Denis V. Antonets

Antibody developability is determined by multiple physicochemical properties, and the relevance of individual properties varies among experimental assays. Existing computational approaches, however, often provide fixed assessments or rely on computationally intensive descriptors. We present kitAb, an open-source framework for scalable physicochemical characterization of antibody Fv regions and assay-specific developability modeling. kitAb calculates 59 interpretable sequence- and structure-based descriptors and integrates them with automated feature selection and regression. We evaluated kitAb across ten public datasets comprising 46 experimental endpoints, using sequence-aware or predefined splits where feasible. Predictive performance was strongly assay-dependent. In nested evaluations with sequence-aware or predefined data splits, models using kitAb descriptors achieved predictive performance comparable on average to those using PROPERMAB descriptors, while outperforming models based on TAP or sequence-only features. In a single-structure CPU timing benchmark, kitAb descriptor calculation was approximately 11-fold faster than PROPERMAB. Predictive performance was similar across the three structure-generation backends, and broader AutoML searches did not substantially improve mean performance. We further applied the same descriptor representation to approximately 3.4 million paired natural antibodies and 706 Phase II or later therapeutic antibodies. Repertoire-scale analysis revealed systematic differences in surface charge, hydrophobic and aromatic organization, CDR length and germline-associated physicochemical profiles. The kitAb code and analysis workflows are openly available at https://github.com/katyachemistry/kitAb/ .

Background/Objectives: In recent years, researchers have directed considerable attention toward the activation of the T-cell response in the development of vaccines against viral infections, including the tick-borne encephalitis virus (TBEV). A particularly promising approach to developing effective and safe T-cell vaccines involves the use of artificial multi-epitope immunogens. The selection of spacers that link epitopes within a construct can have a significant effect on the immunogenicity of multi-epitope constructs. The objective of this study was to design multi-epitope TBEV immunogens using various spacers and to evaluate their immunogenicity. Methods: The present study involved the design of three multi-epitope immunogens, which were developed based on T-cell epitopes from TBEV proteins. The AG1-ub construct contained optimized alanine spacers, the AG2-ub construct contained GPGPG spacers, and the AG4-ub construct contained no spacers. Three DNA vaccines encoding the designed immunogens were subsequently produced. To assess the immunogenicity of the constructs, BALB/c mice were immunized with the designed DNA vaccines via electroporation. Results: The ELISpot assay demonstrated that DNA vaccines encoding AG1-ub and AG4-ub induced a significant number of IFN-γ-producing cells. The DNA vaccine encoding AG2-ub, a multi-epitope with GPGPG spacers, exhibited low immunogenicity, potentially attributable to inadequate processing or misfolding of the AG2-ub protein, thereby affecting the molecule’s structure. Conclusions: The data provided in this study on the effect of spacers on the immunogenicity of multi-epitope constructs can be used to optimize the design of vaccine candidates. However, further research is needed to assess their clinical potential.

Introduction: Modification of natural enzymes to introduce new properties and enhance existing ones is a central challenge in bioengineering. This study is focused on the development of Taq polymerase mutants that show enhanced reverse transcriptase (RTase) activity while retaining other desirable properties such as fidelity, 5′- 3′ exonuclease activity, effective deoxyuracyl incorporation, and tolerance to locked nucleic acid (LNA)-containing substrates. Our objective was to use AI-driven rational design combined with multiparametric wet-lab analysis to identify and validate Taq polymerase mutants with an optimal combination of these properties.Methods: The experimental procedure was conducted in several stages: 1) On the basis of a foundational paper, we selected 18 candidate mutations known to affect RTase activity across six sites. These candidates, along with the wild type, were assessed in the wet lab for multiple properties to establish an initial training dataset. 2) Using embeddings of Taq polymerase variants generated by a protein language model, we trained a Ridge regression model to predict multiple enzyme properties. This model guided the selection of 14 new candidates for experimental validation, expanding the dataset for further refinement. 3) To better manage risk by assessing confidence intervals on predictions, we transitioned to Gaussian process regression and trained this model on an expanded dataset comprising 33 data points. 4) With this enhanced model, we conducted an in silico screen of over 18 million potential mutations, narrowing the field to 16 top candidates for comprehensive wet-lab evaluation.Results and Discussion: This iterative, data-driven strategy ultimately led to the identification of 18 enzyme variants that exhibited markedly improved RTase activity while maintaining a favorable balance of other key properties. These enhancements were generally accompanied by lower K^d, moderately reduced fidelity, and greater tolerance to noncanonical substrates, thereby illustrating a strong interdependence among these traits. Several enzymes validated via this procedure were effective in single-enzyme real-time reverse-transcription PCR setups, implying their utility for the development of new tools for real-time reverse-transcription PCR technologies, such as pathogen RNA detection and gene expression analysis. This study illustrates how AI can be effectively integrated with experimental bioengineering to enhance enzyme functionality systematically. Our approach offers a robust framework for designing enzyme mutants tailored to specific biotechnological applications. The results of our biological activity predictions for mutated Taq polymerases can be accessed at https://huggingface.co/datasets/nerusskikh/taqpol_insilico_dms

