Development of mRNA rabies vaccines

ABSTRACT

Rabies, primarily transmitted to humans by dogs (accounting for 99% of cases). Once rabies occurs, its mortality rate is approximately 100%. Post-exposure prophylaxis (PEP) is critical for preventing the onset of rabies after exposure to rabid animals, and vaccination is a pivotal element of PEP. However, high costs and complex immunization protocols have led to poor adherence to rabies vaccinations. Consequently, there is an urgent need to develop new rabies vaccines that are safe, highly immunogenic, and cost-effective to improve compliance and effectively prevent rabies. In recent years, mRNA vaccines have made significant progress in the structural modification and optimization of delivery systems. Various mRNA vaccines are currently undergoing clinical trials, positioning them as viable alternatives to the traditional rabies vaccines. In this article, we discuss a novel mRNA rabies vaccine currently undergoing clinical and preclinical testing, and evaluate its potential to replace existing vaccines.

KEYWORDS:

• Rabies
• rabies vaccines
• mRNA vaccines
• immunogenicity
• mRNA

Introduction

Rabies is an acute infectious disease caused by the rabies virus (RABV) and is a zoonotic neurological disease. The most common way for humans to contract rabies is through bites, scratching, licking, or mucosal damage by dogs, cats, wild carnivores, as well as carnivorous and vampire bats infected with the RABV. It is estimated that approximately 59,000 people¹ worldwide die from rabies each year, of which 97% cases occur in Asia and Africa. Rabies remains a serious public health concern.

Rabies can be broadly categorized into two types: furious and paralytic. Furious rabies is characterized by classical symptoms, including severe agitation and hydrophobia. By contrast, paralytic rabies predominantly presents with weakness and flaccid paralysis. Once clinical symptoms appear, the mortality rate of patients with rabies is almost 100%. Timely, standardized, and complete post-exposure prophylaxis (PEP) can provide effective preventive protection for individuals exposed to rabies.² Injection of the rabies vaccine is a key step in PEP. Vaccine immunization can produce neutralizing antibodies against the RABV in the body, which can effectively kill the virus before it enters the central nervous system. However, in the real world, implementing correct PEP for severe bites, scratches, and other injuries close to the nerve center, such as the head and face, often makes it difficult to prevent rabies in all cases. The rate at which vaccines produce effective protective neutralizing antibodies is the underlying reason for PEP failure. Currently, commercially available rabies vaccines are mainly inactivated cell tissue culture vaccines, commonly administered via intramuscular injections following the Essen regimen (1-1-1-1-1), 4-dose Essen regimen (1-1-1-1-0), or Zagreb regimen (2-0-1-0-1). Compliance issues caused by frequent visits and long visit cycles hinder the implementation of the complete PEP. Therefore, the development of vaccines with faster neutralizing antibodies, better compliance, and stronger protection while ensuring safety is an important direction in the field of rabies vaccine development. With the continuous development and progress of science and technology, the revolutionary mRNA biotechnology is sparking a revolution in the vaccine field. mRNA vaccines have broad prospects for the prevention and treatment of infectious diseases, owing to their low risk of insertion mutations, high efficacy, fast development cycles, and low production costs.³ Recently, several mRNA vaccines have entered clinical trials and are expected to combat new and recurrent infectious diseases,^4–6 Recently, the successful application of the COVID-19 mRNA vaccine further verified the advantages of this platform and opened the door for the use of mRNA vaccines in infectious disease prevention.^7–10

This review summarizes recent research on the development of mRNA vaccines for rabies, with a brief review of mRNA rabies vaccine-related studies that have shown to be effective in preclinical animal models or clinical trials, and describes the current research status of mRNA rabies vaccines. In addition, most preclinical animal models for rabies mRNA vaccines in this review have used small animals, but some research organizations have conducted immunogenicity studies in non-human primates, such as cynomolgus macaques, and have obtained favorable immunity results.

Rabies vaccines

Typically, vaccines are administered before exposure to an infectious agent and are not particularly useful after exposure. Historically, rabies has been an exception to most other vaccine-preventable infectious diseases because immunization proceeds as a critical intervention not only before but also after exposure to the virus. Although Louis Pasteur invented the rabies vaccine in 1885¹¹ and effective rabies vaccines have been accessible for a considerable period, existing vaccination regimens require multiple doses to attain elevated neutralizing titers. These vaccination regimens are associated with high costs and pose challenges to developing countries where rabies mortality is high.

