Vetnuus | September 2024 21 the medium to long term. SYS6008 can also provide better protection for mice after being attacked by the RABV from seven epidemic clades in China. Long et al.83 determined the optimal mRNA-B sequence for expressing RABV-G protein and evaluated the immunogenicity and efficacy of the RABV-G mRNA vaccine produced by RABV-G mRNA-B-LNP in various animals. These results indicate that a single dose of mRNA-B-LNP induces rapid and long-term protective antibody responses in mice. Compared with inactivated vaccines, a single dose of the mRNA-B-LNP vaccine induced higher neutralizing antibody titers in dogs, whereas two doses induced persistent humoral responses in dogs. mRNA-B-LNP vaccine can be stored as a liquid formulation at 2–8°C for 2 months. In addition, two doses of the mRNA-B-LNP vaccine showed high immunogenicity, inducing strong immune responses, strong Tfh and GC B cell responses, and Th1 biased cellular immune responses in mice, and protection against the RABV. Bai et al.84 from Fudan University developed a nucleosidemodified rabies mRNA-lipid nanoparticle vaccine (RABV-G mRNA-LNP) encoding a codonoptimized viral glycoprotein and assessed the immunogenicity and protective efficacy of this vaccine in mice compared with a commercially available inactivated vaccine. The results showed that a single vaccination with RABV-G mRNA at a moderate or high dose induced more potent humoral and T-cell immune responses in mice than those elicited by three inoculations of the inactivated vaccine. Importantly, mice receiving a single immunization with RABV-G mRNA, even at low doses, showed full protection against the lethal rabies challenge. The humoral immune response induced by a single RABV-G mRNA vaccination in mice can last for at least 25 weeks, whereas a two-dose strategy can extend the duration of the highly protective response to one year or longer. In contrast, the three-dose regimen of the inactivated vaccine failed to do this. Compared to injecting two doses of Rabipur, the unmodified mRNA rabies vaccine RABV-G mRNA developed by Hellgren et al.85 induced higher levels of RABV-G-specific plasma cells and T cells in the blood, and plasma cells in the bone marrow of non-human primates. The mRNA vaccine also stimulated higher RABV-G binding power and neutralizing antibody titers. Despite the presence of similar somatic hypermutations and clonal diversity, the high total antibody titers of the mRNA vaccines led to improved cross-neutralization of related RABV strains, indicating their potential for developing broadly protective vaccines against these viruses. Qiao et al.86 designed an RABV mRNA vaccine expressing the RABV G protein, encapsulated it with LNPs and different nucleic acid immunostimulators (CPG 1018, CPG 2395, and Poly I:C), and then assessed its immunogenicity and protective capacity in mice. Although RABV mRNA capsulated with LNP and CPG 1018 induced a more potent humoral response with high and durable RABV-G specific IgG titers and virus-neutralizing titers, it also induced stronger RABV G-specific cell-mediated immunity (CMI) responses in mice, including the highest proportions of interferon-γ (IFN-γ) and tumour necrosis factoralpha (TNFα)-producing CD4+/CD8+ T cells as shown using a flow cytometry assay. In addition, in the pre-post-exposure challenge assays, LNP + CPG 1018 encapsulated RABV G mRNA induced 100% protection against 25 LD50 of RABV infection, with the highest inhibition efficacy of viral replication, with a decreased virus genome detected by qRT-PCR. These results showed that RABV G mRNA encapsulated with the LNP immune-stimulating nucleic acid CPG 1018 shows promise as a safe and economical rabies vaccine candidate. Li et al.87 designed a non-replicating mRNA vaccine (RV021) encoding the RABV-G in vitro and evaluated its immunogenicity and protective efficacy against live viruses in mice. A two-dose vaccination with 1 μg of RV021 at 7-day intervals induced a protective level of neutralizing antibody that was maintained for at least 260 days. RV021 induced a robust cellular immune response that was significantly superior to that induced by an inactivated vaccine. Two doses of 1 μg RV021 provided full protection against challenge with CVS of a lethal dose. Vaccine potency testing (according to the National Institutes of Health) in vivo revealed that the potency of RV021 at 15 μg/dose was 7.5 IU/dose, which is substantially higher than the standard for lot release of rabies vaccines for current human use. Wan et al.88 reported an LPP-mRNA-G vaccine composed