Ekaterina A. Evtushenko

Newcastle disease virus (NDV) causes a highly contagious avian infection and is characterised by its high genetic diversity and mutation rate. Currently applied vaccines are based on NDV genotypes I and II, which are considerably distinct from the circulating pathogenic genotypes, of which genotype VII is predominant, and therefore, they may not provide complete protection for birds or stop virus transmission. This work is a proof-of-concept study aiming to develop a genotype VII-focused recombinant antigen for a vaccine targeting relevant NDV strains. The research primarily focuses on novel antigen design based on the bioinformatics analysis of recent circulating isolates. Haemagglutinin-neuraminidase (HN) is one of the two main NDV antigens. Two HN fragments (“1”—341–368 aa and “2”—239–257 aa) were selected due to the presence of linear neutralising epitopes. An approach focusing on using a number of selected-fragment variants simultaneously was applied for the first time. The resulting antigen Castle1 encompasses the sequence diversity of most circulating isolates. Preliminary immunological evaluation showed that antibodies from Castle1-immunised animals possessed haemagglutination inhibition activity and interacted with NDV of genotypes I and VII. That makes Castle1 a promising antigen for the further development of a vaccine for preventing ongoing and incoming NDV outbreaks.

Human papillomavirus (HPV)-associated cervical lesions remain a significant disease burden and minimally invasive blood-based biomarkers could complement cytology and HPV testing. This study aimed to characterize the proteomic composition of plasma-derived CD9⁺ small extracellular vesicles (sEVs) across the morphological spectrum of HPV-associated cervical disease, from histologically normal (NILM) through low-grade (LSIL) and high-grade (HSIL) lesions to invasive squamous cell carcinoma (SCC). Plasma samples from 34 women (NILM, LSIL, HSIL, SCC) were pooled per group, and CD9⁺ sEVs were isolated using an electrochemically controlled immunoaffinity capture method, followed by nanoparticle tracking analysis, transmission electron microscopy, Western blotting, and label-free LC–MS/MS proteomic profiling. The core sEV proteome comprised 258 shared proteins. LSIL showed the most pronounced changes with broad enrichment of complement and coagulation components and acute-phase reactants alongside depletion of immunoglobulin chains and complement C1r-like protein (C1RL). HSIL exhibited few differential proteins, dominated by neutrophil degranulation and retinoid metabolism pathways. SCC demonstrated extensive cargo depletion (22 downregulated proteins) and a nearly sevenfold upregulation of C1RL. Five proteins (including immunoglobulin chains and GPLD1) correlated positively with lesion severity. Pathway analysis consistently implicated platelet activation, lipoprotein remodeling, and insulin-like growth factor signaling. We conclude that plasma CD9⁺ sEVs carry stage-specific proteomic signatures distinguishing HPV-associated cervical lesions, with C1RL emerging as a candidate biphasic marker warranting further validation.

Currently, there is a need to improve the cryopreservation process for male gametes, especially for patients with low cryotolerance during sperm cryopreservation. Methods: The content and size of donor extracellular vesicles (EVs) in seminal plasma (SP) were assessed using nanoparticle tracking analysis (NTA), CD marker analysis, and transmission electron microscopy (TEM). Patient ejaculates were exposed to cryopreservation with or without prior co-culture with SP EVs and were not exposed to cryopreservation. The interaction of SP EVs with spermatozoa was assessed by TEM. Apoptotic, necrotic and late apoptotic cells, and mitochondrial functional activity were detected by flow cytometry. Results: NTA showed the highest concentration of SP EVs with a size of 80 nm, corresponding to small EVs. The binding of SP EVs to spermatozoa occurred along the entire plasma membrane, with an increased concentration of SP EVs at the neck and upper third of the sperm head. A significant increase in sperm motility was observed in the EVs co-culture group after cryopreservation/thawing. Flow cytometry showed a significant difference in the JC-1 Red/JC-1 Green ratio, indicating a higher mitochondrial membrane potential in the EVs exposure group. Conclusions: SP EVs have a protective function during human sperm cryopreservation.

