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Neutralizing Epitopes of Type O Foot-and-Mouth Disease Virus. II. Mapping Three Conformational Sites with Synthetic Peptide Reagents
Abstract
Four neutralizing monoclonal antibodies (MAbs), recognizing three functionally independent, conformational sites on type O foot-and-mouth disease virus (FMDV) failed to react with immobilized structural proteins or synthetic peptides but bound to the isolated capsid protein VP1 and peptides in solution. Inhibition ELISA techniques were, therefore, applied using peptide antigens and anti-peptide sera to block MAb binding to virus particles, permitting the identification of those portions of the VP1 protein contributing to the epitopes. The binding site of one MAb, which neutralized a range of type O FMDV isolates, was shown to have components within regions 146 to 150 and 200 to 213 of VP1 with a critical involvement of the amino acids at positions 146 and 206 or 207. The determinants recognized by two other MAbs which were directed at similar, but not identical, epitopes from a second site included components from the 200 to 213 and 143 to 146 regions with amino acids 143 and 144, respectively, appearing critical for the inhibition of the virus binding of the two antibodies. These results demonstrate that the two previously identified immunogenic tracts of VP1 are brought into proximity in the quaternary structure of the virion to form an antigenic domain containing several conformational epitopes, some of which are functionally independent. A fourth, strain-specific MAb was effectively blocked from reacting with virus by peptides corresponding to residues 161 to 180 and 200 to 213.
... The likelihood that the residue change at position 159 of VP1 forms part of an antigenic site on the SAT2 capsid is supported by the fact that residue 159 of VP1 forms part of the base of the G-H loop and the G-H loop residues 140-160 have been shown to play an important role in antigenicity in most FMDV serotypes Bolwell et al., 1989;Crowther et al., 1993a;Parry et al., 1989;Pfaff et al., 1988;Thomas et al., 1988). The antigenic sites Ia and Ib of SAT1 viruses, for example, involve residues on both sides of the RGD motif in the G-H loop (Grazioli et al., 2006). ...
... Comparison of the P1 amino acid sequences of the SAT2 viruses indicated that the variation observed was confined to hypervariable loops of which five were identified in VP1 and one each in VP3 and VP2. The structural orientation of these variable loops on the capsid correlates strongly with previously identified neutralizing epitopes of type A (Baxt et al., 1989;Thomas et al., 1988) and O Crowther et al., 1993a;Kitson et al., 1990;Parry et al., 1989) viruses. The hypervariable regions may act as topographical regions for binding of soluble scFv1 or scFv3 on the virus capsid and the variation observed in reactivity of these soluble scFvs to the panel of SAT2 virus might be a reflection of the amino acid variation in these regions. ...
Recombinant single-chain variable fragments (scFvs) of antibodies make it possible to localize antigenic and immunogenic determinants, identify protective epitopes and can be exploited for the design of improved diagnostic tests and vaccines. A neutralizing epitope, as well as other potential antigenic sites of a SAT2 foot-and-mouth disease virus (FMDV) were identified using phage-displayed scFvs. Three unique ZIM/7/83-specific scFvs, designated scFv1, scFv2 and scFv3, were isolated. Further characterization of these scFvs revealed that only scFv2 was capable of neutralizing the ZIM/7/83 virus and was used to generate neutralization-resistant virus variants. Sequence analysis of the P1 region of virus escaping neutralization revealed a residue change from His to Arg at position 159 of the VP1 protein. Residue 159 is not only surface exposed but is also located at the C-terminal base of the G-H loop, a known immunogenic region of FMDV. A synthetic peptide, of which the sequence corresponded to the predicted antigenic site of the VP1 G-H loop of ZIM/7/83, inhibited binding of scFv2 to ZIM/7/83 in a concentration-dependent manner. This region can therefore be considered in the design of SAT2 vaccine seed viruses for the regional control of FMD in Africa.
... Antigenic diversity is one of the major obstacles to vaccinating against FMD. Some antigenic sites have been defined by the selection and sequence analysis of monoclonal antibodies (MAbs) escape mutants FMDV type O (Crowther et al., 1993; Parry et al., 1989; Pfaff et al., 1988), type A (Moore et al., 1989; Saiz et al., 1991) and type C (Lea et al., 1994). For type Asia1 FMDV, four functionally independent neutralizing antigenic sites have been detected (Butchaiah and Morgan, 1997; Marquardt et al., 2000; Sanyal et al., 1997). ...
... Thus, discontinuous epitopes are relatively difficult to map using synthetic peptides or selection of escape mutants. Functional mimicry of FMDV discontinuous antigenic sites could be achieved by means of synthetic peptide constructions (Parry et al., 1989; Villen et al., 2001 Villen et al., , 2002) and selection of neutralization escape variants (Barnett et al., 1989; Saiz et al., 1991). However, random peptide phage display and reverse genetic system as powerful means for FMDV epitope mapping have rarely been used. ...
Although neutralizing antigenic sites of foot-and-mouth disease virus (FMDV) can be defined by selection of monoclonal antibody (MAb) escape mutants, no conformational neutralizing epitope on the major antigenic site located on the G-H loop of type Asia1 FMDV has been precisely mapped. In this study, we generated a potent neutralizing MAb 3E11, which recognized a conformation-dependent epitope and neutralized FMDV Asia1/YS/CHA/05 in vitro. Importantly, a dose of 5.5 NT(50) of the MAb 3E11 completely protected suckling mice from a dose of 10 LD(50) of homologous virus challenge in vivo. Through a 12-mer random peptide phage display, synthetic peptide analysis and constructing a series of FMDV Asia1/YS/CHA/05 mutants using reverse genetic system, we finely mapped the neutralizing epitope as the 12-amino acid peptide (141)SXRGXLXXLXRR(152). These results provide additional insights into the virus-MAb interaction at the amino acid level and may help in the development of an epitope-based Asia1 FMDV vaccine.
... As mentioned before, a sequential epitope may represent a component of the complete epitope as would be observed by X-ray crystallography. This is exemplified by two discontinuous epitopes of foot-and-mouth disease virus [87]. A similar situation may exist for site IV or D of transmissible gastroenteritis virus [88]. ...
... Recently [87], the use of combinations ofpeptides to delineate discontinuous epitopes of foot-and-mouth disease virus was reported. This method, if generally applicable, would be a useful alternative to the analysis of MAb-resistant or non-binding mutants, which in principle only gives information about antigenic determinants. ...
Several systems are available for the expression of foreign gene sequences in Escherichia coli. We describe the use of prokaryotic expression products of viral gene fragments in order to identify the regions that specify the binding sites of antibodies. This approach is particularly successful if the antigenicity does not depend on the native protein, but only on the amino acid sequence, i.e., if the epitope is sequential. Combining prokaryotic expression with the use of synthetic peptides often permits a fast and accurate mapping of an epitope. The occurrence of immunodominant sequential epitopes on the surfaces of viruses seems to be a widespread phenomenon.
... However, studies have indicated that while RGD is involved in induction of effective virus neutralizing antibodies, it is not the sole motif involved in the blocking of viral entry into host cells. Indeed, virions with mutated RGD motifs are still capable of entering and infecting cells in vitro, and display similar growth kinetics to virions with intact RGD motifs [3,6,18,37,39,46]. The capability of RGDmutant virions to infect cells provides evidence to suggest that there are other mechanisms involved in cell entry and virus neutralization. ...
Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV)possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMD xeno , or PBS, followed by intradermal challenge with FDMV strain O 1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMD xeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMD xeno , when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMD xeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity.
... A structural feature of the outer capsid surface is a long, conformationally flexible loop of the VP1 protein (Sobrino et al 1989, Xie et al 1987. This loop, namely G-H loop, forms a major antigenic site on the virus and includes at its apex an Arg -Gly -ASP motif (Strohmaier et al 1982, Xie et al 1987 amino acids in this loop contain both immunedominant T and B epitopes that can elicits neutralizing antibody (NA) response (Logan et al 1993, Parry et al 1989. The abundant efforts has been carried out to determine the potential of using peptides, particularly those containing the amino acid sequence of the G-H loop, for the vaccination of naturally susceptible animals. ...
... A structural feature of the outer capsid surface is a long, conformationally flexible loop of the VP1 protein (Sobrino et al 1989, Xie et al 1987. This loop, namely G-H loop, forms a major antigenic site on the virus and includes at its apex an Arg -Gly -ASP motif (Strohmaier et al 1982, Xie et al 1987 amino acids in this loop contain both immunedominant T and B epitopes that can elicits neutralizing antibody (NA) response (Logan et al 1993, Parry et al 1989. The abundant efforts has been carried out to determine the potential of using peptides, particularly those containing the amino acid sequence of the G-H loop, for the vaccination of naturally susceptible animals. ...
