©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Production of Human Secretory Component with Dimeric IgA Binding Capacity Using Viral Expression Systems (*)

Lorenz Rindisbacher (1), Sandra Cottet (1), Riccardo Wittek (1), Jean-Pierre Kraehenbuhl (2), Blaise Corthésy (1)(§)

From the (1) Institut de Biologie Animale, Btiment de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland and the (2) Swiss Institute for Experimental Cancer Research and Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The cDNA encoding the NH-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.


INTRODUCTION

Mucosal epithelia of the human body, including the linings of the digestive, respiratory, and urogenital systems, comprise a vast surface permanently vulnerable to attack by outside pathogens. Protection of mucosal surfaces against colonization and possible invasion by pathogenic microorganisms is mediated by a special class of antibodies known as secretory immunoglobulin A (sIgA)() (Mestecky and McGhee, 1987; Brandtzaeg, 1989; Kraehenbuhl and Neutra, 1992a). The antibodies are believed to act by agglutinating potential invaders and facilitate their clearance by peristaltic or mucociliary movement.

The IgA moiety is produced by local mucosal and glandular plasma cells. Two immunoglobulin A (IgA) antibodies are dimerized via J chain in a tail to tail arrangement; the addition of J chain occurs in plasma cells just before secretion (Parkhouse and Della Corte, 1973; McCune et al., 1981). To interact with pathogens on the luminal side of the mucosa, the dimeric IgA (dIgA) antibodies have to be transported across the epithelium barrier by the poly-immunoglobulin receptor (pIgR). The receptor, a type I transmembrane protein, is expressed by the epithelial cells on the basolateral side of the epithelia lining mucosal and glandular surfaces. Upon binding to the receptor, the receptor-ligand complex is internalized and transcytosed (Schaerer et al., 1991); during transport or at the apical cell surface, the pIgR is cleaved and the extracellular portion of the molecule, termed secretory component (SC), is released free or bound to IgA (for review, see Mestecky et al.(1991), Kraehenbuhl and Neutra (1992b), and Neutra et al.(1994)). Studies on the stoichiometry of SC-IgA association suggested that either one or two molecules of SC are contained per sIgA complex (Kühn and Kraehenbuhl, 1982).

The initial binding of SC to dimeric IgA in vitro is noncovalent, and interchain disulfide bridges are formed in a second step (Lindh and Björk, 1977; Garcia-Pardo et al. 1979; Eiffert et al., 1984). The noncovalent interaction has been shown to be mediated by the amino-terminal domain of rabbit SC (Frutiger et al., 1986) and human SC (Bakos et al., 1991). Binding of SC to IgA confers resistance to proteolytic enzymes, including Pronase, papain, trypsin, and pepsin (Underdown and Dorrington, 1974; Lindh, 1975). Brown et al. (1970) have shown that sIgA in duodenal secretions is more resistant than monomeric IgA to the action of proteases. Recently it was reported that human SC was able to compete the binding of Helicobacter pylori to a gastric epthelial receptor by virtue of fucosyl residues associated with SC carbohydrate side chains (Falk et al., 1993; Boren et al., 1993).

Despite the discovery of SC almost three decades ago, little is known about the properties the molecule confers to dimeric IgA antibodies, except for resistance to proteolysis. In order to analyze the molecular role of SC on IgA stability and determine its contribution to immune protection, both monoclonal IgA antibodies and the same antibodies reassociated with SC have to be available. Although protocols have been established for the production of monoclonal dimeric IgA antibodies (Lee et al., 1994), their combination to SC has been hampered by the difficulty to produce sufficient amounts of recombinant SC. The aim of this study was therefore to produce milligram quantities of hSC using recombinant virus expression systems and insect or mammalian cells.

We report here that recombinant hSC (a) is produced and secreted with high efficiency by both insect and mammalian cells, (b) is glycosylated by both insect and mammalian cells, yet to a different extent, (c) is readily isolated from serum-free culture medium by lectin or ion-chelate affinity chromatographies, and (d) binds to dimeric IgA, indicating that the molecule is properly folded and retains its biological activity. This is the first report on the production of biologically active recombinant hSC, that can now be used to dissect the properties it confers to dimeric IgA of defined specificity in in vitro systems (Hirt et al., 1993) or upon mucosal administration.


EXPERIMENTAL PROCEDURES

Cell Culture Conditions

African green monkey kidney CV-1 cells (ATCC CCL70) were grown in Dulbecco's modified Eagle's medium supplemented with 8% fetal calf serum containing 50 units/ml penicillin and 50 µg/ml streptomycin. The cells were cultivated to confluency in 175-cm flasks to a final concentration of 2 10 cells/flask in a 5% CO atmosphere at 37 °C. Human TK 143B cells (ATCC CRL 8303) and human HeLa cells (ATCC CCL 2) were grown under the same conditions. Human HeLa S3 cells (ATCC CCL 2.2; a clonal derivative of the parent HeLa cell line CCL2) were grown in spinner bottles at 37 °C, in minimal essential medium for suspension culture supplemented with 8% fetal calf serum, to a concentration of 6 10 cells/ml.

Spodoptera frugiperda (Sf9) insect cells (ECACC 89070101) were cultured at 28 °C in TC100 medium (Life Technologies, Inc.) containing 10% fetal calf serum or in SF-900 II medium (Life Technologies, Inc.) in the absence of serum. Cells grown as monolayers in T-flasks were passaged at confluency. Cells in suspension were grown as shaker cultures at 125 rpm in Erlenmeyer flasks and were diluted with fresh medium to 5 10 cells/ml twice a week.

Engineering of Human Secretory Component Sequences and Cloning into Viral Transfer Vectors

Human pIgR cDNA was obtained from Dr. Charlotte Kaetzel, University of Cleveland, OH. Since Glu is suggested to represent the authentic carboxyl terminus of human SC (Krajci et al., 1989; corresponding to Glu according to their numbering), we introduced a stop codon immediately downstream of this position in the recombinant protein. An EcoRI-BamHI fragment containing the hSC Kozak (1986) sequence, the hSC ATG initiation codon, the signal sequence for secretion, and the five extracellular Ig-like domains (Mostov et al., 1984), including Asp, was subcloned into pBluescriptII KS+ (Stratagene). The sequence corresponding to Pro to Glu, including a stop codon and an XbaI restriction site, was subsequently introduced 3` to the BamHI site as a double-stranded oligonucleotide,

On-line formulae not verified for accuracy

The resulting construct was referred to as pBS-hSC. The same strategy using the oligonucleotides,served to generate pBS-hSC:6xHis. This sequence encoding the cleavage site for Factor Xa, six consecutive histidines and a stop codon was inserted downstream of the codon for Glu.

