| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 International Centre for Genetic Engineering and Biotechnology, AREA Science Park, Padriciano 99, 34012 Trieste, Italy
2 Department of Molecular and Cell Biology, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Campus Univ. Autonoma Madrid, Darwin 3, Cantoblanco, 28049 Madrid, Spain
3 Fort-Dodge Veterinaria SA, Department of Research and Development, Vall de Bianya, 17813 Girona, Spain
Correspondence
Oscar R. Burrone
burrone{at}icgeb.org;
Luis Enjuanes
L.Enjuanes{at}cnb.uam.es
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
and 
isotypes, by exploiting the dimerizing domains CH4 and CH3 of human and swine origin. Transfected cells secreted these recombinant mini-antibodies efficiently, mainly as dimers stabilized covalently by inter-chain disulphide bridges. The specificity and functionality of the recombinant TGEV-specific SIPs were determined by in vitro binding, neutralization and infection-interference assays. The neutralization indices of the TGEV-specific SIPs were all very similar to that of the original TGEV-specific mAb, thus confirming that the immunological properties have been preserved in the recombinant SIPs. In vivo protection experiments on newborn piglets have, in addition, demonstrated a strong reduction of virus titre in infected tissues of animals treated orally with TGEV-specific SIPs. It has therefore been demonstrated that it is possible to confer passive immunization to newborn pigs by feeding them with recombinant SIPs.
These authors contributed equally to this work. 
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
). TGEV structure includes four structural proteins: three of them, the spike (S), the membrane (M) and the envelope (E) proteins, are embedded in the virus envelope, whilst the nucleoprotein (N) binds to the RNA genome to constitute the nucleocapsid (Escors et al., 2001). The major antigenic sites of TGEV involved in the induction of virus-neutralizing antibodies are located in the globular portion of the S protein (Correa et al., 1990; Gebauer et al., 1991).
Investigations into the mechanisms of TGEV neutralization (Suñé et al., 1990) and of antigenic and genetic variability (Jiménez et al., 1986; Sánchez et al., 1990, 1992) have led to the identification of an S protein-specific mouse monoclonal antibody (mAb), 6A.C3, that neutralized all TGEV isolates tested, as well as TGEV-related coronaviruses infecting other animal species such as dogs and cats. mAb 6A.C3 recognizes a complex, conformation- and glycosylation-dependent antigenic determinant that appears to be essential for virus replication, as no neutralization-escape mutants (mar mutants) were found when it was employed in the selection of resistant mutants (Gebauer et al., 1991).
Passive immunization has proved effective in protecting piglets against infection. Lactation from immune sows, as well as oral administration of serum from immunized animals, confers protection against challenge by virulent virus (Torres et al., 1995). Transgenic mice secreting TGEV-neutralizing chimeric human or porcine recombinant antibodies with IgG or IgA isotypes, derived from mAb 6A.C3, in their milk have been obtained (Castilla et al., 1998; Sola et al., 1998). Chimeric IgA antibodies have been demonstrated to neutralize virus infectivity more efficiently than recombinant IgG with the same specificity in in vitro neutralization assays, probably because of the tetravalent nature of secretory IgAs (Sola et al., 1998). In addition, the IgA isotype is found naturally in mucosal secretions, where it is particularly stable and resistant to proteolytic degradation.
Chimeric antibodies, although efficient in protecting against virus infections, are nevertheless difficult to generate, as their production involves the engineering of two transcriptional units, encoding immunoglobulin heavy and light chains. Single-chain fragments (scFv), obtained by joining the light- and heavy-chain variable regions (VL and VH) from a mAb, reconstitute the original VL–VH association and retain the binding specificity of the original mAb in a single polypeptide (Winter & Milstein, 1991). To improve the affinity of monovalent scFv, dimeric single-chain mini-antibody molecules, named minibodies or SIPs (small immunoproteins), have been generated by connecting an scFv to the dimerizing domain of immunoglobulin heavy chains. These recombinant proteins are efficiently assembled and secreted in dimeric form by mammalian cells (Hu et al., 1996; Li et al., 1997; Borsi et al., 2002; Occhino et al., 2004). It has been demonstrated that scFv multimerization results in an increase of the apparent affinity (avidity) through an overall increase in valency (Pack et al., 1995).
SIPs are very attractive molecules for both diagnostic and therapeutic purposes, because in vivo they exhibit higher tissue penetration than full-length antibodies, whilst at the same time showing a slower clearance than scFvs (Borsi et al., 2002).
