Note: Descriptions are shown in the official language in which they were submitted.
CA 02286301 1999-11-O1 -
The Filamentous Phage as a Multivalent Scaffold for Coupling Synthetic
Peptides for
Immunization
Michael B. Zwick' and Jamie K. Scott'"
' Institute of Molecular Biology and Biochemistry, Simon Fraser University,
8888 University
Drive, Burnaby, B.C., Canada, VSA 156. Tel: (604) 291-5658, Fax: (604) 291-
5583, email:
jkscott@sfu.ca
"Corresponding author
Aug. 19, 1999
CA 02286301 1999-11-O1
J .1 . . . ,.
Introduction
The filamentous bacteriophage is a long flexible rod, approximately 1 um in
length and
6.5 nm in diameter. A circular, single strand of DNA in the interior of the
virus is encapsulated
by five phage-encoded proteins. Four minor coat proteins are found in pairs at
either end of the
phage at five copies each (i.e., pIII and pVI at one end, and pVII and pIX at
the opposite end). In
particular, pIII is a 406 amino acid protein that projects as a knob-like
structure from one tip of
the phage. The bulk of the phage surface consists of the major coat protein
(pVIII), an alpha-
helical protein that coats the length of the phage in a shingled, or fish-
scale-like pattern
(Glucksman et al., 1992; Marvin et al., 1994). ??Figure of pha,~e showing
recombinant fusion
proteins??
Many groups have used the filamentous bacteriophage as an immunological
carrier for
the purpose of eliciting anti-peptide antibody (Ab) responses in animals. In
all of these systems,
the peptide is fused by recombinant methods to a coat protein and is thus
displayed on the
surface of the phage. The first to use a recombinant peptide displayed by the
phage as an
immunogen were de la Cruz et al. (1988 j. These authors used pIII-display in
which only about 5
copies of peptide are displayed per phage. They found that a potent anti-phage
Ab response was
induced, but that only marginal titers of Ab were generated against the
peptide. Soon thereafter,
pVIII-display was pioneered for immunogenic purposes (Greenwood et al., 1991),
and, because
pVIII offers the possibility of having many more copies of recombinant peptide
per phage, it was
found that phage displaying recombinant peptides on pVIII could elicit a
strong anti-peptide Ab
3
CA 02286301 1999-11-O1
'a
' response in the absence of adjuvant. Further work by the same group (Willis
et al., 1993) also
showed that the Ab responses against these phage were T-cell dependent. At the
same time,
Minenkova et al. (1993) immunized rabbits with phage bearing a peptide from
the HIV-1 p17
Gag protein on pVIII and found that Ab was elicited which recognized the
target antigen.
Subsequently, there have been a multitude of studies in which random peptide
libraries (Scott &
Smith, 1990) are screened with Abs that are specific for a target antigen, and
the phage clones
thus selected are then used to immunize animals (for examples see Motti et
al., 1994; Zhong et
al., 1994; Demangel et al., 1996; for reviews see Smith & Petrenko, 1997;
Zwick et al., 1998a).
The goal in these experiments is usually to elicit cross-reactive Abs that
bind both the peptide-
bearing phage clone and the target antigen.
For pVIII-display, a hybrid system is employed, in which the peptide:pVIII
fusion is
supplemented with wild-type pVIII, either in traps, as in the "type 8+8"
system, or in cis, as in
the "type 88" system used in this study (see Smith & Petrenko, 1997, for types
of phage display
systems). The resulting hybrid virion bears both wild type and the
peptide:pVIII fusion on its
surface. For some peptides, as much as 30-40% of the total pVIII on the phage
coat may be
recombinant, whereas for others only a few copies of peptide may be detected
per phage (Malik
et al., 1996/1998). We wanted to test the anti-peptide Ab response elicited in
mice by phage
bearing a pVIII-displayed peptide, designated NANP, corresponding to the major
antigenic
repeat of the malarial circumsporozoite protein. We used as a vector, f88-4
(Zhong et al., 1994),
since our panel of random peptide libraries (Bonnycastle et al., 1996) are
displayed by this phage
vector. Before immunizing, we measured the copy number of the NANP peptide on
the phage
coat and found that it was especially low, even if the phage were amplified
while inducing the
4
CA 02286301 1999-11-O1
..- , _,
;i
tac promoter of recombinant gene 8 (~0.4 % of wild type pVIII). It was later
determined that the
peptide copy-number was low in general for the f88-4 system (~0.4 - S % of
wild type pVIII).
This makes such phage good for affinity-selection experiments, since low
densities of
recombinant peptide would allow for the selection of high affinity clones from
a library.
However, we assumed that the low peptide copy number would make the phage a
poor
immunogen.
To solve the problem of low copy number of peptide on phage, we had the idea
of using
the phage, which have a repetitive multivalent array of pVIIIs, as a classical
immunological
earner to which many synthetic peptides could be coupled -- probably more so
than for
recombinant display on pVIII. We ofren desire to optimize, characterize and
measure the affinity
of synthetic peptide analogs of peptides derived from our libraries, as is
commonly practiced,
because such peptides are free of the confounding effects of multivalency and
possible
contributions from the phage coat to binding. Naturally, we were aware that
conjugation may
affect the affinity of the Ab for the peptide. It is also true, however, that
at least in some cases,
immobilization of a peptide at one end can induce a persistence in its overall
structure that does
not exist when the peptide is free in solution, as was shown by Jelinek et al.
(1997) for an HIV-
derived peptide displayed on the phage coat. Given the advantages of free
peptide model
systems and the problem of low copy number of recombinant peptide, we decided
to use the
phage as a classical protein earner for coupling with synthetic peptide, and
to compare this
immunogen to phage bearing recombinant peptide on either pIII or pVIII.