In order to insert SC sequences into the vaccinia transfer vector p11K (Bertholet et al., 1986), the cDNAs were excised from pBS-hSC and pBS-hSC:6xHis by EcoRI/XbaI double digestion, Klenow filled-in, and blunt-end ligated into BamHI/EcoRI cut and Klenow filled p11K. Clones carrying the insert in either orientation were recovered for the generation of recombinant vaccinia viruses. The constructs were called p11K-hSC, p11K-hSC (reverse orientation), p11K-hSC:6xHis, and p11K-hSC:6xHis, respectively.

Human SC was also cloned into the vaccinia transfer vector pHGS-1 containing the TAAATG element including the translation start codon from the vaccinia 11K promoter (Bertholet et al., 1985). The 5` end of the gene was modified using a ClaI/AccI fragment generated by recombinant PCR (Higushi, 1990), using oligonucleotides 5`-AATAATTTCGCGCGGCCCATTTATAGCATAGAAA-3` and 5`-TTTCTATGCTATAAATG GGCCGCGCGAAATTATT-3` as ``inside'' primers and oligonucleotides 5`-CCATCGATG AAGGACAGTTCTTTCCAG-3` and 5`-GGGGTACCGGTCACCGTTCTGCCCAGGTCC-3` as ``outside'' primers. The PCR products were sequenced by the method of Sanger et al. (1977). Plasmids pHGS1-hSC and pHGS1-hSC:6xHis were constructed via a four piece ligation including: (a) vector pHGS-1 ClaI/EcoRI; (b) the ClaI/AccI PCR fragment containing the modified 5` end of the hSC gene; (c) the AccI/KpnI fragment from pCB6-hpIgR containing the central portion of the gene; and (d) the KpnI/XbaI fragment from pBS-hSC or pBS-hSC:6xHis containing the respective 3` ends.

Cloning of hSC into the baculovirus insertion vector pVL1392 (Invitrogen) was carried out by ligating the EcoRI/XbaI fragments containing the modified cDNAs, excised from pBS-hSC or pBS-hSC:6xHis, into the corresponding restriction sites of pVL1392. The resulting constructs were termed pVL1392-hSC and pVL1392-hSC:6xHis, respectively.

Generation of Vaccinia and Baculovirus Recombinants

The hSC cDNA under control of the 11K promoter was incorporated into the genome of wild-type vaccinia virus strain WR (ATCC VR1354) by homologous recombination into the thymidine kinase (TK) gene. Briefly, subconfluent CV-1 cell monolayers were infected with the temperature-sensitive vaccinia virus ts7 (Drillien and Spehner, 1983) and transfected with WR vaccinia virus DNA and the p11K- or pHGS1-hSC constructs according to Bertholet et al.(1985). Thymidine kinase-negative recombinant viruses were selected from the progeny virus by two rounds of plaque purification on TK 143B cells in the presence of 75 µg/ml bromodeoxyuridine. Recombinant virus plaques were isolated, and positive clones were identified by PCR screening with primers TKL+ (5`-CGGAACGGGACTATGGACGC-3`) and TKR- (5`-GTCCCATCGAGTGCGGCTAC-3`) specifically hybridizing to regions in the left and right portion of the TK gene flanking the insertion site. Recombinant viruses were amplified by infecting TK 143B cell monolayers in the presence of 50 µg/ml bromodeoxyuridine, and large stocks were prepared in CV-1 cells without selection. Recombinant viruses were named after the DNA inserted into the TK locus.

The engineered hSC gene was introduced into the polyhedrin gene of Autographa californica multiple nuclear polyhedrosis virus (baculovirus), essentially as described by Summers and Smith(1988), with modifications. Briefly, wild-type baculovirus DNA and transfer plasmids containing the hSC DNA under control of the polyhedrin promoter were cotransfected into Sf9 cells using cationic liposomes (Felgner et al., 1987): 1 ml of TC100 medium without serum containing 1 µg of baculovirus DNA prepared as described (Piwnica-Worms, 1990), 10 µg of pVL1392-hSC or pVL1392-hSC:6xHis, and 30 µl of DOTAP transfection reagent (Boehringer Mannheim) were vigorously mixed for 15 s, incubated at room temperature for 15 min, and added to 2 10 adherent Sf9 cells in a 60-mm plate, previously washed with serum-free medium. After 4 h of incubation on a rocking platform at room temperature, 1 ml of complete TC100 medium was added and incubation continued at 28 °C in a humidified environment. Five days later, the medium was harvested, and recombinant virus was plaque-purified by optical screening for plaques with occlusion negative phenotype (Summers and Smith, 1988). After two rounds of purification, the presence of recombinant and the absence of wild-type virus was confirmed by PCR as described (Malitschek and Schartl, 1991), using the polyhedrin gene flanking primers 5`-TTTACTGTTTTCGTAACAGTTTTG-3` (forward) and 5`-CAACAACGCACAGAATCTAG-3` (reverse).

Production of Recombinant hSC in Mammalian and Insect Cells

Stationary phase CV-1, HeLa, or TK 143B cells at a density of 2.0-2.5 10 cells/175 cm T-flask in 20 ml of medium were washed with PBS (Sambrook et al., 1989) and infected with recombinant vaccinia virus at the indicated multiplicity of infection (m.o.i.). Trial expression assays were performed with cells cultivated in 6-well dishes in 1 ml of medium. Infected cells were cultured in Dulbecco's modified Eagle's medium in the absence of serum and antibiotics. To assay for secreted hSC, 20-50-µl aliquots of culture medium were removed at defined intervals and replaced by fresh medium in order to avoid changes in the starting culture volume. Culture supernatants originating from cells grown in the absence of virus, infected with wild-type virus, or infected with recombinant virus carrying the engineered hSC DNA in reverse orientation with respect to the promoter were included as controls.

