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Polymerisation of a T cell epitope with an immunostimulatory C3d peptide sequence
enhances antigen specific T cell responses
Neil M. O’Brien-Simpson, Troy J. Attard, Baihui Zheng, Katrina A. Walsh, Eric C.
Reynolds*
Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, 720
Swanston Street, Melbourne, Victoria, 3010, Australia.
Running title: Enhancing peptide antigenicity
*Corresponding author: Professor Eric C Reynolds, The University of Melbourne, Oral
Health CRC, Melbourne Dental School, Bio21 Institute, 720 Swanston Street, Victoria 3010,
Australia.
Tel +61 3 9341 1447, Fax +61 3 9341 1599. Email: [email protected]
Acknowledgements
We also wish to thank Ms Jenny Davis for the animal welfare and handling. This work was
supported by NHMRC grants: APP1029878 and APP1008106.
1
Abstract
The complement protein C3d and C3d derived peptides that bind CD21 are known to enhance
immunity to co-immunised antigens. In this study we have synthesised the minimal CD21
binding sequence of C3d (1227LYNVEA1232) as mono, di and tri tandem repeats and
derivatised the N-terminus with an acryloyl moiety. These acryloyl-(C3d)n peptides were copolymerised with a acryloyl-T cell epitope (PAS1K) from the Porphyromonas gingivalis
antigen the RgpA-Kgp proteinase-adhesin complex. The ability of C3d containing polymers
to enhance T cell immunity in-vitro and in-vivo was evaluated. When used to stimulate invitro PAS1K-primed or RgpA-Kgp complex-primed T cells the C3d containing PAS1K
polymers induced a mixed and significantly (p<0.05) higher IL-4 and IFNγ T cell response
compared to that induced by the PAS1K peptide or polymer. PAS1K polymers containing
tandem repeats of C3d induced a significantly (p<0.05) stronger maximal proliferative
response, at the same antigenic dose, compared to that induced by the PAS1K peptide or
polymer. When used as immunogens to prime T cells all of the C3d containing PAS1K
polymers induced a dominant IFNγ T cell response and reduced the antigen dose required for
maximal proliferation 150-fold compared to that required for the PAS1K- peptide or polymer
primed T cells. In conclusion, the 6 residue sequence LYNVEA from C3d is sufficient to
enhance immunity to an antigen and that the effect is more pronounced when C3d is part of
the immunising antigen rather than an in-vitro stimulating antigen.
Keywords: C3d complement; peptide synthesis; peptide polymers; T cell immune responses
2
Introduction
Complement is typically regarded as a component of the innate immune system, however,
degradation products particularly of the third complement factor, C3, are known to enhance
the adaptive immune response to foreign antigens (Toapanta and Ross 2006). Activation of
C3 leads to the generation of C3dg and its degradation product C3d which form covalent
bonds with foreign antigens, thus providing a ligand for complement receptor 2 (CD21).
CD21 is expressed on B cells, follicular dendritic cells (FDCs), epithelial cells and subsets of
T cells (Hourcade et al. 1989). CD21 engagement with C3d is known to lower the antigen
activation threshold in B cells and induce proliferation as well as playing a role in B cell
memory and tolerance and immunoglobulin class switching (Fearon 1998; Frade et al. 1992;
Mongini et al. 1997; Toapanta and Ross 2006). C3d-antigen complexes have been reported to
enhance antigen processing and presentation by FDCs and they are retained on the cell
surface for extended periods of time which has been shown to play a role in enhancing B cell
activation, antibody production and memory (Bergmann-Leitner et al. 2006; Roozendaal and
Carroll 2007; Zabel and Weis 2001). Although, CD21 is expressed on 1-23% of human and
4-5% of mouse T cells the functional role played by CD21 on T cells is yet to be fully
elucidated (Levy et al. 1992; Molnar et al. 2008). However, CD21 expressing T cells have
been shown to adhere strongly to C3d/CD21 bearing target cells and this enhanced binding is
suggested to increase T cell activation and differentiation (Levy et al. 1992; Masilamani et al.
2002; Sinha et al. 1993). In mice, antigen immunisation has been shown to result in the
increase in lymph node CD21+ T cells which is enhanced by the presence of C3d/CD21
bearing B cells (Kerekes et al. 1998; Molnar et al. 2008).
C3d is a 35 kDa protein and although several binding sites for CD21 (CR2) have been
postulated a 28 amino acid sequence (1209KFLTTAKDKNRWEDPGKQLYNVEATSYA1236)
3
was found to contain the minimal binding domain 1227LYNVEA1232 (Lambris et al. 1985).
Dempsey et al. (1996) have shown that the fusion of C3d to a model antigen, hen egg
lysozyme, enhanced the immune response to the antigen and that including 2 or 3 copies of
C3d resulted in a 1,000 and 10,000 fold increase in the antibody response, respectively.