In this report, we applied SIP technology to the TGEV-neutralizing mAb 6A.C3 to generate stable and easily produced TGEV-specific mini-antibodies of and isotypes, by exploiting the dimerizing domains CH4 and CH3 of human and swine origin. A chimeric human IgA with the same specificity was also produced to compare its in vitro and in vivo efficacy with that of SIPs. Here, we show that passive immunization can be achieved by oral administration of TGEV-specific SIPs, resulting in significant protection against TGEV infection.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
) were grown in DMEM supplemented with 5 % FCS and 1.5 mg G-418 ml–1 (Geneticin; Invitrogen) as a selection agent. The TGEV strain PUR46-MAD (Sánchez et al., 1990) and the related, more virulent strain PUR46-SW11-ST2-C11 (Sánchez et al., 1999) were grown and titrated by plaque formation on ST cells as described previously (Jiménez et al., 1986).
Construction of vectors for recombinant TGEV-specific antibody expression.
To generate TGEV-specific SIPs, mAb 6A.C3 VL and VH genes were amplified by PCR from the previously described vectors pIN-SLC6A and pIN-SHC6A (Sola et al., 1998) with primer pairs 6A.C3-VL-ApaLI (5'-ACAGGTGTGCACTCGGACATTGTGATGACC)/6A.C3-VL-SpeI (5'-CTACCACTAGTGCTGCCTTTTATTTCCAGTTTGG) and 6A.C3-VH-XhoI (5'-ATCCTCGAGCAAAGGAGAGGTTCAGCTGCAGCA)/6A.C3-VH-BspEI (5'-AGTTCCGGAGGAGACTGTGAGAGTGGT), respectively. The amplified fragments were assembled in the pUT-SIP vector (Borsi et al., 2002), which provides the VL–VH unit with a sequence encoding a secretion signal, the hydrophobic leader peptide required for secretion of proteins in the extracellular medium (Li et al., 1997), and contains sequences encoding an 18 aa linker (to join VL with VH) and the CH4 domain of human IgE secretory isoform IgE-S2 (S2CH4) (Batista et al., 1996). The resulting 6A.C3-SIP gene (Fig. 1a) was then excised with HindIII/EcoRI and inserted in the eukaryotic expression vector pcDNA3 (Invitrogen), under the control of the cytomegalovirus (CMV) promoter, to yield the construct pcDNA-6A.C3-hu-SIP. Plasmids pcDNA-6A.C3-hu-SIP and pcDNA-6A.C3-sw-SIP were obtained from pcDNA-6A.C3-hu-SIP after substituting the BspEI–EcoRI fragment containing the S2CH4 coding sequence with fragments encoding the human and swine IgA1 CH3 domains (hu-CH3 and sw-CH3), respectively. Human CH3 cDNA was generated by RT-PCR amplification from human lymphocyte mRNA with primers 5A1 (5'-TACTCCGGAGGCTCTGGCGGAAACACATTCCGGCCCGA) and 3AS (5'-TCTGAATTCTCAGTAGCAGGTGCCGTCCA). Swine CH3 cDNA was amplified from plasmid pIN-SHC6A (Sola et al., 1998) with primers sw5A1 (5'-TATTCCGGAGGCTCTGGCGTGAACACGTTCCGGCCCCA) and sw3AS (5'-TCTGAATTCTCAGTAGCAGATGCCCTCTGCCT).
|
1 heavy-chain constant region cDNA was amplified by RT-PCR from human lymphocyte mRNA with primers hIgA1-Nhe/Hind (5'-TCTAAGCTTCTCTGCTAGCCCGACCAGCCCCAA) and hIgA1-EcoRI (5'-TCTGAATTCTCAGTAGCAGGTGCCGTCCACCT) and inserted into the pcDNA3 expression vector (Invitrogen), cut at HindIII/EcoRI restriction sites, to generate the vector pcDNA-hu-C. The 6A.C3 VH gene was amplified with primers 6A.C3-VH-ApaLI (5'-TCTTGTGCACTCTGAGGTGCAGCTGCAGCA) and 6A.C3-VH-Nhe/Xba (5'-AATTCTAGAAGGGCTAGCGGAGCTCACTGTGAGAGT) and provided with the secretion signal-encoding sequence by inserting it in the vector pUT-SEC (Li et al., 1997), cut with ApaLI/XbaI restriction endonucleases. From the resulting pUT-SEC-6A.C3-VH plasmid, a HindIII/NheI fragment was excised and cloned into the vector pcDNA-hu-C to generate the plasmid pcDNA-6A.C3-hu-C. For light-chain preparation, the 6A.C3 VL-encoding fragment was amplified with the already-described primer 6A.C3-VL-ApaLI and primer 6A.C3-VL-BsiWI (5'-TTCCGTACGTTTTATTTCCAGTTTGGTCC) and inserted in the previously described vector pUT-SEC-hu-C
(Borsi et al., 2002), containing the secretion sequence and the human constant region cDNA, cut at ApaLI/BsiWI sites. The complete chimeric 6A.C3 light-chain gene was then excised by HindIII/XhoI digestion and cloned into the expression vector pCMV2
(Borsi et al., 2002) to generate the plasmid pCMV2-6A.C3-hu-C. Equimolar amounts of both plasmids pcDNA-6A.C3-hu-C and pCMV2-6A.C3-hu-C were used to co-transfect Sp2/0 cells.