In the present study the effect of the locally produced genetic adjuvant of ginea pig specific CpG-motif-containing oligodeoxynucleotide (CpG-ODN) in an inactivated FMD virus vaccine was evaluated. Boosting the ginea pigs with FMD vaccine along with CpG-ODN adjuvant produced relatively higher ratio (5-fold) of FMDV-specific IgG 2a / IgG 1 than those vaccinated in the absence of CpG-ODN. The neutralizing antibody (NA) titer induced by FMD vaccine along with CpG-ODN adjuvant was significantly higher (8-fold) than NA titer induced by the classical FMD vaccine in Alum adjuvant. The titer of NA and virus clearance from serum was consistently and significantly higher in animals primed with FMD vaccine and boosted by CpG-ODN than the classical FMD vaccine. The results of this study showed the potential of CpG-ODN as a genetic adjuvant to FMD vaccine in the development of Th1 responses.
... N 143 → S substitution was fixed in IND 271/2001 (NPP) virus under mBVS. This residue has been reported to be critical by Parry et al. (1989) using synthetic peptides. In IND 120/2002 (NPP) virus under either mBVS or tBVS, V 144 → A substitution was fixed, which has been shown to be antigenically critical with MAR mutants (Kitson et al., 1990). ...
... This suggests that the three proteins expressed and purified from E. coli are properly folded in the VLP. In addition, the data available from mapping of the neutralizing antibody epitopes in serotype O, the most extensively studied FMDV [51,52] and in Asia 1 by Butchaiah and Morgan [53] demonstrate that the major immunologic epitopes of serotype Asia 1 are on the surface oriented interconnecting loops between structural elements. Our observation that potent neutralizing antibody responses were induced by FMD VLP in all three vaccinated animal species demonstrates that the three proteins were not only correctly assembled into VLP but the neutralizing epitopes were also presented correctly on the surface of virus capsids. ...
Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-gamma. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.
... It has been reported that VP1 C terminus (aa sequence 200-213) contributes to the formation of a discontinuous site together with site 1 in the FMDV serotype O (Xie et al., 1987;Parry et al., 1989), but it may also behave as a topologically independent site in both serotypes O (site 5, Aktas & Samuel, 2000) and C (site C, Mateu et al., 1990). Similarly, both situations have been observed in different strains of serotype A (Baxt et al., 1989;Thomas et al., 1988). ...
Knowledge on the antigenic structure of foot-and-mouth disease virus (FMDV) has relevance in the development of diagnostic assays, in the evaluation of the antigenic variability and in the selection of appropriate vaccine strains. Antigenic sites have been investigated only in FMD viruses of serotypes O, A and C, while it would be valuable to extend studies also to other serotypes. This paper reports on the identification of antigenic sites involved in virus neutralisation in the FMDV serotype Asia 1 using a new panel of monoclonal antibodies and their relation with sites described in other serotypes is discussed. Out of 24 monoclonal antibodies raised against the FMDV serotype Asia 1, 10 neutralise viral infectivity and were used to select FMDV mutants resistant to neutralisation. On the basis of their reactivity profile with virus mutants, the 10 neutralising monoclonal antibodies were clustered in four groups corresponding to four independent antigenic sites. By comparing the amino acids sequence of the parental virus and of virus mutants, the amino acids crucial for the four sites were mapped at the following positions: VP1 140-142, VP2 67-79, VP3 58/59, VP3 218. Three of the four neutralising sites identified and mapped on FMDV serotype Asia 1 correspond structurally and functionally to analogous sites described in FMDV serotypes O, A and C, enforcing the evidence that these are dominant antigenic sites in FMDV structure. The fourth site, located at C-terminus of VP3, is a new independent site, described for the first time in FMDV.
... Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines [7]. Although extensive studies have been carried out to examine the epitopes in the viral capsids of serotype O [8,10,21] and serotype A FMDV [11,22,23], there are few reports on Asia1 serotype FMDV [24]. In this study, we explored the molecular basis of the antigenic difference between two viruses, Asia1/YS/CHA/05 from cows and Asia1/1/YZ/CHA/06 from pigs, which react differently against MAb 1B4. ...
Background
Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV.
Methods
Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice.
Results
Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain.
Conclusions
These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.
... Another example is the C-terminal region of the VP1 protein (residues 200-213) of foot-and-mouth disease virus, which is able to elicit high levels of neutralizing antibodies [168]. Residues 200-213 of one VP1 subunit are located next to the immunodominant disordered loop (residues 141-160) of an adjacent subunit [169], and both regions contribute to a single discontinuous neotope [170]. The ability of the terminal regions of proteins to induce antibodies that crossreact with the native protein is partly due to the fact that these regions possess an intrinsic disorder which makes them very similar to the unstructured peptides [171]. ...
The claim that it is possible to rationally design a structure-based HIV-1 vaccine is based on misconceptions regarding the nature of protein epitopes and of immunological specificity. Attempts to use reverse vaccinology to generate an HIV-1 vaccine on the basis of the structure of viral epitopes bound to monoclonal neutralizing antibodies have failed so far because it was not possible to extrapolate from an observed antigenic structure to the immunogenic structure required in a vaccine. Vaccine immunogenicity depends on numerous extrinsic factors such as the host immunoglobulin gene repertoire, the presence of various cellular and regulatory mechanisms in the immunized host and the process of antibody affinity maturation. All these factors played a role in the appearance of the neutralizing antibody used to select the epitope to be investigated as potential vaccine immunogen, but they cannot be expected to be present in identical form in the host to be vaccinated. It is possible to rationally design and optimize an epitope to fit one particular antibody molecule or to improve the paratope binding efficacy of a monoclonal antibody intended for passive immunotherapy. What is not possible is to rationally design an HIV-1 vaccine immunogen that will elicit a protective polyclonal antibody response of predetermined efficacy. An effective vaccine immunogen can only be discovered by investigating experimentally the immunogenicity of a candidate molecule and demonstrating its ability to induce a protective immune response. It cannot be discovered by determining which epitopes of an engineered antigen molecule are recognized by a neutralizing monoclonal antibody. This means that empirical immunogenicity trials rather than structural analyses of antigens offer the best hope of discovering an HIV-1 vaccine.
... In foot-and-mouth disease virus particles, two flexible regions corresponding to residues 141-160 and 200-213 of the VP1 protein were initially considered to be distinct epitopes but were later found to constitute a single neutralization neotope recognized by the same Mab. [21] The loop corresponding to residues 141-160 of one viral subunit is located close to the C-terminal residues 200-213 of an adjacent subunit and a hybrid peptide immunogenic construct comprising both regions was able to elicit a stronger neutralizing antibody response than the loop 141-160 on its own. [22] In the case of some HIV-1 neotopes, it has been suggested that the epitope may have arisen from residues of two adjacent glycoprotein subunits since it involved loops located on opposite sides of one subunit monomer. ...
In spite of 25 years of intensive research, no effective human immunodeficiency virus type 1 (HIV-1) vaccine has yet been developed. One reason for this is that investigators have concentrated mainly on the structural analysis of HIV-1 antigens because they assumed that it should be possible to deduce vaccine-relevant immunogens from the structure of viral antigens bound to neutralizing monoclonal antibodies. This unwarranted assumption arises from misconceptions regarding the nature of protein epitopes and from the belief that it is justified to extrapolate from the antigenicity to the immunogenicity of proteins.
Although the structure of the major HIV-1 antigenic sites has been elucidated, this knowledge has been of little use for designing an HIV-1 vaccine. Little attention has been given to the fact that protective immune responses tend to be polyclonal and involve antibodies directed to several different epitopes. It is concluded that only trial and error, empirical investigations using numerous immunization protocols may eventually allow us to identify which mixtures of immunogens are likely to be the best candidates for an HIV-1 vaccine. Copyright
... Elucidation of the three-dimensional structure of the virus showed that these two regions are in close 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]. ...
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.
... The region of the FMDV capsid that is considered immunodominant includes the central region of loop G- H within protein VP1 (site A, around amino acids 135 to 160; Strohmaier et al., 1982; Bittle et al., 1982; Pfaffet al., 1982 ). This domain constitutes a protruding, disordered loop on the virion (Acharya et al., 1989), involved in delineating multiple continuous and/or discontinuous epitopes in serotypes O, A and C (Xie et al., 1987; Mateu t Present address: Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, U.S.A. et al., 1987 Thomas et al., 1988; Parry et al., 1989a; Bolwell et al., 1989). Site A is the most variable part of the capsid, and the correspondingly extensive antigenic heterogeneity, even within a serotype, has been well documented (Mateu et al., 1988; Martinez et al., 1991). ...
The antigenic sites A and C (the G-H loop and the C terminus, respectively) in VP1 of foot-and-mouth disease virus (FMDV) have been considered the immunodominant regions of the virus involved in the induction of protection. Other antigenic sites have been described but their involvement in protection has not been established. Here we report that two closely related but serologically different FMDVs (the field isolate C3 Argentina/84 and the vaccine strain C3 Resende Br/55) have identical A and C sites but differ as other antigenic sites. Such differences have been documented by reactivity with a panel of 28 monoclonal antibodies (MAbs). The two viruses reacted to the same extent with each of 13 MAbs which recognized epitopes within sites A or C, but reacted differently with six out of 15 MAbs that recognized other sites. Accordingly, sequencing of the entire region coding for the capsid proteins, for both viruses, revealed four amino acid substitutions at three antigenic sites other than A and C. The results suggest that identity of sites A and C may not be sufficient to induce cross-protection, and provide the first evidence of significant antigenic diversification of FMDV in the field mediated by amino acid substitutions outside sites A or C.