Inclusion of multiple copies of C3d in either DNA, protein or polysaccharide based vaccines
has been shown in several studies to enhance immunogenicity to the vaccine antigen, and a
recent study suggests that 6 or more copies of C3d are necessary for efficient enhancement of
the immune response (Green et al. 2003; Haas et al. 2004; Lou and Kohler 1998; Mitsuyoshi
et al. 2005; Mkrtichyan et al. 2008; Suradhat et al. 2001; Toapanta and Ross 2006; Wang et
al. 2006; Zhang et al. 2011). Although, the 28 amino acid C3d binding region peptide is
known to have reduced affinity for CD21, its incorporation into a vaccine construct does
enhance the immune response to the vaccine antigen and multiple copies of the peptide, up to
three have been shown to induce stronger immunity and CD21 affinity than one or two copies
of the peptide (Bergmann-Leitner et al. 2007; Bower and Ross 2006; Lou and Kohler 1998;
Servis and Lambris 1989; Tsokos et al. 1990). Frade et al. (1992) have shown that a 16
residue synthetic peptide of C3d containing the minimal binding sequence 1227LYNVEA1232
does induce proliferation. In another study the multi-valency of a 13 residue C3d peptide
(P13) containing the LYNVEA sequence was found to be essential for activation and
proliferation as the multi-valent (four copies of P13) antigen was able to cross link CD21
bearing effector and target cells (Servis and Lambris 1989).
In this study we investigated whether the minimal C3d binding sequence LYNVEA
when polymerised with a T cell epitope from the oral bacterium Porphyromonas gingivalis is
sufficient to enhance antigen specific T cell proliferative and cytokine responses.
4
Materials and Methods
Chemicals and Reagents
Unless stated otherwise, chemicals used for peptide-resin assembly were of peptide synthesis
grade or its equivalent. O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT), diisopropylethylamine
(DIPEA), dimethylformamide (DMF), trifluoroacetic acid (TFA), piperidine, 9flourenylmethloxycarbonyl (Fmoc)-protected amino acids, Fmoc-6-amino hexanoic acid
(Ahx) and Fmoc-L-Ala-WANG resin were purchased from Auspep Pty. Ltd., Melbourne,
Australia while Fmoc-PAL-PEG-PS resin was purchased from PerSeptive Biosystems, Inc.,
Framingham, MA. Dichloromethane (DCM), diethyl ether, phenol,
trishydroxymethylaminomethane (Tris), ethylenediamine tetraacetic acid (EDTA), methanol
(MeOH) and ammonium chloride were purchased from Ajax Finechem, New South Wales,
Australia. Acrylamide was obtained from BioRad Laboratories, New South Wales, Australia.
Acryloyl chloride, acetonitrile, sodium bicarbonate, sodium hydroxide (NaOH) and sodium
chloride (NaCl) were obtained from MERCK, Victoria, Australia. Triisopropylsilane (TIPS),
guanidine-HCl, ammonium persulfate, tetramethylethylenediamine (TEMED), Dulbecco's
phosphate buffered saline (PBS), complete Freund’s adjuvant (CFA), incomplete Freund’s
adjuvant (IFA), Bovine Serum Albumin (BSA), L-glutamine, sodium pyruvate, gentamicin,
penicillin, streptomycin, concanavalin A (ConA), N-lauroylsarcosine, and polyoxyethylene
sorbitan monolaurate (Tween 20) were purchased from Sigma-Aldrich, New South Wales,
Australia. Dulbecco's modified eagle's medium (DMEM) was obtained from SAFC
Bioscience, Victoria, Australia. Fetal bovine serum (FBS) was purchased from GIBCO
Invitrogen Corporation, Victoria, Australia. The antibodies (Abs) used in ELISPOT assay
were bought from eBiosciences, San Diego, USA. 2-Mercaptoethanol and ammonium
5
bicarbonate were obtained from ICN Biomedicals, Inc., Costa Mesa, USA. 2,4,6Trinitrobenzene sulfonic acid (TNBSA) was kindly provided in powder form by Dr Denis
Scanlon.
Synthesis and purification of Acryloyl-Peptides
All peptides were synthesised as acryloyl-Ahx-(Lys)4 derivatives and PAS1K (Acryloyl-Ahx(Lys)4-NYTAHGSETAWADPL, MW: 2311.6 Da) and C3d-based peptides, C3d (AcryloylAhx-(Lys)4-LYNVEA, MW: 1387.7 Da), [C3d]2 (Acryloyl-Ahx-(Lys)4-LYNVEALYNVEA,
MW: 2077.5 Da) and [C3d]3 (Acryloyl-Ahx-(Lys)4-LYNVEALYNVEALYNVEA, MW:
2767.2 Da) were synthesized using the LibertyTM Microwave Peptide Synthesizer (CEM
Corporation, Matthews, NC, USA). Fmoc chemistry using Fmoc-protected amino acids and
Fmoc-PAL-PEG-PS resin (0.20 mmol/g) or Fmoc-L-Ala-WANG (0.70 mmol/g) as the solid
support was employed throughout the synthesis, with the completed peptides assembled as
their C-terminal carboxyamide (PAS1K) or carboxyl forms (C3d-based peptides). FmocAhx-OH was manually coupled as a spacer (O'Brien-Simpson et al. 1997) to the N-terminus
of each peptide via the addition of HBTU (5 equivalents), HOBT (5 equivalents) and DIPEA
(9 equivalents) in DMF. Fmoc-Ahx-OH coupling was monitored by the TNBSA test
(Hancock and Battersby 1976) and following a negative TNBSA test, the resin was washed
with DMF (1 x 10 ml) and DCM (5 x 10 ml).
