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Annu, Rev, Immunol. 1990,8:737-71
STRUCTURAL BASIS OF
IMMUNE RECOGNITION OF
INFLUENZA VIRUS
HEMAGGLUTININ!
Ian A. Wilson
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037
Nancy J. Cox
Influenza Branch, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333
KEY WORDS: antigenic variation, influenza hemagglutinin, synthetic peptides, vaccines, immune response,
INTRODUCTION
Influenza is a highly contagious acute respiratory illness that appears to have caused serious disease in humans since ancient times. Many early accounts of epidemics of respiratory disease describe typical features of influenza including the clinical symptoms, short incubation period, high attack rates, and rapid progression of the disease through the population ( 1 ). Certain well-documented features of modern epidemics of influenza also emerge from these early accounts. Epidemics of varying severity occurred at regular intervals, caused the highest mortality in the elderly, and were thought to have first appeared in Asia. The quest for the causative agent of influenza was intensified after the devastating pandemic of "Spanish influenza" in 1 9 1 8 and 1 9 1 9, to which was attributed 20 to 40 million deaths ( 1). The first isolation from humans of influenza A virus occurred in 1933 and of influenza B in 1940. Although more than 50 years have passed, influenza viruses continue to cause considerable excess mortality
I The US Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.
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738 WILSON & COX
and widespread morbidity world-wide; they may be the most infectious disease agent that we cannot yet control. This is' the result of the ability of the virus to alter its antigenic properties in unpredictable ways and of our inability to formulate vaccines universally protective against all strains of influenza. To better understand why this virus has eluded our control efforts, it is necessary to understand the three-dimensional structure and viral function of the hemagglutinin (HA) (the viral surface glycoprotein primarily responsible for antigenic variation) and the immune response to this protein. The purpose of this article is to review the structural basis of immune recognition of the hemagglutinin (for previous reviews see 2, 3). Because more is understood about the structure and function of the influenza hemagglutinin than about any other viral membrane glycoprotein, it is appropriate that the HA be considered a model for examining other variable glycoprotein antigens such as the envelope glycoprotein (gp 120) of the human immunodeficiency or AIDS virus.
Influenza is an enveloped animal virus with five internal nonglycosylated proteins (nucleoprotein, matrix protein, and three polymerase proteins) and two integral membrane-surface antigens-the hemagglutinin (HA) and the neuraminidase (NA) (for review see 4). Influenza viruses are divided into three types-A, B, and C-based on antigenic differences in nucleoprotein and matrix proteins. Influenza A viruses are further divided into subtypes based on differences in their surface glycoproteins. Influenza virus nomenclature includes the type of influenza, the host of origin for nonhuman strains, the geographic origin, the strain number, and the year of isolation. An antigenic description of the HA and NA is given in parentheses after the year of isolation. So, for example, the A/Sichuan/2/87 (H3N2) virus was typical of viruses causing epidemic activity in the United States during the 1987-1988 influenza season. Thirteen subtypes of HA (HI -H I 3) and nine subtypes of NA (NI-N9) have been described. Viruses with HA of the H I, H2, and H3 subtypes and NA of the N I and N2 subtypes have caused epidemic activity in humans since 1 933. All subtypes of influenza A viruses have been isolated from birds, and a variety of subtypes have been isolated from swine, horses, seals, and whales. With this background, we now review the structure and function of the HA, natural variation of the HA, B-cell and T-cell responses to HA, viral neutralization, and immunity to influenza vaccines.
STRUCTURE AND FUNCTION OF THE
HEMAGGLUTININ
Three-Dimensional Structure
Influenza virus has its two surface antigens, the HA and NA glycoproteins, inserted in its viral membrane (Figure 1 ) . Some years ago the hem-
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INFLUENZA HEMAGGLUTININ 739
agglutinin was observed by electron microscopy to project from the viral membrane as spikes of approximately 140 A by 40 A, whereas the neuraminidase was more mushroom-shaped with an 85 A wide cap attached to a filamentous stem ( "-' 100 A) (5). Analysis showed the hemagglutinin to have a trimeric head structure whereas the neuraminidase was tetrameric (6). The number of surface antigens on the influenza virus has been difficult to ascertain exactly in part due to the pleomorphic nature of influenza particles. Estimates of 400-600 spikes on the more regular spheroidal viral particles have been reported (6, 7, 8) with the HA approximately five times more prevalent than the NA (9). The HA has been reported to be uniformly distributed on the virion whereas the NA may occur in discrete patches ( 1 0) . The intact hemagglutinin (HA) can be isolated from the virus by detergent solubilization, or as the extracellular fragment (BRA) by proteolytically cleaving it from the viral surface by bromelain digestion ( 1 1 ) . The HA is a homo trimer ( 12) and is synthesized as a single polypeptide (HAO) of around 550 amino acids which is subsequently cleaved into two polypeptide chains, HAl and HA2, by a host cell protease (Figure 2, 3). This cleavage activation is essential for activating the fusion properties of the virus. Both HAO and HA can bind to cells, but viruses with uncleaved HAO have no fusion activity and do not cause infection (Figure 1) .
221
CHO CHO CHO CHO
-1�6���� __ r-� __ � __ � __ � __ -;� __ � __________ ,
211
Signal Peptide
Uricharged M:�br.n�· Domain; c,}I:;'y-
S I S
Fusion Peptide
CHO
s-s 139 165 CHO
Figure 3 Schematic representation of the primary structure of the A/Hong Kong/68 hae
magglutinin. CHO indicates glycosylation sites and S-S disulphide bridges.
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740 WILSON & COX
To understand the structure and function of a viral antigen and its role in immune recognition and viral neutralization, the HA of the A/Hong Kong/1968 virus was crystallized (13) and its three-dimensional structure determined to 3 A resolution (2, 14). The trimeric HA is 224,640 daltons and has a large ectodomain of 503 residues, a short uncharged hydrophobic membrane-spanning domain (24-28 residues), and a small, internal hydrophilic domain (10-15 residues) (Figure 3). The HA has six disulfide bridges and seven N-linked glycosylation sites ( 19% total carbohydrate by weight). The x-ray structure showed the BHA to be 135 A long and 14-40 A in triangular cross-section ( 14). The HA is folded into two distinct domains, a globular head and a fibrous tail (Figures 4, 5). The globular head is entirely composed of HAl residues and is mainly f3-structure with an eightstranded Swiss-roll or jelly-roll type structure. Similar structures have been observed in the capsid proteins of plant and animal viruses such as tomato bushy stunt ( 15) and polio virus ( 16) as well as in tumor necrosis factor ( 17). This framework supports the receptor-binding site which is surrounded by highly variable antigenic loop structures (Figure 5). The fibrous stem region, more proximal to the viral membrane, consists of residues from both HAl and HA2. Three 76 A long helices form a triple coiled-coil structure which is tightly packed at its amino end ( 10 A helix to helix), the helices twist 100 A around each other in a left-handed superhelix. The helices finally splay out at the end closest to the membrane (22 A helix to helix) such that the helical contacts must be mediated through ions or solvent molecules ( 14; Figures 6, 7). Three short helices are packed antiparallel to the larger helices (HA2 38-56) and also twist around the larger triple helices (Figures 6, 7). The amino terminus of the HA2 is inserted between the long helices in such a way that the hydrophobic fusion peptide is buried in the trimeric structure (Figure 7). Similarly, a loop (HAl 27-33) is also inserted between the helices, although it does not make as close three-fold contacts as the fusion peptide (Figure 7). The C-terminus of HA is on the trimer surface and is some 20 A from the terminus of HA2, indicating that a significant rearrangement and conformational change must have occurred after the cleavage and removal of a single arginine residue of HAO. Parallel and antiparallel f3-structures are also present in this stem region (Figure 4). In addition, the location of the N-terminus of
Figure 4 Secondary structure of the A/Aichi/68 HA. The regular parallel and antiparallel p-structure (D) and helices (rods) with hydrogen bonds (::) are shown with the 7 glycosylation sites (*) as from analysis of the coordinates of Wilson et al (14). Recent HA refinements should be consulted for current coordinates (30, 107). HA I is numbered from 1-328 and HA2 from 1001-1 1 75.
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742 WILSON & COX
HAl indicates that the signal peptide may have remained attached to the HA during its biosynthesis and folding, before being removed ( 14). The trimeric structure is principally stabilized by the fibrous stem region with a rather loose association of the globular heads. The relative stability of the HA was calculated ( 1 8) and indicates that the tails contribute'" 28.4 kcal mol-I, and the heads only 5.3 kcal mol-I, to the stability of the intact trimer (46.7 kcal mol-I).
