Toward a SARS-CoV-2 VLP Vaccine: HBc/G as a Carrier for SARS-CoV-2 Spike RBM and Nucleocapsid Protein-Derived Peptides.

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2-220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.

Establishment and Characterization of Free-Floating 3D Macrophage Programming Model in the Presence of Cancer Cell Spheroids.

Reprogramming of tumor-associated macrophages (TAMs) is a promising strategy for cancer immunotherapy. Several studies have shown that cancer cells induce/support the formation of immunosuppressive TAMs phenotypes. However, the specific factors that orchestrate this immunosuppressive process are unknown or poorly studied. In vivo studies are expensive, complex, and ethically constrained. Therefore, 3D cell interaction models could become a unique framework for the identification of important TAMs programming factors. In this study, we have established and characterized a new in vitro 3D model for macrophage programming in the presence of cancer cell spheroids. First, it was demonstrated that the profile of cytokines, chemokines, and surface markers of 3D-cultured macrophages did not differ conceptually from monolayer-cultured M1 and M2-programmed macrophages. Second, the possibility of reprogramming macrophages in 3D conditions was investigated. In total, the dynamic changes in 6 surface markers, 11 cytokines, and 22 chemokines were analyzed upon macrophage programming (M1 and M2) and reprogramming (M1→M2 and M2→M1). According to the findings, the reprogramming resulted in a mixed macrophage phenotype that expressed both immunosuppressive and anti-cancer immunostimulatory features. Third, cancer cell spheroids were shown to stimulate the production of immunosuppressive M2 markers as well as pro-tumor cytokines and chemokines. In summary, the newly developed 3D model of cancer cell spheroid/macrophage co-culture under free-floating conditions can be used for studies on macrophage plasticity and for the development of targeted cancer immunotherapy.

Recombinant Viral Vectors for Therapeutic Programming of Tumour Microenvironment: Advantages and Limitations.

Viral vectors have been widely investigated as tools for cancer immunotherapy. Although many preclinical studies demonstrate significant virus-mediated tumour inhibition in synergy with immune checkpoint molecules and other drugs, the clinical success of viral vector applications in cancer therapy currently is limited. A number of challenges have to be solved to translate promising vectors to clinics. One of the key elements of successful virus-based cancer immunotherapy is the understanding of the tumour immune state and the development of vectors to modify the immunosuppressive tumour microenvironment (TME). Tumour-associated immune cells, as the main component of TME, support tumour progression through multiple pathways inducing resistance to treatment and promoting cancer cell escape mechanisms. In this review, we consider DNA and RNA virus vectors delivering immunomodulatory genes (cytokines, chemokines, co-stimulatory molecules, antibodies, etc.) and discuss how these viruses break an immunosuppressive cell development and switch TME to an immune-responsive "hot" state. We highlight the advantages and limitations of virus vectors for targeted therapeutic programming of tumour immune cell populations and tumour stroma, and propose future steps to establish viral vectors as a standard, efficient, safe, and non-toxic cancer immunotherapy approach that can complement other promising treatment strategies, e.g., checkpoint inhibitors, CAR-T, and advanced chemotherapeutics.

Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model.

Capsid assembly modulators (CAMs) have emerged as a promising class of antiviral agents. We studied the effects of twenty-one newly designed and synthesized CAMs including heteroaryldihydropyrimidine compounds (HAPs), their analogs and standard compounds on hepatitis B virus (HBV) capsid assembly. Cytoplasmic expression of the HBV core (HBc) gene driven by the exogenously delivered recombinant alphavirus RNA replicon was used for high level production of the full-length HBc protein in mammalian cells. HBV capsid assembly was assessed by native agarose gel immunoblot analysis, electron microscopy and inhibition of virion secretion in HepG2.2.15 HBV producing cell line. Induced fit docking simulation was applied for modelling the structural relationships of the synthesized compounds and HBc. The most efficient were the HAP class compounds-dihydropyrimidine 5-carboxylic acid n-alkoxyalkyl esters, which induced the formation of incorrectly assembled capsid products and their accumulation within the cells. HBc product accumulation in the cells was not detected with the reference HAP compound Bay 41-4109, suggesting different modes of action. A significant antiviral effect and substantially reduced toxicity were revealed for two of the synthesized compounds. Two new HAP compounds revealed a significant antiviral effect and a favorable toxicity profile that allows these compounds to be considered promising leads and drug candidates for the treatment of HBV infection. The established alphavirus based HBc expression approach allows for the specific selection of capsid assembly modulators directly in the natural cell environment.

UV inactivation of Semliki Forest virus and E. coli bacteria by alternative light sources.

