Rev. sci. tech. Off. int. Epiz., 2000,19 (3), 744-753

Identification of antigenic epitopes on the foot and mouth disease virus isolate O1/Manisa/Turkey/69 using monoclonal antibodies S.Aktas(

1)

& A.R. S a m u e l (

2)

(1) Sap Foot and Mouth Disease Institute, PK 714 06044, Ulus, Ankara, Turkey (2) Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom Submitted for publication: 21 December 1999 Accepted for publication: 4 February 2000

Summary A panel of mouse monoclonal antibodies (MAbs) w a s produced against a strain of type 0 foot and mouth disease virus (FMDV) from the Middle East, 01/Manisa/Turkey/o9. Seven neutralising MAbs w e r e fully characterised and all w e r e found to react with conformational epitopes. Monoclonal antibody neutralisation-resistant mutants (MARMs) w e r e generated from the parental virus stock and the complete capsid sequences of these M A R M s w e r e determined. Sequence analysis revealed that five of the M A R M s had amino acid substitutions at either residue 72 or 73 of VP2 (ßB-ßC loop), indicating that five of the MAbs w e r e directed against antigenic site 2. The sixth M A R M contained a single amino acid substitution at position 198 of VP1 (carboxy-terminal region). The seventh M A R M contained two amino acid substitutions, at position 72 of VP2 and position 149 of VP1 (ßG-ßH loop). These findings indicate that MAbs directed against a type 0 FMDV from the Middle East recognise residues in the same structural features to those raised against strains from Europe of the same serotype.

Keywords Antigenic sites - Escape mutants - Foot and mouth disease virus - Middle East Monoclonal antibodies - Sequencing.

Introduction Antigenic sites and epitopes which form the neutralising sites of Picornaviridae have been studied extensively using monoclonal antibodies (MAbs) ( 2 3 , 2 6 , 3 6 ) . The isolation of neutralising MAb escape mutants and sequence analysis is the most informative technique for identifying neutralising epitopes. The deduced amino acid sequence of a mutant is compared to that of the parental virus, thus allowing identification of the substitutions that permit evasion of MAb binding. These techniques have been used to identify the neutralising antigenic sites of foot and mouth disease virus (FMDV). Much of this work has been performed using hybridomas derived from the mouse, although both the

mouse and bovine immune systems recognise similar structural features on the surface of the virion of FMDV (3, 2 5 ) . Given that a MAb reacts with a definable sequence (epitope) on the virus surface, reactivity or non-reactivity of a MAb in immunoassays can be used as an indication of antigenic homology for that site when heterologous viruses are profiled ( 1 6 ) . Sequencing efforts may then be directed towards areas of the genome coding for antigenic sites. Several enzyme-linked immunosorbent assay (ELISA) methods have been described for screening isolates of FMDV for reactivity with MAbs ( 1 1 , 3 1 ) . However, although the importance of some individual residues within an epitope has been documented ( 8 , 1 4 , 24), the relative importance of individual neutralising antigenic sites (excluding linear epitopes within the ßG-ßH loop of VP1) has not been unequivocally defined.

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The data from MAb analysis has been augmented by X-ray crystallographic structural studies for several Picornaviridae which indicate that amino acid substitutions in the capsid proteins are found in clusters in loops linking ß-sheets which project from the virus surface ( 1 , 13, 29). Numerous epitope mapping studies have been performed with Aphthovirus ( 2 , 4 , 6, 8, 12, 19, 2 1 , 2 8 , 3 3 , 3 5 , 3 7 ) . Kitson et al identified four independent antigenic sites involving the three capsid proteins of the O1/Kaufbeuren/FRG/66 strain of FMDV (15) and later, a fifth neutralising site on type O was described (8). Site 1, located in the centre of the protomer includes the ßG-ßH loop (residues 1 4 4 - 1 5 4 ) and the carboxy terminal region (residue 2 0 8 ) of protein VP1. Structurally, the carboxy terminus of one VP1 molecule lies across an adjacent protomer, bringing these residues near to the base of the ßG-ßH loop to form one antigenic site. Initially, the structure of the ßG-ßH loop was not resolved in crystallography studies due to an area of diffuse electron density at the position the loop usually occupies. This was due to the highly flexible nature and the ability of the loop to adopt a number of positions above the capsid (1). This flexibility has been postulated as a mechanism to effect antigenic variation (27). The structure of the loop was resolved by study under reducing conditions ( 2 0 ) . Site 3, involving the ßB-ßC loop (residues 4 3 - 4 5 ) of VP1, is located near the five-fold axis of the virus particle. Site 2, at the three-fold axis, involves the ßB-ßC loop (residues 7 0 - 7 7 ) and the adjacent ßE-aB loop (residue 1 3 1 ) of VP2. Site 4 , also close to the three-fold axis, consists of residues 5 6 - 5 8 of VP3, located at the top of the insertion within ßB. Site 5 is a conformational epitope which is comprised of, at least in part, the ßG-ßH loop of VP1 ( 8 ) . Most of the MAbs used to map the neutralising epitopes of serotype O FMDV have been elicited against O strains from Europe ( 2 , 6 , 8 , 1 5 , 1 8 , 2 1 , 2 8 , - 3 3 ) . The present paper reports the results of the production and characterisation, by escape mutant analysis, of MAbs raised against a reference strain which represents a genetic lineage from the Middle East, namely, O1/Manisa/Turkey/69. This virus strain has been used extensively as a vaccine strain, not only in the Middle East, but also elsewhere. The complete sequence of the capsid coding region of O /Manisa/Turkey/69 is published for the first time and is compared to that of O /Kaufbeuren/FRG/66 protein sequence (10). This is the first report of this type of study for a virus representing a genetic lineage from the Middle East. l

1

Immunisation protocol Six-week-old BALB/c mice were inoculated subcutaneously, with 10 µg of inactivated, purified 146S O /Manisa/ Turkey/69 virus particles emulsified in an equal volume of Freund's complete adjuvant. After twenty-eight days, the immunisation was repeated with Freund's incomplete adjuvant (FICA). On day fifty-five, the mice were inoculated a third time with antigen alone, both intravenously and intraperitoneally (i.p.). Three days after the final inoculation, the mice were killed and the spleens removed. Test bleeds were taken on days fourteen, twenty-eight and fifty-eight post inoculation. 1

Fusion of immune lymphocytes and myeloma cells The fusion and cloning were based on the method described by Butcher et al. (7) with amendments as described by Thevasagayam (34).

Production of monoclonal antibody ascitic fluid BALB/c mice were sensitised i.p. with 0.3 ml of FICA to avoid the formation of solid tumours. Three days later, the mice were inoculated i.p. with 1 x 10 /ml hybridoma cells. After approximately ten days, the ascitic fluids were removed from the abdominal cavity by syringe, clarified by centrifugation at 200 g for 5 min at room temperature and stored at —20°C. 6

Isolation of monoclonal antibody resistant mutants Seed stocks of the parental virus, O1/Manisa/Turkey/69, were grown to a titre of > 1 0 cell culture infective dose ( C C I D ) , in IB-RS-2 cells. The monoclonal antibody resistant mutants (MARMs) were then isolated and plaque purified according to the technique described in Crowther et al. ( 8 ) . The culture supernatants were collected and clarified, and reactivity with the selecting MAbs was determined by the antigenic profiling ELISA (see below) ( 3 1 ) . Stable mutants were stored in Dulbecco modified Eagle's medium and glycerol (1:1) at -20°C. 6

50

1

Materials and methods Preparation of virus for monoclonal antibody production O1/Manisa/Turkey/69 was grown in IB-RS-2 cells, inactivated with binary ethylenimine and purified on a 1 5 % - 4 5 % sucrose density gradient (9).

Sequencing Ribonucleic acid (RNA) extraction, reverse transcription of virion RNA, polymerase chain reaction (PCR) and cycle sequencing using an automated sequencer were all performed as described by Knowles et al. (17). The PCR utilised primers located in the 2B and L genes (NK61 and L-463F, respectively) to amplify a 2 , 4 7 4 base pair product ( 3 0 ) . Sequences were aligned manually and translated using the programme Episeq (written by N.J. Knowles, Pirbright). The program RASMOL ( 3 2 ) was employed to visualise the surface accessibility of the antibody contact residues, using data from Acharya et al. (1) and Logan et al. ( 2 0 ) .

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Rev. sci. tech. Off. int. Epiz., 19 (3)

that they recognised conformational epitopes. Only the binding of MAbs SA176 and SA177 were sensitive to proteolytic cleavage of the virus by trypsin, which suggests that these MAbs recognise epitopes located in VP1 (Table I).

Antigenic profiling enzyme-linked immunosorbent assay using monoclonal antibodies The phenotype of the escape mutants was checked by a trapping ELISA using a previously described method (31). Briefly, plates were sensitised with a limiting dilution of a serotype-specific rabbit antisera. After washing, tissue culture supernatants from FMDV-infected IB-RS-2 cells were added to the plates and incubated for 1 h. After incubation and washing, a panel of eight neutralising mouse MAbs were added to the plates. Monoclonal antibody binding was then detected by the addition of rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase, o-phenylenediamine dihydrochloride chromogen and H (substrate). Colour development was halted after 10 min using 1.25 M H S O and the absorbance of each well was measured spectrophotometrically at 4 9 2 nm.

Escape mutants were isolated using seven of the neutralising O1/Manisa/Turkey/69 MAbs produced. Three plaques were selected for each mutant and these were analysed by nucleotide sequencing. On examination of the deduced amino acid sequences (Fig. 1), the substitutions detailed in Table I were apparent. The MAbs SA113, SA107, SA98, SA127 and SA84 recognise a conformational epitope which involves the ßB-ßC loop of VP2. The escape mutant to MAb 85 has two substitutions, one at V P 2 and the other at V P 1 . The escape mutant to MAb 176 has a substitution at the carboxy terminus of VP1 at residue V P 1 . Binding data for MAb 177 suggested that this MAb recognised an identical or similar epitope to MAb 176 (data not shown). 7 2

2 O 2

2

1 4 9

1 9 8

4

Effects of trypsin treatment and denaturation of the virus on monoclonal antibody binding Assessment of the reactivity of the MAbs with trypsin-treated and denatured virus was performed according to the method described in Crowther et al. (8). For trypsin treatment, 5 0 µl of 2 mg/ml trypsin was added to 100 µg sucrose density gradient purified O /Manisa/Turkey/69 FMDV and incubated at 37°C for 15 min. To prepare denatured virus, 10 mg (1%) sodium dodecyl sulphate and 20 µl ( 2 % ) of 2-mercaptoethanol were added to 1 ml of 5 0 µg/ml O1/Manisa/Turkey/69 FMD, and the mixture incubated for 3 min at 95°C. These antigen preparations were then used in an indirect ELISA (22).

The ELISA binding data suggested that MAbs SA84, SA98, SA107 and SA127 recognised similar, if not identical, epitopes. The same is true for SA176 and SA177. However, MAbs 85 and SA113 recognise epitopes which are distinct from the other groups and from each other (Fig. 2 ) .

1

Results Although many clones were successfully propagated after the fusion, seven MAbs were selected for more extensive study. None of these MAbs reacted with denatured virus, indicating

Discussion The MAbs produced and characterised in this study react with antigenic features analogous to those identified in previous studies using type O MAbs (8, 15, 3 7 ) (Table II). Most of the antibodies characterised react with site 2, the ßB-ßC loop of VP2. Surprisingly, none were found to act against linear epitopes on the ßG-ßH loop of VP1, which has always been considered as the main antigenic determinant on the surface of the virus. However, site 2, the ßB-ßC loop of VP2, has recently been reported to be an immuno-dominant antigenic site for FMDV type O in some individual animals (3, 3 0 ) .