There are several advantages of using the phage over other classical carrier
proteins (e.g.,
tetanus toxoid, keyhole limpet hem.ocvanin, and ovalbumin). The phage are
''antigenically-
S
CA 02286301 1999-11-O1
,. .....,.,
. ,s
homogeneous" in the sense that with many, many repeating copies of the same,
small molecular-
weight protein (i.e., pVIII), they will likely induce a restricted Ab
response. In support of this, it
was recently shown that the preponderance of pVIII-reactive Ab raised against
the phage
recognize only about the first 12 amino acids on pVIII (Kneissel et al.,
1999). Another
advantage of the phage is that the repetitive arrangement of pVIII on the
phage coat would allow
for multivalent display of synthetic peptides in the same context, as opposed
to a large, complex
protein that would display each peptide differently on various portions of the
protein surface (the
same advantage exists for recombinant peptide-display on pVIII). Other
advantages of the phage
are that they are easily produced and purified. They are easy to engineer by
recombinant
methods, so the "immunogenic landscape" of the phage can be tailored for a
particular purpose.
Finally, the large size of the phage may lend to it adjuvant-like effects, and
better approximates
the size and composition of pathogens (especially viruses) than do the more
commonly used
protein carriers. Recently, the utility of the phage as an effective
immunologic carrier was shown
by Meola et al. (1995), who found that recombinant peptide displayed on pVIII
was the best
mode of immunization when it was compared to the same peptide displayed on
pIII, recombinant
human H-ferritin, the hepatitis B virus core peptide, and as a synthetic
multiple antigenic peptide
(M.AP; Tam, 1988). The results of M~ola et al. (1995), and others clearly
demonstrate that the
phage is an excellent recombinant carrier for peptide immunizations. For all
of the above
reasons, we hypothesized that the phage would be an excellent choice as a
classical
immunological carrier.
On the other hand, there may be drawbacks of using the phage as a classical
protein
carrier, and thus other modes of peptide display may be preferable. For
example, because of the
6
CA 02286301 1999-11-O1
predominance of the small sized pVIII on the phage coat, there may be a
limited T-cell response
against the phage. With only 50 residues, the number of good MHC class II
epitopes in pVIII is
probably low, although the studies of Willis et al. (1993) clearly indicate
that the immune
response against bacteriophage, at least in BALB/c mice, is T-cell dependent.
Although too
numerous to mention here, there are many different vehicles for peptide
immunizations involving
both recombinant and synthetic peptides. For recombinant display of peptide,
some have used
various proteins as carriers for a fused peptide. We chose to assess whether
peptides displayed
on monomeric maltose-binding protein (MBP; Zwick et al., 1998b) would serve as
a suitable
immunogen, as has been previously demonstrated for a peptide from the
hepatitis B virus
(Martineau et al., 1991/1992). In the case of synthetic peptide-carrier
conjugates, a protein
carrier is typically crosslinked to a large molar excess of peptide, thus
creating a multivalent
immunogen. We also included as an immunogen in this study the NANP peptide
conjugated to
MBP for comparison to its phage counterparts.
In order to couple peptides specifically to MBP and the phage, the water-
soluble,
heterobifunctional crosslinker sulfo-succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) was used. There is
a
maleimide moiety on one end of sulfo~SMCC that reacts with free sulflrydryls
to form a covalent
bond, and a succinimidyl group on the opposite end that reacts with primary
amines also to form
a covalent linkage. Thus, any peptide containing a free sulfhydryl, in theory,
can be coupled to
every copy of pVIII that contains an exposed lysine residue, and the peptide
be displayed in only
one orientation. Accordingly, if a lysine residue were exposed on every copy
of pVIII, thousands
of nucleophilic, amine functionalities would be available for the covalent
ligation of peptidic, or
7
CA 02286301 1999-11-O1
p. ,w o . . ~ . ..-.
'-.
haptenic molecules to produce a highly polyvalent immunogen. The first lysine
residue in pVIII
appears at position 8, which is partially exposed on the phage surface
(Armstrong & Perham
1983), but resides in a region that has alpha-helical structure so there are
restrictions in mobility
at this position, as opposed to residues 1 to S which are more disordered
(Glucksman et al.,
1992). As Lys8 may not be sufficiently exposed for efficient chemical coupling
to bulky
peptides, we decided to engineer a second lysine residue closer to the N-
terminus of pVIII on fl
phage. The resulting phage, designated fl-K, was compared to wild type phage
for efficiency of
peptide coupling, and both conjugates were used in immunization experiments.
Thus, in this study, we decided to test the immunogenicity of the synthetic
version of the
malarial peptide, using two different variants of the filamentous
bacteriophage as carriers, and
compare it to the immunogenicity of the recombinant counterpart fused to
either pIII or pVIII. In
addition, the same peptide was displayed as a recombinant fusion to MBP (Zwick
et al., 1998b),
and the synthetic version of the peptide was also chemically conjugated to
MBP, in order to
compare these MBP conjugates with their phage counterparts, and determine the
best
immunogen for eliciting anti-peptide Abs. We found that for a 24-mer peptide
containing the
major repeat sequence of the malarial circumsporozoite protein, the optimal
anti-peptide
response was achieved by challenpng mice with synthetic peptide-carrier
conjugates rather than
with recombinant display on either pllI, pVIII, or MBP. Both phage and MBP
appeared to be
equally effective as carriers for synthetic peptide as the anti-peptide Ab
response was similar in
these conjugates. The use of chemical conjugation for peptide display on phage
increases the
possibilities for the phage as a carrier for immunizations, and could present
new options for
vaccine design in the future.
8
CA 02286301 1999-11-O1
Abbreviations
Ab, antibody; sulfo-SMCC, sulfo-succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate; MAP, multiple antigenic
peptide; MBP,
maltose-binding protein; RF, replicative form; PEG, polyethylene glycol; PBS,
phosphate
buffered saline; TBS, Tris buffered saline; SDS-PAGE, solium dodecyl sulfate;
ELISA, enzyme-
linked immunosorbent assay; ABTS, 2 2'-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid; r,
recombinant; s, synthetic; N, NANP peptide, NANPNVDP(NANP)3;
Materials and methods
Materials
The oligonucleotide Primer 1 has the sequence 5'-GCCGCTTTTGCGGGATC GTCCGAAGCT
TTNGMACCCTCAGCA GCGAAAGAC-3' (where N = A, C, G, T and M = C or A; GibcoBRL
Life Technolo;~ies. Inc., Gaithersbure. MD). The E. coli strain, K91, the f88-
4 (Zhong et al.,
1994) and the fUSEl7 (pIII-displayed NANP) vectors were all kindly supplied by
G.P. Smith
(University of Missouri-Columbia). ?? supplied the fl vector (Hill & Petersen,
1983).
