Introduction. An uncontrolled flow of migration and powerful anti-vaccination propaganda have led to an outbreak of vaccine-controlled infections among the Russian population in recent years.

The purpose of this study is to assess the dynamics of the incidence of whooping cough, measles, and rubella among the population in the Southern Federal District of the Russian Federation in the period of 2010 to 2023.

Methods. Thi study is based on data from the federal statistical observation form "Information on the number of diseases registered in patients living in the service area of a medical organization" for the period of 2015 to 2023.

Results. Between 2010 and 2023, the number of whooping cough cases amounted to 9199. The lowest number of whooping cough cases in the Southern Federal District was registered in 2021 and amounted to 15 (0.16%). The largest number of whooping cough cases was registered in 2023 and amounted to 4511 (49.04% of all detected cases since 2010). Krasnodar had the highest number of cases (2027—24.7%), followed by Rostov in second place (635 cases—6.9%) and Volgograd in third place (473 cases—5.14%).

The number of cases of rubella in the period from 2010 to 2023 in the Southern Federal District amounted to 108. The smallest number of cases was 0, and this was registered in the period from 2017 to 2023. The largest number was 30 cases, and thiswas registered in 2013 (27.78% of all detected cases since 2010). In first place for the number of cases of rubella was Krasnodar (23 cases—21.3%) in 2013, in second place was Volgograd (13 cases—12.04%) in 2010, and in third place was Astrakhan (12 cases—11.11%) in 2014.

Conclusions. During the studied period, the largest number of cases of whooping cough and rubella was registered in the Krasnodar region. The lowest number of cases of all nosologies was registered in 2020--2021, which was probably due to social isolation during the COVID-19 pandemic.

Introduction. The activation of the adaptive immune system requires the engagement of co-stimulatory pathways in addition to primary antigen receptor signaling, and adjuvants play a central role in this process. Surprisingly, many bacterial polysaccharide vaccines, including typhoid Vi polysaccharide and quadrivalent meningococcal conjugate (MCV4) vaccines, do not incorporate adjuvants, and induce highly variable antibody responses. We found that Toll-Like Receptor 4 ligands present in typhoid vaccines account for the immunogenicity of typhoid vaccines. Based on this rationale, we developed a non-toxic TLR4 ligand, a monophosphoryl lipid A-based adjuvant formulation called Turbo.

Methods. The adjuvanticity of Turbo was tested with WHO-prequalified or FDA-approved typhoid and MCV4 vaccines, as well as a haptenated antigen system (NP-CGG) in several inbred and outbred strains of mice of all ages. The methodology employed included transgenic mouse systems, challenge models, serum bactericidal assays, ELISA, ELISpot, flow cytometry, and immunohistochemistry.

Results: Turbo promoted antibody responses across all ages and eliminated or minimized the booster requirement. In contrast to the commonly used adjuvant alum, Turbo promoted a superior germinal center response accompanied by affinity maturation and class switching to all four IgG isotypes. The magnitude of these IgG responses was sustained for more than one year in mice, which is concurrent with long-lived plasma cells present in bone marrow. Unlike alum, Turbo adjuvanticity is not dependent on canonical or non-canonical inflammasome activation or pyroptosis. Turbo upregulated the expression of the co-stimulatory molecules CD86 and CD40 on B cells, and Turbo adjuvanticity is dependent primarily on the TLR4-MyD88 axis and is lost in mice deficient in CD86 or CD40.

Conclusion: Turbo efficiently engages the required co-stimulatory pathways and promotes long-lasting antibody responses across all ages. Therefore, Turbo can be incorporated into a wide range of mono and multivalent polysaccharide vaccines to enhance immunogenicity and maximize efficacy.

Dengue, a major public health issue in tropical and subtropical regions, currently has no specific treatment and only one licensed vaccine, with limited use in endemic areas. The lack of antiviral drugs and the challenges in understanding dengue’s pathophysiology stem from the absence of an effective animal model that mirrors human infection in terms of viraemia peaks, clinical presentation, and immune response. Humanized mouse models have been useful in viral disease research, but dengue studies often use NOD-scid IL2R gamma null (NSG) immunodeficient mice, which focus on preventing graft rejection rather than fully developing human hematopoiesis. In this study, we compare four humanized mouse models derived from NOD mice expressing human cytokines, which were xenotransplanted and infected with DENV-2 (New Guinea C) intravenously. The models (Hu-NSG, hu-EXL, hu-SGM3) received xenotransplants of CD34+ fetal cord blood, while one model (Hu-SGM3-PBMCs) was transplanted with human PBMCs. We found that models expressing human cytokines had higher viremia compared to the NSG model. All the models showed infectious virus during viremia, confirmed by means of indirect plaque assays. The Hu-SGM3-PBMCs model, in particular, developed a severe infection with signs of bleeding and intestinal necrosis, leading to the death of all mice by day 18 post-infection, with viremia levels twice as high as in the other models according to RT-qPCR. The models using hCD34+ cells showed low or undetectable levels of human proinflammatory cytokines during infection, measured by means of flow cytometry Bead Arrays. In contrast, the hu-mice model with human PBMCs exhibited detectable and decreasing levels of IFN-g, IL-10, IP10, and TNF, which is consistent with the immune modulation seen in DENV-2 New Guinea C strain infections. These results indicate that humanized mice models derived from NOD mice expressing human cytokines enhance viremia and offer a relevant platform for studying dengue and potential treatments.