A comparison of the anatomy of the two influenza virus surface antigens is shown in Figure 5. The NA is composed of a tetrameric globular head of mainly [3-structure containing the sialidase catalytic site which is also surrounded by hypervariable loops ( 1 9, 20, 2 1 ). In this very general sense the neuraminidase (NA) has an anatomy similar to that of the HA globular heads. However, although the HA and NA both bind sialic acid (SA), there is no other obvious resemblance in their detailed structures. In addition, the HA has a substantial fibrous stem domain containing the fusion peptide, whereas the NA has a long filamentous tail.
Receptor Binding
The hemagglutinin receptor binding site is located in a shallow, concave pocket at the distal end of the molecule (Figure 8) and contains residues from throughout the HAl polypeptide chain, including Tyr 98, Trp 1 53, His 1 83, GIn 1 90, Leu 1 94, and Tyr 1 95 that are highly conserved in both A and B strains of influenza virus ( 14). Human and animal isolates of A viruses bind preferentially to sialated oligo saccharides containing SA a2-6Gal or SA a2-3Gal linkages (22, 23). Virus specificity for these different sialylated receptors was identified by selecting variants of H3 strains with different binding specificities. H3 strains specific for a2-6 linkages had Leu at HAl 226 whereas variants with a2-3 specificity differed only by a substitution of GIn at 226 (24). Variants of an avian isolate A/Duck/ Ukraine/63 were selected where the specificity of the parent virus was reversed to oc2-6 with GIn at 226 from oc2-3 with Leu 226 (25). A detailed analysis of the AjUSSR/90/77 strain by site-directed mutagenesis of HAl GIn 226 showed that two mutants, Asn and Met, retained their receptor binding activity whereas several others, including Glu, Leu, Val, and Thr, lost activity (26). Residues on the periphery of the binding site can also affect receptor binding either positively or negatively (27), although surprisingly the deletion of several residues of HAl 224-230 constituting one side of the binding pocket permits virus viability but with altered binding character (28).
Two HAs of different specificity that differ only in position 226-wild type Leu and mutant Gln�have been crystallized and their structures determined in the presence of either oc2-6 or oc2-3 sialyl lactose (29, 30). Only
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INFLUENZA HEMAGGLUTININ 743
sialic acid was visible in the electron density in the binding site, and hence sialic acid alone is identified as the viral receptor (29). The interaction of the sialic acid moiety with these two HAs is generally similar, and as yet no structural explanations exist for the preference of a2-3 versus a2-6 linkages (29, 30a). From these x-ray and NMR studies, rationale design of receptor binding sialic acid analogues is now possible and could provide valuable alternatives to conventional vaccines (29, 30a).
Why a humoral immune response is not directed at the receptor binding site, given its relative accessibility may be partly explained by the binding site being a concave although shallow depression masked by highly immunogenic and antigenic loops which surround the pocket. Nevertheless, antibodies can have ridges which protrude into antigens, as in the HyHEL-1 0 lysozyme-Fab antibody-antigen complex (3 1) .
Membrane Fusion
Viral entry and membrane fusion is also mediated through the hem-. agglutinin. Cleavage maturation of the HA is necessary for infectious virus (Figures 1 , 2) (32, 33). Removal of a single arginine residue by host cell proteases generates a hydrophobic peptide sequence, analogous to the Sendai virus fusion peptide, at the amino terminus of HA2 (Figure 3; 34, 35). In contrast to the single arginine linking HA 1 and HA2 of all known mammalian and apathogenic avian viruses, pathogenic avian strains have several basic amino acids at their cleavage sites (36-38). Glycosylation in the vicinity of these cleavage sites can interfere with normal cleavage and viral pathogenicity (39). The HA2 fusion peptide is buried in the trimer interface such that Leu 1 and Phe 2 of HA2 make contacts around the three-fold of the trimer by inserting between the 76 A long triple helix (Figures 5, 7). The location of the fusion peptide strongly suggests a significant local change in the HA structure after proteolytic HAO cleavage (14).
Unlike Sendai virus, influenza virus does not fuse membranes at neutral pH but rather at a more acid pH. Influenza entry into cells by an endocytotic pathway results in the fusion of the viral membrane with the host cell endosomal membrane in its low pH environment (40-42a). Fusion of the influenza virus occurs over a narrow range of pH, generally around pH 5.0-5.5 (reviewed 41) where fusion with liposomes, lysing of erythrocytes, and cell fusion in culture can occur (43; see also 44). A change in the HA conformation at low pH provided direct evidence for its involvement in the membrane fusion mechanism (45). Between pH 5.0 and 5.5 an irreversible change occurs such that the hydrophobic amino terminus ofHA2 becomes accessible and is released from the trimer interface (Figure 9; 4 1 , 45, 46). Viruses selected in amantadine hydrochloridl�
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744 WILSON & COX
treated cells, in which the endosomal pH is raised, have altered pH dependence for hemolysis and for the pH-triggered conformational change (47). The selected variants have amino acid differences in the vicinity of the fusion peptide as well as in the trimer interface not only of the fibrous stem but also of the globular heads. More detailed evidence for HA structural changes is indicated by altered antigenicity of the pH-5 conformation in antigenic sites B and D which are close to the trimer interface (47-50). Changes in the HA structure are also observed by electron microscopy where the HA shows a marked elongation and thinning in the stem region at pH 5.0 with the globular heads coming apart (5 1 , 52). After low pH treatment, the HA is sensitive to trypsin and can be cleaved at Lys 27 of HAl into soluble "tops" consisting of HAl 28-328 and an insoluble tail made up of HA2 and HAl 1-27 (45). Proteolytic removal of the amino terminal HA2 1-22 by thermo lysin resolubilizes the tails (52). Some perturbation in the triple helices has been observed at pH 5 (53), but little change in overall secondary structure has been noted for either the heads or tails. The use of antipeptide antibodies (54) at low pH, provided evidence for the disassociation of the globular heads and for conformational changes in the stem region, including the exposure of the fusion peptide and a loop peptide which contains the low pH susceptible trypsin cleavage site, Lys 27 (Figure 9). Exposure of both the fusion and loop pep tides significantly alters the HA trimeric association due to their close interactions with the triple helices around the trimer three-fold axis (Figures 7, 9). Additional evidence for conformational change in the stem region has been provided for other HA mutants (55).
How the pH-triggered conformational change in the HA is related to membrane fusion is less clear. The fusion peptide is close to its own membrane (�35 A), and extrusion at low pH would still leave it some distance from the host cell membrane (Figure 9). Conformational changes such as those proposed for the opening of the trimeric globular heads (45, 49, 52,54) would presumably reduce this distance, especially if the globular heads were clipped off in the endosome by pro to lytic cleavage. The role of the hydrophobic fusion peptide in membrane fusion has been demonstrated by selecting fusion mutants (47) or by site-directed mutagenesis (56). In addition, synthetic peptide analogues of the fusion peptide have been shown to promote membrane fusion, with peptide helicity correlating somewhat with fusogenicity (57-59). As many as 2 1 amino acids of the HA2 amiho terminal sequence have been proposed to interact with lipid membranes in an amphipathic helical manner (60). Hence, even with our considerable knowledge of the pH activation of the virus and associated HA conformational changes, major questions still remain as to the actual mechanism of membrane fusion. Whether the HA fusion peptide
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INFLUENZA HEMAGGLUTININ 745
inserts its own membrane into the endosomal membrane, or causes aggregation of HAs on the viral surface perhaps to expose free lipid patches remains unresolved.
NATURAL VARIATION
Gene and Amino Acid
One of the hallmarks of influenza virus is its ability to undergo unpredictable and rapid antigenic variation. This variation causes periodic world-wide pandemics as well as almost annual epidemics (Figure 10). Although antigenic variation occurs for several proteins of influenza virus (for review see 61, 62), only variation of the HA is considered here. Antigenic variation in HA involves two separate processes, antigenic shift and antigenic drift. Antigenic shift occurs only for type-A influenza viruses, when a virus HA of a novel subtype is newly introduced into the human population. The amino acid homologies between the HAl domains of different subtypes that infect humans range from 35 to 60%, with the least variation occurring between HAs of the Hl and H2 subtypes. Shifts may
8�----------------------------------------------�
6
o
-1�----------------------------------------------� 88 89 70 71 72 73 74 75 78 77 78 79 80 81 8283 84 86 88 87 88 89
Year Figure 10 Estimated excess mortality due to influenza in 12 1 US cities from 1968 to 1 989. Strains predominating in pandemic (A/Hong Kong/68) or epidemic years are indicated. Both A type (HI and H3 subtypes) and B type influenza are presently circulating.