Cell transfection.
SIP constructs were expressed in both Sp2/0 and CHO cells. About 10 µg BglII-linearized plasmids and 1x106 cells were used for each transfection. Cells were resuspended in 0.7 ml cold PBS [10.1 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 3 mM KCl (pH 7.2)] and electroporated, after the addition of the linearized plasmids, with a Bio-Rad Gene Pulser equipped with a capacitance extender in a 0.4 cm gap cuvette. Sp2/0 cells were pulsed at 960 µF and 250 V and CHO cells at 25 µF and 1000 V. After electroporation, cells were resuspended in 10 ml culture medium and seeded in a 96-well plate. After 24 h, selective medium containing 400 µg G-418 ml–1 (Geneticin; Invitrogen) was added. Selected clones were then screened for the secretion of recombinant proteins by ELISA.
Evaluation of SIPs by ELISA.
Polystyrene Maxisorp ELISA plates (Nunc) were coated with goat antibodies, either anti-swine IgA (Serotec), anti-human IgA or anti-human IgE (KPL), for detection of sw-SIP, hu-SIP/chimeric IgA or hu-SIP, respectively, dissolved at 1 µg ml–1 in 50 mM Na2CO3/NaHCO3 buffer (pH 9.5). Supernatants from transfected cells were added and incubated for 2 h at room temperature. Serial dilutions of commercial human IgA (Sigma) or human IgE (Chemicon) were used as positive controls. The assay was developed with horseradish peroxidase (HRP)-conjugated antibodies, either anti-swine IgA (Serotec), anti-human IgA, anti-human IgE (KPL) or anti-human light chain (Dako) to detect sw-SIP, hu-SIP, hu-SIP or chimeric IgA, respectively. The substrate for peroxidase, TMB (3,3',5,5'-tetramethylbenzidine) solution (Sigma), was added and A450 was read with a microplate reader (Bio-Rad 550).
Western blotting.
ELISA-positive supernatants from stably transfected cells were subjected to SDS-PAGE (10 % gel) under reducing or non-reducing conditions (with or without 
-mercaptoethanol in the loading buffer), then electroblotted to Immobilon-P PVDF membrane (Millipore). Membranes were blocked with 50 g non-fat dry milk l–1 in PBS containing 0.1 % Tween 20 (PBS/Tween) and incubated with HRP-conjugated anti-swine IgA (Serotec), anti-human IgA or anti-human IgE (KPL) antibodies to detect sw-SIP, hu-SIP, chimeric IgA or hu-SIP, respectively. Membranes were washed extensively with PBS/Tween and incubated with ECL detection reagent (Amersham Biosciences), according to the manufacturer's instruction.
When required, samples were treated with the enzyme PNGase F (peptide N-glycosidase F; New England Biolabs) before SDS-PAGE separation.
ELISA titration and virus neutralization.
TGEV-specific SIPs expressed by Sp2/0 or CHO cells were analysed by ELISA, using purified TGEV as the antigen, and virus neutralization following procedures described previously (Correa et al., 1988). The antibody titre as determined by ELISA was defined as the antibody dilution factor giving a binding to TGEV threefold higher than the background. The neutralization index (NI) was defined as the logarithm of the ratio of the p.f.u. on ST cells after virus incubation in the presence of 50 µl medium or of the indicated antibody solution. The antisera used in ELISA to detect TGEV-specific SIPs were goat anti-human IgE (Nordic), goat anti-human IgA (Pierce) and rabbit anti-swine IgA (Bethyl Laboratories). All antisera were diluted 1 : 500 in PBS containing 0.3 % BSA and 0.1 % Tween 20. Bound antibodies were detected with HRP-conjugated rabbit anti-goat (Sigma) or HRP–protein A (Bio-Rad) diluted 1 : 1000.