Conventional rabies vaccines

Over the past century, various substrates such as whole-animal tissues, primary cell cultures, diploid cells, and continuous cell lines have been widely used for virus propagation. Based on the different culture media, the development of traditional rabies vaccines has gone through three stages: animal nerve tissue vaccines (NTV), avian embryo vaccines, and cell culture vaccines (CCV). Table 1 summarizes the history of rabies vaccine development and lists the major currently available CCV. Many laboratory-fixed RABV strains have been used to prepare rabies vaccines over the past century.¹²

Table 1. Major past and present rabies vaccines for human prophylaxis.

	Vaccine Name	Manufacturers	Type	Substrates	Viral strains	Remarks	Locations
Nerve Tissue Vaccines	Pasteur	Many, through the mid-20th century	Partially inactivated by drying	Rabbit spinal cord	Pasteur fixed strain	Contained adult animal nerve tissue and residual live RABV	NLU
	Fermi	Many, throughout the 20th century	Phenolised, live virus	Sheep, goat, or rabbit brains	CVS	Contains adult nerve tissue and residual live RABV	NLU, except in Ethiopia
	Semple	Many, throughout the20th century	Phenol inactivated	Sheep, goat, or rabbit brains	PV-11	Contained adult animal nerve tissue	NLU
Avian Embryo Vaccines	PDEV	Several (e.g., Berna, Zydus Cadila)	β-propiolactone inactivated (BPL)	Duckembryo	PM	Purified byultracentrifugation	Europe, worldwide
	DEV	Several (e.g., Eli Lilly)	BPL	Duck embryo	PM	Allergy to avian antigens	NLU
	HEP	Lederle Laboratories	Attenuated RABV	Chick embryo cells	PM	Poorly immunogenic compared to other biologics	NLU
Cell Culture Vaccines	HDCV	Several (e.g., Chengdu Kanghua, Sanofi Pasteur, Serum Institute of India)	BPL	Human cultured fibroblasts	PM	Expensive, standard for rabies vaccine	United States, Europe, worldwide
	RVA	Local (e.g., Michigan Department of Health, Bioport)	BPL	Foetal rhesus cell culture	CVS-122	Fewer allergic reactions	United States (NLU)
	FBKC	Local (e.g., Pasteur Institute)	BPL	Foetal bovine kidney cell	PV-31	Concentrated and purified by zonal centrifugation	Several countries in Europe, Africa (NLU)
	PHKCV	Local	Typically formalin inactivated	Primary Syrian hamster kidney cell culture	SAD, Ag	Used historically in the People’s Republic of China and former USSR	China, Russia
	PCECV	Several (e.g., Bavarian Nordic, Bharat Biotech)	BPL	Chick embryo cell culture	PM	Purified by ultracentrifugation	United States, Europe, worldwide
	PVRV	Several (e.g., Bharat Biotech, Indian Immunologicals, Sanofi Pasteur)	BPL	Vero cell line	PV206, CTN-1, aG	Purified by ultracentrifugation	Europe, worldwide

DEV, duck embryo vaccine; HEP, Flury high egg passage vaccine; HDCV, human diploid cell vaccine; NLU, no longer used; PCECV, primary chick embryo cell vaccine; PDEV, purified DEV; PHKCV, primary hamster kidney cell vaccine; PVRV, purified Vero rabies vaccine; RVA, rhesus cell rabies vaccine.

The early rabies vaccines were modified versions of the Pasteur vaccine. They were produced in nerve tissues and inactivated with phenol. For PEP, 14–21 daily doses were required to induce adequate antibody titers. The potency of Semple vaccines was highly variable; these vaccines contained residual nerve tissue, and neurological complications such as neuropathy, Guillain–Barré syndrome, meningitis or encephalitis were common.¹³ NTVs are no longer recommended by the WHO, which endorses the use of vaccines grown in cell culture or embryonated eggs.¹⁴

Duck embryo vaccines (DEV) were prepared from a virus propagated in embryonated duck eggs. Although it reduced the number and severity of post-vaccine reactions, DEV was less immunogenic than the brain tissue vaccines.¹⁵ For DEV vaccines, 14–23 daily inoculations were recommended, but even this “heroic” dosage did not always protect against rabies after severe exposure. Problems, such as high adverse reaction rates and low immunization protection rates, have led to the widespread use of embryo avian vaccines. Thus, there had long been a pressing need for a highly immunogenic rabies vaccine that can be safely and effectively used at low doses for primary immunization and prevention after rabies exposure.¹⁶