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746 WILSON & COX
occur through the process of genetic reassortment. For example, considerable evidence suggests that the Hong Kong influenza epidemic of 1968 was caused by a virus that was a reassortant derived during mixed infection with a human H2N2 virus and an avian or Equine H3 virus (63�65). Another way viruses bearing HAs of a new subtype for humans might emerge is through mutation of an animal or avian virus, causing it to become infectious for humans without reassortment. Viruses that caused epidemics in humans previously can reemerge after remaining relatively unchanged for decades. The reemergence of influenza A (HINl ) viruses in 1 977 is an example. This virus was almost identical to a virus that had caused influenza epidemics in 1 950 (66, 67); how it remained in a genetically "frozen" state for 27 years is not understood.
Antigenic drift occurs in influenza types A, B, and C. Information about the mechanism of antigenic drift has come primarily from sequence analysis of naturally occurring, antigenically drifted field isolates and of laboratory variants selected in the presence of monoclonal antibodies. Considerable data now exist for human field isolates of influenza A (68� 78, N. J. Cox, unpublished data), influenza B (79�8 1) , influenza C (82), and for binding of monoclonal antibodies to variants of influenza A and B (83-89). The rapid rate of variation of the HA is due, in part, to the fact that influenza virus genes, like the genes of other RNA viruses (and perhaps DNA viruses) evolve more rapidly than the DNA genomes of their hosts (82, 90-92). Where detailed comparisons have been made, it has been found that the rate of silent nucleotide substitution was higher than the rate of coding nucleotide substitutions for all genes of influenza virus including the HA (80). However, the elevated rate of coding nucleotide substitution in the HA gene compared to other genes has been taken as evidence that immune selection is an important factor in its evolution (62).
For influenza A viruses of the H3 and HI subtypes isolated during their current eras of circulation, amino acid changes have occurred in the HA I domain at a rate of approximately 0.8% and 1 .0%, per year, respectively. Nucleotide substitutions have occurred at a rate of approximately 4-5 x 1 0-3 nucleotide substitutions per site per year. The hemagglutinin of type-B influenza viruses reportedly varies at a slower rate and is characterized by cocirculating lineages (80). However, for HAl domains of type-B viruses isolated between 1 979 and 1 988 (8 1 ), the rate of change is approximately 0.5% amino acid change per year and 4 x 1 0-3 nucleotide substitutions per site per year, rates similar to those for the HAl domain of type-A influenza. It is now clear for both type-A and type-B influenza that the HAs of successive epidemic strains do not always evolve from the HAs of the previous epidemic strain, especially for influenza B (71 , 78, 80, 8 1) . While the molecular epidemiology and evolution of influenza C are
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INFLUENZA HEMAGGLUTININ 747
less well characterized, natural variation of the HA gene of influenza C occurs more slowly, and distinct strains cocirculate (82). The observed differences may be due to the fact that influenza causes primarily mild respiratory infections in the young and the elderly, and thus influenza C may escape the immune selective pressure exerted on influenza A and B viruses.
Antigenic drift may be imitated in the laboratory by growing influenza viruses in the presence of monoclonal antibodies to HA. Mutants that do not bind to the antibody used for selection occur at a frequency of between 1 0-4 and 1 0-5 (93). The majority of such variants have single amino acid substitutions in the HAl polypeptide chain. That the single amino acid substitution is part of the antibody binding site has been directly demonstrated for both HA and neuraminidase by observations that changes in the crystal structure of two such mutants were confined solely to the region of substitution (3, 94, 94a).
Serologic studies of influenza viruses from animals and birds have shown that variation of the hemagglutinin is less extensive than in human viruses. Sequencing studies of equine viruses of the H3 subtype have shown that the HA of these viruses evolved in a pattern very similar to that for human viruses of the H3 subtype, albeit at a slower rate of approximately 0.3% amino acid change per year (65, 74). The amino acid substitutions occurred at positions corresponding to antigenic sites on the HA of human H3 viruses, suggesting that immunological selection by the host may be involved in the selection of new variants. In contrast, the HA genes of the H3 subtype isolated from ducks are conserved antigenically and genetically with a markedly lower rate of silent and coding nucleotide substitution than for human H3 strains, with amino acid substitutions occurring outside recognized antibody binding sites (95). It has been suggested that immune selection is not a factor in evolution of the HA during virus replication in ducks because their antibody response is weak and shortlived (96).
Amino acid substitutions occur throughout the HA sequence with only a few stretches of conserved amino acids (71 ). In A/Hong Kong/68 HA the variation occurs predominantly in the HAl domain of the HA (83). In fact, much of the surface of the distal domain of the H3 molecule has been altered by amino acid substitutions during the 20-year circulation of the H3 subtype in humans (Figure 1 1); this is also true for the HI subtype over the past ten years (Figure 1 2). These facts illustrate the plasticity and evolutionary potential of the HA that makes it so difficult to predict the molecular nature of future epidemic strains. In the 34 sequences of strains from 1968-1987, a total of 76 different amino acids have changed in the HA 1. If one considers only epidemic years (Figure 1 0), 50 different amino
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acid substitutions are located on HAl in nine reference strains from 1 968-1 987. For 24 sequences of HI strains 1 977-1 988, a total of 45 residues have changed in HAL Amino acid residues have changed more than once at positions 2, 137, 138, 144, 145, 155, 1 56, 159, 1 60, 1 86, 1 88, 1 89, 1 93, 1 98, 244, 246, and 248 for viruses of the H3 subtype isolated between 1 968 and 1988. For viruses of the HI subtype isolated between 1 977 and 1988, similar sequential changes have occurred at amino acids 31, 1 63, 1 89, 1 90, 1 97, and 225.
A number of these amino acids are located in the vicinity of the receptor binding pocket and have been associated with heterogeneity observed in egg-grown influenza viruses (97-101) . The H3 substitutions have been categorized into five antibody combining sites designated A to E (83), though it is clear that some subdivision and overlap of these areas occur (Figures 13, 14). Each new drift variant of epidemiologic importance has generally had four or more amino acid substitutions located in two or more of the antigenic sites. For example, the influenza A (HIN1) epidemic strain A/Chile/83 had six amino acid changes, located in sites B, D, and E, when compared with the previous HI epidemic strain A/England/80 (Figure 12). The influenza A (H3N2) epidemic strain A/Sichuan/87 had seven amino acid changes in sites A, B and E when compared with the previous H3 epidemic strain A/Mississippi/85 and nine amino acid changes located in sites A, B, and E when compared with the vaccine strain A/ Leningrad/86 (Figure 1 5) .
As one would expect, the sequence differences found between field isolates of HA are located primarily at the surface of the molecule, where they are exposed to both solvent and antibodies. Surface accessibility calculations performed using the program ACCESS ( 1 02) demonstrate that the mutated sequence positions seen in H3 are 80Cl'0 more accessible to solvent than is the average residue in the 1968 Hong Kong x-ray structure, while those mutations in HI are only 5 1 % more accessible when mapped onto the Hong Kong trimer structure (Table 1). A total of 27% of the surface of the H3 Hong Kong 1 968 HA has changed within H3 strains up to 1987, while 13% of accessible area of the HI HA has changed from 1977-1988. Analysis of both H I and H3 sequences isolated between influenza epidemic years indicate that on average seventeen unique mutations have occurred which are associated with 3100 A2 of solvent accessible area that is being changed in the trimer or approximately 1000 A per monomer. A similar analysis of hen eggwhite lysozyme residues found in contact with monoclonal antibodies D 1 .3 ( 1 03), HyHEL5 ( 104), and HyHELlO (31 a) ( 16, 13, and 13 contact residues, respectively) reveals 1050, 1090, and 980 A2, respectively. One can therefon� approximate that on average one antibody epitope of hemagglutinin is completely changed
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Table 1 Predicted changes in the HA solvent accessible surface of H3 strains from 1969-1988 and HI strains from 1977-1988 based on the Hong Kong/68 structure (14)
Solvent accessible Percentage Number Solvent area per buried in
of unique accessible mutated residue trimer changesa area (A **2)b (A**2) interfaceC
H3 1968-72 30 (10) 6670 ( 1880) 74 (63) 6. 1 (17.0) 1972-73 12 (6) 3680 (1540) 102 (86) 4.3 (9.7) 1973--75 14 (11) 2850 (2290) 68 (69) 8.9 (10.7) 1975-77 17 (16) 2670 (2480) 52 (52) 13.7 '(14.7) 1977-79 5 (5) 1 400 ( 1400) 93 (93) 0.0 (0.0) 1979-82 19 (8) 2630 (420) 46 (17) 1.5 (7.5) 1982-85 24 (9) 3500 ( 1230) 49 (45) 14.9 (0.0) 1985--87 27 (8) 4520 (1700) 56 (71) 12.0 (0.0)
1968-87 76 (50) , 14,400 (9310) 63 (62) 8,4 (8.8)
HI 1977-78 1 0 1240 41 16.4 1978-80 11 1 940 59 15.9
1980--83 1 8 2820 52 12.1
1983-86 17 2930 57 9.1 1986-88 21 3650 58 10.4
1977-88 45 7120 53 1 4.0
X31/68 53,11 0 3 5 23.2
a Unique changes were determined by the number of positions identified as containing mutations in HA 1 for sequences between the epidemic years indicated.
• Solvent accessible area was calculated (see ref. 102) by rolling a sphere of 1.7 A radius over the trimeric x-ray structure and summing up the contribution from each of the mutation positions.
C The area buried in the trimer interface was calculated as the difference in accessibility of the changed residues in the trimer versus the three separated monomers.
d The number of changed sequence positions found in the HAt's of all sequenced strains between the years indicated are tabulated. Numbers in parenthesis are for differences between only the H3 epidemic sequences Hong Kong/68 (X31/68), ENG/42/72, PC/I/73, VIC/3j75, TEX/I/77, BK/I/79, PHIL/2/82, MISS/I/85C & SC/87.
between epidemic years, or alternatively 20% of each of the five major epitopes. Significantly, epidemic strains which show the largest changes in solvent accessible area from the preceding epidemic strain (see Figure 1 0) were responsible for high influenza mortality rates, independent of the number of actual mutations. Indeed, in cases where the number of sequence mutations in H3 was low, namely 1972-73 and 1977-79, the per residue solvent accessibilities were the highest, indicating that the necessary surface modifications to escape neutralization were done in an economical manner.
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The analysis of surface accessibility of mutant positions illustrates an interesting difference between the natural variants of H I and H3-namely the greater tendency for HI mutations to occur in the trimer interface region. In the Hong Kong x-ray structure, 23% of each monomer's solvent accessible area is buried upon trimer formation. For sequences investigated here, HI variations occurred 14% of the time in regions buried in trimer contacts, while only 8 .4% of H3 replacements were lo,;;ated there. One possible effect of mutations located at the boundary of adjacent monomers is the slight rearrangement of the trimer packing which in turn could be responsible for the generation of a different molecular recognition surface for epitopes located between separate chains. Clearly until one has an xray structure of an actual HI HA, one is limited to a comparison of these mutations on the H3 structure. Nevertheless, no gross changes in overall three-dimensional H I structure are expected from the H3 HA structure.
In spite of the extent of the variation, conservation of certain amino acid residues in the HAl domain has occurred. In the globular head domain, the receptor binding pocket, including residues Tyr 98, Trp 1 53, Glu 190, Leu 194, and His 183, has not changed during drift of the H3 subtype ( 14, 29; Figures 8, 13, 16). A second shell of conserved residues behind the pocket (Cys 97, Pro 99, Cys 139, Phe 147, Tyr 195, Arg 229) seems to stabilize the architecture of the binding site (29) in both H3 and HI subtypes. Another area of largely conserved residues in either the HI or the H3 subtypes lies between amino acids 280 and 328. These amino acids form part of the stalk structure that supports the globular region of HAL A study of the natural variation of influenza B HA from 1940-1987 also shows that the variation occurs in the globular head region in generally the same areas as for influenza A HA although with substantial insertions and deletions of residues compared to influenza A HA (Figure 17; 79-81).
A second type of selective pressure affecting variation of the HA is exerted by the host cells in which influenza viruses are grown (97). For example, antigenically distinguishable viruses can be isolated from mammalian and avian cells infected with a single clinical specimen of influenza A (HI or H3 subtype) or influenza B. This variability may arise as a result of amino acid changes affecting receptor binding in the two host systems (98-100). Initial studies using multiple isolates from a single clinical specimen suggested that isolates obtained in mammalian cells are more homogeneous and may be more representative of the population of virus that replicates in humans than are isolates obtained in eggs (98-100). However, in a recent study with a larger number of clinical specimens, it was shown that the most common subpopulation of egg-grown virus has the same antigenic properties as mammalian cell grown virus (101). These findings should diminish concern that viruses isolated in eggs are not good candidates for the majority virus chosen as a vaccine candidate.
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Glycosylation
INFLUENZA HEMAGGLUTININ 751
Glycosylation of viral antigens is also important in antigenic variation by masking or unmasking of antigenic sites (2, 3, 49, 75, 83, 86, 1 05, 1 10). Possibly, carbohydrate may also stabilize the trimeric structure or mask potential sites of proteolytic cleavage (2, 14, 105). However, the location and number of glycosy1ation sites are not conserved among HAs of different strains and subtypes ( 105, 1 06). In all currently available influenza A HAl sequences, some 27 different residues (26 HAl , l HA2) have potential N-linked glycosylation Asn-X-Ser/Thr sequences, although not all of them may be utilized (Figure I S). These sites are scattered throughout the HA, but tend to cluster around the antigenic sites on the globular heads (Figure IS). From 5 to I I sites are present in the HAs of individual strains, with the glycosylation sequence around residue 20-22 absolutely conserved in all A strains sequenced to date.
In the Hong Kong 1965 HA structure approximately 17-20% of the total protein surface could be covered by carbohydrate. The carbohydrate was difficult to interpret in the electron density map, presumably due to positional disorder or sequence heterogeneity ( 1 4, 1 05). In the refined structure, only 1 2 of around 67 potential sugars have been placed ( 1 07). Carbohydrate primary structure analyses show considerable sequence heterogeneity for the A/Hong Kong/68 strain ( 1 0S) and for the A/Leningrad/3S5/S0 strain ( 109) and depend not only on the virus strain but also on the host cell ( 1 09, 1 1 1 , 1 1 2). Glycosylation can also block the cleavage ofHAO into HAl and HA2 for avian viruses and consequently can regulate virulence (113, 114). Addition of novel carbohydrate sites by site-directed mutagenesis has been shown to affect the biosynthesis and activity of the HA as well as to mask its antigenic sites ( 1 1 5; Figure I S).
Clear evidence for the importance of carbohydrates in modulating antigenicity was provided by selection of a mutant HA (Asp- > Asn 63) in which a new glycosylation site prevented antibody binding and viral neutralization, whereas the mutant virus grown in the presence oftunicamycin was antigenically indistinguishable from the wild type X: 3 1 (Aichi/68) virus ( 1 10). A similar result was obtained when both A/Eng/878/69 and A/Vic/3j75 viruses which had each acquired an additional glycosylation site at residue 63 were grown in the presence of tunicamycin (3, 1 10).
HUMORAL RESPONSE TO HA
Antiviral and Anti-HA
Although it is generally accepted that local humoral immunity in the respiratory tract plays an important role in preventing respiratory viral infections, characterization of the B-cell response to influenza has been
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limited primarily to the measurement of antibody in serum and respiratory secretions. Thus, while there are significant gaps in our knowledge of the overall B-cell response, considerable knowledge does exist concerning the humoral response to infection by influenza viruses (for review see 116). The role of serum antibody to HA in protection against infection has been demonstrated both by the longstanding observation that resistance to infection is correlated with serum anti-HA antibody levels ( 1 1 7- 1 1 9) and by the demonstration of protection against homotypic challenge after passive transfer of immune serum in a mouse model ( 120, 1 2 1 ), More recent studies have shown that protection from live virus challenge is associated with local neutralizing antibody and secretory IgA as well as serum anti-HA antibody ( 1 22, 1 23). The relative roles of local and serum antibodies in preventing influenza are not known, however. Because of the difficulty in obtaining specimens, little is known about humoral immunity in the lower respiratory tract of humans. In one study a low dose of live attenuated H I Nt virus vaccine was used to challenge a small number of adults. An IgG response was detected most frequently in bronchoalveolar lavage fluids of individuals with preexisting serum antibody (124).
