Vetnuus | September 2024 19 during the COVID-19 pandemic.59 Compared with other vaccines, these vaccines have several advantages,60,61 such as low production cost, high efficacy, and rapid production. Moreover, mRNA does not integrate into the host cell genome and there is no risk of insertion mutations or carcinogenesis, nor does it produce infectious particles. In addition, the production of mRNA vaccines does not require cell culture or toxic chemicals. Therefore, it does not result in chemical or biological contamination. Multiple modifications make mRNA more stable and easier to translate. Owing to their safety, effectiveness, and large-scale production capability, mRNA vaccines are receiving increasing attention, and an increasing number of studies have shown that new and effective mRNA vaccines can be developed against any pathogen,62,63 Forms of mRNA vaccines Currently, there are two types of mRNA vaccines: nonamplifying RNA and self-amplifying RNA (saRNA).64 The non-amplifying mRNA consists of a 5′ cap, 5′ UTR, the gene of interest encoding region, 3′ UTR, and PolyA tails. Its main role is to activate the immune system to produce antibodies and induce cellular immune responses against specific pathogens by encoding target proteins.65 saRNA have complex structures. In addition to containing a 5′ cap, PolyA tail, and 5′ and 3′ UTR, similar to those of non-amplifying RNA, saRNA contains a large ORF (autonomous replicating transcription element), four non-structural proteins (nsP1–4), and subgenomic promoter (SGP).66 They are derived from the Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), or Semliki Forest virus (SFV). Viral genes encoding structural proteins originally located behind the subgenomic promoter (SGP) were replaced with heterologous genes encoding target proteins (GOI). This means that in saRNA, the viral structural protein genes are removed; therefore, mRNA is unable to produce infectious viruses. The design of saRNA vaccines gives them the ability to replicate autonomously, self-replicate, and express target proteins within host cells, thereby activating a more robust immune response,67,68 Owing to their high immunogenicity and efficacy, saRNA vaccines are considered promising vaccines with great potential for preventing infectious diseases and responding to outbreaks. Delivery system of mRNA vaccines When utilizing exogenous mRNAs for vaccine design, several challenges must be overcome, one of which is the efficient delivery of the mRNA into the host cell to ensure its conversion into an immunogenic protein.67 Due to the large size and dense negative charge of the mRNA molecule, it is difficult for naked mRNA to cross the cell membrane. In addition, as an exogenous nucleic acid, naked mRNA is readily recognized by pattern recognition receptors (PRRs) within the host cell, which trigger an interferon (IFN) response, leading to the rapid degradation of mRNA,67,69 To solve these problems, it is necessary to improve the delivery efficiency and stability of mRNA using delivery vectors. Several mRNA vaccine delivery systems have been extensively studied and applied. Each of these delivery systems has its own characteristics that can be selected and optimized according to specific vaccine design requirements and application scenarios to improve the delivery efficiency and immunogenicity of mRNA vaccines and achieve the goal of immune protection.69 Among these, lipid nanoparticles (LNPs) are the most widely used mRNA vaccine carriers.70 LNPs are usually composed of four components: an ionizable cationic lipid that facilitates self-assembly into virus-sized particles and allows the release of mRNA into the cytoplasm; lipid-linked polyethylene glycol (PEG), which extends the half-life of the formulation; cholesterol, which acts as a stabilizer of the formulation; and phospholipids, which maintain the lipid bilayer structure,71,72 LNPs play important roles in the delivery of mRNA vaccines and induction of immune responses. Dendritic cells (DCs) have shown high efficiency in receiving mRNA transfection; thus, they are an effective pathway for mRNA vaccine transfection in vivo and in vitro. This method of mRNA delivery is widely used because it does not require vector molecules to achieve high transfection efficiency.73 In addition to the two systems described above, commonly used mRNA