In the current global influenza surveillance system, influenza viruses are characterized antigenically based on ferret serum hemagglutinin-inhibition (HAI) antibody cross-reactivity. We first screened publications for influenza H3N2 virus cross-reactive antibody data. Then, we searched the H3N2 viruses with cross-reactive antibody data for their amino acid sequences of the HA1 polypeptide (www.flu.lanl.gov) (8). Table 1 shows the full name, abbreviation, identification (ID) by type, and accession code of the H3N2 viruses (12–16). Six sets of ferret serum HAI cross-reactivity data were available for analysis. The first set included 11 viruses (55 pairwise comparisons, virus ID: A to K) isolated from 1971 to 1979 (12). The second set included 8 viruses (28 pairwise comparisons, virus ID: J, L to R) isolated from 1979 to 1987 (17). The third set included 10 viruses (45 pairwise comparisons, virus ID: S to AB) isolated from 1989 to 1994 (13). The fourth set included 8 viruses (28 pairwise comparisons, virus ID: AC to AJ) isolated from 1994 to 1996 (18). The fifth set included 5 viruses (10 pairwise comparisons, virus ID: AE, AK to AN) isolated from 1995 to 1999 (15). The sixth set included 6 viruses (15 pairwise comparisons, virus ID: AN to AT) isolated from 1999 to 2002 (16). A mathematical method had been proposed to calculate "antigenic relatedness" between two viruses (presented as a percentage) as a geometric mean of two ratios between the heterologous and homologous antibody titers (19,20).

Since our study investigates the relationship between antigenic difference and amino acid changes in the HA1 polypeptide, the mathematical method was modified to calculate "antigenic distance" (i.e., reciprocal of antigenic relatedness). For example, if homologous titers of two viruses are 640 and 640 and two heterologous titers against each other are 320 and 320, the antigenic relatedness between these two viruses is ([320 x 320]/[640 x 640])^½ = 50%, and the antigenic distance between these two viruses is ([640 x 640]/[320 x 320])^½ = 2. Table 2 shows the antigenic distances of the 55 pairwise comparisons among the 11 viruses in the first set. In total, 181 pairwise comparisons among 45 viruses were available for analysis. Among the 181 pairwise comparisons, 56 (31%) have an antigenic distance <4 (i.e., similar antigenicity), and 125 (69%) have an antigenic distance >4 (i.e., antigenic variant) (21).

Amino acid sequences of the HA1 polypeptide were downloaded from the Los Alamos Influenza Sequence Database (8) or entered from the original publications if they were not available from the Los Alamos Influenza Sequence Database. Amino acid sequences of the 45 viruses were harmonized to same length (329 residues) and were numbered according to A/Aichi/2/68 HA1 sequence because the 3-D structure of the A/Aichi/2/68 hemagglutinin protein has been determined (4–6). Pairwise alignments among the 45 sequences were conducted by using S-Plus 2000 (Insightful Corporation, Seattle, WA). Pairwise-aligned amino acid sequence data were transformed into 0 (without change) and 1 (with change) and were further linked with the pairwise antigenic distance data for predicting analyses.

The first model was based on amino acid differences in the whole HA1 polypeptide (329 residues). The second model was based on amino acid differences in the five antigenic sites (131 residues) (Appendix) (7,8). The third model was based on the 20 positions related to mouse monoclonal antibody binding (Appendix) (9,10). The fourth model was based on the 18 positions under positive selection (Appendix) (7). The fifth model was based on the 32 codons of substantial diversity (Appendix) (11). For evaluating the qualitative performance of the five prediction models, an antigenic variant was defined as antigenic distance >4 (21). Positive predictive value (PPV), negative predictive value (NPV), and agreement of the five prediction models were calculated, and different cutoff levels of amino acid differences were compared by using the receiver-operating characteristic analysis (22).

Figure A shows the scatterplot between antigenic distance and number of amino acid changes in the HA1 peptide (328 residues). Among the 181 pairwise comparisons, the antigenic distance ranged from 1 to 181, and the number of amino acid changes in the HA1 peptide ranged from 1 to 36. Overall, the antigenic distance correlated to the number of amino acid changes in the HA1 polypeptide (R = 0.74, p < 0.001). Different cutoffs of amino acid changes in the HA1 polypeptide were evaluated for predicting antigenic variants. The highest agreement was found with a cutoff of >7 amino acid changes, which shows that the NPV, PPV, and agreement were 66% (31/47), 81% (109/134), and 77% (140/181), respectively (Figure A).