The serum antibody response to HA is subtype specific. Epidemiologic studies in humans have shown that infection by one subtype confers little or no protective immunity to other subtypes, and sera from humans or experimentally infected animals do not cross-react with viruses of different subtypes ( 125). This is also likely to be true for secretory antibody. After infection, individual sera contain antibodies to determinants on the HA of the infecting strain that are strain-specific as well as cross-reacting antibodies to determinants shared by variants of that subtype. Passive transfer of antibody in the mouse model has demonstrated that heterotypic serum is less protective than homotypic serum ( 120, 1 2 1 ). In vitro the cross-reactive antibody is less effective than strain-specific antibody in neutralizing virus ( 126). In human immune sera that have been virusabsorbed the proportions of strain-specific and cross-reactive antibodies depend on the individual's previous experience with influenza infection. In sera from unprimed naturally infected children, the predominant antibodies are strain-specific with only a small amount of cross-reactive antibody present ( 127). In adults who had previously been exposed to an earlier variant, the predominant antibodies after infection were crossreacting and strain-specific for the previous variant (128). These studies demonstrate that antibodies induced by infection with influenza are primarily directed toward those determinants shared between viruses. This is the phenomenon of "original antigenic sin" ( 1 29), a selective anamnestic response during infection by influenza such that the immunologic response is oriented toward the antigens experienced during the original infection. This probably occurs because multiple antibody combining sites exist, and
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INFLUENZA HEMAGGLUTININ 753
sequential subtype variants arising during antigenic drift have amino acid changes in only one or two of them (see section on Natural Variation). That the specificity of the antibody response to HA in humans is limited and varies considerably from individual to individual (130-132), may explain the frequent emergence of antigenic variants that can infect a majority of the population. Such variants could arise in individuals who become only partially immune (i.e. do not develop antibody to all antibody binding sites) and are therefore susceptible to an antigenic mutant changed in only one or two sites. Variation in the range of specificities of anti-HA antibodies in individual mice has also been demonstrated ( 133, 134).
The most detailed studies to determine the dynamics of class-specific antibody response to HA in serum and nasal secretion have been reported by Murphy and his colleagues, who used live attenuated vaccines. Serum antibody responses typical for primary viral infections were detected in antibody-free children using the ELISA technique (135). IgM, IgA, and IgG antibodies appeared in the serum within two weeks after inoculation of virus, although IgA responses occurred less frequently and to lower titers. The maximum serum IgG response was detected at approximately six weeks, while IgM and IgA antibody levels declined after two weeks. In nasal secretions, IgA was the predominant antibody and was present in the majority of individuals within two weeks of inoculation. IgG and IgM responses in nasal secretions occurred less frequently and to lower titers. Young adults primed by natural infection and challenged with live attenuated vaccine were examined in another study ( 136). Most individuals mounted serum IgG and IgA responses, with a correlation between serum and secretory IgA; however, IgM responses were rare.
Serum antibody to HA can persist for decades, and retrospective serosurveys suggest that a limited number of influenza A subtypes recycle (137). That immunity can also persist for decades was dramatically shown when influenza A (HINI) strains, similar to viruses that circulated previously in 1950, spread throughout the world in 1977 to 1978. Few infections occurred in individuals born during or before 1950, demonstrating that substantial immunity remained after almost 30 years. The duration of homologous immunity has been examined for individuals with documented infection with A/Hong Kong/68 (H3N2) virus; resistance to infection was found to last at least four years (138). Other studies have demonstrated that immunity to influenza A (H3N2) in adults extended from four to seven years and included two or more variants of the H3N2 subtype (118).
Monoclonal Antibodies
Immunoglobulins IgG, IgA, secretory (s) IgA, and IgM can neutralize virus, and it is likely that each contributes to protection (118). Not all
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antibodies that bind to HA neutralize the virus ( l39), and it is clear that neutralizing antibodies bind to specific regions. The probable locations of these specific sites for both the HINI and H3N2 subtypes of influenza A have been identified by determining the sites of single amino acid changes in mutants selected in the presence of monoclonal antibodies (Figure 19; 83, 86), along with the locations of sequence variation in the HAs of field strains of influenza (see section on Natural Variation; Figures 1 1 -17). The natural variant substitutions are in five somewhat overlapping regions (designated A to E for the H3 strains (2, 3, 83) and Cal , Ca2, Cb, Sa, and Sb for the HI strains (86)). It is currently believed that each of these regions is an antibody binding area against which neutralizing antibodies are produced during virus infection. The variants selected by monoclonal antibodies have amino acid changes which are also located on the globular heads of the HA and generally cluster around the conserved receptor binding site (83) for both HI and H3 strains (Figure 19; 83, 86). It might be expected that neutralizing antibody would prevent entry of influenza virus into susceptible cells by blocking virus binding to host cell receptors, as has been shown with reovirus ( 140). However, Dimmock and his colleagues have shown that neutralization of influenza virus by polyclonal or monoclonal IgG did not inhibit attachment, penetration or uncoating and transport of the viral genome to the nucleus, but it did inhibit primary transcription ( 14 1 , 142). On the other hand, neutralizing IgM and IgA prevented attachment of up to half of the virus and rendered the other half unable to be internalized .( 143, 144). A quantitative study of the interaction of influenza virus with neutralizing antibody has suggested that only a small proportion of the HA spikes are responsible for neutralization ( 145).
Antipeptide Antibodies
To investigate immune recognition of influenza virus further, synthetic peptides of the hemagglutinin have been used extensively as immunogens to produce HA reactive antibodies. Green et al ( 146) first showed that a battery of antipeptide antibodies raised against 20 different peptides comprising almost the entire sequence of the AjVicj3j75 HAl could react with HA or virus in contrast to anti-HA or antiviral antibodies whose reactivity is generally confined to the hypervariable regions of the HAl globular heads. The opportunity of raising antibodies against areas of the HA which are not normally immunogenic when either the HA or intact virus is used as the immunogen raised possibilities of using synthetic peptides as vaccines ( 147, 148). However, it appears that antipeptide antibodies against influenza are not particularly effective for viral neutralization, although in one case, marginally lower virus titers and partial
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INFLUENZA HEMAGGLUTININ 755
protection were shown in mice immunized with a peptide corresponding to HAl 138-1 64 (site B) ( 149). Antipeptide antibodies that specifically react with the major antigenic sites have been difficult to obtain particularly against the "loop" peptide ( 140- 146) of site A ( 146, 1 49, 1 50, 1 5 1 ). Nevertheless, cyclization of the loop peptide was effective in inducing antibodies that were reactive with virus, possibly by stabilizing the peptide loop conformation. However, these antibodies did not neutralize ( 152). Several antipeptide antibodies have now been shown to bind better to the pH-5 conformation than to the neutral HA structure (54, 1 53), presumably because the pH-triggered conformational change exposes previously buried peptide sequences. For example, the C-terminus of HAl (305-328) elicits antibodies that react well with the peptide, HA, and intact virus at both neutral and low pH, with significantly enhanced reactivity following pH-5 treatment of the HA (54, 1 46, 1 53). Extensive mapping of the determinants of this peptide has shown 3 1 4-318 LKLAT to be the immunodominant epitope for monoclonal antipeptide antibodies, whereas another site 320-328 was recognized by both polyclonal sera and monoclonal antibodies ( 154). Two of these monoclonal antibodies recognize different epitopes that are separated only by three residues, and both can simultaneously bind to the 24 amino acid peptide ( 1 55). The fine specificity of the T- and B-cell immune response to this 24-mer peptide in Balb/c mice has been reported ( 1 56).
