Virus-like Particles (VLPs) are unique biomaterials that structurally mimic the morphology and configuration of native viruses, yet they contain no viral genetic material such as DNA or RNA, and thus possess no infectivity. Owing to this characteristic, VLPs exhibit enormous potential in vaccine research and development, particularly in the design of vaccines against Hepatitis B, Human Papillomavirus (HPV), and the emerging Coronavirus Disease 2019 (COVID-19).
VLPs are capable of triggering robust immune responses, as they can be recognized as foreign substances by the host immune system, thereby eliciting specific immune reactions including antibody production and cell-mediated immunity. This renders VLPs a safe and effective vaccine platform for the prevention of multiple viral diseases.
In addition, VLPs can serve as delivery vehicles to transport specific molecules or drugs to targeted cells in vivo. By mimicking the entry pathways of native viruses, VLPs can be utilized to investigate viral life cycles and develop novel antiviral therapeutics.
In recent years, VLP technology has achieved rapid advancement in preventive medicine, driving the research and development of numerous highly efficacious vaccine candidates against various infectious diseases and substantially expanding the preventive capacity of the vaccine industry.
1. Development History of VLP Vaccines
As highly structured protein particles, VLPs are readily internalized by antigen-presenting cells, enabling the activation of both innate and adaptive immune responses in hosts. Over the past three decades, the application of VLPs has expanded extensively, especially in the field of vaccines. A number of VLP-based vaccines have been commercially launched or advanced into different stages of clinical research. The Hepatitis B Virus (HBV) VLP vaccine represents the first approved VLP-based vaccine, followed by the successive licensing of Human Papillomavirus (HPV) VLP and Hepatitis E Virus (HEV) VLP vaccines. In 2021, the malaria vaccine was approved for marketing. Furthermore, Norovirus and influenza VLP vaccines are currently in clinical trials, while Hepatitis C virus and dengue virus VLP vaccines remain in the preclinical stage. Meanwhile, VLP vaccines targeting animal viruses such as porcine parvovirus are under continuous development.
2. Diversity of VLPs
2.1 Structural Diversity
The structural diversity of VLPs endows them with versatile functional applications. VLPs are categorized into non-enveloped and enveloped viral particles. Non-enveloped VLPs are further divided into single-capsid protein VLPs and multi-capsid protein VLPs. Single-capsid VLPs consist of a single capsid protein and can be produced in both prokaryotic and eukaryotic expression systems, as exemplified by licensed HPV VLP vaccines. The production of multi-capsid protein VLPs is more complex, which relies on the co-expression of distinct capsid proteins and subsequent intracellular complex assembly in eukaryotic expression systems.
Enveloped VLPs feature more sophisticated structures compared with non-enveloped counterparts and can only be generated in eukaryotic expression systems. They are encapsulated by host-derived glycoprotein-containing lipid membranes in a matrix form, and these glycoproteins serve as key immunogenic antigens targeted for neutralizing antibody production. Such VLPs are characterized by intricate structures and high construction difficulty.
Possessing favorable immunogenicity and high safety profiles, VLPs have gradually become a research hotspot in modern pharmaceutical and vaccine development. Studies have demonstrated that the mechanism of action of VLPs is primarily associated with immune stimulation. Firstly, VLPs display conformationally native antigens similar to authentic viruses, and surface antigens of VLPs enhance B cell activation by crosslinking multiple B cell receptors[3]. Secondly, VLPs are internalized by dendritic cells via phagocytosis and permeation. The intracellular VLPs are processed and presented by Major Histocompatibility Complex (MHC) class II molecules to activate helper T cells. Finally, the highly ordered structure of VLPs enables recognition by specific receptors, facilitates MHC class I antigen presentation, and further triggers cytotoxic T lymphocyte responses.
3. VLP Expression Platforms
A variety of expression platforms including prokaryotic and eukaryotic systems are available for VLP production, such as yeast, baculovirus/insect cell, mammalian cell and plant systems. Moreover, cell-free expression systems have also been successfully applied to VLP expression.
Yeast Expression System
Ease of expression: High (+++)
Scalability: High (+++)
Production yield: High (+++)
Post-translational modification: Low (+)
Baculovirus-Insect Cell Expression System
Ease of expression: Moderate (++)
Scalability: Moderate (++)
Production yield: Moderate (++)
Post-translational modification: High (+++)
Mammalian Cell Expression System
Ease of expression: Moderate (++)
Scalability: Moderate (++)
Production yield: Low (+)
Post-translational modification: Very high (++++)
Plant Expression System
Ease of expression: Moderate (++)
Scalability: Moderate (++)
Production yield: Low (+)
Post-translational modification: Very high (++++)
3.1 Bacterial Expression System
Bacteria rank among the most widely used expression hosts. Due to incomplete disulfide bond formation, poor protein solubility and other limiting factors, bacterial systems are unsuitable for the production of enveloped VLPs, but applicable to non-enveloped VLPs composed of one or two viral structural proteins.
Escherichia coli is the most prevalent bacterial host for VLP production, featuring low production cost, rapid cell growth, high protein expression level and easy scale-up. Multiple VLP vaccines generated by E. coli expression systems have entered clinical trials. Successful VLP formation has also been observed in other bacterial strains, including Lactobacillus casei and Pseudomonas fluorescens.
