Development of MVA-d34 Tetravalent Dengue Vaccine: Design and Immunogenicity.

Dengue fever, an infectious disease that affects more than 100 million people every year, is a global health problem. Vaccination may be the most effective prevention strategy for the disease. However, the development of vaccines against dengue fever is complicated by the high risk of developing an antibody-dependent increase in infection. This article describes the development of an MVA-d34 vaccine against the dengue virus based on a safe and effective MVA viral vector. The DIII domains of the envelope protein (E) of the dengue virus are used as vaccine antigens, as antibodies against these domains do not cause an enhancement of infection. The use of the DIII domains of each of the four dengue virus serotypes made it possible to generate a humoral response against all four dengue virus serotypes in immunized mice. We also showed that the sera of vaccinated mice present virus-neutralizing activity against dengue serotype 2. Thus, the developed MVA-d34 vaccine is a promising candidate vaccine against dengue fever.

The Succession of the Cellulolytic Microbial Community from the Soil during Oat Straw Decomposition.

The process of straw decomposition is dynamic and is accompanied by the succession of the microbial decomposing community, which is driven by poorly understood interactions between microorganisms. Soil is a complex ecological niche, and the soil microbiome can serve as a source of potentially active cellulolytic microorganisms. Here, we performed an experiment on the de novo colonization of oat straw by the soil microbial community by placing nylon bags with sterilized oat straw in the pots filled with chernozem soil and incubating them for 6 months. The aim was to investigate the changes in decomposer microbiota during this process using conventional sequencing techniques. The bacterial succession during straw decomposition occurred in three phases: the early phase (first month) was characterized by high microbial activity and low diversity, the middle phase (second to third month) was characterized by low activity and low diversity, and the late phase (fourth to sixth months) was characterized by low activity and high diversity. Analysis of amplicon sequencing data revealed three groups of co-changing phylotypes corresponding to these phases. The early active phase was abundant in the cellulolytic members from Pseudomonadota, Bacteroidota, Bacillota, and Actinobacteriota for bacteria and Ascomycota for fungi, and most of the primary phylotypes were gone by the end of the phase. The second intermediate phase was marked by the set of phylotypes from the same phyla persisting in the community. In the mature community of the late phase, apart from the core phylotypes, non-cellulolytic members from Bdellovibrionota, Myxococcota, Chloroflexota, and Thermoproteota appeared. Full metagenome sequencing of the microbial community from the end of the middle phase confirmed that major bacterial and fungal members of this consortium had genes of glycoside hydrolases (GH) connected to cellulose and chitin degradation. The real-time analysis of the selection of these genes showed that their representation varied between phases, and this occurred under the influence of the host, and not the GH family factor. Our findings demonstrate that soil microbial community may act as an efficient source of cellulolytic microorganisms and that colonization of the cellulolytic substrate occurs in several phases, each characterized by its own taxonomic and functional profile.

Dynamic of the Soil Microbiota in Short-Term Crop Rotation.

Crop rotation is one of the oldest and most effective methods of restoring soil fertility, which declines when the same plant is grown repeatedly. One of the reasons for a reduction in fertility is the accumulation of pathogenic and unfavorable microbiota. The modern crop rotation schemes (a set of plant species and their order in the crop rotation) are highly effective but are designed without considering soil microbiota dynamics. The main goal of this study was to perform a short-term experiment with multiple plant combinations to access the microbiological effects of crop rotation. It could be useful for the design of long-term crop rotation schemes that take the microbiological effects of the crop rotation into account. For the analysis, five plants (legumes: vetch, clover, and cereals: oats, wheat, and barley) were used. These five plants were separately grown in pots with soil. After the first phase of vegetation, the plants were removed from the soil and a new crop was planted. Soil samples from all 25 possible combinations of primary and secondary crops were investigated using v4-16S rDNA gene sequencing. It was shown that the short-term experiments (up to 40 days of growing) are effective enough to find microbial shifts in bulk soil from different plants. Both primary and secondary cultures are significant factors for the microbial composition of microbial soil communities. Changes are the most significant in the microbial communities of vetch soils, especially in the case of vetch monoculture. Growing clover also leads to changes in microbiota, especially according to beta-diversity. Data obtained can be used to develop new crop rotation schemes that take into account the microbiological effects of various crops.