The first attempts to develop a tissue culture vaccine were made by Kissling¹⁷ in 1958 and Fenje¹⁸ in 1960. Both investigators used primary hamster kidney (PHK) cells for the production of the RABV. In the early 1960s, staff at the Wistar Institute in Philadelphia, Pennsylvania, developed a human diploid cell vaccine (HDCV) using the WI-38 human diploid cell line for viral propagation, which avoids side effects such as animal protein allergies produced by primary tissue culture.¹⁹ HDCV contains concentrated and purified viruses, and the immune response in experimental animals and humans is much better than that of DEV, neonatal mouse brain, and adult animal brain tissue vaccines.²⁰ Although HDCV has good immunogenicity, its production costs and price are high. Therefore, the world has been striving to produce rabies vaccines at lower costs while achieving or exceeding the efficacy, safety, and immunogenicity levels achieved by HDCV.

A rabies vaccine developed by Sanofi Pasteur in Europe and many developing countries based on virus cultivation in Vero cells (African green monkey kidney cells) has been licensed.²¹ A purified Vero cell rabies vaccine (PVRV) for human use has also been launched and used by multiple factories in India (e.g. Bharat Biotechnology, Wockhardt, and the Institute of Human Bioproducts) and China (e.g. Chengdu Biotechnology, Wuhan Institute of Bioproducts, Liaoning Yisheng Biopharmaceutical Co., Ltd., and Liaoning Chengda Biotechnology Co., Ltd.,).¹² During the production of PVRV, Vero cells can grow and infect microcarrier beads and can be cultured in fermentation tanks to produce a large amount of tissue culture medium containing the RABV. Clinical studies have shown that the RABV neutralizing antibody (RVNA) response after initial and intensified injections of PVRV is comparable with that after pre-exposure prophylaxis (PrEP) or PEP vaccination with HDCV.²²

New rabies vaccines

Currently, commercially available rabies vaccines are mainly CCV; however, the immunization process is complex, with multiple visits and long visit cycles, which objectively hinders the implementation of complete PEP, resulting in occasional reports of vaccine failure.²³ In addition, vaccine production and immunization costs are high and immunization procedures are complex, making it far beyond the reach of using a single immunization regimen to expand PrEP and include it in children’s immunization plans in high-risk areas.²⁴ Therefore, the development of vaccines with good immunogenicity, faster production of effective protective neutralizing antibodies, better compliance, low cost, and stronger protection while ensuring safety is an important direction for new rabies vaccine research and development.

In recent years, novel adjuvant vaccines, protein and peptide vaccines, genetically modified vaccines, virus-like particle vaccines (VLPs), viral vector vaccines, and nucleic acid vaccines (DNA and RNA vaccines) have been the focus of search for new rabies vaccines.

The rabies vaccine is not only effective in preventing rabies but also a therapeutic vaccine that requires the production of sufficient antibodies and immune defense in the shortest possible time after exposure to prevent RABV infection. Although Alum has been approved as the first adjuvant for human use and is widely used, some believe that it delays the early production of antibodies and cannot effectively induce cellular immunity,^25–27 which is detrimental to the post-exposure preventive effect against rabies. On 30 June 2005 the China Food and Drug Administration banned the use of aluminum hydroxide (Al (OH)₃) adjuvants in human rabies vaccines.²⁸ Efficient, safe, and low-cost rabies adjuvant vaccines must not only reduce vaccine injection volume, but also enhance the immediate immune response, produce higher and more persistent levels of antibodies, and trigger strong cellular and humoral immunity by enhancing antigen presentation to antigen-specific immune cells, which is crucial^27,29–31 Second-generation adjuvants based on ligands for Toll-like receptors (TLR) appear to perform better.²⁷ One adjuvanted rabies vaccine called the PIKA vaccine, composed of Rabipur and a polyinosinic-polycytidylic acid-based TLR-3-activating adjuvant, has completed a phase II clinical trial.³² This vaccine is composed of Rabipur and a TLR-3 activation adjuvant based on polyinosine cytidine. The vaccine was administered in an accelerated regimen, with two doses on day 0, two doses on day 3, and one dose on day 7, and was compared with the conventional Rabipur regimen administered on days 0, 3, 7, and 14. The results showed that the PIKA rabies vaccine acceleration regimen triggered a protective immune response as early as day 7. All subjects in the PIKA group reached a protective rabies virus neutralizing antibody titers (0.5 IU/ml) on day 14, and the antibody titer increased faster than that in response to the control vaccine,^33–35