3.2 Yeast Expression System
Yeast is commonly employed for recombinant protein expression as well as VLP manufacturing, predominantly for the production of non-enveloped VLPs. In particular, Saccharomyces cerevisiae and Pichia pastoris are favored for their advantages of rapid cell proliferation, high protein yield, favorable scalability and moderate post-translational modifications (PTMs). Currently, two FDA-approved VLP-based vaccines, Engerix-B (HBV vaccine) and Gardasil (HPV vaccine), are manufactured using yeast expression systems. Nevertheless, the lack of complex PTM pathways constitutes a major drawback of yeast systems, restricting their broader application in VLP production.
3.3 Baculovirus-Insect Cell Expression System
The baculovirus-insect cell expression system is one of the most commonly used platforms for VLP production. It enables rapid and convenient VLP expression, making it ideal for developing viral vaccines with rapidly mutable surface antigens such as influenza vaccines. Insect cell expression systems offer multiple strengths including high recombinant protein yield, complete complex PTM pathways and the capability to assemble multi-protein VLPs. Widely utilized insect cell lines include Sf9, Sf21, Tn-368 and High Five cells.
A potential limitation of this system is that the N-glycosylation patterns of expressed glycoproteins are relatively simpler compared with mammalian cells, which may compromise its applicability for certain VLP-related research. With optimized insect cell glycosylation profiles, the baculovirus-insect cell system is expected to become the optimal platform for industrial VLP vaccine production.
3.4 Plant Cell Expression System
Compared with traditional expression platforms, plant expression systems exhibit superiorities such as high recombinant protein yield, low cost and high-efficiency production processing. Accordingly, plant systems have emerged as a cost-effective and scalable alternative for manufacturing various pharmaceutical proteins including vaccines. More than 55 distinct plant viruses have been exploited to construct antigen expression platforms, including Tobacco Mosaic Virus, Alfalfa Mosaic Virus, Cowpea Mosaic Virus and Papaya Mosaic Virus. Among them, the Agrobacterium-mediated Tobacco Mosaic Virus vector represents one of the most efficient plant expression systems.
3.5 Mammalian and Avian Cell Expression System
Animal cell expression systems are the most valuable and attractive platforms capable of producing both non-enveloped and enveloped VLPs with multiple structural proteins. They are recognized as the most efficient systems for VLP production owing to their capacity to conduct sophisticated and precise PTMs, which are indispensable for proper protein folding.
A variety of mammalian cell lines are widely adopted for VLP manufacturing, including CHO, BHK-21, HEK293 and CAP-T cells. CHO cells are the most commonly utilized cell line, with a non-human origin that lowers the risk of human viral contamination. HEK293 cells are extensively used to produce VLPs against HIV, influenza and rabies viruses, and CAP-T cell lines have also been validated as efficient platforms for HIV VLP production.
However, animal cell expression systems have inherent drawbacks: low protein yield, high production cost, long culture cycle, and potential risk of contamination by endogenous mammalian pathogens.
3.6 Cell-Free Expression System
Cell-free systems provide an alternative strategy for in vitro VLP production via recombinant protein synthesis. These systems are generally derived from bacterial or yeast cell lysates for the biosynthesis of viral capsid proteins. Compared with cell-based expression platforms, cell-free systems feature time efficiency, high protein yield, minimal cellular contaminants, and the capability to generate VLPs containing unnatural amino acids or toxic protein intermediates. Conversely, high manufacturing costs greatly limit their commercial large-scale application.
4. Optimization of VLPs
Although multiple VLP expression platforms have been established and preliminarily optimized, their biological potency still fails to meet future market and clinical demands. To address these limitations, extensive efforts have been devoted to VLP optimization, including cell culture medium optimization, cell line engineering, rational design via design of experiments (DOE), and culture medium composition modification.
Furthermore, a more sophisticated strategy adopts constraint-based modeling for bioprocess optimization. Research indicates that constraint-based modeling centered on genome-scale metabolic networks serves as a core approach for in-depth understanding of upstream bioprocesses. A genome-scale metabolic network is a mathematical representation of cellular metabolism that quantifies the stoichiometry of all metabolic transformation processes. Based on this network, diverse metabolic models can be constructed to tackle key challenges in bioprocess optimization, such as medium optimization and engineering target identification. Therefore, in silico simulation tools can be applied to optimize medium composition by analyzing intracellular metabolic flux distribution, thereby reducing by-product accumulation and enhancing cellular productivity.
Conclusion
VLP-based vaccines are capable of inducing potent humoral and cellular immune responses against multiple antigens, while demonstrating superior safety profiles relative to conventional vaccines. With inherent similarities to parental viruses in particle size and morphology, VLPs have evolved into a widely applied biotechnology over the past decades.
Meanwhile, the structural diversity and functional versatility of VLPs endow them with tremendous research potential for further development. Robust and flexible production platforms have broadened the application prospects of VLP technology. With the latest technological advances, the inherent advantages of VLPs in vaccine production and preparation processes provide a critical research platform and promising prospects for the development of novel vaccines.