Transient expression of TGEV-specific SIPs in BHK-pAPN cells and interference with TGEV infection.
BHK-pAPN (8x105) cells were transfected with 5 µg pcDNA-6A.C3-hu-SIP, pcDNA-6A.C3-hu-SIP, pcDNA-6A.C3-sw-SIP or plasmids encoding isotype-matching SIPs of irrelevant specificity, and 10 µl Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications. Twenty-four hours after transfection, cells were infected with TGEV PUR46-MAD at an m.o.i. of 0.5. After 1 h virus adsorption, the inoculum was replaced with culture medium at 37 °C. Supernatants from infected cells were analysed for virus titre at 0, 3, 18, 24 and 48 h post-infection (p.i.).
In vivo protection assays.
Two-day-old newborn piglets at the beginning of the experiment, which were obtained from sows that tested seronegative for TGEV and porcine respiratory coronavirus, were used in the immune-protection assays. The animals were treated three times per day with 4 ml cell-culture supernatant containing the different TGEV-specific SIPs or an sw-SIP with an irrelevant specificity as a negative control, diluted in milk. As a positive control, 0.4 ml ascitic fluid from the original mAb 6A.C3, also diluted in milk, was administered. With the first SIP administration, piglets were also infected with the virulent TGEV strain PUR46-SW11-ST2-C11 at 107 p.f.u. per animal, diluted in the same solution of SIP in milk. Four animals were tested with each construct. For a deeper characterization of passive protection provided in vivo by TGEV-specific SIPs, 6A.C3 sw-SIP and the negative control sw-SIP were administered to three additional groups of three piglets each, according to three different procedures: on day 0, the first dose of SIP-containing supernatant was administered 6 h before (A), at the same time as (B) or 6 h after (C) virus challenge. In the A and B situations, a new dose of SIP was also administered on day 0, 6 h after virus inoculation. In the following 2 days (days 1 and 2 p.i.), a dose of SIP was administered three times per day to the piglets. At 3 days p.i., piglets were sacrificed and virus titres were evaluated in gut and lung tissues, as p.f.u. (g tissue)–1 on ST cells.
Protection against TGEV infection was expressed as reduction in virus titre, corresponding to the ratio of the p.f.u. obtained after administering the negative-control SIP to those obtained when the SIP-6A.C3 mini-antibodies were administered.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
CH3 domains (hu-CH3 and sw-CH3) or the human S2CH4 domain (hu-S2CH4) (Batista et al., 1996) were inserted, separated by a short linker of 4 aa (GGSG, Fig. 1
). All constructs contained at the 5' end a secretion signal-encoding sequence and they were finally assembled into a eukaryotic expression vector under the control of the CMV promoter.
Human S2CH4 was chosen because it contains a cysteine residue at its carboxy terminus (Fig. 1a). It has already been demonstrated that SIP dimers are stabilized covalently by disulphide bridges between these two carboxy-terminal cysteines (Borsi et al., 2002). Both hu-CH3 and sw-CH3 contain cysteine residues at the penultimate position before the carboxy terminus; it is therefore expected that SIPs also form covalent dimers through inter-chain disulphide-bond formation (Fig. 1b).
SIPs were produced by transfection of CHO and Sp2/0 cells. Both cell lines expressed recombinant SIPs with high efficiency and clones producing up to 10 µg ml–1 were obtained. No significant differences in expression level were found among the different SIP isotypes.
As a control, a chimeric human IgA with the same specificity was generated by inserting the 6A.C3 VL and VH genes into expression vectors containing the human light-chain and heavy-chain constant-region cDNAs. These plasmids were then co-transfected into Sp2/0 cells and clones expressing complete IgAs were selected.
Biochemical properties of TGEV-specific SIPs
The TGEV-specific SIPs were analysed by SDS-PAGE followed by Western blotting. As expected, the hu-SIP showed a molecular mass of 40 kDa when analysed under reducing conditions, whilst under non-reducing conditions, it mainly migrated as a dimer of 80 kDa (Fig. 2a), because of the inter-chain disulphide-bridge formation between the carboxy-terminal cysteine residues of the hu-S2CH4 domain. hu-SIP and sw-SIP also migrated mainly as dimers under non-reducing conditions (Fig. 2a). This indicates that inter-chain disulphide bonds are also formed between cysteine residues located in the carboxy-terminal tails of human and swine CH3 domains.
|
SIP and sw-SIP showed molecular masses slightly higher than the expected 42 kDa, due to the presence of an N-glycosylation site in the sequences of both hu-CH3 and sw-CH3 domains (Fig. 1a). Indeed, treatment of hu-SIP and sw-SIP with the enzyme PNGase F, which removes all N-linked oligosaccharides from N-glycosylated proteins, produced a clear shift in the mobilities of both monomeric and dimeric SIPs (Fig. 2b). In reducing SDS-PAGE, the chimeric human IgA showed the expected two bands of 28 and 62 kDa for the light and heavy chains, respectively (Fig. 2a).
