Isolation of foot-and-mouth disease virus specific bovine antibody fragments from phage display libraries

Journal of Immunological Methods 286 (2004) 155 – 166 www.elsevier.com/locate/jim

Research Paper

Isolation of foot-and-mouth disease virus specific bovine antibody fragments from phage display libraries Yong Joo Kim a,b, Francßoise Lebreton a, Claude Kaiser a, Catherine Crucie`re a, Michelle Re´mond a,* a UMR 1161 INRA-AFSSA-ENVA de virologie-Agence francßaise de se´curite´ sanitaire et alimentaire, Laboratoire d’e´tudes et de recherches en pathologie animale et zoonoses, 94703 Maisons-Alfort, France b Foreign Animal Disease Research Division, National Veterinary Research and Quarantine Services, An Yang, South Korea

Received 18 September 2003; received in revised form 9 December 2003; accepted 5 January 2004

Abstract Foot-and-mouth disease virus (FMDV) is an important veterinary pathogen which can cause widespread epidemics. Due to the high antigenic variability of FMDV, it is important to undertake mutation analysis under immunological pressure. To study the bovine antibody response at a molecular level, phage display technology was used to produce bovine anti-FMDV Fabs. CH1-VH chains with FMDV specific binding could be isolated after selection from a library made from vaccinated cattle. Though their involvement in the bovine immune response remains to be ascertained, it is planned to express the five different selected VH domains in bacterial or insect systems as sequence homologies with integrin h6 chain could shed light on the basis of FMDV type receptor specificities. D 2004 Elsevier B.V. All rights reserved. Keywords: Phage display library; Bovine antibody VH fragment; Foot-and-mouth disease virus

1. Introduction Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of livestock. It affects a wide variety of cloven-hoofed

Abbreviations: BSA, bovine serum albumin; CDR, complementary determining region; FBS, foetal bovine serum; OD, optical density; OPD, O-phenylenediamine; PCR, polymerase chain reaction; PFU, plaque forming unit; TBS, tris-buffer saline. * Corresponding author. Tel.: +33-1-49-77-13-17; fax: +33-143-68-97-62. E-mail address: [email protected] (M. Re´mond). 0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.01.002

domestic and wild animals. The etiological agent of this disease is foot-and-mouth disease virus (FMDV) which belongs to the Aphthovirus genus of the Picornaviridae family. The viral particle contains a positive-strand RNA genome of about 8500 nucleotides, enclosed within a protein capsid (Belsham, 1993). Its structural proteins exhibit multiple linear and conformationally highly immunogenic epitopes (Bachrach, 1985). Seven serotypes of FMDV have been identified by cross-protection and serologic tests. Each type has been further subtyped on the basis of quantitative differences in cross-protection and serological tests (Kitching et al., 1989). Antigenic variation within a

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type occurs as a continuous process of antigenic drift without clear-cut demarcations between subtypes. This antigenic heterogeneity has important economic implications for vaccine development and selection, since immunity acquired through infection or the use of current vaccines is strictly type-specific and, to a lesser degree, subtype-specific (Brown, 1999). For FMD, it is widely accepted that antibodies have an important role to play in the protection and recovery of cattle from the disease (McCullough et al., 1992). The antigenic characterization of the virus which is essential for the design of a vaccine has often relied on the use of murine monoclonal antibodies which have been extensively studied using neutralizing monoclonal antibody escape mutants and sequence analysis (review in Mateu, 1995). A few studies have focused on the relevance of this approach. Experiments, either with viral mutants (Crowther et al., 1993), peptide analysis (Mateu et al., 1995a), bovine monoclonal antibodies derived from heterohybridomas (Barnett et al., 1998) and competition experiments (Aggaral and Barnett, 2002) suggest the recognition of similar antigenic features by the two species, though the relative importance of the individual neutralizing sites and the key residues within an epitope may differ (Krebs et al., 1993; Rieder et al., 1994; Mateu et al., 1995b). Because immunological pressure has been shown to be critical for the generation of antigenic variants, the determination of amino acid replacements during in vitro selection under bovine polyclonal sera immune pressure was thoroughly investigated (Xie et al., 1987; Borrego et al., 1993; Mateu et al., 1994; Schiappacassi et al., 1995). Comparison with mutations arising in variants selected in vivo is complicated by the natural genetic variation and the quasi species structure of FMD isolates (Sobrino et al., 1989; Martinez et al., 1992; Feigelstock et al., 1996; Holguin et al., 1997). Antibody engineering technology is a new area in the field of molecular immunology for the production of libraries of Fab fragments by bacterial expression of the genes coding for the VH domain of the heavy chain and the VL domain of the light chain and display of the fragments on the surface of filamentous phage (Hoogenboom et al., 1998; Winter et al., 1994). Such phage display methodology permits rapid construction of large combinatorial antibody libraries and has been used successfully to produce

