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Diagnostic and immunoprotective potential of influenza virus hemagglutinin stalk peptide: generation of novel vaccine prototypes

 

 

Project Title:Diagnostic and immunoprotective potential of influenza virus hemagglutinin stalk peptide: generation of novel vaccine prototypes

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Industry-Driven Research”

Project Nr.: 1.1.1.1/16/A/054

Period: 1st April 2017 – 31st March 2020

Project costs: 648 643,69 EUR

Principle Investigator: Dr. biol. A. Kazāks

 

The lack of universal influenza vaccine is a global health problem. Novadays, a special interest is attended to structurally conserved protein domains capable to elicit protection against broad range of influenza viruses. Hemagglutinin (HA) is a homotrimeric viral major surface protein which is the main target of virusneitralizing antibodies. A membrane-proximal HA stalk domain is structurally highly conservative which allow to consider it as a promising universal vaccine candidate. However, seasonal influenza vaccine or natural virus infection leads to very weak stalk-specific antibody response. A well-known strategy to improve immune response is exposition of peptides on the surface of virus-like particles (VLPs). One of the most conservative HA stalk region is so called large antigenic helix (LAH). Our recent study has revealed that this 57 aa-long peptide in E.coli forms stable α-helical trimer which is relatively easy to purify. First, project aims to generate a novel broad protection range vaccine prototype by exposition of LAH trimer on VLP surface, which is achievable by genetic fusion of the LAH gene with the coat protein gene from T=3 symmetry-derived virus. Secondly, LAH trimer will be used as antigen to develop a serodiagnostic assay for identification of people with increased anti-stalk antibody titers.

Information published: 03.04.2017.


Progress of the project

1 April 2017 - 30 June 2017

Project is realized according to proposed time scale. First, a number of original DNA constructions have been generated. They encode large antigenic helix (LAH) domain from infuenza hemagglutinin stalk which is genetically fused to coat protein genes from various RNA phages and separated by flexible linker sequences. Chimeric genes were expressed in E.coli resulting in high-level synthesis of recombinant proteins. While majority of target protein remained in debris fraction, some were partially soluble and will be used as templates for further optimization studies.

Secondly, a novel vector was constructed for expression of single LAH3 gene. Initially, no production of target protein was detected, however, after codon optimization sufficient synthesis level was obtained. Purification procedure for LAH3 has been developed. Like LAH1 this protein also forms trimer, with some differences revealed in purification. Conditions for efficient long-term storage of LAH trimers were established at -20°C. Obtained proteins will be used for structural studies as well as for development of novel immunological tests.

Information published: 30.06.2017.

 

Progress of the project

1 July 2017 - 30 September 2017

Project is realized according to proposed time scale. Currently work is in progress simultaneously in all four work packages (WPs).

WP1 activity 1.1 has been finished where a number of chimeric LAH-CP genes were tested. From four phage carrier candidates two most perspective were selected to continue optimization of constructs. Within the frames of 1.2 activity  phage AP205 and PP7 carriers are now under investigation with different linker sequences separating LAH domain from CP part. Developed constructs are under comparative expression, solubility and VLP assembly studies.

WP2 activity 2.1 has been finished where technology for purification of LAH1 and LAH3 trimers has been developed and improved. Purified trimers will be used to carry out immunologic tests in mouse and human model systems within the frames of activities 2.2 and 2.3. According to work plan, the respective activities will be realized through the outsourcing and according procurement procedure is in progress.

WP3. Determination of crystal structure of LAH trimers has been started and the first crystals are already obtained at low pH conditions. First structural data are expected at the end of the year.

WP4 activity 4.1 has been started. Mice were immunized with purified LAH trimers to collect B-cells for investigation of LAH-specific antibodies within frames of  upcoming activities 4.2 and 4.3.

Information published: 29.09.2017.

 

Progress of the project

1 October 2017 - 31 December 2017

Project is realized according to proposed time scale. Currently work is in progress simultaneously in all four work packages (WPs).

WP1. Within the frames of activity 1.2 phage AP205 and PP7 carriers served as templates to test different linker sequences separating LAH domain from CP part. A number of new constructs have been created to investigate solubility and VLP assembly. It was found that solubility of chimeras is highly dependent on choice of linker sequence, however, VLP assembly has not been detected so far. Thus, in parallel with optimization of current constructs screening of new carrier proteins for LAH trimer exposition is planned.

WP2. Purified LAH trimers are currently used as antigens for development of novel immunologic tests in mouse and human model systems within the frames of activities 2.2 and 2.3. According to work plan, the respective activities are realized through the outsourcing which is ensured by iQur company in England.

WP3. For determination of crystal structure of LAH trimers crystals have been obtained in different buffer systems at low pH conditions. First x-ray diffraction spectra have been generated in synchrotron to be used for structure calculations.

WP4. Within the frames of activity 4.1 mice were immunized with purified LAH trimers and spleens collected. Single B-cell sorting has been performed by FACS to isolate LAH-specific B-cells. These cells will be used for amplification, cloning and sequencing of genes encoding variable antibody regions.

Information published: 02.01.2018.

 

Progress of the project

1 January 2018 - 31 March 2018

 WP1. Within the frames of activity 1.2 phage PP7 has been considered as the most perspective candidate among RNA phages tested and attempts for exposition of LAH trimers on its surface are in progress. In parallel screening of other carrier proteins as ferritin nanoparticles and hepatitis B core protein is underway.

WP2. Purified LAH trimers are being used as antigens for novel immunologic ELISA tests provided by iQur company as Outsourcing partner. A novel method for detection of anti-stalk antibodies in mice sera has been developed within the frames of activity 2.2. Development of similar method in human sera (activity 2.3) is currently in progress.

WP3. To obtain three-dimensional structure of LAH trimers processing of X-ray data is underway. The primary results obtained suggest that at low pH conditions trimers adopt conformation corresponding to post-fusion form of hemagglutinin.

WP4. Amplification by RT-PCR and sequencing of genes encoding variable antibody regions from single LAH-specific B-cells is currently in progress.

Information published: 29.03.2018.

 

Progress of the project

1 April 2018 – 30 June 2018

Project is being realized according to time scale within all four work packages.

WP1. Attempts to incorporate LAH trimers within virus-like particles are in progress (activity 1.2). Novel data suggest that such chimeric particles could be obtained on the base of hepatitis B virus core protein.

WP2. In activity 2.3 continues development of ELISA-based method for detection of anti-stalk antibodies in human sera. These tests are realized by iQur company as Outsourcing partner.

WP3. To obtain three-dimensional structure of LAH trimers processing of X-ray data is underway. Results confirm that at low pH condition trimers adopt conformation corresponding to post-fusion form of hemagglutinin.

WP4. Several variable antibody regions from both heavy and light chains were PCR-amplified and sequenced from single LAH-specific B-cells. These data will be used to continue investigation in activity 4.2.

Information published: 29.06.2018.




Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".