The rabies virus glycoprotein (RABV-G) is the only protein that exists on the surface of viral particles and is responsible for the binding of the virus to host cells. It stimulates humoral immunity to produce RVNA and T cells to induce cellular immunity. It assembles into homologous trimeric proteins on the surface of viral particles. Many expression systems have been explored for glycoprotein expression, such as mammalian expression systems based on human embryonic kidney (HEK) 293,³⁶ juvenile hamster kidney (BHK) 21,³⁷ or Chinese hamster ovary (CHO)³⁸ cell lines, which have been successfully tested to varying degrees. Depending on the cell medium and culture conditions,³⁹ they can glycosylate G proteins to varying degrees. Insect cell expression systems based on recombinant rod-shaped viruses⁴⁰ or transfected Schneider’s Drosophila 2 cells⁴¹ can express proteins at higher production sites, but typically only add shorter polysaccharides, fucose, and xylose residues. Yeast expression systems are also cost-effective; however, they undergo glycosylation with only mannose-containing polysaccharides, which is very different from glycosylation in mammalian cells. Therefore, yeast-derived RABV-G exhibit poor immunogenicity.⁴² RABV-G has also been expressed in plants such as tomatoes,⁴³ carrots,⁴⁴ and corn,⁴⁵ with the aim of developing an edible rabies vaccine. Although the production of exogenous proteins in plants is cost-effective, it is hindered by purification issues. To date, the immune response triggered by oral immunization with plant-derived RABV glycoproteins has been variable and is usually weaker than the immune response required to achieve reliable protection.⁴⁶

The RABV can be modified through reverse genetics⁴⁷ by deleting genes encoding phosphoprotein (P)⁴⁸ or matrix protein (M),⁴⁹ thereby weakening the virus virulence. This modified RABV does not replicate in animals (including immunodeficient animals); therefore, it may be safe. Rabies viruses with a matrix protein deficiency are more immunogenic than wild-type viruses with a phosphate protein deficiency or inactivation.⁵⁰ However, the modified virus has less reproductive power in cell culture than the wild-type virus, which may increase the cost of vaccine production. Researchers have also developed vaccine viruses carrying two or three copies of glycoprotein genes.⁵¹ In mouse studies, inactivated multi-G-protein RABV vaccines showed stronger immunogenicity than wild-type viruses.

The safest and most immunogenic of the different types of viral vector vaccines are adenoviruses, based on E1 deficiency.⁵² Adenoviruses cause replication defects by inserting sequences into the deleted E1 domain, which encodes proteins necessary for the transcription of other viral genes. Removing the adenoviral vector E1 induces a strong immune response in T and B cells. Due to the low persistence of the viral vector, this response can last for a long time.⁵³ Because of the strong innate immune response induced by adenoviruses, high doses can cause serious side effects. The main disadvantage of adenoviral vectors is that their immunogenicity and efficacy are disrupted by neutralizing antibodies targeting the vector in the body^54,55 Adenoviruses are widely present, and most people are infected with different adenoviral serotypes in early childhood. The presence of neutralizing antibodies in the body depends on the serotype and geographical location of the virus. Viruses isolated from non-human primates such as chimpanzees have been used as vectors. The serotypes of these viruses are extremely similar to those of human adenoviruses, but they do not spread among humans.⁵⁶ Therefore, most adults lack neutralizing antibodies against monkey adenovirus, and even those with antibodies often have low titers. An E1-deficient chimpanzee adenovirus, SAdV-25 (also known as AdC68), expressing the RABV-G, has been widely tested in mice and non-human primates,^56,57 After a single intramuscular injection, the virus can induce an effective and sustained neutralizing antibody response. Animals (including non-human primates) achieve complete protection against the RABV 1 year after vaccination. The adenovirus vector vaccine for the RABV will be highly cost-effective, as it is estimated that the cost of a single-dose vaccine is only $1.