The crosslinker, sulfo-SMCC was purchased from Pierce (Pierce, Rockford, IL).
The
9
CA 02286301 1999-11-O1
i, ~. ~..
monoclonal Ab Pf2A10 (Wirtz et al., 1987) was a gift from R. Wirtz (WRAIR,
Washington, DC;
SmithKline Beecham and New York University). Female BALB/c mice (6 weeks old)
were
purchased from Charles River (St. Constant, Quebec).
fl -K Vector Construction
fl phage (Hill & Petersen, 1982) was subjected to site-directed mutagenesis
(Sambrook et
al., 1989) using Primer 1: This oligonucleotide contains a codon with one
degenerate, and one
partially-degenerate position that encodes either a serine or an alanine
residue. This partial
degeneracy was included in the design of the primer in case of the unlikely
event that if one of
the residues prevented phage production, the other would be selected. Thus,
300 ng purified, fl
ssDNA was mixed with 35 ng Primer 1 in 10 uL 10 mM Tris-HCl (pH 7.5), 2 mM
MgCl2 and 50
mM NaCI. The annealing mixture was heated at 70°C for 10 min, allowed
to cool to room
temperature over 45 min, and put on ice for 10 min. The annealing mixture was
mixed, on ice,
with 10 uL reaction mix containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM
dithiothreitol, 1 X bovine serum albumin, 1 mM ATP, 0.25 mM dNTP, 4 units T7
DNA
polymerase (New England Biolabs Ltd. (NEB), Mississauga, ON), and 4 units T4
DNA ligase
(NEB). The mixture was incubated for 5 min on ice, 5 min at room temperature,
and then 60 min
at 37 °C. The fill-in reaction was stopped by heating the mixture to 70
° C for 10 min. The fill-in
products were separated on a 0.8% agarose gel in 4X GBB buffer (168 mM Tris,
80 mM sodium
acetate, 7.2 mM Na2EDTA and pH adjusted to 8.3 with glacial acetic acid). The
presence of a
CA 02286301 1999-11-O1
band, approximately 6.4 kb in size, indicated the presence of replicative form
(RF) circularized,
fl dsDNA. An aliquot (1 uL) of the fill-in product was used to transform 100
uL of Escherichia
coli (K91) cells following the heat shock method described in Sambrook et al.
(1989). The
transformed cells were mixed with top agar (-, and maintained at 50°C),
and quickly poured
onto agar plates containing NZY medium. Single colonies were picked,
amplified, and the RF
DNA was isolated and tested for the presence of the desired mutation by
digestion with the
restriction endonuclease HindIII (NEB) following the manufacturer's
instructions. A single
clone was selected that contained the HindIII site, and the correct sequence
was verified by DNA
sequence analysis (Bonnycastle, 1998).
Large-scale preparation offl-Kphage
Three liters of NZY medium (Smith & Scott, 1993) were inoculated with a
single, well
isolated colony of K91 cells infected with fl-K phage, ~~d the culture was
shaken at 250 rpm for
21 h at 37°C. The culture was centrifuged at 4500 x g (rma,~ for 10 min
at 4°C , and the
supernatant spun again at 11 000 x g for 10 min at 4°C. The supernatant
containing the phage
was mixed with 0.1 S vol of PEG/IVaCI ( 16.7% polyethylene glycol (PEG 8000,
Sigma, St. Louis,
MO), 3.3 M NaCI) by inverting 100 times, and left to precipitate overnight at
4°C. The phage
were centrifuged at 11 000 x g for -10 min at 4 °C, the supernatant
removed, and the pellet was
resupended in 100 mL PBS (23 m_'~f I~zaHzP04, 77 mM Na,HP04, 1 SO mM NaCI, pH
7.4). The
resuspended phage were cleared by centrifugation at 20 000 x g for 10 min at
4°C, and then
11
CA 02286301 1999-11-O1
~ ,, .. , .J
precipitated with PEG/NaCI as described. The phage were resuspended in 50 mL
PBS, cleared
again, then solid CsCI (C-3139, Sigma) was added to a concentration of 31%
(w/v). The phage
mixture was transferred to two 39-mL quick-seal polyallomer tubes (#342414,
Beckman
Instruments Inc., Palo Alto, CA), and centrifuged at 57 000 rpm in a 70Ti
rotor on an
ultracentrifuge (L8-80, Beckman) for 21 h at 5°C. A large, diffuse,
bluish band containing the
phage was removed from the tubes using a 18-gauge needle equipped with a 10-mL
syringe, and
distributed between two 32-mL polycarbonate tubes (#355631, Beckman). The
tubes were filled
to the shoulder with PBS, and spun at 50 000 rpm for 4 h at 4°C in a
70Ti rotor. The phage
pellets were resuspended in a total of 28 mL PBS, heated to 70°C for 30
min, and cleared by
spinning at 20 000 x g for 20 min at 4 ° C. The supernatant was
transferred to a fresh tube, and
the phage concentration, determined by absorbance measurement,was 2.8 x 10'4
phage
particles/mL (Day, 1969). The phage stock was checked by DNA gel
electrophoresis, and a
single band was observed. The sequence in the region of the mutagenesis was
reconfirmed by
DNA sequence analysis.
Production of f88, rN.~pIII, and r14'.pYlll phage
The recombinant, NANP peptide-bearing phage (rN:pIII and rN:pVIII phage), and
the
parent vector f88-4 (Zhong et al., 1994; also referred to as f88 in the text),
were amplified and
purified by a method similar to that used for fl-K prage, except that, during
cell growth the
12
CA 02286301 1999-11-O1
culture medium contained tetracycline (15 ug/uL) to sei_ect for E. coli cells
infected with phage,
which harbor tetracycline resistance genes.
