. The analysis of serum antibodies revealed that small Aß1-42 oligomers (1-2 nm in size) are highly immunogenic. They induced predominantly IgG2b and IgG2a responses. In contrast, larger Aß1-42 oligomers and monomers induced weaker IgG response in immunized mice. The monoclonal antibody against 1-2 nm Aß1-42 oligomers was generated and used for antigenic characterization of Aß1-42 oligomers. Epitope mapping of both monoclonal and polyclonal antibodies demonstrated that the main immunodominant region of the 1-2 nm Aß1-42 oligomers is located at the amino-terminus (N-terminus) of the peptide, between amino acids 1 and 19. CONCLUSIONS: Small Aß1-42 oligomers of size 1-2 nm induce the strongest immune response in mice. The N-terminus of Aß1-42 oligomers represents an immunodominant region which indicates its surface localization and accessibility to the B cells. The results of the current study may be important for further development of Aß-based vaccination and immunotherapy strategies.