Team:TU Darmstadt/Project/Outlook

TU Darmstadt

Outlook

Achieving higher modularity

During the development of The Real MVP platform, we considered how we can improve the modularity to cover more potential applications. With the sortase, we established a solution to modify assembled Virus-like particles (VLPs) with any desired cargo to the P22 capsid. But whether it is the size or the field of application other candidates for virus capsids can fit the purpose better. For example, the polyomavirus JC VLPs are promising for gene therapy as their high availability of positively charged amino acids in the VLP enables efficient DNA packaging. Some VLPs like the plant virus CPMV can be widely chemically modified. [1] Enveloped VLPs have the advantage of being able to integrate the displayed particle in the envelope. Thus, the native conformation of an antigen is maintained and the uptake by antigen-presenting cells is enhanced. [2] To achieve higher modularity, investigating in other viral capsids with exposed C- or N-terminus might be interesting. If so, the sortase reaction can also be used for other VLPs, which has already been shown to work on other capsids than P22, for example on hepatitis B virus core protein (HBc) VLPs [3] or M13 capsid proteins VLPs. [4]

Engineering Sortase and improving the linking reaction

During our research we were amazed by the potential of the Sortase A7M. It turned out that this variant was the perfect candidate to modify the exterior of VLPs in vivo. In our modeling, we discovered that this Sortase performs a flap movement with a flexible loop during the linking reaction. This movement might direct the bound polyG towards the active site leading to a decrease in activation energy. With this knowledge we could try to improve the efficiency by protein design or alter the LPXTG motif. Furthermore, other linking enzymes like Sortases B having an NPX[T/S] [N/G/S] motif [5, 6] might be interesting as another approach to achieve higher modularity.

What will be the future expressionsystem?

Through our research about VLPs and the talks to many experts, we have noticed that there are many different expression systems that could be used for the VLP production. So why did we prefer E. coli instead of other possible expression systems?

E. coli has many advantages fitting well for Modular Virus-like particles (MVP). It is well understood, easily cultivated and cheap to grow even in industrial scale. Simultaneously, E. coli reaches high expression levels. The yield of E. coli strains is up to 3 times higher than in yeast. Middelberg et al. showed viral capsomere concentrations at gram per liter levels, with the potential of upscaling, using industrial bioreactors.[7] Well known protocols in combination with fast growth enable quick adaption to upcoming problems while holding the costs at a minimum. All these criteria are reasons why E. coli is well suited as an expression platform for MVPs.[8]

Of course, apart from all the advantages E. coli also has its downsides. Contamination with endotoxins during protein expression and VLP assembly (as it can be seen here) demand higher efforts in protein purification to guarantee safe products. This was also stressed by many experts we talked to. Another problem regarding E. coli includes the solubility of recombinant proteins, especially proteins with an eukaryotic background. E. coli is unable to post-translationally modify synthesized proteins for example via glycosylation. Disulfide bonds may not be generated, and the correct protein folding may not be possible. Nevertheless, approaches are already available to improve the right folding by disulfide bonds. For example, Ono et al. described disulfide shuffling with persulfides that enables soluble expression of recombinant stem cell factors. [9] Altogether, we have to conclude that the protein space that our MVP platform covers is limited.[3]

However, our key solving the latter is the core of our MVP. Being able to produce high amounts of VLPs while adjusting the possible modification degree during production, we can first produce and purify correctly assembled VLPs. Afterwards, we can modify them with complicated proteins as well as glycoproteins and primary amines using Sortase A. A huge variety of compatible capsids guarantees the solubility of chosen capsomers. The modification of VLPs is not limited by E. coli and we can use the strength of E. coli advantages as an expression system!

Nevertheless, there are still modifications that cannot be realized using the Sortase A and E. coli. Modifications that are not based on proteins or primary amines are not accessible for the Sortase A, for example glycosylation or phosphorylation. In these cases, a chemical modification could further increase modularity. Other limits are complex modifications. For those, more complex expression systems are needed. To give an example: vaccines against the influenza virus are expressed in mammalian cells as matrix proteins and surface glycosylation from different types of influenza have to be combined in order to assemble functional VLPs. [3]

In the following table, you can see a brief overview of other possible expression systems. Those can further increase the power of MVP in cases E. coli does not work.