Coupling synthetic peptide to fl-K and f88-4 phage
About 2.5 x 10'3 physical particles of fl-K or f88-4 phage were mixed with 0.6
mg of
sulfo-SMCC crosslinker (Pierce) in a total vc.lunle of 500 uL PBS. The final
concentration of
phage was 5 x 10'3 particles/mL, and was chosen because, at higher
concentrations of phage (i.e.,
101'' particles/mL or greater), the suspension becomes gel-like, and the
addition of crosslinker
causes significant precipitation. At ~ x 10'3 particles/mL, there was still a
small amount of
precipitate after mixing with sulfo-SMCC; however, samples more dilute than
this were avoided
since lower coupling efficiencies occur under these conditions. The mixtures
of phage and sulfo-
SMCC were rotated slowly at 37°C for 45 min, brought to 1 mL with PBS,
and precipitated by
mixing with 0.15 vol of PEG/NaCI and incubating on ice nor 15 min. The samples
were spun at
15 000 x g for 15 min at 4°C, and all traces of the supernatants were
removed. The pellets were
resuspended in 1 mL PBS, and the phage were precipitated a second time with
PEG/NaCI as
before. The SMCC-activated phage w ere considerably more susceptible to PEG-
precipitation
than untreated phage, since the phage solutions immediately turned cloudy upon
addition of
PEG/h,TaCl, hence, extensive incubations at 4°C, and lor_g
centrifugation times were unnecessary.
The activated phage were resuspended in 1 mL PBS, and mixed with 1.35 mg NANP
peptide
(HZN-IVANPNVDPNANPNANPI~ ~-DD-Orn{bio}-C-CONHz; where HZN- represents a free N-
13
CA 02286301 1999-11-O1
:, >, . , . ,
terminus, -DD- was added as a spacer and to increase solubility, Orn{bio}
represents a
biotinylated ornithine residue, and -CONH2 indicates that the C-terminus is
blocked by amidation;
Alberta Peptide Institute, Edmonton, AB). At 2700 pVIII molecules per phage
particle and a
total of 2.5 x 10'3 phage, there are 112 nmol pVIII available for coupling.
Thus, 1.35 mg (S00
nmol) of the NANP peptide (mol. wt. 2701 g/mol) produces ~4.5 times molar
excess of peptide
over pVIII molecules. The mixtures of peptide and activated phage were rotated
slowly
overnight at 4°C, and the samples were purified twice by PEG-
precipitation (as above) to
remove free peptide. The final pellets, containing the synthetic NANP peptide-
phage conjugates
(sN-f1K and sN-f88), were resuspended in 900 uL PBS, and stored frozen at -
20°C. Aliquots
were removed and run on a 0.8% agarose gel in 4X GBB buffer, and DNA from the
peptide-
phage conjugates appeared as single bands that were identical in mobility to
those of untreated
phage.
Production of recombinant NANP peptide: ~LIBP fusion protein (rN.~MBP), and
preparation of
.rynthetic NANP peptide-MBP conjugate (sAr MBP)
The rN:MBP fusion protein. as well as control MBP (i. e., recombinant. MBP
fused to an
unrelated peptide sequence, YDVPD~-A, and purified under the same conditions
as the
rN:~P), were both produced from t~. pMal-X v~c.-~;ter iii E. coli, and
purified as described
previously (Zwick et al., 1998b). To prepare the synthetic sN-MBP, 2 mg
purified control MBP
was mixed with 2 mg sulfo-SMCC in 300 uL PBS and rocked slowly for 40 min at
37°C. The
14
CA 02286301 1999-11-O1
sample was diluted to 2 mL with PBS, transferred to a Centricon-30
ultrafiltration device
(Amicon, Inc., Beverly MA), and then washed 4 times in PBS at 4°C
following the
manufacturer's instructions. The SMCC-activated MBP was divided, and
approximately 1 mg
was mixed with 1.35 mg synthetic NANP peptide in 185 uL PBS containing 5 mM
EDTA,
whereas 0.8 mg SMCC-activated MBP was taken through the conjugation steps in
150 uL PBS
containing 5 mM EDTA, in the absence of peptide. The samples were rocked at
4°C for 2 h 15
min to allow coupling, and washed in PBS four times as befi;re. The samples
were back-eluted
from the centricon device in a final volume of 300 uL and were stored frozen
at -20°C.
SDS-PAGE
Phage proteins were separated u~ ~ng a mod~riec~ SDS-PAGE system (pers. comm.,
R.N.
Perham) based on that of Schagger & von Jagow (1987). The stacking gel
consisted of 16.6%
acrylamide, 0.17% bis, 19% glycerol, 1 M Tris-HCl (pH 8.3), and the separating
gel consisted of
4.7% acrylamide, 0.048% bis, 0.72 M Tris-HCl (pH 8.3). The upper (cathode)
chamber was
almost completely filled with buffer containing 0.1 M Tris, 0.1 M Tricine (no
pH adjustment
necessary; cat#T-7911, Sigma, St. Louis, MO), and 0.1% SDS. The buffer in the
lower (anode)
chamber was 0.2 M Tris-HCl (pH 8.9~_ In pr~.parati~n for loading, phage
samples were diluted in
PBS and mixed with 1/3 volumes .1X gei loading buffer (8% SDS, 40% glycerol,
68 mM Tris-
HCI, pH 6.8, 0.008% bromophenol bhne). All samples were boiled for 5 min
immediately before
loading. The MBP proteins were run on a 12% acrylamide gel according to the
method of
CA 02286301 1999-11-O1
Sambrook et al. (1989). SDS-PAGs containing MBP samples were stained with
Coomassie blue
following the procedure of Sambrook et al. (1989), whereas the SDS-PAGs
containing phage
samples were silver-stained essentially following the method of Morrisey (
1981 ).
Mouse immunizations
The mice were immunized as summarized in Table ~. All injections were done
intraperitoneally
(i.p.), and diluted in a total of 100 uL PBS containing 1 mg/mL AdjuvaxTM
adjuvant (Alpha-Beta
Technology, Worcester, MA). The mice were bled from the tail vein on Days 0,
14, and 28, just
prior to injection. On Day 42, the mice were bled by cardiac puncture under
COZ anaesthesia.
