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Epitope analysis for influenza vaccine design

Until now, design of the annual influenza vaccine has relied on phylogenetic or whole-sequence comparisons of the viral coat proteins hemagglutinin and neuraminidase, with vaccine effectiveness assumed to correlate monotonically to the vaccine-influenza sequence difference. We use a theory from statistical mechanics to quantify the non-monotonic immune response that results from antigenic drift in the epitopes of the hemagglutinin and neuraminidase proteins. The results explain the ineffectiveness of the 2003--2004 influenza vaccine in the United States and provide an accurate measure by which to optimize the effectiveness of future annual influenza vaccines.


Introduction
Antigenic variation constitutes one mechanism employed by influenza viruses to evade the adaptive response of the

Methods
We have developed a theory of the immune response to an antigenic drift strain after vaccination based on statistical mechanics, Figure 1 for a second antigen, after exposure to an original antigen whose epitope region differs by probability p epitope . The key measure of antigenic drift in the theory is p epitope , the fractional change between the dominant epitope regions of the vaccine and the circulating strain, defined by the equation p epitope = number of mutations within the epitope number of amino acids within the epitope .
This characterization of antigenic drift in our theoretical model emphasizes the experimental fact that only the epitope regions are significantly involved in immune recognition, as shown by immunoassays and crystallographic images [4].
In the theory, it is the percent of the epitope that changes that characterizes antigenic drift. To provide additional empirical support for the theory, we performed an historical analysis of the influenza seasons between 1991-2000 when 4 the H3N2 virus suptype was dominant [5,6,7,8,9,10,11,12,13]. For every season, hemagglutinin sequences [14] were compared between the vaccine strain and the predominant circulating strain. A quantitative scale was defined to measure the seasonal flu severity as follows: low (1), mild (2) and high (3).
The values of seasonal flu severity were correlated with the calculated p epitope values. In addition, to make clear that the epitope region is primarily responsible for immune recognition, we also correlated the seasonal flu severity with antigenic drift of the entire hemagglutinin sequence, normalized by the total number of amino acids in the protein p sequence . The results presented in Figure   2 show that seasonal flu severity is correlated with p epitope rather than p sequence , thus favoring the epitope analysis approach. Therefore, it is both logical and consistent with the observed data to characterize antigenic drift by the number of mutations within the epitope regions, as we do in the present work.
According to historic clinical experience, and to our model, when the antigenic drift between the vaccine and circulating strain, characterized by the p epitope value, is small, exposure to the vaccine antigen leads to a higher affinity constant than without exposure. This result is why immune system memory and vaccination are generally effective. When the antigenic drift between the vaccine and circulating strain is large, the vaccine antigen is uncorrelated with the circulating strain antigen, and so immune system memory does not play a role. When the antigenic drift between the vaccine and circulating strain epitopes is modest (0.23 < p epitope < 0.6), our theory predicts that memory response may be worse than the naive response (the solid curve lies below the dashed curve in Figure 1), which means that the immunological memory from the vaccine exposure actually gives worse protection, i.e., a lower affinity constant, than would no vaccination whatsoever. This result is the original 5 antigenic sin phenomena for influenza: vaccination creates memory sequences that for some mutation rates of influenza may increase susceptibility to future exposure [15,16]. Parenthetically, not every infectious disease exhibits original antigenic sin, with measles one such example. The measles virus does not undergo antigenic drift, and despite approximately eight different subtypes that have been identified to date [17], the HA and NA genetic variation among them does not exceed 7% on a nucleotide basis [17], or roughly 2% on an amino acid basis. Accordingly, within the context of our model, there is no possibility of original antigenic sin for measles (since p epitope < 0.02, see Figure   1).
Original antigenic sin stems from localization of the immune system response

Results
We compared the epitope sequences of the HA protein to look for mutations in the A/Fujian/411/2002 strain [14] with respect to the A/Panama/2007/99 strain [18]. The hemagglutinin H3 protein has five epitope regions (A, B, C, D, E) that have been identified and sequenced [19], among which A and B are usually dominant [20,21]. There exists experimental and clinical evidence that epitope regions mutate much faster than other regions in the viral proteins, presumably due to antibody selective pressure [22,23,24], with the dominant epitopes mutating most rapidly [25]. Therefore, in the absence of more detailed information, we take an observed high mutation rate (i.e., a high p epitope value)

Discussion
In Figure 1  Taken in aggregate, these results, Figure 1, suggest that the 2003-2004 flu vaccine would have essentially no effect against the circulating A/Fujian/411/2002 strain, in agreement with clinical findings [1] and in disagreement with early expectations [2].
In conclusion, we suggest that strains related by antigenic drift be compared by measuring differences in the epitope regions of the hemagglutinin and neuraminidase proteins and not by differences in the whole sequence or phylogeny as is presently done. This particular point is supported by the correlations of seasonal flu severity with epitope antigenic drift shown in Figure 2. Thus, there is a need for a detailed characterization of the epitope regions in the different influenza strains. In particular, a precise determination of which epitopes are dominant in the proposed vaccine strain and an experimental measure of p epitope and of cross activity between the proposed vaccine strain and the circulating strains would be highly productive. In absence of this determination, we suggest to estimate the dominant epitope as that which shows the most antigenic drift [22,23,24,25], as we do in the present work. From either epitope sequence drift or cross activity, Figure 1 can be used to estimate the degree of the immune response, which is a non-linear and non-monotonic function of the measured data. We believe that this quantitative epitope analysis should be incorporated as part of the regular protocol for construction of the annual influenza vaccine.

Acknowledgments
We thank Nancy Cox Correspondence and requests for materials should be addressed to mwdeem@rice.edu. Fig. 1. The evolved affinity constant to a second antigen after exposure to an original antigen whose epitope region differs by probability p epitope (solid line