Modification of natural enzymes to introduce new properties and enhance existing ones is a central challenge in bioengineering. This study is focused on the development of Taq polymerase mutants that show enhanced reverse transcriptase (RTase) activity while retaining other desirable properties such as fidelity, 5′-3′ exonuclease activity, effective deoxyuracil incorporation, and tolerance to locked nucleic acid (LNA)-containing substrates. Our objective was to use AI-driven rational design combined with multiparametric wet-lab analysis to identify and validate Taq polymerase mutants with an optimal combination of these properties. The experimental procedure was conducted in several stages: 1) On the basis of a foundational paper, we selected 18 candidate mutations known to affect RTase activity across six sites. These candidates, along with the wild type, were assessed in the wet lab for multiple properties to establish an initial training dataset. 2) A ridge regression model was trained on this dataset to predict the enzymes’ properties. This model enabled us to select 14 new candidates for further experimental testing. 3) We refined our predictive model using Gaussian process regression and trained it on an expanded dataset now including 33 data points. 4) Leveraging the refined model, we screened in silico over 27 million potential mutations, thus selecting 16 for detailed wet-lab evaluation. Through this iterative data-driven approach, we identified 18 enzymes that not only manifested considerably enhanced RTase activity but also retained a balance of other required properties. These enhancements were generally accompanied by lower K _d , moderately reduced fidelity, and greater tolerance to noncanonical substrates, thereby illustrating a strong interdependence among these traits. Several enzymes validated via this procedure were effective in single-enzyme real-time reverse-transcription PCR setups, implying their utility for the development of new tools for real-time reverse-transcription PCR technologies, such as pathogen RNA detection and gene expression analysis. This study illustrates how AI can be effectively integrated with experimental bioengineering to enhance enzyme functionality systematically. Our approach offers a robust framework for designing enzyme mutants tailored to specific biotechnological applications. The results of our biological activity predictions for mutated Taq polymerases can be accessed at https://huggingface.co/datasets/nerusskikh/taqpol_insilico_dms .

The Colorado potato beetle is one of the most devastating potato pests widespread in the world. However, its viral pathogens remain highly unexplored. Here using SISPA high-throughput sequencing of Colorado potato beetle (CPB) samples derived from prepupal larvae that died from an unknown infection, we have identified two previously unknown viruses and assembled their full-length genomic sequences. The subsequent genetic and phylogenetic analysis of the obtained sequences demonstrated that the isolated viruses, named Leptinotarsa iflavirus 1 and Leptinotarsa solinvi-like virus 1 , are the novel representatives of Iflaviridae and Solinviviridae viral families, respectively. To the best of our knowledge, these are the first sequencing-confirmed insect viruses derived directly from CPB samples. And we also propose that Leptinotarsa iflavirus 1 may be associated with lethal disease in CPB.

The Colorado potato beetle (CPB) is one of the most serious insect pests due to its high ecological plasticity and ability to rapidly develop resistance to insecticides. The use of biological insecticides based on viruses is a promising approach to control insect pests, but the information on viruses which infect leaf feeding beetles is scarce. We performed a metagenomic analysis of 297 CPB genomic and transcriptomic samples from the public National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database. The reads that were not aligned to the reference genome were assembled with metaSPAdes, and 13314 selected contigs were analyzed with BLAST tools. The contigs and non-aligned reads were also analyzed with Kraken2 software. A total of 3137 virus-positive contigs were attributed to different viruses belonging to 6 types, 17 orders, and 32 families, matching over 97 viral species. The annotated sequences can be divided into several groups: those that are homologous to genetic sequences of insect viruses (Adintoviridae, Ascoviridae, Baculoviridae, Dicistroviridae, Chuviridae, Hytrosaviridae, Iflaviridae, Iridoviridae, Nimaviridae, Nudiviridae, Phasmaviridae, Picornaviridae, Polydnaviriformidae, Xinmoviridae etc.), plant viruses (Betaflexiviridae, Bromoviridae, Kitaviridae, Potyviridae), and endogenous retroviral elements (Retroviridae, Metaviridae). Additionally, the full-length genomes and near-full length genome sequences of several viruses were assembled. We also found sequences belonging to Bracoviriform viruses and, for the first time, experimentally validated the presence of bracoviral genetic fragments in the CPB genome. Our work represents the first attempt to discover the viral genetic material in CPB samples, and we hope that further studies will help to identify new viruses to extend the arsenal of biopesticides against CPB.