antibodies against a variety of pathogens in different species such as man (Marks et al., 1991; Barbas et al., 1993; Schmitz et al., 2000), chimpanzee (Schofield et al., 2000), mouse (Clackson et al., 1991), cattle (O’Brien et al., 1999), sheep (Li et al., 2000), camel (Arbabi Ghahroudi et al., 1997), rabbit (Lang et al., 1996) and chicken (Davies et al., 1995; Yamanaka et al., 1996). The restricted number of genes used for generating antibody diversity in cattle leads to technical simplifications in applying this technology in this species (Sinclair et al., 1995; Parng et al., 1996, Saini et al., 1997; Lopez et al., 1998). The antibody genes can be recovered from lymphocyte mRNA by RT-PCR using only one set of specific primers for each single family of VH and VL genes. Then, they can be conveniently cloned into a specially engineered vector pCombBov (O’Brien et al., 1999). Because, within this vector, the gene of the Ig first constant domain CH1 is cloned in frame with a portion of the M13 bacteriophage gene III, expression results in a fusion protein of CH1-VH1 and the carboxyl-terminal portion of minor coat protein III. Fab can also be expressed as soluble Fabs after removal of the gene III region from the phagemid constructs. The purpose of our study was to generate FMDV specific bovine Fabs from libraries expressed on phages and thereafter to select FMDV neutralization resistant variants. Though we succeeded in the construction of a library, incomplete Fabs lacking a VL domain could only be isolated. Study of their biological properties is described and discussed.

2. Materials and methods 2.1. Animals Six cattle were vaccinated by intramuscular injections in the neck three times over a period of 2 months each with one dose of trivalent (O1 Manisa, A22, A Iran 96) FMD vaccine (Merial, Lyon, France). Three and five days after the last injection, spleen, prescapular lymph node, bone marrow and blood lymphoid cells were removed in the slaughter house and snap frozen in liquid nitrogen.

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2.2. RNA isolation and PCR amplification Total RNA was extracted from a mixture of different organs using Trizol (Gibco BRL, Invitrogen, Cergy Pontoise, France) and 15 Ag were reversetranscribed to cDNA using an oligo(dT) primer and AMV reverse transcriptase (Qbiogene, Montreal, Canada). VH with the amino-terminal heavy chain constant region CH1 and VL regions were amplified by polymerase chain reaction (PCR) using Taq polymerase and oligonucleotide primers as already described (Table 1, O’Brien et al., 1999). PCR products were purified by gel electrophoresis and extraction, then digested with excess restriction enzymes. 2.3. Construction of antibody phage library The method of library construction was essentially as described by O’Brien et al. (1999). The pComBov vector was digested with an excess of SacI and BstEII, treated with phosphatase, and then purified by agarose gel electrophoresis. Two micrograms of plasmid was ligated to 330 ng of SacI/BstEIIdigested VL fragments with 20 units of T4 DNA ligase. Ligated DNA was electroporated into Escherichia coli XL1Blue (Stratagene, La Jolla, CA, USA) using a Bio-Rad Gene Pulser and amplified. The size of the light chain library was determined by plating aliquots of the culture on Luria-Beriani agar plates containing 100 Ag/ml of carbenicillin. Phagemid DNA containing the light chain library was prepared