... The region around amino acids 140 to 160 of the structural protein VP1 contains a major antigenic determinant for the neutralization of FMDV (Strohmaier et al., 1982; Pfaff et al., 1982; Bittle et al., 1982; Rowlands et al., 1983; Geysen et al., 1984 Geysen et al., , 1985). This region includes several epitopes (Pfaff et al., 1988; Stave et al., 1988; Thomas et al., 1988; Bolwell et al., 1989; Mateu et al., 1989) and for FMDV of serotype O some of the epitopes also contain residues of VP1 segment 200- 213 (Parry et al., 1985Parry et al., , 1989 Xie et al., 1987), thus indicating a complex antigenic structure. Peptides representing this antigenic site may constitute synthetic vaccines for FMD (reviews in Brown, 1988 Brown, , 1989), but a major drawback for their efficacy is the remarkable potential for antigenic variation of FMDV (Mateu et al., 1987Mateu et al., , 1988Mateu et al., , 1989 Gebauer et al., 1988; reviewed by Domingo et al., 1990). ...
Neutralizing monoclonal antibodies (nMAbs) elicited against foot-and-mouth disease virus (FMDV) of serotype C were assayed with field isolates and variant FMDVs using several immunoassays. Of a total of 36 nMAbs tested, 23 recognized capsid protein VP1 and distinguished at least 13 virion conformation-independent epitopes involved in neutralization of FMDV C. Eleven epitopes of FMDV C-S8c1 have been located in segments 138-156 or 192-209 of VP1 by quantifying the reactivity of nMAbs with synthetic peptides and with nMAb-resistant mutants of FMDV C-S8c1 carrying defined amino acid substitutions. The main antigenic site of FMDV C-S8c1 (VP1 residues 138 to 150) consists of multiple (at least 10), distinguishable, overlapping epitopes. Some amino acid replacements abolished one of the epitopes, whereas other replacements affected several epitopes in this region. The conservative substitution His(146)----Arg, found in many nMAb-resistant mutants analysed, abolished the reactivity of the virus with all nMAbs that recognized epitopes in the main antigenic site of FMDV C-S8c1. This indicates that a minimum genetic change can result in a highly amplified phenotypic effect, as regards the antigenicity of FMDV.
... Sequence data on a num- MAbs it was reported (Parry et a/., 1989) that the MAbs ber of MAb selected resistant variants of 01 BFS 1860 selecting these variants recognized peptides from the have also recently been published (Barnett eta/., 1989; 1143-l 150 and 1200-l 213 regions (site 1). The Parry et al., 1989). Seven of the 11 MAbs recognized parent conflict is difficult to explain; however, it is worth trypsin-treated virus (this process cleaves within the noting that early studies on poliovirus antigenic sites 1 140-l 160 region and prevents anti-l 140-l 160 pepusing synthetic peptides yielded results which have not tide antibodies binding to the virus) and variants se- subsequently been supported by knowledge of the three-dimansjonal structure of the virus (see review by Minor, 1 Q90). ...
Sequence analysis of monoclonal antibody resistant mutants of type O foot and mouth disease virus has been performed. Distinct clusters of amino acid substitutions conferring resistance to neutralization at each of the four previously defined antigenic sites (McCahon et al., 1989, J. Gen. Virol. 70, 639-645) have been identified. One site corresponds to the well-known 140-160 region of VP1, a second site is also on VP1, one site is on VP2, and the fourth site is on VP3. All of the amino acid substitutions identified are located on the surface of the virus. Despite the differences in three-dimensional structure between FMDV and other picornaviruses the neutralizing antigenic sites occur in analogous positions on the capsid surface.
... These data show that site A is complex and that it is formed by residues located in distal segments of the S glycoprotein. This seems to be the most frequent situation in epitopes from other systems studied in detail (site B reported in this manuscript; DiMarchi et al., 1986;Parry et a/., 1989;Davies et al., 1988;Posthumus et al., 1990;Wiegers et al., 1990). Although we have defined precise amino acids contributing to the different antigenic sites, the participation of other residues to the antigenicity of the subsites is not excluded. ...
The S glycoprotein of transmissible gastroenteritis virus (TGEV) has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. The residues that contribute to these sites were localized by sequence analysis of 21 mutants that escaped neutralization or binding by TGEV-specific monoclonal antibodies (MAbs), and by epitope scanning (PEPSCAN). Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. In addition, mar mutant 1B.H6 with residue 586 changed had partially altered both subsite Aa and Ab, indicating that these subsites overlap in residue 586; i.e. this residue also is part of site A. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). The location of site D is in agreement with PEPSCAN results. Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus. Its accessibility can be modulated by treatment at pH greater than 11 (at 4 degrees) and temperatures greater than 45 degrees. Sites A and B are fully dependent on glycosylation for proper folding, while sites C and D are fully or partially independent of glycosylation, respectively. Once the S glycoprotein has been assembled into the virion, the carbohydrate moiety is not essential for the antigenic sites.
In 2019, multiple FMD outbreaks occurred in swine farms vaccinated against FMDV in southern Vietnam. This study investigated the genotypic characteristics of FMDV strains from these outbreaks. Seven samples were collected from pigs exhibiting FMD clinical signs. All FMDV-positive samples were amplified and sequenced for the gene encoding the VP1. Results were analyzed and compared with sequences of reference strains and vaccine strains on GenBank. Phylogenetic analysis showed that all seven field isolates belonged to serotype O, topotype SEA/Mya-98. These strains shared high homology with strains from Vietnam (2018), Korea, and China, but low homology with vaccine strains. Moreover, 21 amino acid substitutions were found in the VP1 protein of the FMDV field strains, many of which were crucial antigenic determinants involved in the neutralization of FMDV. These findings suggest that the current vaccine may not be effective against the emerging FMDV strains in southern Vietnam.
Foot-and-mouth disease (FMD) is an economically devastating animal disease. There are seven serotypes, A, O, C, Asia 1, South African Territories 1, 2, and 3 (SAT1, SAT2, and SAT3), among which serotype O shows the greatest distribution worldwide. Specifically, the O/ME-SA/Ind-2001 lineage, which was reported in India in 2001, has since emerged worldwide, with the O/ME-SA/Ind-2001d and O/ME-SA/Ind-2001e sublineages recently emerging in North Africa, Middle East Asia, Southeast Asia, and East Asia. The antigenic relationship (r1) value for the O1 Manisa and O/Mya-98 lineage inactivated vaccine against various O/ME-SA/Ind-2001 lineages of FMDV isolates, were matching (r1 > 0.3) or non-matching (r1 < 0.3), indicating that the vaccine based on the O/ME-SA/Ind-2001 lineage FMDV, is valuable. In this study, we developed a new vaccine strain, O/SKR/Boeun/2017 isolate, belonging to the O/ME-SA/Ind-2001e sublineage as an outbreak of this sublineage occurred in 2017 in the Boeun county of the Republic of Korea (O/SKR/Boeun/2017). This experimental vaccine exhibited high immunogenicity in pigs and cattle and was antigenically matched with representative FMDV lineages (ME-SA, O/ME-SA/PanAsia, O/SEA/Mya-98, and O/Cathay) in Asia, as demonstrated by two-dimensional virus neutralization tests (2D-VNT). In addition, a 100% survival rate in C56BL/6 mice vaccinated with 1/15 of a pig dose was observed following challenge with FMDV O/VIT/2013 (O/ME-SA/PanAsia) at 10 days post-vaccination. Further, we analyzed the major antigenic sites of the O/SKR/Boeun/2017 vaccine strain as well as other viruses, by 2D-VNT. These results suggest that the O/ME-SA/Ind-2001e sublineage is a promising vaccine strain candidate in Asia, and other countries, for protection against the emerging FMDV.
Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the Nkuku® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.
In spite of 25 years of intensive research, no effective human immunodeficiency virus type 1 (HIV-1) vaccine has yet been developed. One reason for this is that investigators have concentrated mainly on the structural analysis of HIV-1 antigens because they assumed that it should be possible to deduce vaccine-relevant immunogens from the structure of viral antigens bound to neutralizing monoclonal antibodies. This unwarranted assumption arises from misconceptions regarding the nature of protein epitopes and from the belief that it is justified to extrapolate from the antigenicity to the immunogenicity of proteins.
The claim that it is possible to rationally design a structure-based HIV-1 vaccine is based on misconceptions regarding the nature of protein epitopes and of immunological specificity. Attempts to use reverse vaccinology to generate an HIV-1 vaccine on the basis of the structure of viral epitopes bound to monoclonal neutralizing antibodies have failed so far because it was not possible to extrapolate from an observed antigenic structure to the immunogenic structure required in a vaccine. Vaccine immunogenicity depends on numerous extrinsic factors such as the host immunoglobulin gene repertoire, the presence of various cellular and regulatory mechanisms in the immunized host and the process of antibody affinity maturation. All these factors played a role in the appearance of the neutralizing antibody used to select the epitope to be investigated as potential vaccine immunogen, but they cannot be expected to be present in identical form in the host to be vaccinated. It is possible to rationally design and optimize an epitope to fit one particular antibody molecule or to improve the paratope binding efficacy of a monoclonal antibody intended for passive immunotherapy. What is not possible is to rationally design an HIV-1 vaccine immunogen that will elicit a protective polyclonal antibody response of predetermined efficacy. An effective vaccine immunogen can only be discovered by investigating experimentally the immunogenicity of a candidate molecule and demonstrating its ability to induce a protective immune response. It cannot be discovered by determining which epitopes of an engineered antigen molecule are recognized by a neutralizing monoclonal antibody. This means that empirical immunogenicity trials rather than structural analyses of antigens offer the best hope of discovering an HIV-1 vaccine.
Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, P < 0.05). The ear fibroblasts of the transgenic lambs transfected with the PsiCheck2-VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the “Sleeping Beauty” transposon system is an efficient method to produce transgenic animals.
Residues identified as part of epitopes on structural proteins across the six tested serotypes of FMDV, along with corresponding positions on all serotypes.
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Despite the impressive advances that have been made in recent years in our understanding of the mechanisms of immune responses to pathogenic organisms and the antigenic and chemical structures of the organisms themselves, these have yet to result in the widescale introduction of novel vaccines. In both veterinary and human fields the vaccines in use today are basically similar to those developed at the dawn of the era of vaccination and fall into two groups: killed vaccines, in which the virulent pathogen is rendered innocuous by chemical or physical inactivation, and attenuated vaccines, in which the virulence of the pathogen is reduced by laboratory culture in a largely empirical fashion. One exception is the surface antigen vaccine against hepatitis B virus, which is expressed in yeast and is the only practical vaccine to have emerged so far from the application of recombinant DNA techniques (57).
Foot-and-mouth disease (FMD) is an acute systemic disease of cloven-hooved animals, including cattle, swine, sheep, and goats. Despite mortality rates being generally below 5%, FMD severely decreases livestock productivity and trade. It is considered the economically most important disease of farm animals. Near two thousand million doses of vaccine are used annually to try to control FMD, which, nevertheless, is enzootic in most South American and African countries, parts of Asia, the Middle East, and the south of Europe. The causative agent, foot-and-mouth disease virus (FMDV), is an aphthovirus of the family Picornaviridae, a historically important virus as it was the first recognized viral agent (Loeffler and Frosch, 1898). In this chapter we review briefly the structure of FMDV and the organization and expression of its genome (Section II) and, in more detail, recent results on genetic variability (Section III) and antigenic diversity (Section V), reflected in several serotypes and subtypes of the virus. Such a diversity has implications for vaccine design and disease control, as discussed in Section V. Finally, we propose a model of evolution of FMDV and discuss its implications (Sections VI and VII).
Antigenic variants offoot-and-mouth disease virus (FMDV)were generated andfrequently becamedominant inclonal populations ofFMDV (clone C-S8cl) grown intheabsence ofanti-FMDV antibodies. We havenow passaged eight samples ofthesame FMDV clone inthepresenceofalimited amountofneutralizing polyclonal antibodies directed tothemajorantigenic site A ofcapsid protein VP1.Complexpopulations ofvariants showing increased resistance topolyclonal seraandtosite A-specific monoclonal antibodies were selected. Some populations exhibited markeddecreases inviral fitness. Multiple aminoacidreplacements within site A-and alsoelsewhere inVP1-accumulated upon passageofthevirus ineither theabsence or thepresenceof neutralizing antibodies. However, antigenically critical replacements atone position insite A occurred repeatedly inFMDV passaged underantibody selection, buttheywere never observed inmany passages carried outeither intheabsence ofantiviral antibodies or inthepresenceofan irrelevant antiviral serum. Thus, even though antigenic variation ofFMDV can occurintheabsence or presenceofimmuneselection, critical replacements whichlead toimportant changes inantigenic specificity wereobserved onlyasa result of selection byneutralizing antibodies. RNA virus populations replicate ascomplex distributions ofnonidentical butclosely related genomestermedviral quasispecies (13-19, 21,26,27). Extreme genetic heteroge- neity istheresult ofhighmutation rates-estimated on average as10-to10-' substitutions pernucleotide site per roundofcopying-during RNA genomereplication and retrotranscription
IntroductionThe Uniqueness of the Autoantibody-defined Antigenic Determinant or the Autoepitope The Highly Conserved Nature of the AutoepitopeThe Autoepitope Resides at or in Close Proximity to the Functional Region or Binding Site of the AntigenThe Autoepitope is Composed of Conformation-dependent Discontinuous Sequences of the AntigenConclusions The Highly Conserved Nature of the AutoepitopeThe Autoepitope Resides at or in Close Proximity to the Functional Region or Binding Site of the AntigenThe Autoepitope is Composed of Conformation-dependent Discontinuous Sequences of the Antigen
Structural studies of foot-and-mouth disease virus (FMDV) have largely focused on the mature viral particle, providing atomic
resolution images of the spherical protein capsid for a number of sero- and sub-types, structures of the highly immunogenic
surface loop, Fab and GAG receptor complexes. Additionally, structures are available for a few non-structural proteins. The
chapter reviews our current structural knowledge and its impact on our understanding of the virus life cycle proceeding from
the mature virus through immune evasion/inactivation, cell-receptor binding and replication and alludes to future structural
targets.
A set of four monoclonal antibodies which neutralized the infectivity of Mengo virus was used to select 20 non-neutralizable (escape) mutants. Altered amino acids were identified by sequence analyses of the capsid-coding regions of the mutant virus genomes. Mutations were found predominantly in proteins VP2 and VP3, while mutations in VP1 were detected only as second mutations. The Mengo virus VP2 mutations at amino acid residues 2144, 2145, 2147, and 2148 align with site Nlm II in human rhinovirus-14 and site 2 in polioviruses 1 and 3. The mutation at 2075 as well as those at 3057, 3061, and 3068 in VP3 correspond to site 3 in poliovirus. These alignments notwithstanding, the results of cross-neutralization experiments indicate the existence of a single composite neutralization site on the Mengo virion. Considering the three-dimensional structure of the Mengo capsid, the amino acids which are altered in the escape mutants are all exposed on the outer surface and none are found in the “pit,” the probable site for binding of a cellular receptor. The VP3 mutations are located in the VP3 “knob” and the VP2 mutations on a nearby ridge. Together these mutations define a set of epitopes within a single composite antigenic determinant which forms a crescent-shaped area around the three-fold icosahedral axes of the Mengo virion.
Many RNA viruses encode error-prone polymerases which introduce mutations into B and T cell epitopes, providing a mechanism for immunological escape. When regions of hypervariability are found within immunodominant epitopes with no known function, they are referred to as "decoy epitopes," which often deceptively imprint the host's immune response. In this work, a decoy epitope was identified in the foot-and-mouth disease virus (FMDV) serotype O VP1 G-H loop after multiple sequence alignment of 118 isolates. A series of chimeric cyclic peptides resembling the type O G-H loop were prepared, each bearing a defined "B cell xenoepitope" from another virus in place of the native decoy epitope. These sequences were derived from porcine respiratory and reproductive syndrome virus (PRRSV), from HIV, or from a presumptively tolerogenic sequence from murine albumin and were subsequently used as immunogens in BALB/c mice. Cross-reactive antibody responses against all peptides were compared to a wild-type peptide and ovalbumin (OVA). A broadened antibody response was generated in animals inoculated with the PRRSV chimeric peptide, in which virus binding of serum antibodies was also observed. A B cell epitope mapping experiment did not reveal recognition of any contiguous linear epitopes, raising the possibility that the refocused response was directed to a conformational epitope. Taken together, these results indicate that xenoepitope substitution is a novel method for immune refocusing against decoy epitopes of RNA viruses such as FMDV as part of the rational design of next-generation vaccines.
The development of vaccines for new and re-emerging pathologies and infections is based on the ability to define immunogenic epitopes. An immunogenic B-cell peptide epitope is a specific restricted antigen region that is capable of eliciting a humoral immune response and of combining with a specific site on antibodies. Using a number of experimental models and based on data from several literature reports, we identified low levels of sequence similarity to the host proteome as one of the main factors modulating the B-cell epitope repertoire in the humoral immune response. In point of fact, a low level of sequence identity to the host proteins is a common denominator unifying the composite, disparate assembly of linear peptide B-cell epitopes that has been experimentally validated and described in the literature. Here, we explore the proteomic similarity of conformational epitopes experimentally validated and described in published reports. Again, discontinuous epitopic structures formed by non-contiguous amino acid residues were found to define peptide sequences with a low level of similarity to the host. The present meta-analysis adds further significance to the immunological low-similarity theory and its clinical implications. Potentially, low-similarity peptide epitopes pave the way for novel effective vaccines in cancer, autoimmunity, and infectious diseases.
