Enhanced Bovine Herpesvirus Type 1 Neutralization by Multimerized
Single-Chain Variable Antibody Fragments Regardless of Differential
Glycosylation
Yfke Pasman,a Eva Nagy,b and Azad K. Kaushika
Departments of Molecular and Cellular Biologya and Pathobiology,b University of Guelph, Guelph, Ontario, Canada
T
he remarkable specificity to a desired epitope of monoclonal
antibodies (Abs) (12) or their variable-region fragments (7)
makes these proteins suitable for clinical diagnosis, immunodiagnostics, and therapeutics. A single-chain variable fragment (scFv)
is an example of a monovalent Ab fragment where the aminoterminal variable heavy-chain and variable light-chain domains
are held together through an artificial flexible peptide linker to
form a stable antigen (Ag)-binding site against an epitope. For
example, certain viral epitopes provide a suitable target for the
binding of scFv to neutralize virus (7). Antibody fragments against
human or animal pathogens can be produced economically in
plants, mammalian cells, or microorganism-based expression systems such as Escherichia coli or Pichia pastoris. Previously, our
laboratory constructed neutralizing scFvs with 7- and 18-aminoacid linkers against bovine herpesvirus type 1 (BoHV-1) (14, 15).
The short linker size (0 to 2 amino acids) has been suggested to
form trimers, tetramers, and/or higher multimers (6, 13), as it
influences the configuration of the variable domains. The increased avidity as a result of such scFv multimerizations may potentially enhance virus neutralization abilities.
BoHV-1 is an etiological agent of infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle (19, 23).
Subsequent to primary viral infection, a lifelong latent infection is
established in affected cattle, which can be reactivated during
stress (weaning, transport, or crowding) or as a result of immunosuppression. BoHV-1 is also involved in bovine respiratory disease complex (BRDC) or shipping fever, a disease caused by a
synergistic effect of viral and bacterial infections leading to severe
pneumonia, which may be fatal. The economic losses due to
BRDC alone cost the U.S. cattle industry up to 3 billion dollars
annually (10). The currently used vaccines do not provide adequate protection against BoHV-1 infection due to associated
problems such as viral shedding, abortions, immunosuppression,
and reversion to virulence (22). Therefore, passive immunization
with neutralizing Abs or their Ag-binding fragments potentially
provides an adjunct disease prevention approach for the eradication of BoHV-1 from a herd. In addition, these Ag-binding fragments will provide useful reagents for the specific detection of
BoHV-1 in an immunoassay, for example, by immunofluorescence (14, 15).
We demonstrate here the development of a functional multimeric scFv with a 2-amino-acid linker against BoHV-1 with improved in vitro virus neutralization potency, as a result of increased avidity via multimerization. The scFvs were expressed in
Pichia pastoris, a eukaryotic microorganism capable of expressing
scFvs in the secreted form and performing posttranslational modifications such as folding, the formation of disulfide bridges, and
glycosylation (4). Little is known about the glycosylation of Ab
variable regions, although it has been suggested that it may affect
antigen recognition negatively or positively (8). The recombinant
multimerized scFvs against BoHV-1 described here are heterogeneous proteins due to differential cleavage and glycosylation, but
these proteins retain virus recognition and neutralization functions.
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p. 1150 –1157
Clinical and Vaccine Immunology
Received 2 March 2012 Returned for modification 26 March 2012
Accepted 27 April 2012
Published ahead of print 13 June 2012
Address correspondence to Azad K. Kaushik, akaushik@uoguelph.ca.
Supplemental material for this article may be found at http://cvi.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/CVI.00130-12
August 2012 Volume 19 Number 8
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Single-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1) in vitro were constructed and expressed in Pichia pastoris. In contrast to
the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv
demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or
-gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected MadinDarby kidney (MDBK) cells in vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major
peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which
copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant
scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high
avidity, resulting in increased virus neutralization in vitro compared to that of monovalent scFv with a long (18-amino-acid)
flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.