Table 1 Primers for cloning bovine V genes (from O’Brien et al., 1999) VL primers Forward TAG AGC TCC GTG TCC GTS WMY CTG GG Reverse GTA GAG GTA GGT CAC CGA AGG TGG GGA CTT GGG VH + CH1 primers Forward ATA TAT ACG GAC CGA GCC TGG TGA AGC CCT CACAGA CC Reverse TGG GCA ACT AGT AAC AGC CTT GTC CAC CTT GGT GC Restrictions sequences are italicized.

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using Maxiprep columns (Qiagen, Courtaboeuf, France), then digested with an excess of RsrII and SpeI and purified as above. Two micrograms of the VL library were ligated to 660 ng of RsrII/SpeI-cut VH PCR product, transformed into XL1Blue cells, amplified and titered as above. To produce recombinant phage, 1012 plaque forming unit (PFU) of VCSM13 helper phage (Stratagene) were added and the cultures amplified overnight. The phage suspension was precipitated by PEG/NaCl solution (Kay et al., 1996). 2.4. Biopanning of library The library phage stocks were reamplified as follows: 20 Al of stored phage were added to 10 ml of log-phase XL1Blue cell culture, incubated at 20 jC for 15 min, before adding 10 ml Superbroth containing 10 Ag/ml of tetracycline and 20 Ag/ml of carbenicillin (SB/tet10/carb20). After 1 h at 37 jC with vigorous shaking, the concentration of carbenicillin was increased to 50 Ag/ml. Additional incubations, the addition of VCSM13, overnight incubation and phage precipitation were as described above. Panning of the library was performed essentially as described by Burton et al. (1991). Immunotubes (Nunc) were coated with monoclonal antibody 3H4 against O1 Manisa in carbonate buffer (15 mM Na2CO3/35 mM NaHCO3, pH 9.6) overnight at 4 jC, and incubated with purified FMDV O1 Manisa in 50 mM Tris-buffered saline pH 8 (TBS, Sigma, Saint Louis, USA). A volume of 1 ml of phage library suspension containing 10 12 – 1013 phages was bound for 2 h at 37 jC, then the tubes were washed in TBS/0.5% Tween 20 for 5 min. Tubes were washed twice in round 1, five times in round 2, and 10 times in round 3 and any subsequent rounds. Phages were eluted in 0.1 M HCl, pH 2.2/1 mg/ml bovine serum albumin (BSA) for 10 min, and neutralized immediately with 2 M Tris base. Then, phages were added to 2 ml of fresh log-phase XL1Blue cells for 15 min, and amplified. Phage output was titered by infecting aliquots of XL1 blue cells with dilutions of eluted phage and plating on Luria-Beriani agar/100 Ag/ml of carbenicillin. The phage input at each round was also determined. Panning was repeated for four to five rounds over consecutive days.

