Virus-like particle

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.[1][2][3][4] Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs derived from the Hepatitis B virus (HBV) and composed of the small HBV derived surface antigen (HBsAg) were described in 1968 from patient sera.[5] 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), Paramyxoviridae (e.g. Nipah) and bacteriophages (e.g. Qβ, AP205).[1] VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells.[6][7]

VLPs can also refer to structures produced by some LTR retrotransposons (under Ortervirales) in nature. These are defective, immature virions, sometimes containing genetic material, that are generally non-infective due to the lack of a functional viral envelope.[8][9] In addition, wasps produce polydnavirus vectors with pathogenic genes (but not core viral genes) or gene-less VLPs to help control their host.[10][11]

Applications

Therapeutic and Imaging Agents

VLPs are a candidate delivery system for genes or other therapeutics.[12] These drug delivery agents have been shown to effectively target cancer cells in vitro.[13] 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 [14]

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.[15]; the particles' small radius of roughly 20-200 nm allows for sufficient draining into lymph nodes. 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, which are commercially available. There are currently a selection of vaccines against Human Papilloma Virus (HPV) such as Cervarix by GlaxoSmithKline along with Gardasil and Gardasil-9, produced by Merck & Co. Gardasil consists of recombinant VLPs assembled from the L1 proteins of HPV types 6, 11, 16, and 18 expressed in yeast and is adjuvanted with aluminum hydroxyphosphate sulfate. Gardasil-9 consists of L1 epitopes of 31, 33, 45, 52 and 58 in addition to the listed L1 epitopes found in Gardasil. Cervarix consists of recombinant VLPs assembled from the L1 proteins of HPV types 16 and 18, expressed in insect cells, and is adjuvanted with 3-O-Desacyl-4-monophosphoryl lipid (MPL) A and aluminum hydroxide [16]

The most up-to-date vaccine against Hepatitis B Virus (HBV) is Sci-B-Vac, manufactured by VBI Vaccines Inc. It is produced by expression in Chinese hamster ovary (CHO) cells. The three epitopes of hepatitis B surface antigen: S, Pre-S1 , and Pre-S2 in their glycosylated and non-glycosylated forms, are displayed on a phospholipid matrix adjuvanted by aluminum hydroxide. This is considered to be direct competitor to Engerix-B manufactured by GlaxoSmithKline, which consists of Hepatitis B surface antigen adsorbed to aluminum hydroxide.

The first VLP vaccine that addresses malaria, Mosquirix, (RTS,S) has been approved by regulators in the EU. It was expressed in yeast. RTS,S is a portion of the Plasmodium falciparum circumsporozoite protein fused to the Hepatitis B surface antigen (RTS), combined with Hepatitis B surface antigen (S), and adjuvanted with AS01 (consisting of (MPL)A and saponin).

Research suggests that VLP vaccines against influenza virus could provide stronger and longer-lasting protection against flu viruses than conventional vaccines.[17] 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.[18] Novavax and Medicago Inc. have run clinical trials of their VLP flu vaccines.[19][20]

VLPs have also been used to develop a pre-clinical vaccine candidate against chikungunya virus.[15]

Lipoparticle technology

The VLP Lipoparticle was developed to aid the study of integral membrane proteins.[21] 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.[22] [23]

Expression Host Systems

The first step to creating a VLP is cloning and expressing the structural genes of interest.[1] 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.[1] 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.[7] 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[1]
  • Inability to generate proper disulfide bonds within proteins[1]
  • Other recombinant proteins of interest, particularly from eukaryotic cells, may be insoluble in an E. colisystem[1]
  • Presence of endotoxins in generated proteins[1][24]

Research has suggested that culturing the cells at a low temperature or use of a fusion protein system can increase solubility for other proteins.[1]

Yeast systems have been used to express structural genes of bacterial, yeast, plant and mammalian origin.[1] 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.[1] 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[1] and 2) Research has suggested that VLP assembly may occur more efficiently during the purification stage, instead of the cultivation stage.[1] Pichia and Hansenulaare 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 successfully create the VLP.[1][24] If more than one protein is required, the cell can be coinfected 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.[1] Although glycosylationis present, the patterns differ from that of mammalian cells, leading to a slightly different product.[25]

