What are VLPs?

VLPs (Virus-Like Particles) are highly structured protein particles self-assembled from one or more structural proteins of a virus. Most VLPs have icosahedral or helical shell structures composed of several specific structural proteins. They possess characteristics very similar to real viruses, making them easily recognizable by the immune system. VLPs present viral antigens in a manner similar to their native conformation, thereby inducing a strong immune response. However, they do not contain the genetic material of the virus, thus cannot replicate or cause disease^[1]. In simple terms, VLP vaccines provide an opportunity for the immune system to "simulate and practice," enabling an immediate counterattack when encountering the actual virus. Moreover, VLP vaccines have inherent adjuvant effects, comparable to "playing a game with built-in cheats." Have you ever imagined a type of vaccine that could combat various deadly viruses without worrying about potential side effects? The answer is VLPs (Virus-Like Particles) vaccines.

As a type of subunit vaccine, VLPs have been considered the most promising candidate to replace traditional inactivated vaccines in recent years due to their outstanding advantages of safety, efficacy, and the ability to enable differential diagnosis. The preparation process of VLP vaccines generally includes the following steps: viral structural gene construction and cloning, host expression system selection, isolation and purification, and analytical identification^[2].

Classification of VLP Vaccines

Based on the structure of the original virus, VLP vaccines can be classified into two categories: enveloped and non-enveloped. Non-enveloped VLP vaccines contain only particles formed by viral proteins, such as those for norovirus and hepatitis E virus, and are generally produced using prokaryotic expression systems or lower eukaryotic expression systems. Enveloped viruses, in addition to the virus's own proteins, also include the host cell membrane, examples being influenza A virus and hepatitis B virus. These are generally produced using eukaryotic expression systems.

Fig. 1  Schematic diagram of Hepatitis B virus

Advantages of VLP vaccines

1) Safety: VLPs do not contain replicase or nucleic acids encoding viral structural proteins, lacking the ability to replicate.

2) Highly effective immune protection: VLP self-assembling vaccines can activate the body's immune system, producing specific antibodies and cellular immune responses, providing long-term, lasting immune protection.

3) Wide adaptability: VLP self-assembling vaccines can be designed for different pathogens, including viruses and bacteria, offering new options for the prevention and treatment of various infectious diseases.

4) Diversity of expression systems: Multiple expression systems including E.coli, yeast, mammalian cells, insect cells, and plants can be used to express VLPs.

5) Stability and ease of preparation: VLP vaccines possess good physical and chemical stability, can be preserved under extreme conditions, are easily prepared through engineering methods for large-scale production, and can achieve stable mass production.

Groundbreaking Vaccine: Why Now?

Since the 1980s, VLPs have gradually replaced traditional vaccines to become the preferred choice for vaccine preparation^[3], and as therapeutic vaccines for inflammation, pain, allergies, tumors, etc., they have long been integrated into our daily lives. For example, hepatitis B vaccine, hepatitis E vaccine, cervical cancer vaccine, and influenza vaccine are all "active" classic VLP vaccines. The hepatitis B virus (HBV) vaccine developed by Merck, as the world's first VLP vaccine, was approved for marketing as early as 1986. Subsequently, preventive vaccines for human papillomavirus, hepatitis E virus, and other indications were successively launched. The following figure shows the statistics of currently marketed and in-development VLP vaccines.

Fig. 2   Marketed and In-Development VLP Vaccines

Expression Systems for VLP Vaccines

Common expression systems for VLPs include Escherichia coli, baculovirus/insect cells, yeast, mammalian cells, and cell-free expression systems. Approximately 170 different host expression systems are available. Among the currently reported VLP vaccines, about 70% are based on eukaryotic expression systems, while 30% are based on prokaryotic expression systems. Over 100 different types of VLPs have been prepared and characterized.

The advantages and disadvantages of different expression systems are compared in the figure below.

Fig. 3   Comparison of different expression systems

Purification and Assembly of VLP Vaccines

The purification of virus-like particles (VLPs) primarily involves four steps: lysis, crude extraction, concentration, and refinement^[4].

1) Lysis: When host-expressed assembled VLPs cannot be secreted extracellularly, cell lysis is required for purification^[5]. Mammalian and insect cells are generally treated with detergents, while high-pressure homogenization, sonication, grinding, and freeze-thaw methods^{[6, 7]} are more commonly used for bacteria, yeast, and plant cells. The most crucial aspect during lysis is preventing protein degradation. Therefore, it is necessary to select an appropriate buffer system based on protein properties and add protease inhibitors and protein stabilizers^[8].

