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.

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.

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.

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.