Rabies Virus Glycoprotein‑Based DNA Vaccines: Current Status and

Future Directions

Yuqin Wang

Changchun American International School, Changchun, Jilin, China

Keywords: Rabies DNA Vaccine, Glycoprotein, Immunogenicity.

Abstract: Rabies is a very virulent viral infection, and rabies DNA vaccines are a novel approach to its prevention.

These vaccines are prepared by placing the gene that codes for a key rabies virus antigen in a recombinant

DNA plasmid under the control of eukaryotic ex-pression elements. When given to the body, they cause

humoral and cell-mediated immune reactions with the following benefits: broad-spectrum immunity, long-

term im-munity, fewer side - effects, easy production, and absence of requirements for cryopreservation.

Nonetheless, DNA vaccines are limited by relatively weak immunogenicity relative to protein-based vaccines,

uncertain long-term protection, and restricted mass production and regulatory approval. Ongoing research is

aimed at enhancing immunogenicity, optimizing dosing regimens, simplifying production processes, and

establishing appropriate regulatory infrastructures. These efforts will maximize the application of rabies DNA

vaccines in rabies control globally.

1 INTRODUCTION

Rabies remains a critical public health threat, with

nearly 100% mortality once clinical symptoms

appear. The rabies virus is a neurotropic pathogen that

primarily invades and replicates within the nervous

system. Transmission occurs when infected animals

bite, scratch, or lick broken skin or mucous

membranes of humans (WHO, 2019). Upon entry, the

virus’s glycoprotein spikes bind specifically to

receptors on host neuronal cells, facilitating viral

entry. Inside the cell, the nucleoprotein orchestrates

transcription and replication of the viral genome,

leading to rapid viral proliferation. As viral

replication disrupts neuronal function, impaired nerve

signal transmission manifests clinically as

hydrophobia, dysphagia, and respiratory difficulties.

Progressive neuronal damage ultimately results in

respiratory and circulatory failure and death. Rabies

remains widely endemic across multiple regions,

imposing significant health, economic, and social

burdens globally (Whitehouse et al., 2023).

Rabies vaccines include human diploid cell

vaccines, Vero cell vaccines, and primary cell

vaccines, such as those derived from hamster kidneys

and chicken embryos. While these vaccines have

been instrumental in controlling the disease, they

mainly stimulate humoral immunity and offer limited

activation of cellular immune responses. The

reliance on humoral immunity alone may not provide

sufficient protection against viral infection,

particularly in the context of emerging viral variants.

This demonstrated the inefficacy of traditional rabies

vaccines. DNA vaccines, on the other hand, showcase

a stable alternative with the potential to stimulate both

humoral and cellular immune responses.

Furthermore, DNA vaccines are less expensive, more

stable, easy to prepare, do not need cryogenic

preservation, and are easy to popularize in developing

countries (Fisher et al., 2018). This review aims to

explore the key technologies and challenges in the

development of rabies virus DNA vaccines and assess

their potential in global vaccination. The

comprehensive analysis of the existing literature is

aimed to provide theoretical support for the

improvement and popularization of the rabies virus

vaccine in the future.

330

Wang, Y.

Rabies Virus Glycoproteinâ

SBased DNA Vaccines: Current Status and Future Directions.

DOI: 10.5220/0014488000004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 330-334

ISBN: 978-989-758-789-4

2 STRUCTURE AND IMMUNE

EVASION STRATEGIES OF

THE RABIES VIRUSS

2.1 Structure and Mechanism of

Infection

The rabies virus is a bullet‑shaped, single‑stranded,

negative‑sense RNA virus of the Rhabdoviridae

family. It has a structure built up of a nucleocapsid

core, with a surrounding lipid bilayer envelope. The

surface glycoprotein is vital in the viral entry into the

host cell. There are glycoprotein spikes that can

specifically bind to receptors on the surface of host

nerve cells, such as the nicotinic acetylcholine

receptor. This binding event induces conformational

changes of the glycoprotein involved in the fusion of

the virus with the cell membrane and the entry of the

virus into the cell by endocytosis. Inside the cell, the

nucleoprotein releases the viral genome that starts

viral transcription and replication. The virus hijacks

the host’s transcriptional machinery to produce viral

proteins, ultimately leading to the assembly of new

virions and their spread through neural pathways.

This efficient invasion mechanism contributes to the

high lethality of rabies infections (Sugiyama et al.,

2025).

2.2 Immune Evasion Strategies

The rabies virus has developed sophisticated

strategies to escape the host immune system. One

primary mechanism involves its ability to persist

within nerve cells, where immune surveillance is

inherently limited. Unlike other tissues, the nervous

system has restricted access to circulating immune

cells, allowing the virus to replicate and spread

largely undetected. Apart from this, the antigenic

structure is continuously modulated by the virus.

