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Seasonal influenza remains a serious global health problem, leading to high mortality rates among the elderly and individuals with comorbidities. Vaccination is generally accepted as the most effective strategy for influenza prevention. While current influenza vaccines are effective, they still have limitations, including narrow specificity for certain serological variants, which may result in a mismatch between vaccine antigens and circulating strains. Additionally, the rapid variability of the virus poses challenges in providing extended protection beyond a single season. Therefore, mRNA technology is particularly promising for influenza prevention, as it enables the rapid development of multivalent vaccines and allows for quick updates of their antigenic composition. mRNA vaccines have already proven successful in preventing COVID-19 by eliciting rapid cellular and humoral immune responses. In this study, we present the development of a trivalent mRNA vaccine candidate, evaluate its immunogenicity using the hemagglutination inhibition assay, ELISA, and assess its efficacy in animals. We demonstrate the higher immunogenicity of the mRNA vaccine candidate compared to the inactivated split influenza vaccine and its enhanced ability to generate a cross-specific humoral immune response. These findings highlight the potential mRNA technology in overcoming current limitations of influenza vaccines and hold promise for ensuring greater efficacy in preventing seasonal influenza outbreaks.

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

An excessive inflammatory response to SARS-CoV-2 is thought to be a major cause of disease severity and mortality in patients with COVID-19. Longitudinal analysis of cytokine release can expand our understanding of the initial stages of disease development and help to identify early markers serving as predictors of disease severity. In this study, we performed a comprehensive analysis of 46 cytokines (including chemokines and growth factors) in the peripheral blood of a large cohort of COVID-19 patients (n=444). The patients were classified into five severity groups. Longitudinal analysis of all patients revealed two groups of cytokines, characterizing the “early” and “late” stages of the disease course and the switch between type 1 and type 2 immunity. We found significantly increased levels of cytokines associated with different severities of COVID-19, and levels of some cytokines were significantly higher during the first three days from symptom onset (DfSO) in patients who eventually required intensive care unit (ICU) therapy. Additionally, we identified nine cytokines, TNF-α, IL-10, MIG, IL-6, IP-10, M-CSF, G-CSF, GM-CSF, and IFN-α2, that can be used as good predictors of ICU requirement at 4-6 DfSO.