HeLaS3 cells grown to a density of 6 10 cells/ml in suspension minimal essential medium were transferred under sterile conditions to centrifuge bottles, pelleted 120 g for 10 min at room temperature, then washed twice with PBS and once with serum-free suspension minimal essential medium. After resuspension at a density of 1.0 10 cells/ml, they were poured into a spinner bottle, infected with an m.o.i. of 10 for 1 h, diluted to 4 10 cells/ml in serum-free suspension minimal essential medium, and then incubated for 21-24 h. Culture supernatant was harvested by centrifugation for 15 min at 120 g.

Baculovirus inocula for infection of Sf9 cells were obtained from cell culture supernatants recovered at least 4 days post-infection at m.o.i.'s larger than 1 with master stock virus. Sf9 cells at densities of 1 10 cells/ml in shaker cultures or 5 10 cells/25-cm T-flask were infected at estimated m.o.i.'s of 1 or 5 by addition of a corresponding volume of virus inoculum directly to the cultures. Four days post-infection (or as indicated), cells were centrifuged for 10 min at 600 g and the supernatants containing secreted recombinant protein were harvested. Expression of secreted and intracellular recombinant SC from mammalian or insect cells was monitored by immunoblotting.

Affinity Purification of Recombinant Human Secretory Component

Nickel Chelate Affinity Chromatography

Protein recovered from mammalian cell culture medium was dialyzed/concentrated (100-fold) in 20 mM sodium phosphate, 500 mM NaCl, pH 7.8 (Ni binding buffer), in a Spectrum Micro-ProDiCon system model FS-15 using 25-kDa molecular weight cut-off membranes. This material was applied to a Hitrap chelating matrix (Pharmacia Biotech; 1-ml bed volume/ml of concentrated culture supernatant), charged with nickel ions and equilibrated with Ni binding buffer. Culture supernatants originating from infected Sf9 cells were loaded three times onto Ni-nitrilotriacetic acid-agarose columns (Qiagen; 300-µl bed volume for 1 ml of culture supernatant for analytical, and 2-ml bed volume per 100 ml of supernatant for preparative assays), equilibrated with Ni binding buffer. Columns were subsequently washed with 20 mM sodium phosphate, 500 mM NaCl, pH 6.3 (Ni washing buffer), followed by the same buffer containing 20 mM imidazole. Bound protein was eluted with washing buffer containing 100 mM imidazole and fractions were collected in siliconized polypropylene tubes. Eluted proteins were subjected to SDS-PAGE, and purity as well as lack of degradation were assayed by silver staining and immunoblotting.

Concanavalin A Affinity Chromatography

Mammalian cell culture supernatant were dialyzed and concentrated as above, in 10 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl, 1 mM MnCl (ConA-binding buffer). This material or unconcentrated insect cell culture supernatant were passed over ConA-agarose beads (Vector Laboratories, 500-µl bed volume per ml of loaded material), equilibrated with ConA binding buffer. The columns were extensively washed with ConA binding buffer, and bound material was eluted with the same buffer containing 500 mM methyl--D-mannopyranoside (Fluka). Collection and subsequent analysis of fractions were carried out as above.

Enzymatic Deglycosylation

Recombinant hSC produced in various host cell types was treated with the endoglycosidases PNGase F (EC 3.2.2.18 and 3.5.1.52) or EndoH (EC 3.2.1.96; both Boehringer Mannheim) according to the manufacturer's instructions. Briefly, 150 µl (insect) or 600 µl (mammalian) cell culture supernatant or, as controls, appropriate amounts of human sIgA (Sigma) or purified nonglycosylated hSC produced in bacteria, were extracted with a mixture of methanol and chloroform and precipitated with methanol, as described (Wessel and Flügge, 1984). The precipitates were solubilized and denatured by boiling for 2 min in 50 µl of 1% SDS. 15 µl of this solution were added to 135 µl of PNGase F buffer (20 mM sodium phosphate, 50 mM EDTA, 10 mM sodium azide, 0.5% Nonidet P-40, 0.1 M -mercaptoethanol, pH 7.2) for mock treatment and PNGase F digestion, or to 135 µl of the same buffer, but adjusted to pH 5.5, for EndoH digestion. The samples were again boiled for 2 min, then 4 µl of PNGase F (0.2 unit/µl), EndoH (1 milliunit/µl), or HO were added to the corresponding tube. Digestions and control samples were incubated at 37 °C for 16 h, then extracted and precipitated using the methanol/chloroform procedure. The resulting pellets were dissolved in sample buffer for analysis by immunoblotting.

Immunoblotting

Proteins were subjected to PAGE on 6 or 8% resolving gels under denaturing (0.1% SDS) and reducing (100 mM dithiothreitol) conditions, together with prestained molecular weight markers (Bio-Rad). Separated proteins were then transferred to nitrocellulose or polyvinylidine difluoride membranes (Bio-Rad) according to the manufacturer's recommendations. Nonspecific binding sites were saturated for 1 h at room temperature by incubation in a blocking buffer made of TBS (25 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, pH 7.5), 5% non-fat dry milk, and 0.05% Tween 20 (Bio-Rad). The membrane was probed for 1 h at room temperature with either a rabbit antiserum against recombinant hSC() or a mouse monoclonal antibody to hSC (Sigma), diluted 1:3,000. Bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin antibodies (Sigma) and the enhanced chemiluminescence kit from Amersham.

Dot Blot Reassociation Assay

Dimeric IgA specific for mouse mammary tumor virus gp52 protein were purified from mouse hybridoma clone MB2 (Weltzin et al., 1989) by size exclusion chromatography on a Sepharose CL-4B column (2.2 100-cm; Pharmacia Biotech Inc.). Mouse IgG were obtained from Sigma. Binding of recombinant hSC to dIgA antibodies was tested as follows. 500 ng of mouse dIgA, or as a control 200 ng of mouse IgG in 10 µl of PBS containing 6.25% glycerol, were spotted onto 24-mm diameter nitrocellulose membranes (0.2-µm pore size; Schleicher & Schuell) and allowed to air dry. Filters were then transferred to 6-well tissue culture dishes and blocked with TBS containing 5% non-fat dry milk and 0.05% Tween 20 for 1 h at room temperature. After removal of the blocking solution, the dIgA-carrying filters were overlaid with 100 ng of recombinant hSC protein (recovered from crude culture supernatant, or purified by affinity chromatography) in 1.5 ml of fresh blocking solution for 1 h at room temperature. After three 10-min washes with TBS, 0.05% Tween 20, recombinant hSC bound to the dIgA on the membranes was detected using monoclonal anti-hSC antibodies (Sigma) under the conditions given for immunoblotting. Similarily, samples of recombinant hSC containing 100 ng of protein were immobilized onto nitrocellulose and overlaid with mouse dIgA or IgG under the conditions given above. Detection of the bound component was performed with anti-mouse IgA or anti-mouse IgG Fc specific-monoclonal antibodies (Sigma), respectively.