Single-Chain Fv against BoHV-1
against BoHV-1. (a) Lane 1, molecular weight (in thousands) ladder; lane 2, HB9907 hybridoma VJ amplicon; lane 3, HB9908 VJ positive control; lane 4,
negative control. (b) Lane 1, molecular weight ladder; lane 2, HB9907 VHDHJH amplicon; lane 3, HB9908 VHDHJH positive control; lane 4, negative control. (c)
Lane 1, molecular weight ladder; lane 2, VJ–2-codon-linker–VHDHJH amplicon; lane 3, negative control.
MATERIALS AND METHODS
Hybridoma. A mouse ⫻ cattle xenogeneic hybridoma, HB9907 (ATCC,
Rockville, MD) (17), secreting monoclonal IgG1 antibody against
BoHV-1, provided cDNA, the genetic source of rearranged VHDHJH and
VJ genes. The hybridomas were grown in RPMI 1640 (Gibco Canada)
supplemented with 10% horse serum, 5 mM sodium pyruvate, 0.5 M
minimal essential medium (MEM) nonessential amino acids, 1 mM Lglutamine, gentamicin (50 g/ml), and 5 M 2-mercaptoethanol (14).
cDNA synthesis, PCR, and cloning. First-strand cDNA was synthesized by using SuperScript II reverse transcriptase (Invitrogen) from 5 g
of total RNA (TRIzol; Invitrogen) isolated from HB9907 hybridomas. The
V-J recombinations were PCR amplified from cDNA using a sense
primer corresponding to framework 1 (FR-1) with a built-in SfiI restriction site (underlined) (sVJ-s [5=-GTGGCCCAGCCGGCCCAGGCTGTG
CTGACTCAG-3=]) and an antisense primer corresponding to FR-4 with
the designed flexible linker (VJL-as [5=-AGAACCTAGGGACGGTCAGT
GTGGTCCCGCT-3=]) (Mobix, McMaster University, Hamilton, Ontario, Canada). Platinum Taq high-fidelity polymerase (Invitrogen) was
used to amplify the targeted cDNA using PCR conditions that included
hot-start denaturation at 95°C for 30 s followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 64°C for 30 s, and extension at
72°C for 1 min, with a final extension step at 72°C for 7 min. Similarly,
VHDHJH recombinations were amplified by using sense (LVDJ-s [5=ACACTGACCGTCCTAGGTTCTCAGGTGCAGCTGCG-3=]) and antisense (VDJS-as [5=-CTGGCCGGCTTGGCCACTAGTGGAGGAGACGGTGACCAG-3=]) primers (Mobix, McMaster University, Hamilton,
Ontario, Canada) designed from the linker and FR-1, and FR-4 with an
SfiI restriction site (underlined), respectively. The PCR conditions were
similar to those used for VJ amplifications except for annealing at 68°C
for 30s. The cDNA derived from HB9908 hybridomas (ATCC, Rockville,
MD) (17) provided the positive control for both VJ and VHDHJH amplification reactions.
The purified VJ and VHDHJH amplicons were combined in an overlap PCR (9, 14) including the nucleotide sequence encoding the 2-aminoacid linker (glycine-serine), using sense (SVJ-s) and antisense (VDJS-as)
primers. The PCR conditions included a hot start at 94°C for 30 s followed
by 25 cycles of denaturation at 94°C for 15 s, annealing at 65°C for 15 s,
and extension at 68°C for 2 min, followed by a final extension step at 68°C
for 30 min. The overlap SfiI-digested VJ-2L-VHDHJH amplicon was
ligated into the SfiI-digested and dephosphorylated (calf intestinal alka-
August 2012 Volume 19 Number 8
line phosphatase; Invitrogen) pPICZ␣ expression vector. The ligate was
used to transform E. coli One Shot TOP10 cells (Invitrogen), and plasmids
isolated from zeocin-resistant colonies (QIAprep miniprep; Qiagen
Inc.) were sequenced (Mobix, McMaster University, Hamilton, Ontario,
Canada).
