Virus-Like Particles (VLPs): Understanding their Structure, Expression, and Role in Immune Response

What are virus-like particles (VLPs)?

Virus-like particles (VLPs) are nanoparticle structures (50-300nm diameter) that mimic the organization and conformation of authentic native viruses but lack the viral genome, making them non-infectious. They are composed of viral capsid proteins that self-assemble into structures resembling the natural virus. Since they do not contain genetic material, they cannot self-replicate and cause disease. VLPs, due to the virus-like structure and shape of a virus, can elicit a strong immune response, similar to the actual virus, making them an ideal carrier for immunization, and therefore, a powerful tool for antibody discovery. Since VLPs do not contain any viral genetic material, they are inherently safer than live attenuated or inactivated viruses. VLPs are produced using various expression systems, such as bacteria, yeast, insect cells, and mammalian cells, depending on the type of viral proteins required and the complexity of the VLP structure.

Recently, VLPs have been leveraged to display target antigens that would otherwise be difficult to express. This includes transmembrane proteins such as G protein-coupled receptors (GPCRs) or solute carrier proteins (SLCs), which have multiple transmembrane regions and are hard to express in a native conformation without a natural membrane structure. Additionally, VLPs can be used to boost the immunogenicity of target antigens that would otherwise be difficult to generate antibodies against. At KACTUS, we use VLPs to display different types of transmembrane or poor immunogenicity proteins to empower antibody discovery research. 

Figure 1. Transmission electron microscopy (TEM) images of Claudin 18.2, CD20, and GPRC5D VLPs. 

The Structure of VLPs

The main structural components of VLPs are the capsid proteins, which self-assemble into a shell or coat that mimics the outer layer of the virus. These proteins are crucial for maintaining the integrity and shape of the VLP. VLPs are often made of one or more proteins from a single virus type but can also comprise capsid proteins from two or more viruses. VLPs can be divided into enveloped or non-enveloped subtypes, depending on the presence of a lipid bilayer.

Enveloped VLPs: 

Enveloped VLPs contain a lipid bilayer encapsulating one or more capsid protein layers underneath. The capsid layer provides a scaffold for the VLP. The lipid bilayer is obtained during exocytosis from the cell in which the VLP is produced. Target antigens can be displayed in the lipid bilayer for detection by the immune system. Envelope VLPs are ideal for displaying multi-pass transmembrane proteins such as Claudins or GPCRs. The lipid bilayer allows for the expression of full-length transmembrane proteins in their natural conformation. 

Figure 2. Enveloped VLP displaying multi-pass transmembrane protein

Non-Enveloped VLPs:

Non-Enveloped VLPs contain one or more layers of capsid proteins without any lipid bilayer. Target antigens can be displayed on the capsid protein layer for detection by the immune system. The viral capsid proteins increase the immune response to the target antigen which makes non-envelope VLPs ideal for displaying proteins with poor immunogenicity. This may include proteins with high homology (GPC3), small size (BCMA), or glycosylation (CD24). Non-envelope VLPs can also be used to display important epitopes of small domains of larger proteins. 

Figure 3. Non-envelope VLP displaying surface protein. 

How VLPs are expressed

VLPs are produced using various expression systems that facilitate the assembly of viral proteins into structures resembling native viruses. This can include bacterial, mammalian, insect, yeast, or plant expression systems. The choice of expression system depends on the type of viral proteins, the complexity of the VLP structure, and the intended application. Bacterial and mammalian expression systems are most commonly used for VLP (virus-like particle) production, each with distinct advantages and limitations. Bacterial expression systems, such as Escherichia coli, offer high yield, low cost, and simplicity in genetic manipulation, making them suitable for producing simpler VLPs that do not require extensive post-translational modifications (PTMs). However, their inability to perform complex PTMs, like glycosylation, limits their use for more intricate VLPs. In contrast, mammalian expression systems, including HEK293 and CHO cells, excel in producing VLPs with authentic mammalian PTMs, ensuring high fidelity in protein folding and assembly. While this makes them ideal for therapeutic applications and vaccines requiring precise PTMs, they come with higher costs, slower growth rates, and more complex cultivation requirements.

