Three Phages One Host: Isolation and Characterization of Pantoea agglomerans Phages from a Grasshopper Specimen.

The bacterial genus Pantoea comprises species found in a variety of different environmental sources. Pantoea spp. are often recovered from plant material and are capable of both benefitting the plants and acting like phytopathogens. Some species of Pantoea (including P. agglomerans) are considered opportunistic human pathogens capable of causing various infections in immunocompromised subjects. In this study, a strain of P. agglomerans (identified by 16S rRNA gene sequencing) was isolated from a dead specimen of an unidentified Latvian grasshopper species. The retrieved strain of P. agglomerans was then used as a host for the potential retrieval of phages from the same source material. After rounds of plaque purification and propagation, three high-titer lysates corresponding to putatively distinct phages were acquired. Transmission electron microscopy revealed that one of the phages was a myophage with an unusual morphology, while the two others were typical podophages. Whole-genome sequencing (WGS) was performed for each of these isolated phages. Genome de novo assembly and subsequent functional annotation confirmed that three different strictly lytic phages were isolated. Elaborate genomic characterization of the acquired phages was performed to elucidate their place within the so-far-uncovered phage diversity.

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.

Effects of urinary extracellular vesicles from prostate cancer patients on the transcriptomes of cancer-associated and normal fibroblasts.

BACKGROUND: Increasing evidence suggests that cancer-derived extracellular vesicles (EVs) alter the phenotype and functions of fibroblasts and trigger the reprogramming of normal fibroblasts into cancer-associated fibroblasts (CAFs). Here, we for the first time studied the effects of urinary EVs from PC patients and healthy males on the transcriptional landscape of prostate CAFs and normal foreskin fibroblasts. METHODS: Patient-derived prostate fibroblast primary cultures PCF-54 and PCF-55 were established from two specimens of PC tissues. EVs were isolated from urine samples of 3 patients with PC and 2 healthy males and used for the treatment of prostate fibroblast primary cultures and normal foreskin fibroblasts. The EV-treated fibroblasts were subjected to RNA sequencing analysis. RESULTS: RNA sequencing analysis showed that the fibroblast cultures differed significantly in their response to urinary EVs. The transcriptional response of foreskin fibroblasts to the urinary EVs isolated from PC patients and healthy controls was very similar and mostly related to the normal functions of fibroblasts. On the contrary, PCF-54 cells responded very differently - EVs from PC patients elicited transcriptional changes related to the regulation of the cell division and chromosome segregation, whereas EVs from healthy males affected mitochondrial respiration. In PCF-55 cells, EVs from both, PC-patients and controls induced the expression of a number of chemokines such as CCL2, CCL13, CXCL1, CXCL8, whereas pathways related to regulation of apoptotic signaling and production of cell adhesion molecules were triggered specifically by EVs from PC patients. CONCLUSION: This study demonstrates that urinary EVs from PC patients and healthy controls elicit distinct transcriptional responses in prostate CAFs and supports the idea that EVs contribute to the generation of functional heterogeneity of CAFs. Moreover, this study suggests that the changes in the gene expression pattern in EV recipient cells might serve as a novel type of functional cancer biomarkers.

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.

COVID-19: IMPACT ON HUMAN RIGHTS FROM THE HEALTHCARE PERSPECTIVE IN THE CASE OF PATIENTS TRIAGE.