The blood samples were allowed to clot overnight at 4°C, and then
centrifuged at 12 000 x g for
min. The serum supernatants were trmsfei:ed to fresh microfuge tubes, and, for
the tail-bleed
samples, mixed with an equal volume of Tris buffered saline (TBS; 100 mM Tris-
HCI, pH 7.5,
150 mM NaCI) containing 2% BSA and 0.04% sodium azide. For the serum samples
from the
final bleeds, sodium azide (Sigma) was added to a final concentration of 0.02%
(w/v). All of the
serum samples were heated to 52 °C in a water bath for 20 min to
inactivate complement, and
stored at 4°C. The mice sera from within each group were pooled before
use in the ELISA
experiments.
ELISA and KinExA
16
CA 02286301 1999-11-O1
Microwells (EasyWash, Costar, Corning Inc., Corning, NY) were coated with 35
uL TBS
containing either 5 x 109 f88 particles, 200 ng MBP, or 1 ug streptavidin
(Roche Diagnostics,
Laval, Quebec), and gently rocked overnight at 4°C. For all the washing
steps, 200 uL TBS
containing 0.1% Tween 20 (ICN Biomedicals Inc., Aurora, OH) were dispensed by
an EL-403
plate washer (Bio-Tek Instruments, Inc., Winooski, VT); the plate was shaken
for 5 sec on low
setting, and set to stand S sec between each wash. The wells were washed
twice, and 35 uL TBS
containing 10'z molecules of biotinylated NANP peptide was added to the
aspirated wells
containing immobilized streptavidin; all other wells received 200 uL TBS
containing 2% BSA
(Fraction V, Sigma). After incubating the microplate for 30 min at room
temperature, wells
containing peptide were topped with 200 uL TBS containing 2% BSA, and the
plate was blocked
for 1 h at 37°C. The wells were washed twice, and 35 uL pooled serum
from each group (Table
1) diluted in TBS containing 1% BSA and 0.1% Tween 20 were added. The Abs were
incubated
in the appropriate wells for 2 h at room temperature. The wells were washed
five times, and 35
uL goat anti-mouse IgG (H+L):horseradish-peroxidase conjugate (Pierce),
diluted 1:500 in TBS
containing 0.1% Tween 20, was added. ABTS solution was prepared by mixing 3.07
ml 0.1 M
citric acid and 1.93 ml 0.2 M NazHPO~, and adding 2 mg of 2 2'-azino-bis(3-
ethylbenzthiazoline-
6-sulfonic acid) (Sigma) and 5 uL 30°/'~ (w/wi IIZOZ (BDH Inc.,
Toronto, ON). After 30 min at
room temperature, the wells were «-as~ed six times and 35 uL freshly-prepared
ABTS solution
was added. After 45 min, the absorbance at 405 nm and 450 nm was measured on
an EL 312e
Bio-Kinetics Reader (Bio-Tek), and the results were reported as A4p5-A~9o.
17
CA 02286301 1999-11-O1
The KinExA immunoassay is an in-solution assay for Kd determination, and has
been
described in detail by Blake et al. (1997). The procedure used in this study
was essentially that
of Craig et al. ( 1998), with the modification that the secondary (detection)
Ab was Cy5-
conjugated goat anti-mouse F(ab')Z (Jackson Immunoresearch Laboratories Inc.,
West Grove,
PA). The assay was conducted using the KinExA instrument (Sapidyne
Instruments, Inc., Boise,
ID), and the data was analyzed using the software provided by the manufacturer
(Sapidyne).
Amino acid analysis
All amino acid analyses were performzd at the Alberta Peptide Institute (U.
Alberta, Edmonton,
AB).
Results
Characterization of fl -K phage, and immunogen conjugates
The nucleotide and amino acid sequences of the N-terminal region of mature
pVIII, for
both wild-type fl and the phage bearii~g the extra Lys residue near the N-
temlinus of pVIII (fl-
K) are shown in Fig. ~ . The insertion. of four residues into the N-terminal
segment of pVIII was
well tolerated by fl -K phage, since the phage yield was very high (~2 x 10' '
phage/L), and no
18
CA 02286301 1999-11-O1
'..
mutations were discovered by DNA sequencing in the insert region following
large-scale
amplification of the phage. Phage that were coupled to the NANP peptide with
sulfo-SMCC, or
treated with SMCC alone, were subjected to DNA-gel electrophoresis, and the
DNA from the
chemically-modified phage appeared as a single band that was identical in
mobility to that of
untreated phage, indicating that the ssDNA of the phage was not modified by
the crosslinking
(data not shown). When the same samples were subjected to SDS-PAGE, the effect
of the
2~
crosslinker could be seen (Fig.. The appearance of multiple bands with a
slower mobility than
pVIII, indicated that the NANP peptide was successfully coupled to the major
coat protein of fl-
K. It appeared that the side-chain of Lys8 in the mature pVIII of f88-4 was
su~ciently exposed
to react with the succinimidyl moiety of sulfo-SMCC (the amino acid sequence
of mature pVIII
for f88 is identical to fl in the region shown in Fig.l), or, that the less-
reactive amino terminus of
pVIII is participating. In the case of fl-K treated with SMCC alone, some
crosslinking of pVIII
can be seen, which resulted in a ladder-like effect. Although sulfo-SMCC-
activated phage are
supposed to couple specifically to sulfliydryls, in their absence, the
maleimide moiety of sulfo-
SMCC is known to react with free primary amines at a 1000-fold lower rate than
with
sulfhydryls (Hermanson, 1996). Thus, for sulfo-SMCC-treated fl-K, the free E-
amino group of
the lysine residue near the N-terminus of pVIII likely crosslinked to itself
in the absence of a
Cys-containing peptide. The ladderin~ effect observed with SMCC-treated fl-K
does not occur
with the f88 counterpart, although some dimer can be seen, indicating there is
some adventitious
crosslinking occurring as well, albeit to a lesser degree.