Antipeptide antibodies against another peptide HAl 75-1 10 of the A/Vic/3/75 strain have been extensively studied. A majority of a panel of 2 1 monoclonal antibodies ( 1 57) reacted with a single immunodominant determinant corresponding to residues 98- 106 ( 1 58). The fine specificity of the determinant defined by antibody reaction with a myriad of synthetic peptides ( 1 58-1 6 1 ) was shown to be residues 10 1- 106 (DVPDYA Ka '" 10-8 M). This determinant lies in the trimer interface ( 158), consistent with antibody reactivity only to the monomeric tops (Ka '" 10-6 M) but not to the trimeric structure (54, 1 59, 162). However, antipeptide antibody studies have shown that this determinant becomes exposed in the pH-5 treated BRA, which is consistent with the trim eric heads coming apart in its proposed fusion reactive conformation (54). Why this peptide is so immunogenic stimulated NMR studies of its conformation in solution. The free peptide HA 1 (98- 106) has a surprisingly high percentage of type-II f3-turn structure in water ( 1 63, 1 64). Further observations showing that immunologically reactive pep tides have conformational preferences in aqueous solution have led to suggestions that such peptides may be more likely than others to induce protein reactive antibodies ( 1 64). It is also interesting to note that the precise determinant specified by immunological mapping is entirely accessible on the surface on HAl
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monomer but has a {3-turn at the end opposite to that shown by the peptide in solution ( 1 58; Figure 20). Two different Fabs ( 1 7/9 and 26/9) and Fabs complexed with pep tides containing the HAl 10 1-106 epitope have been crystallized ( 165; E. A. Stura, I. A. Wilson, unpublished). The structure of one free Fab 1 7/9 has been solved by x-ray diffraction to 2.3 A resolution (1. Rini, 1. A. Wilson, unpublished) and the structure of two complexes to 3 A resolution (D. Schulze-Gahmen, 1. A. Wilson, unpublished; E. A. Stura, I. A. Wilson, unpublished). A peptide containing this determinant, 9 1- 108, has been found to elicit an anti-influenza immum; response ( 166).
Antipeptide antibodies can then be markedly different from either antiprotein or antiviral antibodies. Antipeptide antibodies can be generated against parts of the structure not normally antigenic when the intact protein is used as the immunogen. Several of these antibodies react better with the pH-5 conformation and to parts of the molecule conserved in sequence as is, for example, the entire HAl 98-1 1 0 sequence in H3 strains. If such antibodies could be targetted against a conformational form that appears during either receptor binding, or fusion, then possibly one could produce antibodies that would react with functionally conserved regions and lead to a more general vaccine. We still do not understand in structural terms how anti peptide antibody can recognize a peptide antigen as well as its cognate sequence in the intact antigen. An answer to this fascinating but as yet unanswered question (see Figure 20) should be available soon ( 165, 165a; R. Stanfield, I. A. Wilson, unpublished observations).
CELLULAR RESPONSE TO HA
The role of T lymphocytes in influenza virus infection has only recently been examined in detail because of the technical difficulty in studying T lymphocyte responses as compared to studying antibody response, and because antibodies had been shown to neutralize influenza infection. During the past five years a great deal has been learned about T-cell function, in general, and T-cell responses to influenza, in particular. It is now clear the T lymphocyte response can occur after stimulation by any of the influenza structural proteins. However, we confine our remarks here to T-cell response to the hemagglutinin.
T Helper (Th) cells
Th cells recognize foreign antigens on the surface of antigen presenting cells only when the foreign processed antigen is present on the cell surface in association with HLA class-II la-region major histocompatibility complex gene products ( 167). In humans, the HLA-DR molecules are involved ( 168). Studies with proteins such as lysozyme ( 169), myoglobin ( 1 70), and
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INFLUENZA HEMAGGLUTININ 757
ovalbumin ( 1 7 1 ) indicate that Th recognition of large globular antigens requires a processing step during which denaturation and fragmentation occur in the lysosomal compartment of the antibody-presenting cell. These and other studies suggest that Th cells recognize continuous determinants as peptide fragments of protein, in contrast to B cells that see discontinuous determinants of the intact protein. Similar observations were made for B-ceU and T-cell responses to influenza virus hemagglutinin. Whereas antibodies to HA recognize conformational determinants on the variable HA head region, T h clones stimulated by HA can also recognize pep tides from conserved regions of the molecule. An early study showed pep tides taken from all regions of HA 1 of the H3 subtype could stimulate T h cells, and peptide HA l 306-328 was immunodominant ( 1 72, 1 73) (Figure 2 1 ). The HA2 chain of the molecule or peptides from it can restimulate T cells from virus primed mice ( 1 74, 1 75). More recent studies with H3 HA
demonstrated recognition of peptides HA 1 48-68 (Figure 21) and 1 28-148 by Th from infected mice. These studies showed that clones of different specificity could recognize antigenic variants, suggesting that conformational epitopes can be important in T-cell recognition and that Tcell recognition includes specificity for determinants in the variable regions of the molecule to which antibodies also are directed ( 1 76, 1 77). Studies on the T-cell response to HA of the H I subtype in mice following infection with AjPRj8j34 virus showed that Th cells recognized peptides HAl 1 09-1 20 ( 1 78) and 290-3 1 0 ( 1 79; Figure 2 1 ) . Residues HAl 1 1 5 and 1 36 have been identified as being particularly important in T-cell recognition of the H I subtype ( 1 80).
Fewer studies have been done on T-cell memory. A recent study ( 1 8 1 , 1 82) showed that after murine infection by influenza virus o f the H3 subtype, the majority of memory T-cell clones recognize antibody binding regions, specifically HA l 1 77-1 99 and 1 82-1 99, which are within site B and HA l 56-76 around sites C and E (Figure 2 1 ) . Residues 63, 1 89, 1 93 and 198, all important in antibody recognition of HA, were shown to affect T-cell recognition ( 1 8 1 , 1 82). The studies indicate that both B cells and T cells may recognize similar sites on the HA and therefore both systems may provide immune selective pressure.
Cytotoxic T (Tc) Cells
Cytotoxic T lymphocytes (CTL) specifically recognize and lyse virusinfected target cells, generally in conjunction with class-I MHC molecules. For both mice and humans, infection by influenza A viruses is known to produce a CTL response thought to be important in limiting the spread of infection and in clearing of the virus. The majority of Tc cells in most influenza infections recognize type-specific antigens ( 183). However, there
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is a population of Te cells that recognizes regions of HAl or HA2 ( 1 84). Induction of cytotoxic T cells was reported with fragments HA2 103-123 and HAl 1 8 1-204 ( 1 85, 1 86), with the HAl peptide inducing Tc at a molar efficiency ten times greater than the HA2 peptide. During infection with H2N2 virus AjJapanj305j57, about 45% of the cytotoxic T cells induced are HA specific ( 1 87). Use of recombinant vaccinia virus containing truncated forms of the HA polypeptide from AjJapanj305j57 has made it possible to define a site in the transmembrane anchor region that is recognized by class-I MHC-restricted CTL. This site (HA 523--545) along with a site in the HAl domain (202-221 ) has been defined both by peptides and by truncated molecules expressed in vaccinia (Figure 2 1 ). Expression of a truncated HA gene encoding only the transmembrane and cytoplasmic tail domains of HA resulted in recognition by CTL ( 1 88 , 1 89). These results indicate that a processed form of HA is recognized by class-I specific CTL.
VACCINATION AGAINST INFLUENZA
Inactivated Vaccines
Inactivated influenza vaccines are routinely used in many countries worldwide. Because they are the only vaccines available in a number of countries, they are the primary focus of prevention efforts. Present day vaccines contain strains of the three influenza viruses circulating in humans: two influenza A viruses (HI N l and H3N2) and one influenza B virus. Vaccine strains are selected on the basis that inactivated vaccines should be formulated to contain viruses with surface antigens (HA and NA) most like those of the epidemic strains in circulation. In primed young adults, parenteral vaccination with inactivated vaccines results, in up to 90% of recipients with what is considered to be a protective level of serum hemagglutination-inhibition (HAl) antibody ( 190, 19 1 ) ; however, the proportion of unprimed individuals who mount a protective antibody response is reduced and adequate responses are achieved only after two doses of vaccine ( 190, 1 92). A rise in nasal wash antibody is detected in 25 to 50% of primed vaccinees, but the proportion of unprimed individuals who mount a local antibody response is also lower than for primed individuals ( 193). Serum antibody levels remain at what an� considered to be protective levels for at least a year or longer in primed young adults ( 192, 1 94). However, antibody levels decline more rapidly after vaccination of unprimed individuals ( 192), and antibody levels decline more rapidly in vaccinated than in infected young children ( 195).
While the specificity of the antibody response after vaccination appears to be similar to that following natural infection ( 128), antibody induced by vaccination with a new virus subtype may be less cross-reactive with
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Figure I Schematic representation of inHuenza virus. Binding to the cell surface is followed
by a membrane fusion event in the low pH of the endosome. HA and NA are embedded in
the viral membrane with the underlying M (matrix protein) which surrounds the RNP (ribonucleo
protein) and eight negative sense RNA strands . The HA is known to change conformation at
low pH.
Figure 2 Schematic representation of the viral HA polypeptide chain. HAO is cleaved to
HA 1 and HA2 by host cell proteases.
