# Combination of molecular approaches to access homogeneous glycoconjugates for structure-immunogenicity relationship studies

Laura Laneque, Typhaine Violo, Aline Pillot, Annie Lambert, Johann Hendrickx, Emilie Camberlein, Cyrille Grandjean

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Laura Laneque, Typhaine Violo, Aline Pillot, Annie Lambert, Johann Hendrickx, et al.. Combination of molecular approaches to access homogeneous glycoconjugates for structure-immunogenicity relationship studies. 3rd Chemical Biology Symposium, Jan 2023, Paris, France. hal-04857628

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Distributed under a Creative Commons Attribution 4.0 International License
# Combination of molecular approaches to access homogeneous glycoconjugates for structure-immunogenicity relationship studies

Laura Laneque¹, Typhaine Violo¹, Aline Pillot¹, Annie Lambert¹, Johann Hendrickx¹, Emilie Camberlein¹ and Cyrille Grandjean*,¹

¹ Nantes Université, CNRS, US2B, UMR 6286, 44000 Nantes, France.

## INTRODUCTION

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Glycoconjugate vaccines consist of the conjugation of bacterial surface polysaccharides (PS) to a carrier protein. The latter allows the induction of antibodies directed against the carbohydrate antigen thanks to the recruitment of CD4+ T lymphocytes to elicit a protective immune response.

PS length, carrier protein/PS ratio and bioconjugation chemistry have been established as key factors that have a direct impact on the immune response. In addition, it has long been assumed that the sites to which the PS is attached to the carrier protein (connectivity) may also have an impact.

Impact of connectivity on the immune response can now be addressed through a combination of molecular tools such as site-selective mutagenesis, incorporation non-canonical amino acids (ncAA) and bioorthogonal conjugation strategies leading to fully-controlled, homogeneous glycoconjugates.

## OBJECTIVES AND STRATEGIES

In our study, pneumococcal surface adhesin A (PsaA), a highly conserved immunogenic protein expressed by *Streptococcus pneumoniae* and a synthetic dodecasaccharide mimicking the capsule of Pn serotype 14 have been selected as model carrier protein and carbohydrate antigen, respectively. Taking advantage of the absence of cysteine in PsaA, up to four lysine residues have been mutated to cysteine for subsequent bioconjugation. Site-specific ncAA incorporation approach has also been envisaged as an alternative.

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## FUTURE DIRECTIONS

Conjugation sites were targeted to key locations within or outside T-helper epitopes to investigate how connectivity impact the recruitment of CD4+ T lymphocytes and beyond the level and the quality of humoral response induced against the carbohydrate antigen and the PsaA.

## RESULTS

### Directed-site mutagenesis strategy :

* Mutated genes were verified by Sanger sequencing.

* Presence of cysteines in the modified proteins was confirmed by mass spectrometry.

* The secondary structure of the protein after cysteine mutation(s) is conserved.

<figure><img src="image_4.png" /><figcaption>Overlay of circular dichroism spectra of PsaA mutants (mono-, di-, tri-, tetra-mutants) against the wild type.</figcaption></figure>

### Non-canonical amino acid insertion approach :

* Presence of the ncAA in the protein was confirmed by mass spectrometry.

* Purity was verified by electrophoresis.

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12% SDS-Page of the collected fractions. Line FB: crude fraction from cell lysis; FNR: fraction not retained by the column; FL₁, FL₂ and FL₃: start, middle and end of wash. FE₁ and FE₂: gradient of elution (0 to 1M of MgCl₂). FE₃ and FE₄: elution (2M of MgCl₂). Ffin: end of the program.

UMR CNRS 6286
Unité des Sciences Biologiques et des
Biotechnologies
2 rue de la Houssinière
44322 Nantes FRANCE

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