The Newcastle disease virus (NDV), an avian paramyxovirus, induces the highly contagious Newcastle disease in poultry. Newcastle disease outbreaks, common in many developing countries, have been recorded worldwide for a century. Poultry, even vaccinated stocks, together with wild and synanthropic birds, serve as reservoirs of NDV. Despite the extensive use of commercial NDV vaccines, Newcastle disease outbreaks frequently occur in vaccinated chickens, resulting in great economic losses. The primary limitation of commercial Newcastle disease vaccines is their restricted compatibility with emerging novel NDV strains. The advancement of vaccines and vaccination techniques is anticipated to reduce the propagation of pathogenic NDV strains and consequently alleviate losses in poultry production. This review examines the NDV genotypes and strains implicated in both current and historical Newcastle disease outbreaks, and evaluates existing and candidate NDV vaccines, emphasizing recent innovations and novel techniques. Our aim was to delineate critical subjects for future inquiry and to furnish extensive data that could aid researchers in understanding the current advancements and existing problems in Newcastle disease vaccination prophylactics. The emergence of a new generation of vaccines employing advanced technologies may substantially improve the efficacy of Newcastle disease prevention and control.

Background. Biological fluids contain diverse extracellular vesicle (EV) populations differing in size, origin, composition, and function. This study pioneers asymmetric depth filtration for isolating seminal plasma (SP) EVs, bypassing ultracentrifugation (current gold standard). Objective. Validate the novel asymmetric depth filtration method for SP EV isolation as an alternative to ultracentrifugation. Design and methods. Twelve samples of seminal plasma were collected from young men and patients of advanced reproductive age with mild teratozoospermia. EVs were isolated from the SP using the proposed method. Western blot analysis for vesicle membrane markers CD9, CD63, and CD81 confirmed the presence of SP EVs in the studied samples. Transmission electron microscopy further validated the presence of SP EVs. Morphological characteristics of SP EVs from men of different age groups were described. Quantitative characteristics of the SP EV samples were assessed using Nanoparticle Tracking Analysis (NTA). Results. The obtained particle size and concentration distributions revealed age-related changes in SP EVs: in the advanced reproductive age group, a shift in the dis tribution peak toward larger particles was observed compared to the peak distribution in the young men group. Conclusion. The results provide a basis for further investigation of SP EVs and exploration of their potential clinical applications in assisted reproductive technologies, such as in vitro fertilization programs.

Background. In assisted reproductive technology (ART) programs, not all oocytes obtained during transvag inal puncture (TVP) are suitable for fertilization, as not all of them are at MII stage. Oocytes at germinal vesicle (GV) and MI stages are discarded because, after cumulus cells (CC) removal, their maturation and fertilization become impossible. Study proposes method for oocytes maturing by introducing extracellular vesicles (EVs) from follicular fluid (FF) into the perivitelline space. Objective. To evaluate the clinical feasibility of GV/MI oo cytes maturing technology without cumulus cells by injecting donor FF EVs under the zona pellucida. Materials and methods. FF (5 ml) was collected from 4 donors. EVs were isolated using sequential centrifugation. Portion of EVs was analyzed using Nanoparticle Tracking Analysis (NTA), while another portion was examined using transmission electron microscopy (TEM). Total of 53 immature oocytes were selected for the main group and 18 for the control group. Donor FF EVs were injected 4 hours after CC removal by introducing EVs suspension under zona pellucida. After 17 hours, oocyte maturity was assessed in both groups. Results. Oocytes maturation rate in the main group was statistically significantly higher than in the control group, indicating the potential effectiveness of the EV-IVM method for maturing GV-stage oocytes. Conclusion. Obtained data provide hope for the development of a new method for in vitro oocyte maturation.