P. pastoris transformation. Electrocompetent P. pastoris strain
KM71H (Muts Arg⫹; Invitrogen) cells were prepared according to the
manufacturer’s instructions for EasySelect. For transformation, 80 l of
P. pastoris was mixed with 5 l SacI-linearized recombinant plasmid (⬃1
g/l) and incubated for 5 min at 0°C in a cuvette (0.2-cm gap; Bio-Rad).
Electroporation conditions were a voltage of 1.5 kV, a field strength of 7.5
kV/cm, a capacity of 25 F, a resistance (pulse controller) of 400 ⍀, and a
time constant of 8.1 ms (Genepulser; Bio-Rad). After the cells were pulsed,
1 ml 1 M sorbitol was added, followed by incubation at 30°C for 1.5 h. The
transformants were plated onto 2% yeast extract-peptone-dextrose
(YPD) agar supplemented with 1 M sorbitol and 100 g zeocin/ml and
incubated for 4 days at 30°C. Single colonies were grown at 30°C in buffered minimal glycerol complex (BMGY) medium for 18 to 24 h to an
optical density at 600 nm (OD600) of ⬎2.0, and protein expression was
induced in buffered minimal methanol complex (BMMY) medium containing 0.5% methanol. Methanol (0.5%, vol/vol) was added every 24 h to
maintain induction until supernatant collection at 96 h postinduction.
scFv purification. The His-tagged recombinant protein secreted by P.
pastoris KM71H was purified on a nickel-charged affinity column (ProBond; Invitrogen) under native conditions, as described previously (14).
The purified recombinant protein was concentrated by using a centrifugal
10,000 molecular-weight cutoff (MWCO) filter device (Millipore, Bedford, MA), and protein concentrations were determined by the use of a
Bio-Rad protein assay kit.
Western immunoblotting. Purified scFv was fractionated in 12%
SDS-PAGE gels (15a) and stained with Coomassie blue (0.15%, wt/vol)
(Brilliant Blue R-250; Fisher). The recombinant protein was electrophoretically transferred onto a nitrocellulose membrane (Protran; Schleicher
& Schuell Inc.) and immunodetected by using a mouse anti-His antibody
conjugated with alkaline phosphatase (AP) (1:2,000; Invitrogen) and
5-bromo-4-chloro-3-indolyl phosphate (BCIP)–nitroblue tetrazolium
(NBT) chromogen (Promega). The negative methodological control included the pPICZ␣ (without insert)-transformed P. pastoris KM71H (P4
clone) supernatant that did not express the recombinant protein, according to the manufacturer’s protocol.
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FIG 1 PCR amplification of V-J (a), VH-DH-JH (b), and overlap V-J-2L-VH-DH-JH (c) from cDNA from HB9907 xenogeneic hybridomas secreting IgG1
Pasman et al.
according to the IMGT numbering system (22, 23), and the linker is shown in boldface type.
Mass spectrometry. Samples of purified and desalted recombinant
protein were run on a Waters/Micromass Ultima quadrupole time of
flight (Q-TOF) mass spectrometer operated in the positive-ion mode
(McMaster Regional Centre of Mass Spectrometry, Hamilton, Ontario,
Canada).
Virus neutralization assay. A plaque reduction assay was performed
with recombinant scFv to determine its BoHV-1 virus neutralization ability (14). Recombinant scFv3-18L (15) and bovine serum positive for neutralizing antibodies against BoHV-1 (titer, 1:48; Animal Health Laboratory, Ontario Veterinary College) were used as positive controls. The
negative controls included fetal bovine serum (FBS), bovine serum albumin
(BSA), phosphate-buffered saline (PBS), and Dulbecco’s minimal essential
medium (DMEM). To determine if the recombinant protein caused a significant plaque reduction (ⱖ50%), a one-sample t test (two-tailed P value) was
performed (GraphPad Prism, version 4.03; GraphPad Software).
Immunodetection of the BoHV-1 epitope. BoHV-1 (Cooper strain)infected MDBK cells were grown in DMEM (4% FBS and 1 mM L-glutamine) until approximately a 90 to 100% cytopathic effect was achieved.
