Virus-like particles (VLPs) are unique biomaterials that have a similar appearance and structure to real viruses, but essentially do not contain viral genetic material, such as DNA or RNA. Therefore, they do not have the ability to infect. Due to this property of VLPs, they have shown great potential in vaccine development, especially in designing vaccines against hepatitis B, human papillomavirus (HPV), and the latest new coronavirus (COVID-19).

Development of VLP vaccines

As highly structured protein particles, VLPs are conducive to the uptake of antigen-presenting cells and can stimulate the host's innate and adaptive immune response functions. In the past three decades, VLPs have been widely used, especially in the field of vaccines. Some VLP-based vaccines have been used in commercial production or have entered different stages of clinical research (Figure 1). Hepatitis B virus (HBV) VLP was the first VLP-based vaccine approved, followed by human papilloma virus (HPV) VLP and hepatitis E virus (HEV) VLP. In 2021, a malaria vaccine was approved for marketing. In addition, Norwalk virus VLP vaccines and influenza virus VLP vaccines are undergoing clinical trials, and hepatitis C virus VLP vaccines and dengue virus VLP vaccines are in the preclinical trial stage. At the same time, animal viruses based on VLPs are also being developed, such as porcine parvovirus VLP vaccines.

Figure 1. Developmental milestones of currently approved VLP-based vaccines. (Lua LH, et al. 2014)

Mechanism of Action of VLP

Due to its good immunogenicity and high safety, VLP has gradually become a hot topic in drug and vaccine research today. Studies have shown that the mechanism of action of VLP is mainly related to immune stimulation. First, VLP natural viruses have antigens with similar conformations, and VLP surface antigens can enhance B cell activation by cross-linking multiple B cell receptors. Secondly, VLP is taken up by dendritic cells through mechanisms such as phagocytosis and penetration. The internalized VLP is processed and presented by the Major Histocompatibility Complex (MHC) II for activation of helper T cells (Figure 2). Finally, due to the highly ordered structure of VLP, it can be recognized by specific receptors and increase MHC class I antigen presentation, thereby inducing T lymphocyte toxicity.

Figure 2. Immunostimulation by VLPs and virosomes. (Mellid-Carballal, Rocio, et al. 2023)

VLP expression platform

Various expression platforms, including prokaryotic and eukaryotic systems, can be used to produce VLPs, such as yeast, baculovirus/insect cell systems, mammalian cells, and plant systems. In addition, cell-free expression systems have also been successfully applied to the expression of VLPs.

• Bacteria

Bacteria are one of the most widely used expression systems. They are not suitable for the production of enveloped VLPs due to various factors such as incomplete disulfide bond formation and protein solubility issues. However, they can be used to produce non-enveloped VLPs with one or two viral structural proteins. Escherichia coli is the most common bacterial host cell for VLP production. Various VLP vaccines generated using E. coli expression systems have entered clinical trials due to low production costs, fast cell growth, high protein expression levels, and ease of scale-up. In addition to E. coli, successful VLP formation has also been observed in other bacteria, such as Lactobacillus casei and Pseudomonas fluorescens.

• Yeast

Yeast is commonly used for recombinant protein expression and also for the production of VLPs, usually for the production of non-enveloped VLPs. In particular, Saccharomyces cerevisiae and Pichia pastoris are favored for their advantages such as fast cell growth, high protein expression yields, scalability, and a certain degree of post-translational modifications (PTMs). Currently, two FDA-approved vaccines based on VLPs, Engerix-B (HBV vaccine) and Gardasil (HPV vaccine), have been produced in yeast expression systems. However, the lack of complex PTM pathways is a major disadvantage of yeast expression systems, which limits their application in VLP production.

• Baculovirus/Insect Cells

The baculovirus/insect cell expression system is the most commonly used expression system for producing VLPs. Baculovirus-based VLP expression is rapid and convenient, so this system is suitable for the production of viral vaccines that rapidly change their surface antigens (such as influenza virus vaccines). The insect cell expression system has several advantages, such as high yields of expressed proteins, the presence of complex PTM pathways, and the formation of multi-protein VLPs. Commonly used insect cell lines include Sf9 cells, Sf21 cells, Tn-368 cells, and High Five cells. The main potential disadvantage of the baculovirus/insect cell expression system is that the N-glycosylation pattern of the expressed glycoprotein is simpler than that of mammalian cells, which may be a disadvantage for some VLP applications. If the insect cell glycosylation pattern is improved, the baculovirus/insect cell system may be the best expression system for VLP-based vaccine production.

• Plant cells

Compared with traditional methods, plant expression systems have advantages such as high protein expression levels, low cost and high-performance expression processing. Therefore, plant expression systems are currently a cost-effective and scalable alternative for the production of different pharmaceutical proteins, including vaccines. More than 55 different plant viruses have been used to construct antigen expression platforms, including tobacco mosaic virus, alfalfa mosaic virus, cowpea mosaic virus and papaya mosaic virus. Among them, one of the most effective plant expression systems is based on Agrobacterium-mediated tobacco mosaic virus expression vector.

• Mammalian and avian cells

Animal cell expression systems are the most valuable and attractive platforms for the production of a variety of structural proteins for both non-enveloped and enveloped VLPs. Animal cell expression systems are the most efficient systems for VLP production because they are able to make complex and precise PTMs, which are essential for correct protein folding. Currently, a variety of animal cell lines are widely used to produce VLPs, including CHO, BHK-21, HEK293, and CAP-T. Among them, CHO is the most commonly used cell line, which is not derived from human cells and has a lower risk of contamination by human viruses. HEK293 has been widely used to produce VLPs against HIV, influenza, and rabies viruses. The CAP-T cell line has also been shown to be an efficient expression system for the production of HIV VLPs. However, animal cell expression systems also have certain drawbacks. Low protein yield, high production cost, long expression time, and the possibility of cell lines carrying mammalian pathogens are the main potential disadvantages.

• Cell-free systems

Cell-free systems provide an alternative to VLP production by expressing recombinant proteins in vitro. These systems usually consist of bacterial or yeast cells that synthesize viral capsid proteins. Compared with cell-based protein expression platforms, cell-free systems save time, provide high protein yields, have limited cellular contaminants, and can selectively generate VLPs containing unnatural amino acids or toxic protein intermediates. However, cell-free systems have high production costs and significant limitations in commercial applications.

VLP-based vaccines are believed to induce strong humoral and cellular immunity against a variety of antigens. At the same time, they are safer than traditional vaccines, and due to their similarity to the parent virus in size and morphology, VLP has developed into a widely used biotechnology in the past few years. At the same time, the diversity of VLP structure and the versatility of its function provide the potential for rapid development of research. With the latest development of VLP technology, its advantages in vaccine production and vaccine preparation technology provide an important research platform and broad application prospects for the development of new vaccines.

Virus-Like Particles (VLPs) Service at Creative Biogene

VLP development faces multiple challenges, including high requirements for expression system folding and assembly capabilities, production yield fluctuations and consistency control difficulties, separation and purification challenges, and lack of unified quality control standards. Creative Biogene, combining Continuous Bioprocessing Manufacturing (CBM) concepts and Advanced Process Control (APC) strategies, has achieved significant breakthroughs in improving production efficiency and ensuring product quality consistency.

Our process development team builds customized VLP process development plans based on client needs, from prokaryotic/eukaryotic expression system selection and upstream expression optimization to downstream purification strategy establishment, ensuring controllable, scalable, and transferable processes: