FEMS Microbiology Letters 100 (1992) 483-488 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier

483

FEMSLE 80012

The conformational specificity of viral epitopes Marc H.V. Van Regenmortel Institut de Biologic Mol~culaire et Cellulaire, CNRS, Strasbourg, France

Received 12 June 1992 Accepted 29 June 1992

Key words: Virus; Virus antigenicity; Epitope; Tobacco mosaic virus; Influenza virus; Antigen conformation

1. SUMMARY Four types of antigenic sites found in viruses are discussed: cryptotopes, neotopes, metatopes and neutralization epitopes. The role played by conformation on the specificity of viral epitopes is illustrated in the case of tobacco mosaic virus and influenza virus. It appears that mechanisms reminiscent of induced fit contribute to the recognition of viral epitopes by antibodies.

2. INTRODUCTION The antigenic sites or epitopes of a virus particle correspond to those parts of the capsid or envelope proteins that are recognized by the combining sites or paratopes of antibody molecules. In the absence of further qualification, the term epitope refers to a B cell epitope, i.e. recognized by a B cell receptor and by an antibody. In the present review, only B cell epitopes

Correspondence to: M.H.V. Van Regenmortel, Institut de Biologic Mol6culaire et Cellulaire, CNRS, 15 rue Ren6 Descartes, 67084 Strasbourg Cedex, France.

will be considered. A second type of epitope known as T cell epitope corresponds to peptide fragments of viral proteins that have undergone processing within an antigen-presenting cell. These T cell epitopes are recognized by T cell receptors in association with proteins of the major histocompatibility complex [1]. During the development of an immune response to a virus, B cell receptors recognize the intact tertiary and quaternary structures of the capsid and envelope proteins of the virus. Likewise the antibody molecules that are subsequently produced by plasmocytes are also specific for the intact three-dimensional structure of virus particles. In addition, however, antibodies may also be produced against the dissociated or denatured viral subunits (the so-called soluble antigens) that often accompany the intact virions used for immunization. Furthermore, some viruses such as enteroviruses may have their capsid proteins selectively cleaved by proteases during replication in the gut and this may lead to an alteration of the viral epitopes [2]. This is probably the reason why the distribution of epitopes recognized by neutralizing type 3 poliovirus antibodies in children immunized with chemically inactivated poliovirus vaccine (containing un-

484

F

A

F A

ope

erypti~t°

B

J m etatope

E

E

Fig. 1. Schematic model of the protein subunits of TMV as monomer and double layer disk showing the location of different types of epitopes. Surface A' harbours neotopes while surfaces B, C and D possess cryptotopes. Metatopes are present on the bottom surface E of the subunit [7].

cleaved particles) differs from that seen by antibodies resulting from oral poliovirus vaccination or natural infection [3]. It is important to recognize that the structure of the antigen that gives rise to an immunogenic stimulus in immunized animals or in virus-infected individuals remains clouded in the mystery of the 'black box' and that only indirect inferences are possible.

3. T Y P E S OF V I R A L E P I T O P E S Four types of viral epitopes have been distinguished, called cryptotopes, neotopes, metatopes and neutralization epitopes [4]. The first three types are illustrated in Fig. 1 in the case of the protein subunits of the rod-shaped plant virus: tobacco mosaic virus (TMV). This virus consists of about 2100 identical protein subunits arranged as a helix around an R N A molecule. The protein subunits which contain 158 amino acid residues can also associate to form a two-layer disk as shown in Fig. 1. Cryptotopes are found on the surfaces B, C and D that become buried when the subunits polymerize to form helical rods or disk aggregates. As a result, the cryptotopes present on subunits can bind antibodies only after dissociation or denaturation of the virus particle. In contrast, neotopes are absent in the con-

stituent monomeric subunits and exist only in particles with an intact quaternary structure. Neotopes are found on surface A' of the polymer (Fig. 1) and arise either as a result of conformational changes in the monomers induced by intersubunit bonding or through the juxtaposition of residues from neighbouring subunits. The label m e t a t o p e was introduced to refer to epitopes present in both dissociated and polymerized forms of the viral coat protein [5]. In a study of 22 m o n o c l o n a l antibodies specific for metatopes of TMV, it was found that all of them reacted with only one of the two extremities of viral rods containing the 5' end of the R N A [6]. This means that the antibodies reacted with the bottom face of the protein subunit (E in Fig. 1) containing the two a-helices made up of residues 73-89 and 111-135 [7]. The ability of antimetatope antibodies to react with only one extremity of rod-shaped virus particles is not a general phenomenon, since in the case of beet necrotic yellow vein virus, antibodies were found to react with epitopes located on both extremities of the particles [8]. Neutralization epitopes correspond to epitopes of the virus that are recognized by antibody molecules able to neutralize the infectivity of the virus. Such epitopes can only be identified and measured in a functional assay,, the neutralization test, and their properties cannot be analysed outside of the operational context of neutralizing antibodies and potentially infectable cells [4]. Not every antibody capable of binding to a virion is able to interfere with the process of viral infectivity and there is little information on which structural features differentiate neutralization epitopes from other epitopes present on the surface of virus particles. With the view of developing synthetic viral vaccines, there have been many attempts to mimic neutralization epitopes by means of linear synthetic peptides. For instance, peptides corresponding to residues 141-160 and 200-213 of VP1 protein of foot-and-mouth disease virus (FMDV) have been shown to be able, when injected singly or together, to induce neutralizing antibodies in immunized animals. Elucidation of the three-dimensional structure of the virus showed that these two regions are in close

