One of the key stages in the development of mRNA vaccines is their delivery. Along with liposome, other materials are being developed for mRNA delivery that can ensure both the safety and effectiveness of the vaccine, and also facilitate its storage and transportation. In this study, we investigated the polyglucin:spermidine conjugate as a carrier of an mRNA-RBD vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The conditions for the self-assembling of mRNA-PGS complexes were optimized, including the selection of the mRNA:PGS charge ratios. Using dynamic and electrophoretic light scattering it was shown that the most monodisperse suspension of nanoparticles was formed at the mRNA:PGS charge ratio equal to 1:5. The average hydrodynamic particles diameter was determined, and it was confirmed by electron microscopy. The evaluation of the zeta potential of the investigated complexes showed that the particles surface charge was close to the zero point. This may indicate that the positively charged PGS conjugate has completely packed the negatively charged mRNA molecules. It has been shown that the packaging of mRNA-RBD into the PGS envelope leads to increased production of specific antibodies with virus-neutralizing activity in immunized BALB/c mice. Our results showed that the proposed polycationic polyglucin:spermidine conjugate can be considered a promising and safe means to the delivery of mRNA vaccines, in particular mRNA vaccines against SARS-CoV-2.
Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A—H1N1 and H3N2—and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0–M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.
After the genome sequence of SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus 2) was published and the number of infected people began to increase rapidly, many global companies began to develop a vaccine. Almost all known approaches to vaccine design were applied for this purpose, including inactivated viruses, mRNA and DNA-vaccines, vaccines based on various viral vectors, synthetically generated peptides and recombinant proteins produced in cells of insects and mammals. This review considers one of the promising vaccine platforms based on messenger RNA. Until recent years, mRNA-vaccination was out of practical implementation due to high sensitivity to nuclease degradation and consequent instability of drugs based on mRNA. Latest technological advances significantly mitigated the problems of low immunogenicity, instability, and difficulties in RNA-vaccine delivery. It is worth noting that mRNA-vaccines can efficiently activate both components of the immune system, i. e. T-cell and humoral responses. The essential advantage of mRNA-vaccines includes fast, inexpensive, scalable and uniform production providing a large output of desirable products in vitro. Synthesis and purification processes significantly simplify the process technology of mRNA drugs with injectable purity. Thus, mRNA production via in vitro transcription is more advantageous as compared with DNA-vaccines since it is a chemical process without the use of cells. mRNA techniques make it possible to pass all the phases of vaccine development much faster in comparison with the production of vaccines based on inactivated viruses or recombinant proteins. This property is critically important when designing vaccines against viral pathogens as the main problem of disease control includes a time gap between an epidemic and vaccine development. This paper discusses studies on the development of vaccines against coronaviruses including SARS-CoV-2 with special attention to the mRNA technique.
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Цель. Определение особенностей развития групповой заболеваемости энтеровирусной инфекцией (ЭВИ) на территориях Уральского федерального округа (УФО) и Западной Сибири в 2017 г. Материалы и методы. Проводился ретроспективный эпидемиологический анализ групповой заболеваемости ЭВИ по данным форм федерального статистического наблюдения и информации, предоставленной территориальными органами Роспотребнадзора. Индикация и идентификация энтеровирусов проводилась молекулярно-генетическими методами. Результаты. Был зарегистрирован 51 очаг групповой заболеваемости ЭВИ. Интенсивность и динамика формирования вспышек коррелирует с уровнем (r = 0,65; p < 0,05) и сезонной динамикой (r = 0,80; p < 0,05) спорадической заболеваемости ЭВИ на данной территории. Воздушно-капельный путь в качестве основного пути передачи был указан в 68,2% случаев, как дополнительный в 22,0%, а как единственный в 14,6%. Контактно-бытовой путь как единственный был указан только в 9,8% очагов. Генотип этиологического агента удалось определить в 82,2% вспышек. В этиологической структуре доля энтеровирусов вида А составила 51,4% (Coxsackievirus A6 (CV-A6)-43,2%), энтеровирусов вида В-70,3% (Echovirus 30 (E30)-48,6%). Заключение. В эпидемический процесс при групповой заболеваемости ЭВИ в УФО и Западной Сибири в 2017 г. преимущественно были вовлечены дети до 6 лет из детских дошкольных учреждений, где формировались небольшие и непродолжительные вспышки. В этиологической структуре групповой заболеваемости доминировали два генотипа энтеровирусов: E30 и CV-A6, что определяло преобладание в структуре клинических форм энтеровирусного менингита и экзантематозного поражения слизистых оболочек и кожи.