The Structure of Stable Cellulolytic Consortia Isolated from Natural Lignocellulosic Substrates.

Recycling plant matter is one of the challenges facing humanity today and depends on efficient lignocellulose degradation. Although many bacterial strains from natural substrates demonstrate cellulolytic activities, the CAZymes (Carbohydrate-Active enZYmes) responsible for these activities are very diverse and usually distributed among different bacteria in one habitat. Thus, using microbial consortia can be a solution to rapid and effective decomposition of plant biomass. Four cellulolytic consortia were isolated from enrichment cultures from composting natural lignocellulosic substrates-oat straw, pine sawdust, and birch leaf litter. Enrichment cultures facilitated growth of similar, but not identical cellulose-decomposing bacteria from different substrates. Major components in all consortia were from Proteobacteria, Actinobacteriota and Bacteroidota, but some were specific for different substrates-Verrucomicrobiota and Myxococcota from straw, Planctomycetota from sawdust and Firmicutes from leaf litter. While most members of the consortia were involved in the lignocellulose degradation, some demonstrated additional metabolic activities. Consortia did not differ in the composition of CAZymes genes, but rather in axillary functions, such as ABC-transporters and two-component systems, usually taxon-specific and associated with CAZymes. Our findings show that enrichment cultures can provide reproducible cellulolytic consortia from various lignocellulosic substrates, the stability of which is ensured by tight microbial relations between its components.

Development of a Universal Epitope-Based Influenza Vaccine and Evaluation of Its Effectiveness in Mice.

Vaccination is an effective and economically viable means of protection against the influenza virus, but due to rapid viral evolution, modern seasonal vaccines are not effective enough. Next-generation vaccines are designed to provide protection against a wide range of influenza virus strains, including pandemic variants. In our work, we made an epitope-based universal vaccine, rMVA-k1-k2, against the influenza virus based on the modified vaccinia Ankara (MVA) vector and using our own algorithms to select epitopes from conserved fragments of the NP, M1 and HA proteins of influenza A and B. We show that double immunization protects mice with a 67% or greater efficiency against viral influenza pneumonia when infected with various strains of the H1N1, H2N2, H3N2 and H5N1 subtypes of influenza A. In animals, the level of protection provided by the rMVA-k1-k2 vaccine was comparable to that provided by the universal M001 and MVA-NP+M1 (Invictus) vaccines, which have shown success in clinical trials, against strains of the H1N1 and H3N2 subtypes.

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda.

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.

Essential function of VCP/p97 in infection cycle of the nucleopolyhedrovirus AcMNPV in Spodoptera frugiperda Sf9 cells.

The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degradation pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed expression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin-proteasome system.

Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 ß subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

Egress of budded virions of Autographa californica nucleopolyhedrovirus does not require activity of Spodoptera frugiperda HSP/HSC70 chaperones.

The induction of heat shock proteins in baculovirus infected cells is well documented. However a role of these chaperones in infection cycle remains unknown. The observation that HSP70s are associated with virions of different baculoviruses reported by several researchers suggests that HSPs might be structural components of viruses or involved in virion assembly. These hypotheses were examined by using a novel inhibitor of the ATPase and chaperoning activity of HSP/HSC70s, VER-155008. When VER-155008 was added early in infection, the synthesis of viral proteins, genome replication and the production of budded virions (BV) were markedly inhibited indicating the dependence of virus reproduction on host chaperones. However, BV production was unaffected when VER-155008 was added in the mid-replication phase which is after accumulation of products required for completion of the viral DNA replication. These results suggest that the final stages in assembly of BV and their egress from cells do not depend on chaperone activity of host HSP/HSC70s.

Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells.

Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

New insights into the induction of the heat shock proteins in baculovirus infected insect cells.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.