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Latest revision as of 16:14, 26 October 2012

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Applications

The goal of our project is to construct standardized self-assembling particles with a simple and versatile attachment system for either packaging molecules, presenting ligands/epitopes, or both. By combining the PnA (Plug ‘n Apply) System and Virus-Like Particles (VLPs), we create a tool that can be applied in numerous applications. Below are some amazing examples.

Site-specific drug delivery

Most conventional medicines will spread throughout a patient and are always active, leading to unwanted side-effects in healthy tissue. A universal method to administer drugs in a site-specific manner would be greatly beneficial to patients of various diseases all over the world. At present, realization of this dream is not yet within limits of technology and finances. We believe, however, that our tools can be an important step towards achieving this major goal.

Molecules like medicinal drugs can be packed within VLPs and specific receptor-binding domains have been fused to VLP exteriors. Using this, proofs of concept for site-specific drug delivery using VLPs have already been presented in vitro [1, 2]. However, fusing the receptor-binding domains (ligands) to the viral Coat Proteins poses problems regarding VLP formation and stability. For this reason, research institutes need to spend a lot of time and money in fusing each separate ligand to a VLP.

We believe that the VLP PnA System we present here can overcome this problem by facilitating easier, cheaper and standardized fusion of ligands to our modified VLPs. Instead of fusing specific ligands directly to the viral coat proteins, they can be fused to one end of our PnA System. This can be done either by traditional fusing methods or by a relatively simple PCR procedure. Because standardized attachment anchors have already been fused to the exterior of our VLPs, the viral coat proteins need no longer be modified in any way. This eliminates the problems regarding VLP stability and enables a straightforward manner of attaching ligands.

Figure 1: Producing site-specific drug delivery system using VLPs: functional cargo, in this case medicine, is loaded inside of the VLP and targeting ligand is attached on the outside [2]

Standardized vaccine platforms

Viruses are recognized by the immune system by their physical appearance. The receptors protruding from the outside of a virus particle play a crucial role in this: Their accessibility makes them ideal targets for the immune system. Vaccines often make use of this by presenting pieces of these receptors (epitopes) to the immune system, allowing the body to produce antibodies and become resistant to these parts of the virus. For fear of viral activity though, traditional vaccines must rely on incomplete viruses for immunization. Virus-Like Particles (VLPs) on the other hand may display all virus-specific epitopes without risk of viral activity and are thus ideal platforms for vaccine production. This is why many modern ‘Quality by Design’ approaches nowadays use VLPs for vaccine production [3-6]. Because of their ideal shape and size, ease of production and high immunogenicity, VLPs can even be used for vaccination against viruses that aren’t related to the coat protein’s origin, or against bacteria and other parasites. This is done by presenting different epitopes, for example on a VLP made from Hepatitis B core antigen [7].

But in the world of vaccines, the story of production is highly complex and different for every vaccine. Vaccines are produced with approaches fixed on the disease. The lack of flexibility in the production of novel vaccines makes it possible for new pathogens to arise as pandemic diseases. A good example of this is the case of the 2009 Mexican pig flu. We know that influenza viruses can combine from a collection of 19 different Heamaglutinin (H) with 9 different Neuraminidase (N) receptors, used in the viral infection cycle and by the immune system for recognition. While the H1N1 viral variant was lab confirmed as early as April 2009, it was not until November 2009 that vaccination started. This meant that even though knowledge about the virus was pre-existent, much of the world was forced to combat the new flu virus with inhibiting medicines instead of protective immunization. Reason for this is the slow production and verification of vaccines in response to a viral outbreak.

To aid in a better and quicker response to newly emerging pathogens, a more standardized approach of vaccine production could help in containing pandemic outbreaks. Faster reaction in vaccine development and production could be achieved with a universal approach and giving vaccine developers the opportunity to better adapt their process to the demand using the newly developed Quality by Design approach.