Plant systems are less popular, but are good for the creation of VLPs with specific characteristics.[1] 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.[26] 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,[1] oral delivery was found to provide some protection against enteric pathogens, but not with any other body system,[26] lack of antigen accumulation in the plant,[26] and the avoidance of digestive acid and degrading enzymes.[26]

The gene(s) for the protein(s) of interest are most commonly introduced using Agrobacterium[1][26].Once introduced, the gene can incorporate in either the nuclear or chloroplast genome.[6] Although chloroplast transformation leads to very high copy numbers, it is a prokaryotic genome, so no glycosylation is observed.[26] Genetic material can be introduced into the capsid during or after its assembly.[27]

Mammalian systems are one of the most popular choices for researchers, making more than half of the recombinant proteins used in the pharmaceutical industry.[1] 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.[1][24][28] 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:[28]

  • 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.[28]

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.[28]

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.[29] Study also reveals that in vitro assembly of VLPs competes with aggregation[30] and certain mechanisms exist inside the cell to prevent the formation of aggregates while assembly is ongoing.[31]

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.[32] 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,[33] reactive unnatural amino acids[34] or SpyTag/SpyCatcher reaction[35][36] in order to covalently attach the molecule of interest. This method is effective at directing the immune response against the attached molecule, thereby inducing high levels of neutralizing antibody and even being able to break tolerance to self-proteins displayed on VLPs.[36]

Purification of non-enveloped VLPs

After the proteins of interest have been cloned and expressed in one of the above-mentioned systems, they must be purified to get the final VLP product. Purification of non-enveloped VLPs generally involves four basic steps:

  1. Cell Lysis - cells are broken to release VLPs into solution[1]
  2. Cell Clarification - 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[37]
  4. Cell Polishing - removal of residual impurities[37]