The Colorado potato beetle (CPB) is one of the most serious insect pests with high ecological plasticity and ability to rapidly develop resistance to insecticides. The use of biological insecticides based on viruses is a promising approach to control insect pests, but the information on viruses, which infect leaf feeding beetles, is scarce. We performed the metagenomic analysis of 297 CPB genomic and transcriptomic samples from public NBCI SRA database. The reads that were not aligned to the reference genome were assembled with metaSPAdes and 13314 selected contigs were analyzed with BLAST tools. The contigs and non-aligned reads were also analyzed with Kraken2 software. 3137 virus-positive contigs were attributed to different viruses belonging to 6 types, 17 orders and 32 families, matching over 97 viral species. The annotated sequences can be divided into several groups: homologous to genetic sequences of insect viruses ( Adintoviridae, Ascoviridae, Baculoviridae, Dicistroviridae, Chuviridae, Hytrosaviridae, Iflaviridae, Iridoviridae, Nimaviridae, Nudiviridae, Phasmaviridae, Picornaviridae, Polydnaviriformidae, Xinmoviridae etc.), plant viruses ( Betaflexiviridae, Bromoviridae, Kitaviridae, Potyviridae ), and endogenous retroviral elements ( Retroviridae, Metaviridae ). Also, the full-length genomes and near-full length genome sequences of several viruses were assembled. We have also found the sequences belonging to Bracoviriform viruses and for the first time experimentally validated the presence of bracoviral genetic fragments in CPB genome. Our work is the first attempt to discover the viral genetic material in CPB samples and we hope that further studies will help to identify new viruses to extend the arsenal of biopesticides against CPB. The analytical pipeline and additional materials are available at https://github.com/starchevskaya-maria17/uncoVir

С момента начала пандемии COVID-19 в 2020 году в России наблюдалось пять волн циркуляции SARS-CoV-2. Наблюдалась быстрая эволюция SARS-CoV-2, которая привела к появлению новых вариантов вируса, которые последовательно сменялись более адаптированным вариантом. Вариант Омикрон, выявленный в ноябре 2021 года, быстро распространяется в мире и в России и вытесняет предыдущий вариант Дельта. Целью данного исследования было описание результатов мониторинга и проведение сравнительной генетической характеристики изолятов варианта Омикрон вируса SARS-CoV-2, выделенных в России и секвенированных во ФБУН ГНЦ ВБ “Вектор” Роспотребнадзора. Материалы и методы: в качестве материала для исследования использовали мазки из рото- и носоглотки, собранные Центрами Гигиены и Эпидемиологии Роспотребнадзора. Секвенирование образцов с использованием Illumina Miseq. Биоинформатический анализ геномных данных проводился с помощью ряда программ, таких как FastQC (v.0.11.9), MAFFT (v.7.475), IQ-TREE, Pangolin (v.3.17), Minimap2 (v.2.17-r941), SnpEff (v.5.0e). Результаты и выводы: В работе было отсеквенировано и проанализировано 324 изолятов варианта Омикрон вируса SARS-CoV-2, выделенных в России в период с 13.12.2021 по 17.01.2022. Выделенные изоляты имели большинство основных характерных мутаций варианта Омикрон, включая 32 мутации в S-белке, которые могут быть ассоциированы с повышенной трансмиссивностью, уклонением от иммунного ответа и изменением тропизма и патогенности. Изоляты варианта Омикрон, выявленные в России, по основным генетическим маркерам сходны со штаммами, циркулирующими в мире, для которых было показано преимущество в заражении по сравнению с вариантом Дельта, снижение эффективности вакцин, но при этом, возможно, менее тяжелое течение заболевания. Вариант Омикрон на данное время считается опасным патогеном и необходимые меры предосторожности, профилактики и лечения являются актуальными на данном этапе пандемии.

The aim of the study was to create a highly immunogenic vaccine construct based on a recombinant variant of a replication-defective MVA strain of vaccinia virus, expressing virus-like particles that mimic natural infection with Marburg virus. Materials and methods. The recombinant virus was obtained through recombination between homologous viral DNA sequences and the insertion plasmid pDel2-GP-VP-Pat which carries transgenes of the structural proteins GP and VP40 of Marburg virus, flanked by fragments of MVA strain genome. Structure of the recombinant virus was confirmed in PCR and using sequencing, transgenes expression was analyzed by Western blotting, viruslike particles formation was recorded using electron microscopy. Evaluation of immunogenicity and protectivity was carried out using a guinea pig model. The antibody titer was determined in enzyme-linked immunosorbent assay. To assess T-cell response, the intracellular staining of cytokines was used, followed by analysis of samples on a flow cytometer. Results and discussion. On the basis of highly attenuated MVA strain of vaccinia virus a recombinant variant MVA-GP-VP40-MARV has been constructed, carrying a cassette of transgenes, GP and VP40, of Marburg virus in the region of deletion II of the genome. The expression of transgenes in MVA-permissive CER cells infected with recombinant MVA-GP-VP40-MARV strain and secretion of GP and VP40 proteins into culture medium have been demonstrated. Electron microscopy analysis has revealed the presence of Marburg virus-like particles in the culture medium of cells 12 hours after infection. Double vaccination of guinea pigs with MVA-GP-VP40-MARV strain at a dose of 108 PFU/animal induced the formation of antibodies to Marburg and vaccinia viruses, as well as 100 % protection against lethal Marburg virus infection (50 LD50). Using original TEpredict software, the structure of T-helper epitopes of GP protein has been predicted. Using the ICS method, the biological activity of these epitopes has been experimentally confirmed and it was shown that they provide the induction of a T-cell immune response as part of the MVA-GP-VP40-MARV vaccine construct.