Following removal of the Fmoc group [20% piperidine/DMF (v/v; 20 min) the
peptide-resin was washed with DMF (6 x 5ml), DCM (6 x 5ml) and diethyl ether (6 x 5ml),
then dried under reduced pressure (18h). Acryloylation of the dried Ahx-peptide-resins (0.1
mmol) were carried out manually using 8 fold molar excess of acryloyl chloride (0.8 mmol)
and 16 fold molar excess of DIPEA (1.6 mmol) in anhydrous DMF (4 ml) under nitrogen for
1 hour and the reaction monitored using the TNBSA test.
6
The assembled acryloyl-Ahx-peptides were cleaved from the resin support and side
chain deprotected using a mixture containing TFA/H2O/TIPS/phenol (92.5:2.5:2.5:2.5;
v/v/v/v; 5 ml) for 2 hours. The TFA/peptide solution was collected by filtration and
evaporated to approximately 1 mL under a stream of nitrogen at room temperature and the
crude peptides precipitated in ice cold diethyl ether, centrifuged (4,000g), washed with
diethyl ether (3 x 40ml) and dried under reduced pressure.
The dried crude peptides were then dissolved in 0.1% (v/v) TFA in milliQ water and
purified using a semi-preparative ZORBAX 300 SB-C18 column (9.4 mm x 25 cm) installed
in a High Performance Liquid Chromatography (HPLC) 1100 system (Agilent Technologies
Pty. Ltd., Victoria, Australia) under a flow rate of 4 ml/min using buffer A (0.1% v/v TFA in
milliQ water) and buffer B (0.1% TFA in 90% acetonitrile, 10% milliQ water, v/v) as the
limiting solvent. Peptide detection was performed by absorbance at 214 nm. Fractions were
collected and identified using electrospray mass spectrometry (Esquire LC-MS, Bruker
Daltonics, NSW, Australia).
Polymerization of N-acryloyl-peptides
N-acryloyl-Ahx-(Lys)4-PAS1K was polymerised singularly and with equimole equivalents of
either N-acryloyl-Ahx-(Lys)4-C3d, N-acryloyl-Ahx-(Lys)4-[C3d]2 or N-acryloyl-Ahx-(Lys)4[C3d]3 in the presence of 50 mole equivalents of acrylamide as a co-monomer to produce the
peptide polymers; poly-PAS1K, poly-PAS1K+C3d, poly-PAS1K+[C3d]2 and polyPAS1K+[C3d]3, respectively. Monomers (acryloyl-peptides and acrylamide) were dissolved
in de-gassed polymerisation buffer (6 M guanidine-HCl containing 2 mM EDTA and 0.5 M
Tris buffer, pH 8.) to yield a total monomer concentration of 2.5% w/v. Polymerization was
initiated at room temperature and under nitrogen by the addition of 4% (w/w) ammonium
7
persulfate of the molar concentration of monomers present and 5 μL of a 20% v/v TEMED
solution in polymerization buffer.
After 18 hours the peptide polymers were dissolved in 50 mM ammonium bicarbonate
and buffer exchanged using a Vivaspin 20 membrane column (GE Healthcare Life Sciences,
NSW, Australia) at 8000 x g. The polymers were then purified via Gel Permeation
Chromatography (GPC) using a Superdex 200 column (1.6 x 60 cm, GE Healthcare Life
Sciences, NSW, Australia) installed in a 1100 HPLC system (Agilent Technologies Pty. Ltd.,
Victoria, Australia) with a flow rate of 0.5 ml/min, using an isocratic gradient of 50 mM
ammonium bicarbonate as the running buffer. Detection was performed by UV absorbance at
214 nm. Isolated peptide polymers (>95% purity) were lyophilized using the Christ Freeze
Dryer (Osterode am Harz, Germany) and stored at -20°C.
Immunisation Protocols
Animal experimentation was approved by the University of Melbourne animal ethics
committee. For the T-cell activation studies, BALB/c mice were immunised with 60μg of
acryloyl-Ahx-(Lys)4-PAS1K peptide or 50μg of RgpA-Kgp complex (prepared as described
by Pathirana et al. (Pathirana et al. 2006) in complete Freund’s adjuvant (CFA) in the hock of
the hind legs. In further experiments BALB/c mice were immunised with 50μg of peptide or
peptide polymer antigens: acryloyl-Ahx-(Lys)4-PAS1K peptide, poly-PAS1K, polyPAS1K+C3d, poly-PAS1K+[C3d]2, poly-PAS1K+[C3d]3 in CFA.
T-cell Proliferation Assays
Seven days after immunisation lymphocytes were prepared from pooled inguinal and
popliteal lymph nodes of BALB/c mice. Lymph nodes and spleens were collected in
enriched Dulbecco’s Modified Eagles Medium/Ham’s Nutrient Mixture F12 (DMEM)
8
supplemented with 10% (v/v) heat inactivated (56ºC, 30 mins) foetal bovine serum (FBS), 2
mM glutamine, 2 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 30 μg/mL gentamicin,
100 I.U./mL penicillin and 100 μg/mL streptomycin (JRH Biosciences, Parkville, Australia),
L-arginine (116 mg/mL), L-asparagine (36 mg/mL), and folic acid (6 mg/mL, Sigma,
Melbourne, Australia) and single cell suspensions made by passing the lymph nodes or
spleens through a wire mesh (200 μm).