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2.5. Production of phage clones Eluted clones from the final round of panning were picked into 200 Al of SB/tet10/carb50/1% glucose in 96-well round bottom plates and grown at 37 jC overnight. Aliquots of each culture (5 Al) were added to 200 Al of medium as above but containing 0.1% glucose and grown for an additional 2 –4 h, after which 109 VCSM13 helper phage were added. After 15 min at 20 jC, the cultures were grown for an additional 2 h at 37 jC. Finally, kanamycin was added to a final concentration of 70 Ag/ml, and the plates were incubated at 37 jC overnight. 2.6. ELISA screening Culture supernatants were added to immunoplates (Maxisorp, Nunc, Roskilde, Denmark) coated with O1 Manisa virus bound to the plate via a monoclonal antibody and blocked with 5% powder milk (Marvel) in TBS-Tween (McCullough et al., 1985). The phage suspensions (107 PFU per well) were bound for 2 h at 37 jC, followed by extensive washing of the wells with TBS/0.1% Tween 20. Bound phages were detected with peroxidase conjugated anti-fd bacteriophage antibody (Amersham Biosciences, Saclay, France), followed by enzyme substrate O-phenylenediamine (OPD). Absorbance values were read at 490 nm. Selected phages were further tested against O BFS, A22, C Noville, and Asia 1 using the same protocol. 2.7. Restriction mapping and sequencing Individual clones were grown in SB/carb50/1% glucose overnight at 37 jC, and plasmid DNA was isolated using Qiaprep spin miniprep columns (Qiagen). DNA was digested with EcoRII and analyzed by electrophoresis on an 8% polyacrylamide gel. Individual restriction digestion patterns were visualized after staining in ethidium bromide. Plasmid DNA from selected clones was sequenced in the VH and VL regions using the previously described primers.

digestion followed by religation. The resulting phagemid was electroporated into XL1 Blue cells to produce clones secreting soluble Fab fragments. Clones were further grown as described in O’Brien et al. (1999). Fab fragments were concentrated from supernatants by ammonium sulfate precipitation at 50% concentration. 2.9. Plaque-reduction neutralization test To test virus neutralization by both antibody fragments expressed on the phage particle and soluble fragments, titered FMDV suspension was incubated with twofold dilutions of the recombinant clones for 1 h at 37 jC. The reaction mixtures were allowed to adsorb to confluent IBR’S2 cell monolayers for 1 h and then discarded. The cells were overlaid with 1% low melting agarose in MEM 5% foetal bovine serum (FBS). After 20 h, the plates were fixed under agarose and stained. Assays were repeated at least on two occasions. 2.10. Blocking ELISA Ten different (on the basis of their biological properties) FMDV mouse monoclonal antibodies were used as competitors in a blocking ELISA to investigate the presence of potentially shared epitopes between the different groups of recombinant clones and mouse monoclonal antibodies. A blocking ELISA with a positive serum from a vaccinated cow was also performed. Plates coated with viral particles as previously described were first incubated with the inhibiting bovine serum (diluted 1/ 4) or the different mouse monoclonal antibodies (diluted 1/10 and 1/100). A single dilution of the recombinant phage suspension was then added to all wells for 1 h at 37 jC. Bound phages were revealed with peroxidase conjugated anti-M13. The percentage of phage binding inhibition was assessed by comparison with controls including negative sera and irrelevant monoclonal antibodies. 2.11. Western blotting

2.8. Expression of soluble Fab fragments The gene III fragment was removed from plasmid DNA of selected clones by NheI and SpeI enzyme

The reactivity of antibody fragments against denatured antigen was evaluated by Western blotting. Detergent treated, sucrose gradient-purified FMDV

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O1 Manisa was electrophoresed on a 16% SDSpolyacrylamide gel in Laemmli buffer (Sambrook et al., 1989) and transferred electrophoretically to nylon membrane Hybond P (Amersham Biosciences). The filter was blocked in PBS/0.1% Tween/5% powder milk and strips were incubated with recombinant phages in blocking buffer overnight at 4 jC. After washing, peroxidase conjugated anti-M13 (Amersham) was added. After additional washings, strips were developed with a chemiluminescence kit (ECL, Amersham) and autoradiographed.

after the last vaccination. The initial light chain libraries consist in 2  105 recombinants and formed the basis for insertion of heavy chain genes yielding a final library of, respectively, 5  105 and 1  105 transformed bacteria. Enrichment of antigen specific binding phage was measured through the four and five rounds of library panning and resulted in 1.2  107 and 2.8  107 pfu of eluted phage (Table 2).

2.12. Protein sequence analysis

Seven hundred clones were randomly selected and tested by ELISA. Of these, 43 clones were demonstrated to react specifically with FMDV O1 Manisa (Table 2).