OBJECTIVE: The aim: To analyse human right issues in health care during the SARS CoV-2 pandemic from the perspective of patients' and medical practitioner's rights. PATIENTS AND METHODS: Materials and methods: Multidisciplinary research will be conducted as part of national as well as international health care policy documents, international human right instruments, including case law decisions, and also major scientific articles on human rights from the healthcare perspective will be analysed. The comparative, Research synthesis method and systemic analyses will be made during the research. RESULTS: Results: The study confirmed that human right issues in the context of health care in case of Patient triage become relevant and transformed. The situation of COVID-19 has created new precedents for human rights, with a particular impact on the rights of doctors and patients. CONCLUSION: Conclusions: Human rights from the healthcare perspective become the topical issue during the COVID 19 pandemic. The legal practice in human rights had several transformations and it provides new challenges from patients as well as medical practitioners' rights. Important problem, from the ethical -legal perspective is the Patient's triage. The situation on patients triage or ranking shows importance and possibility of each member state to act fast, taking into consideration fast changing situation in medical health system. Sorting patients whose lives are at risk according to certain criteria for assessing their lives is not acceptable. In order to reduce events of violations of human rights during COVID 19 in healthcare, the emphasis should be placed for explanation of decisions made in state level.

Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.

BACKGROUND: Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs. RESULTS: Approximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging. CONCLUSIONS: Compared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs.

Isolation and Characterization of a Novel Aeromonas salmonicida-Infecting Studiervirinae Bacteriophage, JELG-KS1.

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage-JELG-KS1-infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture.

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.

Identifying Key Drivers of Efficient B Cell Responses: On the Role of T Help, Antigen-Organization, and Toll-like Receptor Stimulation for Generating a Neutralizing Anti-Dengue Virus Response.

T help (Th), stimulation of toll-like receptors (pathogen-associated molecular patterns, PAMPs), and antigen organization and repetitiveness (pathogen-associated structural patterns, PASPs) were shown numerous times to be important in driving B-cell and antibody responses. In this study, we dissected the individual contributions of these parameters using newly developed "Immune-tag" technology. As model antigens, we used eGFP and the third domain of the dengue virus 1 envelope protein (DV1 EDIII), the major target of virus-neutralizing antibodies. The respective proteins were expressed alone or genetically fused to the N-terminal fragment of the cucumber mosaic virus (CMV) capsid protein-nCMV, rendering the antigens oligomeric. In a step-by-step manner, RNA was attached as a PAMP, and/or a universal Th-cell epitope was genetically added for additional Th. Finally, a PASP was added to the constructs by displaying the antigens highly organized and repetitively on the surface of CMV-derived virus-like particles (CuMV VLPs). Sera from immunized mice demonstrated that each component contributed stepwise to the immunogenicity of both proteins. All components combined in the CuMV VLP platform induced by far the highest antibody responses. In addition, the DV1 EDIII induced high levels of DENV-1-neutralizing antibodies only if displayed on VLPs. Thus, combining multiple cues typically associated with viruses results in optimal antibody responses.

Is There a Future for Traditional Immunogens When We Have mRNA?

As the SARS-CoV-2 pandemic ends and we enter into a post-pandemic world, it is the time to reflect on the lessons learned [...].

Modulation of Hybrid GRM2-type Bacterial Microcompartment Shells through BMC-H Shell Protein Fusion and Incorporation of Non-native BMC-T Shell Proteins.

Bacterial microcompartments (BMCs) are organelle-like structures in bacteria that facilitate a wide range of enzymatic reactions. The microcompartment shell contains an encapsulated enzymatic core and, in contrast to phospholipid-based eukaryotic organelle membranes, has a pseudoicosahedral shape composed of BMC-H, BMC-T, and BMC-P proteins with conserved structures. This semipermeable microcompartment shell delineates the enzymatic core assemblies and the intermediates from the rest of the cell. It is also thought to function as a barrier against toxic intermediates as well as to increase the reaction rate. These properties of BMCs have made them intriguing candidates for biotechnological applications, for which it is important to explore the potential scope of the BMC shell modulation possibilities. In this work, we explore two BMC shell modulation mechanisms: first, confirming the incorporation of three trimeric BMC-T shell proteins and two truncated BMC-T shell proteins into Klebsiella pneumoniae GRM2-type BMC protein shells containing no representatives of this group, and second, producing BMC particles from double- and triple-fused hexameric BMC-H shell proteins. These results reveal the potential for "mix and match" synthetic BMC shell formation to ensure shell properties specifically suited to the encapsulated cargo and show for the first time the involvement of an essentially dimeric pseudohexameric shell protein in BMC shell formation.

