Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A.

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.

Revaccination in Age-Risk Groups with Sputnik V Is Immunologically Effective and Depends on the Initial Neutralizing SARS-CoV-2 IgG Antibodies Level.

Vaccination against COVID-19 has occurred in Russia for more than two years. According to the Russian official clinical guidelines to maintain tense immunity in the conditions of the ongoing COVID-19 pandemic, it is necessary to use booster immunization six months after primary vaccination or a previous COVID-19 contraction. It is especially important to ensure the maintenance of protective immunity in the elderly, who are at risk of severe courses of COVID-19. Meanwhile, the immunological effectiveness of the booster doses has not been sufficiently substantiated. To investigate the immunogenicity of Sputnik V within the recommended revaccination regimen and evaluate the effectiveness of booster doses, we conducted this study on 3983 samples obtained from individuals previously vaccinated with Sputnik V in Moscow. We analyzed the level of antibodies in BAU/mL three times: (i) six months after primary immunization immediately before the booster (RV), (ii) 3 weeks after the introduction of the first component of the booster (RV1), and (iii) 3 weeks after the introduction of the second component of the booster (RV2). Six months after the primary vaccination with Sputnik V, 95.5% of patients maintained a positive level of IgG antibodies to the receptor-binding domain (RBD) of SARS-CoV-2. The degree of increase in the specific virus-neutralizing antibodies level after revaccination increased with a decrease in their initial level just before the booster dose application. In the group of people with the level of antibodies up to 100 BAU/mL six months after the vaccination, a more than eightfold increase (p < 0.001, Wilcoxon criterion with Bonferroni adjustment) in the level of specific antibodies was observed (Me = 8.84 (IQR: 3.63−30.61)). A significant increase in the IgG level after receiving both the first and the second booster doses occurred at the initial titer level up to 300 BAU/ mL (p < 0.001) in those who did not contract COVID-19 in the past and up to 100 BAU/mL (p < 0.001) in those who were previously infected with SARS-CoV-2. A significant increase in the antibody level after the first dose of the booster was noted for people who had up to 500 BAU/mL (p < 0.05), regardless of the previous COVID-19 infection. Thus, revaccination is most effective in individuals with an antibody level below 500 BAU/mL, regardless of the vaccinee age and COVID-19 contraction. For the first time, it has been shown that a single booster dose of the Sputnik vaccine is sufficient to form a protective immunity in most vaccinees regardless of age and preexisting antibody level.

Avidity of IgG to SARS-CoV-2 RBD as a Prognostic Factor for the Severity of COVID-19 Reinfection.

The avidity index (AI) of IgG to the RBD of SARS-CoV-2 was determined for 71 patients with a mild (outpatient) course of COVID-19, including 39 primarily and 36 secondarily reinfected, and 92 patients with a severe (hospital) course of COVID-19, including 82 primarily and 10 secondarily infected. The AI was shown to correlate with the severity of repeated disease. In the group of outpatients with a mild course, the reinfected patients had significantly higher median AIs than those with primary infections (82.3% vs. 37.1%, p < 0.0001). At the same time, in patients with a severe course of COVID-19, reinfected patients still had low-avidity antibodies (median AI of 28.4% vs. 25% in the primarily infected, difference not significant, p > 0.05). This suggests that the presence of low-avidity IgG to RBD during reinfection is a negative prognostic factor, in which a patient's risk of developing COVID-19 in a severe form is significantly increased. Thus, patients with IgG of low avidity (AI ≤ 40%) had an 89 ± 20.5% chance of a severe course of recurrent COVID-19, whereas the detection of high-avidity antibodies (AI ≥ 50%) gave a probability of 94 ± 7.9% for a mild course of recurrent disease (p < 0.05).

A Deep Look Into COVID-19 Severity Through Dynamic Changes in Blood Cytokine Levels.

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.