Team:Wageningen UR/ModifyingtheHepatitisB

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== Part 2: Modifying the Hepatitis B VLP for Vaccine Production ==
 
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==== Introduction ====
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= Hepatitis B and the Hepatitis B core Antigen VLP =
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The focus for the Hepatitis B VLP will be on vaccine production as final application. The final goal is to obtain a modified and bricked VLP containing an epitope. As a proof of principle we will link GFP to the outside and check the GFP with ELISA. GFP will be attached on the outside in two different ways: By fusion to the monomer of the VLP and via the K/E coil system.
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'''Aims''': Attach epitopes to the outside of the Hepatitis B VLP in order to make vaccines for pathogens other than Hepatitis B. Giving a proof of principle with GFP. Compare two different ways of attaching the GFP to the outside of the VLP.
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== Introduction ==
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[[File:HepBMap.png|500px|right|thumb|<p align="justify">''Figure 1: Map of the prevalance of Hepatitis B [2]</p>]]
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The Hepatitis B virus is a widespread virus causing liver infection. The prevalence of Hepatitis B is higher in less developed countries, as can be seen in figure 1. About 1% of the population in Western-Europe and Northern America are infected, and up to 5% in less developed countries are infected[1]. The virus can be spread by blood-to-blood contact or as STD (sexually transmitted disease). This is the reason why we do not use full viruses, but the empty shells only. By using the coat proteins only, we prevent transmission.
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==== Outside Modification of the Hepatitis B VLP ====
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== The VLP ==
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[[File:HepBwtVLP.jpg|500px|left|thumb|<p align="justify">''Figure 2: The full VLP of Hepatitis B from the Viper Database</p>]]
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The empty shell, or Virus-Like Particle, is formed by the Hepatitis B core Antigen (HepBcAg), see figure 2. The VLP has already been used for the vaccination against Hepatitis B itself, back in 1980 [3]. Afterwards, the Hepatitis B VLP has been modified and suggested to be used as carrier for epitopes of other pathogens.
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In this project, we will try to make the attachments of epitopes or ligands even more conveniently. [https://2012.igem.org/Team:Wageningen_UR/OutsideModification#Hepatitis_B The external loop of the Hepatitis B core antigen can be modified], that is attaching the k-coil to the VLP. Using the PnA System, a wide variety of antigens and ligands can be attached.
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[https://2012.igem.org/Team:Wageningen_UR/InsideModification#Hepatitis_B_with_a_K-coil_on_the_inside The inside of the VLP can be modified in a similar way], allowing controlled packaging of proteins inside the VLP.
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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 adapt their process better to the demand using the newly developed Quality by Design approach. The focus will be on providing a universal approach for a connector system. The aim is to create a “Plug 'N Apply System". The concept is that only the epitopes need to be selected and produced, after which they can be applied upon one standard vaccination platform. These epitopes are fused to connector peptides. The production will be one standardized synthetic biology procedure, and produced in a single, production friendly organism. 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. The industry can shift their production according to the demand by solely selecting the epitope that they fuse to the connector. This will provide more flexibility and quicker response to emerging pathogenic threats
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== Methods ==
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<p align="justify">
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The template of the coat protein for the Hepatitis B core antigen was supplied by the John Innes centre, in a plasmid usually used for the expression of this protein in plants. We used primers that already including the prefix and suffix of Standard 10, so after amplification, the gene can be directly ligated into a pSB1C3 backbone. Or, as we did, combined with an inducible promotor and RBS.
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In order to make the modified VLPs, we designed primers with an overhang containing the modifications. A detailed description of the primers and the modifications can be found in the [[Team:Wageningen_UR/Journal/week15|Journal]].
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==== Inside Modification of the Hepatitis B VLP ====
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== Results ==  
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We constructed the BioBrick that contains an inducible promoter, RBS and the gene encoding the wild-type HepBcAg. After expression and purification of the protein, we assembled the VLPs ''in vivo''. The samples were analyzed using Electron Microscopy, which yielded the picture below (figure 3).
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[[File:HepBEM1b.jpg|500px|center|thumb|<p align="justify">''Figure 3: The electron micrograph from wild-type HepBcAg particles assembled after production using ''E.coli'' (BL21) which were transformed with the plasmid containing the [http://partsregistry.org/Part:BBa_K883201 HepBcAg gene]. VLPs are in green circle, debris is in red circle. </p>]]
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== References ==
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<p>
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1. Organization, W.H. Hepatitis B; factsheet No204. 2012 July 2012 [cited 2012 September 23]; Available from: http://www.who.int/mediacentre/factsheets/fs204/en/.
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2. E.H., T. Hepatitis B. Infectious Diseases Related To Travel 2011; Available from: http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter-3-infectious-diseases-related-to-travel/hepatitis-b/uganda.htm.
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3. Liu, F., et al., Virus-like particles: potential veterinary vaccine immunogens. Res Vet Sci, 2012. 93(2): p. 553-9.
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Latest revision as of 02:12, 27 September 2012