Table I Table s h o w i n g reactivity of O1/Manisa/Turkey/69 monoclonal antibodies w i t h various antigen preparations and t h e residues identified as critical for binding by e s c a p e mutant analysis Monoclonal antibody

Antigenic site

Trypsin sensitive

Denatured virus

Substitution

Residue

Structure

SA113

Site 2

No

No

S->G

VP2

72

SA107

Site 2

No

No

D->A

VP2

73

ßB-ßC VP2

SA98

Site 2

No

No

D->A

VP2

73

ßFJ-ßC VP2

SA127

Site 2

No

No

D->N

VP2

73

ßB-ßC VP2

SA84

Site 2

No

No

D->N

VP2

73

ßB-ßC VP2

SA85

Site 2/5

No

No

Q->R

VP2 , V P 1

SA176

Site 1

Yes

No

Q->R

VP1

SA177

Site 1

Yes

No

ND

ND: no data A :alanine D : aspartic acid G : glycine

N Q R S

: asparagine :glutamine : arginine : serine

72

1 9 8

ND

1 4 9

ßB-ßCVP2

ßG-ßH VP1 Carboxy terminus VP1 Possibly carboxy terminus VP1

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- denotes that the sequence is the same as the parent virus * denotes an ambiguity where determination of the sequence was not possible

Fig. 1 Sequence a l i g n m e n t s h o w i n g t h e d e d u c e d amino acid s e q u e n c e of the e s c a p e mutants to O 1 / M a n i s a / T u r k e y / 6 9 monoclonal antibodies

Certainly, there is no reason to believe that the method of screening the hybridomas discriminated against clones secreting antibody to the ßG-ßH loop. Given that inactivated antigens were used to immunise the mice for MAb production, the possibility that the ßG-ßH loop had undergone some proteolytic degradation either before or after inoculation cannot be excluded. However, the reason this

should have happened in these experiments and not others is unclear. Residues V P 2 and V P 2 have already been implicated as part of site 2 (15) and the fact that these residues are also important for the O1/Manisa/Turkey/69 vaccine strain would suggest that, although sequence differences exist between the ßB-ßC loops of O1/Manisa/Turkey/69 and O1/Kaufbeuren/FRG/66, the accessibility of specific residues 7 2

7 3

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Rev. sci. tech. Off. int. Epiz., 19 (3)

the MAb SA113, and a Q—>R (glutamine—marginine) substitution is also evident at V P 2 . The latter has also been implicated as part of site 2 of type O FMDV. This confirms that subtle changes in sequence within an antigenic site can have very specific effects on the binding of antibodies which are elicited against that site.

Monoclonal antibody

1 3 3

Mutant

All the MAbs were found to be directed against conformational epitopes, although only single amino acid substitutions were evident for most of the MARMs. The exception to this is the MARM selected with MAb SA85, which has two substitutions, one at V P 2 and the other at VP1 . Monoclonal antibody SA85 recognises a conformational epitope and both substitutions may be required to abrogate binding and neutralisation by SA85. Alternatively, the substitutions could be the result of one random mutation and one mutation selected for by the MAb. The fact that trypsin treatment does not affect binding of SA85 might make the random accumulation of a mutation at VP1 more likely. The MAb directed against site 5 (characterised in Crowther et al. [8]) whose critical residue was at V P 1 , was sensitive to proteolytic cleavage by trypsin. Re-cloning MAb SA85 and the isolation and characterisation of further MARMs may help to resolve this enigma. 7 2

1 4 9

Monoclonal antibody binding as a percentage of the reaction with the parental isolate O1/Manisa/Turkey/69:

Fig. 2 Reactivity of O 1 / M a n i s a / T u r k e y / 6 9 monoclonal antibodies w i t h the

1 4 9

e s c a p e mutant viruses in antigenic profile e n z y m e - l i n k e d immunosorbent assay

1 4 9

to the immune system is unchanged. Binding assays by ELISA demonstrated that an escape mutant produced using MAb SA176 no longer bound MAbs SA176 and SA177. However, a mutant produced against either MAb SA84, 9 8 , 107 or 127 affected the binding of all of these MAbs. Although the critical residue for MAbs 107, 9 8 , 127 and 8 4 is V P 2 , subtle differences may exist in the epitopes to which each MAb is elicited. The use of a D—>A (aspartic acid—»alanine) substitution to evade binding of MAbs 107 and 9 8 and a D—»N (aspartic acid—>asparagine) substitution for MAbs 127 and 8 4 indicates that different mechanisms or affinities of MAb binding may have a role to play. Further binding studies reveal that these MAbs display some small differences in reactivity with field isolates (data not shown). 7 3