ELISA

Two-antibody sandwich assay was performed according to Harlow and Lane(1988). The first antibody was mouse monoclonal antibody to hSC (Calbiochem) at a dilution of 1:500, the second antibody was rabbit polyclonal antiserum to hSC used at a dilution of 1:1,000, and detection was carried out using a goat anti-rabbit IgG peroxidase conjugate antibody (Sigma; dilution of 1:1,000) with 1,2-phenylenediamine as the chromogene. These conditions were optimized to detect as little as 0.25 ng of hSC in a native conformation.

High Pressure Gel Filtration Chromatography

In a total volume of 100 µl of PBS, varying amounts of purified recombinant hSC were incubated with a constant amount of dimeric IgA, yielding solutions of 0.5:1, 1:1, and 2:1 µM, respectively. The solutions were incubated for 16 h at ambient room temperature and then passed over a 1-cm 30-cm Superose 12 HR 10/30 column (Pharmacia) coupled to a FPLC system (Pharmacia) at 0.3 ml/min. Elution was effected with PBS, with continuous monitoring performed at 278 nm. The presence of dimeric IgA and recombinant SC in peak fractions was confirmed by Western blotting.


RESULTS

Construction of Recombinant Virus

In order to determine the role of SC in IgA antibody stability and its contribution to protection against microbial pathogens, we have produced recombinant human SC that retains its capacity to bind dimeric IgA using two viral expression systems. Since SC is generated by proteolytic cleavage of the pIgR during transepithelial transport we introduced a stop codon in the receptor's cDNA at a position corresponding to the putative natural cleavage site. The 5` region encoding the hSC signal peptide was preserved in order to direct the protein into the secretory pathway and to subsequently recover the recombinant protein from the culture medium. Finally, to facilitate the purification of the protein, a histidine hexamer tag preceded by a factor Xa cleavage site was inserted upstream of the artificial stop codon, encoding six consecutive histidines preceeded by a Factor Xa cleavage site. The resulting recombinant genes were subcloned into vaccinia and baculovirus transfer vectors (Fig. 1).


Figure 1: Schematic representation of cloning and engineering of hSC sequence into baculovirus and vaccinia virus expression vectors. The pVL1392 construct contains the hSC cDNA fragment encoding amino acid Met to Glu downstream of the baculovirus polyhedrin promoter (pPH). A stop codon (End) or an artificial sequence coding for 6 histidines and a stop codon (6xHisEnd) was introduced 3` of amino acid 589. The naturally occurring Kozak sequence in hSC was conserved in the cloning process. Constructs p11K and pHGS1 contain the same segment of hSC cDNA, with the presence or absence of codons for a 6xHis tail. Both constructs are under the control of the vaccinia virus late 11K promoter (p11K). Construct p11K carries the hSC Kozak sequence, while in construct pHGS1, the highly conserved vaccinia virus promoter element TAAAT(G) is coupled to the initiation ATG codon as in the natural situation. At the level of the junctions between viral and hSC sequences, the hSC 5` region is shown in bold characters. Sequences involved in homologous recombination are depicted as open rectangles, promoters as dot-filled arrows, and the ss box upstream of the hSC gene stands for signal sequence.



The foreign DNA was introduced into the double-stranded DNA genome of vaccinia or baculovirus following published procedures (Mackett et al., 1984; Summers and Smith, 1988). The foreign genes require viral promotors to drive efficient transcription. The transfer plasmids p11K and pHGS-1 contain the vaccinia late promoter P11K (Hänggi et al., 1986) flanked by vaccinia TK sequences to direct recombination into the TK locus of the viral genome. In the p11K recombinants, the Kozak sequence (Kozak, 1986) was derived from the hSC gene itself, while pHGS-1 recombinants contain viral transcriptional and translational regulatory sequences originating from the 11K viral gene. Recombinant hSC DNAs containing their own translation initiation signals were subcloned into the baculovirus transfer vector pVL1392, downstream of the promoter governing expression of the viral polyhedrin gene. The presence of the hSC cDNA insert in the viral genomes, as well as the absence of contaminating wild-type virus, was ascertained by PCR utilizing hSC sequence- or transfer vector-specific primers, respectively, that generated unique amplification products of the expected sizes (data not shown).

Optimization of Recombinant Human Secretory Component Expression

We first determined the kinetics of hSC production after infection at a multiplicity of infection (m.o.i.) of 1 or 5. CV-1 cells were infected with recombinant vaccinia viruses p11K-hSC:6xHis and pHGS1-hSC:6xHis. To facilitate subsequent purification of the product, the cells were cultured in serum-free Dulbecco's modified Eagle's medium during infection and the production phase. Aliquots of culture supernatant were removed at 3, 6, 9, 20, and 23 h post-infection, separated by polyacrylamide gel electrophoresis, and immunoblotted with hSC antiserum. The recombinant protein appeared 9 h after infection using the p11K-hSC:6xHis recombinant virus (Fig. 2A). Increasing the m.o.i. from 1 to 5 accelerated the production without increasing the final yield of hSC by 20 and 24 h after infection (). Addition of serum to the culture medium did not enhance SC production (data not shown).


