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

Funding: Measure 1.1.1.1 “Industry-Driven Research” of specific objective 1.1.1 “To increase the research and innovation capacity of scientific institutions of Latvia and their ability to attract external funding by investing in human resources and infrastructure”

Project No: 1.1.1.1/16/A/054

Period: 1 April 2017 – 31 March 2020

Project costs: 648 643,69 EUR

Principle Investigator: Dr. biol. A. Kazāks

Summary:

The main aim of the project is to develop a novel broad protection range influenza vaccine prototype. This will be achieved by targeted combination of modern technologies in gene and protein engineering with novel scientific findings in appropriate field. Thus, project contributes to realization of Latvian Smart Specialization Strategy-directed growth priorities and transformation of economics, with more narrow specialization in biomedicine, medical technologies, biopharmaceutics un biotechnology. Project aims to combine novel scientific data on current influenza vaccine candidates with high-resolution structural data of influenza HA protein and RNA bacteriophage shells. Using gene and protein engineering methods, it is planned to connect two structures: LAH trimer in its native pre-fusion form carrying conformational epitopes exposed on surface of highly immunogenic VLPs. Recent studies were targeted either to use of free HA stalk peptide trimers (contain conformational epitopes but are not immunogenic enough), or attempted to incorporate these peptides in VLPs as monomers (proteins are highly immunogenic but do not carry conformational epitopes). There is high probability that combination of both native structures (LAH trimer + VLP) will result in generation of broad protection range vaccine prototype.

Project applicants has all the necessary information and resources to complete the proposed tasks according to the time schedule. There are LAH-encoding DNA templates available as well as cloned genomes and structural data of 8 RNA bacteriophages. Project personal has a long-time experience in VLP biotechnology and structural biology. There is also all the necessary technical equipment along with a qualified staff to accomplish the tasks to be realized in BMC. The lab has standard equippment for cloning and cultivation of recombinant E. coli. For protein purification, AKTA FPLC chromatography device and numerous purification columns are available. ABI PRISM 3130xI sequencer is available for verification of DNA constructs, and MALDI-TOF masspectrometer – for protein identification and verification. Development of serodiagnostic assays, protection tests, as well as isolation of stalk-specific B-cells will be realized by Outsourcing Partner.

Information published 01.04.2017.

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.

Project progress

1 July 2018 – 30 September 2018

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

WP1. Activity 1.2 has been finished with successful incorporation of LAH trimers within virus-like particles derived from hepatitis B virus core protein. Optimization of purification of appropriate VLPs will be accomplished in activity 1.3.

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

WP3. Data compilation to obtain three-dimensional structure of LAH trimers was finished. Results confirm that trimers adopt conformation corresponding to post-fusion form of hemagglutinin at low pH conditions. Appropriate structure file has been deposited in PDB databank under accession code 6GOL.

WP4. Within activity 4.2 variable antibody regions from both heavy and light chains isolated from single LAH-specific B-cells are being cloned in expression vector pVITRO-IgG1/κ for synthesis of appropriate antibodies in mammalian cells.

Information published 28.09.2018.

Project progress:

1 October 2018 – 31 December 2018

• In WP1 activity 1.3 optimization of purification for chimeric LAH trimer containing VLPs is in progress.
• WP2 was finished as within activity 2.3 an ELISA-based method for detection of anti-stalk antibodies in human sera has been developed by iQur company as Outsourcing partner.
• WP2 was finished as three-dimensional structure of LAH trimers has been deposited in PDB database under accession number 6GOL as well as described in scientific publication.
• In WP4 activity 4.2 DNA constructions to obtain LAH-specific antibodies have been generated. Currently appropriate constructs are being expressed in mammalian cells.

Information published 28.12.2018.

Project progress

1January 2019 – 31 March 2019

• WP1 activity 1.3 was finished. Chimeric LAH trimer containing VLPs as vaccine prototype candidates were purified. The antigenic and immunogenic properties of respective chimeric VLPs in mice model are currently being examined within activity 1.4. These tests are provided by Institute of Experimental Medicine in St Petersburg, Russia, as Outsourcing Partner.
• WP4 activity 4.2 was finished. In this activity secretory expression of LAH-specific antibodies has been established in mammalian suspension cell culture. Purification method for appropriate antibodies with affinity chromatography was developed. Functional characterization of purified antibodies is in progress.

Information published 29.03.2019.

Project progress:

1 April 2019 – 30 June 2019

• The antigenic and immunogenic properties of selected vaccine prototype VLPs in mice model are currently being examined within activity 1.4. These tests are provided by Institute of Experimental Medicine in St Petersburg, Russia, as Outsourcing Partner. The primary protection test is finished which allowed to establish the most prospective vaccine candidate. Currently the second protection test is ongoing with several influenza virus strains.
• LAH-specific antibodies were produced in mammalian suspension cell culture and purified by affinity chromatography. Functional characterization of purified antibodies by ELISA using different recombinant LAH proteins as antigens is in progress.

Information published 28.06.2019.

Project progress

 1 July 2019 – 30 September 2019

• The antigenic and immunogenic properties of selected vaccine prototype VLPs are currently being examined in mice model within activity 1.4. These tests are provided by Institute of Experimental Medicine in St Petersburg, Russia, as Outsourcing Partner. Secundary protection test with different influenza viruses allowed to characterize immunoprotective potential of the most prospective vaccine candidate. Currently within activity 1.5 several T-cell activity tests will be performed to complete experimental part of the project.
• Functional characterization of purified LAH-specific antibodies is being continued by ELISA tests using different recombinant LAH proteins and chimeric VLPs as antigens.

Information published 30.09.2019.

Project progress

 1 October 2019 – 31 December 2019

• The properties of selected vaccine prototype VLPs have been examined in mice model within activities 1.4 and 1.5. These tests were provided by Institute of Experimental Medicine in St Petersburg, Russia, as Outsourcing Partner. Experimental part of the project is now finished with characterization of immunoprotective potential of the most prospective vaccine candidate. Currently obtained data are being collected and processed for scientific publication.
• Activity 4.3 is finished with functional characterization of purified LAH-specific antibodies by ELISA tests using different recombinant LAH proteins and chimeric VLPs as antigens. It was detected that obtained antibodies are subtype-specific and can be successfully used for localization of LAH sequence within chimeric VLPs.

Information published 30.12.2019.

Project progress

1 January 2020 – 31 March 2020

At this time, the final activity of the project 1.6 was carried out, dedicated to gathering and preparing the data generated in activities 1.3-1.5 for publication. High quality results demonstrating the immunoprotective potential of the selected vaccine prototype were obtained. Over the course of the project, a new method was developed for acquiring hypero VLP, where a genetic merge with chemical attraction was combined. This allows multiple antigens to be included in one part at the same time. The manuscript is prepared for submission to Vaccines magazine.

Information published 31.03.2020.