Bacterial expression systems based on Tymovirus-like particles for the presentation of vaccine antigens.

Virus-like particles (VLPs) are virus-derived artificial nanostructures that resemble a native virus-stimulating immune system through highly repetitive surface structures. Improved safety profiles, flexibility in vaccine construction, and the ease of VLP production and purification have highlighted VLPs as attractive candidates for universal vaccine platform generation, although exploration of different types of expression systems for their development is needed. Here, we demonstrate the construction of several simple Escherichia coli expression systems for the generation of eggplant mosaic virus (EMV) VLP-derived vaccines. We used different principles of antigen incorporation, including direct fusion of EMV coat protein (CP) with major cat allergen Feld1, coexpression of antigen containing and unmodified (mosaic) EMV CPs, and two coexpression variants of EMV VLPs and antigen using synthetic zipper pair 18/17 (SYNZIP 18/17), and coiled-coil forming peptides E and K (Ecoil/Kcoil). Recombinant Fel d 1 chemically coupled to EMV VLPs was included as control experiments. All EMV-Feld1 variants were expressed in E. coli, formed Tymovirus-like VLPs, and were used for immunological evaluation in healthy mice. The immunogenicity of these newly developed vaccine candidates demonstrated high titers of Feld1-specific Ab production; however, a comparably high immune response against carrier EMV was also observed. Antibody avidity tests revealed very specific Ab production (more than 50% specificity) for four out of the five vaccine candidates. Native Feld1 recognition and subclass-specific antibody tests suggested that the EMV-SZ18/17-Feld1 complex and chemically coupled EMV-Feld1 vaccines may possess characteristics for further development.

PreS1 Containing HBc VLPs for the Development of a Combined Therapeutic/Prophylactic Hepatitis B Vaccine.

The available HBV vaccines based on the HBV surface protein are manufactured in yeasts and demonstrate excellent prophylactic but no therapeutic activity and are thus ineffective against chronic HBV infection. Five different HBV core proteins (HBc)-full length and C-terminally truncated-were used for the insertion of the short, preS1,aa 20-47 and long, preS1phil, aa 12-60 + 89-119 fragments. Modified virus-like particles (VLPs) were compared for their biotechnological and immunological properties. The expression level of HBc-preS1 proteins was high for all investigated proteins, allowing us to obtain 10-20 mg of purified VLPs from a gram of biomass with the combination of gel filtration and ion-exchange chromatography to reach approximately 90% purity of target proteins. The immunogenicity of chimeric VLPs was tested in BALB/c mice, showing a high anti-preS1 response and substantial T-cell proliferation after stimulation with HBc protein. Targeted incorporation of oligonucleotide ODN 1668 in modified HBc-preS1 VLPs was demonstrated.

Validation of potential RNA biomarkers for prostate cancer diagnosis and monitoring in plasma and urinary extracellular vesicles.

