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Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs derived from the Hepatitis B virus and composed of the small HBV derived surface antigen (HBsAg) were described in 1968 from patient sera. VLPs have been produced from components of a wide variety of virus families including Parvoviridae (e.g. adeno-associated virus), Retroviridae (e.g. HIV), Flaviviridae(e.g. Hepatitis C virus) and bacteriophages (e.g. Qβ, AP205). VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells.

Applications

Virus research

VLPs are used in studies to identify viral protein components.

Therapeutic and Imaging Agents

VLPs are a candidate delivery system for genes or other therapeutics. These drug delivery agents have been shown to effectively target cancer cells in vitro. It is hypothesized that VLPs may accumulate in tumor sites due to the enhanced permeability and retention effect, which could be useful for drug delivery or tumor imaging

Vaccines

VLPs are useful as vaccines. VLPs contain repetitive, high density displays of viral surface proteins that present conformational viral epitopes that can elicit strong T cell and B cell immune responses. Since VLPs cannot replicate, they provide a safer alternative to attenuated viruses. VLPs were used to develop FDA-approved vaccines for Hepatitis B and human papillomavirus. More recently, VLPs were used to develop a pre-clinical vaccine against chikungunya virus.

Research suggests that VLP vaccines against influenza virus could provide stronger and longer-lasting protection against flu viruses than conventional vaccines. Production can begin as soon as the virus strain is sequenced and can take as little as 12 weeks, compared to 9 months for traditional vaccines. In early clinical trials, VLP vaccines for influenza appeared to provide complete protection against both the Influenza A virus subtype H5N1 and the 1918 flu pandemic. Novavax and Medicago Inc. have run clinical trials of their VLP flu vaccines.

Mycoviruses

Some fungi contain mycoviruses that lack the ability to be transmitted in cell free preparations and may be classified as VLPs. These are important in phytopathology, as they can cause hypovirulence in some species of phytopathogenic fungi.^{[citation needed]}

Lipoparticle technology

The VLP Lipoparticle was developed to aid the study of integral membrane proteins. Lipoparticles are stable, highly purified, homogeneous VLPs that are engineered to contain high concentrations of a conformationally intact membrane protein of interest. Integral Membrane proteins are involved in diverse biological functions and are targeted by nearly 50% of existing therapeutic drugs. However, because of their hydrophobic domains, membrane proteins are difficult to manipulate outside of living cells. Lipoparticles can incorporate a wide variety of structurally intact membrane proteins, including G protein-coupled receptors (GPCR)s, ion channels and viral Envelopes. Lipoparticles provide a platform for numerous applications including antibody screening, production of immunogens and ligand binding assays.

Expression Host Systems

The first step to creating a VLP is cloning and expressing the structural genes of interest. There are many systems to choose for expression. The chosen expression system can determine the limitations and effectiveness of the resulting VLP. The most well established expression systems being used today are as follows:

Bacterial systems are one of the most widely used, and are often based on the very well studied bacteria, Escherichia coli. This is a preferred method for production of recombinant proteins on a global scale due to the low cost and rapid nature of production, ease of scaling up, and high levels of expression. It is also possible to construct a VLP with multiple types of structural proteins. However, there are several disadvantages that come with use of this system:

• Inability to produce post-translational modifications
• Inability to generate proper disulfide bonds within proteins
• Other recombinant proteins of interest, particularly from eukaryotic cells, may be insoluble in an E. coli system
• Presence of endotoxins in generated proteins

Research has suggested that culturing the cells at a low temperature or use of a fusion protein system can increase solubility for other proteins.

Yeast systems have been used to express structural genes of bacterial, yeast, plant and mammalian origin. Unlike bacterial systems, it is possible to introduce post-translational modifications and there is no endotoxin presence. Another disadvantage is that this system only allows for the creation of non-enveloped viruses. Yeast expression is unique in two ways: 1) to successfully produce a VLP using a yeast expression system, the bacteria must be propagated in bacteria before being introduced to the cell to create a stable transgene product and 2) Research has suggested that VLP assembly may occur more efficiently during the purification stage, instead of the cultivation stage. Pichia and Hansenula are the most commonly used yeast strains.