Red blood cells were removed from the spleen cell suspension by treatment with ammonium
tris chloride buffer (17 mM Tris-HCl, 140 mM ammonium chloride in MilliQ water, pH 7.2)
for five minutes at room temperature, and then washed three times in enriched DMEM (500
g, 5 mins at room temperature, IEC Centra GP8R refrigerated centrifuge, Thermo Electron
Corporation, Melbourne, Victoria, Australia.).
Monocytes and dead cells were removed from the lymph node cell suspension using
Lympholyte®-M (Cedarlane® Laboratories Limited, Ontario, Canada) as per the
manufacturers’ instructions. Lymph node T-cells were separated using mouse CD 90
(Thy1.2) magnetic micro beads (catalogue number 130-049-101, Miltenyi Biotech, Germany)
and the AutoMACs (Miltenyi Biotech, Germany) as per the manufacturers’ instructions. Tcells were cultured in enriched DMEM at a concentration of 3 x 105 lymph node T-cells/well
in a 96 well microtitre plate (Nunc, Denmark) in the presence of syngeneic γ-irradiated (2200
Rads) spleen cells (3 x 105cells/well) together with antigen: either acryloyl-Ahx-(Lys)4PAS1K peptide or polymer antigens, poly-PAS1K, poly-PAS1K+C3d, poly-PAS1K+[C3d]2,
poly-PAS1K+[C3d]3 (concentration 80 – 0.04 nmol/mL) or RgpA-Kgp complex
(concentration 25 – 0.01 μg/well) in a total volume of 250 μL. T-cells were incubated for
four days at 37°C in an atmosphere of 5% CO2 in air. One μCi 3H-thymidine (Amersham
Pharmacia Biotech, UK) was added per well in the final 18 hours of incubation. Cells were
then lysed with cell lysis buffer (30 mM Tris-HCl, 100 mM EDTA, 1% N-Lauroylsarcosine,
9
pH 8) and harvested onto glass fibre filter mats using a Tomtec Harvester96® MACH III cell
harvester (Tomtec, Connecticut, USA). The glass fibre filter mats were then air dried, and
sealed in plastic bags containing 5 mL of Betaplate Scint (Perkin Elmer, N.S.W, Australia)
and incorporation of 3H-thymidine was measured using a Wallac Microbeta Trilux Liquid
Scintillation Counter (Perkin Elmer, N.S.W, Australia). Data is expressed as stimulatory
index (S.I.) ± SD where S.I. is calculated as the counts per minute (cpm) divided by the
negative control cpm and statistically analysed using one-way ANOVA and Dunnett’s 3T test
(SPSS for Windows, Release 6.0; SPSS)..
ELISPOT Assay
ELISPOT assay was performed using the Millipore Multiscreen 96-well filtration plates
(MAHAS450, Millipore, N.S.W, Australia). Plates were coated with anti-mouse cytokine
capture antibodies (eBiosciences, San Diego, CA, USA), specific for IL-4 (catalogue number
14-7041, clone 11B11) and interferon-gamma (IFN-γ, catalogue number 14-7312, clone R46A2), at a concentration of 4 μg/mL in 0.1 M sodium bicarbonate buffer (pH 9.5) and
incubated overnight at 4ºC.
The ELISPOT plates were then washed with Dulbecco’s PBS and blocked with
enriched DMEM for one hour at 37ºC. Lymph node T-cells and spleens were prepared as
above. Antigen-primed lymph node cells (3 x 105/well) were incubated with γ-radiated (2200
Rads) syngeneic spleen cells as a source of APCs (3 x 105cells/well) and antigen: either
RgpA-Kgp complex (5 μg/well) or acryloyl-Ahx-(Lys)4-PAS1K peptide or polymer antigens,
poly-PAS1K, poly-PAS1K+C3d, poly-PAS1K+[C3d]2, poly-PAS1K+[C3d]3 (20 nmol/well).
Plates were incubated at 37ºC in an atmosphere of 5% CO2 in air for 48 hours in a humidified
incubator. The plates were then washed with phosphate buffered saline (PBS) containing
0.05% v/v Tween 20 (PBST) three times and once with MilliQ water. Cytokine-specific
10
biotinylated antibodies (eBiosciences, San Diego, CA, USA) specific for IL-4 (catalogue
number 13-7042, clone BVD6-24G2) and IFN-γ (catalogue number 13-7311, clone XMG
1.2) were added at a concentration of 2 μg/mL in Dulbecco’s PBS/enriched DMEM (1:1, v/v)
and incubated at room temperature for two hours. Plates were washed six times with PBST
and Streptavidin-Alkaline Phosphatase conjugate (Roche, Germany) was added to the plates
at a 1:1000 dilution in Dulbecco’s PBS/enriched DMEM (1:1, v/v) and incubated for one
hour at room temperature. The plates were washed six times with PBST and three times with
PBS, and 50 μL of substrate buffer (one tablet of 5-Bromo-4-chloro-3-indolyl
phosphate/Nitro blue tetrazolium; BCIP/NBT, catalogue number B-565, Sigma-Aldrich, St
Louis, MO, USA dissolved in 10 mL MilliQ water) was added per well. Spots were allowed
to develop for 20 – 30 minutes, before stopping the reaction by washing with water. The
spots were counted using EliSpot Reader Lite (version 2.9. Autoimmun Diagnostika GmbH
Ebinger Strasse 4, Strassberg, Germany). Data is expressed as spot forming cells per million
(SFC/million) and statistically analysed using one-way ANOVA and Dunnett’s 3T test (SPSS
for Windows, Release 6.0; SPSS).