mRNA vaccines

Nucleic acid vaccines contain both DNA and RNA. The principle involves connecting antigen genes with eukaryotic expression vectors and introducing them into the body to induce an immune response against the expressed proteins. Its advantage is that it can simultaneously activate both humoral and cellular immunity, and the body produces only an immune response to the inserted antigen, thereby reducing side effects. DNA vaccines are highly efficient and inexpensive, which makes them a good choice for use in developing countries. Human clinical trials have been disappointing in terms of their immunogenicity, although various efforts have been made to improve the efficacy of these vaccines. Several DNA vaccines have been licensed for veterinary use, such as the vaccines against the West Nile virus in horses and infectious hematopoietic necrosis virus in salmon, but no DNA vaccines have been licensed for human use; thus, the focus of nucleic acid vaccine research and development has shifted to RNA vaccines.⁵⁸ mRNA vaccines have been the most popular nucleic acid technology, particularly 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 results 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: non-amplifying 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 protein67. 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 has been considerably promoted.

Preclinical trials

Currently, there are several preclinical studies on mRNA rabies vaccines have shown that RNA vaccines administered to mice, dogs, pigs, rats, monkeys, and other animals can protect against the RABV (Table 2).

Table 2. Preclinical trials.

Year	Vaccine Platform	Vaccine	Forms	Delivery Platform	Preclinical Setting	Ref.
2009	Division of Animal Biotechnology, Indian Veterinary Research Institute	Sin-Rab-G-RNA	self-amplifying Mrna	/	mice	78
2016	CureVac	RABV-G mRNA	non-amplifying mRNA	Protamin	mice and pigs	61
2020	GSK	RG SAM (NEG)	self-amplifying mRNA	Cationic nanoemulsion	rats	62
2022	Liverna	LVRNA001	non-replicating mRNA	LNPs	mice and dogs	79
2022	GSK	RVG-saRNA	self-amplifying mRNA	SLNs; PNPs; LNPs	mice	82
2023	CSPC	SYS6008	non-replicating mRNA	LNPs	mice and cynomolgus macaques	83
2023	AMMS	mRNA-B-LNP	/	LNPs	mice and dogs	84
2023	Fudan University	RABV-G mRNA-LNP	non-amplifying mRNA	LNPs	mice	85
2023	Karolinska Institutet	RABV-G mRNA	/	LNPs	Chinese rhesus macaques	86
2023	Chengdu Qingbaijiang District People’s Hospital	RABV G mRNA	non-amplifying mRNA	LNP and nucleic acid immunostimulator (CPG 1018, CPG 2395 and Poly I:C)	mice	87
2023	Vaccines R&D Department, Changchun Institute of Biological Products,	RV021	non-amplifying mRNA	LNPs	mice	88
2023	Huazhong Agricultural University	LPP-mRNA-G	non-amplifying mRNA	LPP	mice and dogs	89

Saxena et al.⁷⁹ developed a self-amplifying RNA vaccine (SAM) that utilizes the SINV RNA replicon. In vitro-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 non-replicating 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 these tissues decreased, and it was still detected on day.⁶⁰ Overall, the animals showed good tolerance to the RG SAM (CNE) vaccine, which triggered the expected IgG immune response.

Since 2020, there has been a surge of interest in mRNA rabies vaccine research, driven by interest in mRNA COVID-19 vaccines. In 2022, Lavida developed a non-replicating mRNA rabies vaccine LVRNA001 based on the Therapeutics platform (Chinese patent ZL201911042634.2).⁸⁰ The ORF of the vaccine mRNA encodes the glycoprotein (RABV-G) of the CTN-1 strain (GenBank: ARC39382.1), which has been used to produce human rabies vaccines in China,^89,90 The vaccine uses LNPs as a delivery system. The results showed that LVRNA001 can induce sustained neutralizing antibodies and strong Th1 cell immune responses in mice and maintain high antibody levels for at least 6 months after immunization. The induced antibodies can effectively prevent the replication of the virus in mice and dogs, and have a protective effect against challenge with 50LD₅₀ RABV. Compared to a 3-day or 7-day dosing interval, extending the dosing interval in mice (14 days) can produce more antibodies. In addition, the immune protective effect in dogs after exposure showed that the survival rate after two doses of LVRNA001 was 100%, while the three-month survival rate of the inactivated vaccine control group was only 33.33%. LVRNA001 induces strong protective immune responses in both mice and dogs.