All of the chemically-coupled conjugates were examined by amino acid analysis
in order
to determine the number of NANP peptides displayed per carrier molecule
(results shown in
19
CA 02286301 1999-11-O1
w, ..
Table 2). Table 2 clearly shows that the chemically coupling of synthetic
peptide to a protein
Garner results in a much higher number of peptides per carrier molecule than
does recombinant
display. The amino acid analysis of sN-f88 and sN-fl K, revealed that a large
percentage of
pVIIIs were coupled to the peptide on the phage (527% and 96114%,
respectively). The
enhancement of coupling of synthetic NANP to fl-K, above that of f88-4, can be
attributed to the
existence of the engineered Lys residue near the N-terminus of pVIII on fl-K
(see above). The
peptide to carrier ratio for sN-MBP is also very high (318 to 1). The number
of Lys residues in
MBP is 35, so the coupling ratio of the NANP peptide to MBP must be near the
limit.
As mentioned above, the recombinant display of the NANP peptide on the phage
and
MBP was of lower density. The copy number of the NANP peptide on the phage was
determined visually by SDS-PAGE (Fig. and found to be very low (~16 copies per
phage,
assuming 3900 copies of pVIII for f88-4 phage, or 0.4% of the total pVIII).
Our experience with
recombinant pep:pVIII expression (i.e-, with clones from f88-based vectors)
has been that
roughly 0.5%-5% of the major coat proteins are recombinant. Thus, the NANP
peptide is poorly
expressed in our recombinant pVIII-display system, and may reflect the
immunogenicity of
other, poorly expressed pep:pVIII fasions. The copy number of peptides was
assumed to be 5 per
phage for rN:pIII, and 1 copy per hiBP molecule for rN:MBP (data summarized in
Table 2).
The recombinant phage, rN:pIII and rN:pIII, and the recombinant MBP displaying
the
NANP peptide, rN:MBP, were all probed with the NANP-specific, monoclonal Ab
Pf2A.10
(Wirtz et al., 1987), and all the recombinant constructs were found to be
positive by ELISA (data
not shown). Immobilized, synthetic N.AIv'p peptide also produced a strong
signal by ELISA
CA 02286301 1999-11-O1
J ~u
probing with Pf2A.10 (data not shown). By KinExA analysis, the free NANP
peptide bound to
Pf2A 10 with a Kd = 130 nM (Error bounds: Kd High = 173 nM, and Kd low = 81
nM).
Mouse immunizations
Six groups of Balb/c mice (groups 1-6) were immunized with different
recombinant, and
chemically-coupled peptide-phage conjugates, and their sera were tested for
anti-peptide and
anti-carrier Ab responses by ELISA. 'Ve found from a previous dosage
experiment (data not
shown) that 20 ug of phage was sufficient to elicit an anti-peptide Ab
response in BALB/c mice,
as well as a strong anti-phage response; thus, the mice were injected with an
equivalent of 25 ug
of phage protein (see Table 1). In most cases, the mice were primed and
boosted with the same
immunogen. In two groups (3 and 4), however, we tried a heterologous
immunization in which
the priming and boosting immunogens were different (see Table 1). The purpose
of the
heterologous immunizations were to sew if the differences in pVIII would alter
the
immunogenicity of the phage such that, more of the Ab response would be
directed toward the
peptide than the phage coat in the boosting conjugate (see Discussion). The
mice in groups 9
and 10 were immunized with SMCC-treated f88-4, and SMCC-treated fl-K,
respectively. They
were added as controls in case there was a large difference in the Ab response
against the phage
carrier due to the alteration of the phag~ coat by (i), the addition of the
four amino acid residues
in the pVIII of fl-K and, (ii), the modification caused by the crosslinker,
sulfo-SMCC.
21
CA 02286301 1999-11-O1
The mouse sera were tested by ELISA for their reactivity with biotinylated
NANP
peptide that was captured on immobilized streptavidin (results shown in Fig:.
Most striking, is
that the the anti-peptide response was greatest with the chemically-conjugated
peptide
immunogens, and it did not matter significantly whether the carrier was f88,
fl-K or MBP. At
serum dilutions less than the 1:800 dilution shown in Fig., there was a
detectable IgG response
against NANP peptide for mice immunized with rN:pIII and rN:pVIII (i. e., O.D.
~0.2 at 1:50
serum dilution), indicating that pVIII display was not significantly better
than pIII display
despite having a 3-fold difference in the peptide to phage ratio. On the other
hand, there was a
significant ELISA signal against peptide at a 1:800 serum dilution for groups
1-4, 7 and 8. The
order of serum-reactivity of these groups of mice with the NANP peptide,
correlates with the
number of peptides per kDa of carver as shown in the right-hand column of
Table 2. Thus, our
results support the strategy of increasing the copy number of peptide on a
given carrier to achieve
the maximum anti-peptide Ab response. Again, the strongest anti-peptide
responses were from
the synthetic peptide conjugates. Given that the results are from pooled sera,
it was not seen how
the responses varied within a group. Thus, it is difficult to make inferences
about the smaller
differences in ELISA signals such as amnong the groups receiving the different
synthetic peptide
conjugates. Nonetheless, it remains char that the anti-peptide response was
best with the
chemically-conjugated peptide immure~gens, and it did not matter significantly
whether the
carrier was f88, fl-K or MBP.
We questioned whether the lower reponse against the recombinant counterparts
was due
to a generally low response against the entire immunogen (perhaps the chemical
coupling itself
enhanced the immunogenicity of these immunogens). Thus, we tested the response
against
22
CA 02286301 1999-11-O1
Garner and saw that the carrier responses were comparable, and thus the
response against peptide
S
relative to carrier was enhanced with synthetic conjugates. Fig. ~ shows the
results of ELISAs
comparing the IgG response against f88-4 phage for all the phage-immunized
mice. Apart from
some fluctuations in ELISA signals, most of the conjugates had similar
reactivities toward f88-4
phage. This was an important result because it shows that even with high
levels of crosslinking
with peptide, a potent anti-phage Ab response is elicited, and that the Abs
against either fl-K or
f88 phage are largely cross-reactive with one another. This does not
necessarily mean that the
Ab specificities against pVIII in either phage are identical, as there could
be very little similarity
in the pVIII-specific Abs toward the ri~-o phage. (See Western blot results)
However, a
significant portion of the Ab response is directed toward the minor coat
proteins (i.e., pIII, pVI,
pVII and pIX), and these Abs will recognize both phage with identical affinity
and specificity.