vaccine delivery systems include protamine and cationic nanoemulsion (CNE). Protamine plays multiple roles in mRNA vaccine delivery. First, it effectively protects the mRNA from degradation by serum RNA enzymes, thereby improving the stability and durability of the vaccine. Protamine also acts as an immune activator, which helps enhance the immune response of the body.74 CNE are commonly used to deliver self-amplifying mRNA vaccines. Nanoemulsions play an important role in vaccine delivery, not only to improve the transfection efficiency of mRNA but also to enhance the immune effect.75 Application of RNA technology to rabies The gene-coding region of the rabies vaccine mRNA mainly expresses RABV glycoprotein, which is the only protein present on the surface of RABV virus particles and is the main target for neutralizing antibodies,76–78 From 2020 to 2023, many mRNA vaccines have been developed to prevent COVID-19. For example, the BNT162b1/2 mRNA vaccine developed by Pfizer and BioNTech and mRNA–1273 jointly developed by Moderna and NIH. With the help of the mRNA COVID-19 vaccine production platform, research and development of mRNA rabies vaccines have been considerably promoted. Preclinical trials Currently, there are several preclinical studies on mRNA rabies vaccines that have shown that RNA vaccines administered to mice, dogs, pigs, rats, monkeys, and other animals can protect against RABV (Table 2). Saxena et al.79 developed a self-amplifying RNA vaccine (SAM) that utilizes the SINV RNA replicon. Invitro-transcribed RNA (Sin-Rab-G RNA) was transfected into mammalian cells, and the self-amplification and expression of rabies glycoproteins were analyzed. To evaluate the immunogenicity of mRNA rabies vaccine, mice were immunized with 10 μg Sin Rab-G RNA, rabies DNA vaccine, or Rabipur. The results showed that Sin Rab-G RNA produced cellular and humoral IgG responses similar to those produced by the DNA rabies vaccine. After administration of the 20 LD50 RABV CVS strain, the protection rate of Sin Rab-G RNA in mice was similar to that of the DNA rabies vaccines, both of which were 80%. This study indicates that this self-amplifying RNA vaccine can effectively induce an immune response and provide protection comparable to that of a rabies DNA vaccine, thereby effectively responding to the challenge posed by the RABV. Unfortunately, the protective effect of the Sin-Rab -G RNA vaccine after viral challenge was lower than that of Rabipur. In 2016, CureVac used an optimized non-replicative mRNA vaccine (Pasteur strain, GenBank number: AAA47218.1) encoding the RABV-G in animal experiments to demonstrate its immunogenicity and protective efficacy.62 In mice, this vaccine-induced potent RVNA and effectively prevented rabies infection. Tracking the titers of functional antibodies in mice for up to one year revealed that the titers in all dose groups remained stable throughout the observation period. T-cell analysis revealed that RABV-G mRNA induced specific CD4+ and CD8+ T cells, with CD4+ T cells induced more by the vaccine than by inactivated control vaccines. It is worth noting that the RABV-G mRNA vaccine also showed immunogenicity in domesticated pigs, reaching protective antibody titers after the initial immunization, and further increasing antibody titers after enhanced immunization. The kinetics of the virus-neutralizing antibody reactions in pigs were comparable to those of the control vaccines. The above studies demonstrate the feasibility of using nonreplicating mRNA rabies vaccines in small and large animals as well as their strong potential as candidate rabies vaccines. In 2020, Stokes et al.63 used self-amplifying mRNA technology to deliver an RG SAM (CNE) vaccine with RABV-G as an antigen and cationic nanoparticles as the delivery system. Repeated-dose toxicity and biological distribution studies were conducted to understand the local tolerance, potential systemic toxicity, and biological distribution of the vaccine. In the repeated-dose toxicity studies, rats were intramuscularly injected with the RG SAM (CNE) vaccine every two weeks, for a total of four injections, with a recovery period of four weeks. On the second day, rabies RNA was detected at the injection site and in the lymph nodes. Over time, the distribution of rabies RNA in Article
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