To explore the impact of administration route on the efficacy of RNA vaccines, Anderluzzi et al.⁸¹ investigated the immunogenicity of self-amplifying mRNA encoding RABV-G on different nanoparticle platforms (solid lipid nanoparticles, polymer nanoparticles, and lipid nanoparticles). The results indicated that among the four nanoparticle formulations, intramuscular and intradermal injections initially showed similar immunogenicity, but ionizable lipid nanoparticles exhibited higher long-term IgG responses. However, intranasal administration can lead to a lower immune response and rapid clearance, regardless of the formula used. This indicates that both the route of administration and the form of nanoparticles can affect the efficacy of saRNA vaccines.

By 2023, there were numerous preclinical studies on mRNA rabies vaccines. For example, a non-replicating mRNA vaccine (SYS6008)⁸² encoding the RABV (CTN-1 strain) RABV-G and tested its immunogenicity and immune protection in mice, as well as its immunogenicity and immune persistence in cynomolgus macaques. SYS6008 stimulated mice to produce higher levels of RVNA faster than the Rabipur-inactivated vaccine. In a PrEP mouse model, intramuscular injection with a 1/30 human dose of SYS6008 alone was able to resist the attack of a lethal dose of RABV. In the PEP model, the immunoprotective effect of SYS6008 was greater than that of the control vaccine. In addition, SYS6008 can generate a good immune response in cynomolgus macaques, and RVNA levels can be maintained at effective levels in 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 nucleoside-modified rabies mRNA-lipid nanoparticle vaccine (RABV-G mRNA-LNP) encoding a codon-optimized 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 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 tumor necrosis factor alpha (TNFα)-producing CD4+/CD8+ T cells as shown using a flow cytometry assay. In addition, in the pre- and post-exposure challenge assays, LNP + CPG 1018 encapsulated RABV G mRNA induced 100% protection against 25 LD50 of RABV infection, with 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 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 sequence-modified 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 LPP-mRNA-G promoted vaccine uptake and exhibited an ideal biological distribution 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 vaccine 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 promoted 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 freeze-drying, 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 next-generation 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-mRNA-GH270P 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 complexed forms with the cationic protein protamine.⁷³ CV7202 is a novel mRNA-LNP formulation (Table 3).

Table 3. Clinical trials.

Time	Vaccine Platform	Vaccine	Forms	Delivery Platform	Clinical Setting	Immunization Methods	Immune Response	Ref.
2017	CureVac	CV7201	Non-replicating mRNA	Cationic protein protamine	Human	Volunteers (18–40 y) with no history of rabies vaccination were recruited and received three doses of CV7201 intradermally or intramuscularly via a needle syringe or one of three needleless devices. Subsequent cohorts received incremental doses, with one cohort receiving a booster dose one year later.	The mRNA-based prophylactic vaccine candidate induces enhanced functional antibodies against viral antigens when injected using a needleless device, but not when injected via a needle syringe. The vaccine was generally safe and reasonably well tolerated.	92
2021	CureVac	CV7202	Non-replicating mRNA	LNPs	Human	Enrolled 55 healthy participants (18–40 y) to receive intramuscular injections of 5 μg (n = 10), 2 μg (n = 16), or 1 μg (n = 16) CV7202 on day 1; subsets (n = 8) of 1 μg and 2 μg groups received second doses on day 29. Controls (n = 10) received rabies vaccine, Rabipur, on days 1, 8 and 29.	Two 1 μg or 2 μg doses of CV7202 were well tolerated and elicited rabies neutralizing antibody responses that met WHO criteria in all recipients, but 5 μg had unacceptable reactogenicity for a prophylactic vaccine.	93

Between 21 October 2013 and 11 January 2016 CureVac⁹³ enrolled and vaccinated 101 participants with 306 doses of mRNA (80–640 µg) using needle syringe (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 intradermally 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. One year later, eight (57%) of 14 participants boosted with an 80 µg needle free intradermal dose of CV7201 achieved titers of 0·5 IU/mL or more. Conversely, intradermal or intramuscular needle syringe injection was ineffective, with only one participant (who received 320 µg intradermally) showing a detectable immune response. This first-ever demonstration in humans shows that a prophylactic mRNA-based candidate vaccine can induce functional antibodies against a viral antigen when administered with a needle-free device, but not when injected with a needle syringe. The vaccine was generally safe with a reasonable tolerability profile.