The process of VLP expression and purification begins with gene cloning, where genes encoding viral structural proteins and the target antigen are inserted into an expression vector. This vector is then introduced into host cells (such as bacteria, yeast, insect, mammalian, or plant cells) through transformation or transfection. The viral proteins and target antigen are expressed and self-assembled into VLPs within the host cells. The cells may be lysed or the VLPs obtained from the extracellular media. The VLPs are then purified using a combination of ultrafiltration, diafiltration, and chromatography, and further polished to remove host cell proteins and nucleic acids. Depending on the application, the final product may have buffer optimization or the addition of an adjuvant.

Figure 4. Workflow for Virus-Like Particle (VLP) Production and Formulation. Viral structural proteins and antigenic proteins are encoded in plasmids. After plasmid transfection and protein expression, VLPs self-assemble, characterized by the presence of antigenic proteins and a natural lipid bilayer, without infectious machinery. The VLPs undergo purification, including clarification, purification, and polishing steps, resulting in purified VLPs. Finally, the purified VLPs are sometimes formulated by adding adjuvants and excipients to prepare the final product.

Immune Response to VLPs

The multimeric structure of VLPs mimics the native virus from which the capsid proteins are derived. This allows them to be recognized by the host immune system via structures similar to pathogen-associated molecular patterns (PAMPs), which are typically seen in native viruses. VLPs are recognized by pathogen recognition receptors (PRRs) on the surface or within endosomes of dendritic cells (DCs), which leads to the uptake and processing of VLPs by antigen-presenting cells (APCs). This processing results in the presentation of VLP-derived antigens on the surface of APCs, where they are recognized by T cells. The engagement of T cells with these complexes initiates an adaptive immune response, including the activation and proliferation of specific T cells. Additionally, the multimeric nature of VLPs allows them to efficiently cross-link B cell receptors (BCRs), which can be sufficient to prime B cells and initiate antibodies. This cross-linking triggers B cell activation, leading to the internalization and processing of VLPs by B cells, which then present VLP-derived antigens on MHC class II molecules to CD4+ T cells. This interaction further enhances the humoral immune response by promoting antibody affinity maturation and class switching. Because they can engage both B cells and T cells, VLPs are capable of inducing robust and balanced humoral and cellular immune responses. 

This is an important consideration for antibody discovery, where potent target antigens are necessary to generate high-quality antibodies during an immunization campaign. Envelope VLPs can be used to increase the immunogenicity of membrane proteins such as GPCRs, and allow for generating antibodies against the membrane protein in its native conformation. Non-envelope VLPs can generate a stronger immune response for antigens or epitopes that normally have poor immunogenicity, such as BCMA. Moreover, the multimeric nature of VLPs often obviates the need for adjuvants, although adjuvant co-administration can further enhance their immunogenicity. 

Figure 5. Schematic representation of the interaction between pattern recognition receptors (PRRs) from dendritic cells (DCs) and VLPs. Source

VLP Product Catalog

KACTUS offers a selection of various full-length multi-transmembrane proteins displayed on VLPs for antibody discovery and screening. The VLPs provide a native-like environment for the transmembrane proteins, preserving their structural integrity and functional epitopes, which are crucial for accurate antibody binding studies. By utilizing VLP-displayed proteins, researchers can achieve more reliable and physiologically relevant results in their antibody development workflows, for complex and difficult-to-express antibody targets. Our product portfolio consists of various transmembrane targets or low immunogenicity antigens including GPRC5D, CD24, Claudin 6, CD20, and Claudin 18.2. Browse our VLP catalog products, request custom VLP services, or contact a team member to learn more. 

References

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