Figure 2: Time course of hSC expression in mammalian and insect cells as a function of multiplicity of infection. Panel A, cultures of CV-1 cell monolayers were infected either with vaccinia virus recombinant p11K-hSC:6xHis (lanes 1-5) or vaccinia virus recombinant pHGS1-hSC:6xHis (lanes 6-10) at m.p.i. of 1 plaque forming unit/cell (top) and 5 plaque forming units/cell (bottom). At 3, 6, 9, 20, and 23 h post-infection, aliquots were taken up, separated by SDS-PAGE (8% separating gel; reducing conditions), transferred to blotting membrane, and hSC was detected immunochemically as described under ``Experimental Procedures.'' Molecular size markers are given in kilodaltons. Panel B, refined time course analysis of hSC expression in HeLa cells infected with vaccinia virus recombinant p11K-hSC:6xHis at a m.o.i. of 5 plaque forming units/cell. Aliquots of supernatant were collected 3, 6, 9, 12, 15, 27, 30, and 33 h post-infection. Protein accumulation in the culture medium was monitored using Western blot. Panel C, analysis of hSC synthesis during recombinantbaculovirus infection. Sf9 cell grown in suspension (lanes 1-6) or as monolayers (lanes 7-12) were infected with approximately 1 and 5 plaque forming units/cell of baculovirus recombinant pVL1392-hSC:6xHis. Culture medium of cells grown in suspension or as a monolayer were taken up at day 0, 1, 2, 3, 4, and 5, and analyzed by immunodetection.



The kinetics of hSC production reflected that observed with other heterologous cDNAs driven by the vaccinia late promoter (Hänggi et al., 1986). To test whether the rate of hSC production and secretion observed in CV-1 cells was similar in another cell line, we infected HeLa cells at an m.o.i. of 5 with the p11K-hSC:6xHis recombinant virus, and looked for hSC production at shorter intervals. The appearance of hSC was assessed by immunoblotting (Fig. 2B), and quantitative analysis was based on ELISA (). Secreted recombinant hSC could be observed 6 h after infection, starting to accumulate after 9 h.

Following infection of insect cells with recombinant baculovirus, expression of the corresponding recombinant protein is known to follow slower kinetics when compared to vaccinia virus (Summers and Smith, 1988). Similar slow kinetics of hSC secretion were observed in Sf9 cells infected with pVL1392-hSC:6xHis recombinant baculovirus (Fig. 2C). Suspension or monolayer cultures were infected at estimated m.o.i.s of 1 and 5, and aliquots of culture medium were removed at 0 through 5 days post-infection in 24-h intervals and analyzed by immunoblotting. Suspension cultures start to accumulate recombinant protein in the supernatant 2 days post-infection. As compared to monolayer cultures, production in suspension yielded higher amounts of hSC.

On immunoblots, recombinant hSC produced from recombinant vaccinia virus was detected in as little as 2.5 µl of cell culture supernatant, whereas the baculovirus product was revealed in less than 1 µl of supernatant. The protein expressed by each of the systems was quantitated densitometrically by comparing the signals obtained from increasing amounts of culture supernatant with known quantities of an sIgA standard on the linear part of the response curve of the Western blot (data not shown). The concentration of hSC recovered in culture supernatants from the vaccinia virus/mammalian cell system ranged from 5 to 10 mg/liter, while the baculovirus/insect cell system yielded up to 50 mg/liter.

Addition of Sequences to the COOH Terminus of hSC Does Not Alter Expression

The cDNAs introduced into the viral genomes contained initiation sites for translation derived either from the hSC gene itself or, in the case of vaccinia pHGS-1 recombinants, from the viral 11K gene. In addition, a 6xHis tag preceded by a Factor Xa cleavage site were fused to the artificial carboxyl termini of the recombinant proteins (Fig. 1). To determine whether these minor differences in nucleotide and amino acid sequence altered hSC expression, CV-1 cell monolayers were infected with the various vaccinia virus recombinants at an m.o.i of 1. Twenty hours post-infection, equal amounts of supernatant were collected, and the presence of hSC protein was assessed by Western blotting. As shown in Fig. 3A, comparable signal intensities on the Western blot and similar patterns of migration of recombinant hSC production were observed. With both the hSC and 11K regulatory sequences upstream of the ATG codon, equivalent amounts of protein were synthesized as assayed by ELISA (), reflecting comparable translation initiation efficiencies. The addition of six histidyl residues together with the target sequence for Factor Xa at the carboxyl terminus of the protein did not alter the secretion of recombinant hSC using either viral expression systems (Fig. 3, A and B). No hSC production was detected when CV-1 cells were infected with a vaccinia recombinant carrying the hSC gene in reverse orientation with respect to the viral promoter. Accordingly, no recombinant protein was produced by CV-1 or Sf9 infected with wild-type virus or by non-infected cells (data not shown).


Figure 3: Western blot analysis of engineered hSC produced in CV-1 and Sf9 cells. Panel A, expression of hSC as a function of engineered cDNA inserts. CV-1 cell monolayers were infected either with recombinant vaccinia virus p11K-hSCEnd, vaccinia virus recombinant p11K-hSC, two individual vaccinia virus recombinants p11K-hSC:6xHis, recombinant vaccins virus pHGS1-hSC, three individual vaccinia virus recombinants pHGS1-hSC:6xHis at a multiplicity of 1 plaque forming unit/cell. 20 h post-infection, cell culture media were collected and 20 µl of each supernatant were analyzed by immunoblotting as described under ``Experimental Procedures.'' Molecular size markers are given in kDa. Panel B, expression of hSC carrying or lacking a 6xHis tag in Sf9 cells infected for 4 days with baculovirus recombinant pVL1392-hSC or pVL1392-hSC:6xHis, respectively. No expression of hSC occurs in Sf9 cells infected with wild type BV.



The Glycosylation Pattern of hSC Differs Depending on the Cell Line and the Recombinant Viral Expression System