Contents

Hepatitis B and the Hepatitis B core Antigen VLP

Introduction

Figure 1: Map of the prevalance of Hepatitis B [2]

The Hepatitis B virus is a widespread virus causing liver infection. The prevalence of Hepatitis B is higher in less developed countries, as can be seen in figure 1. About 1% of the population in Western-Europe and Northern America are infected, and up to 5% in less developed countries are infected[1]. The virus can be spread by blood-to-blood contact or as STD (sexually transmitted disease). This is the reason why we do not use full viruses, but the empty shells only. By using the coat proteins only, we prevent transmission.













The VLP

Figure 2: The full VLP of Hepatitis B from the Viper Database

The empty shell, or Virus-Like Particle, is formed by the Hepatitis B core Antigen (HepBcAg), see figure 2. The VLP has already been used for the vaccination against Hepatitis B itself, back in 1980 [3]. Afterwards, the Hepatitis B VLP has been modified and suggested to be used as carrier for epitopes of other pathogens. In this project, we will try to make the attachments of epitopes or ligands even more conveniently. The external loop of the Hepatitis B core antigen can be modified, that is attaching the k-coil to the VLP. Using the PnA System, a wide variety of antigens and ligands can be attached. The inside of the VLP can be modified in a similar way, allowing controlled packaging of proteins inside the VLP.













Methods

The template of the coat protein for the Hepatitis B core antigen was supplied by the John Innes centre, in a plasmid usually used for the expression of this protein in plants. We used primers that already including the prefix and suffix of Standard 10, so after amplification, the gene can be directly ligated into a pSB1C3 backbone. Or, as we did, combined with an inducible promotor and RBS.

In order to make the modified VLPs, we designed primers with an overhang containing the modifications. A detailed description of the primers and the modifications can be found in the Journal.

Results

We constructed the BioBrick that contains an inducible promoter, RBS and the gene encoding the wild-type HepBcAg. After expression and purification of the protein, we assembled the VLPs in vivo. The samples were analyzed using Electron Microscopy, which yielded the picture below (figure 3).

Figure 3: The electron micrograph from wild-type HepBcAg particles assembled after production using E.coli (BL21) which were transformed with the plasmid containing the [http://partsregistry.org/Part:BBa_K883201 HepBcAg gene]. VLPs are in green circle, debris is in red circle.

References

1. Organization, W.H. Hepatitis B; factsheet No204. 2012 July 2012 [cited 2012 September 23]; Available from: http://www.who.int/mediacentre/factsheets/fs204/en/.
2. E.H., T. Hepatitis B. Infectious Diseases Related To Travel 2011; Available from: http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter-3-infectious-diseases-related-to-travel/hepatitis-b/uganda.htm.
3. Liu, F., et al., Virus-like particles: potential veterinary vaccine immunogens. Res Vet Sci, 2012. 93(2): p. 553-9.

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