Figure 3a presents images constructed using RASMOL (33) where the residues critical for binding the O /Manisa/ Turkey/69 MAbs have been highhghted on the structure of O1/BFS 1860/UK/67. For comparison, Figure 3b shows the same image with the residues defining the five antigenic sites highhghted ( 8 , 14). The loops to which the MAbs have been mapped are assumed to have similar structures in both viruses, due to the necessity of surface accessibility of the residues for antibody binding. Figure 4 is an alignment of the deduced amino acid sequences of the two strains, which are 9 4 % conserved at the protein sequence level. Residues V P 1 and V P 1 are aspartic acid (D) and glutamine (Q) in O1/Manisa/Turkey/69, and threonine (T) and glutamic acid (E) in O /Kaufbeuren/FRG/66. These changes in 1

1 9 7

1 9 8

However, an escape mutant against MAb SA113 utilises a S—>G (serine—>glycine) change at V P 2 to escape binding of 7 2

1

Table II Characteristics of the m o n o c l o n a l antibodies used to define t h e five neutralising antigenic sites of O1/Kaufbeuren/FRG/66 ( 8 , 1 5 )

C D E F

Monoclonal antibodies

Antigenic site

Trypsin sensitive

Denatured virus

Substitution

Residues

B2

Site 1

Yes

Yes

L->S

VP1

D9

Site 1

Yes

Yes

L->S, L->R, K-»M

VP1 , VP1

C6

Site 2

No

No

V - » G , T - » N , Dh>H

VP2 , V P 2 , V P 2

C9

Site 2

No

No

V-»F,

C8

Site 3

No

No

T->P, P->T,LQ

VP1 , VP1

14EH9

Site 4

E->V,K

VP3

0C3

Site 5

-Yes

No No

Q-»H

VP1 "

M : methionine N :asparagine P : proline Q : glutamine

R S T V

: cysteine : aspartic acid : glutamic acid : phenylalanine

G H K L

: glycine : histidine : lysine : leucine

S-»C, F-»L, S-»H

Structural feature

1 4 4

144

ßG-ßH loop VP1 1 4 8

, VP1

70

71

73

70

72

75

43

44

1 5 4

VP2 , VP2 , VP2 , V P 2

58

1

ßG-ßH loop VP1 ßB-ßC loop VP2 77

ßFJ-ßC loop VP2 ßB-ßC loop VP1 ßG, VP3

9

: arginine : serine threonine : valine

ßG-ßH loop VP1

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Rev.sci.tech.Off.int.Epiz..19{3)

a) Residues identified as critical for the binding of the O^Manisa/Turkey/69 monoclonal antibodies (MAb)

MAb SA107 and 98 73YP2asp-ala(D-A)and MAbSA127and84 73 VP2fasp-asn (D-N)

MAb SA176 198VP1 gln-arg (Q-R)

MAb SA113 72VP2 ser-gly(S-G]

MAb SA85 72VP2ser-asn(S-N)and 149 VP1 gln-arg (Q-R)

b) Residues (highiighted in yellow) identified as critical for the binding of monoclonal antibodies defining the five neutralising antigenic sites of type 0 foot and mouth disease virus by Kitson étal. (15) and Crowther étal. (8) (Table II)

Blue : VP1 Red :VP2 Green: VP3 Fig.3 Images created using the program RASMOL The ribbon and spacefill plot depicts a crystallographic protomer and is mapped onto the structure of 0,/BFS 1860/UK/67 using the data from Acharya et al. and Logan efa/. (20)

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- denotes that the sequence is the same as the parent virus * denotes an ambiguity where determination of the sequence was not possible

Fig. 4 Sequence alignment s h o w i n g the deduced amino acid s e q u e n c e of the capsid region of 0 , / M a n i s a / T u r k e y / 6 9 and O1/Kaufbeuren/FRG/66