Table 3 shows some unique pairwise comparisons with unusual patterns between antigenic distances and amino acid changes. A/Shanghai/11/87 and A/Victoria/7/87 were antigenically different (antigenic distance = 5.7), but they had only one amino acid difference (R247S). The position 247 is located at the antigenic site D. In addition to the amino acid change at position 247, A/Shanghai/11/87 had two more amino acid differences from A/Sichuan/2/87 (E156K, S186V) and A/Sydney/1/87 (A138S, N193K), but these three viruses were antigenically similar (antigenic distance <4). A/Victoria/7/87 had only two amino acid differences from A/Sichuan/2/87 (K156E, V186S) and A/Sydney/1/87 (S138A, K193N), but A/Victoria/7/87 was antigenically different from these two viruses (Table 3). The positions 156, 186, and 193 are located at the antigenic site B and the position 138 is located at the antigenic site A. Moreover, the positions 156 and 193 are also located at the mouse monoclonal antibody-binding sites (Table A1).

The unusual patterns between antigenic distances and amino acid differences may be due to interaction between amino acid changes in the hemagglutinin or laboratory variability, which needs further experiments to clarify. In addition, A/Victoria/3/75 and A/Victoria/112/76 had only two amino acid differences (L3F, R229G), but they were antigenically different (antigenic distance = 5.7) (Table 3), which also requires further experiments to clarify. The position 3 is not located at any antigenic site, and the position 229 is located at the antigenic site D. We found that 3 of 80 pairwise comparisons with >12 amino acid changes had antigenic distance <4 (Figure A).

A/Sydney/5/97 and A/Panama/2007/99 had 12 amino acid differences, but these two viruses were antigenically similar (antigenic distance = 1.4) based on ferret serum HAI titers (Table 3). However, inactivated vaccines containing A/Sydney/5/97 induced low serum antibody titers against A/Panama/2007/99 in humans; therefore, A/Sydney/5/97 was replaced by A/Panama/2007/99 as the vaccine strain for the 2000–01 season (3). A/HK/1550/2002 had 12 amino acid differences from A/Chile/6416/01 and 14 amino acid differences from A/Fujian/140/2000, but A/HK/1550/2002 was antigenically similar to these two viruses (Table 3). These three comparisons may indicate that interaction of multiple amino acid changes could potentially preserve the 3-D structure of HA1. Alternatively, the ferret serum HAI assay system is not sensitive enough to detect the antigenic difference.

Figure B shows the scatterplot between antigenic distance and number of amino acid changes in the five antigenic sites (131 amino acid positions). Among the 181 pairwise comparisons, amino acid changes in the five antigenic sites ranged from 1 to 32. Overall, the antigenic distance correlated to number of amino acid changes in the five antigenic sites (R = 0.77, p < 0.001). Different cutoffs of amino acid changes in the five antigenic sites were evaluated for predicting antigenic variants. The highest agreement was found by using a cutoff of >7 amino acid changes, which shows that the NPV was 71% (42/59), PPV was 89% (108/122), and agreement was 83% (150/181) (Figure B).

Figure C shows the scatter plot between antigenic distance and number of amino acid changes in the 20 amino acid positions related to mouse monoclonal antibody binding. Overall, the antigenic distance correlated to number of amino acid changes in the 20 amino acid positions (R = 0.74, p < 0.001). Different cutoffs of amino acid changes in the previously defined 20 amino acid positions were evaluated for predicting antigenic variants. The highest agreement was found by using a cutoff of >2 amino acid changes, which shows that the NPV was 64% (32/50), PPV was 82% (107/131), and agreement was 77% (139/181) (Figure C).

Figure D shows the scatterplot between antigenic distance and number of amino acid changes in the 18 amino acid positions under positive selection. Overall, the antigenic distance correlated moderately to number of amino acid changes in the 18 amino acid positions (R = 0.43, p < 0.001). Different cutoffs of amino acid changes in the 18 amino acid positions were evaluated for predicting antigenic variants. The highest agreement was found by using a cutoff of >1 amino acid changes, which shows that the NPV was 55% (6/11), PPV was 71% (120/170), and agreement was 70% (126/181) (Figure D).

Figure E shows the scatter plot between antigenic distance and number of amino acid changes in the 32 codons with substantial diversity. Overall, the antigenic distance correlated moderately to number of amino acid changes in the 32 codons (R = 0.68, p < 0.001). Different cutoffs of amino acid changes in the 32 codons were evaluated for predicting antigenic variants. The highest agreement was found by using a cutoff of >2 amino acid changes, which shows that the NPV was 72% (13/18), PPV was 74% (120/163), and agreement was 74% (133/181) (Figure E). Overall, the model based on the number of amino acid changes in the five antigenic sites has the highest correlation to the antigenic distance (R = 0.77) and the best performance for predicting antigenic variants (agreement = 83%).