Structural basis of immune recognition of influenza virus hemagglutinin.

Annu, Rev, Immunol. 1990,8:737-71

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STRUCTURAL BASIS OF IMMUNE RECOGNITION OF INFLUENZA VIRUS

Annu. Rev. Immunol. 1990.8:737-787. Downloaded from www.annualreviews.org by University of Illinois - Urbana Champaign on 04/16/13. For personal use only.

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 "Span ish 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 1 933 and of influenza B in 1 940. Although more than 50 years have passed, influenza viruses continue to cause considerable excess mortality The US Government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper. I

737


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 influ enza 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 1 987-1 988 influenza season. Thirteen subtypes of HA (H I -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-

739

INFLUENZA HEMAGGLUTININ

agglutinin was observed by electron microscopy to project from the viral membrane as spikes of approximately 1 40 A by 40 A, whereas the neur aminidase 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 pro teolytically cleaving it from the viral surface by bromelain digestion ( 1 1). The HA is a homo trimer ( 1 2) 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).


"-'

CHO

CHO

CHO

-16� ��__r-�

__

CHO

�

�__

�

__

-;�

__

�

__

__________

,

139

s-s Signal Peptide

S I S

165

CHO

Uricharged M:�br.n�· Domain; c,}I:;'y-

Fusion Peptide

221 211 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.


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 hydro philic 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 eight stranded 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 anti parallel 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 2733) 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.


�--'------.... 741


742

WILSON & COX

HAl indicates that the signal peptide may have remained attached to the HA during its biosynthesis and folding, before being removed ( 1 4). 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 ( 1 4). Human and animal isolates of A viruses bind preferentially to sialated oligo saccharides containing SA a26Gal 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 H A l 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 H A l 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 sur prisingly 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



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 immu nogenic and antigenic loops which surround the pocket. Nevertheless, antibodies can have ridges which protrude into antigens, as in the HyHEL1 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 endo cytotic 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 of HA2 becomes accessible and is released from the trimer inter face (Figure 9; 4 1 , 45, 46). Viruses selected in amantadine hydrochloridl�


744

WILSON & COX

treated cells, in which the endosomal pH is raised, have altered pH depen dence 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 H A l 1-27 (45). Proteolytic removal of the amino terminal HA2 1-22 by thermolysin resolubilizes the tails (52). Some per turbation 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 peptides significantly alters the HA trimeric association due to their close inter actions 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 protolytic cleavage. The role of the hydrophobic fusion peptide in membrane fusion has been dem onstrated 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


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 unpre dictable 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 7273 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 1 2 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.


746

WILSON & COX

occur through the process of genetic reassortment. For example, con siderable evidence suggests that the Hong Kong influenza epidemic of 1 968 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 45 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 char acterized 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 HA l 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



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 H A l polypeptide chain. That the single amino acid substitution is part of the antibody binding site has been directly demon strated 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 geneti cally with a markedly lower rate of silent and coding nucleotide sub stitution than for human H3 strains, with amino acid substitutions occur ring 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 HA l 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 1 968-1987, 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


748

WILSON & COX

acid substitutions are located on HAl in nine reference strains from 1 9681 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, 1 37, 1 38, 1 44, 1 45, 1 55, 1 56, 1 59, 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 1 988, 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 1 3, 1 4). 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 1 968 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 1 000 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 HyHELl O (31 a) ( 1 6, 13, and 1 3 contact residues, respectively) reveals 1050, 1 090, and 980 A2, respectively. One can therefon� approximate that on average one antibody epitope of hemagglutinin is completely changed


749

Table 1

Predicted changes in the HA solvent accessible surface of H3 strains from 19691988 and HI strains from 1 977-1988 based on the Hong Kong/68 structure (14)


Number of unique changesa

Solvent accessible area (A **2)b

Solvent accessible area per mutated residue (A **2)

Percentage buried in trimer interfaceC

H3 1968-72 1972-73 1973--75 1975-77 1977-79 1979-82 1982-85 1985--8 7

30 12 14 17 5 19 24 27

1968-87

76 (50)