Blast, Fasta, and Matcher programs available on line at ‘‘www.pasteur.fr’’ were used for homology research, comparisons and alignments. 2.13. Peptide experiments Two peptides with the following sequence LALKLRPGL-CoNH2 and CVCRSGWTG-CoNH2 with and without an N-terminal biotin group linked via a six carbon atom chain were synthesized by Epytop (Nimes, France). Immulon B microplates (Dynatech) were coated with streptavidin (100 Ag/ ml) overnight and then blocked with 4% bovine serum albumin in carbonate buffer. Biotinylated peptides (1 to 100 Ag/ml) were added for 1 h at room temperature. After washings, O1 Manisa FMDV suspension was added and the bound virus was revealed with anti-O guinea-pig serum and peroxidase conjugated antiguinea pig immunoglobulin. Competition experiments aimed at inhibiting the phage binding were designed as described above using 25 to 200 Ag/ml of peptide before adding the phage suspension to the plate. Peptides in the same range of concentration as above were also introduced in a neutralization assay using 100 UFP of FMDV for 1 h before adsorption using the IBR’S2 cell line.

3.2. Selection of clones

3.3. Nucleic acid sequencing and restriction enzyme analysis Sequence analysis was performed on 14 of the 43 clones. From these 14 clones, five unique heavy chain sequences were identified (Fig. 1). The EcoRII restriction enzyme pattern observed with plasmid DNA of selected clones was used to assign the non-sequenced clones within sequence types. The clones were distributed across three major and two minor groups (Table 4). It is worth noting that the predominant sequences (types III and IV) were also those which displayed the longest complementary determining region (CDR)3. The sequence of the four selected clones in the second library belonged to the same group (group V). Though it was checked by means of PCR and sequencing that during the library construction, the DNA coding for VL was inserted at its site (66% of clones taken at random possessed the insert), none of the selected clones displayed the VL DNA insert after rounds of biopanning. The search for VL DNA in 10 random nonbinding clones was also negative for 9 of them, suggesting that the insert was lost during the rounds of biopanning.

3. Results 3.4. ELISA analysis 3.1. Biopanning of libraries Two libraries were generated from lymphoid cell RNA taken, respectively, from cattle 3 and 5 days

Positive clones were further tested against O1 BFS, A22, C Noville and Asia1 strains of FMDV (Fig. 2). The strong reactivity with both O1 Manisa

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Table 2 Enrichment of libraries to FMDV O1 Manisa Number of eluted phage (PFU)

ELISA screening against O1 Manisa

Library

First biopanning

Second biopanning

Third biopanning

Fourth biopanning

Fifth biopanning

Number of clones tested

Number of positive clonesa

3 days after last injection 5 days after last injection

2.1  102 2.0  103

1.7  103 1.6  105

9.0  103 1.0  105

7.2  104 2.8  107

1.2  107

660 40

39 4

a

Clones with corrected OD>0.1 were recorded positive.

and O BFS but not with the other FMDV types indicated that all the recombinant phages were FMDV type specific. 3.5. Neutralization tests Recombinant phages and soluble antibody fragments were screened for their ability to neutralize FMDV in vitro. A low neutralizing activity against FMDV type O was observed with all group of recombinant phages except group II. No neutralization of other FMDV types was recorded, thus confirming the specificity of the binding (Table 3A). Some soluble antibody fragments showed slight neutralizing activity when used undiluted or diluted 1/ 2 (Table 3B). 3.6. Western blotting A Western blot assay was used to determine the nature of the epitopes recognized by the antibody

fragments. Three out of the five types of recombinant phage visualized one protein band against denatured FMDV (Table 4). This protein was of the same size (23 kDa) as that revealed by the FMDV guinea-pig antiserum which was included as a control and could be assigned to VP1. This indicated that these antibody fragments most likely recognized linear epitopes within the protein. 3.7. Blocking ELISA Binding inhibition of the recombinant phage with a positive bovine serum was only observed with type IV (40% inhibition) although a smaller reduction (12% and 17%, respectively) was noted for types III and V. Binding of the minority clones belonging to types I and II could not be inhibited (Table 4). Although we tried to perform blocking ELISA with five neutralizing and five non-neutralizing mouse monoclonal antibodies, only two of them inhibited the binding of types IV and V (Table 4). These two neutralizing monoclonal antibodies were