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

References
  1. Zeltins A (January 2013). "Construction and characterization of virus-like particles: a review". Molecular Biotechnology. 53 (1): 92–107. doi:10.1007/s12033-012-9598-4. PMC 7090963. PMID 23001867.
  2. Buonaguro L, Tagliamonte M, Tornesello ML, Buonaguro FM (November 2011). "Developments in virus-like particle-based vaccines for infectious diseases and cancer". Expert Review of Vaccines. 10 (11): 1569–83. doi:10.1586/erv.11.135. PMID 22043956.
  3. "NCI Dictionary of Cancer Terms". National Cancer Institute. 2011-02-02. Retrieved 2019-04-19.
  4. Mohsen MO, Gomes AC, Vogel M, Bachmann MF (July 2018). "Interaction of Viral Capsid-Derived Virus-Like Particles (VLPs) with the Innate Immune System". Vaccines. 6 (3): 37. doi:10.3390/vaccines6030037. PMC 6161069. PMID 30004398.
  5. Bayer ME, Blumberg BS, Werner B (June 1968). "Particles associated with Australia antigen in the sera of patients with leukaemia, Down's Syndrome and hepatitis". Nature. 218 (5146): 1057–9. Bibcode:1968Natur.218.1057B. doi:10.1038/2181057a0. PMID 4231935.
  6. Santi L, Huang Z, Mason H (September 2006). "Virus-like particles production in green plants". Methods. 40 (1): 66–76. doi:10.1016/j.ymeth.2006.05.020. PMC 2677071. PMID 16997715.
  7. Huang X, Wang X, Zhang J, Xia N, Zhao Q (2017-02-09). "Escherichia coli-derived virus-like particles in vaccine development". NPJ Vaccines. 2 (1): 3. doi:10.1038/s41541-017-0006-8. PMC 5627247. PMID 29263864.
  8. Beliakova-Bethell N, Beckham C, Giddings TH, Winey M, Parker R, Sandmeyer S (January 2006). "Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components". RNA. 12 (1): 94–101. doi:10.1261/rna.2264806. PMC 1370889. PMID 16373495.
  9. Purzycka KJ, Legiewicz M, Matsuda E, Eizentstat LD, Lusvarghi S, Saha A, et al. (January 2013). "Exploring Ty1 retrotransposon RNA structure within virus-like particles". Nucleic Acids Research. 41 (1): 463–73. doi:10.1093/nar/gks983. PMC 3592414. PMID 23093595.
  10. Burke, Gaelen R.; Strand, Michael R. (2012-01-31). "Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors". Insects. 3 (1): 91–119. doi:10.3390/insects3010091. PMC 4553618. PMID 26467950.
  11. Leobold, Matthieu; Bézier, Annie; Pichon, Apolline; Herniou, Elisabeth A; Volkoff, Anne-Nathalie; Drezen, Jean-Michel; Abergel, Chantal (July 2018). "The Domestication of a Large DNA Virus by the Wasp Venturia canescens Involves Targeted Genome Reduction through Pseudogenization". Genome Biology and Evolution. 10 (7): 1745–1764. doi:10.1093/gbe/evy127. PMC 6054256. PMID 29931159.
  12. Petry H, Goldmann C, Ast O, Lüke W (October 2003). "The use of virus-like particles for gene transfer". Current Opinion in Molecular Therapeutics. 5 (5): 524–8. PMID 14601522.
  13. Galaway, F. A. & Stockley, P. G. MS2 viruslike particles: A robust, semisynthetic targeted drug delivery platform. Mol. Pharm. 10, 59–68 (2013).
  14. Kovacs, E. W. et al. Dual-surface-modified bacteriophage MS2 as an ideal scaffold for a viral capsid-based drug delivery system. Bioconjug. Chem. 18, 1140–1147 (2007).
  15. Akahata W, Yang ZY, Andersen H, Sun S, Holdaway HA, Kong WP, et al. (March 2010). "A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection". Nature Medicine. 16 (3): 334–8. doi:10.1038/nm.2105. PMC 2834826. PMID 20111039.
  16. Zhang X, Xin L, Li S, Fang M, Zhang J, Xia N, Zhao Q (2015). "Lessons learned from successful human vaccines: Delineating key epitopes by dissecting the capsid proteins". Human Vaccines & Immunotherapeutics. 11 (5): 1277–92. doi:10.1080/21645515.2015.1016675. PMC 4514273. PMID 25751641.
  17. "Creating a Mutant Strain of Streptococcus Free of All Integrated Viruses" (Press release). American Society for Microbiology. May 27, 2010. Retrieved June 8, 2010.
  18. Perrone LA, Ahmad A, Veguilla V, Lu X, Smith G, Katz JM, et al. (June 2009). "Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge". Journal of Virology. 83 (11): 5726–34. doi:10.1128/JVI.00207-09. PMC 2681940. PMID 19321609.
  19. John Gever (12 September 2010). "ICAAC: High Antibody Titers Seen With Novel Flu Vaccine".
  20. Landry N, Ward BJ, Trépanier S, Montomoli E, Dargis M, Lapini G, Vézina LP (December 2010). Fouchier RA (ed.). "Preclinical and clinical development of plant-made virus-like particle vaccine against avian H5N1 influenza". PLOS One. 5 (12): e15559. Bibcode:2010PLoSO...515559L. doi:10.1371/journal.pone.0015559. PMC 3008737. PMID 21203523.