/Manisa/Turkey/69 are characteristic of this virus, and being located near the carboxy terminus of VP1, would be expected to affect the antigenicity of the virus. This is demonstrated by the glutamine—marginine (Q—>R) substitution which abrogates binding of MAb SA176. Structural studies have revealed that the carboxy terminus of VP1 lies close to the VP1 ßG-ßH loop of an adjacent protomer (1). The sequences of the ßG-ßH loops of VP1 in O1/Manisa/Turkey/69 and O /Kaufbeuren/FRG/66 also exhibit diversity on the amino (N) terminal side of the arginine-glycine-aspartic acid (RGD) motif. This tri-peptide motif is highly conserved in FMDV isolates as it functions as a ligand for the cellular receptor, integrin a V ß 3 (5). The ßB-ßC loop of VP1 (site 3) has different residues at V P 1 and V P 1 in comparison with O1/Kaufbeuren/FRG/66, and residue V P 1 has been demonstrated to be critical for the binding of a bovine MAb (3). Residues previously mapped at sites 4 and 5 are the same in both virus strains.

type O FMDV (18, 2 7 ) . However, the exact effects cannot be predicted without further structural or functional data for the O1/Manisa/Turkey/69 strain.

In addition to substitutions on loops joining ß-sheets at previously defined neutralising antigenic sites, a number of other substitutions are distributed throughout the structure. Many of these substitutions probably have no net effect on the antigenicity of the virus. However, some substitutions, although not likely to be exposed on the capsid, may influence the antigenicity of the virus by perturbing or distorting loop structures which lie close to them. These types of effects have previously been reported for substitutions on the ßB-ßC of VP1 and residues at the carboxy terminus of

Acknowledgements

O1

1

4 5

4 6

4 5

Many field isolates display antigenic characteristics which, in serological tests, are similar to either O FMDV from Europe (O1/BFS 1860/UK/67; O1/Kaufbeuren/FRG/66; O1/Lausanne/ Switzerland/65) or O /Manisa/Turkey/69 (a vaccine strain from the Middle East). The availability of MAbs to /Manisa/Turkey/69 and O1/Lausanne/Switzerland/65 will now enable direct comparison of the defined sites in field isolates, by ELISA. Antigenic variation is reported to be limited at the known sites of type O ( 3 0 ) , these MAbs may therefore help to define the extent and effect of the permissible variation in protein sequence at the individual sites. The importance of these epitopes in the development of immunity following the infection of natural hosts is currently under investigation. 1

1

O1

The authors would like to thank Mr N.J. Knowles for critical review of the manuscript. This work was partially funded by the Ministry of Agriculture Fisheries and Food, United Kingdom. Dr S. Aktas was supported by the Turkish Ministry of Agriculture and Rural Affairs.

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Identification d'épitopes antigéniques sur l'isolat O1/Manisa/Turkey/69 du virus de la fièvre aphteuse à l'aide d'anticorps monoclonaux S. Aktas & A.R. Samuel Résumé Une batterie d'anticorps monoclonaux de souris dirigés contre une souche du virus de la fièvre aphteuse de type 0 du Moyen-Orient, O1/Manisa/Turkey/69, a été obtenue. Sept anticorps monoclonaux neutralisants ont été intégralement typés et tous ont réagi avec des épitopes conformationnels. Des mutants résistants à la neutralisation par les anticorps monoclonaux ont été produits à partir de la souche virale parentale et les séquences complètes des capsides de ces mutants résistants ont été déterminées. L'analyse des séquences a révélé que cinq de ces mutants présentaient des substitutions d'acides aminés, au niveau soit du résidu 72, soit du résidu 73 de la VP2 (boucle ßB-ßC), ce qui montre que cinq des anticorps monoclonaux étalent dirigés contre le site antigénique 2. Le sixième mutant comportait une seule substitution d'acide aminé à la position 198 de la VP1 (région carboxy-terminale). Le septième mutant comprenait deux substitutions d'acides aminés, l'une en position 72 de la VP2 et l'autre en position 149 de la VP1 (boucle ßG-ßH). Ces résultats montrent que les anticorps monoclonaux dirigés contre le virus de la fièvre aphteuse de type 0 du Moyen-Orient reconnaissent des résidus selon un schéma structurel Identique à celui observé pour les anticorps dirigés contre les souches européennes du même sérotype.