Fig~shows that pooled sera from group 9 had a significantly lower ELISA signal
against phage
than all the others. It is unclear as to why this is, since SMCC-f88 is less
modified than sN-f88;
the latter gave a strong ELISA signal against f88-4 phage. Possibly, the group-
size (4 mice) was
not be representative of the average anti-phage Ab response with this
immunogen. The carrier
response was also checked for the mice immunized with the recombinant, and
chemically-
coupled peptide-MBP conjugates. The ELISA results showing the IgG response of
pooled sera
from mice immunized with sN-MBP (gzoup 7), rN:MBP (group 8) or S14ZCC-MBP
(group 11)
6
are shown in Fig.~C As can be seen, the anti-MBP IgG response was greater for
mice immunized
with rN-MBP. This was not unexpected because the mice in group 8 received more
protein (33
ug) than did groups 7 and 11 (10 ug), amd the latter two were immunized with
chemically-
modified MBP.
23
CA 02286301 1999-11-O1
i
'" ., , , . _ .,~
Discussion
In this study, it was determined that the phage can be used as an effective
classical carrier
for the preparation of synthetic peptide conjugates, capable of eliciting good
anti-peptide Ab
responses in BALB/c mice. We have shown this also for two other peptides (data
not shown),
which, similar to the NANP peptide, conjugated to the fl-K phage at high
density (more than one
peptide per copy of pVIII, data not shown). We also showed that the potency of
the anti-peptide
Ab response elicited by a conjugate of the NANP peptide with MBP, matched that
of the phage
conjugates, all of which were superior to recombinant phage and MBP in
eliciting an Ab
response against peptide.
Hey et al. ( 1994) used what they termed "a two fusion partner system" for
eliciting anti-
peptide Abs in rabbits. Their results indicated that, in some cases, the
peptide response could be
increased relative to the carrier response if the same peptide were used to
boost the animal but
that a different carrier be used for the prime and boosts. In their study,
however, T-cell help
probably derived, at least in part, from the fusion peptides themselves, which
were fairly large
(39 to 96mers). Because the sera v~~e used for each group were pooled from 4
mice each, there
lacks the replicates to do sensitive statistics. Thus, we can be confident of
large differences, but
we have reservations about smaller ones such as in the heterologous
immunizations. With this in
mind, there does appear to be slightly utter ELISA signals against peptide for
serum from
heterologously-immunized mice (groups 3 and 4), as compared to those that were
immunized
homologously (groups 1 and 2; see Fi~~. It must be pointed out, however, that
the anti-phage
24
CA 02286301 1999-11-O1
Ab response appears to be stronger in the heterologous group as well (Fig.. It
also appears
that sN-f88 was somewhat better than sN-fl K at eliciting an anti-NANP
response in mice. This
may suggest that the density of the NANP peptide on sN-fl K is too high, and
that the optimal
ratio of peptide to pVIII for eliciting a potent anti-peptide response in mice
is less than 1 to 1. In
any event, the fl -K phage may have had an enhancing effect on anti-peptide Ab
production in the
heterologous immunizations. Iin order to improve upon this effect, further
mutagenesis of fl-K
would be necessary to reduce the cross-reactive response against phage
proteins. The
filamentous phage are particularly well-suited to mutagenesis, and
heterologous immunization
experiments with divergent phage clones may be a particularly attractive means
of eliciting anti-
peptide Ab responses, as well as examining the behaviour of the Ab response
following
heterologous challenge.
In summary, we found that the lack of a potent anti-peptide Ab response
induced by
phage displaying recombinant peptide on pIII, or by phage with a low copy
number of
recombinant peptide on pVIII, can be overcome by the multivalent display of
synthetic peptide
chemically-coupled to the phage coat proteins. Our results must be taken with
some caution,
however, if a synthetic peptide is derived from an eptitope mimic or
"mimotope". If it is
determined that a peptide derived from a phage-display library intimately
depends on the phage
coat for its activity, the synthetic version may be a poor candidate for
immunizations, at least
without further optimization. The addition of a Lys residue in fl-K, permitted
superior levels of
peptide crosslinking using the heterobifunctional crosslinker, sulfo-SMCC. The
correlation
beriveen the surface density of a peptide on an immunogen and the
immunogenicity of the
peptide may, however, have a limit. since serum from mice immunized with sN-
f88 gave a
CA 02286301 1999-11-O1
stronger anti-peptide Ab response than did the serum against sN-fl K. Because
phage are so
ideally-suited to mutagenesis, further modifications to pVIII may be easily
achieved. We chose
to position the extra Lys residue in fl-K a few residues downstream of the
leader peptidase
cleavage site, since Lys residues positioned immediately adjacent to this site
may inhibit the
insertion of promature proteins in the inner membrane of E. coli (Andersson &
von Heijne,
1993). It may be possible, however, to shift the lysine residue introduced in
fl-K even closer to
the N-terminus of pVIII. Moreover, the pre-existing lysine residue in fl-K,
corresponding to
position 8 in fl, could be mutated to an arginine or another polar residue, to
reduce side-reactions
from occurring. The outer domains of pIII could be removed to lower the
complexity and cross-
reactivity in the Ab response against heterologous challenge with phage. The
phage could also
be genetically engineered to display a T-cell epitope, or another reactivity
prior to the chemical
conjugation step. Experiments involving the interplay between recombinant
technology and
chemical-conjugation should yield some interesting results for vaccine
research in the future.
Acknowledgements
We thank Scott Diguistini for technical assistance, and Loeki Van der Waal and
the staff at the
SFU Animal Care Facility for the care and handling of the mice used in this
study. This work
was supported by
26
CA 02286301 1999-11-O1
IJ .~ . . ,
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31
CA 02286301 1999-11-O1
;.