BACKGROUND: Nucleic acid-based prevention tools provide a promising platform for developing vaccines, including those against COVID-19. Previously, we developed the pVAXrbd DNA vaccine encoding the receptor-binding domain (RBD) of SARS-CoV-2, which, when administered intramuscularly to animals, induced a relatively weak immune response. The next stage of the study is to increase the immune response, in particular, using electroporation as one of the methods for increasing the immunogenicity of DNA vaccines. AIM: The aim of this article is to evaluate the immune response using electroporation in mice after immunization with pVAXrbd. MATERIALS AND METHODS: BALB/c mice were immunized with pVAXrbd using direct and reverse polarity square wave direct current electroporation with three pulses of 12 V for 30 ms and an interval of 950 ml with a current limit of 45 mA. RESULTS: BALB/c mice were immunized twice with an interval of three weeks with a dose of 100 g of DNA. Using ELISA, the titers of RBD-specific antibodies in the group of animals immunized with pVAXrbd using electroporation were 1:109350, which is 16 times higher than in the group of animals that received the DNA vaccine only intramuscularly (titers 1:6750). IFN ELISpot analysis showed that the largest number of cells (2434 spots/splenocytes, million) producing IFN in response to stimulation with peptides from the RBD protein was registered in the group of animals immunized with pVAXrbd using electroporation. For comparison, in the control group, the number of cells is 6.5 times lower: 380 spots / splenocytes, mln. CONCLUSIONS: Administration of the pVAXrbd DNA vaccine to laboratory animals by electroporation significantly enhances both the humoral and cellular specific immune response compared to intramuscular administration of the naked DNA vaccine.
Constant antigenic drift of circulating influenza viruses leads to inefficiency of seasonal influenza vaccines, thus requiring annual re-design of these vaccines. Therefore, the development of a universal influenza vaccine is of particular relevance. A promising line of research in this area is to design the immunogens consisting of conserved protein fragments from different influenza viral strains. The aim of this work was to assess immunogenicity of DNA vaccines and mRNA vaccines encoding artificial antigens consisting of conserved hemagglutinin stem fragments and conserved M2 protein. We have obtained DNA vaccine constructs encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stalk from the two subtypes of influenza A H1N1 and H3N2, and conserved M2 protein. These DNA vaccines were used as templates for the synthesis of mRNA vaccines. To assess immunogenicity of the obtained constructs, BALB/c mice were immunized with DNA and mRNA vaccines by i/m administration. Assessment of the humoral immune response was carried out by ELISA, using influenza viruses A/Aichi/2/68(H3N2), A/California/07/2009 as antigens and the ULTRIX vaccine containing purified antigens of H1N1 and H3N2 influenza viruses. T cell immune response was assessed using two methods: intracellular cytokine staining (ICS) and ELISpot. ICS was performed to determine CD8+ and CD4+T-lymphocytes producing IFN. ELISpot was carried out using the mouse IFN ELISpot kit (BD). A peptide mixture which included composition of the target antigens, was used for cell stimulation. The results showed that the designed DNA vaccine constructs induce virus-specific humoral and cellular responses in immunized BALB/c mice. Intramuscular administration of the naked mRNA vaccine constructs induced a weak humoral immune response, thus suggesting a need for further work to improve the delivery approaches.
BACKGROUND: RBD, receptor-binding domain, a key region of the SARS-CoV-2 surface glycoprotein for virus binding to host cell receptors and one of the targets of virus-neutralizing antibodies. That is why RBD is a promising immunogen for the development of vaccines that can provide protection against COVID-19. Vaccine mRNA is one of the new and rapidly developing vaccine platforms, and the delivery system is a very important component of it. AIM: The aim of this work was to present the results of a study of the antigenic properties of mRNA encoding the receptor-binding domain of SARS-CoV-2 when administered in combination with a polycationic carrier. MATERIALS AND METHODS: Dynamic and electrophoretic light scattering were used to characterize mRNA complexes with a polyglucin-spermidine conjugate. To assess the immunogenicity of mRNA were immunized BALB/c mice. The specific activity of the sera was assessed using enzyme immunoassay. RESULTS: As a result, the sizes and surface charge of the RBD-encoding mRNA complexes with the polyglucin-spermidine conjugate were determined. It has been shown that wrapping mRNA in a polyglucin-spermidine conjugate shell leads to an increase in the induction of RBD-specific antibodies in BALB/c mice compared to naked mRNA. CONCLUSIONS: An mRNA encoding the receptor-binding domain of SARS-CoV-2 has been obtained. It has been shown that the packaging of mRNA into the polyglucin-spermidine conjugate shell leads to an increase in immunogenic properties.
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