Mots-clés Anticorps monoclonaux - Moyen-Orient - Mutants échappant à la neutralisation Séquençage - Sites antigéniques - Virus de la fièvre aphteuse.

Identificación de epitopos antigénicos de la cepa O1/Manisa/Turkey/69 del virus de la fiebre aftosa utilizando anticuerpos monoclonales S. Aktas & A.R. Samuel Resumen Los autores describen un proceso experimental por el que se elaboró en primer lugar una batería de anticuerpos monoclonales de ratón contra una cepa de tipo 0 del virus de la fiebre aftosa, concretamente la cepa O1/Manisa/Turkey/og, de Oriente Medio. Se caracterizaron por entero siete anticuerpos monoclonales neutralizantes, que en todos los casos resultaron reactivos ante epitopos conformativos. A partir del linaje vírico parental se generaron mutantes resistentes a la neutralización por anticuerpos monoclonales, cuyas secuencias de nucleocápside completas se determinaron posteriormente. El análisis de esas secuencias reveló que cinco de las cepas mutantes presentaban una sustitución de aminoácidos en el residuo 72 o en el residuo 73 de la proteína VP2 (hélice ßB-ßC), de lo que se deduce que cinco de los anticuerpos monoclonales estaban dirigidos contra el sitio antigénico 2. El sexto mutante contenía una sola sustitución de aminoácidos en la posición 198 de la proteína VP1 (región carboxiterminal), y el séptimo dos sustituciones, una en la posición 72 de la VP2 y

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otra en la posición 149 de la VP1 (hélice ßG-ßH). Estos resultados ponen de manifiesto que el reconocimiento de los residuos se produce según un mismo patrón estructural tanto en anticuerpos dirigidos contra una cepa de tipo 0 de Oriente Medio como en anticuerpos reactivos ante cepas europeas del mismo serotipo.

Palabras clave Anticuerpos monoclonales - Mutantes resistentes - Oriente Medio - Secuenciación Sitios antigénicos-Virus de la fiebre aftosa.

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Barnett P.V., Ouldridge E.J., Rowlands D.J., Brown F. & Parry N.R. (1989). - Neutralizing epitopes on type O foot-and-mouth disease virus. I. Identification and characterisation of three functionally independent, conformational sites. J. gen. Virol, 70, 1483-1491.

3.

Bamett P.V., Samuel A.R., Pullen L., Ansell D., Butcher R.N. & Parkhouse R.M.E. (1998). - Monoclonal antibodies against O serotype foot-and-mouth disease virus, from a natural bovine host, recognize similar antigenic features to those defined by the mouse. J. gen. Virol, 79, 1687-1697.

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Ferris N.P., Donaldson A.I., Bamett I.T.R. & Osborne R.W. (1984). - Inactivation, purification and stability of 146S antigens of foot and mouth disease virus for use as reagents in the complement fixation test. Rev. sci. tech. Off. int. Epiz., 3 (2), 339-350.

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Haresnape J.M. & McCahon D. (1983). - Four independant antigenic determinants on the capsid polypeptides of aphthovirus. J. gen. Virol, 64, 2345-2355.

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Hogle J.M., Chow M. & Filman D.J. (1985). - The three-dimensional structure of poliovirus at 2.9 Â resolution. Science, 229, 1358-1365.

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Kitson J.D.A. (1990). - Antigenic studies of type O FMD virus. PhD Thesis. University of Reading, United Kingdom, 224 pp.

15.

Kitson J.D.A., McCahon D. & Belsham G.J. (1990). Sequence analysis of monoclonal antibody resistant mutants of type O foot-and-mouth disease virus: evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites. Virology, 179, 26-34.

16.

Knowles N.J., Marquardt O. & Samuel A.R. (1988). Antigenic and molecular characterization of isolates from recent outbreaks of foot-and-mouth disease in the Federal Republic of Germany. In Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, 20-23 September, Prague. Food and Agriculture Organization, Rome, Appendix 24, 149-154.

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