Workpackage 1

 

Work package number

1

Start date or starting event:

M1

Work package title

Design of universal influenza VLPs based on tandem core technology.

Activity Type[1]

RTD

Participant number

1

2

3

4

5

 

 

Participant short name

iQur

UoL

LBMC

UCL

CRP-Santé

 

 

Person-months per participant:

51.0

3.0

40.5

73.8

1.1

 

 

                   

 

Objectives

To design and produce VLPs based on HBc tandem core carrying at least two known, “universal” non-polymorphic antigens including, but not limited to, M2, NP, HA stalk and others from IAV.

 

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

 

Introduction

Influenza antigens will be cloned into the tandem core vector and expressed in Pichia pastoris. The consortium will develop an optimised generic purification strategy which will be applicable to this and all future versions. Material will be characterised for VLP formation and antigen expression prior to in vivo testing.

 

Task 1.1: Design and construction of VLP constructs (iQur M1-M5)

In a first round VLPs will be designed with two copies of the same antigen (one for each MIR). For each of the different “universal” antigens different variants (eg. sequence length, variants etc) will be designed to optimize their immunogenicity in the context of the tandem core constructs.  It is anticipated that for each universal antigen 2 to 4 variant constructs will be designed. 

In a second round, combinations of two or more “universal” antigens will be designed. It is anticipated that at least 5 combination constructs will be produced.

The tandem core antigen fusion proteins will be constructed by conventional molecular biology. A codon optimization step for optimal expression in Pichia pastoris may be included.  

 

Task 1.2: Expression of recombinant VLPs (iQur M1-M3)

The transformation of yeast will be outsourced to Mologic Limited, UK., who will produce plates of recombinant Pichia pastoris. These will then be screened by PCR (IQUR) and positive clones will be provided to iQur, UCL and LBMC, who will provide purified VLPs to the consortium.

 

Task 1.3: Optimisation of purification  and batch characterisation (iQur, LBMC, UCL, M3-M9)

 iQur, LBMC and UCL will use their complementary expertise to optimise the purification of the recombinant VLPs and to develop a robust protocol suitable for all VLP constructs. High-throughput microscale methods will be applied to evaluate several lead clones for optimisation and their ‘processability.’ Previous experience indicates that changes to the expression strain and chimeric VLP will lead to slight changes in the process characteristics, such as their propensity to aggregate, adhere to membranes, fold and denature and contaminant profile. Therefore UCL will apply their scale down methodology to screen for the optimum clone with respect to titre and VLP folding then begin investigation of optimal feed strategies for VLP expression. This will be followed by primary recovery and monitoring the process impact of homogenisation on centrifugation solids carry-over, tangential flow filtration and chromatography. During this process the critical process parameters will be identified to maintain the key quality attributes of the target VLP. The production groups will provide material to UoL for analysis (WP2) and then release purified VLPs to the consortium for further studies. Studies will include;

Task 1.3.1: Initial biomass generation.

Task 1.3.2: Clone selection

Task 1.3.3: Induction optimisation using Design of Experiments methodology

Task 1.3.4: Testing product for susceptibility to shear

Task 1.3.5: Effects of homogenisation and centrifugation on fermentation harvest windows

Task 1.3.6: Optimisation of cross-flow filtration

Task 1.3.7: Integration of centrifugation and cross-flow filtration

Task 1.3.8: Pilot scale model verification

 

Task 1.4: Redesign/optimisation (iQur M8-M14)

Preclinical data (immunogenicity efficacy in mice and ferrets; stability etc)  will be assessed (WPs 3 & 4) and will guide further optimization steps of the VLP constructs in an iterative process of testing and redesigning, as may be necessary. Again, these new constructs will be outsourced for yeast transformation before being supplied again to the consortium.

 

Task 1.5: Scale up development (LBMC, UCL, M3-M9)

Lead candidates will be scaled-up in fermenters by LBMC and UCL. Samples will be sent to UoL for analysis. Lead candidate will be scaled up to 20L bioreactors. Geometric and kinematic basis for scaling as well as requirements for oxygen blending will be established before technology transfer to 3P. Material generated here will feed into Work Package 4. This material will then be processed in the UCL Biochemical Engineering Pilot plant, where pilot scale continuous homogenisation, centrifugation, tangential flow filtration and chromatography will be applied to elude optimal operating parameters for the process and final technology transfer to LBMC and 3P.

 

Task 1.6: Further development (iQur, LBMC, UCL M11-M36)

UCL will use their bespoke microtitre plate methods to optimise buffers and resins that may lead to increased product recovery and purity. Similarly, LBMC will use their experience of yeast expression of monomeric HBc to improve yields and recoveries using a variety of new constructs. Both groups will also examine methods to reduce aggregation and develop a vaccine formulation strategy.

 

RISK MITIGATION STRATEGY: iQur, LBMC and UCL will continue to develop new constructs, even when first lead constructs have emerged, to develop further valuable intellectual property and providing a back-up strategy in case the lead construct will fail to reach commercial maturity.

 

 

Deliverables** (brief description and month of delivery)

 

D1.1 100mg of purified VLPs with single “universal” antigens for each of the three universal antigens (2-4 variants for each universal antigen) (M5)

D1.2 100mg of purified VLPs with combinations of 2-4 universal antigens  (3-5 combination constructs) (M15)

D1.3 Robust  method for efficient production of recombinant VLPs purification (M9)

D1.4 Robust method for efficient purification of recombinant VLPs suitable for transferring to a contract manufacturer. (M12)

 



[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).