The objective of this study was to screen and identify the B cell epitopes of structural proteins of foot-and-mouth disease virus (FMDV) serotype Asia1. The complete amino acid sequence of all the four structural proteins (P1 region) was analyzed using the DNAStar Protean system. Seventeen peptides were predicted and selected as potential B cell epitopes. The potential B cell epitope genes were cloned into the pGEX-6P-1 plasmid, then expressed and purified. The resulting 17 glutathione S-transferase (GST) fusion peptides were detected by Western blot and ELISA for evaluation of their antigenicity. Six of the 17 fusion peptides were identified successfully by sera from rabbits immunized with the purified P1 polyprotein of FMDV type Asia1. The six fusion proteins were epi1-1 (VP1:(1)TTTTGESADPVT(12)), epi1-2 (VP1:(17)NYGGETQTARRLH(29)), epi1-6 (VP1:(194)TTQDRRKQEIIAPEKQTL(211)), epi2-2 (VP2:(40)EDAVSGPNTSG(50)), epi3-1 (VP3:(26)YGKVSNPPRTSFPG(39)), and epi4-2 (VP4:(30)YQNSMDTQLGDN(41)). The results of this study lay a foundation for further study of the structure and function of the structural proteins and may aid in the design of an epitope vaccine against foot-and-mouth disease (FMD) type Asia1. This study has also shown that the bioinformatics method, in combination with molecular biology methods can be used to map the B cell epitopes on viral proteins.
A significant frequency of amino acid substitutions, that affect important antigenic sites on capsid protein VP1, has been found among viral preparations used for the production and for challenge, in protection assays, of foot-and-mouth disease (FMD) vaccines. The amino acid substitutions present in one of the viruses studied abolished its reactivity with two neutralizing monoclonal antibodies that recognized different epitopes on VP1. Thus, the results obtained illustrate the high potential for antigenic variation introduced by the multiple cycles of growth usually undergone by the strains used for the production and challenge of FMD vaccines.
The term “quasispecies” describes complex distributions of replicating molecules subject to mutation and competitive selection (Eigen 1971; Eigen and Schuster 1979; Eigen and Biebricher 1988). The original theoretical concept of Eigen and colleagues concerned populations of infinite numbers of individual molecules and ideal, steady-state equilibrium conditions (recent review on the theoretical concept in Eigen et al. 1989). It is clear that in spite of their large population size, RNA viruses deviate from such idealized behaviour. However, several key features of RNA viruses such as nucleotide sequence heterogeneity, generally high mutation rates, and potential for very rapid evolution are best understood in the framework of the quasispecies concept (see the chapter by Holland et al.). Viral isolates, either in their natural niche or disturbed by adaptation to growing in cell culture, consist of a multitude of viable and defective mutants termed the “mutant spectrum” of the population. During replication, each genomic distribution is dominated by one (or several) “master sequence (s),” that generally coincides with the average or consensus sequence of the population. With the levels of genetic heterogeneity for foot- and-mouth disease virus (FMDV) documented in the following paragraphs, the master sequence often represents as little as 1% or less of the population of molecules, and it may have a brief life span. Here we review the evidence for the quasispecies structure of FMDV and its biological implications, notably the antigenic diversity of this widespread pathogen.
We studied proliferative responses of bovine T lymphocytes to foot-and-mouth disease virus (FMDV) serotypes A, O and C as well as to three peptides including the two major B-cell epitopes of FMDV (VP1[141-156] and VP1[200-213]). Peripheral blood mononuclear cells (PBMC) from cattle previously vaccinated with monovalent vaccine responded to both homotypic and heterotypic virus strains. Of 14 FMDV-specific bovine T-cell clones, which were prepared from PBMC of an animal vaccinated with the trivalent vaccine, 11 reacted to each of the three serotypes A, O and C. This indicates that several T-cell epitopes might be conserved among these serotypes. PBMC from one of two cattle immunized with VP1[141-156]KLH, one of two cattle immunized with VP1[200-213]KLH and two of three cattle immunized with CC-VP1[200-213]-PPS-VP1[141-156]-PCG responded to the homotypic virus strain. After immunizations with VP1[200-213]KLH also heterotypic responses were found. Thus, it appears that these two B-cell sites include T-cell determinants that are recognized by some cattle. However, when proliferative responses of PBMC from an animal vaccinated with the trivalent vaccine were tested, no responses were found to VP1[141-156] and VP1[200-213], whereas the response was very poor to CC-VP1[200-213]-PPS-VP1[141-156]-PCG. These results suggest that these sequences do not represent dominant T-cell epitopes and/or that T-cell reactivity towards these synthetic peptides does not completely cover the T-cell reactivity towards the fragments present after processing of the whole virus.
Foot-and-mouth disease virus (FMDV) was analysed using 30 monoclonal antibodies (MAbs) obtained from Balb/c mice immunized with FMDV C3 Resende (C3R) subtype 7 and 14 days before fusion No. 15 and 16 respectively. Fourteen MAbs were neutralizing and by means of competition radioimmuno assay it was possible to classify them into four groups. The first group consisted of MAbs specific for three sequential and three conformational epitopes. The second group consisted of MAbs specific for two conformational and for one sequential epitope. The third and the fourth groups consisted only of one MAb each, being specific for conformationally and one sequentially dependent epitope, respectively.
Six synthetic peptides corresponding to regions of bovine enterovirus (BEV), strain VG-5-27, elicited antibodies in mice which reacted with the virus in various assays. These antibodies have been characterised on the basis of their ability to (1) neutralize the virus, (2) bind to the intact virus particle in an immunoprecipitation test, (3) react with the denatured viral proteins, and (4) give immunofluorescent staining of virus infected cells. We have also determined the proportion of antipeptide antibody which binds to the virus in each case. All of the sera immunoprecipitated the virus and neutralized its activity to varying extents. Two of the sera specific for VP 1 sequences failed to react with denatured VP 1 whereas all the other antisera reacted with their respective parental proteins. All of the sera reacted with VG-5-27 infected cells in an immunofluorescence test. The proportion of antibodies to each peptide recognizing intact virus was variable and did not appear to correlate with neutralizing activity. In addition, the ability of each of the sera to react with and neutralize three other strains of the virus was analysed. With one of these strains significant cross-neutralization was observed.
This manuscript describes the design of new vaccines based on synthetic peptides. To this end, we first analyze the structural basis of antigenic reactivity and specificity and the various types of epitopes that form the mosaics of macromolecular antigens, as well as the regulatory mechanisms involved in immune recognition. A distinction is made between sequential or continuous epitopes, and discontinuous or conformational ones, which are the majority of epitopes in globular proteins. In this context it is of particular interest to identify epitopes reacting with B cells and T cells, respectively, or with cytotoxic T cells, in association with the major histocompatibility cell-surface antigens, and the role of these interactions in protective immunity. Identification of such epitopes in proteins of viral, bacterial, or parasitic organisms led to the synthesis of peptides, which when used in conjunction with appropriate carriers and/or adjuvants induced neutralizing antibodies. Particular examples are described, including: bacterial epitopes and mainly those of toxins of diphtheria, cholera, and shigella, leading not only to neutralizing antibodies but also to protective immunity against the deleterious effects of the respective toxins; parasite epitopes, such as those leading to anti-malaria vaccine, based on either the sporozoite or the merozoite stage antigens; viral epitopes leading to protective immunity, with special emphasis on influenza virus where induction of CTL is crucial; and finally, synthetic peptide vaccines against HIV, which should lead to broad specificity protective immunity while avoiding the risks of a vaccine based on the infectious agent. The rapid recent progress in this field, as described in this review, increases the prospect of constructing successful synthetic peptide vaccines in the not too distant future.
Specificity of anti-p25 antibodies produced against either whole Human Immunodeficiency Virus type 1 (HIV-1) particles in humans and chimpanzees, or against soluble forms of the protein in chimpanzees and rabbits was analyzed by ELISA using a panel of 37 long (> or = 30 residues) or shorter (9-21 residues) overlapping peptides covering the entire p25 sequence. Antibodies elicited by intact virions presented similar reactivity patterns in HIV-1-infected humans and in HIV-1-infected or immunized chimpanzees and recognized only a limited region mostly the C-terminus of the molecule. Moreover, 8 of the human sera (36%), which nonetheless reacted with high titers and avidity with native p25, did not bind to any long or short peptide. These results suggest that the majority of antibodies elicited by viral particles are presumably directed to conformational epitopes. In contrast, antibodies raised against soluble forms of p25 could react against all long peptides but one (residues 211-245) and against some short peptides, indicating that most of p25 sequence may be immunogenic under these conditions. These results suggest that the reactivity spectrum of anti p25 antibodies is rather different if they are produced against intact HIV-1 particles or the soluble protein. They also indicate that it may be possible to manipulate the specificity of the humoral immune response by using either intact virions or purified proteins.
The amino acid replacements that underlay the diversification of the main antigenic site A (VP1 residues 138 to 150) of foot-and-mouth disease virus (FMDV) of serotype C have been identified. Sixteen new VP1 sequences of isolates from 1926 until 1989 belonging to subtypes C1, C2, C3, C4, C5, and unclassified are reported. The reactivities in enzyme-linked immunoelectrotransfer blot assays of capsid protein VP1 with a panel of neutralizing monoclonal antibodies that recognize sites A or C (the VP1 carboxy-terminus) have been correlated with the amino acid sequence at the relevant epitopes. The analyses involving the immunodominant site A reveal two mechanisms of antigenic change. One is a gradual increase in antigenic distance brought about by accumulation of amino acid replacements at two hypervariable segments within site A. A second mechanism consists of an abrupt antigenic change manifested by loss of many epitopes, caused by one replacement at a critical position (particularly Ala (145)----Val or His (146)----Gln). The identification of the amino acid substitutions responsible for such large antigenic changes provides new information for the design of synthetic anti-FMD vaccines. However, the screening of isolates from six decades suggests that the virus, even within the confines of a single serotype, has exploited a minimum of its potential for antigenic variation.