The clarified supernatant was spun at 115,460 ⫻ g for 1 h at 4°C (Optima
L-80XP ultracentrifuge; Beckman Coulter) on 30% sucrose in a TNEbuffer layer (10 mM Tris, 100 mM NaCl, 1 mM EDTA [pH 7.4]), and the
viral pellet was resuspended in Tris-EDTA (TE) buffer (pH 8.0).
Purified BoHV-1 was separated into envelope and capsid protein fractions (6a) and subjected to immunodetection by recombinant scFv in a
Western blot. Briefly, BoHV-1 (250 g) was resuspended in 250 l viral
lysis buffer (10 mM Tris buffer [pH 7.5] [Fisher Scientific], 1% Igepal-630
1152 cvi.asm.org
[Sigma]) and incubated at 20°C for 1 h, followed by centrifugation at
13,148 ⫻ g for 90 min at 4°C. The pellet comprising the capsid fraction was
washed with viral lysis buffer and resuspended in loading buffer (100 mM
Tris Cl [pH 6.8], 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol
blue, 20% glycerol). The envelope protein fraction in the supernatant was
precipitated with trichloroacetic acid (TCA) (100%, wt/vol) for 10 min at
4°C, and the pellet was washed three times with 200 l cold acetone, dried,
and resuspended in loading buffer. The samples were boiled for 10 min
prior to fractionation on 12% SDS-PAGE gels and transferred onto a
nitrocellulose membrane (Protran; Schleicher & Schuell Inc.). The nitrocellulose membrane was washed (0.05% Tween 20 in PBS) and blocked
(2.5% Tween 20 in PBS), and the viral epitope was immunodetected by
using recombinant scFv (5 g/ml), followed by mouse anti-His-AP antibody (1:2,000; Invitrogen) and NBT-BCIP chromogen (Promega).
BoHV-1 gB and gE enzyme-linked immunosorbent assays (ELISAs).
gB and gE enzyme immunoassays (Herdcheck IBRgB; Idexx, Switzerland)
for the detection of BoHV-1-specific antibodies were used to determine
whether recombinant scFv recognized the gB or gE immunodominant
epitopes of BoHV-1. Briefly, 50 l of phosphate wash buffer (Idexx, Switzerland) was added to each well coated with BoHV-1 antigen, followed by
50 l of scFv3-18L (40 g/ml) or scFv5-2L (40 g/ml). The controls
included negative BoHV-1 gB/gE (negative) and positive BoHV-1 gB/gE
(positive) sera. The plates were incubated for 2 h at 37°C, followed by the
incubation of BoHV-1 gB- or BoHV-1 gE-specific monoclonal antibody
conjugated to horseradish peroxidase (HRP) for 1 h at 20°C. After the
plates were washed, TMB substrate solution (3,3=,5,5=-tetramethylbenzi-
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FIG 2 Nucleotide sequence of the scFv5-2L variable-region construct in the pPICZ␣ vector. Complementarity-determining regions (CDRs) are underlined
Single-Chain Fv against BoHV-1
expressed in Pichia pastoris. Hydrophobic amino acids (I, V, L, F, C, M, A, and W) in -strands are shown in blue; proline (P) is shown in yellow. The loops are
limited by amino acids shown in squares (anchor positions). Arrows indicate the direction of the -strands. Blue, light green, and dark green loops represent
CDR1L, CDR2L, and CDR3L, respectively; red, yellow, and purple loops represent CDR1H, CDR2H, and CDR3H, respectively; and hatched circles correspond
to missing positions according to the IMGT numbering system for V and V-like domains (11, 16). The linker composed of glycine and serine amino acids is
shown in the green box.
dine and H2O2) (Idexx, Switzerland) was added, and the reaction was
stopped with 1 M HCl after 10 to 15 min. The absorbance was measured at
650 nm (Titertek Multiscan). The blocking of the gB and gE dominant
epitopes was calculated as a percentage of the absorbance by the sample
relative to the average absorbance value of the negative control (NCav):
[(NCav ⫺ ODsample)/NCav] ⫻ 100%. For the detection of the gB and the
gE dominant epitopes, blocking percentages of ⬍45% and ⬍30%, respectively, were considered negative.