Over a period, the glycoprotein mutates, which causes

changes in the epitopes recognized by the immune

cells of the host, such as antibodies and T

lymphocytes. Therefore, the immune system is not

able to clearly recognize the virus, which allows the

virus to carry on with its infection cycle and cause

disease. This antigenic variation, combined with the

virus’s neurotropic nature, made the rabies able to

start the infection while evading effective immune

responses (Guo et al., 2019).

3 DNA VACCINE TECHNOLOGY

FOR RABIES CONTROL

3.1 Principles of DNA Vaccines

DNA vaccines are extremely efficient in rabies

prevention, offering advantages over traditional

vaccines. DNA vaccines for rabies are constructed by

placing the gene encoding a key antigen (which is

often the glycoprotein of the rabies virus) under the

control of eukaryotic expression elements that form a

recombinant DNA plasmid. When this plasmid is

introduced into the animal body, it enters host cells.

Inside the host cell, the plasmid utilizes the cell's

endogenous transcription and translation machinery.

The eukaryotic promoter in the plasmid initiates the

transcription of the inserted antigen-encoding gene

into messenger RNA (mRNA). This mRNA is then

translated into the antigen protein by ribosomes in the

cytoplasm. The newly synthesized antigen protein is

then presented on the cell surface or released into the

extracellular environment. This process of the antigen

activates the host's immune system, which then leads

to the production of both humoral and cellular

immune responses. B cells recognize the antigen and

differentiate into plasma cells, which secrete

antibodies. T cells, including cytotoxic T

lymphocytes (CTLs), are also activated, which can

directly kill virus-infected cells. This dual activation

of humoral and cellular immunity provides broad and

long-lasting protection against rabies virus (Mlingo et

al., 2025).

3.2 Advantages of DNA Vaccines

Firstly, the DNA vaccine will have a benefit in terms

of immunogenicity compared with traditional rabies

vaccines. While traditional vaccines would mostly

exert humoral immunity, DNA vaccines are capable

of additionally activating cellular immune responses.

Based on such broad immune activation, DNA

vaccines provide greater protection against any viral

infection, especially in the presence of their variants.

Secondly, in terms of efficacy, DNA vaccines should

give long-lasting protection, mainly due to the

possibility of prolonged antigen production within the

host cell. Traditional vaccines may have to be given

repeatedly, for example, by multiple boosters, to

maintain some immunity. In addition, DNA vaccine

side effects are generally expected to be fewer than

those inherited from traditional vaccines. They do not

contain live or attenuated viruses, consequently

Rabies Virus Glycoproteinâ

SBased DNA Vaccines: Current Status and Future Directions

331

reducing the likelihood for the vaccine to cause some

form of the disease itself or severe allergic reactions.

Moreover, the DNA vaccines are relatively easy to

manufacture. What is required is mainly plasmid

amplification in bacteria, which is much more

convenient and less costly compared to production

processes of traditional vaccines, like growing viruses

in cell cultures (Porter et al., 2017). Finally, in

contrast to other vaccines, one does not have to worry

about cryogenic preservation for DNA vaccines,

which makes them warranted for use in developing

countries, owing to the limited cold-chain structure.

4 OPTIMIZATION STRATEGIES

FOR RABIES VIRUS DNA

VACCINES

4.1 Enhancing Glycoprotein

Immunogenicity

The glycoprotein is one of the most immunogenic

antigens of the DNA vaccine for rabies and is thus the

vaccine target since it is the main protein that the

immune system recognizes in natural infection (Chen

et al., 2025). Various optimization approaches have

been employed to increase the immunogenicity of the

rabies virus glycoprotein. Codon optimization has

been employed to enhance translation efficiency in

host cells, while fusion with immune-stimulating

cytokines, such as granulocyte-macrophage colony-

stimulating factor (GM-CSF), has been investigated

to enhance antigen presentation. Structural

modifications, such as stabilizing the glycoprotein in

its native trimeric conformation, have also been

explored to improve its recognition by the immune

system and increase the production of neutralizing

antibodies (Ng et al., 2022).

4.2 Plasmid Design and Promoter

Selection

Selecting the right plasmid plays an important role in

the functionality of the rabies virus DNA vaccines.

Different plasmids have different properties and

making a well-considered choice can significantly

affect the success of the vaccine. Promoters are yet

another game-changer in plasmid-based vaccines.