of a sequencemodified messenger ribonucleic acid encoding RABV-G glycoprotein (RABV-G) packaged in lipopolysaccharide (LPP) nanoparticles with a core-shell structure. The double-layered LPP structure improves the protection and delivery of RABV-G mRNA and allows for the gradual release of mRNA molecules as the polymer degrades. The unique core-shell-structured nanoparticles of LPPmRNA-G promoted vaccine uptake and exhibited an ideal biological tribution pattern of low liver targeting during intramuscular immunity. Single-dose administration of low-dose LPP-mRNA -G in mice triggered a strong humoral immune response and provided complete protection against fatal RABV brain attacks. Similarly, single immunization with low-dose LPP-mRNA-G induced high virus-neutralizing antibody titers in dogs. The above findings demonstrate the potential of LPP-mRNA-G as a promising next-generation rabies vaccine for both humans and companion animals. They also developed a circRNA cine called circRNA-G that targets lymph nodes expressing RABV-G. By directly introducing a mannose modification into the synthesis of PEG lipids, the resulting PEG mannose can be used to prepare mannose LNPs (mLNPs) targeting dendritic cells, thereby promoting the specific distribution of circRNA- G to the lymph nodes (mLNP circRNA-G). They demonstrated that mLNP circRNA-G sustained antigen availability and moted the production of mouse follicular helper T cells, germinal center B cells, long-lived plasma cells, and memory B cells. Moreover, vaccines with this targeted modification remained stable after being stored at 4°C for at least 24 weeks after freezedrying, and their immunogenicity was also maintained. This study provides a universal platform for designing freeze-dried vaccines with targeted stability and demonstrates the potential of lymph node-targeted circRNAs as a nextgeneration vaccine.91 H270P targeted mutation stabilizes RABV-G in its pre-fusion conformation. In 2024, Cao et al.92 reported the development of a highly promising rabies mRNA vaccine consisting of H270P targeted mutations encapsulated in LNP, named LNP-mRNA -G-H270P. They evaluated the humoral and cellular immunity of the vaccine against unmodified LNP-mRNA-G and commercially available inactivated vaccines in a mouse model. The RABV-G-specific IgG titer and RVNA in the LNP-mRNA -G-H270P group were significantly higher than those in the LNP- mRNA-G and inactivated vaccine groups. Similarly, the LNP- mRNAGH270P group IFN- γ IL-2 levels in splenic cells and supernatant of splenic cells, as well as the production of IFN- γ CD4+T cells were significantly higher than those in the other two vaccine groups. These results indicate that targeting the H270P mutation in RABV-G using the mRNA LNP vaccine platform is a promising strategy for the development of more effective rabies vaccines. Clinical trials Although many preclinical studies on mRNA rabies vaccines have been conducted, only CureVac CV7201 and CV7202 have undergone phase I clinical studies on their safety, tolerance, and immunogenicity in humans. CV7201 and CV7202 are mRNA vaccines targeting RABV-G. CV7201 is a lyophilized, temperature-stable mRNA candidate vaccine composed of mRNA encoding the RABV-G in free and complex forms with the cationic protein protamine.73 CV7202 is a novel mRNA-LNP formulation (Table 3). Between 21 October 2013 and 11 January 2016, CureVac93 enrolled and vaccinated 101 participants with 306 doses of mRNA (80–640 μg) using needle syringes (18 intradermally and 24 intramuscularly) or needle-free devices (46 intradermally and 13 intramuscularly). Seven days post-vaccination, 60 (94%) of 64 intradermally vaccinated participants and 36 (97%) of 37 intramuscularly vaccinated participants reported solicited injection site reactions, whereas 50 (78%) of 64 dermally vaccinated participants and 29 (78%) of 37 intramuscularly vaccinated participants reported solicited systemic adverse events, including 10 grade 3 events. One unexpected, possibly related, serious adverse reaction that occurred 7 days after a 640 μg intramuscular dose resolved without sequelae. mRNA vaccination by needle-free intradermal or intramuscular device injection induced virus neutralizing antibody titers of 0·5 IU/mL or more across dose levels and schedules in 32 (71%) of 45 participants administered 80 μg or 160 μg CV7201 doses intradermally and six (46%) of 13 participants administered 200 μg or 400 μg CV7201 doses intramuscularly. Development of mRNA rabies vaccines <<<20
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