RESULTS
Construction of anti-BoHV-1 scFv with a 2-amino-acid linker.
The variable lambda light- and heavy-chain rearrangements amplified from HB9907 hybridoma cDNA were combined in an
overlap PCR (Fig. 1) together with a 2-codon (GGT TCT) linker
and cloned into the pPICZ␣ vector in the VL-VH configuration
with a built-in c-myc epitope and His tag targeted for expression
under the influence of the AOX1 promoter. Upon the screening of
recombinant clones, the scFv2L5 clone was isolated and analyzed
for nucleotide sequence (Fig. 2). The deduced protein sequence
and secondary structure, presented as IMGT Collier de Perles in
Fig. 3, demonstrated the characteristic strands, loops, and turns
typical of variable domains. The supernatant of P. pastoris
(KM71H strain) transformed with the scFv2L5 plasmid, expressing the recombinant protein after 96 h of induction with 0.5%
methanol, was subjected to immunochemical analysis. The re-
August 2012 Volume 19 Number 8
combinant protein secreted by P. pastoris, designated scFv5-2L,
demonstrated the presence of an approximately 34-kDa protein
(Fig. 4) in a Western blot, consistent with the expected theoretical
molecular mass of 30.3 kDa. These experiments demonstrated
the stable expression of scFv with a 2-amino-acid linker in a
VL-linker-VH configuration in P. pastoris that could be purified via
a His tag on nickel-charged affinity resin.
Differential glycosylation in recombinant scFvs expressed in
P. pastoris. Mass spectrum (Q-TOF) analysis of scFv5-2L showed
multiple peaks at around 27 kDa and weaker signals at around
30.3 kDa (Fig. 5a). A careful analysis of the data, taking into consideration a 5-Da margin for readout accuracy (see Table S1 in the
supplemental material), showed differential cleavage that resulted
in a loss of the His tag on scFvs. Nevertheless, these scFvs without
a His tag copurified on Ni-chelating resin with properly expressed
scFvs with a His tag via noncovalent multimerization. This consistent observation provides indirect evidence for scFv5-2L multimerization per se as a result of the 2-amino-acid linker. The
addition of 1 to 9 mannose residues and other unidentified small
adducts (Fig. 5a) seemed to provide further scFv heterogeneity.
Mass spectrum analyses of monomeric scFv3-18L showed the
most prominent signals at around 31.7 kDa (Fig. 5b; see also Table
S1 in the supplemental material), where recombinant protein heterogeneity was due to the differential cleavage of the ␣-signal se-
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FIG 3 Two-dimensional IMGT Collier de Perles of the amino acids (shown as one-letter abbreviations) of the VL-linker-VH region of recombinant scFv5-2L
Pasman et al.
DISCUSSION
FIG 4 Coomassie blue-stained 12% SDS-PAGE gel (a) and Western immunoblot (b) of recombinant protein scFv5-2L. Lane 1, negative control (PBS);
lane 2, molecular mass marker (kDa); lane 3, scFv5-2L supernatant (10⫻ concentrate); lane 4, purified scFv5-2L protein; lane 5, negative-control recombinant P. pastoris P4 (without insert) supernatant.
quence by the Ste13 endoprotease together with a lack of glycosylation (31.552 kDa) or variable 1- to 8-mannose-residue
(31.712 to 32.844 kDa) additions. The possible cleavage and
posttranslational modification sites of recombinant scFv5-2L
expressed in P. pastoris are diagrammatically shown in Fig. 6.
As would be expected, His tag cleavage was not observed in the
mass spectrum of recombinant scFv3-18L, consistent with its
monomeric form.