Fig. 1. Alignment of deduced amino acid sequences of bovine VH selected by panning against O1 Manisa FMDV. The framework regions (FR) were identified by homology to a known bovine sequence (ref. Genbank no. U36824). The residues are numbered according to Kabat et al. (1991). A dash ( – ) indicates identity to uppermost sequence. *Indicates no amino acid at this position.

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Fig. 2. Graphical representation of ELISA results. Reactivity of selected clones to O1 Manisa, O BFS, A22, C Noville, Asia1 fixed on plastic wells by monoclonal antibody and BSA as control. Recombinant clones are classified in groups I – V according to VH types.

further analyzed by sequencing viral antibody escape mutants according to the method described by Aktas and Samuel (2000). The resulting mutated residues were located in VP2 and belonged to

neutralization site 2 (Aktas and Samuel, 2000). It was concluded that types IV and V antibody fragment were directed to at least part of site 2 epitope.

Table 3 Neutralization tests A. Neutralizing activity and specificity of recombinant phages FMDV type

O1 Manisa

Phage dilution (log10 inverse) Group I (clone A3) Group II (clone A37) Group III (clone A36) Group IV (clone A4) Group V (clone BD)

1

1.3

O BFS

64%a (28/77) 5% (40/42) 42% (54/92) 52% (37/77) 48% (38/72)

25% (80/107) 12% (37/42) 16% (78/92) 0% (28/25) 34% (71/107)

1.6

NT 2% (90/92) NT NT

C Noville

A22

Asia 1

1

1.3

1.6

1

1

1

42% (105/180) 10% (163/180) 68% (51/157) 40% (68/114) 80% (13/65)

17% (150/180) 10% (162/180) 46% (73/133) 40% (65/107) 48% (34/65)

0% (180/180) 7% (168/180) 37% (85/133) 32% (73/107) NT

NT

0% (27/27) 3% (70/72) 0% (33/27) 0% (29/27) 7% (67/72)

5% (104/119) NT

NT 7% (40/43) 0% (43/43) 0% (32/30)

0% (122/119) 8% (110/119) NT

B. Neutralizing activity of soluble fragments FMDV type

O1 Manisa

Dilution log10 inverse Group I (clone A3) Group II (clone A37) Group III (clone A7)

0.3 0% 0% 31% (78/132) 0% 30% (93/132)

Group IV (clone A4) Group V (clone BD)

O BFS 0.6 0% 0% 0% (130/132) 0% 0%

0.3 0% 0% 80% (39/188) 0% 67% (81/188)

C Noville 0.6 0% 0% 44% (106/188) 0% 20% (58/72)

0.3 0% 0% 0% 0% 0%

Although assays were performed on different phage clones in each group, results for only one clone of each group are presented for the sake of clarity. From control data analysis, only inhibition higher than 15% is significant. NT: not tested. a Results are expressed as the percentage of plaque inhibition. The data in brackets are numbers of plaques observed relative to the mean number of plaques in the virus control.

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Table 4 Characteristics of recombinant phage clones Groups of recombinant phage

Number of clones obtained and frequency

Western blot

Neutralization tests (FMDV O) Recombinant phage

Soluble fragment

Positive bovine sera

Monoclonal antibody

I II III IV V

1, 2% 1, 2% 22, 42% 20, 38% 8, 16%

+(VP1) +(VP1) +(VP1) 0 0

+ 0 + + +

0 0 + 0 +

0 0 12% 40% 17%

0 0 0 60% (10B4)a 25% (13G11)a

a

10B4, 13G11: monoclonal antibodies which recognized neutralization site 2 (Aktas and Samuel, 2000).