The sublingual vaccine introduced in this study induces mucosal antibody-mediated protection against SARS-CoV-2 and influenza viruses, which infect via the upper respiratory tract, mouth, and nose. Poly(I:C) is an adjuvant used to activate TLR3-mediated immune responses, but it remains unapproved due to its proinflammatory side effects. The mucosae of the oral cavity are the primary target of the sublingual vaccination, although this poses a practical limitation due to the inhibitory mucin barrier.

We developed a sublingual SARS-CoV-2 vaccine using the SARS-CoV-2 RBD antigen and Poly(I:C) adjuvant, as well as an influenza vaccine including the HA antigen. These vaccines were tested in non-human primates, macaque monkeys, utilizing N-acetyl cysteine to disintegrate the mucus layer. The sublingual Poly(I:C)-adjuvanted vaccine (SPAV) elicited mucosal and systemic immune responses, including antigen-specific secretory IgA in saliva and nasal washing, as well as specific IgA and IgG in blood. These antibodies neutralized SARS-CoV-2, suggesting that the SPAV protects against the SARS-CoV-2 virus.

SPAV appeared to be safe judging from the results of the blood tests, the plasma inflammatory cytokines, the gene expression of the proinflammatory factor in the WBC, and a direct comparison to AddaS03, which is an emulsion adjuvant viewed as being safe. In mice, an intranasally administrated Poly(I:C)-adjuvanted vaccine had a potent unfavorable effect in the olfactory bulb, causing the upregulated expression of nine proinflammatory genes, but the same was not true in monkeys. Thus, the previously reported detrimental effects of Poly(I:C)-adjuvanted vaccines in mice are overstated due to differences in nose structure and function, as well as immune response, between mice/rodents and macaques/primates.

DNA microarray analysis revealed that the SPAV mediates atypical up- or down-regulated gene expression associated with immune suppression/tolerance, leading to incomplete Treg differentiation and T-cell exhaustion. Possibly, the PASV could induce previously unknown effects on balancing stimulation and inhibition, like the “Yin and Yang" concept, in immune responses.

The Bacillus Calmette–Guérin (BCG) vaccine, the only licensed tuberculosis (TB) vaccine, has limited effectiveness in preventing pulmonary TB in adults. Our study explores using the mammary gland of non-transgenic goats to produce an oral vaccine candidate, combining secretory IgA (SIgA) with epitopes from TB. This approach aims to enhance mucosal immunity and provide comprehensive protection across different stages of TB infection. The vaccine candidate was developed by engineering recombinant epitopes including Antigen 85b (Ag85b) for active TB, alpha-crystallin (Acr) for latent TB, and resuscitation-promoting factor E (RpfE) for reactivated TB combined with SIgA. We evaluated the immunological response by administering goat milk containing the recombinant protein directly as oral immunization. Five groups of Balb/C mice (n=5) were categorized as follows: recombinant milk (RM), normal milk (NM), BCG prime with RM boost (BCG-RM), BCG prime with NM boost (BCG-NM), and BCG alone (BCG). The RM and NM groups received daily oral immunizations with RM or NM for two weeks. The BCG-primed groups received booster doses of RM or NM daily for two weeks, one month after the initial BCG vaccination. Two weeks after the final immunization, the mice were sacrificed, and IgA levels in the saliva and lung lavage were measured using enzyme-linked immunosorbent assay (ELISA). Mice immunized with recombinant vaccine-containing milk, especially those primed with BCG, showed a significant increase in IgA levels in the saliva and lung lavage compared to the normal milk and BCG-only groups. These results indicate that the recombinant vaccine-containing milk can effectively induce a strong mucosal immune response against TB. The significant increase in IgA levels in mice, particularly in BCG-primed groups, highlights the potential of this vaccine as a booster candidate with BCG priming.