HI 1977-78 1978-80 1980--8 3 1983-86 1986-88

10 11 18 17 21

1240 1 940 2820 2930 3650

41 59 52 57 58

16.4 1 5.9 12.1 9.1 10.4

1977-88

45

7120

53

1 4.0

53,11 0

35

23.2

X31/68

(10) (6) (11) (16) (5) (8) (9) (8)

6670 3680 2850 2670 1 400 2630 3500 4520

( 1880) (1540) (2290) (2480) ( 1400) (420) ( 1230) (1700)

, 14,400 (9310)

74 102 68 52 93 46 49 56

(63) (86) (69) (52) (93) (17) (45) (71)

63 (62)

6. 1 (17.0) 4.3 (9.7) 8.9 (10.7) 13.7 '(14.7) 0.0 (0.0) 1.5 (7.5) 14.9 (0.0) 12.0 (0.0) 8,4

(8.8)

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. CThe area buried in the trimer interface was calculated as the difference residues in the trimer versus the three separated monomers. dThe 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.


750

WILSON & COX

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 x ray 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; 79-8 1). 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 mam malian 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 speci men suggested that isolates obtained in mammalian cells are more homo geneous 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 can didates for the majority virus chosen as a vaccine candidate.


751


Glycosylation 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 differ ent strains and subtypes ( 1 05, 1 06). In all currently available influenza A HA l sequences, some 27 different residues (26 HA l , 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 I S). 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 het erogeneity for the A/Hong Kong/68 strain ( 1 0S) and for the A/Lenin grad/3S5/S0 strain ( 1 09) 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 H A l 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 anti genicity was provided by selection of a mutant HA (Asp- > Asn 63) in which a new glycosylation site prevented antibody binding and viral neu tralization, whereas the mutant virus grown in the presence of tunicamycin was antigenically indistinguishable from the wild type X: 3 1 (Aichi/68) virus ( 1 1 0). 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 ( 1 20, 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 ( 1 25). 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 ( 1 20, 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 virus absorbed 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 anti bodies are strain-specific with only a small amount of cross-reactive anti body present ( 127). In adults who had previously been exposed to an earlier variant, the predominant antibodies after infection were cross reacting and strain-specific for the previous variant (128). These studies demonstrate that antibodies induced by infection with influenza are pri marily 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



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 ( 1 33, 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 atten uated vaccine were examined in another study ( 1 36). 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 sero surveys 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 pre viously in 1950, spread throughout the world in 1977 to 1978. Few infec tions 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 docu mented infection with A/Hong Kong/68 (H3N2) virus; resistance to infec tion was found to last at least four years (138). Other studies have dem onstrated 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 ( l 39), 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 1 9; 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 ( 1 40). However, Dimmock and his col leagues 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 ( 1 4 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 ( 1 43, 1 44). 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 ( 1 45).

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 ( 1 46) first showed that a battery of antipeptide antibodies raised against 20 different peptides comprising almost the entire sequence of the AjVicj3j75 H A l 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 H A l globular heads. The opportunity o f raising antibodies against areas o f 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 ( 1 47, 148). However, it appears that antipeptide anti bodies against influenza are not particularly effective for viral neutral ization, although in one case, marginally lower virus titers and partial