Functional analysis of TGEV-specific SIPs
The functionality of TGEV-specific SIPs, expressed stably by both CHO and SP2/0 mammalian cells, was analysed by ELISA on virus-coated plate, and virus-neutralization assays.
The secreted chimeric human IgA and SIPs (hu-SIP, hu-SIP and sw-SIP) showed good binding affinities for TGEV, as their ELISA-determined titres (i.e. the highest dilution giving a threefold increase over background) were all around 103 (Table 1).
|
). Virus-neutralizing activity was TGEV-specific, as the unrelated Vesicular stomatitis virus (VSV) was not neutralized by the chimeric 6A.C3 antibodies (data not shown).
No differences in activity were observed between TGEV-specific hu-SIP expressed in epithelial CHO cells or in myeloma Sp2/0 cells. Moreover, no significant differences in binding or neutralizing activity were observed between SIPs with different isotypes of human and swine origin. In addition, both binding and neutralizing activities were similar to those observed for the complete human IgA with the specificity of mAb 6A.C3, suggesting that the apparent affinity of the original antibody has not changed significantly in SIP mini-antibodies.
Interference with TGEV infection in BHK-pAPN cells transiently expressing TGEV-specific SIPs
BHK-pAPN cells (Delmas et al., 1995) stably express porcine aminopeptidase N, the cellular receptor for TGEV (Delmas et al., 1992), and are therefore susceptible to TGEV infection. These cells were transfected with either TGEV-specific SIPs or SIPs with similar isotype and antigenically irrelevant specificity and then infected with TGEV (m.o.i. 0.5). Cells expressing TGEV-specific SIPs were resistant to TGEV infection, as no cytopathic effect was observed at 48 h p.i., whereas cell monolayers expressing the irrelevant SIPs were lysed almost completely (Fig. 3a). The protection experiment was not continued for a longer time period because BHK-pAPN cells detach from culture plates after reaching confluence. The virus titre in the supernatants of infected cells was evaluated at 24 h p.i., as maximum virus titres are reached at this time point during infection of BHK cells by TGEV. A strong reduction in virus titre (105- to 106-fold) was observed in the supernatants of cells expressing TGEV-specific SIPs, in comparison with cell cultures producing SIPs with irrelevant specificity (Fig. 3b). The decrease in infectivity was specific, as cells expressing an irrelevant SIP synthesized TGEV efficiently. Extracellular neutralization is likely to be responsible for the strong decrease in virus production, as only about 20 % of the cells became transfected and secreted TGEV-specific SIPs into the medium, protecting the whole cell culture against infection.
|
). From in vitro neutralization experiments, it has been calculated that this amount of antibody solution can neutralize approximately 106 p.f.u., only about 10 % of the viral load administered. In all analysed cases, in fact, the infectivity of the virus inoculum after mixing with the antibody was always higher than 104.5 p.f.u., the lethal dose 50 (LD50) estimated for TGEV in newborn animals (L. Enjuanes, unpublished results). As a positive control, 0.4 ml mAb 6A.C3 ascitic fluid, with an NI higher than 5, was administered.
Previous results from our laboratory have shown that production of virus in piglets reached a maximum between days 2 and 4 p.i. (Sánchez et al., 1999). Therefore, it was decided to evaluate the potential production of virus within the animals at the plateau, i.e. at day 3 p.i., by determining virus titres in the gut and lung, which constitute the two main target organs of the virus. A clear reduction in virus titre was observed in both lung and gut tissues when compared with virus produced in animals administered with SIPs of irrelevant specificity (Fig. 4). Virus-titre reduction was always of at least 12-fold and it reached a maximum of 106-fold with SIP in lung, comparable to what was obtained with the original full-length mAb 6A.C3. Histopathological analysis of tissues from sacrificed animals showed that the most relevant signs of TGEV infection, i.e. atrophy of intestinal villi and interstitial pneumonia (measured as alveolar-wall thickening and the presence of exudates containing leukocytes), were absent from animals treated with TGEV-specific SIPs or the control mAb 6A.C3. Only mild pulmonary lesions were observed in one piglet treated with hu-SIP-6A.C3, in which a lower reduction in TGEV titre was detected. In contrast, control piglets infected with TGEV in the absence of antibody or treated with the antigenically irrelevant SIP showed signs of interstitial pneumonia and villous atrophy. The extent of immune protection was followed by studying the onset of atrophy of intestinal villi and interstitial pneumonia because these are the most reliable markers of TGEV-induced disease, as opposed to appearance of diarrhoea. This clinical sign is very frequent in TGEV-infected piglets, but it is also a very common episode in newborn animals as a consequence of artificial feeding, obligatory in in vivo experiments.