The quick spreading of the SARS-CoV-2 virus, initiating the global pandemic with a significant impact on economics and health, highlighted an urgent need for effective and sustainable restriction mechanisms of pathogenic microorganisms. UV-C radiation, causing inactivation of many viruses and bacteria, is one of the tools for disinfection of different surfaces, liquids, and air; however, mainly mercury 254 nm line is commonly used for it. In this paper, we report our results of the experiments with newly elaborated special type polychromatic non-mercury UV light sources, having spectral lines in the spectral region from 190 nm to 280 nm. Inactivation tests were performed with both Escherichia coli (E.coli) bacteria and Semliki Forest virus (SFV) as a representative of human enveloped RNA viruses. In addition, the effect of prepared lamps on virus samples in liquid and dry form (dried virus-containing solution) was tested. Reduction of 4 log10 of E.coli was obtained after 10 min of irradiation with both thallium-antimony and arsenic high-frequency electrodeless lamps. High reduction results for the arsenic light source demonstrated sensitivity of E. coli to wavelengths below 230 nm, including spectral lines around 200 nm. For the Semliki Forest virus, the thallium-antimony light source showed virus inactivation efficiency with a high virus reduction rate in the range of 3.10 to > 4.99 log10 within 5 min of exposure. Thus, the new thallium-antimony light source showed the most promising disinfection effect in bacteria and viruses, and arsenic light sources for bacteria inactivation, opening doors for many applications in disinfection systems, including for pathogenic human RNA viruses.

Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor.

Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases-N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11-as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central "hub" protein that regulates the activity of at least seven methyltransferases.

Alphavirus-Driven Interferon Gamma (IFNg) Expression Inhibits Tumor Growth in Orthotopic 4T1 Breast Cancer Model.

Interferon gamma (IFNg) is a pleiotropic cytokine that can potentially reprogram the tumor microenvironment; however, the antitumor immunomodulatory properties of IFNg still need to be validated due to variable therapeutic outcomes in preclinical and clinical studies. We developed a replication-deficient Semliki Forest virus vector expressing IFNg (SFV/IFNg) and evaluated its immunomodulatory antitumor potential in vitro in a model of 3D spheroids and in vivo in an immunocompetent 4T1 mouse breast cancer model. We demonstrated that SFV-derived, IFN-g-stimulated bone marrow macrophages can be used to acquire the tumoricidal M1 phenotype in 3D nonattached conditions. Coculturing SFV/IFNg-infected 4T1 spheroids with BMDMs inhibited spheroid growth. In the orthotopic 4T1 mouse model, intratumoral administration of SFV/IFNg virus particles alone or in combination with the Pam3CSK4 TLR2/1 ligand led to significant inhibition of tumor growth compared to the administration of the control SFV/Luc virus particles. Analysis of the composition of intratumoral lymphoid cells isolated from tumors after SFV/IFNg treatment revealed increased CD4+ and CD8+ and decreased T-reg (CD4+/CD25+/FoxP3+) cell populations. Furthermore, a significant decrease in the populations of cells bearing myeloid cell markers CD11b, CD38, and CD206 was observed. In conclusion, the SFV/IFNg vector induces a therapeutic antitumor T-cell response and inhibits myeloid cell infiltration in treated tumors.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Characterization of Novel Biopolymer Blend Mycocel from Plant Cellulose and Fungal Fibers.

In this study unique blended biopolymer mycocel from naturally derived biomass was developed. Softwood Kraft (KF) or hemp (HF) cellulose fibers were mixed with fungal fibers (FF) in different ratios and the obtained materials were characterized regarding microstructure, air permeability, mechanical properties, and virus filtration efficiency. The fibers from screened Basidiomycota fungi Ganoderma applanatum (Ga), Fomes fomentarius (Ff), Agaricus bisporus (Ab), and Trametes versicolor (Tv) were applicable for blending with cellulose fibers. Fungi with trimitic hyphal system (Ga, Ff) in combinations with KF formed a microporous membrane with increased air permeability (>8820 mL/min) and limited mechanical strength (tensile index 9-14 Nm/g). HF combination with trimitic fungal hyphae formed a dense fibrillary net with low air permeability (77-115 mL/min) and higher strength 31-36 Nm/g. The hyphal bundles of monomitic fibers of Tv mycelium and Ab stipes made a tight structure with KF with increased strength (26-43 Nm/g) and limited air permeability (14-1630 mL/min). The blends KF FF (Ga) and KF FF (Tv) revealed relatively high virus filtration capacity: the log10 virus titer reduction values (LRV) corresponded to 4.54 LRV and 2.12 LRV, respectively. Mycocel biopolymers are biodegradable and have potential to be used in water microfiltration, food packaging, and virus filtration membranes.

Application of Alphaviral Vectors for Immunomodulation in Cancer Therapy.

BACKGROUND: The lack of specific and efficient cancer therapies has influenced the development of novel approaches, such as immunotherapy, which from its original application of immunogenic protein delivery has developed into the use of more sophisticated recombinant gene delivery methods to achieve better safety and efficacy profiles. This approach involves viral and non-viral delivery systems. METHODS: Expression vectors have been engineered for alphaviruses, including Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus. For immunotherapeutic applications, recombinant particles, RNA replicons and layered DNA vectors that express tumor-associated antigens (TAAs) and cytokines have been studied in animal models and in a few clinical trials. RESULTS: Immunization studies with TAAs and cytokines have elicited strong antibody responses and vaccination has provided protection against challenges with tumor cells in mouse models. Furthermore, the combination of TAAs and cytokines, antibodies and growth factors and the co-administration of chemotherapeutics and bacteriabased adjuvants have enhanced immunogenicity. Intratumoral and systemic delivery of recombinant alphavirus particles has demonstrated significant tumor regression and prolonged survival rates in rodent tumor models. CONCLUSION: Alphavirus-based immunotherapy represents a rapid and efficient method for prophylactic and therapeutic applications in animal models.