The capsid protein VP1-encoding RNA regions of the foot-and-mouth disease virus isolates O1Lombardy/1946 and O2Brescia/1947 were sequenced and found to be closely related to each other and to O2Normandy/1949, despite some sequence differences. The O1Lombardy sequence was expected to be more closely related to those of the subtype O1 isolates of 1965 and later (e.g., O1Kaufbeuren/1966), but this was not the case. The serological subtyping of both the Lombardy and the Kaufbeuren isolate as O1 strains was possibly due to identical VP1 C-terminal sequences, since all the subtype O2 isolates differ here from the O1 isolates at residue 209. Considerable dissimilarity of other O1Lombardy and O2Brescia genome parts to those of O1Kaufbeuren was qualitatively shown by analyzing the sizes of RNase-treated hybrids formed with virus RNA and defined subgenomic fragments of O1Kaufbeuren-specific antisense cRNA. These hybrids were fragmented into oligonucleotides, but others containing O1Kaufbeuren virus RNA were protected.
Changes resulting in altered antigenic properties of viruses nearly always occur on their surface and have been attributed to the substitution of residues directly involved in binding antibody. To investigate the mechanism of antigenic variation in foot-and-mouth disease virus (FMDV), variants that escape neutralization by a monoclonal antibody have been compared crystallographically and serologically with parental virus. FMDVs form one of the four genera of the Picornaviridae. The unenveloped icosahedral shell comprises 60 copies each of four structural proteins VP1-4. Representatives from each of the genera have similar overall structure, but differences in the external features. For example, human rhinovirus has a pronounced 'canyon' that is proposed to contain the cell attachment site, whereas elements of the attachment site for FMDV, which involves the G-H loop (residues 134-160) and C-terminus (200-213) of VP1, are exposed on the surface. Moreover, this G-H loop, which is a major antigenic site of FMDV, forms a prominent, highly accessible protrusion, a feature not seen in other picornaviruses. It is this loop that is perturbed in the variant viruses that we have studied. The amino acid mutations characterizing the variants are not at positions directly involved in antibody binding, but result in far-reaching perturbations of the surface structure of the virus. Thus, this virus seems to use a novel escape mechanism whereby an induced conformational change in a major antigenic loop destroys the integrity of the epitope.
Five neutralizing monoclonal antibodies (nMAbs) obtained against type A5 Spain-86 foot-and-mouth disease virus were used to generate a series of neutralization-resistant variants. In vitro and in vivo assays showed that the variants were fully refractory to neutralization by the selecting nMAb. On the basis of cross-neutralization and binding assays, two neutralizing antigenic sites have been located on the virus surface; one, located near the C-terminus of VP1, displayed a linear epitope, and the second, located on VP2, displayed two conformational epitopes. Nucleotide sequencing of RNA of the parental and variant capsid protein-coding region P1 has placed the amino acid changes at position 198 of VP1 for the first site and at positions 72 and 79 of VP2 for the related epitopes in the second site. The relative importance of these two sites in the biological properties of foot-and-mouth disease virus is discussed.
Polyclonal antisera were generated against bacterially derived fusion proteins of the open reading frames (ORFs) of the capsid proteins of cottontail rabbit papillomavirus (CRPV). The carboxy-terminal two-thirds of CRPV L1 and the carboxy-terminal half of CRPV L2 were cloned into a bacterial expression vector and induced proteins were used as antigen and immunogen. The polyclonal antisera were tested in a series of immunological assays, including ELISA, Western blot, and neutralization of CRPV. ELISA demonstrated that the polyclonal antisera raised against expressed L1 proteins reacted strongly to disrupted CRPV virion antigen and weakly both to intact CRPV virion and disrupted BPV-1 virion. Anti-CRPV L2 antisera reacted strongly only to intact and disrupted CRPV virion antigen. Viral capsid proteins of CRPV were detected in Western blots of HPV-11, BPV-1, and CRPV virus particles by these polyclonal antisera. The anti-L1 sera recognized the major capsid protein (60 kDa) and the anti-L2 sera identified a 76-kDa viral protein of CRPV. Only the antisera generated against expressed L2 neutralized CRPV. The neutralizing titer of the anti-L2 sera, however, was several orders of magnitude lower than the titer of a neutralizing polyclonal antiserum that was generated by immunizations with intact CRPV virions.
Epitopes defined by monoclonal antibodies (mAb) specific for the Bordetella pertussis outer membrane protein P.69 (pertactin) were mapped using a series of amino- and carboxy-terminal deletion mutants expressed in Escherichia coli. mAb were found to bind predominantly to a region of pertactin spanning a (Pro-Gln-Pro)5 repeat motif and one mAb was found to bind to another region spanning a (Gly-Gly-Xaa-Xaa-Pro)5 repeat motif. To localize further the mAb-binding sites, a panel of synthetic peptides, a series of 94 overlapping hexameric peptides, and a P.69 30-amino acid fusion to a hepatitis B core protein (HBcAg-69), were synthesized. This combined approach has identified the binding site for the mAb BBO5: Pro-Gly-Pro-Gln-Pro-Pro; mAb BBO7, E4A8 and E4D7: Ala-Pro-Gln-Pro-Pro-Ala-Gly-Arg; and mAb BPE3: Thr-Leu-Trp-Tyr-Ala-Glu-Ser-Asn-Ala-Leu-Ser-Lys-Arg. We have used a non-lethal murine respiratory model of B. pertussis infection to investigate the ability of a peptide containing the epitope of the mAb BBO5 to elicit protective immunity. Immunization of mice with the HBcAg-69 protein prevented growth of B. pertussis in the lungs compared to mice receiving HBcAg alone, and protection correlated with high titers of anti-P.69 antibodies.
Five poliovirus recombinants containing sequences corresponding to foot-and-mouth disease virus (FMDV) antigenic sites were constructed. Viable virus was recovered from four of these plasmids, in which the VP1 beta B-beta C loop (antigenic site 1) of poliovirus type 1 Sabin had been replaced with sequences derived from the VP1 beta G-beta H loop (antigenic site 1) of FMDV O1 Kaufbeuren (O1K), chimera O1.1 (residues 141 to 154), chimera O1.2 (residues 147 to 156), and chimera O1.3 (residues 140 to 160) or from the beta B-beta C loop of VP1 (antigenic site 3) in chimera O3.1 (residues 40 to 49). One chimera (O1.3) was neutralized by FMDV-specific polyclonal serum and monoclonal antibodies directed against antigenic site 1 of FMDV. Chimeras O1.3 and O3.1 induced site-specific FMDV-neutralizing antibodies in guinea pigs. Chimera O1.3 was capable of inducing a protective response against FMDV challenge in some guinea pigs.
Four categories of viral epitopes can be distinguished that have been designated cryptotopes, neotopes, metatopes and neutralization epitopes. Specific examples of each epitope type are presented and the methods used for locating their positions in viral proteins are described. The epitopes of four well-characterized viruses, namely poliovirus, foot-and-mouth disease virus, influenza virus and tobacco mosaic virus are briefly described.
A novel yet simple method is described that facilitates the synthesis of large numbers of peptides to the extent that the synthesis process need no longer be the limiting factor in many studies involving peptides. By using the methods described, 10-20 mg of 248 different 13-residue peptides representing single amino acid variants of a segment of the hemagglutinin protein (HA1) have been prepared and characterized in less than 4 weeks. Through examination of the binding of these analogs to monoclonal antibodies raised against residues 75-110 of HA1, it was found that a single amino acid, aspartic acid at position 101, is of unique importance to the interaction. Two other residues, aspartic acid-104 and alanine-106, were found to play a lesser but significant role in the binding interaction. Other single positional residue variations appear to be of little or no importance.
A collection of 35 mouse monoclonal antibodies, raised against human rhinovirus 14 (HRV-14), was used to isolate 62 neutralization-resistant mutants. When cross-tested against the antibodies in a neutralization assay, the mutants fell into four antigenic groups, here called neutralization immunogens: NIm-IA, -IB, -II, and -III. Sequencing the mutant RNA in segments corresponding to serotype-variable regions revealed that the amino acid substitutions segregated into clusters, which correlated exactly with the immunogenic groups (NIm-IA mutants at VP1 amino acid residue 91 or 95; NIm-II mutants at VP2 residue 158, 159, 161, or 162; NIm-III mutants at VP3 residue 72, 75, or 78; and NIm-IB mutants at two sites, either VP1 residue 83 or 85, or residue 138 or 139). Examination of the three-dimensional structure of the virus (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London], 317:145-153, 1985) revealed that each of the substitution clusters formed a protrusion from the virus surface, and the side chains of the substituted amino acids pointed outward. Moreover, four of the amino acid substitutions, which initially appeared to be anomalous because they were encoded well outside the cluster groups, could be traced to surface positions immediately adjacent to the appropriate viral protrusions. We conclude that three of the four antigens, NIm-IB, -II, and -III, are discontinuous. Thus, the amino acid substitutions in all 62 mutants fell within the proposed immunogenic sites; there was no evidence for alteration of any antigenic site by a distal mutation.