Introduction: Prostate cancer (PCa), one of the most prevalent malignancies affecting men worldwide, presents significant challenges in terms of early detection, risk stratification, and active surveillance. In recent years, liquid biopsies have emerged as a promising non-invasive approach to complement or even replace traditional tissue biopsies. Extracellular vesicles (EVs), nanosized membranous structures released by various cells into body fluids, have gained substantial attention as a source of cancer biomarkers due to their ability to encapsulate and transport a wide range of biological molecules, including RNA. In this study, we aimed to validate 15 potential RNA biomarkers, identified in a previous EV RNA sequencing study, using droplet digital PCR. Methods: The candidate biomarkers were tested in plasma and urinary EVs collected before and after radical prostatectomy from 30 PCa patients and their diagnostic potential was evaluated in a test cohort consisting of 20 benign prostate hyperplasia (BPH) and 20 PCa patients' plasma and urinary EVs. Next, the results were validated in an independent cohort of plasma EVs from 31 PCa and 31 BPH patients. Results: We found that the levels of NKX3-1 (p = 0.0008) in plasma EVs, and tRF-Phe-GAA-3b (p < 0.0001) tRF-Lys-CTT-5c (p < 0.0327), piR-28004 (p = 0.0081) and miR-375-3p (p < 0.0001) in urinary EVs significantly decreased after radical prostatectomy suggesting that the main tissue source of these RNAs is prostate and/or PCa. Two mRNA biomarkers-GLO1 and NKX3-1 showed promising diagnostic potential in distinguishing between PCa and BPH with AUC of 0.68 and 0.82, respectively, in the test cohort and AUC of 0.73 and 0.65, respectively, in the validation cohort, when tested in plasma EVs. Combining these markers in a biomarker model yielded AUC of 0.85 and 0.71 in the test and validation cohorts, respectively. Although the PSA levels in the blood could not distinguish PCa from BPH in our cohort, adding PSA to the mRNA biomarker model increased AUC from 0.71 to 0.76. Conclusion: This study identified two novel EV-enclosed RNA biomarkers-NKX3-1 and GLO1-for the detection of PCa, and highlights the complementary nature of GLO1, NKX3-1 and PSA as combined biomarkers in liquid biopsies of PCa.

Plasma and urinary extracellular vesicles as a source of RNA biomarkers for prostate cancer in liquid biopsies.

Introduction: Extracellular vesicles (EVs) have emerged as a very attractive source of cancer- derived RNA biomarkers for the early detection, prognosis and monitoring of various cancers, including prostate cancer (PC). However, biofluids contain a mixture of EVs released from a variety of tissues and the fraction of total EVs that are derived from PC tissue is not known. Moreover, the optimal biofluid-plasma or urine-that is more suitable for the detection of EV- enclosed RNA biomarkers is not yet clear. Methodology: In the current study, we performed RNA sequencing analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer. Results and Discussion: The most abundant RNA biotypes in EVs were miRNA, piRNA, tRNA, lncRNA, rRNA and mRNA. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype. The levels of 63 mRNAs, 3 lncRNAs, 2 miRNAs and 1 piRNA were significantly increased in the tumors and decreased in the post-operation urinary EVs, thus suggesting that these RNAs mainly originate from PC tissue. No such RNA biomarkers were identified in plasma EVs. This suggests that the fraction of PC-derived EVs in urine is larger than in plasma and allows the detection and tracking of PC-derived RNAs.

Styrylpyridinium Derivatives for Fluorescent Cell Imaging.

A set of styrylpyridinium (SP) compounds was synthesised in order to study their spectroscopic and cell labelling properties. The compounds comprised different electron donating parts (julolidine, p-dimethylaminophenyl, p-methoxyphenyl, 3,4,5-trimethoxyphenyl), conjugated linkers (vinyl, divinyl), and an electron-withdrawing N-alkylpyridinium part. Geminal or bis-compounds incorporating two styrylpyridinium (bis-SP) moieties at the 1,3-trimethylene unit were synthesised. Compounds comprising a divinyl linker and powerful electron-donating julolidine donor parts possessed intensive fluorescence in the near-infrared region (maximum at ~760 nm). The compounds had rather high cytotoxicity towards the cancerous cell lines HT-1080 and MH-22A; at the same time, basal cytotoxicity towards the NIH3T3 fibroblast cell line ranged from toxic to harmful. SP compound 6e had IC50 values of 1.0 ± 0.03 µg/mL to the cell line HT-1080 and 0.4 µg/mL to MH-22A; however, the basal toxicity LD50 was 477 mg/kg (harmful). The compounds showed large Stokes' shifts, including 195 nm for 6a,b, 240 nm for 6e, and 325 and 352 nm for 6d and 6c, respectively. The highest photoluminescence quantum yield (PLQY) values were observed for 6a,b, which were 15.1 and 12.2%, respectively. The PLQY values for the SP derivatives 6d,e (those with a julolidinyl moiety) were 0.5 and 0.7%, respectively. Cell staining with compound 6e revealed a strong fluorescent signal localised in the cell cytoplasm, whereas the cell nuclei were not stained. SP compound 6e possessed self-assembling properties and formed liposomes with an average diameter of 118 nm. The obtained novel data on near-infrared fluorescent probes could be useful for the development of biocompatible dyes for biomedical applications.