The apparent molecular weight of secreted recombinant hSC varied significantly depending on the host cell type used for expression (Fig. 4A). When produced in insect cells which are unable to generate fully glycosylated forms, the baculovirus hSC product migrated faster than natural hSC on SDS gels. When recovered from human HeLa or TK 143B cells, hSC significantly and reproducibly migrated at a slightly higher M than natural human SC, or recombinant hSC produced by monkey CV-1 cells. In order to correlate these size differences with glycosylation, we examined the susceptibility of recombinant hSC to the action of endoglycosidase PNGase F, which removes high mannose, hybrid, and complex type N-linked carbohydrates from glycoproteins (Tarentino and Plummer, 1994). Upon PNGase F treatment, two bands were observed with recombinant hSC produced in mammalian cells (Fig. 4B); a 66-kDa band comigrating with the unglycosylated bacterial product, and a 60-kDa band corresponding to the deglycosylated ``natural'' product. In Sf9 insect cells, only the 60-kDa band could be detected. We explain this digestion pattern as follows. Following transcytosis, the pIgR is trimmed of both its transmembrane and cytoplasmic domains by a so far unidentified protease activity, leading to the release of SC. The site of protease cleavage has not been defined with dependable accuracy. Our constructs are based on cleavage at Glu (see``Experimental Procedures''), leading to the synthesis of a recombinant protein with a calculated molecular mass of 66,026 daltons in the absence of any glycosylation. Assuming that the band at 60-kDa represents the deglycosylated, naturally cleaved pIgR, a portion of the hSC proteins expressed in mammalian cells and all of the protein produced in insect cells have somehow been trimmed to the size of natural hSC. On the other hand, a proportion of the mammalian products remains intact and migrates at the expected position for a fully deglycosylated recombinant hSC, such as the unglycosylated recombinant bacterial product. Such proteolytic cleavage within the COOH-terminal region of the recombinant protein would also remove the 6 x histidine tag, thus affecting the yield of hSC purified on Ni affinity resins (see below). Endoglycosidase H, an enzyme that does not cleave complex type N-linked carbohydrate (Elbein et al., 1982), did not induce any significant change in the molecular weight of recombinant hSC (Fig. 4B), suggesting that the bulk of carbohydrates N-linked to recombinant hSC is of the complex type.


Figure 4: Enzymatic deglycosylation of recombinant hSC expressed in different cell lines. Panel A, whole cell lysate from E. coli M15 strain transformed with expression vector pQE9-hSC6xHisEnd (B), crude supernatants from baculovirus recombinant pVL1392-hSC:6xHis-infected Sf9 cells (Sf9), vaccinia virus recombinant p11K-hSC:6xHis-infected HeLaS3 cells (S3), HeLa cells (H), TK 143B cells (TK), CV-1 cells (C), and standard human secretory IgA (IgA) were separated on a reducing 8% SDS-polyacrylamide gel, blotted to polyvinylidine difluoride membrane, and location of hSC was identified using anti-hSC monoclonal antibody. Note the difference in the patterns of migration of the overproduced hSC proteins compared to the natural hSC. Panel B, supernatants shown in panel A were incubated in the presence of PNGase F (F), endoglycosydase H (H), or the corresponding buffer alone (-). The resulting samples were electrophoresed on 6% polyacrylamide gel, and processed for immunodetection.



Purification of Recombinant Human Secretory Component

Recombinant hSC protein carrying a carboxyl-terminal 6xHis tag produced by HeLaS3 or Sf9 cells maintained in serum-free medium was purified from 20-h or 4-day post-infection supernatants, respectively, by Ni-chelate affinity chromatography. Small volumes of about 100-fold concentrated HeLa or unconcentrated Sf9 supernatant were applied onto equilibrated in binding buffer at pH 8.0. In order to remove unspecifically bound material, the column was washed stepwise with washing buffer (pH 6.3), and subsequently with washing buffer containing a subelution concentration (20 mM) of imidazole competitor. Recombinant hSC was eluted at 100 mM imidazole, indicating high binding specificity to the Ni resin. Purified hSC from Sf9 cells migrated on SDS-PAGE as a single band of about 70 kDa (Fig. 5C, lane 4) with no degradation products as revealed by immunodetection (Fig. 5C, lane 5) and the recovery was estimated to be 80-90% of the amount of recombinant hSC loaded onto the column. The recovery of purified hSC produced using the vaccinia system reached almost 100% with several SC-unrelated proteins probably of viral origin contaminating the preparation (Fig. 5A, lanes 4 and 5).


Figure 5: Purification of recombinant hSC by affinity chromatography. Analysis by SDS-PAGE (8% separating gel, reducing conditions) of purified hSC produced either in HeLaS3 cells infected with vaccinia virus recombinant p11K-hSC:6xHis (panels A and B), and in Sf9 cells infected with baculovirus recombinant pVL1392-hSC:6xHis (panels C and D). Panels A and C correspond to samples resulting from affinity chromatography on Ni-chelate column, whereas panels B and D contain fractions from the ConA affinity column. Samples in panels A-D were loaded in the following order: lane 1, cell culture supernatant (SN); lane 2, column flow-through (FT); lane 3, column fraction containing the first wash (W); lanes 4 and 5, column fraction containing the elution peak of hSC (E). Proteins were visualized by silver staining (lanes 1-4) and immunodetection (lane 5). Molecular size markers are given in kilodaltons (kDa).



This drawback prompted us to evaluate a different affinity chromatography protocol. Since recombinant hSC produced in mammalian and Sf9 cells is glycosylated (Fig. 4), we explored the possibility of purifying the recombinant protein on a lectin column. Dialyzed/concentrated CV-1 supernatant or unconcentrated Sf9 supernatant were loaded onto the ConA column and, after extensive washing, recombinant hSC was eluted with 0.5 M methyl--D-mannopyranoside. Human SC produced by the vaccinia/HeLaS3 system eluted as a single M 85,000-90,000 band on silver-stained SDS-PAGE (Fig. 5B, lane 4) or immunoblot (Fig. 5B, lane 5). The recovery was over 90%. The protein expressed in Sf9 cells was also efficiently purified (Fig. 5D), but the elution was over a broader range of fractions with a final recovery below 60%

