No abstract

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between wild-type IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

Graphical abstract The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N -linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of the SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low-abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low-abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA outperforms DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03643-7.

Turning to historic times it would appear that from A.D. ioo to A.D. 8oo the country was ruled by a Gaelic aristocracy and it may be that this rule was over peoples of many distinct races. Prior to the Norse invasions of about 8oo A.D. there was a unity of civilisation, law and language under a Gaelic High Kingship. Ireland was not conquered by the Romans. The Norse invaders established colonies and fortresses round the coast and these were especially strong at Dublin, Wexford, Waterford, Cork and Limerick. For two hundred years they tried to subdue the country but their attempts ended when Brian Boru routed their Dublin garrison in i 014. With the centre of their power destroyed the Norsemen's coastal settlements became isolated communities enjoying citizen rights under the High Kingship. After a period of unsettled Gaelic rule, Ireland experienced a tide of Anglo-Norman invasion and settlement from i i66 to about 1300. This flow was mainly into south and east Louth, Meath, the eastern half of Westmeath,