|
SIP-6AC3 was selected, as its constant region is derived from porcine IgA and it had shown good in vitro neutralization properties (Table 1). In addition, IgA is the predominant immunoglobulin isotype in respiratory and enteric mucosa, where protection against TGEV infection is expected. Three time points in the administration of SIPs were evaluated, including administration of the recombinant antibody before, at the same time as or after virus challenge. In all cases, a significant reduction in virus titre, mainly in lung tissues, was observed as a consequence of SIP administration, in comparison either with the administration of a SIP with irrelevant specificity or with the lack of SIP administration (Fig. 5). In lung tissues, the highest virus reduction (>8x105-fold) was associated with the administration of neutralizing SIPs before TGEV infection (procedure A). The administration of SIP either simultaneously with (procedure B) or 6 h after (procedure C) virus infection also reduced virus titres in the lung, although more moderately (8x103-fold in procedure B; 3x103-fold in procedure C). Virus-titre reduction in gut tissues was noticeable in all cases, although lower than that observed in lung (6x101- to 1.5x102-fold).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
S2CH4, which has already been exploited as a dimerizing domain (Borsi et al., 2002; Occhino et al., 2004). The other two SIP versions incorporated the CH3 dimerizing domain from either human or swine IgA. All of these recombinant mini-antibodies were secreted successfully from transfected cells, mostly as covalently stabilized dimers, demonstrating that cysteine residues in the carboxy-terminal tails of CH3 domains can form inter-chain disulphide bonds, as observed previously for SIPs (Borsi et al., 2002; Occhino et al., 2004). The specificity of the recombinant TGEV-specific SIPs was determined by in vitro neutralization and cell-protection assays. The NIs of the TGEV-specific SIPs were very similar to that of the parental 6A.C3 mAb, thus confirming that the biological activity of the TGEV-specific mAb was preserved in the recombinant SIPs. Protection assays against TGEV infection in cell cultures showed that the transient expression of TGEV-neutralizing SIPs reduced virus titres dramatically (up to 106-fold) to undetectable levels. Still, immunofluorescence experiments showed the presence of residual virus antigens in a minor fraction (<0.1 %) of cells (data not shown), suggesting that the presence of neutralizing SIPs in the medium prevented virus propagation in the cell culture until virus infection was extinguished.
In vivo protection experiments have, in addition, demonstrated that it is possible to confer passive immunization to newborn piglets by feeding them with recombinant SIPs diluted in milk. It has been demonstrated previously that SIPs display an extended half-life in the bloodstream when compared with monomeric scFv (Borsi et al., 2002). Present in vivo results indicate that SIPs are also stable when administered orally and, therefore, that they are sufficiently resistant to the enzymic and pH conditions of the enteric tract. This is especially relevant to validate the efficiency of recombinant SIPs in order to provide protection against enteric infections. In these experiments, no toxic effect related to SIP administration has been observed, suggesting that SIPs are safe molecules for the passive protection of piglets.
Although neutralizing SIPs were administered orally, a significant reduction in virus titre was observed not only in enteric tissues, but also in lungs. This could be due to the prevention of virus shedding, which results in a reduction of virus dissemination by the faecal–oral route. Alternatively, SIPs might prevent the internal distribution of the virus to respiratory organs, because of their high tissue-penetration properties.
In contrast to diarrhoea, histopathological lesions such as interstitial pneumonia and atrophy of intestinal villi are more specific signs associated with TGEV infection. Data from in vivo protection experiments showed that these signs were absent or reduced dramatically in piglets treated with TGEV-neutralizing SIPs, whereas they were evident in negative-control animals, either infected in the absence of antibody or those to which an irrelevant SIP was administered. The reduction in histopathological lesions correlated with virus titres recovered from lung and gut tissues.