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protection were shown in mice immunized with a peptide corresponding to HAl 1 38-1 64 (site B) ( 1 49). Antipeptide antibodies that specifically react with the major antigenic sites have been difficult to obtain particularly against the "loop" peptide ( 1 40- 1 46) of site A ( 1 46, 1 49, 1 50, 1 5 1 ). Never theless, 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 deter minants of this peptide has shown 3 1 4-3 18 LKLAT to be the immu nodominant epitope for monoclonal antipeptide antibodies, whereas another site 320-328 was recognized by both polyclonal sera and mono clonal antibodies ( 1 54). 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 1 0 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- 1 06 ( 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 1 0 1 - 1 06 (DVPDYA Ka '" 1 0-8 M). This determinant lies in the trimer interface ( 1 58), con sistent with antibody reactivity only to the monomeric tops (Ka '" 1 0-6 M) but not to the trimeric structure (54, 1 59, 1 62). However, antipeptide antibody studies have shown that this determinant becomes exposed in the pH-5 treated BRA, which is consistent with the trimeric 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- 1 06) has a surprisingly high percentage of type-II f3-turn structure in water ( 1 63, 1 64). Further observations show ing that immunologically reactive peptides have conformational pref erences 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 peptides containing the HAl 1 0 1 - 1 06 epitope have been crystallized ( 1 65; 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- 1 08, has been found to elicit an anti-influenza immum; response ( 166). Antipeptide antibodies can then be markedly different from either anti protein 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 antipeptide 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 ( 1 65, 1 65a; 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. Dur ing 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 ( 1 68). Studies with proteins such as lysozyme ( 169), myoglobin ( 1 70), and



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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, Th clones stimulated by HA can also recognize peptides from conserved regions of the molecule. An early study showed peptides taken from all regions of HA 1 of the H3 subtype could stimulate Th 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 28148 by Th from infected mice. These studies showed that clones of different specificity could recognize antigenic variants, suggesting that con formational epitopes can be important in T-cell recognition and that T cell 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 H A l 1 091 20 ( 1 78) and 290-3 1 0 ( 1 79; Figure 2 1 ) . Residues H A l 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 1 9 8 , 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 virus infected 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 1 03-123 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 trun cated forms of the HA polypeptide from AjJapanj305j57 has made it possible to define a site in the transmembrane anchor region that is recog nized by class-I M HC-restricted CTL. This site (HA 523--545) along with a site in the HAl domain (202-22 1 ) 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 world wide. 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 (H I N l and H3N2) and one influenza B virus. Vaccine strains are selected on the basis that inactivated vaccines should be for mulated 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 hem agglutination-inhibition (HAl) antibody ( 1 90, 1 9 1 ) ; however, the pro portion of unprimed individuals who mount a protective antibody response is reduced and adequate responses are achieved only after two doses of vaccine ( 1 90, 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 indi viduals ( 193). Serum antibody levels remain at what an� considered to be protective levels for at least a year or longer in primed young adults ( 1 92, 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 ( 1 28), antibody induced by vaccination with a new virus subtype may be less cross-reactive with


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.

HA

NA

....._---

-+---

Voriable Loops

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.


Figure 6

Stereo views of the triple helices in the fibrous stem of the HA region (HA2 37126). (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

H6 Hemagglutinin.

Unique Structural Features of Influenza Virus H15 Hemagglutinin.

Structural basis of antigenic variation: studies of influenza virus neuraminidase.

The structural basis of the function of influenza virus glycoproteins.

Mechanisms of hemagglutinin targeted influenza virus neutralization.

Antigenic determinants in influenza virus hemagglutinin.

Structural features influencing hemagglutinin cleavability in a human influenza A virus.

Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor.

Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity.

Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

Identification of hemagglutinin and neuraminidase genes of influenza B virus.

The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin.

Structural insights into the membrane fusion mechanism mediated by influenza virus hemagglutinin.

Lymphocyte responses to hemagglutinin and neuraminidase subunits of influenza virus.

Specificity of cytotoxicity T cells directed to influenza virus hemagglutinin.

Studies on the primary structure of the influenza virus hemagglutinin.

Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus.

Refinement of the influenza virus hemagglutinin by simulated annealing.

Biogenesis of influenza a virus hemagglutinin cross-protective stem epitopes.

The Molecular Determinants of Antibody Recognition and Antigenic Drift in the H3 Hemagglutinin of Swine Influenza A Virus.

Structural basis of nectin-1 recognition by pseudorabies virus glycoprotein D.

Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8.

Structural basis for histone mimicry and hijacking of host proteins by influenza virus protein NS1.

Alternative recognition of the conserved stem epitope in influenza A virus hemagglutinin by a VH3-30-encoded heterosubtypic antibody.