11
Results and Discussion
Synthesis and polymerisation of peptide polymers
To investigate the ability of the C3d minimal binding epitope LYNVEA (C3d) to act as a
molecular adjuvant we initially synthesised via solid phase peptide chemistry using standard
FMOC chemistry protocols the C3d peptide monomers as acryloyl-Ahx- derivatives. Based
on the reports that multi-sequential copies of C3d enhance immunity, mono- di and trisequential LYNVEA repeats were produced. These acryloyl-Ahx-[C3d]n peptides although
successfully synthesised were difficult to solubilise and the yield of purified peptides was less
than 1 mg from a 0.1 mmole scale synthesis. To address the solubility issue the peptides were
synthesised with a four lysine spacer between the acryloyl-Ahx moiety and the N-terminus of
the [C3d]n peptide. The four lysine [(Lys)4)] spacer has been used in a number of studies to
improve the solubility of peptides but also has an added benefit in that it functions as a
cathepsin B cleavage site thus enhancing antigen processing at a site removed from the
peptide epitope when it is incorporated into a macro-molecule (Rejmanova et al. 1983;
Sarobe et al. 1993). With these features of the (Lys)4 motif in mind we synthesised the [C3d]n
and PAS1K peptides as acryloyl-Ahx-(Lys)4 derivatives to enhance solubility and
antigenicity, respectively. Furthermore, acryloyl-peptide monomers that are highly soluble in
aqueous buffers were found to incorporate into polymers more readily (Jackson et al. 1997;
O'Brien-Simpson et al. 1997). Figure 1A shows the HPLC chromatogram of the crude
acryloyl-Ahx-(Lys)4-PAS1K (Ac-PAS1K) with the highlighted peak being the desired
peptide and the inserted analytical HPLC chromatogram of the purified Ac-PAS1K (observed
mass = 2312.7 Da, calculated mass = 2311.6 Da). The secondary product eluting earlier at
14.8 minutes was found to be a lysine deletion of Ac-PAS1K. The HPLC chromatogram of
the crude acryloyl-Ahx-(Lys)4-[C3d]n (Ac-[C3d]n) peptide mixture for Ac-[C3d], Ac-[C3d]2
12
and Ac-[C3d]3 are shown in Figure 1B-D and clearly show one major synthetic product
which corresponded to the desired peptide. For each sequential addition of C3d sequence
(LYNVEA) there was an increase in the retention time of the peptide (Figure 1B-D)
indicating an increasing hydrophobic nature of the minimal C3d binding sequence.
The free radical polymerisation approach using ammonium persulphate and TEMED
to initiate and activate co-polymerisation of the acryloyl-Ahx-(Lys)4-peptides and acrylamide
was used and the reaction conditions were based on earlier reports (Jackson et al. 1997;
O'Brien-Simpson et al. 1997). Similar to previous reports all of the peptide polymers eluted
in the void volume of a size exclusion Superdex 200 column, which has an exclusion
molecular weight of approximately 1.3 x 106 Da for globular structures (Figure 2A) (Jackson
et al. 1997; O'Brien-Simpson et al. 1997). Amino acid analysis of the purified peptide
polymers showed that the peptides contributed to 10-15% w/w of the polymers and the ratio
of PAS1K to C3d peptide was similar to the peptide monomer ratio at the initiation of
polymerisation (i.e 12.6% for Poly-PAS1K; 10.3% for Poly-PAS1K+[C3d] ratio 1:1.1;
14.7% for Poly-PAS1K+[C3d]2 ratio 1:0.9 and 12.8% for Poly-PAS1K+[C3d]3 ratio 1:0.9).
These incorporation rates of peptides into acrylamide polymers are similar to those found by
others {Jackson, 1997 #1231;O'Brien-Simpson, 1997 #1154}. Figure 2B shows a
representative size exclusion profile of a PAS1K and [C3d]n co-polymer (PolyPAS1K+[C3d]n) eluting in the void volume (14.8 mins) and the unreacted monomers eluting
in the inclusion volume (38-46 mins).
Although all of the acryloyl-Ahx-(Lys)4-peptides could be polymerised with a small
molecular weight co-monomer such as acrylamide none of the peptides formed polymers or
oligomers in the abscence of a small molecular weight co-monomer as determined by size
exclusion chromatography using Superdex 200 and Superdex-peptide columns to detect
peptide polymers or oligomers, respectively (data not shown). Different acryloyl/vinyl
13
monomers are known to preferentially form block co-polymers (Sandler and Karo 1992) and
steric hindrance is considered to be a major factor for monomers not forming single monomer
species polymers. Each of the acryloyl-Ahx-(Lys)4-peptides eluted at high acetonitrile (buffer
B) concentrations, indicating significant hydrophobicity (Figure 1). In the aqueous
polymerisation buffer, this high level of hydrophobicity may result in increased steric
hindrance about the acryloyl moiety, effectively suppressing free radical transfer in the
polymerization reaction (Zou et al. 1993). As a consequence, each of the peptide polymers either containing a single peptide (PAS1K) or the heteropolymeric (PAS1K+[C3d]n) - are
likely to be block co-polymers.