Insect cell systems have fast growth rates in media without animal products, capacity for large scale cultivation, and the possibility of introducing post translational modifications. A baculovirus vectors always needs to succesfuly create the VLP. If more than one protein is required, the cell can be coinfecte with a polycistronic vector, or infected with multiple monocistronic vectors. The latter method is preferred, because it allows for manipulation of individual protein levels, and identification of which ones are necessary. Although glycosylation is present, the patterns differ from that of mammalian cells, leading to a slightly different product.

Plant systems are less popular, but are good for the creation of VLPs with specific characteristics. Initially, plant-based expression systems gained popularity because they were attached to the idea of edible vaccines. It was thought that if an antigen was recombinantly expressed in a plant, ingestion of it would cause an immune response and effectively vaccinate the patient. Research has since moved away from edible vaccines for several reasons: administration of the vaccine by a medical professional is more likely to yield reproducible results, oral delivery was found to provide some protection against enteric pathogens, but not with any other body system, lack of antigen accumulation in the plant, and the avoidance of digestive acid and degrading enzymes.

The gene(s) for the protein(s) of interest are most commonly introduced using Agrobacterium. Once introduced, the gene can incorporate in either the nuclear or chloroplast genome. Although chloroplast transformation leads to very high copy numbers, it is a prokaryotic genome, so no glycosylation is observed. Genetic material can be introduced into the capsid during or after its assembly.

Mammalian systems are one of the most popular choices for researchers, making more than half of the recombinant proteins used in the pharmaceutical industry. While the complexity of construction and applications can often be a problem, it also leads to expression of highly efficient, high quality, complex VLPs that also have the correct glycosylation pattern. This system is useful for using a single polycistronic vector, as described above for the insect expression system. The recombinant VLPs are usually achieved using one of two methods:

• Adhesion Culture - cells are seeded onto a surface and given proper nutrients
• Suspension Culture - cells are grown suspended in some type of culture media

The latter method is more widely used when using mammalian cells to create VLPs.

Cell Free Systems

Cell-Free Protein Systems (CFPS) are sometimes used to create VLPs. The following CFPS are commercially available: E. coli, wheat germs, insect cells, and rabbit reticulocytes.

Purification of VLPs

After the proteins of of interest have been cloned and expressed in one of the above mentioned systems, they must be purified to get the final VLP product. Purifaction generally involves 4 basic steps:

1. Cell Lysis - cells are broken to release VLPs into solution
2. Cell Clarifiaction - cellular debris is removed, leaving behind VLPs
3. Cell Concentration - The cell lysate (in this case, VLPs) are brought up to higher concentration in solution
4. Cell Polishing - removal of residual impurities

These steps can be repeated multiple times in cycles depending on which protocol is used.

Assembly

The understanding of self-assembly of VLPs was once based on viral assembly. This is rational as long as the VLP assembly takes place inside the host cell (in vivo), though the self-assembly event was found in vitro from the very beginning of the study about viral assembly. Study also reveals that in vitro assembly of VLPs competes with aggregation and certain mechanisms exist inside the cell to prevent the formation of aggregates while assembly is ongoing.

Linking targeting groups to VLP surfaces

Attaching proteins, nucleic acids, or small molecules to the VLP surface, such as for targeting a specific cell type or for raising an immune response is useful. In some cases a protein of interest can be genetically fused to the viral coat protein. However, this approach sometimes leads to impaired VLP assembly and has limited utility if the targeting agent is not protein-based. An alternative is to assemble the VLP and then use chemical crosslinkers, reactive unnatural amino acids or SpyTag/SpyCatcher reaction in order to covalently attach the molecule of interest. This method has shown to be very effective at directing the immune response against the attached molecule, thereby inducing high levels of neutralizing antibody titers and breaking immune self-tolerance

(((Will fix the date error thingies in citations - Science Direct links were being weird with automatic citation)))

(((Do i lean too heavily on source 1 throughout the paper?)))