Periodic Reporting for period 4 - Sugar-Enable (Influenza Virus - Sugar Interactions, From Glycan Arrays To Better Vaccines)
Periodo di rendicontazione: 2023-06-01 al 2023-11-30
Summary for Publication
Achievements:
AIM 1:
The biological relevant receptor.
This aim entailed the development of two different array techniques, glycan-arrays to determine receptor specificity and tissue-arrays to determine tissue and host tropism. Both approaches have been developed.
For the glycan array, we are now close to the 200 proposed structures that include symmetric and asymmetric N-glycans capped with different sialic acids in different linkages (Broszeit et al, 2019; Broszeit et al, 2020). We also elaborated using LacNAc modifications (Gagarinov et al, 2019), glycolipids (Arunkumar et al, 2021), and for SARS-CoV-2 we employed our heparan sulfate array (Liu et al, 2021).
The tissue arrays have been created and been employed for a wide variety of projects in which we use tissue staining (Bouwman et al, 2020; Bouwman et al, 2021; Nemanichvili et al, 2021; Spruit et al, 2021).
AIM 2:
Determining infectivity.
To determine which exact structures, confer efficient infection of different influenza A viruses we created several N-glycan containing sialic acids that can be transferred to cells. However, while working on our novel glycan arrays, we observed a distinct novel minimal receptor specificity of contemporary H3N2 viruses. These viruses are extremely hard to characterize because they don’t hemagglutinate erythrocytes and hardly infect standard tissue culture cells. To provide a proof of principle that specificity on a glycan array can be translated to that on a cell surface we opted to extend LacNAc repeats on erythrocytes to make them amendable for antigenic characterization of H3N2 viruses. Indeed, as determined by mass spec and phenotypically we now have created such erythrocytes and are currently employed by WHO surveillance centers.
The importance of sialic acid modifications
We described the presence of Neu5Gc in different animal models and published the results of the CMAH-/- mice infection experiment (Spruit et al 2021). We identified a set of amino acids and this manuscript is published in the Journal of Virology (Spruit et al, 2022). Finally we observed that these mutations also confer sialyl-lewis X specificity and described this and the in vivo experiments of these mutants (Spruit et al 2024)
AIM 3:
Challenges in flu vaccine development.
This project is published (Spruit et al, 2023)
Achievements:
AIM 1:
The biological relevant receptor.
This aim entailed the development of two different array techniques, glycan-arrays to determine receptor specificity and tissue-arrays to determine tissue and host tropism. Both approaches have been developed.
For the glycan array, we are now close to the 200 proposed structures that include symmetric and asymmetric N-glycans capped with different sialic acids in different linkages (Broszeit et al, 2019; Broszeit et al, 2020). We also elaborated using LacNAc modifications (Gagarinov et al, 2019), glycolipids (Arunkumar et al, 2021), and for SARS-CoV-2 we employed our heparan sulfate array (Liu et al, 2021).
The tissue arrays have been created and been employed for a wide variety of projects in which we use tissue staining (Bouwman et al, 2020; Bouwman et al, 2021; Nemanichvili et al, 2021; Spruit et al, 2021).
AIM 2:
Determining infectivity.
To determine which exact structures, confer efficient infection of different influenza A viruses we created several N-glycan containing sialic acids that can be transferred to cells. However, while working on our novel glycan arrays, we observed a distinct novel minimal receptor specificity of contemporary H3N2 viruses. These viruses are extremely hard to characterize because they don’t hemagglutinate erythrocytes and hardly infect standard tissue culture cells. To provide a proof of principle that specificity on a glycan array can be translated to that on a cell surface we opted to extend LacNAc repeats on erythrocytes to make them amendable for antigenic characterization of H3N2 viruses. Indeed, as determined by mass spec and phenotypically we now have created such erythrocytes and are currently employed by WHO surveillance centers.
The importance of sialic acid modifications
We described the presence of Neu5Gc in different animal models and published the results of the CMAH-/- mice infection experiment (Spruit et al 2021). We identified a set of amino acids and this manuscript is published in the Journal of Virology (Spruit et al, 2022). Finally we observed that these mutations also confer sialyl-lewis X specificity and described this and the in vivo experiments of these mutants (Spruit et al 2024)
AIM 3:
Challenges in flu vaccine development.
This project is published (Spruit et al, 2023)
Work performed
-Set up tissues and glycan microarrays as described
-developed seel technology and created glyco-remodeled erythrocytes
-studies on the contribution of NeuGc in the receptor repertoire of influenza A viruses in detail and expanded on this as we observed sialyl lewis X specificities.
-Created cell lines with significantly increased galactose and N-acetylglucosamine transferase expression and analyzed the resulting functionalities for influenza A virus infection.
-Set up tissues and glycan microarrays as described
-developed seel technology and created glyco-remodeled erythrocytes
-studies on the contribution of NeuGc in the receptor repertoire of influenza A viruses in detail and expanded on this as we observed sialyl lewis X specificities.
-Created cell lines with significantly increased galactose and N-acetylglucosamine transferase expression and analyzed the resulting functionalities for influenza A virus infection.
Expected progress
- We aim to further elaborate on our glycan array setup with more asymmetric N-glycan structures to probe the contributions of different arms and LacNAc modifications like fucose and sulfates
- Another aim is to answer multiple biological questions regarding receptor specificity.
- We aim to further elaborate on our glycan array setup with more asymmetric N-glycan structures to probe the contributions of different arms and LacNAc modifications like fucose and sulfates
- Another aim is to answer multiple biological questions regarding receptor specificity.