Introduction: Tuberculosis (TB) remains one of the leading infectious diseases worldwide. The Bacillus Calmette-Guérin (BCG) vaccine, the only currently available TB vaccine, offers limited protection in adults. Therefore, it is critical to develop new effective vaccines. Considering Mycobacterium tuberculosis (Mtb) primarily infects the lungs, mucosal vaccines may be a promising strategy, as they can stimulate both systemic and mucosal immunity. This study explores the development and evaluation of a novel mucosal TB vaccine utilizing TB multi-epitopes by producing a recombinant chimeric protein in the mammary gland of goat, and in vivo mucosal immunization by utilizing the stable properties of the chimeric protein.

Methods: An AAV vector carrying TB multi-epitopes and secretory IgA-J chain were co-transduced into the mammary gland of goat, and milk was collected daily. The chimeric protein containing the TB multi-epitopes antigen and secretory components in milk was detected by Western blot against anti-His and anti-Ag85b. The protein was then purified from the milk using the Akta Prime purification system. The mice were immunized using purified protein intranasally, and their immunogenicity responses were evaluated by flow cytometry.

Results: In milk containing chimeric protein, bands corresponding to murine secretory component (75 kDa) and multi-epitopes Antigen Fc-alpha (75 kDa) could be detected against anti-His and anti-Ag85b, respectively. After the chimeric protein was purified, a purity of 95% of chimeric protein was obtained and detected at 75 kDa. Mice immunized with the vaccine candidate were shown to elicit immune response by producing a higher level of activated and memory T cells compared to the control group.

Conclusion: The protein complex formed by TB multi-epitopes-Fc alpha and the murine secretory component can be obtained, purified and detected in milk samples, provided that the mammary gland acts as a potential bioreactor for the production of the recombinant vaccine candidate. The immune response was shown to be induced by the protein complex in mice by producing more activated and memory T cells.

Today, an ideal adjuvant must meet several essential criteria: it must be safe, biocompatible, and capable of directing the immune response and enhancing antigen presentation. ASC16 (GRAS and FDA Inactive Ingredients Database) is a derivative of vitamin C that can form viscoelastic hydrogels or act as a toxin inhibitor depending on whether it is present at high or low concentrations, respectively. For these reasons, we proposed to evaluate the effect of dispersed ASC16 (low concentration; Mo) as an additive in a hydrogel (high concentration; Pa40) to produce experimental antivenom. For the formation of the hydrogel, first, ASC16 (25 mg) and PEG₄₀₀ solution (0.750 mL) were mixed and heated to 63 °C until complete solubilization. Then, this dispersion was cooled to 40 °C, and then it was incorporated into 0.250 mL of PBS solution with or without dispersed ASC16 (120 µM) and finally venom proteins (6-10 µg/mice). Afterward, BALB/c mice were inoculated with hydrogel in the absence or presence of additive (Pa40 and Pa40Mo). Fourteen days after the inoculation, skin and blood samples were taken for histological analysis of local injury and to determine the titre and specificity of the antibodies by ELISA and Immunoblotting tests. We used t-tests for two independent samples to verify whether there were significant differences. Our results show that Pa40Mo induced the highest titres (3.75) and had the highest Western blot signal, followed by Pa40 (3.27), with significant differences (p≤0.05). Histological analyses indicate that both formulations caused tissue alterations by the observation of regenerating muscle cells. This study shows that both formulations provide evidence of acute injury and that formulations with dispersed ASC16 as an additive induce higher antibody titres and have greater specificity than formulations without the additive. However, future studies are needed to determine the signalling mechanism of dispersed ASC16 .

Introduction: Foot-and-mouth disease (FMD) is a highly contagious viral infection affecting cattle, pigs, and other cloven-hoofed animals. The disease, caused by FMD virus (FMDV), poses a severe threat to the livestock industry due to its ability to spread rapidly and cause high productivity losses. In India, to prevent outbreaks and safeguard animal health and productivity, a trivalent vaccine containing the inactivated virus antigens of three serotypes (O, A, and Asia-1) is employed. While humoral immunity, characterized by antibody production, is essential for defending against FMDV, a comprehensive understanding of protection also requires an investigation of cell-mediated immune (CMI) responses.

Methods: Indian cattle aged 6 to 9 months were immunized with a trivalent FMD vaccine in a water-in-oil-in-water (W/O/W) formulation. We evaluated the virus neutralizing antibody and CMI responses following vaccination. The cattle were challenged with homologous FMDV serotype O at six months post vaccination.