T cell proliferative and cytokine responses induced by the peptide polymers
A number of studies have shown that incorporation of C3d tandem repeats of either the whole
protein sequence or peptides incorporating the CD21 binding site sequence into DNA-,
recombinant protein-, or peptide- based immunogens enhances immunity to the coimmunised antigen (Bergmann-Leitner et al. 2007; Bower and Ross 2006; Lou and Kohler
1998; Servis and Lambris 1989; Tsokos et al. 1990; Zhang et al. 2011). Furthermore, Servis
and Lambris (1989) have shown that four copies of a 13 residue C3d peptide pendent from εamino groups of lysine residues in a linear peptide template was sufficient to enhance T cell
proliferation by cross-linking the CD21 receptor on the T cell and the antigen presenting cell.
Free radical polymerisation of the minimal CD21 binding sequence LYNVEA from C3d and
tandem repeats of it would incorporate into one immunogen both valency features known to
enhance immunity. Using the PAS1K+[C3d]n polymers we were able to investigate whether;
the minimal CD21 binding sequence is sufficient to enhance immunity and if copy number or
tandem repeats of LYNVEA induce a larger effect.
14
To investigate whether the peptide polymers would enhance antigen specific T cell
responses, mice were immunised with either the PAS1K peptide or the native antigen the
RgpA-Kgp complex and after 7 days the isolated lymph node T cells were stimulated in-vitro
with peptide and peptide polymers. Figure 3 shows the T cell proliferative responses of
antigen primed and antigen stimulated T cells with the typical proliferative profiles of a
maximal stimulation at a certain antigen concentration followed by inhibition of T cell
stimulation at higher antigen doses via anergy and/or apoptosis {Falk, 2000 #8519;LaSalle,
1994 #8520}. Figure 3A shows that PAS1K peptide primed T cells when incubated with
PAS1K peptide had a maximal proliferative response at an antigen concentration of 40 nmol.
Similarly, Poly-PAS1K+[C3d]2 and Poly-PAS1K+[C3d]3 both induced a maximal T cell
response at an antigen concentration of 40 nmol, with Poly-PAS1K+[C3d]2 inducing a
significantly (p<0.05) stronger T cell response compared to PAS1K peptide and PolyPAS1K+[C3d]3 stimulated T cells. Interestingly, Poly-PAS1K and Poly-PAS1K+[C3d] did
not induce a T cell response in the PAS1K primed T cells. For RgpA-Kgp complex primed T
cells Poly-PAS1K+[C3d]2 and Poly-PAS1K+[C3d]3 both induced a significantly (p<0.05)
stronger T cell response compared to that induced by the PAS1K peptide (Figure 3B).
Although Poly-PAS1K did not induce a T cell response in the RgpA-Kgp complex primed T
cells, Poly-PAS1K+[C3d] induce a T cell response at an antigen concentration of 20 nmol,
albeit, significantly (p<0.05) weaker than that induced by the other stimulating antigens.
The lack of stimulation of T cells with Poly-PAS1K and Poly-PAS1K+[C3d] may be
due to antigen processing of the PAS1K peptide from the polymer back bone, resulting in the
cleavage of the PAS1K peptide within the T cell epitope, thus reducing the amount of antigen
available. Several studies have shown that conjugation of a T cell epitope into a construct
reduces T cell stimulation as the epitope in the peptide construct is enzymatically hydroysed
during antigen processing, not necessary for free peptide, which reduces the amount of
15
stimulatory peptide presented via MHC class II to T cells {Dyrberg, 1986 #8515;Fitzmaurice,
2000 #8518;Lu, 1991 #8516;Partidos, 1992 #8517}. The C3d tandem repeat peptide
polymers Poly-PAS1K+[C3d]2 and Poly-PAS1K+[C3d]3 both induced a strong proliferative
response. Several studies have shown that tandem repeats of C3d peptides enhances
immunity to the co-immunised antigen by lowering the antigenic stimulation threshold
(Bower and Ross 2006; Haas et al. 2004; Servis and Lambris 1989; Zhang et al. 2011).
Assuming that PAS1K is processed in a similar way for all of the peptide polymers, the
inclusion of at least two tandem repeats of the minimal C3d sequence LYNVEA into the
peptide polymers rather than a single copy were sufficient to reduce the antigenic threshold
so that the available peptide (PAS1K) was more efficient at stimulating a T cell proliferative
response.
To investigate whether the C3d peptide polymers could enhance immunity in-vivo,
mice were immunised with each peptide polymer and the PAS1K peptide and the ability of
the antigen primed T cells to proliferate was evaluated using the native antigen the RgpAKgp complex in-vitro. Figure 3C shows that the PAS1K peptide and Poly-PAS1K primed T
cells had maximal proliferative responses at 3.13 and 1.56 μg/well of RgpA-Kgp complex,
respectively and that the PAS1K peptide primed T cells had a significantly (p<0.01) stronger
response compared to Poly-PAS1K primed T cells. All of the PAS1K+C3d peptide polymer
primed T cells had maximal proliferative responses at a stimulating antigen concentration of
0.02 μg/well which was significantly (p<0.05) lower concentration than that of the maximal
proliferative response by the PAS1K peptide or Poly-PAS1K-primed T cells. Furthermore,
the total T cell numbers isolated from the PAS1K+C3d peptide polymer immunised mice was
consistently 10% higher in each experiment compared to T cell numbers isolated from
PAS1K peptide and Poly-PAS1K immunised mice.