Interaction Between Recombinant hSC and Dimeric IgA

The IgA binding activity of recombinant SC was assessed by a dot blot reassociation assay (DORA). The principle illustrated in Fig. 6A is described under ``Experimental Procedures.'' After immobilization of one of the interacting partners on a nitrocellulose filter, the other component is added as an overlay in a buffered solution in the presence of nonspecific protein competitor. Association is determined by immunodetection of the component in the overlay phase. Formation of a specific SCdIgA complex was observed with hSC recovered from the supernatant of insect and mammalian cells infected with the appropriate recombinant virus (Fig. 6B, lanes 1-5 and 7-11). Both interacting partners retained their binding capacity indicating that the functional structure of the immobilized protein was preserved. This was further supported by the recognition of recombinant SC by SC-specific antibodies (Fig. 6D, lane 3). No binding was detected in supernatants from cells infected with recombinant vaccinia expressing SC in the reverse orientation (p11K-hSC:6xHis; Fig. 6B, lanes 6 and 12), with wild-type virus or from non-infected cells (data not shown). In order to further demonstrate the specificity of the interaction, we repeated the DORA with purified hSC produced in three different cell lines (Fig. 6C). Consistently, immobilized SC bound dimeric IgA (lanes 1-3) and recombinant hSC bound to immobilized dimeric IgA (lanes 4-6). Recombinant SC did not bind IgG (Fig. 6D, lanes 1 and 2). To determine which proportion of recombinant hSC is capable of associating with dimeric IgA, we analyzed by ELISA the hSC recovered in the overlay after incubation with the nitrocellulose filters carrying the polymeric immunoglobulins (Fig. 7A). No more than 5% of the recombinant hSC protein input can be detected when an equimolar mixture of the partners is mixed (compare lanes 1 and 3 with lanes 2 and 4), reflecting both quantitative binding to dimeric IgA and correct three-dimensional folding. In the absence of dimeric IgA (lane 6), or when monomeric IgA was spotted (lane 8), recombinant hSC remained free in the overlay. These series of binding data demonstrate the usefulness of the DORA test for rapid and sensitive assessment of SCdIgA reassociation in vitro.


Figure 6: In vitro determination of binding specificity between recombinant SC and dimeric IgA. Panel A, cartoon representation of DORA. Details are given under ``Experimental Procedures.'' Panel B, binding of mouse dimeric IgA (IgA) to human SC secreted in culture supernatant (SN) of recombinant virus-infected cells (lanes 1-6). Sf9 cells were infected with baculovirus recombinant pVL1392-hSC:6xHis, and mammalian cells with vaccinia virus recombinant p11K-hSC:6xHis. SN corresponds to supernatant of CV-1 cell infected with vaccinia virus recombinant p11K-hSC:6xHis. Lanes 7-12 show binding of the same set of SC-containing SN to spotted dIgA. SC and dIgA were present at a 1:1 molar ratio. S3, HeLaS3 cells; anti--chain, affinity purified antibody against the heavy chain of mouse IgA; anti-SC, monoclonal antibody against human SC. Panel C, binding of dIgA to purified recombinant SC produced in culture SN of Sf9, HeLaS3, and CV-1 cells infected as for the experiments in panel B (lanes 1-3). Association between purified SC and immobilized dIgA is shown in lanes 4-6. Panel D, lack of interaction between purified recombinant SC produced in virus-infected Sf9 and HeLaS3 cells and mouse IgG (lanes 1 and 2). Binding of anti-SC antibody to spotted SC is not abolished after incubation with IgG (lane 3). Anti--chain, affinity purified antibody against the Fc domain of mouse IgG.




Figure 7: In vitro determination of binding efficiency between recombinant SC and dimeric IgA. Panel A, over 95% of recombinant hSC input binds dimeric IgA (dIgA). DORA was performed as described under ``Experimental Procedures,'' and unbound hSC in the supernatant was analyzed by ELISA. Lanes 1,3,5, and 7, input hSC; lanes 2,4,6, and 8, hSC in the overlay after a 1-h incubation. Lanes 1 and 2, hSC expressed in Sf9 cells and dIgA; lanes 3 and 4, hSC expressed in HeLa cells and dIgA; lanes 5 and 6, hSC expressed in HeLa cells and no dIgA; lanes 7 and 8, hSC expressed in HeLa cells and monomeric IgA (mIgA). The ELISA read-out corresponding to unbound hSC is expressed as 100% on the vertical axis. Panel B, high pressure gel filtration chromatography of varying ratios of recombinant hSC to dIgA. The reactants were present at a 0.5:1 molar ratio (-), a 1:1 molar ratio (- - -), and a 2:1 molar ratio () of hSC to dimeric IgA, respectively. The position of the sIgA complex and free SC are indicated on the top of the peaks.



SCdIgA complex formation was further demonstrated by high pressure gel filtration chromatography experiments. Different molar ratios of recombinant hSC and dimeric IgA were reacted at room temperature for 16 h, and their products were resolved by size fractionation (Fig. 7B). Observation of a shift in the position of elution of hSC to that of dimeric IgA indicates that the two proteins can recognize each other in this dynamic assay totally performed in buffered solution. At a 0.5:1 ratio of hSC to IgA, only the high molecular weight complex peak is present, indicating that the reformation of hSCIgA complex occurs, and accordingly, that recombinant hSC is properly folded. At a 1:1 ratio, the hSC peak is again totally shifted to the position of the high molecular mass species. At a 2:1 ratio, the hSCIgA complex peak remains the same, but a free hSC peak is now observed.


DISCUSSION

Secretory IgA, the major immunoglobulin class in mucosal and glandular secretions, consists of one dimeric IgA unit and two additional polypeptide chains, J chain and SC. The heavy, light, and J chains are synthesized and dimeric IgA is assembled in plasma cells, whereas SC is contributed by the epithelial cells of mucosal and glandular tissues. To produce large amounts of sIgA for passive oral (mucosal) immunization (Apter et al., 1993a, 1993b; Lee et al., 1994), it is necessary to synthesize both SC and dimeric IgA in different cells and subsequently properly associate the two components. While large amounts of dIgA can be produced by and purified from hybridoma clones, preparation of complete sIgA has been so far prevented by the lack of efficient production systems for SC. As a first step toward in vitro reconstitution of sIgA antibody molecules, we evaluated two viral expression systems for the production of human SC that retains its capacity to bind and stabilize dimeric IgA molecules.

The insect cell-based baculovirus system is well suited for high-level expression of heterologous genes (Miller, 1993). Appropriate folding, assembly, and targeting of recombinant proteins by insect cells, as well as their capacity to perform many of the post-translational modifications of higher eukaryotes, usually allowed the recovery of large amounts of biologically active product. Complex molecular structures such as murine immunoglobulin heterodimers have been successfully produced utilizing this system (Putlitz, 1990). Vaccinia virus has been used to express foreign genes in mammalian cells for more than 10 years (Mackett et al., 1982; Panicali and Paoletti, 1982). Due to the extended host range of vaccinia, the heterologous gene contained in one single virus recombinant can be expressed in almost any mammalian cell type. Therefore, infection with recombinant vaccinia virus usually generates high amounts of a mammalian protein that most closely resembles its natural counterpart in terms of structure and function.