While strong promoters such as the cytomegalovirus

promoter are effective for high-level expression of the

antigen-encoding gene, there are specific contexts in

which tissue-specific promoters may be more

appropriate since they target expression of the antigen

in specific types of cells to enhance the immune

response. Some additional enhancers can also be

transfected to help regulate gene expression through

the plasmid. These elements work together to

improve the production of the antigen protein within

host cells that enhances the immune effect of the

vaccine (Disis et al., 2023).

4.3 Delivery Systems for DNA Vaccines

Efficient delivery of DNA vaccines into host cells is

critical for achieving robust immune responses. There

are a number of delivery methods that can be utilized

for DNA vaccines. Viral vectors, including

adenoviruses, and lentiviruses, can be used to deliver

the DNA plasmid. These vectors can successfully

penetrate the host cell and release the plasmid inside.

However, they may activate existing immune

responses against the vector itself. Other methods of

delivery involve the use of liposomes to encapsulate

the DNA plasmid to protect it from degradation and

assist with its entry into cells. Relatively safe, they

can also be altered to target specific cell types.

Electrically, this method provides a field in which

temporary pores are produced by electric pulses

transmitted between electrodes into the membranes of

the cells; through the pores, DNA plasmids can get

into the cells. All the different delivery approaches

each have their inherent strengths and weaknesses,

leading researchers to sporadically amuse themselves

with techniques to optimize them with the hope of

further rousing rabies virus DNA vaccine

effectiveness.

5 CHALLENGES AND FUTURE

PATHWAYS

5.1 Mitigation of Immunogenicity

Constraints

Although promising possibilities of DNA vaccines,

these mainly produce more timid immune responses

as opposed to protein-based ones. The primary reason

lies in intricate processes of antigen expression and

presentation after the DNA vaccine has achieved

entry into cells and are guided by different variables.

In addressing the weakness, scientists turn towards

several avenues. On the one hand, more optimization

of the antigen gene design is underway. For example,

modification of the amino acid makeup of the antigen

BEFS 2025 - International Conference on Biomedical Engineering and Food Science

332

can enhance the immune system recognition. On the

other hand, studies on new adjuvants are ongoing.

The adjuvants have the potential to enhance the

ability of the immune cells to absorb and process

antigens and to make the immune response stronger.

5.2 Fulfilling Long: Term Protection

and Booster Needs

While DNA vaccines can extend antigen expression,

the long - term duration of immunity is an issue. The

research today aims to identify the best dosing

regimen and if booster doses should be administered

periodically to ensure protective immunity.

Researchers perform animal and clinical studies,

varying dosing frequencies, dosage, and route of

administration, to monitor variations in the immune

response. Meanwhile, they are studying vector

systems that can stably and continuously express

antigens, so the immune system is always stimulated

and maintains a high level of immune protection.

Meanwhile, research on the generation and

maintenance mechanism of memory immune cells is

carried out to provide a theoretical basis for making

reasonable booster immunization schedules.

5.3 Large-Scale Production and

Regulatory Considerations

In order to apply them on a large scale, DNA vaccines

must be produced inexpensively in large lots and

undergo stringent regulatory evaluations to establish

efficacy as well as safety. For now, the vast majority

of DNA vaccine production relies on bacterial

fermentation for plasmid amplification, which

however still remains challenging such as cost-

prohibitive and yield-fluctuating protocols. In the

future, there is a necessity to optimize the production

process, increase the yield and purity of plasmids, and

reduce production costs. Existing regulatory

frameworks may not fully accommodate DNA

vaccines, given their status as a novel vaccine class.

Therefore, there is a need to create a specialized

regulatory system for DNA vaccines, balance safety

and efficacy assessment, and simplify production and

the approval process, which will be paramount for

their global application.

6 CONCLUSION

Rabies is a deadly viral infection that dangerously

threatens human and animal health. Rabies vaccines

have played an important role in preventing and

controlling rabies, but with certain drawbacks. DNA

vaccines, a new form of rabies protection, possess the

following unusual strengths. By positioning the gene

responsible for coding an important antigen (such as

glycoprotein) of the rabies virus onto a recombinant

DNA plasmid governed by eukaryotic regulatory

elements, they can activate both the cellular and

humoral host immune responses once inside the

organism and thereby bestow broad-spectrum and

durable immunity. Relative to traditional vaccines,

DNA vaccines possess significant advantages in

immunogenicity, efficacy, side - effects, ease of

production, and storage needs. However, currently,

DNA vaccines also possess many issues in practical

applications, such as comparatively weak

immunogenicity, requiring extending the duration of

immunity, and encountering difficulties in mass

production and approval by regulatory agencies.

Future research needs to be more profound, like

maximizing vaccine design, increasing production

processes, and streamlining the regulatory system, to

maximize the full potential of DNA vaccines in

prevention and control of global rabies and safeguard

the life and health of human beings and animals.

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