Workpackage 2

 

 

Work package number

2

Start date or starting event:

M3

Work package title

Characterisation of VLPs

Activity Type[1]

RTD

Participant number

1

2

 

 

 

 

 

Participant short name

iQur

UoL

 

 

 

 

 

Person-months per participant:

2.0

36.0

 

 

 

 

 

                   

 

Objectives

To provide a central resource for the analysis of purified VLPs (provided by other members of the consortium).

Application and development of our existing methods for determination of the long-term stability of VLPs.

 

Description of work (possibly broken down into tasks), and role of participants

 

UoL will act as a central point for structural analysis of VLPs provided by other partners in the consortium. Their expertise will be used to determine the correct folding and morphology of VLPs. They will also develop stability assessment methods and determine the effects of buffers and other excipients. Purified samples will be sent to UoL by participants throughout the course of the project.

 

Task 2.1: Electron microscopy (EM): (UoL M3-M36)

In order to assess correct VLP assembly, the group will carry out electron microscopic analysis of VLP morphology using samples provided by other partners in the consortium. Method development will include immunogold staining (with appropriate antibodies) which can be used to determine the location of inserted IAV antigens within the VLP.

 

Task 2.2: Further structural analysis (UoL M3-M36)

Analytical ultracentrifugation will be used to provide information on VLP mass and shape under different buffer/storage conditions.  Circular dichroism can be used on nucleic acid-free samples in order to characterise folding. This will give information on the proportion of alpha helices to beta sheets etc, again under a range of buffer conditions. In addition, we have experience of successful disassembly and reassembly of VLPs (using size exclusion chromatography and mass spectrometry) and will explore this in order to generate ultra-pure VLPs. We have the option of working with our collaborators at the University of Oxford on crystallographic structure determination of selected VLPs, should this be appropriate.

 

Task 2.3: Stability studies (UoL, M3-M36)

Using the methodologies developed above, UoL will carry out long-term stability analysis on samples provided by consortium partners. This will allow an accurate shelf-life to be ascribed to the product. In addition, accelerated stability assays which we have developed elsewhere will be employed. These involve EM and/or differential hydrophobic dye binding. In addition, we will employ a range of antibodies in order to correlate the above with antigenic integrity.

 

 

Task 2.4: Biochemical analysis (UoL, M3-M36)

Using gel electrophoresis, we will assess the biochemical homogeneity of the VLPs, including the extent of post-translational modifications e.g. protein phosphorylation.

 

 

Deliverables** (brief description and month of delivery)

D.2.1 Method for routine electron microscopic analysis of VLPs (M4)

The scientist employed at UoL will be trained on current methods of VLP electron microscopy.

 

D.2.2 Method for further characterisation of VLPs (M12)

This will involve methods for immunogold staining for antigen insertion (immunoEM) and characterisation of VLPs e.g. by circular dichroism, analytical centrifugation, gel electrophoresis, disassembly/reassembly.

 

D.2.3 Long-term stability methods for VLPs (M18)

Structural information will be used to develop methods for assaying stability and antigenic integrity. Existing thermal stability assays involving hydrophobic dye binding will be applied to provide rapid preliminary data.

 

D.2.4 Long-term stability data for VLPs (M30)

The above methods will be used to carry out a long-term stability study on material supplied throughout the course of the project. These data will allow a final shelf-life to be attributed to the final product.

 



[1]   Please indicate one activity per work package: RTD = Research and technological development; DEM = Demonstration; MGT = Management of the consortium; OTHER = Other specific activities, if applicable (including any activities to prepare for the dissemination and/or exploitation of project results, and coordination activities).