Reduction in virus titre by administration of the positive-control mAb 6A.C3 to TGEV-infected piglets was more efficient than that observed after administering the 6A.C3-derived SIPs. This probably reflects a higher stability of the association of V regions, and therefore higher affinity for the antigen, in the full-length antibody compared with the SIPs, in which V regions are in the scFv format. It could also be related to the very high NI (>5) shown by mAb 6A.C3 in cell-culture neutralization assays (Table 1), which may correspond to an antibody titre higher than estimated.
Cell-culture neutralization assays have shown previously that a significant amount of virus remained infectious after mixing the virus and the SIP (see Results). Therefore, co-administration of virus and SIP still represents the administration of an amount of infectious virus sufficient to cause disease and death in piglets. Furthermore, the detection of virus in lung and gut tissues at day 3 p.i. cannot have its origin in the inoculum administered, because if the virus does not replicate in vivo in the enteric tract, it is cleared out within the first 2 days after inoculation (L. Enjuanes, unpublished results).
In addition, the effect of SIP administration at different time points revealed that efficient protection can also be achieved when virus and antibody are not administered simultaneously, thus ruling out the possibility that the protective effect was due to the in vitro neutralization of virus inoculum. The most efficacious method consisted of the prior administration of neutralizing SIPs in relation to virus challenge, providing a protective environment that prevents TGEV infection.
In general, virus-titre reduction in lung was significantly higher than that detected in gut tissues. Virus replication in intestine cells could be more variable than in pulmonary cells, depending on the age of piglets and the maturation and differentiation status of the organ, as it has been described that cells susceptible to TGEV infection are mostly present in newborn piglets (Enjuanes & Van der Zeijst, 1995). These differences might explain a higher variability in in vivo results obtained from gut tissues, leading sometimes to a lower virus-titre reduction.
Although a virus-sterilizing protection was not achieved in vivo, virus titres recovered from animal tissues after treatment with TGEV-neutralizing SIPs (<104.5) were lower than those inducing diarrhoea and death. As the virus doses infecting animals in field conditions are probably lower than those provided experimentally, the present results suggest that passive immunization with neutralizing SIPs might be a potential therapy to protect piglets from TGEV-induced disease under field conditions. Moreover, considering that 2-day-old piglets are the most susceptible animals to TGEV infection, the results obtained in the in vivo assay could anticipate the success of this protection approach in older piglets. In spite of the difficulties of in vivo experiments with piglets, this work provides the proof of principle of protection against virus infection by the oral administration of neutralizing SIPs. Further characterization of this therapeutic approach is in progress.
The transient expression of virus-neutralizing SIPs in mucosal surfaces could be used to provide immediate protection against virus infections that use these areas as the main port of entrance to the body. TGEV-specific SIPs are expressed equally well by myeloma Sp2/0 and epithelial CHO cells, indicating that the DNA constructs could be used to transform epithelial cells at mucosal surfaces for somatic gene therapy. This type of therapy could be of potential benefit to protect non-vaccinated individuals, such as newborn animals. Alternatively, the oral administration of virus-neutralizing SIPs might be particularly relevant when an immediate intervention is required to protect against bacterial or virus infections. For example, this technology could be applied to protect health-care workers against infections by severe acute respiratory syndrome (SARS) coronavirus, for which an efficient vaccine has not yet been obtained.
The easy manipulation of SIP genes makes possible their expression by using a variety of delivery vectors. One interesting possibility is the expression of SIPs in plants to achieve passive immunization upon feeding of animals, in order to develop cost-effective immune-protection strategies.
|
ACKNOWLEDGEMENTS |
|---|
|
NOTE ADDED IN PROOF |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
; Monger et al., 2006), and they were shown to be effective at protecting piglets against TGEV infection.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
|---|
Batista, F. D., Efremov, D. G. & Burrone, O. R. (1996). Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly. Proc Natl Acad Sci U S A 93, 3399–3404.
Borsi, L., Balza, E., Bestagno, M., Castellani, P., Carnemolla, B., Biro, A., Leprini, A. Sepulveda, J., Burrone, O. & other authors (2002). Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int J Cancer 102, 75–85.[CrossRef][Medline]
Castilla, J., Pintado, B., Sola, I., Sánchez-Morgado, J. M. & Enjuanes, L. (1998). Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk. Nat Biotechnol 16, 349–354.[CrossRef][Medline]
Correa, I., Jiménez, G., Suñé, C., Bullido, M. J. & Enjuanes, L. (1988). Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus. Virus Res 10, 77–93.[CrossRef][Medline]
Correa, I., Gebauer, F., Bullido, M. J., Suñé, C., Baay, M. F., Zwaagstra, K. A., Posthumus, W. P. A., Lenstra, J. A. & Enjuanes, L. (1990). Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus. J Gen Virol 71, 271–279.