Our aim is to construct a VLP-based universal connector system called "Plug ‘n Apply (PnA) System". We believe that the PnA System we present here can provide such a standardized vaccine production approach. The concept is that only the epitopes need to be selected and fused to a connector peptide. The connector peptide is able to “plug onto” a standardized platform, which then provides an expression surface ready for immunization of subjects. The standardized platform can be produced as a bulk reservoir to provide massive reserves of backbone for a sudden explosive demand of vaccine. Because these platforms have already proven to be suitable for use in human vaccines, the industry can shift their production according to the demand by solely selecting the epitope that they fuse to the connector. In the case of a well-known virus like influenza, there may already be a bulk production protocol for each of the (i.e. 28) virus-associated receptors linked to the PnA System, so that any specific combination may be rapidly produced. We believe that this will provide more flexibility and thus a cheaper and quicker response to emerging pathogenic threats.

Although based on virus-like particles, our system is not limited to viruses in its vaccination abilities. Proofs of concept have already been delivered for VLP-based vaccination against bacteria and other pathogens, and even cancers [7, 8]. We believe that with our VLP PnA System, any future VLP-based vaccine can be developed more cost- and time-efficiently.

Table 1: Examples of VLPs used for vaccines and vaccine development. Abbreviations: HBV, hepatitis B virus; HPV, human papilloma virus; HEV, hepatitis E virus; HCV, hepatitis C virus; HIV, human immunodeciency virus; SARS, severe acute respiratory syndrome[4]

Nanomaterials, nanoreactors and more

Although our team is registered in iGEMs medical track, we believe that our product has high potential in a wide range of both scientific and industrial applications. In material sciences for example, VLPs are investigated as a potential nano-scale building block because of their extraordinary self-assembly properties. Our PnA System could well serve here as a way of attaching ‘third party’ molecules to the material. In biochemistry, VLPs are being used for their potential as biological nanoreactors or enzyme carriers [9]. The major challenge here is to attach the selected enzymes to the VLPs. With our Plug ‘n Apply System, this should be far easier. The same could even be true for less obvious applications such as use of VLPs in electrode assemblies or loading of contrast-generating materials in VLPs [10-11].

Figure 2: An Example of loading nano-material into VLPs: DNA micelle-templated virus-like particle formations. (A) Loading of hydrophobic molecules (green) into the core of the micelle. (B) Equipping the micelles with hydrophilic molecules (red) attached to complementary DNA strands by hybridization[2]

By standardizing our tools in a ‘BioBrick’ format and submitting them to the Registry of Standardized Parts, we make them available for future optimizations and uses, to ultimately lead to a limitless range of applications. It is our hope that one day, this range of applications will far exceed the boundaries of our current imagination!

References

• 1. Garcea, R.L. and L. Gissmann, Virus-like particles as vaccines and vessels for the delivery of small molecules. Current Opinion in Biotechnology, 2004. 15(6): p. 513-517.

• 2. Ma, Y., R.J.M. Nolte, and J.J.L.M. Cornelissen, Virus-based nanocarriers for drug delivery. Advanced Drug Delivery Reviews, 2012. 64(9): p. 811-825.

• 3. Roldao, A., et al., Virus-like particles in vaccine development. Expert Review of Vaccines, 2010. 9(10): p. 1149-1176.

• 4. Grgacic, E.V.L. and D.A. Anderson, Virus-like particles: Passport to immune recognition. Methods, 2006. 40(1): p. 60-65.

• 5. Roy, P. and R. Noad, Virus-like particles as a vaccine delivery system - Myths and facts. Human Vaccines, 2008. 4(1): p. 5-12.

• 6. Noad, R. and P. Roy, Virus-like particles as immunogens. Trends in Microbiology, 2003. 11(9): p. 438-444.

• 7. Yang, X.-Y., H. Bo, and Y.-L. Shu, Hepatitis B virus core antigen as a carrier for virus-like partical vaccine: a review. Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui], 2012. 28(3): p. 311-6.

• 8. Ramqvist, T., K. Andreasson, and T. Dalianis, Vaccination, immune and gene therapy based on virus-like particles against viral infections and cancer. Expert Opinion on Biological Therapy, 2007. 7(7): p. 997-1007.

• 9. Cardinale, D., N. Carette, and T. Michon, Virus scaffolds as enzyme nano-carriers. Trends in Biotechnology, 2012. 30(7): p. 369-376.

• 10. Ki, T.N., et al., Stamped microbattery electrodes based on self-assembled M13 viruses. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(45): p. 17227-17231.

• 11. Huh, Y.M., et al., Hybrid nanoparticles for magnetic resonance imaging of target-specific viral gene delivery. Advanced Materials, 2007. 19(20): p. 3109-3112.