Multimerized scFvs have higher BoHV-1-neutralizing potency. The virus-neutralizing ability of scFv5-2L was determined
by a plaque reduction assay and evaluated in comparison to that of
scFv3-18L. Recombinant scFv5-2L inhibited BoHV-1 replication
in MDBK cells in a dose-dependent manner (Fig. 7). An scFv5-2L
concentration as low as 1 M neutralized (⬎50%) BoHV-1 replication in MDBK cells in vitro (P ⬍ 0.05). In contrast, a 2-fold
amount (2 M) of monomeric scFv3-18L was needed to achieve a
comparable level of virus neutralization. Clearly, the in vitro virus
neutralization potency of scFv5-2L is much higher (75%) than
that of monomeric scFv3-18L (59%) at a 10 ⌴ concentration
(Fig. 7). While no nonspecific plaque reduction was observed with
a heterologous protein, bovine serum albumin (BSA), antiBoHV-1 bovine serum significantly inhibited BoHV-1 replication
(65%; dilution, 50⫻). High-titer serum with anti-BoHV-1 antibodies achieved 100% plaque reduction in virus neutralization
assays (data not shown). These observations suggest a 2-foldhigher BoHV-1 neutralization potency of multimerized scFv5-2L
than that of monomeric scFv3-18L, apart from a higher degree of
virus neutralization ability.
1154 cvi.asm.org
With the objective to enhance the BoHV-1 neutralization function of an scFv by multimerization to increase avidity, a recombinant scFv with a 2-amino-acid linker (scFv5-2L) was constructed and expressed in P. pastoris under the influence of the
AOX1 promoter. The scFv was expressed as a secreted fusion
protein with c-myc and His tags for subsequent purification
and immunodetection. The presence of additional residues of a
vector origin or the 2-amino-acid linker did not affect Ag recognition or hinder the multimerization required to form an
Ag-binding site (6, 13). The functionality of scFv5-2L was confirmed by its BoHV-1 neutralization ability in vitro and antigen
recognition in a Western blot. The scFv with a linker shorter
than 12 amino acids cannot fold to form a functional domain
but needs to pair with another scFv in order to form a functional Ag-binding site (2, 13). In addition to the linker size,
scFv multimerization is influenced by factors such as the VLJLVHDHJH interface stability, concentration, and ionic strength
(1, 5). The noncovalent formation of multivalent multimers
clearly increased the in vitro BoHV-1 neutralization potency, as
it was achieved at a lower molar concentration (1 M) by
scFv5-2L, similarly to scFv1-7L (14) but in contrast to monovalent scFv3-18 (2 M). Whether the formation of trimers,
tetramers, or higher multimers generates only functional Agbinding sites is not known, but the increased avidity of such
multimers likely enhances the virus neutralization potency. Evidence for scFv5-2L multimerization leading to increased avidity, however, is largely circumstantial, relying on mass spectrum analyses and scFv functionality per se. Direct evidence for
scFv5-2L multimerization using structural approaches, e.g.,
electron microscopy or crystallography, is needed to confirm
the degree of multimerization in the context of valency. The in
vivo protective ability of the scFvs is being experimentally
tested in rabbit animal models and bovine calves.
The recombinant protein scFv5-2L, including monomeric
scFv3-18L (data not shown), recognized the 32-kDa protein present in the envelope fraction of BoHV-1. The neutralizing epitope
on BoHV-1 recognized by monoclonal IgG (HB9907) or the derived scFvs has not yet been identified. Based on theoretical molecular modeling (14), a probable carbohydrate moiety in the
BoHV-1 epitope is predicted to be recognized by scFv5-2L or
scFv3-18L present on the envelope glycoprotein. However, both
scFv5-2L and scFv3-18L did not recognize immunodominant
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Recombinant scFv against BoHV-1 recognizes an epitope on
the 32-kDa envelope protein. In a Western blot, recombinant
scFv5-2L recognized an approximately 32-kDa protein in the envelope fraction of BoHV-1 (Fig. 8). Such an immunodetection of
the 32-kDa protein in the BoHV-1 envelope is specific, as it was
absent in the capsid fraction (Fig. 8). The scFvs with 7- and 18amino-acid linker sizes are known to specifically recognize
BoHV-1 antigen in infected but not uninfected MDBK cells in an
immunofluorescence assay (14, 15). Furthermore, two immunodominant envelope glycoproteins, gB and gE, of BoHV-1 were
not recognized by either multimeric scFv5-2L or monomeric
scFv3-18L in a blocking ELISA (Fig. 9). These findings suggest
that neutralizing recombinant scFvs against BoHV-1 likely recognize a non-gE or -gB immunodominant epitope on the viral
envelope.
