Workpackage 3

 

Work package number

3

Start date or starting event:

M5

Work package title

Immunogenicity studies in mice

Activity Type[1]

RTD

Participant number

1

5

 

 

 

 

 

Participant short name

iQur

CRP-Santé

 

 

 

 

 

Person-months per participant:

2.0

32.6

 

 

 

 

 

                   

 

Objectives

To examine the immunogenicity of VLPs expressing invariant IAV antigens and to make informed selections of lead candidates

 

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

It will be crucial to demonstrate a strong and comprehensive immune response in animal models prior to entering the clinical phase of the programme. iQur has considerable experience in generating VLP antibody responses, whilst Luxembourg can significantly augment immune profiling by examining both T and B cell responses. Both will be required for an optimal vaccine. VLPs developed in WP1/WP 2 will be tested for their protective immune responses in mice. The results will guide several rounds of iterative improvements of the VLP constructs. First, each of the three universal antigens (HA stalk, M2, NP) will be optimized in association with the tandem core VLPs (Task 3.1-3). Then, combinations of the most promising antigens will be tested (Task 3.4-8).

 

Task 3.1: Testing of VLPs carrying variants of the HA stalk antigen: (IQUR/CRP-SANTÉ M5-M12)

Initial vaccinations will be carried out by iQur in order to identify active vaccine candidates. Once immunogenicity has been confirmed, this material will be transferred to Luxembourg for further testing. These tests will include: serum antibody levels against the carrier HBc and the IAV antigen (ELISA), neutralizing activity against different influenza subtypes (seasonal H1N1, H3N2, pandemic H1N1, avian and pig viruses as appropriate), T cell proliferation assays, IFNγ ELISPOTs, cytotoxic T cell assay, antibody mediated cytotoxicity, challenge-protection experiments with H1N1 A/PR/8/34 (parameters monitored: body weight, body temperature, viral lung titres and survival). A small panel of adjuvants licenced in humans will be tested in association with the best HA stalk antigen to identify the most effective adjuvant.    

 

Task 3.2: Testing of VLPs carrying 1 influenza M-derived antigen (IQUR/CRP-SANTÉ, M5-M12).

Methods as described for Task 3.1 (except neutralization assays). The most effective adjuvant with respect to the best M-derived antigen will be determined. 

 

Task 3.3: Testing of VLPs carrying 1 influenza NP-derived antigen (IQUR/CRP-SANTÉ, M5-M12).

Methods as described for Task 3.1 (except neutralization assays). The most effective adjuvant with respect to the best NP-derived antigen will be determined. 

 

Task 3.4: Testing of VLPs carrying different combinations of 2 to 4 of the lead antigens from task 3.1-3.3 (IQUR/CRP-SANTÉ, M15-M20).

Methods as described for Task 3.1. The most effective adjuvant with respect to the best antigen combination will be determined. 

 

Task 3.5: Challenge/protection experiments against different subtypes (CRP-SANTÉ, M15-M18).

The most promising single and multiple antigen VLP-constructs will be further tested in mice against different seasonal H1N1, H3N2 subtypes and pandemic H1N1 (2009) virus, using the most promising adjuvant. 

 

Task 3.6: Investigation of the B cell repertoire to determine signatures of protective ADCC responses (CRP-Santé, M16-M26).

Isolation of DNA/RNA from lymphoid organs and bone marrow from vaccinated and control mice Amplification of the heavy chain CDR3 region (and other variable regions if necessary) followed by next generation multiple parallel sequencing. Bioinformatics processing of sequencing data and identification of characteristic signatures of a protective antibody and ADCC responses

 

Task 3.7: Investigation of the T cell repertoire to determine T cell receptor (TCR) usage in protective responses (CRP-Santé, M16-M26).

Isolation of DNA/RNA from T helper cells and CTL cultures of vaccinated and control mice Amplification of selected variable regions of the T cell receptor. Bioinformatics processing of sequencing data and analysis of TCR usage in a protective immune response.

 

Task 3.8: Investigation of virus isolates in mice with partial protection (CRP-Santé, M16-M26)

Virus will be isolate from lungs of (partially) protected mice. HA, NP, M sequences as appropriate will be amplified using established NGS protocols. The libraries will be submitted to NGS (Ion Torrent) for deep-sequencing.

Identification of major or minor virus populations with potential vaccine escape mutations    .

 

Task 3.9: Compiling a short list of (mixed)  VLP constructs for further investigation (IQUR/CRP-SANTÉ, M26)

Go/NoGo decision for the ferret experiments on the basis of the above immunogenicity studies and challenge protection experiments

 

 

Deliverables** (brief description and month of delivery)

 

D.3.1: Identification of the most immunogenic/protective VLP-HA construct in mice (M12)

Antigen selection

D.3.2: Identification of the most immunogenic/protective VLP-M2 construct in mice (M12)

Antigen selection

D.3.3: Identification of the most immunogenic/protective VLP-NP construct  in mice (M12)

Antigen selection

D.3.4: Go/NoGo decision based on a short list of the most immunogenic/protective VLP-HA/M2/NP in mice (M15)

Combined antigen selection

D3.5: Identification of a protective CDR3 heavy chain signature in B cells  (M22)

Evidence of dominant immunoglobulin generation.

D3.6: Identification of the protective TCR usage signature (M22)

Evidence of T-cell activation.



[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).