Host actual usage for industrial VLP production advantages disadvantages

Yeast[3, 10]

20%
  • possibility of post-translational modifications
  • production of RNA bacteriophage VLPs
  • secretion of proteins eases purification
  • more complicated because of the need of yeast shuttle-vectors
  • lower yields

Insect cells[3, 11]

28%
  • fast growth in animal product free medium
  • post-transcriptional modification similar to mammalian cells
  • able to express six proteins in the same cell which self assemble to VLPs
  • possibility to use larvae to further decrease production costs
  • lower yields than yeast and E. coli
  • production process needs much more time (e.g. 12 weeks )
  • downstreaming process is still challenging in order to obtain high yield/high purity

Plants[3, 12]

9%
  • cell cultures can be handled similar to bacterial cultures
  • possibility of post-transcriptional modifications
  • complex designs of expression
  • plant transformation methods are more difficult
  • early selection of transformed cells from non-transformed tissue is difficult
  • homologous recombination has not been achieved efficiently
  • unstable gene expression
  • long developing and harvesting times

Mammalian cells[3, 13]

25%
  • highest complexity
  • efficient folding
  • accurate and authentic post-transcriptional modification
  • really sensitive
  • lowest yields

A further alternative to increase production could be Vibrio natriegens described by iGEM Marburg 2018. However, as characterization and strains to produce high amounts of protein are not fully developed yet, E. coli stays the expression system of our choice.

Empowering The Real MVP for drug delivery

With the collaboration with iGEM team Freiburg and their project Reflect we tried to prove our platform can be used for targeted drug delivery systems (DDS). But empowering The Real MVP platform for DDS in terms of modularity and reproducibility based on our modification systems needs further investigation and research. Current studies focus on the encapsulation of small molecules into the interior of VLPs by varying the pH, temperature or ion concentrations. Thereby, a dynamic structure of the VLP is achieved and the nanoparticle can be loaded through small openings. [14] Another option to modify VLPs with drugs is by chemical reactions.[15] For instance, by the Cu(I)-catalyzed azide-alkyne cycloaddition fluorescence proteins have been attached to the VLPs. [16] We conceptualized a different approach to attach small molecules to the interior and exterior of the VLP using our LPETGG-linking system. With this, we try to transfer the idea of full modularity on the development of drug delivery systems approaches. As described in the literature chemical linking reactions can be used to connect our LPETGG-sequence to small drug molecules. [16] These fused drugs can then be presented on the VLP and delivered to the specific target.

Speedup of vaccine development

VLPs have already been used for vaccine development for years. At this time, vaccination displays the most effective immunization method. [17] Therefore, the VLP surface is being modified with virulent epitopes, mostly peptides or proteins. Two big examples of approved VLP-based vaccines are those against Hepatitis B and against HPV (human papillomaviruses). [17] Compared to other vaccination methods, VLPs have many advantages. The biggest lead is that the particles only consist of the viral capsid but lack the viral genomic material on the inside. Comparing to other vaccines that, for instance, contain inactivated or attenuated viruses and therefore always carry the risk to reconvert into pathogenic species, VLPs pose a lower risk for negative side effects. [18] Yet VLPs still have the ability to enter target cells and generate strong immune responses. [18] Moreover, unlike real viruses they are not capable of replication in the human body. [17] Consequently, they pose a massively safer method than other vaccination procedures, not only for the recipient but for the engineer who comes in contact with VLPs as well. [17] Due to their structure, the particles are able to activate B-cells and therefore provoke very high humoral and cellular immune responses for viral and non-viral diseases.[17,19] Triggering of the immune system happens through the recognition of the repetitive subunits VLPs consist of. Having a VLP-based modular platform would definitely improve the development of vaccines, as we have learned to talk to the immunology expert Dr. Stefan Schülke. Thereby, the testing and approval of vaccines would be accelerated.