485

proximity on the surface of the virion and contribute to a single, discontinuous epitope [9]. The two regions 141-160 and 200-213 that combine to form a single epitope originate from two neighbouring subunits and constitute a neotope recognizable by a neutralizing antibody [10]. The loop comprising residues 133-158 of VP1 of FMDV forms a particularly disordered protrusion on the virus surface which prevented this region from being located with precision by X-ray crystallography [9]. The considerable flexibility of the polypeptide backbone in this region may be responsible for the finding that the corresponding synthetic peptides were able to mimic the viral epitope very effectively. These results underline the functional relevance of segmental mobility in antigenic regions in proteins [11,12]. Another example of a discontinuous neutralization epitope is the cluster of residues 221-226 of VP1 and residues 164-170 of VP2 of poliovirus 1 which together form a neotope comprising residues from two separate structural proteins [13].

4. CONFORMATIONAL VIRAL EPITOPES

FEATURES

OF

The neotopes described above in the case of FMDV and poliovirus illustrate the fact that viral epitopes may arise in the quaternary structure of the virion by juxtaposition of residues that belong to separate coat protein subunits. In general most epitopes are made up of residues that are brought together by the folding of a single polypeptide chain, thereby creating so-called discontinuous epitopes. When the tertiary structure of the protein is disrupted by denaturation or fragmentation of the molecule, the residues that made up the discontinuous epitope are scattered and each component usually is no longer individually recognized by theantibody. However, a significant proportion of neutralizing monoclonal antibodies (mAbs) recognizing native viral proteins also bind to short peptide fragments (7-20 residues) of the protein although with lower affinity than to the virus [14]. Such fragments are given the status of continuous epitope of the protein. The ability of antipeptide antibodies to neutralize the infectiv-

ity of many different viruses also demonstrates that synthetic peptides and native viral proteins are able to be recognized by the same antibodies [15]. These findings refute the claim [16] that all instances of cross-reactivity between peptides and proteins are due to the presence in the assay of antibodies specific for denatured protein molecules. It should be emphasized that the existence of antigenic cross-reactivity between a short peptide and its cognate protein does not necessarily establish the presence of a so-called continuous epitope in the corresponding region of the protein. Not every amino acid in a peptide of 7-20 residues is a contact residue interacting directly with the antibody paratope since some residues can usually be replaced by any of the other 19 amino acides without affecting the antigenicity of the peptide [17]. Furthermore, the antigenic reactivity of a peptide also depends on its conformation, as shown by the influence of assay format on peptide-antibody recognition [18]. Antibodies do not recognize, in the same manner, a peptide absorbed to a solid-ph~/se, conjugated to a carrier or free in solution, and even lengthening a peptide by a few residues may abolish its antigenic reactivity [14]. Several approaches have been used to increase the degree of conformational mimicry between peptide and intact protein [19]. A particularly useful approach is cyclization of the peptide either by a disulfide bridge or through lactame formation using linkers of different length [20]. For instance, in the case of antigenic site A of influenza virus haemagglutinin, antibodies produced against cyclic peptides were able to protect mice against an intranasal challenge with influenza virus whereas the corresponding linear peptide did not [21]. However, cyclizing peptides corresponding to loops and turns in viral proteins does not always lead to improved antigenic cross-reactivity between peptide and virus, as shown in a recent study of a comovirus structurally related to picornaviruses [22]. The binding of a peptide to antiprotein antibodies may also be facilitated by the induction of a native-like conformation in the peptide during formation of the peptide-antibody complex

486 Table 1 Results of two-site binding assays between mAbs and T M V protein measured in a BIAcore biosensor system [25] mAb2

mAbl Anticryptotope

Antineotope Anticryptotope Antimetatope

263P 174P 161P 6P 16P 151P

Antimetatope

Antineotope

174P

161P

6P

16P

151P

. + -

+

+

+

+

+ +

+ + -

.

. + + +

.

253P

. + -

m A b l : Immobilized antibody on sensor chip used to trap T M V protein. Only antimetatope and certain anticryptotope antibodies were able to trap T M V protein. Negative results with antineotope m A b l are due to the inability of these antibodies to capture the viral protein. mAb2: Second antibody tested for its ability to bind T M V protein captured by m A b l . + = pair of mAbs that bind concurrently to the viral protein. = pair of mAbs that do not bind concurrently. -

[23,24]. In a similar fashion, it has been shown that a neotope conformation could be induced in viral coat protein by the binding of an antibody molecule. This was demonstrated in a recent study of TMV mAbs [25]. When certain anti-cryptotope or anti-metatope mAbs were immobilized on the sensor chip to capture TMV coat protein monomers, a neotope conformation was induced in the protein as revealed by the binding of anti-neotope mAbs (Table 1). It is not clear if the induction of a neotope-like conformation in the monomeric subunit was brought about by the capturing antibody or by the second, anti-neotope antibody. In conclusion, the conformational plasticity of viral epitopes present in short peptides or in intact viral particles is extensive, and this allows the occurrence of induced-fit type phenomena that facilitate antigenic cross-reactions between peptide and cognate protein. Additional structural studies of antigen-antibody complexes [26] combined with quantitative binding measurements [27] will be necessary to elucidate more fully how such plasticity can be reconciled with the high specificity of immunological interactions. As demonstrated by mutagenesis studies of antibody combining sites [28] the nature of the epitope-paratope interaction cannot be predicted from either structural or binding data alone. As discussed elsewhere, both structural and func-

tional approaches are necessary to gain an understanding of the nature of immunological specificity [29].

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