3.8. Homology with bovine integrins As these antibody fragments were able to link the virus as single chains, we hypothesised that they might bind the virus in the same way as integrins, the virus cellular receptors. The search for homologies between the sequences of bovine integrins and the antibody chain VH domains resulted in four homologies only found with the sequence of the h6 subunit (Table 5). Of these four homologies, two seemed meaningful (ALKXRPG and CRSGW) owing to the low probability that they might be due to random matching, 5  10 5 and 4  10 5 respectively, as calculated by a previously described method (Germaschewski and Murray, 1996; Chen et al., 1996) and their location in the CDR3 segment. 3.9. Experiments with synthetic peptides No viral capture, phage binding inhibition or neutralization effect was observed with the peptide

Table 5 Sequence homologies between VH domains and bovine integrin

N

Blocking ELISA % binding inhibition by:

LALKSRPGL. On the contrary, the peptide CVCRSGWTG displayed nonspecific binding in the ELISA test. Although phage binding could not be abolished, 50% to 90% plaque inhibition (according to peptide concentration) was observed with all FMDV types and swine vesicular disease virus (an unrelated enterovirus) in the neutralization test.

4. Discussion The aim of this work was to study the bovine FMDV immune response at a molecular level and eventually to find out either new epitopes or important residues involved in bovine antibody responses to previously known epitopes. We constructed a bovine Fab library expressed on phages. After rounds of biopanning, we selected single chains composed of CH1-VH1 which confer binding properties to the recombinant phages as revealed in ELISA, western blotting and viral neutralization tests. The lack of VL was attributed to a recombination-deletion mechanism

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during the enrichment cycles as only 10% of the negative clones possessed a VL insert after four cycles against 66% before. In an attempt to express complete Fab, a third library was made by inserting the VH DNA from the previously selected binding clones into a VL library. Clones were directly tested without biopanning. Only 10% of the clones displayed binding properties though 60% of them had incorporated VH DNA and none of these binding clones had a complete VL ORF. It was concluded that most of the VL partners for the previously selected VH would probably be functionally incompatible. This was already noted in previous experiments where different VL domains were conjugated to binding VH domains. Most of the VL domains lowered the affinity or were inhibitory (Collet et al., 1992; Cai and Garen, 1996). Naturally occurring functional immunoglobulin composed of heavy chain dimers and devoid of light chains is a well-documented phenomenon in the Tylopoda (camels, dromedaries and llamas). In these species, the VH domains are also characterized by an extended CDR3 segment, possibly compensating for the absence of VL and substitutions of some conserved hydrophobic residues in the framework (Muyldermans et al., 1994; Muyldermans, 2001; HamersCasterman et al., 1993). VH domains with binding properties have also been isolated from the murine and human species (Reiter et al., 1999; Ward et al., 1989; Cai and Garen, 1997). Besides its atypical VH, llama also display conventional VH, able to bind without VL (Tanha et al., 2002). Structural analysis of the antigen– antibody interactions showed that the heavy chain CDR3 form the principal contacts with the antigen and that diversity in the CDR3 region is sufficient for most antibody specificities (Chothia et al., 1985; Chothia and Lesk, 1987; Padlan, 1994; Xu and Davis, 2000). In the bovine immune system, as well as that of other ruminants, antibody diversity relies more on somatic hypermutation and the generation of long CDR3 than on the number of VH genes. These VH genes belong to a single family (Bov VH1) composed of no more than 20 members (Lopez et al., 1998; Aitken et al., 1999). The length of most VH CDR3 ranges from 4 to 26 amino acids. However, some of these long CDR3 appear to have restricted light chain pairing (Kaushik et al., 2002).