2) Crude Extraction: This step involves removing cell debris and large polymers^[9]. Centrifugation is a commonly used method in crude protein extraction, typically using low-speed centrifuges or continuous-flow centrifuges^[5].

3) Concentration: The main purpose of this step is to remove extraneous proteins and increase the concentration of the target protein. There are many concentration methods, including ammonium sulfate precipitation, polyethylene glycol precipitation, sucrose-cesium chloride gradient ultracentrifugation, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography^[10].

4) Refinement: The primary goal of refinement is to remove host-expressed proteins and DNA, as well as impurities introduced during the process. Refinement mainly employs ion-exchange chromatography, membrane devices, and size-exclusion chromatography.

5) Assembly: Since incomplete or irregular assembly can lead to reduced efficacy of VLP vaccines, it is sometimes necessary to disassemble and reassemble VLPs to improve their stability, homogeneity, and immunogenicity^[11]. Moreover, it is essential to select an appropriate buffer system based on the inherent properties of the VLPs. For example, the disassembly of HPV16 L1 VLPs requires a solution with high pH, low salt, and reducing agents, while reassembly needs conditions of low pH, high salt, and oxidizing agents^[9]."

Characterization of Virus-Like Particles

During the preparation of VLPs, it is necessary to evaluate the production process, mainly focusing on four aspects: properties, content, efficacy, and purity.

1) Properties: The characterization of VLP properties primarily includes aspects such as morphology, size, amino acid sequence, relative molecular mass, degradation and modification status, neutralizing epitopes, and antigen specificity^[12]. Methods include SDS-PAGE, Western Blot, ELISA, surface plasmon resonance technology, transmission electron microscopy, immunoelectron microscopy, protein sequencing, and mass spectrometry.

2) Content: The main methods include ELISA, BCA assay, Bradford assay, and UV spectrophotometry.

3) Efficacy: This is crucial for demonstrating the effectiveness of VLP vaccines. It mainly includes in vitro tests, such as hemagglutination inhibition assays, and in vivo tests, such as immunization trials and challenge tests.

4) Purity: This is key to assessing the effectiveness of purification and refinement processes, and is also related to the suitability for clinical trials. Purity testing mainly examines the purity of VLP structural proteins, the assembly rate of VLPs, residual host DNA and proteins, and the amount of protein-nucleic acid complexes. The primary methods include ELISA, SDS-PAGE, DNA staining methods, agarose gel electrophoresis retardation assays, and transmission electron microscopy^[8].

The Future of VLP Vaccines

VLP vaccines, as an inactivated yet highly efficient vaccine preparation method, are transforming our understanding of future immune defense strategies. Moreover, due to their natural affinity for target host cells, VLPs have been applied in cell-targeting applications such as gene therapy and targeted drug delivery. They can also be combined with probes for use in bioimaging or in basic research to elucidate viral infection mechanisms. VLPs represent cutting-edge advances in immunological science, offering a powerful and safe tool to contribute to human health.

References

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[2] Construction and Characterization of Virus-Like Particles: A Review. Molecular Biotechnology, 2013, 53(1): 92-107.

[3] Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: Current advances. Biotechnology Advances, 2011, 29(6):869-878.

[4] Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system. Methods, 2016, 95: 13–25.

[5] Large-scale production and purification of VLP-based vaccines. Journal of Invertebrate Pathology, 2011, 107(supp-S):S42-S48.

[6] Enzymatic lysis of microbial cells[J].Biotechnol Lett,2007, 29(7):985-994.

[7] Preparation of extracts from prokaryotes[J]. Methods Enzymol,1990, 182:147-153.

[8] Construction and characterization of virus-like particles:A review[J[. Mol Biotechnol, 2013, 53(1):92-107.

[9] Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity[J]. Virol J, 2012, 9:52.

[10] Downstream processing of viral vectors and vaccines[J]. Gene Ther, 2005，121:103-110.

[11] Human parvovirus B19 virus-like particles:In vitro assembly and stability[J]. Biochimie, 2012, 94(3):870-878.

[12] Virus-like particle-based human vaccines:Quality assessment based on structural and functional properties[J]. Trends Biotechnol,2013, 31(11):654-663.