16
The ability of C3d containing peptide polymers to enhance immunity to a far greater
extent in-vivo than in-vitro may be due to the immunostimulatory effects of C3d on the
antigen presenting cells. CD21 the receptor for C3d is known to be up regulated on APCs
upon activation, thus enhancing cross-linking of APC’s and T cells via CD21/C3d ligation
and reducing the T cell activation threshold (Allison 1998). This would result in an increase
of the polydiversity of the antigen specific T cell repertoire and their proportion within the
whole T cell population. APC’s in in-vitro assays are treated chemically or with radiation to
prevent them from proliferating and generating proteins. Thus, unlike in-vivo-APC’s, invitro-APC’s would not be able to up regulate CD21 on their surface, which may account for
why the C3d containing peptide polymers were able to enhance a stronger immune response
when used to immunise rather than stimulate.
To further investigate the effects of the C3d containing peptide polymers, T cells
isolated from antigen primed mice were stimulated with antigen in-vitro and the IFNγ and IL4 cytokines secreted were analysed by ELISPOT (Figure 4). PAS1K peptide and PolyPAS1K+[C3d]3 induced a mixed IL-4 and IFNγ response in PAS1K-primed T cells, whereas
Poly-PAS1K+[C3d] and Poly-PAS1K+[C3d]2 induced a significantly (p<0.05) stronger IL-4
response (Figure 4A). In RgpA-Kgp complex primed T cells the PAS1K peptide stimulated a
mixed IL-4 and IFNγ response (Figure 4B). Poly-PAS1K induced a significantly (p<0.05)
weaker IL-4 and IFNγ response compared to that induced by the other peptide polymers or
PAS1K peptide. All of the PAS1K+C3d containing polymers induced a mixed IL-4 and IFNγ
response which was significantly (p<0.05) stronger than the response induced by the PAS1K
peptide with the RgpA-Kgp complex-primed T cells (Figure 4B). Figure 4C shows the IL-4
and IFNγ response induced by the RgpA-Kgp complex on PAS1K peptide and peptide
polymer primed T cells. The RgpA-Kgp complex induced a mixed IL-4 and IFNγ response in
PAS1K primed T cells, whereas Poly-PAS1K primed T cells had a predominant IL-4
17
cytokine response. In contrast, all of the Poly-PAS1K+[C3d]n primed T cells had a
predominantly and significantly (p<0.05) higher IFNγ response compared to the response of
PAS1K peptide or Poly-PAS1K primed T cells, indicating that C3d may promote a Th1 cell
phenotype.
C3d is known to promote and enhance both IL-4 and IFNγ responses and is regarded
as a general immunostimulatory agent (Bower and Ross 2006). However, Wang et al. (2006)
and Suradhat et al. (2001) reported that incorporation of multiple repeats of C3d molecules
into DNA based vaccines resulted in a predominant Th2 phenotype. Alternatively, Allison
(1998) and Knopf et al. (2008) demonstrated that immunising with tandem repeats of C3d
sequences in recombinant protein and peptide based vaccines induced a Th1 cell phenotype.
Furthermore, peptides containing the LYNVEA C3d sequence have been shown to directly
interact with the T cell receptor inducing secretion of IFNγ from T cells (Knopf et al. 2008).
When used as stimulating antigens in-vitro, C3d containing peptide polymers tend to promote
a mixed Th1/Th2 response indicating a general immunostimulatory role of C3d in the
polymers. When used as immunising antigens, however, there is a strong promotion of a Th1
phenotype, which is consistent with the response when immunising with protein/peptide
based vaccines containing C3d sequences (Allison 1998; Knopf et al. 2008). The ability of
Poly-PAS1K and Poly-PAS1K+C3d to induce cytokine secretion in PAS1K primed and
RgpA-Kgp complex primed T cells, while inducing weak or undectable proliferation, could
be attributed to the incubation time and level of sensitivity of each assay. For certain
antigens ELISPOT assays have been shown to be more sensitive compared to a 3H-thymidine
assay and at the point when 3H-thymidine is added to detect proliferation, the response
induced by weakly stimulating antigens is typically poorly detected (Macatangay et al. 2010;
Schultes and Whiteside 2003; Troye-Blomberg et al. 1984). Furthermore, T cell cytokine
secretion can also be independent of proliferation (Jung et al. 1999). These may in part
18
explain the differences in the cytokine and proliferative responses induced when PolyPAS1K and Poly-PAS1K+C3d are used as stimulating antigens in-vitro. According to the
results presented here, however, peptide polymers used to immunise mice promoted strong T
cell proliferative and cytokine secretion responses. When used to immunise mice PolyPAS1K induced a weaker proliferative response than peptide alone indicating that the PAS1K
T cell epitope may not be as biologically available as PAS1K peptide. The reduced
stimulation of T cells by the inclusion of PAS1K into a polymer was overcome by inclusion
of monomeric or tandem repeats of the C3d sequence LYNVEA, which improved the
magnitude of the T cell response and reduced the activation threshold compared to that
induced by peptide polymer without C3d.