Results: The vaccine elicited potent humoral and cellular immune responses. The vaccine-induced virus-neutralizing antibody response peaked around two to four weeks post-vaccination and was maintained for up to six months. Elevated IFN-γ and reduced IL-4 cytokine levels by 14 days post-vaccination (dpv) and the dominance of the IgG2 isotype antibody at 30 dpv suggested a predominantly Th1-type cellular immune response. Interestingly, IFN-γ and IL-4 expression exhibited inverse patterns of regulation, highlighting the precise regulation of Th1 and Th2 responses. The challenging of vaccinated cattle with the homologous FMDV serotype O at six months post-vaccination showed 100% protective efficacy.

Conclusions: The study findings demonstrated that humoral, as well as CMI, responses with a predominant Th1 immune response contributed to the vaccine’s efficacy against the FMDV challenge in cattle.

[Introduction] The vaccinia virus (VACV) LC16m8 strain is a highly attenuated vaccine against smallpox and mpox, licensed in Japan. Unlike its parental strain (LC16mO) or ancestral vaccines, the B5R protein of the LC16m8 strain has a frameshift mutation, resulting in a truncated B5R protein.

[Objective] Our objective was to determine the B5R protein antigenic domains and develop an assay to distinguish LC16m8 vaccine-induced antibodies from orthopoxvirus infections.

[Methods] Four recombinant proteins, located at the N-terminal and C-terminal region of the B5R, were expressed in E.coli, and their antigenicity was examined by immunoblot analysis. They were then used to produce polyclonal antibodies in SPF rabbits. Next, the sensitivity of the anti-B5R antibodies was evaluated by immunofluorescence assay using orthopoxvirus-infected cells, or by expressing their B5R homologous regions. Furthermore, a Luminex bead-peptide assay was developed to target the B5R domains, and its specificity was evaluated using anti-orthopoxvirus antibodies. Finally, we screened the sera of 45 healthy human donors and 5 Mpox patients.

[Results] The expressed B5R N-terminal and C-terminal proteins were specifically recognized by anti-LC16mO B5R antibodies. The anti-B5R N-terminal antibodies cross-reacted with orthopoxvirus-infected cells or their expressed B5R homologous region. Nonetheless, the C-terminal antibody cross-reacted with infected cells of VACV, monkeypox and cowpox virus, but did not with the LC16m8 strain or the expressed LC16m8 B5R protein. Both B5R peptides showed cross-reaction with antibodies against VACV and monkeypox virus (Clade I, IIa & IIb). Furthermore, seven human serum samples showed neutralizing antibodies against VACV, from which two human donors and one Mpox patient showed affinity to the C-terminal peptide and recognized the full-length-B5R protein.

[Conclusion] The expressed B5R proteins, anti-B5R antibodies and Luminex bead-peptide assay might be useful for detecting orthopoxvirus infections among the LC16m8-vaccinated population.

Healthcare workers (HCWs) are at increased risk of occupational exposure to hepatitis B virus (HBV) infection, even if they have received a complete immunization schedule of three doses of hepatitis B vaccine in infancy. According to our Hospital Immunization Program, Hepatitis B surface (anti – HBs) antibody serum levels were determined in all HCWs at the time of hospital admission in order to investigate the HBV immunity state. HCWs with low anti-HBs serum levels ( < 10 mIU/mL) usually received a booster dose of 20 µg/1 ml Engerix B ( GlaxoSmithKline Biologicals, Belgium) vaccine. However, due to a temporary shortage of the 20 µg Engerix B vaccine, we wondered if 10 µg/0,5 ml (pediatric dose) could be equally effective to elicit the immunogenicity in our HCWs. For this reason, we conducted a pilot study on forty homogeneous HCWs with a non-immune level of anti HBs antibody ( < 10 mIU/ml) who were previously vaccinated in infancy. Twenty HCWs (Group A) received a booster dose of Engerix B vaccine 10 µg/0,5 ml, and twenty HCWs (Group B) received the standard dose of 20 µg/1 ml of Engerix B vaccine. One month after the vaccination, the anti-HBs antibody serum levels in all participants in the study were determined. The results showed a significant increase in anti-HBs antibody serum levels both after 10 micrograms and 20 micrograms of Engerix B vaccine. All but two healthcare workers were immunized after their booster dose; no statistically significant difference was found in the mean of anti-HBs antibody serum levels between groups A and B. If confirmed in a large clinical trial, the use of 10 micrograms Engerix B booster dose in adults could represent a preferred choice in terms of cost efficacy, particularly in specific situations, for example in vaccination programs in low-income countries.