Seven neutralizing monoclonal antibodies were used to characterize 30 escape mutants of a type O foot-and-mouth disease (FMD) virus (O1 Kaufbeuren) selected with the five most active antibodies. Three non-overlapping antigenic sites were found by ELISA and cross-neutralization studies. Within two of the sites the epitopes of two or more monoclonal antibodies overlapped. Two of the sites were conformation-dependent and could not be detected on virus subunits or isolated denatured polypeptides. The third site was less conformation-dependent since the appropriate monoclonal antibodies were able to bind to 12S subunits, isolated VP1 protein and a synthetic peptide containing residues 141 to 160 of VP1 in ELISA. Electrofocusing of mutants of that site showed a high frequency of electrophoretic alterations in VP1. The sequence of most or all of the VP1 coding region of 10 escape mutants of that site plus three parental isolates was determined by primer extension sequencing. At least five amino acids were found to be involved but in only one case (residue 148 of VP1) did a change at that residue produce complete resistance to neutralization. Partial resistance was produced by changes at residues 144, 154 or 208 of VP1 or another residue(s), as yet undefined, that is probably in one of the other capsid polypeptides. Thus the site defined by these mutants was made up of at least three regions, the region involving residues 144 to 154 of VP1, the region encompassing residue 208 from the COOH terminus of VP1, plus a region, probably of VP2 or VP3, encompassing the undefined residue(s).
We have molecularly cloned a natural variant of feline leukemia virus subtype B. This isolate is unique in that it is not neutralized by a monoclonal antibody which neutralized all other feline leukemia virus isolates tested, including members of the A, B, and C subtypes. Western immunoblotting indicated that the monoclonal antibody was less able to bind to the gp70 of the resistant isolate (designated lambda B1) than to the gp70s of susceptible viruses. Nucleotide sequence analysis of the envelope gene of lambda B1 revealed a high degree of homology with the susceptible Snyder-Theilen, Gardner-Arnstein, and Rickard subtype B isolates, including the presence of a 5-amino-acid minimal binding epitope required for binding by the neutralizing monoclonal antibody. The only change within the vicinity of this epitope was in a single nucleotide, and this difference changed a proline residue to leucine three amino acids from the N terminus of the binding epitope. Competitive binding studies with synthetic peptides indicated that substitution of leucine for proline resulted in a 10-fold decrease in the ability of the peptide to compete for antibody binding to native antigen. The results are consistent with the interpretation that this amino acid change lowers the affinity of antibody binding, resulting in failure of the antibody to neutralize the variant virus.
Antigenic mutants of poliovirus (Sabin strain, serotype 1) were isolated by the resistance of the virus to anti-Sabin neutralizing monoclonal antibodies. The amino acid replacements within the capsid protein sequence causing the altered antigenicity were identified for each of 63 isolates. The mutations cluster into distinct nonoverlapping peptide segments that group into three general immunological phenotypes on the basis of cross-neutralization analyses with 15 neutralizing anti-Sabin monoclonal antibodies. Location of the mutated amino acid residues within the three-dimensional structure of the virion indicates that the majority of these amino acid residues are highly exposed and located within prominent structural features of the viral surface. Those mutated amino acid residues that are less accessible to antibody interaction are often involved in hydrogen bonds or salt bridges that would stabilize the local tertiary structure of the antigenic site. The interactions of the peptide segments that form these neutralizing sites suggest specific models for the generation of neutralization-resistant variants and for the interaction between the viral surface and antibody.
Foot-and-mouth disease virus challenge experiments in guinea-pigs and immunoassays with a range of peptides equivalent to either or both of the sequences 141 to 158 and 200 to 213 of VP1 showed the most effective structure, in terms of protection, to be one in which both 'sites' were present with a minimum of additional amino acids. An 80 residue peptide comprising amino acids 134 to 213 was considerably less effective than 40 or 45 residue peptides. The major site for the induction of protection was deduced to be in the region 141 to 158. Thus, protection with the 40 or 45 residue peptide did not appear to be due to the presence of antibody directed solely to the 200 to 213 sequence. Finally, induction of antibody to the latter site appeared to be dependent on both the size of the peptide and the disposition of 'sites' within it.
This paper describes the reactions of a number of monoclonal antibodies produced against purified whole virions of foot-and-mouth disease virus in 3 different enzyme immunoassay systems. The first system used whole virus bound non-covalently to microplates; the second used whole virus trapped by a polyclonal antibody which was bound to microplates; and the third allowed the monoclonal antibodies to react with the whole virions in suspension (liquid phase) before trapping by the solid-phase-bound polyclonal antibody. Different reactions with panels of monoclonal antibodies were observed depending on which system was used. Such variations in reactivity give an insight into the alterations in the expression of virus epitopes in the different enzyme immunoassay systems. The reactions of selected monoclonal antibodies were used to illustrate these changes and the results compared to those obtained in similar systems using polyclonal antisera produced against isolated virion polypeptides.
The antigenic structure of tobacco mosaic virus has been analysed by measuring the ability of nine monoclonal antibodies to distinguish between wild-type virus and 13 mutants showing single and double amino acid substitutions in the coat protein. Although the majority of antibodies detected those substitutions that were located at the outer surface of the virion, some of them also recognized conformation alterations induced by exchanges occurring deep inside the subunit. In the case of five mutants, the antibody reactivity was reduced compared with wild-type virus, while in the case of three others, it was significantly higher. Each monoclonal antibody possessed a unique discrimination pattern with respect to the different substitutions. The simultaneous presence of two exchanges led to the complete disappearance of any binding with six of the nine antibodies and to reduced binding with three others. The superior discriminatory capacity of monoclonal antibodies compared with polyclonal antisera was demonstrated by the fact that three exchanges not detected with antisera were found to alter the antigenicity when tested with monoclonal antibodies.
Cloned cDNA molecules from three serotypes of FMDV have been sequenced around the VP1-coding region. The predicted amino acid
sequences for VP1 were compared with the published sequences and variable regions identified. The amino acid sequences were
also analysed for hydrophilic regions. Two of the variable regions, numbered 129-160 and 193-204 overlapped hydrophilic regions,
and were therefore identified as potentially immunogenic. These regions overlap regions shown by others to be immunogenic.
Purified foot-and-mouth disease virus (FMDV) to type O1K was treated with several endopeptidases of differing specificity. The immunizing protein VPThr was cleaved into two detectable fragments by all enzymes except for glutamic acid-specific Staphylococcus aureus V8 protease. The longest fragments were generated by mouse submaxillary gland protease and the shortest by trypsin treatment of the intact virion. Several fragments, including the peptides resulting from the cyanogen bromide (CNBr) cleavage of the isolated protein VPThr were characterized in terms of their molecular weights N- and C-terminal amino acids, and ability to induce virus-specific antibodies. The order of the fragments along the protein was determined, and then located on the amino acid sequence of the protein. Two enzyme-sensitive areas of the protein were found on the surface of the virion: between sequence positions 138 and 154 and between portion 200 and the C terminus. Peptides containing these sections were able to induce neutralizing antibodies against the homologous FMDV. When the virus was treated with trypsin or with chymotrypsin, several amino acids between the detectable fragments were lost and the infectivity of the virus was reduced. The infectivity was retained, however, when the enzyme treatment resulted in cleavage of protein with no loss of amino acids or only the cutting away of the C-terminal section. These results suggest that the property of cell attachment is restricted to small regions of the surface of the virus particle.
The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.
Mutations have been identified in variants of poliovirus, type 1 (Mahoney) on the basis of their resistance to neutralization
by individual monoclonal antibodies. The phenotypes of these variants were defined in terms of antibody binding; the pattern
of epitopes expressed or able to be exploited for neutralization were complex. Single amino acid changes can have distant
(in terms of linear sequence) and generalized effects on the antigenic structure of poliovirus and similarly constituted virions.
Eleven neutralizing monoclonal antibodies (MAbs) were produced to the O1BFS 1860/67 strain of foot-and-mouth disease virus (FMDV), and were characterized for their ability to bind viral and subviral antigens in different ELISA tests and to neutralize heterologous type O isolates. Neutralization escape variants of the homologous virus, isolated under pressure from five of these MAbs, were used in cross-neutralization tests with all of the 11 antibodies. These studies identified three functionally independent, conformational, neutralizing sites. The most conformationally dependent site bound antibody which neutralized a range of type O virus isolates. A second site was less dependent on conformation and was recognized by antibody that was strain-specific. The least conformational site bound MAbs which showed limited cross-neutralization of other type O strains. This latter site appeared to be immunodominant and contained several overlapping epitopes which showed some differences in their specificities. Isoelectrofocusing and sequencing studies of the variants strongly suggested that polypeptide VP2 contributes to the immunodominant site.
The structure of foot-and-mouth disease virus has been determined at close to atomic resolution by X-ray diffraction without experimental phase information. The virus shows similarities with other picornaviruses but also several unique features. The canyon or pit found in other picornaviruses is absent; this has important implications for cell attachment. The most immunogenic portion of the capsid, which acts as a potent peptide vaccine, forms a disordered protrusion on the virus surface.