Single-Chain Fv against BoHV-1
scFv5-2L is 30,261 Da, and that for scFv3-18L is 31,555 Da. A detailed description of separate peaks is provided in Table S1 in the supplemental material.
epitopes on essential gB and nonessential gE envelope proteins
(20). Experiments aimed at an analysis of surface-accessible neutralizing epitopes on the envelope protein, essential for viral replication, are in progress.
FIG 6 Schematic representation of possible cleavage and posttranslational
modification sites of recombinant scFv5-2L expressed in P. pastoris. The signal
sequence necessary for the secreted protein is cleaved by the Kex2 and Ste13
endoproteases; scFv5-2L amino acid positions 2 and 259 are likely sites of
cleavage by an unidentified protease. Yellow boxes represent the sequence
originating from the pPICZ␣ vector. GS, glycine-serine (2-amino-acid linker).
August 2012 Volume 19 Number 8
Mass spectrum analysis revealed multiple scFv5-2L species at
around 27 and 30.3 kDa but none within a 5-Da range of the
expected mass of 30.261 kDa. Several peaks close to the expected
molecular mass can be explained by the differential cleavage of the
signal sequence and variable glycosylation. Some of the peaks representing molecular masses of around 27 kDa were found to be
compatible with cleavage at position 259 of scFv5-2L, which
would result in the loss of the c-myc and His tags, consistent with
Coomassie blue-stained SDS-PAGE and Western blot observations. Purified His-tag-cleaved recombinant scFv5-2L, revealed
during the mass spectrum analysis, could exist only because of
noncovalent multimerization permitting copurification on Nichelating resin with properly His-tagged scFv5-2L. As the built-in
expression vector His tag site resides outside the variable region of
the heavy and light chains of the parental Ab, it does not interfere
with the formation of the functional Ag-binding site. Such a cleavage of the His tag in some scFvs is consistent with the presence of
a single but the absence of a separate protein band in the Western
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FIG 5 Mass spectra of recombinant scFv5-2L (a) and scFv3-18L (b) expressed in P. pastoris. The expected molecular mass for properly processed and unglycosylated
Pasman et al.
BoHV-1 neutralization is indicated by the ⬎50% plaque reduction in 3 to 8
individual experiments. Note that multimerized scFv5-2L significantly inhibits plaque formation by BoHV-1 compared to monomeric scFv3-18L at concentrations of 5 M (P ⬍ 0.05) and 10 M (P ⬍ 0.001). Various controls
included 100 PFU BoHV-1 with media including 2% FBS, BSA (negative), and
1% or 2% anti-BoHV-1 serum (positive). Bars indicate means ⫾ standard
errors (ⴱ, P ⬍ 0.05 [significant]; ⴱⴱⴱ, P ⬍ 0.001 [extremely significant] [determined by an unpaired t test, with a two-tailed P value]).
blot. Mass spectrum analyses of scFv3-18L also showed protein
heterogeneity with molecular masses of around 31.7 kDa containing a peak of 31.552 kDa, which seems to represent the expected
theoretical molecular mass of 31.555 kDa. The observed scFv318L heterogeneity is likely because of differential cleavage, glycosylation, and the addition of minor adducts. The second cluster of
peaks is absent as His-tag-cleaved recombinant scFv3-18L, as it
will not be purified on nickel-charged affinity resin, confirming its
monomeric form per se. Another research group (21) previously
showed a similar cleavage of an scFv at the C terminus, resulting in
the loss of the c-myc and His tags. Such a cleavage at the C terminus is probably caused by an unidentified protease that is not
affected by the presence of the proteinase inhibitor Casamino
Acids.
The scFv5-2L and scFv3-18L glycosylations related to a mass of
1 to 9 mannoses may reflect O-linked glycosylation, since a significantly higher mass would be expected if it were due to N-linked
glycosylation. Since O-linked glycosylation has been described for
P. pastoris on threonine and serine, these residues could also com-
FIG 8 Western immunoblot of BoHV-1 envelope and capsid protein fractions
immunodetected by recombinant scFv5-2L. Lane 1, molecular mass marker;
lane 2, BoHV-1 envelope fraction; lane 3, BoHV-1 capsid fraction.