.. , ,,.,
a Table 1
Mouse Immunization Schedule
Group Number Priming Immunogen Used for Dosage per
of Mice Immunogen First (Day 14) and Injection'
in Group (Day 0) Second (Day 28)
Boosts
Experimental
groups:
1 4 sN-fl K sN-fl K 25 ug
2 4 sN-f88 sN-f88 25 ug
3 4 sN-f88 sN-f1K 25 ug
4 3 sN-fl K sN-f88 25 ug
4 rN:pIII rN:pIII 25 ug
6 4 rN:pVIII rN:pVIII 25 ug
7 4 sN-MBP sN-MBP 10 ug
8 4 rN:MBP rN:MBP 33 ug
Control groups:
9 3 SMCC-f88 SMCC-f88 25 ug
3 SMCC-f1K SMCC-f1K 25 ug
11 3 SMCC-MBP SMCC-MBP 10 ug
' All of the mice were immunized intraperitoneally (i.p.) with 100 uL 1 mg/mL
Adjuvax
adjuvant in PBS. All of the phage-immunized mice received 8.3 x 10" conjugated
phage
particles per injection, which is ~25 ug protein assuming that an O.D. at 269
nm of 30 is 6
mg/mL of phage protein.
CA 02286301 1999-11-O1
Table 2
The number of NANP peptides displayed per carrier molecule for the recombinant
and
synthetic peptide immunogens
Number of
peptides
displayed
/ coupled
Name Description of Peptide-
Carrier Conjugate eer p~u~ per phage'per lcDao
f
p carrier
III or MBP
sN-f88 f88 vector crosslinked 0.52 2030 0.088
to
synthetic NANP peptide
sN-f1K f1K vector crosslinked 0.96 2590 0.16
to
synthetic NANP peptide
rN:pIII recombinant NANP peptide1 5 0.00022
displayed on pIII
rN:pVIII recombinant NANP peptide0.004 16 0.0007
displayed on pVIII
sN-MBP MBP crosslinked to synthetic31 - 0.60
NANP peptide
rN:MBP recombinant NANP peptide1 - 0.019
displayed on MBP
a the number of copies is calculated assuming fl-K (6.4 kb) has 2700 pVIIIs
and f88-4/rN:pVIII
(9.2 kb) and rN:pIII (9.2 kb) have 3900 pVIIIs.
b the molecular-weight of fl-K approximately 16 Mda (Berkowitz & Day, 1980),
and thus f88-4
was calculated to be 23 Mda. The molecular weight of the MBPs produced from
the pMal-X
vector is ~52 kDa.
CA 02286301 1999-11-O1
~ 'd .r . .
Fig.? Cartoon illustration of the filamentous bacteriophage.
Fig.l. Engineering a Lys residue into the N-terminus of the mature, major coat
protein (pVIII)
of fl phage. A. A partial nucleotide and amino acid sequence of pVIII of fl
phage (same as for
fd and its derivative, f88-4 phage). The vertical line (~) indicates the
leader peptidase cleavage
site, and the wedge (") indicates the insertion site of the four residues in
fl-K. The underlined
sequence corresponds to the region of homology to Primerl (see Materials and
Methods). B.
The analogous region in fl-K as is shown for fl in A. The four residues
inserted in the N-
terminal region of mature pVIII are shown between the asterisks (*). The
unique HindIII site of
fl-K is also shown (underlined).
3
Fig.',. Estimating the number of NANP peptide:pVIII fasions per phage
particle. Lane 1: f88-4,
6x10'° phage particles, Lanes 2-9: Decreasing amounts of recombinant
phage (3-fold diltuions).
Lane 2: 6x10'° particles. Lane 3: 2x10'° partciles. Lane 4:
6.7x109 particles. Lane 5: 2.2.x109
particles. Lane 6: 7.4x108 particles. Lane 7: 2.5x108 particles. Lane 8:
8.2x10' particles. Lane
9: 2.7x10' particles. Based on the band intensities of the wild-type pVIII
band in Lane 8 and the
NANP peptide:pVIII band in Lane 3, one can wstimate that the expression of the
recombinant
band is 5 3-fold dilutions, or 243-fold Iower than that of wild-type pVIII.
Given a pVIII copy
number of 3900 for f88-4 phage, the copy number of NANP peptide:pVIII per
phage is ~16.
[Reprinted with permission CSHL?]
2
Fig.: SDS-PAGE analysis of phage proteins (A.) and MBP (B.) before and after
treatment with
CA 02286301 1999-11-O1
sulfo-SMCC and peptide coupling. A. Lane l: f88, no treatment. Lane 2: fl-K,
no treatment.
Lane 3: f88, treatment with sulfo-SMCC. Lane 4: fl-K, treatment with sulfo-
SMCC. Lane S:
fl-K, after coupling with NANP peptide. Lane 6: f88, after coupling with NANP
peptide. B.
MBP
Fig.4. ELISA signals against immobilized NANP peptide for pooled sera from
mice immunized
with different carriers displaying the NANP peptide. The categories (bottom)
indicate the
ELISA signals of pooled serum from each group of mice (see Table ~, following
the prime,
first and second boosts (indicated on the right with the serum dilution used).
The results are the
average from two separate experiments.
Fig.S. ELISA signals against immobilized f88-4 phage for pooled sera from mice
immunized
with different carriers displaying the NANP peptide. The results are the
average from two
separate experiments. The categories (bottom) indicate the ELISA signals of
pooled serum from
each group of mice (see Table ~, following the prime, first and second boosts
(indicated on the
right with the serum dilution used). The results are the average from two
separate experiments.
Fig.6. ELISA signals against immobilized MBP for pooled sera from mice
immunized with
different carriers displaying NANP peptide. The results are the average from
two separate
experiments. The categories (bottom) indicate the ELISA signals of pooled
serum from each
group of mice (see Table ~, following the prime, first and second boosts
(indicated on the right
with the serum dilution used). The resu.~ts are the average from two separate
experiments.
