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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CARBOFLU (Chemoenzymatic synthesis of complex glycans to decipher the interactions between them and Influenza A virus)

Teaser

Summary

Influenza A virus displays in majority in its surface two glycoproteins, Hemagglutinnin (HA) and Neuraminidase (NA). The virus mutate these two glycoproteins very fast what provides to it a via of escape from immune system. HA recognizes sialic acid containing glycans and NA cleaves these sialic acid moieties from the glycans. The affinity of HA for the glycans varies as the mutation occurs and the sialidase activity of NA also changes. In this project, our goal is to describe the chronological order of the mutation as well as to unravel wether there is any cooperation between the two envelope glycoproteins HA and NA.

Influenza A virus is a major threat for the people all over the world. According to the World Health Organization (WHO), between 291,000 and 646,000 deaths worldwide are caused directly by the flu. Knowing more about the evolutionary pathway would increase the success rate of the new vaccines.

a) Chemoenzymatic synthesis of a library of complex glycans.
b) Interaction of the synthetic glycans with viral HA and NA.

Conclusions

Chemoenzymatic approach for the synthesis of glycans has been developed.

The library of synthesized glycans has been used in binding affinity assays with HAs, in addition, the glycans has been employed to calculate the sialidase activity of the NAs.

Work performed

In this project, a chemo-enzymatic syntheses of sialic acid containing glycans have been performed. The library is populated with bi-antennary symmetrical and asymmetrical glycans and also by linear glycans. A LC-MS based method involving the use of an internal standard has been developed to quantify the amount of the sialic acid released by different NAs. Glycan library obtained has been tested by using two different Neuraminidases from the H7N9 influenza A virus. The preferences of Neruaminidases for the different glycans have been described and the results will be published. Preliminary results obtained by the LC-MS-based method were presented at the International Carbohydrate Symposium in Lisbon in July 2018.
In parallel, Computational calculations were developed to assist in a project targeting the exact structure elucidation of Glycans from biological samples by using Ion-Mobility Spectrometry (IMS) and analysis of standards. The computational calculations protocol developed enabled us to predict the CCS values of two positional isomers successfully. This progress will boost the employ of IMS as an identification technique, and the theoretical calculations will reduce significantly the synthetic effort needed. The results of this project were communicated through a flash talk and a poster at the 4th NVMS-BSMS Conference on Mass Spectrometry. The communication was awarded the best poster.

Final results

Neuraminidases from Influenza A virus have been studied predominantly using the MUNANA assay, however, the activity values do not have any significance regarding the glycans found in humans. Other experiments were developed to measure the sialic acid released by the Neuraminidases but they relied on indirect measurements. The LC-MS-based method developed during this project used an internal standard. Through this method we have been able for the first time to profile the glycans preferences of the N9 (WT) and the N9(T401A) from H7N9. In addition, this robust method will be applied to different virus strains, this will have a significant impact on the knowledge of the viral evolutionary pathway and pave the way for more efficient vaccines making people\'s life better.

The development of the computational protocol capable to predict the CCS values of the glycans will reduce dramatically the number of glycans standards needed for the Glycomics analysis of glycoproteins. In this way, Ion-Mobility Spectrometry will turn into a promising tool in the discovery of new biomarkers.