1156 cvi.asm.org
FIG 9 Blocking by recombinant scFv5-2L and scFv3-18L of the BoHV-1 envelope immunodominant epitope on gB using anti-gB-HRP monoclonal Ab
(a) and on gE using anti-gE-HRP Ab (b) in an ELISA. Negative controls included bovine serum without gB or gE antibodies against BoHV-1 or phosphate-buffered saline (PBS), while bovine serum with a positive antibody titer
for gB or gE provided the positive control.
pete for the glycosylation of potential N-glycosylation sites (AsnX-Ser/Thr). O-glycosylation is seen in both mammals and yeast,
but glycosylation in P. pastoris can occur at sites not necessarily
glycosylated in the native protein (4). Furthermore, the phosphorylation of mannose residues is common and could account for
some of the unknown minor adducts (3). The relatively lower level
of glycosylation of proteins expressed in P. pastoris together with
reduced immunogenicity, in comparison to Saccharomyces cerevisiae, make P. pastoris a preferred expression system for proteins
requiring posttranslational modifications (18). Little is known
about the effect of glycosylation on the variable Ab regions, but it
can positively or negatively influence Ag binding (8). Furthermore, glycosylation has been suggested to play an important role
in the prevention of protein degradation (4). The experiments
outlined here demonstrate that the differential glycosylation of
scFvs (scFv5-2L and scFv3-18L) did not affect their antigen recognition or virus neutralization function. Furthermore, the inhibition of scFv glycosylation by the growth of P. pastoris in the
presence of tunicamycin did not affect antigen recognition (data
Clinical and Vaccine Immunology
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FIG 7 Virus neutralization potency of recombinant scFv5-2L and scFv3-18L.
Single-Chain Fv against BoHV-1
ACKNOWLEDGMENTS
This research was supported by an NSERC Canada discovery grant to
Azad K. Kaushik.
We are thankful to M. Kirk Green and Ewan Desilva, McMaster Regional Centre for Mass Spectrometry, McMaster University, Hamilton,
Ontario, Canada, for their support for Q-TOF mass spectrometry.
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not shown). These observations together with mass spectrometry
data suggest an O-glycosylation of recombinant scFv that is not
inhibited by tunicamycin treatment. This is consistent with the
absence of an N-glycosylation site on recombinant scFv. Nevertheless, scFv heterogeneity due to posttranslational modifications
deserves important consideration in the development of standardized therapeutics and immunodiagnostics. Future experiments should aim at the development of growth conditions to
prevent the heterogeneous glycosylation or enzymatic deglycosylation of recombinant proteins. The effect of the differential glycosylation of Ag-binding sites on scFv function needs to be examined.
The recombinant scFvs against BoHV-1 potentially provide an
adjunct immunotherapeutic, together with the available vaccines
against BoHV-1, especially during transportation, when latent
BoHV-1 becomes activated as a result of stress, leading to “shipping fever” and bovine respiratory disease complex. Recombinant
scFv may be used as an intranasal spray in vaccinated cattle during
transportation to prevent BoHV-1 infection by blocking viral
transmission. These immunotherapeutics may also be used to prevent the spread of BoHV-1 infection by artificial insemination by
treating high-quality semen to inhibit BoHV-1 replication. Furthermore, these immunotherapeutics may be used for local application to treat infectious pustular vulvovaginitis caused by
BoHV-1. While antibody-based therapy currently may be costly,
technological advances, e.g., new expression systems, optimized
antibody fragments, or combination therapies, are likely to make
these therapies cost-effective in the future. Apart from their therapeutic significance, recombinant scFvs may be used for the rapid,
specific, and sensitive immunodetection of BoHV-1 infection, for
example, by immunofluorescence (14, 15). Overall, multimerized
scFv5-2L has a high virus neutralization efficacy due to increased
valency and is potentially suitable for passive immunization and
topical application.