19
Concluding Remarks
In conclusion, we have demonstrated that acryloyl-peptides and acryloyl-derivatised
immuno-stimulatory C3d peptide, can be co-polymerised to yield high molecular weight
antigens. We have shown that valency of C3d in terms of tandem repeats but also copy
number in a peptide polymer is important to enhance the immune response to the coimmunised antigen. Previous studies have included the minimal CD21 binding domain of
C3d (LYNVEA) as part of a larger sequence, however, our data has shown that incorporation
of just the minimal C3d binding residues is sufficient to enhance immunity and that the effect
is more pronounced when C3d is part of the immunising antigen rather than an in-vitro
stimulating antigen. Our finding that the 6 residue sequence LYNVEA from C3d is sufficient
to enhance immunity to a co-immunised antigen may further the adoption of C3d as an
immunostimulatory component in vaccine formulations.
20
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Figure Legends
Figure 1:
Analytical Reverse phase-HPLC profiles of synthetic acryloyl-peptides. Reversed phase
HPLC (RP-HPLC) profile of crude acryloyl-peptides with the corresponding analytical RPHPLC of the purified acryloyl-peptide and ES-MS observed mass (insert) of; (A) acryloylPAS1K, expected molecular weight [m/z 2312.6 (M+H)+], (B) acryloyl-C3d, expected
molecular weight [m/z 1388.7 (M+H)+], (C) acryloyl-[C3d]2, expected molecular weight [m/z
2078.5 (M+H)+], and (D) acryloyl-[C3d]3, expected molecular weight m/z 2768.2 (M+H)+].
RP-HPLC was performed using a semi-preparative Zorbax 300 SB-C18 column installed in a
HPLC system. Peptide separation was achieved using 0-100% buffer B (0.1% TFA in 90%
acetonitrile, 10% milliQ water, v/v) gradient over a period of 30 min at a flow rate of 4.0
ml/min.
Figure 2: Size exclusion chromatography profiles of crude synthetic polymers. (A) Size
exclusion chromatography profile of Poly-PAS1K. Elution times of the standards,
Thyroglobulin (A, MW: 669kDa), Ferritin (B, MW: 440kDa) and Catalase (C, MW: 232kDa)
are indicated by vertical arrows. (B) Size exclusion chromatography profile of PolyPAS1K+[C3d]3, collected from time 15.0-17.0 min as indicated by the vertical lines. Size
exclusion chromatography was performed using a Superdex 200 column installed in a HPLC
system. Separation was based on molecular size using an isocratic gradient of 50 mM
ammonium bicarbonate as the running buffer at a flow rate of 0.5 ml/min. Detection was
performed by absorbance at 214 nm.
27
Figure 3: T-cell proliferative responses induced by peptide polymers. T-cells were
isolated from the popilteal and inguinal lymph nodes of BALB/c seven days after
immunisation with antigen (peptide, peptide-polymer or the RgpA-Kgp complex) and then
re-stimulated in-vitro to determine T cell proliferative response. T-cells were isolated from
PAS1K peptide (A) and RgpA-Kgp complex (B) immunised mice and were incubated with γirradiated syngeneic spleen cells and either PAS1K peptide () or peptide polymer; PolyPAS1K (), Poly-PAS1K+C3d (), Poly-PAS1K+[C3d]2 () or Poly-PAS1K+[C3d]3 (),
concentration of the stimulating antigen was based on PAS1K. (C), T-cells isolated from
antigen (PAS1K peptide () or peptide polymer; Poly-PAS1K (), Poly-PAS1K+C3d (),
Poly-PAS1K+[C3d]2 () or Poly-PAS1K+[C3d]3 ()) immunised mice and were incubated
with γ-irradiated syngeneic spleen cells and the native antigen the RgpA-Kgp complex. T-cell
proliferation was measured using 3H-thymidine incorporation for the last 18 hours of the
assay and is expressed as the stimulatory index (S.I.). Data is representative of duplicate
assays ± SD.
Figure 4: T-cell cytokine responses induced by peptide polymers. T-cells were isolated
from the popilteal and inguinal lymph nodes of BALB/c seven days after immunisation with
antigen (peptide, peptide-polymer or the RgpA-Kgp complex) and then re-stimulated in-vitro
to determine the antigen-specific IL-4 and IFNγ secretion response. T-cells were isolated
from the PAS1K peptide (A) and RgpA-Kgp complex (B) immunised mice and were
incubated with γ-irradiated syngeneic spleen cells and either PAS1K peptide or peptide
polymer (Poly-PAS1K, Poly-PAS1K+C3d, Poly-PAS1K+[C3d]2 or Poly-PAS1K+[C3d]3),
concentration of the stimulating antigen was based on PAS1K. (C), T-cells isolated from
antigen (PAS1K peptide or peptide polymer; Poly-PAS1K, Poly-PAS1K+C3d, PolyPAS1K+[C3d]2 or Poly-PAS1K+[C3d]3 immunised mice and were incubated with γ-
28
irradiated syngeneic spleen cells and the native antigen the RgpA-Kgp complex. IL-4 and
IFNγ T-cell cytokine responses were determined after 2 days of incubation. Data is expressed
as spot forming cells per million (SFC) ± SD minus the background, and is representative of
triplicate or duplicate assays.
29