  21. "Integral Molecular" (PDF). Archived from the original (PDF) on 2009-07-31. Retrieved 2010-04-30.
  22. Willis S, Davidoff C, Schilling J, Wanless A, Doranz BJ, Rucker J (July 2008). "Virus-like particles as quantitative probes of membrane protein interactions". Biochemistry. 47 (27): 6988–90. doi:10.1021/bi800540b. PMC 2741162. PMID 18553929.
  23. Jones JW, Greene TA, Grygon CA, Doranz BJ, Brown MP (June 2008). "Cell-free assay of G-protein-coupled receptors using fluorescence polarization". Journal of Biomolecular Screening. 13 (5): 424–9. doi:10.1177/1087057108318332. PMID 18567842.
  24. Fuenmayor J, Gòdia F, Cervera L (October 2017). "Production of virus-like particles for vaccines". New Biotechnology. 39 (Pt B): 174–180. doi:10.1016/j.nbt.2017.07.010. PMC 7102714. PMID 28778817.
  25. Yin J, Li G, Ren X, Herrler G (January 2007). "Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes". Journal of Biotechnology. 127 (3): 335–47. doi:10.1016/j.jbiotec.2006.07.012. PMID 16959350.
  26. Mason, H. S.; Herbst-Kralovetz, M. M. (2012), Kozlowski, Pamela A. (ed.), "Plant-Derived Antigens as Mucosal Vaccines", Mucosal Vaccines: Modern Concepts, Strategies, and Challenges, Current Topics in Microbiology and Immunology, Springer Berlin Heidelberg, 354, pp. 101–120, doi:10.1007/82_2011_158, ISBN 9783642236938, PMC 7122597, PMID 21811930
  27. Lomonossoff, George P.; Evans, David J. (2014), Palmer, Kenneth; Gleba, Yuri (eds.), "Applications of Plant Viruses in Bionanotechnology", Plant Viral Vectors, Current Topics in Microbiology and Immunology, Springer Berlin Heidelberg, 375, pp. 61–87, doi:10.1007/82_2011_184, ISBN 9783642408298, PMC 7121916, PMID 22038411
  28. Wurm FM (November 2004). "Production of recombinant protein therapeutics in cultivated mammalian cells". Nature Biotechnology. 22 (11): 1393–8. doi:10.1038/nbt1026. PMID 15529164.
  29. Adolph KW, Butler PJ (November 1976). "Assembly of a spherical plant virus". Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 276 (943): 113–22. Bibcode:1976RSPTB.276..113A. doi:10.1098/rstb.1976.0102. PMID 13422.
  30. Ding Y, Chuan YP, He L, Middelberg AP (October 2010). "Modeling the competition between aggregation and self-assembly during virus-like particle processing". Biotechnology and Bioengineering. 107 (3): 550–60. doi:10.1002/bit.22821. PMID 20521301.
  31. Chromy LR, Pipas JM, Garcea RL (September 2003). "Chaperone-mediated in vitro assembly of Polyomavirus capsids". Proceedings of the National Academy of Sciences of the United States of America. 100 (18): 10477–82. Bibcode:2003PNAS..10010477C. doi:10.1073/pnas.1832245100. PMC 193586. PMID 12928495.
  32. Wetzel D, Rolf T, Suckow M, Kranz A, Barbian A, Chan JA, et al. (February 2018). "Establishment of a yeast-based VLP platform for antigen presentation". Microbial Cell Factories. 17 (1): 17. doi:10.1186/s12934-018-0868-0. PMC 5798182. PMID 29402276.
  33. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kündig T, et al. (August 2002). "A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses". Vaccine. 20 (25–26): 3104–12. doi:10.1016/S0264-410X(02)00266-9. PMID 12163261.
  34. Patel KG, Swartz JR (March 2011). "Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry". Bioconjugate Chemistry. 22 (3): 376–87. doi:10.1021/bc100367u. PMC 5437849. PMID 21355575.
  35. Brune KD, Leneghan DB, Brian IJ, Ishizuka AS, Bachmann MF, Draper SJ, et al. (January 2016). "Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization". Scientific Reports. 6: 19234. Bibcode:2016NatSR...619234B. doi:10.1038/srep19234. PMC 4725971. PMID 26781591.
  36. Thrane S, Janitzek CM, Matondo S, Resende M, Gustavsson T, de Jongh WA, et al. (April 2016). "Bacterial superglue enables easy development of efficient virus-like particle based vaccines". Journal of Nanobiotechnology. 14 (1): 30. doi:10.1186/s12951-016-0181-1. PMC 4847360. PMID 27117585.
  37. Peixoto C, Sousa MF, Silva AC, Carrondo MJ, Alves PM (January 2007). "Downstream processing of triple layered rotavirus like particles". Journal of Biotechnology. 127 (3): 452–61. doi:10.1016/j.jbiotec.2006.08.002. PMID 16959354.
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