CV7202 is a novel mRNA-LNP formulation. A Phase I clinical study aimed to evaluate the safety, reactivity, and immunogenicity of different doses of CV7202 administered via intramuscular injection.⁹⁴ CureVac recruited 55 healthy adolescents aged 18–40 in Belgium and Germany to receive intramuscular injections of 5 μg (n = 10), 1 μg (n = 16), or 2 μg (n = 16) doses of CV7202 on day 1; The subgroups of the 1 μg and the 2 μg (n = 8) received a second dose of CV7202 on day 29. The control group (n = 10) received Rabipur on days 1, 8, and 29. The 5 μg dose of CV7202 group exhibited unacceptably high reactivity, and a high innate immune response driven by type 1 interferon and cytokines In addition, strong induction of Toll-like receptor signaling pathways were observed in most participants, which may have led to unfavorable reactivity and immunogenicity characteristics. The 1 μg and 2 μg dose groups showed better tolerance. No serious adverse events or vaccine-related discontinuation was observed. Low-dose-dependent neutralizing antibody reactions were detected starting from day 15, and low-dose-dependent RVNA reactions were detected. By day 29, 29%, 31%, and 22% of RVNA in the 1, 2, and 5 μg groups, respectively, were 0.5 IU/mL. After two doses of 1 or 2 μg immunization, all vaccinated individuals had a titer of 0.5 IU/mL on day 43, which was not significantly lower than that in the vaccine control group.

However, some difficulties remain in conducting subsequent phase 2 and phase 3 clinical trials in humans. The strength and durability of the immune response, particularly against rabies, an inevitable fatal infection, are not yet fully understood.

Discussion

Rabies is as a preventable but incurable zoonotic infectious disease, and 99% of human cases are transmitted by dogs. The most effective way to prevent rabies is to vaccinate dogs. Currently, effective management of dogs is not possible in many countries; therefore, standardized PEP is the best choice for preventing human rabies. Among these, immunization with a rabies vaccine is one of the core links in PEP. However, many factors, such as high vaccine costs, complex immunization procedures, and the combined use of passive immunization preparations for rabies, have led to a decrease in the compliance with rabies vaccines. The main objectives of the development of new rabies vaccines are safety, low cost, good immunogenicity, and simple immunization procedures.

First, ensuring that the vaccine has a good safety profile is the key to minimizing adverse reactions and side effects and safeguarding the health and safety of the vaccinated. Second, reducing the cost of vaccine production to make it more affordable will increase vaccination coverage, particularly in less economically developed areas, allowing more people to afford the vaccination. Third, vaccines must be immunogenic, that is capable of inducing a strong and long-lasting immune response that ensures long-term protection. Finally, simplifying the immunization process, including reducing the number and complexity of vaccinations, improves adherence and convenience so that more people are willing and able to complete the full course of vaccination. Achieving these goals will considerably enhance the effectiveness of rabies prevention and contribute to a more effective control of rabies transmission globally, particularly in resource-limited areas.

Over the past two decades, researchers have extensively discussed the potential of mRNA as a preventive means for the treatment of various infectious diseases. Particularly since 2020, with the successful application of mRNA vaccines in the COVID-19 epidemic and potential exploration in other diseases, mRNA vaccines have been receiving increasing attention and research investment and are expected to achieve new breakthroughs and progress in future vaccine research and development. In this article, we discuss various novel mRNA rabies vaccines that have been studied clinically and preclinically. We found that only the candidate rabies vaccines CV7201 and CV7202 developed by CureVac completed Phase I clinical studies. In preclinical studies, two doses of 80 µg CV7201 vaccine separated by 21 days induced high neutralizing antibody titers in mice and pigs, and triggered antigen-specific CD4+and CD8+T cell responses62. Subsequently, CureVac used proprietary LNP produced by Acuitas Therapeutics as a delivery vector for the new rabies candidate vaccine, CV7202. In phase I preclinical studies, CV7202 delivered unmodified mRNA encoding RABV-G and produced strong neutralizing antibodies, as well as CD8+ and CD4+T cell responses. In non-human primates, two doses of 100 µg CV7202 with a 28 d interval were well tolerated, producing antibody titers 20 times higher than commercially available rabies vaccines.⁹⁵ The phase I results showed that two doses of 1 µg produced high titer, strong adaptive immune responses, good tolerance, and significant improvement compared to CV7201 with a protamine delivery system.⁹⁴

