Ainar Kossylganova

Background: Human brucellosis remains an important One Health problem, yet no vaccine is licensed for human use despite decades of antigen discovery and experimental vaccine development. This review examines why promising Brucella vaccine candidates have not progressed toward human application, with particular attention to the relationship between immune-evasion mechanisms, experimental evidence of protection, and the barriers that arise during translation. Methods: A critical narrative review of peer-reviewed literature on Brucella immunopathogenesis, immune evasion, protective immunity, antigen discovery, vaccine platforms, and human translational requirements was conducted. The evidence was interpreted according to experimental maturity, distinguishing computational or hypothesis-generating studies, mechanistic in vitro or ex vivo evidence, animal immunogenicity studies without challenge, and challenge-supported protection. Additional consideration was given to safety, durability, cross-species protection, model relevance, manufacturing, and regulatory requirements. Results: Brucella uses multiple mechanisms to limit innate recognition, alter intracellular trafficking and antigen presentation, and modulate host immune responses. These mechanisms provide a biological rationale for mechanism-informed vaccine design, but direct evidence that individual vaccine strategies overcome specific immune-evasion pathways remains limited. Across the vaccine literature, many candidates remain at the computational, antigen-production, or immunogenicity stages, whereas fewer have demonstrated protection after experimental challenge. Even challenge-supported candidates rarely address durability, cross-species breadth, human-relevant correlates of protection, extended safety, reproducible manufacturing, or readiness for clinical development. Conclusions: The absence of a licensed human brucellosis vaccine is better explained by attrition across successive stages of development than by a shortage of candidate antigens or vaccine platforms. Progress is likely to depend on advancing fewer, better-supported candidates through an integrated evidence pathway that links mechanistic rationale and challenge protection with durability, safety, species breadth, product quality, and human clinical relevance.

The development of viral vector vaccines against brucellosis requires strict control of the authenticity of recombinant strains used as vaccine candidates. One of the key stages in the molecular characterization of such strains is confirmation of the presence of heterologous genetic inserts encoding Brucella antigens. The aim of this study was to assess the authenticity of recombinant influenza strains by detecting brucellosis-related genetic inserts using reverse transcription polymerase chain reaction (RT-PCR). The study objects were recombinant influenza virus strains Flu-NS1-80-Omp16, Flu-NS1-80-L7/L12, Flu-NS1-80-Omp19, and Flu-NS1-80-Cu-Zn SOD, containing genes encoding immunodominant Brucella proteins. Analytical sensitivity was evaluated using serial tenfold dilutions of a positive control with a known RNA concentration. The specificity of the method was confirmed by the presence of amplicons of the expected size, approximately 1250–1300 bp for recombinant constructs and approximately 890 bp for the control virus, as well as by the absence of nonspecific amplification in negative controls. During the analysis of recombinant strains Flu-NS1-80-OMP16, Flu-NS1-80-OMP19, Flu-NS1-80-SOD, and Flu-NS1-80-L7/L12, target fragments were detected in all samples, indicating successful integration of Brucella spp. inserts into the viral genome. Reproducibility assessment showed 100% concordance of results in 10 independent replicates for all tested samples. The positive and negative controls demonstrated the expected results, confirming the correct performance of the reaction and the absence of contamination. The obtained results confirm the authenticity of the recombinant influenza strains and the presence of brucellosis-related genetic inserts in their genome, indicating the correctness of the viral vector constructs and their potential for further use in the development of vaccine preparations against brucellosis.

Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D²) and antigen concentration (R² = 0.956–0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625–10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations.

Introduction. Brucellosis remains one of the most widespread and underdiagnosed zoonotic infections, with over 500,000 new human cases annually and continued endemic circulation in livestock. Brucella melitensis, B. abortus, B. suis, and B. canis are the key pathogenic species causing chronic disease with nonspecific clinical symptoms. Brucella antigens are central to the pathogenesis and immune response, yet current diagnostic tests lack sufficient specificity, and no licensed human vaccine is available. This highlights the urgent need for more accurate antigen-based tools for both diagnosis and prevention. Aim. To review and evaluate the main Brucella antigens with proven or potential diagnostic and vaccine value, focusing on their structural features, immunogenicity, and applicability in translational research. Search strategy: A systematic review was conducted following PRISMA guidelines. Literature searches were performed in PubMed, Scopus, and Web of Science (2018–2025) using predefined keywords related to Brucella antigens, diagnostics, and vaccine development. After multi-stage screening of titles, abstracts, and full texts, 106 studies were included in the final analysis, as illustrated in the PRISMA flow diagram. Results. Outer membrane proteins (Omp16, Omp19, Omp25, Omp31), periplasmic proteins (BP26, Cu/Zn-SOD, L7/L12), heat shock proteins (Hsp60, Hsp70), and Type IV secretion system proteins (VirB) demonstrate distinct immunogenic profiles. Among them, L7/L12, Omp19, and SOD show the highest promise in terms of serological sensitivity and protective immune stimulation. A subset of these antigens is currently being explored as part of recombinant subunit vaccine formulations. Conclusion. Molecularly defined Brucella antigens offer tangible opportunities to improve human brucellosis diagnostics and support the rational development of safer, targeted vaccines. Their documented immunological relevance justifies further preclinical evaluation and standardization in applied immunodiagnostics. Введение. Бруцеллез остаётся одной из наиболее распространенных и недодиагностируемых зоонозных инфекций, с более чем 500 000 новых случаев среди людей ежегодно и устойчивой энзоотией среди сельскохозяйственных животных. Brucella melitensis, B. abortus, B. suis и B. canisявляются основными патогенными видами, вызывающими хронические заболевания с неспецифическими клиническими проявлениями. Антигены Brucella играют ключевую роль в патогенезе и формировании иммунного ответа, однако существующие методы диагностики недостаточно специфичны, а лицензированной вакцины для человека до сих пор не существует. Это подчёркивает необходимость создания более точных антиген-ориентированных средств диагностики и профилактики. Цель. Провести обзор и оценку основных антигенов Brucella spp., обладающих подтвержденной или потенциальной диагностической и вакцинной значимостью, с акцентом на их структурные особенности, иммуногенность и прикладную ценность. Стратегия поиска: Систематический обзор проведён в соответствии с рекомендациями PRISMA. Поиск литературы выполнен в базах PubMed, Scopus и Web of Science за период 2018–2025 гг. с использованием заранее определённых ключевых слов, связанных с антигенами Brucella, диагностикой и разработкой вакцин. После многоэтапного отбора по заголовкам, аннотациям и полным текстам в итоговый анализ было включено 106 исследований, что отражено на схеме PRISMA. Результаты. Наружные мембранные белки (Omp16, Omp19, Omp25, Omp31), периплазматические белки (BP26, Cu/Zn-SOD, L7/L12), белки теплового шока (Hsp60, Hsp70) и компоненты системы секреции типа IV (VirB) демонстрируют разную степень иммуногенности. Среди них белки L7/L12, Omp19 и SOD обладают наибольшим потенциалом как серологические маркеры и компоненты вакцинных конструкций. Часть этих антигенов рассматривается в составе рекомбинантных субъединичных вакцин. Заключение. Молекулярно охарактеризованные антигены Brucella открывают реальные перспективы для совершенствования диагностики и разработки целенаправленных и безопасных вакцин против бруцеллеза человека. Их иммунологическая значимость обосновывает необходимость дальнейшей доклинической оценки и внедрения в прикладную иммунодиагностику. Кіріспе. Бруцеллез – жыл сайын 500...