This review addresses the ongoing global challenge posed by emerging and evolving viral diseases, underscoring the need for innovative vaccine development strategies. It focuses on the modern approaches to creating vaccines based on recombinant proteins produced in different expression systems, including bacteria, yeast, plants, insects, and mammals. This review analyses the advantages, limitations, and applications of these expression systems for producing vaccine antigens, as well as strategies for designing safer, more effective, and potentially ‘universal’ antigens. The review discusses the development of vaccines for a range of viral diseases, excluding SARS-CoV-2, which has already been extensively studied. The authors present these findings with the aim of contributing to ongoing research and advancing the development of antiviral vaccines.

The virions of plant viruses and their structurally modified particles (SP) represent valuable platforms for recombinant vaccine epitopes and antitumor agents. The possibility of modifying their surface with biological compounds makes them a tool for developing medical biotechnology applications. Here, we applied a new type of SP derived from virions and virus-like particles (VLP) of Alternanthera mosaic virus (AltMV) and well-studied SP from Tobacco mosaic virus (TMV). We have tested the ability of SP from AltMV (AltMV SP_V) and TMV virions also as AltMV VLP to bind to and penetrate Ewing sarcoma cells. The adsorption properties of AltMV SP_V and TMV SP are greater than those of the SP from AltMV VLP. Compared to normal cells, AltMV SP_V adsorbed more effectively on patient-derived sarcoma cells, whereas TMV SP were more effective on the established sarcoma cells. The AltMV SP_V and TMV SP were captured by all sarcoma cell lines. In the established Ewing sarcoma cell line, the effectiveness of AltMV SP_V penetration was greater than that of TMV SP. The usage of structurally modified plant virus particles as a platform for drugs and delivery systems has significant potential in the development of anticancer agents.

The development of cross-reactive vaccines is one of the central aims of modern vaccinology. Continuous mutation and the emergence of new SARS-CoV-2 variants and subvariants create the problem of universal coronavirus vaccine design. Previously, the authors devised three recombinant coronavirus antigens, which were based on the sequence collected in 2019 (the Wuhan variant) and produced in an E. coli bacterial expression system. The present work has shown, for the first time, that these recombinant antigens induce the production of antibodies that clearly interact with produced in CHO full-length S-protein of the Omicron variant. The immunogenicity of these recombinant antigens was studied in formulations with different adjuvants: Freund’s adjuvant, Al(OH)₃ and an adjuvant based on spherical particles (SPs), which are structurally modified plant virus. All adjuvanted formulations effectively stimulated Omicron-specific IgG production in mice. These universal coronavirus antigens could be considered the main component for the further development of broad-spectrum coronavirus vaccines for the prevention of SARS-CoV-2 infection. The present work also provides evidence that the synthetic biology approach is a promising strategy for the development of highly cross-reactive vaccines. Moreover, it is important to note that the bacterial expression system might be appropriate for the production of antigenically active universal antigens.

Chlamydia abortus is the etiological agent of abortion and fetal loss in sheep, goats and bovine cattle in many countries. Even though commercially available vaccines can reduce the incidence in sheep, the development of new, safe, and effective vaccines remains high on the agenda. In this study, an evaluation was made of the efficacy of a vaccine candidate, an inactivated antigen based on the extract of outer membrane proteins of a C. abortus strain known as Chlamydia VNITIBP-21, in combination with recombinant flagellin as an adjuvant. Pregnant sheep (n = 43) were divided into three groups: an experimental vaccinated group, a control infected group and a control non-infected group. The sheep were vaccinated twice, with an interval of 3 weeks, then infected with the homologous virulent strain of Chlamydia abortus on pregnancy day 75. The vaccine candidate reduced C. abortus shedding in vaginal swabs considerably, in comparison with the control group. In addition, ewes in the experimental group experienced no abortions, while those in the control group experienced instances of abortion, as well as births of weak and nonviable lambs. The findings show that the vaccine candidate proved itself to be promising in combatting the agent of ovine abortion and fetal loss.