In 2023, many reports related to mRNA rabies vaccines were published. Numerous preclinical studies on mRNA rabies vaccines have shown that, regardless of the delivery system used, whether self-amplifying or non-amplifying, mRNA can cause animals to produce strong neutralizing antibodies. SYS6008 has been studied in mice and non-human primates, and the results showed that SYS6008 can generate a good immune response in crab-eating monkeys and maintain effective RVNA levels in the medium to long term. LVRNA001 has been studied in mouse and dog models. It can induce sustained neutralizing antibodies and strong Th1 cell immune responses in mice and maintain high antibody levels for at least 6 months after immunization. Immunization in dogs showed that the survival rate after the two doses of LVRNA001 was 100%, whereas the survival rate of the inactivated vaccine control group at 3 months was only 33.33%. Although there are many studies on mRNA rabies vaccines, most of the animal models used in these studies were mice or rats. Gene vaccines are generally considered less effective in large animals than in rodents.⁹⁶ Therefore, Yu et al.⁸² conducted a study in a larger species with closer proximity to the human genome (cynomolgus macaques) and evaluated the immunogenicity of the SYS6008 rabies vaccine. Cynomolgus macaques, non-human primates, are the best animal models for human vaccine development, and their vaccine immunization dose used is equivalent to the vaccine immunization dose used in humans. The results showed that all cynomolgus macaques had neutralizing antibody titers above the protective level of 0.5 IU/mL on day 7 after the first immunization, and the RVNA titers remained above 0.5 IU/mL on day 152, suggesting that it is important to conduct immunogenicity and immunoprotection studies in large animals before launching a clinical study of a new gene vaccine. Moreover, the immunization effect in small animal models needs to be further validated in larger animal models.

Most current human rabies vaccines are inactivated vaccines, and the immunization procedure is mostly four or five doses by intramuscular injection, with a long immunization cycle and a high number of visits. The rabies mRNA vaccines discussed in this review mostly use a two-dose immunization procedure, requiring only two doses of immunization, with a short visit cycle and low number of visits, which can considerably reduce the cost of immunization in rabies PEP. Although mRNA vaccines are a promising new platform with high versatility, effectiveness, simplicity, scalability, affordability, and cold chain-free potential, two phase I clinical trials targeting mRNA rabies vaccines have shown that, although they had overall good tolerance and immunogenicity, the response was weaker than expected based on animal results. Therefore, it is necessary to better understand the mechanism of action of mRNA vaccines to understand the impact of innate immune responses generated by mRNA and delivery systems, as well as the different immune responses to mRNA vaccines in different species, to accelerate the development of mRNA vaccines.

Abbreviations

PEP	=	Post exposure prophylaxis
NTV	=	Nerve tissue vaccine
CCV	=	Cell culture vaccines
UTR	=	Untranslated region
RABV-G	=	Rabies virus glycoprotein
PHK	=	Primary hamster kidney
DEV	=	Duck embryo vaccine
HEP	=	Flury high egg passage vaccine
HDCV	=	Human diploid cell vaccine
NLU	=	No longer used
PCECV	=	Primary chick embryo cell vaccine
PDEV	=	Purified duck embryo vaccine
PHKCV	=	Primary hamster kidney cell vaccine
PVRV	=	Purified Vero rabies vaccine
RVA	=	Rhesus cell rabies vaccine
PrEP	=	Pre-exposure prophylaxis
VLPs	=	Virus-like particle vaccines
RABV	=	Rabies virus
TLR	=	Toll-like receptors
RVNA	=	Rabies virus neutralizing antibody
SaRNA	=	Self-amplifying RNA
ORF	=	Open reading frame
NSP	=	Non-structural proteins
SGP	=	Subgenomic promoter
VEEV	=	Venezuelan equine encephalitis virus
SINV	=	Sindbis virus
SFV	=	Semliki Forest virus
SGP	=	Subgenomic promoter
GOI	=	Gene of interest
PRRs	=	Pattern recognition receptors
IFN	=	Interferon
LNPs	=	Lipid nanoparticles
PEG	=	Polyethylene glycol
CNE	=	Cationic nanoemulsion

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