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H A
..... _--- Voriable Loops
NA
-+--- Ii-structure -to- ':M\�� Cor
HA.2
External
Idembro.ne
Inlernal
Figure 5 Anatomy of a viral antigen-the hemagglutinin (HA) and the neuraminidase (NA)
of influenza virus. The schematic drawings are modified, with permission, from the published
structures of the HA and NA antigens solved by x-ray crystallography ( 14, 1 9) . The two structures are shown on approximately the same scale, with arrows repff:senting I3-strands and
cylinders representing at-helices. The locations of the proteolytic cleavage sites used to obtain
soluble antigens for crystallization are shown. The HA is cleaved close to the viral membrane (HA2 175), whereas the NA has a 100 A long stalk removed (NA 1-74177). The HA is
anchored in the membrane by its carboxyl end, whereas the NA is anchored in the membrane by its amino end. The conformations of the remaining membrane-bound structures have not been determined, and their representation here is only schematic . N and C indicate the amino and carboxy termini for the two polypeptide chains of HA, HAl (328 residues), and HA2
(225 residues) and the NA (469 residues). The viral antigens extend approximately 1 35-150 A from the viral membrane. The locations of the HA receptor binding site and NA active site, and fusion peptide, are also indicated. Both binding sites are located in clefts at the ends of conserved �-structure cores and are surrounded by protruding loops, which are the major sites of antigenic variation.
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Figure 6 Stereo views of the triple helices in the fibrous stem of the HA region (HA2 37-126). (a) The three 76 A long helices twist around each other in a super twisted left-handed coiled-coil structure (cyan-mainchain, yellow-sidechains) and are connected by a short extended chain (red) to three short antiparallel helices (red-mainchain, purple-sidechains). (b) Stereo view of the triple helix illustrating the helix-helix packing and side chain interactions. Basic residues are shown in blue, acidic in red, hydrophobic in magenta and hydrophilic in green on the <x-helical backbone (cyan) around the three-fold axis.
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Figure 7 Cross-sectional views of the triple helix perpendicular to the three-fold axis of the trimer. Close packing around the N-terminal end of the helix is maintained predominantly by
hydrophobic sidechains (HA2 77-104). The helices start to splay apart and hydrophilic residues are now also found around the trimer three-fold axis with additional short a-helices (HA2 37-
56), which pack antiparallel to the triple helix. The triple helix around 98-104 and 1 12- 1 18
is penetrated by a loop of HAl (27-33), and by the fusion peptide (HA2 1-5), respectively. The helices are shown in light and dark blue (main chain) and yellow (sidechains) and the loop (HA l 27-33) and fusion peptide residues (HA2 1-5) are shown in red (sidechain) and
purple (main chain).
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Figure 8 (a) Stereo view of the receptor binding site . The sialic acid is shown in yellow interacting with the conserved residues (purple), Trp 153, Tyr 98, His 1 83, Leu 194, Glu 1 90, Thr 155, Ser 1 36 and Leu (Oln) 226 attached to the polypeptide backbone (blue) of the
binding pocket. Coordinates for this figure were obtained from Drs. William Weis and Don Wiley. (b) Solvent accessible surface as a space filling representation (213, 214) of the receptor binding site (blue). The sialic acid (yellow) has been slightly displaced to show the complementarity of fit. Design of modified sialic acids is in progress for use as potential drugs to combat
influenza disease (29, 30a).
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Figure 9 (a) Solvent accessible surface representation of the HA trimer (blue) showing the location of the fusion peptide HA2 1-20 (red-oxygen, yellow-nitrogen, white-carbon). The
fusion peptide is almost completely buried in the trimer interface (left) but is proposed to be
released close to its own membrane end after the pH-triggered conformational change at
pH �5.0. The hydrophobic fusion peptide is shown released from the trimer interface in the
right hand image to simulate this event. (b) Schematic representation of the low pH-induced con
formational changes in the HA detected by anti-peptide antibodies. Depicted are the neu
tral pH form (left) and the low pH form (right). The peptides in the stem region (rods) are proposed to be exposed first (loop peptide, red; fusion peptide, green; C-HAI peptide, cyan) followed by the opening of the trimeric globular heads (spheres) to expose peptides in the interface (purple; pink-HAl 98-106), interface or hinge region (red). Figure modified from
White & Wilson (54).
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B f· ..
1�n E crr
(�
I,
\ \ ,
'} , ,�. ,',
;. '\ ;. , '
.... \ ,/.�;
�
0
\
- � -.,', , .1
0 "
,
'J . • �.4;
I ' ; �
. ,
� , I ,
, , ,� ')
.\�. : ... :" .', ,1 >, ( -..... \.,1, .· ... , �t:!:".
� � � . ,' . ;71
, \
't ,�
') , . ...... ,', , ,( t .� , "
.; ! '
�.� E�
� t;" ,
\;, t' • ----, .. I -' ;> .... -" ( \ > ft ) '.. � � " --: I
'-. "- I <: ':> , � <) "- <; \
.. ��' .. �� ,'". -->\ , t: �\� .J" c.
" , ��::, ..... -
�.).. �� ,p , � . . .
\ 't �'!: \
') " '-.' . .. \.Itt,. . '.: \ .:� . '> '
, . )\ ( ,1 (' .... . "". :" " o _, '
" .
Figure 11 Natural variation in the HA of H3 strains from 1968-1987. (a) Location of amino acid differences shown on the HA monomer in strains from years of significant influenza activity are shown by solvent accessible surfaces to represent the altered HA antigenic surface: site A-red; site B-yellow; site C-magenta; site D-blue. and site E-green (as defined by
Wiley et al 83). Generally when changes occur throughout antigenic sites (A-E) a new epidemic strain emerges (see Figure 13 for the complete representation of all sequence changes in HAl) .
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(b) Cumulative changes in the HA's from AlAichil68 to 1987. The external surface of the
HAl has essentially been completely altered since 1968 except for the receptor binding site and areas around glycosylation sites.
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Figure 12 Natural variation in the HA monomer of HI strains from 1 977-1988. Sequence homology suggests that the HI HA's are structurally similar to the H3 HA's . These different H I antigenic areas are represented on the AlHong Kong/68 structure in Figure 1 1 . The precise
location and definition of antigenic sites varies from HI to H3 due mainly to different glycosylation sites (see Caton et al (86) for H I antigenic site classification). However, as with the H I strains,
the globular head region varies substantially from year to year. (a) Amino acid variation from strain to strain during 1977-1988. (b) Cumulative variation from the AlUSSRl77 strain until 1987 as in Fig. I I .
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Figure 13 (a) Natural variation represented on the HA trimer of H3 strains from 1967-1987. The amino acid variation is represented in a stereo view of the HA trimer by colored beads
site A-red; site B-yellow; site C-magenta; site D-cyan; site E-green. The HA polypeptide
is represented by a Cn alpha trace in dark blue, carbohydrate sites by white beads and other changes in nonassigned antigenic sites by purple. (b) Natural variation shown similarly for HI
strains from 1977-1987.
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Figure 14 (a) Natural variation in H3 strains from 1968-1987. A stereo view of the HA trimer chain trace (blue) with the solvent accessible surface of residues changed from 1 968-1987, represented by a dot surface (21 3). This representation clearly shows that the sites designated A-E tend to merge into one another on the trimer. (b) Natural variation in H I strains from
1977-1988. Antigenic site color representations are as in Fig. 1 1-13 .
Figure 15 Antigenic variation from 1986-1987. The vaccine strain A/Len/86 was ineffective due to the extensive amino acid sequence variation in the NSic/87 strain. Such problems with
strains used in the vaccine occur when such extensive variation occurs. The HAl polypeptide chain is shown in dark blue, HA2 in orange and the amino acid changes are represented as a
solvent accessible dot surface with color representation as in Figure 1 1-13 .
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Figure 16 (a) Surface accessible space filling representation of the HA trimer (red) down the
threefold axis showing the antigenic variation from year to year (blue) , carbohydrate locations
(yellow), and receptor binding site (white) , 1968-1988. The HA varies extensively both in
amino acid residue and carbohydrate location while maintaining a conserved receptor binding site. (b) Stereo view of the receptor binding sites viewed down the trimt:r three-fold axis. The
amino acid variation in mainly the A&B antigenic sites (site A, red; site B , yellow; site E, green; site D, light blue) completely surrounds the sialic acid (purple) in the receptor binding
site (29).