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From the data presented here, we conclude that because of the loss of VL, we selected functional heavy chain with long CDR3 (24 and 27 residues for types 4 and 5, respectively). However, the typical substitutions by hydrophilic residues at positions 44, 45 and 47 of camelid VH segment FR2 were not observed in our sequences (except for Arg 45 in type 5). Compared to the Fabs selected by O’Brien et al. (1999) where the majority of the clones displayed VH CDR3 of five residues length, the question of their involvement in the immune response remains open. It may be noteworthy that because of two Arg substitutions at positions 106 and 108, the FR4 segment of type 3 is the product of the second gene JH which is not preferentially used (Berens et al., 1997). Binding inhibition with a positive bovine serum was only effective, though partial, for VH type 4 and a similar result was obtained with a murine monoclonal directed against site 2 of neutralization on the viral capsid. Four types of recombinant phage repeatedly displayed a low specific neutralizing capacity which could be confirmed for two of them while testing their respective soluble fragments. It should be noted here that the neutralization of FMDV by monovalent antibodies has already been observed, at least when directed to site 1 (Mason et al., 1996; Verdaguer et al., 1997). The discrepancy between the neutralizing property of the recombinant phages types 1 and 4 and their soluble chains could be attributed either to some steric hindrance by the phage particle rather than a true neutralizing effect or a too low concentration of the soluble fragment. The lack of VL, the main target of anti-bovine Fab, prevents any quantification of their production. It is planned to express these single chains as a fusion protein with a marker so that they can be further purified and quantified. As already suggested by Baranowski et al. (2001), we hypothesised that the binding of the virus to some of the selected chains could mimic binding with its cellular receptor, the integrin family. The search for homologies between types 1 and 5 VH sequences and the extracellular domains of h1, h3, h5, h6 and aV bovine integrin chains resulted in the identification of the motifs ALKSRPG and CRSGW exclusively found in the h6 chain, in the hybrid and EGF-3 domains, respectively. The integrin aVh6 was recently recog-

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nized as a specific cellular receptor for FMDV types O and A (Jackson et al., 2000; Duque and Baxt, 2003). The molecular basis for the preferential use of aVh1, aVh3 or aVh6 by the different FMDV serotypes has not yet been determined. Other viruses such as coxsackie A9, HIV, canine adenovirus type 2, rotavirus and hantavirus also interact with aVh5 or aVh3 independently of the RGD binding site (review in Nemerow and Stewart, 2001; Triantafilou et al., 2000). Although like most of the other ligands, the virus binds via the G-H loop on VP1 to the integrin RGD binding site located at the head of the integrin, encompassing the hA domain (Xiong et al., 2001, 2002), the specificity for a particular integrin has been shown to rely on the h subunit, in the Cterminal ectodomain in the case of type A (Neff et al., 2000) and in the ligand binding region of the hA domain (Duque and Baxt, 2003). It was recently demonstrated that in the low affinity state, the extracellular domain of the integrin is in a bent conformation with the ligand binding domain coming close to the EGF domains and to the cellular membrane (Xiong et al., 2001; Travis et al., 2003). In this state, the viral capsid could interact simultaneously with the hA domain and the C-terminal part of the integrin h chain. The putative interactions of the virus and the integrin motifs ALKSRPG and CRSGW could not be confirmed by peptide experiments. The lack of tertiary structure or an inappropriate design of the peptides could partially explain this. The expression of these VH fragments in bacterial or insect systems will allow high concentration to be produced. If their biological properties are maintained, it should be possible to determine first, which parts of the virus are involved in the binding of the phages and secondly whether some of the interactions truly mimic the affinity for integrins.

Acknowledgements We are very grateful to Dr. Philippa O’Brien (Beatson Institute for Cancer Research, Glasgow, Scotland) for generously providing the vector pComBov, Dr. Philippe Dubourget from Merial, Dr. Martel and Dr. Miege from Afssa-Lyon for giving us access

to vaccinated cattle and for help in taking samples. We also thank C. Fays for her excellent technical assistance.

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