Delmas, B., Gelfi, J., L'Haridon, R., Vogel, L. K., Sjöström, H., Noren, O. & Laude, H. (1992). Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357, 417–420.[CrossRef][Medline]
Delmas, B., Kut, E., Gelfi, J. & Laude, H. (1995). Overexpression of TGEV cell receptor impairs the production of virus particles. Adv Exp Med Biol 380, 379–385.[Medline]
Enjuanes, L. & Van der Zeijst, B. A. M. (1995). Molecular basis of transmissible gastroenteritis virus epidemiology. In The Coronaviridae, pp. 337–376. Edited by S. G. Siddell. New York: Plenum Press.
Escors, D., Camafeita, E., Ortego, J., Laude, H. & Enjuanes, L. (2001). Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core. J Virol 75, 12228–12240.
Gebauer, F., Posthumus, W. P. A., Correa, I., Suñé, C., Smerdou, C., Sánchez, C. M., Lenstra, J. A., Meloen, R. H. & Enjuanes, L. (1991). Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein. Virology 183, 225–238.[CrossRef][Medline]
Hu, S., Shively, L., Raubitschek, A., Sherman, M., Williams, L. E., Wong, J. Y., Shively, J. E. & Wu, A. M. (1996). Minibody: a novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res 56, 3055–3061.
Jiménez, G., Correa, I., Melgosa, M. P., Bullido, M. J. & Enjuanes, L. (1986). Critical epitopes in transmissible gastroenteritis virus neutralization. J Virol 60, 131–139.
Li, E., Pedraza, A., Bestagno, M., Mancardi, S., Sanchez, R. & Burrone, O. (1997). Mammalian cell expression of dimeric small immune proteins (SIP). Protein Eng 10, 731–736.
Monger, W., Alamillo, J. M., Sola, I., Perrin, Y., Bestagno, M., Burrone, O. R., Sabella, P., Plana-Duran, J., Enjuanes, L. & other authors (2006). An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts. Plant Biotechnol J 4, 623–631.[CrossRef][Medline]
Occhino, M., Raffaghello, L., Burrone, O., Gambini, C., Pistoia, V., Corrias, M. V. & Bestagno, M. (2004). Generation and characterization of dimeric small immunoproteins specific for neuroblastoma associated antigen GD2. Int J Mol Med 14, 383–388.[Medline]
Pack, P., Müller, K., Zahn, R. & Plückthun, A. (1995). Tetravalent miniantibodies with high avidity assembling in Escherichia coli. J Mol Biol 246, 28–34.[CrossRef][Medline]
Sánchez, C. M., Jiménez, G., Laviada, M. D., Correa, I., Suñé, C., Bullido, M., Gebauer, F., Smerdou, C., Callebaut, P. & other authors (1990). Antigenic homology among coronaviruses related to transmissible gastroenteritis virus. Virology 174, 410–417.[CrossRef][Medline]
Sánchez, C. M., Gebauer, F., Suñé, C., Méndez, A., Dopazo, J. & Enjuanes, L. (1992). Genetic evolution and tropism of transmissible gastroenteritis coronaviruses. Virology 190, 92–105.[CrossRef][Medline]
Sánchez, C. M., Izeta, A., Sánchez-Morgado, J. M., Alonso, S., Sola, I., Balasch, M., Plana-Duran, J. & Enjuanes, L. (1999). Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence. J Virol 73, 7607–7618.
Sola, I., Castilla, J., Pintado, B., Sánchez-Morgado, J. M., Whitelaw, C. B., Clark, A. J. & Enjuanes, L. (1998). Transgenic mice secreting coronavirus neutralizing antibodies into the milk. J Virol 72, 3762–3772.
Suñé, C., Jiménez, G., Correa, I., Bullido, M. J., Gebauer, F., Smerdou, C. & Enjuanes, L. (1990). Mechanisms of transmissible gastroenteritis coronavirus neutralization. Virology 177, 559–569.[CrossRef][Medline]
Torres, J. M., Sánchez, C., Suñé, C., Smerdou, C., Prevec, L., Graham, F. & Enjuanes, L. (1995). Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein. Virology 213, 503–516.[CrossRef][Medline]
Winter, G. & Milstein, C. (1991). Man-made antibodies. Nature 349, 293–299.[CrossRef][Medline]
Received 9 May 2006;
accepted 20 September 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