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Figure 1 7 Natural variation in B influenza virus HA strains from 1940--1 987. Amino acid
differences are compared for three different B strains and represented on the AIHong Kong/68
structure of the monomer (a) and trimer. (b) The color representation is the same as in Figure 1 3 . Influenza B HA's do not accumulate continuous sequential mutations in quite the same way as the influenza A HA's. Also substantial insertions and deletions occur in B HA's relative to A HA's. However the framework structure is likely to be very similar (see also Krystal et al (79)) .
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Figure 18 Stereo view of the location of potential glycosylation sites in all influenza A strains
( 1933-1988) sequenced to date. The location of the Asn in AsnlXlSer or Thr sequences is
indicated by a dot surface and residue number. Location of site-directed mutants with additional glycosylation sequences are shown by circled numbers ( 1 15) .
Figure 19 Monoclonal antibody selected mutants of (a) H3 strains (2, 3 , 83) and (b) H I strains (NPRl8/34, (86)). Only a representative sample i s shown for both. The location and sequence of the escape mutant differences assigned to different antigenic sites is shown on a schematic of the HA monomer. Carbohydrate sites are shown by circles in yellow.
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Figure 20 (a) Stereo representation of the solvent accessible surface of the peptide HAl 98-108 in the conformation it adopts in the crystal structure of the hemagglutinin (14). The conforma
tion the peptide adopts in solution is a type II J3-tum at 98-10 I ( 163 , 164). (b) Close up
stereo view of the solvent accessible molecular surface (214) around the sequence 98-106 in
the hemagglutinin monomer structure displayed on a Raster display unit. A ball-and-stick represen
tation of HAl 98-106 can be viewed through its translucent gray surface and is shown in the same conformation as Figure 20(a). Only residues 101-106 are clearly visible. The C, N and o atoms are colored green, light blue and red, respectively. The HAl non-translucent dark blue surface shows the other surrounding residues of the sequence 75- 1 1 0 (yellow) embedded
the HA l polypeptide chain (yellow).
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Figure 21 Location of T-cell epitopes represented on an HA monomer. Peptides identified as being Th or Tc cell sites are shown by colored portions of the HA chain (grey) for (a) H I ,
(b) H 2 and (c) H3 sUbtypes (for details see text) . The H I , H 3 peptides shown are predominantly
T h sites whereas the H2 peptides are Tc sites.
Printing charges for the color illustrations included in this review were supported by a generous gift from The R. W. Johnson Pharmaceutical Research Institute .
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INFLUENZA HEMAGGLUTININ 759
variants of that subtype than is antibody induced by natural infection ( 1 96). In addition, several studies have shown that antibody induced by natural infection is more protective than that induced by vaccine ( 1 97, 1 98). The protective efficacy of inactivated vaccines as indicated by influenza illness in young adults is high and commonly quoted as being between 60 and 80% ( 199, 200), with the lower value seen after challenge with an antigenically drifted virus and the higher value seen when the virus causing disease is closely related to the vaccine virus. In individuals over 65 years of age (who are a primary target population for vaccination in the United States), vaccine efficacy is apparently lower ( 1 99, 20 1-204). This is most likely a reflection of reduced immune competency in this population (203) in whom the antibody response to HA is considerably reduced. In fact, as many as 50% of elderly vacinees may fail to respond with a four-fold increase in HAl antibodies (204), conventionally accepted as an indication of a significant antibody response. In addition, antibody response in the elderly may be delayed (205) and the HI titer may return to baseline more rapidly than for young adults (206). Despite these problems, there is considerable evidence to suggest that vaccination with inactivated vaccines reduces the incidence of infection, the incidence of lower respiratory tract disease including pneumonia, and the incidence of death due to influenza infection in the elderly (20 1 , 202, 207).
Other Approaches to Prevention and Control
Because of the frequent emergence of antigenic variants, the occasional appearance of totally new pandemic strains of influenza, and the limited immune response of some individuals to inactivated vaccines, special problems not observed with other viral diseases exist in preventing influenza. Therefore alternative vaccine and antiviral strategies are being explored. These alternative strategies include live attenuated influenza vaccines, antiviral compounds, peptide vaccines (see antipeptide Antibodies), sialic acid analogs to bind influenza receptors (see Receptor Binding) and inhibitors of fusion or antibodies that inhibit fusion (see Fusion). Of these possible alternatives, only the antiviral compound amantadine is in current use, although live attenuated vaccines may soon be licensed. Amantadine hydrochloride and rimantidine hydrochloride are antiviral compounds effective in prophylaxis and therapy of influenza A. Both compounds interfere with the replication of influenza A viruses, but the specific mechanisms of their antiviral activity are not fully understood. At high concentrations in vitro, there appears to be a selective effect on the HA protein, while at lower concentrations the M2 protein (and possibly the HA or M l as well) has been affected (208). Mutants that are resistant to these antivirals arise in vitro and in vivo, although the clinical SIgnificance of this is
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760 WILSON & COX
not yet known. Live attenuated influenza vaccines are still under investigation in a number of countries and have been used widely in only a few countries including the USSR and China. These vaccines represent the best studied alternative to inactivated vaccines. Possible advantages of a live attenuated over an inactivated influenza vaccine include the stimulation of a local secretory response, of a cytotoxic T cell response, and of immunity that is of longer duration and cross-protective against a wider range of antigenic variants. At the present, the most promising live attenuated vaccines are prepared by reassortment of a cold-adapted (CA) donor virus and an epidemic strain (209). Reassortants with genes encoding the surface glycoproteins (HA and NA) from the epidemic strain and internal genes conferring attenuation from the parent donor strain have been selected to examine the local and serum antibody response to HA during primary influenza infection (see section on Humoral Response) as well as to compare the protective efficacy of live and inactivated influenza vaccines. Results have been quite promising. A number of CA recombinants bearing surface antigens from a variety of H IN I and H3N2 epidemic strains have been shown to be infectious, immunogenic, areactogenic, and genetically stable when administered intranasally to children and young adults (210). In young adults, protection from virus replication ranged from about 50% to 80% while protection from illness ranged from about 60% to 100% (21 1 ). In older adults with chronic diseases, however, the CA vaccines have been shown to be less infectious and less immunogenic, perhaps because of preexisting immunity in these individuals as well as decreased immunocompetence (2 12). It now appears that vaccination of children with live CA vaccines and of adults with inactivated vaccines may provide advantages for both populations.
CONCLUSIONS
Influenza virus escapes immune recognition and neutralization by constantly varying the surface of its hemagglutinin surface antigen. The hemagglutinin has important biological roles in receptor binding and membrane fusion which require conservation of the functionally important areas of the HA structure. The structural framework of the HA is such that critical residues can be conserved while antigenic variation can occur in the connecting loops and helices. Not only can mutations then accumulate in these hypervariable regions from strain to strain, but also insertions and deletions can be accommodated in different strains, subtypes, and types of influenza. Carbohydrate is also important in antigenic variation by masking or unmasking antigenic sites. New epidemic strains emerge when a significant number of mutations accumulate in these
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INFLUENZA HEMAGGLUTININ 76 1
antigenic sites. Effective vaccination is then possible only if the vaccine strain is antigenically similar to the current circulating strains. These properties of the hemagglutinin of influenza virus have made prevention and control efforts for this disease less effective than for many other viral diseases. Therefore, pursuing novel approaches to preventing influenza is essential. Such approaches include design of drugs to prevent the essential receptor binding or membrane fusion functions of the HA. Understanding the structural basis for immune recognition and of the function of this molecule plays a central role in devising such strategies. In addition, the knowledge gained in studying the hemagglutinin of influenza viruses may provide critical insights for developing strategies for the prevention and control of diseases caused by other viruses with variable membrane glycoproteins such as HIV and other retroviruses.
ACKNOWLEDGMENTS
Support is gratefully acknowledged from NIH grants AI-23498, GM-3841 9, and GM-38794 (1. A. Wilson). We thank Drs. Don Wiley and William Weis for use of their unpublished coordinates of the HA-sialic acid complex and Dr. Howard Gary for Figure 10. This manuscript was significantly enhanced thanks to Patricia O'Reilly who provided excellent computation and graphics expertise for the majority of the color figures and also by Daved Fremont and Dan Bloch for computer analysis and graphics used throughout the tables and figures. We would like to thank Dr. Alan Kendal for help and support, and Michelle Dietrich for excellent assistance in manuscript preparation. This manuscript is publication # 6 120-MB from the Research Institute of Scripps Clinic.
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