Morganella Phage Mecenats66 Utilizes an Evolutionarily Distinct Subtype of Headful Genome Packaging with a Preferred Packaging Initiation Site.

Both recognized species from the genus Morganella (M. morganii and M. psychrotolerans) are Gram-negative facultative anaerobic rod-shaped bacteria that have been documented as sometimes being implicated in human disease. Complete genomes of seven Morganella-infecting phages are publicly available today. Here, we report on the genomic characterization of an insect associated Morganella sp. phage, which we named Mecenats66, isolated from dead worker honeybees. Phage Mecenats66 was propagated, purified, and subjected to whole-genome sequencing with subsequent complete genome annotation. After the genome de novo assembly, it was noted that Mecenats66 might employ a headful packaging with a preferred packaging initiation site, although its terminase amino acid sequence did not fall within any of the currently recognized headful packaging subtype employing phage (that had their packaging strategy experimentally verified) with clusters on a terminase sequence phylogenetic tree. The in silico predicted packaging strategy was verified experimentally, validating the packaging initiation site and suggesting that Mecenats66 represents an evolutionarily distinct headful genome packaging with a preferred packaging initiation site strategy subtype. These findings can possibly be attributed to several of the phages already found within the public biological sequence repositories and could aid newly isolated phage packaging strategy predictions in the future.

Comparison of Bacterial Expression Systems Based on Potato Virus Y-like Particles for Vaccine Generation.

Plant-based virus-like particle (VLP) vaccines have been studied for years, demonstrating their potential as antigen-presenting platforms. In this paper, we describe the development of, and compare between, simple Escherichia coli-based antigen display platforms for the generation of potato virus Y (PVY) VLP-derived vaccines, thus allowing the production of vaccines from a single bacterial cell culture. We constructed four systems with the major cat allergen Fel d 1; namely, direct fusion with plant virus PVY coat protein (CP), mosaic PVY VLPs, and two coexpression variants of conjugates (SpyTag/SpyCatcher) allowing coexpression and conjugation directly in E. coli cells. For control experiments, we included PVY VLPs chemically coupled with Fel d 1. All constructed PVY-Fel d 1 variants were well expressed and soluble, formed PVY-like filamentous particles, and were recognized by monoclonal Fel d 1 antibodies. Our results indicate that all vaccine variants induced high titers of anti-Fel d 1 antibodies in murine models. Mice that were immunized with the chemically coupled Fel d 1 antigen exhibited the highest antibody titers and antibody-antigen interaction specificity, as detected by binding avidity and recognition of native Fel d 1. IgG1 subclass antibodies were found to be the dominant IgG class against PVY-Fel d 1. PVY CP-derived VLPs represent an efficient platform for the comparison of various antigen presentation systems to help evaluate different vaccine designs.

Identification and Full Genome Analysis of the First Putative Virus of Sea Buckthorn (Hippophae rhamnoides L.).

The agricultural importance of sea buckthorn (SBT; Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting SBT have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of SBT for the first time. Sequence analysis of the obtained viral genome revealed high similarity with several viruses belonging to the genus Marafivirus. The genome of the new virus is 6989 nucleotides (nt) in length according to 5', 3' RACE (without polyA-tail), with 5' and 3' 133 and 109 nt long untranslated regions, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1954 amino acids with the characteristic marafivirus non-structural protein domains-methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by 6 nt, encoding the coat protein (CP) with typical signatures of minor and major forms. Both CP forms were cloned and expressed in a bacterial expression system. Only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly.

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.