Changing selective pressure during antigenic changes in human influenza H3

Abstract

The rapid evolution of influenza viruses presents difficulties in maintaining the optimal efficiency of vaccines. Amino acid substitutions result in antigenic drift, a process whereby antisera raised in response to one virus have reduced effectiveness against future viruses. Interestingly, while amino acid substitutions occur at a relatively constant rate, the antigenic properties of H3 move in a discontinuous, step-wise manner. It is not clear why this punctuated evolution occurs, whether this represents simply the fact that some substitutions affect these properties more than others, or if this is indicative of a changing relationship between the virus and the host. In addition, the role of changing glycosylation of the haemagglutinin in these shifts in antigenic properties is unknown. We analysed the antigenic drift of HA1 from human influenza H3 using a model of sequence change that allows for variation in selective pressure at different locations in the sequence, as well as at different parts of the phylogenetic tree. We detect significant changes in selective pressure that occur preferentially during major changes in antigenic properties. Despite the large increase in glycosylation during the past 40 years, changes in glycosylation did not correlate either with changes in antigenic properties or with significantly more rapid changes in selective pressure. The locations that undergo changes in selective pressure are largely in places undergoing adaptive evolution, in antigenic locations, and in locations or near locations undergoing substitutions that characterise the change in antigenicity of the virus. Our results suggest that the relationship of the virus to the host changes with time, with the shifts in antigenic properties representing changes in this relationship. This suggests that the virus and host immune system are evolving different methods to counter each other. While we are able to characterise the rapid increase in glycosylation of the haemagglutinin during time in human influenza H3, an increase not present in influenza in birds, this increase seems unrelated to the observed changes in antigenic properties.

Figure 1. Changes in the number of glycosylation sites with time.

Clusters are indicated as block colouring. Thick red line represents the main branch of the tree. Dates for the internal nodes were estimated based on extrapolating a linear least-squares fit of the acquisition time of the available sequences.

Figure 2. Characteristics of the four substitution matrices.

Each substitution matrix is represented by the Venn diagram of physical properties devised by Taylor . Non disulfide-bonded cysteines have been excluded from the figure, as all cysteines in HA1 are disulfide bridged. Each substitution matrix is characterized by a relative overall substitution rate and different propensities for the various amino acids represented by equilibrium frequencies. Amino acids in this figure are colored according to these equilibrium frequencies compared with the overall average. Blue indicates a frequency is less than the mean, with red amino acids greater. More intense colors are proportionally further from the mean .

Figure 3. A) Log2-odds representation of the propensity of various types of locations for various substitution matrices in HA1, indicating the log2 of the relative frequency of a given substitution matrix in each of the types of locations divided by what would be expected at random.

Substitution-matrix assignments are averaged over all of the internal nodes of the phylogenetic tree. Eight different types of location types are considered: antigenic (‘Antigenic’), receptor binding sites (‘Binding’), non-antigenic non-binding exposed sites (‘Surface’), buried sites (‘Buried’), sites of ‘cluster-difference’ substitutions (Smith et al. 2004) (‘Difference’), and positively-selected sites (‘+Sel’). Substitution matrices are substitution-matrix 1 (horizontal lines), substitution-matrix 2 (stippled), substitution-matrix 3 (diagonal lines), and substitution-matrix 4 (cross-hatched). Locations are considered buried or exposed based on whether their accessible side-chain area is larger or smaller than 10% of the areas calculated by Ahmad et al. . Positively-selected sites were based on the Maximum Likelihood analysis of Yang ; consistently less significant correlations were observed when positively-selected sites identified by Bush et al. were used. B) Log2-odds representation of the propensity of various types of locations for various numbers of substitution-matrix changes. A posteriori probabilities of changes of substitution matrix over the tree are calculated for each site. (Probabilities of substitution-matrix change less than 5% are neglected.) Sites are then assigned into one of three categories of rate of change in selective pressure from the number of changes found: slow (white, 0–1 changes), medium (gray, 1–2 changes), and fast (black, >2 changes). Arrows in the plot refer to log2-odds of negative infinity.