References

  1. ↑ Tang, Shubing, et al. "A modular vaccine development platform based on sortase-mediated site-specific tagging of antigens onto virus-like particles." Scientific reports 6 (2016): 25741. [1]
  2. ↑ Pitoiset, Fabien, Thomas Vazquez, and Bertrand Bellier. "Enveloped virus-like particle platforms: vaccines of the future?." (2015): 913-915. [2]
  3. ↑ Zeltins, Andris. "Construction and characterization of virus-like particles: a review." Molecular biotechnology 53.1 (2013): 92-107. [3]
  4. ↑ Raeeszadeh-Sarmazdeh, Maryam, et al. "Protein nanoparticles as multifunctional biocatalysts and health assessment sensors." Current opinion in chemical engineering 13 (2016): 109-118. [4]
  5. ↑ Jacobitz, Alex W et al. “Sortase Transpeptidases: Structural Biology and Catalytic Mechanism.” Advances in protein chemistry and structural biology vol. 109 (2017): 223-264 [5]
  6. ↑ Naik, Mandar T., et al. "Staphylococcus aureus sortase a transpeptidase calcium promotes sorting signal binding by altering the mobility and structure of an active site loop." Journal of Biological Chemistry 281.3 (2006): 1817-1826 [6]
  7. ↑ M.Liew, A.Rajendran, A. Middelberg, Microbial production of virus-like particle vaccine protein at gram-per-litre levels, Journal of Biotechnology 150, 2010, S. 224-231. [7]
  8. ↑ X.Huang, X. Wang, J.Zhang, N.Xia, Q.Zhao, Escherichia coli-derived virus-like particles in vaccine development, npj Vaccines, 2017 [8]
  9. ↑ T.Ueda, T.Akuta, T. Kikuchi-Ueda, K.Imaizumi, Y.Ono, Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Echendria coli by disulfide shuffling with persulfide, Protein Expression and Purification 120, 2016, 99-105 [9]
  10. ↑ J. Tomé-Amat, L.Fleischer, S.Parker, C. Bardliving, C.Batt, Secreted production of assembled Norovirus-like particles from Pichia pastoris [10]
  11. ↑ T. Vicente, A. Roldão, C. Peixato, M. Carrando, P. Alves, Large-scale production and purification of VLP-based vaccines, Journal of Invertebrate Pathology 107, 2011, S42-S48 [11]
  12. ↑ Naik, Mandar T., et al. "Staphylococcus aureus sortase a transpeptidase calcium promotes sorting signal binding by altering the mobility and structure of an active site loop." Journal of Biological Chemistry 281.3 (2006): 1817-1826 [12]
  13. ↑ L. Santi, Z. Huang, H. Mason, Virus like particles produces in green plants, National Institutes of Health, 2006, 66-76 [13]
  14. ↑ Yujie Ma, et al., Virus-based nanocarriers for drug delivery, Drug Delivery Reviews, 2012 [14]
  15. ↑ E. Strable, M.G. Finn, Chemical Modification of Virus and Virus-Like Particles, Viruses and Nanotechnology. Pp 1-21, 2009 [15]
  16. ↑ Eric Gillitzer, et al., Chemical modification of a viral cage for multivalent presentation, ChemComm, 2002 [16]
  17. ↑ J.Fuenmayor, F.Gòdia, L. Cervera, Production of virus-like particles for vaccines, New Biotechnology, 2017, 39: 174-180 [17]
  18. ↑ Christine Ludwig and Ralf Wagner, Virus-like particles – universal molecular toolboxes, Current Opinion in Biotechnology, 2007, 18: 537-545 [18]
  19. ↑ Kathryn M. Frieze, David S Peabody and Bryce Chackerian, Engineering virus-like particles as vaccine platforms, Current Opinion in Virology, 2016, 18: 44-49 [19]
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