We first established that recombinant SC was produced and transported along the secretion pathway in both systems. The levels of expression for all constructs were indistinguishable and within the expected range, i.e. 5-10 mg of protein/liter of culture medium for the vaccinia system, and 50 mg of protein/liter of culture medium for the baculovirus system. In addition, the use of translation initiation sites of different origins in the vaccinia system did not have any visible effect either. Thus, subtle modifications in the engineered cDNA were very well accommodated by the cellular machinery responsible for transcription, translation, maturation, and secretion of recombinant hSC.

Human SC purified from milk is heavily glycosylated with four N-linked sugar side chains accounting for over 20% of its molecular weight (Mizoguchi et al., 1982). A major disadvantage of the baculovirus system compared to the mammalian cell-based system is its limited ability to terminally glycosylate glycoproteins (Miller, 1993, and references therein). Our results with endoglycosidase treatment indicate that complex-type carbohydrate contributes approximately 10 and 19-24 kDa to the apparent molecular mass of hSC produced in insect and mammalian cells, respectively. hSC from purified sIgA antibodies behaves in a very similar fashion as the recombinant proteins in terms of migration on SDS-PAGE, as well as toward treatment with endoglycosidases, indicating a strong structural relationship between the natural and overproduced species. Recombinant SC produced by insect and mammalian cells were both able to efficiently bind to dimeric IgA, suggesting that slight differences in glycosylation did not affect the interaction, as previously reported by Bakos et al.(1991, 1994). Indeed, these authors showed that deglycosylated SC bound with equal or higher affinity to polymeric IgAs. Whether glycosylation affects the kinetics of SC-IgA association and play a role in protection will require further biochemical and immunological investigations.

Metal-chelate affinity chromatography first described by Porath et al.(1975) is based on the ability of certain amino acids (histidine, tryptophan, and tyrosine) to act as electron donors for reversible binding to transition metal ions immobilized on a solid support. The affinity of histidine residues for immobilized Ni ions allows selective purification of proteins containing a stretch of at least six consecutive histidines in surface-exposed regions of the molecule, such as amino and carboxyl termini (Hochuli et al., 1987). Fusion of six histidines adds only 720 daltons to the protein, and its biological function and immunogenic properties are usually retained (Janknecht et al., 1991; Parvin et al., 1992; Taussig et al., 1993). Efficient single-step purification of baculovirus-expressed hSC with dimeric IgA binding capability further demonstrates the potential of this chromatography. In contrast, hSC protein expressed with the vaccinia system could not be purified to homogeneity as contaminating polypeptides were repeatedly co-eluted at high imidazole concentration. Since the same pattern of contaminants was observed with different cell lines, it is likely that those are of vaccinia viral origin.

ConA affinity chromatography represents a second efficient single-step procedure to purify recombinant hSC. This was facilitated by growing the cells in serum-free conditions. In addition, it appears that glycoproteins secreted by infected cells represents a small proportion of the large number of cellular and viral proteins present in cell culture supernatants (Fig. 5). The recovery of vaccinia-produced hSC is almost complete, whereas baculovirus-expressed hSC is only partially yielded under identical conditions, reflecting a stronger affinity for the lectin due to the lower content of complex sugars in insect cells rendering more terminal -D-mannose residues accessible to immobilized ConA.

We have developed a simple dot blot overlay assay to assess IgA binding of recombinant hSC (Fig. 6A). The recombinant hSC protein binds exclusively to dimeric IgA, and is produced in an active conformation, as less than 5% is found not to be reassociated with IgA (Fig. 6, B and C; Fig. 7A). The specificity of binding is further illustrated in control experiments showing that hSC does not bind to IgG (Fig. 6D). It is worth mentioning at this point of the discussion that we only used purified dimeric IgAs, and not simply the secretion products of hybridoma cultures known to contain monomers, multimers, aggregates, and serum proteins that could nonspecifically trap recombinant hSC in the assay. In addition, the quantitative shift in the position of elution of recombinant hSC upon binding to dimeric IgA observed in gel filtration experiments (Fig. 7B) further validates the filter binding assay as a fast, qualitative, and reliable test. These binding data indicate that, (a) specific recognition between recombinant hSC and dimeric IgA occurs in solution, (b) confirm that all, or nearly all the recombinant hSC protein is competent for binding to dimeric IgA, (c) argue in favor of an 1:1 stoichiometry of association, as suggested by previous studies (Kerr, 1990). The biochemical nature and kinetics of the interaction of heterologous and homologous IgA with recombinant hSC produced by the different expression systems and cell types is currently being analyzed.

In conclusion, the baculo- and vaccinia viral systems are suitable to provide high yields of recombinant hSC. Efficient procedures allowed purification of the recombinant protein in a single step. This material will be used on a preparative scale for reassociation with mouse and human monoclonal IgA, as well as with humanized recombinant antibodies of the A isotype. This approach will allow investigation in vitro of the stabilizing effect of hSC in sIgA complex against protease degradation as well as its contribution to mucosal immune protection (Boren et al., 1993). In addition, thanks to the availability of sufficient amounts of SC, it will be possible to determine in animal models the immunoprotective potential offered by dimeric IgA alone and reconstituted with recombinant SC.

  
Table: Production of recombinant hSC using the vaccinia virus system

Serial dilutions of culture supernatants were assayed by ELISA using purified sIgA as a standard. The linear part of the response curve was used for quantification.



FOOTNOTES

*
This work was supported by research funds from the Swiss National Science Foundation, Biotechnology Priority Program, Grant 5002-034603 (to L. R., J.-P. K., and B. C.) and from the Etat de Vaud (to S. C. and R. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-21-692-41-38; Fax: 41-21-692-41-05.

The abbreviations used are: sIgA, secretory IgA; pIgR, polymeric immunoglobulin receptor; IgA, immunoglobulin A; dIgA, dimeric IgA; hSC, human secretory component; PCR, polynucleotide chain reaction; m.o.i., multiplicity of infection; Endo H, endoglycosidase H; PNGase F, peptide-N-glycosidase F; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TK, thymidine kinase; DORA, dot blot reassociation assay.

L. Rindisbacher and B. Corthésy, unpublished data.


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