The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.
Antisera were raised against the chemically synthesized peptide corresponding to each epitope of three foot-and-mouth disease virus strains. Peptide synthesis was further used to determine which amino acid residues in each epitope are important for the specificity of antisera raised against the whole virus. The specificity of the antibody paratope for its epitope was shown to depend on structure as well as sequence. Anti-virus sera demonstrated a greater specificity for the homologous peptide than did the anti-peptide sera. Two of the three peptides were able to induce neutralizing antibodies against the homologous virus. The specificities of the antibodies present in the anti-peptide sera were also inferred from the reactions of each with related sets of peptides. The cross-reactions observed for the anti-peptide sera were readily explained in terms of the antibody specificities determined to be present. The findings also suggest that the diversity of antibodies raised against small peptides is limited and is determined by the immune system. A similar limited response to the native protein was observed, which may account for the high frequency with which anti-peptide sera react with the native homologous protein.
Foot-and-mouth disease virus has been crystallized with the objectives of (1) determining the composition and conformation of the major immunogenic site(s) and (2) comparing its structure with those of the related polio, rhino and Mengo viruses, representing the other three genera of the picornaviruses. Most of the work has been done with virus strain O1BFS 1860, which crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. Virus recovered from crystals was infectious, and was indistinguishable from native virus both in protein composition and buoyant density. The stability of the crystals in the X-ray beam was comparable with that of other picornavirus crystals and they diffracted to a resolution of better than 2.3 A. Initial analysis of the X-ray diffraction data shows the virus to be positioned on a point of 23 symmetry in a close-packed array so that examples of all the icosahedral symmetry elements, except the 5-fold axes, are expressed crystallographically. The cell dimensions are a = b = c = 345 A, alpha = beta = gamma = 90 degrees, with a space group of I23. The diameter of the virus particle is 300 A. Despite the small size of the crystals, diffraction data have been collected to a reasonable resolution using a synchrotron source. Phasing of the diffraction data will be attempted using the methods of molecular replacement.
Synthetic peptides representing the amino acid sequence 141-160 of the structural protein VP1 of foot-and-mouth disease virus (FMDV) elicit virus-neutralizing antibody. Absorption of anti-peptide sera with purified virus particles removed all detectable virus-binding and neutralizing activity, and reduced the ELISA titres against the homologous peptide by 31-41%. The proportion of anti-peptide antibodies that also recognized virus was unaffected by whether the peptide had been inoculated free, carrier-linked or as part of a fusion protein. The majority of these antibodies reacted with sites composed of residues 142-150. Peptides extended at the amino terminus, into regions shown to be poorly antigenic on the intact virus, induced greater neutralizing responses by increasing the proportion of virus-binding antibodies recognizing region 141-150 from 35% to 70%. However, the total proportion of activity against the longer homologous peptide removed by virus absorption remained within the range 31-41%.
The three-dimensional structure of poliovirus has been determined at 2.9 A resolution by x-ray crystallographic methods. Each of the three major capsid proteins (VP1, VP2, and VP3) contains a "core" consisting of an eight-stranded antiparallel beta barrel with two flanking helices. The arrangement of beta strands and helices is structurally similar and topologically identical to the folding pattern of the capsid proteins of several icosahedral plant viruses. In each of the major capsid proteins, the "connecting loops" and NH2- and COOH-terminal extensions are structurally dissimilar. The packing of the subunit "cores" to form the virion shell is reminiscent of the packing in the T = 3 plant viruses, but is significantly different in detail. Differences in the orientations of the subunits cause dissimilar contacts at protein-protein interfaces, and are also responsible for two major surface features of the poliovirion: prominent peaks at the fivefold and threefold axes of the particle. The positions and interactions of the NH2- and COOH-terminal strands of the capsid proteins have important implications for virion assembly. Several of the "connecting loops" and COOH-terminal strands form prominent radial projections which are the antigenic sites of the virion.
A chemically synthesized peptide consisting essentially of two separate regions (residues 141 to 158 and 200 to 213) of a virus coat protein (VP1) from the O1 Kaufbeuren strain of foot-and-mouth disease virus was prepared free of any carrier protein. It elicited high levels of neutralizing antibody and protected cattle against intradermolingual challenge by inoculation with infectious virus. Comparative evaluation of this peptide with a single-site peptide (residues 141 to 158) in guinea pigs suggests the importance of the VP1 carboxyl terminal residues in enhancing the protective response.
Uncoupled synthetic peptide representing the sequence of amino acids 141-160 of foot-and-mouth disease virus (FMDV) protein VP1 induced a virus-neutralizing antibody response in guinea-pigs. This response required incomplete Freund's adjuvant (IFA) for the primary inoculation and was dependent on the presence of an added cysteine residue with an unblocked sulphydryl group at the carboxy-terminus. Secondary immunization could be carried out in the absence of adjuvant. A study of the relative activities of nested sets of uncoupled peptides from 150-160 to 135-160 and 141-160 to 141-155 indicated that amino acids 146-156 were critical for the induction of virus-neutralizing antibodies and that extension to 137-160 further improved this response. Results of in vitro proliferation studies demonstrated that the carboxy-terminal residues on this peptide may form a T-cell epitope. The significance of these observations in the broader context of synthetic peptide vaccines is discussed.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The epitopes of six monoclonal antibodies generated against type A12 foot-and-mouth disease virus (FMDV) VP1 or its largest cyanogen bromide fragment (13 kd) were characterized. Five of these monoclonal antibodies neutralized viral infectivity. Solid-phase and competitive antigen binding assays using virion-derived antigens or a biosynthetic VP1 polypeptide identified two distinct neutralizing epitopes. One epitope was located between amino acid residues 145-168 of VP1 and the other between amino acids 169-179. The results indicate that antibodies reacting with two distinct areas of the VP1 polypeptide are capable of neutralizing FMD virus.
We have determined a major antigenic site for virus neutralization on the capsid protein VP1 of poliovirus type 3. Antigenic mutant viruses selected for resistance to individual monoclonal antibodies had point mutations concentrated in a region 277-294 bases downstream from the start of the region of viral RNA coding for VP1. These findings provide the basis for an improved understanding of the molecular basis of virus neutralization.
A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.
Chemically synthesized peptides corresponding to two different regions
of the VP1 polypeptide of foot-and-mouth disease virus (FMDV) produced
high levels of serotype-specific virus neutralizing antibody in cattle,
guinea pigs and rabbits. A single inoculation of one of these peptides
protected guinea pigs against subsequent challenge with the virulent
virus.
Investigations on the purification of small quantities of radioactively
labeled foot-and-mouth disease (FMD) virus are described. The results indicated
that there is present in untreated virus suspensions and pellets a complex of the
7-m mu component and cellular material, which has the same sedimentation
characteristics in sucrose density gradients as the 25-m mu infective component
but is disrupted by deoxycholate into smaller components that sediment much more
slowly in the sucrose gradient. The ratio of plaque-forming units and complement-
fixing actlvity to radioactive counts in the individual fractions in sucrose
gradients of deoxycholate-treated pellets was remarkably constant, indicating
that the material in the peak was homogeneous. The experiments provided
substantial evidence that the virus obtained from deoxycholate-treated pellets is
free from substances found in uninfected cells. (P.C.H.)
Neutralisation en culture cellulaire du pouvoir infectieux du virus de la fi6vre aphteuse par des s6rums provenant de pores immunis6s ~ l'aide d'une prot6ine virale purif6e
- J Grosclaude
- L Wantyghem
- S Rouze
LAPORTE, J., GROSCLAUDE,,L, WANTYGHEM, L, BERNARD, S. & ROUZE, P. (1973). Neutralisation en culture cellulaire
du pouvoir infectieux du virus de la fi6vre aphteuse par des s6rums provenant de pores immunis6s ~ l'aide
d'une prot6ine virale purif6e. Comptes rendus hebdomadaires des s~ances de l'Acad~mie des sciences 276D,
3399-3402.
Identification of neutralizing epitopes of foot-and-mouth disease virus. In Vaccines '85; Molecular and Chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases
- N R Ouldridge
- E J Barnett
- P V Rowlands
- D J Brown
- F Bittle
- J Houghten
- R A Lerner
PARRY, N. R., OULDRIDGE, E. J., BARNETT, P. V., ROWLANDS, D. J., BROWN, F., BITTLE, J., HOUGHTEN, R. A. & LERNER,
R. A. (1985). Identification of neutralizing epitopes of foot-and-mouth disease virus. In Vaccines '85;
Molecular and Chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases, pp. 211-216. Edited by
R. A. Lerner, R. M. Chanock & F. Brown. New York: Cold Spring Harbor Laboratory.
Antibodies against a preselected peptide recognise and neutralise foot-and-mouth disease virus
- E Mussgay
- M Bohm
- H O Schulz
- G F Challer
PFAFF, E., MUSSGAY, M., BOHM, H. O., SCHULZ, G. F. & $CHALLER, H. (1982). Antibodies against a preselected peptide
recognise and neutralise foot-and-mouth disease virus. EMBO Journal 1, 869-874.