Download A recombinant human HLA-class I antigen linked to dextran elicits

Journal of Immunological Methods 360 (2010) 1–9
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Journal of Immunological Methods
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Review
A recombinant human HLA-class I antigen linked to dextran elicits innate
and adaptive immune responses
Jorgen Schøller a, Mahavir Singh b, Lesley Bergmeier c, Katja Brunstedt a, Yufei Wang c,
Trevor Whittall c, Durdana Rahman c, J. Pido-Lopez c, T. Lehner c,⁎
a
b
c
Immudex, Copenhagen, Denmark
Lionex GmbH and Helmholtx Center for Infection Research, 38124 Braunschweig, Germany
Kings College London, Mucosal Immunology Unit, Guy's Hospital, London, England, United Kingdom
a r t i c l e
i n f o
Article history:
Received 23 February 2010
Received in revised form 18 May 2010
Accepted 25 May 2010
Available online 10 June 2010
Keywords:
HSP
Dendritic cells
HIV
SIV
a b s t r a c t
The objective of this study was to produce and evaluate the immunogenic potential of a
recombinant HLA-class I antigen linked to dextran. The HLA-A*0201 heavy chain and β2
microglobulin were cloned by PCR amplification of overlapping oligonucleotides and produced
in E. coli. These were assembled with a CMV binding peptide motif, the HLA complex was
biotinylated and bound by streptavidin coated dextran at a ratio of 24 HLA to 1 dextran molecule
(termed Dextramer). Allostimulation of human PBMC in vitro and in vivo immunization of Balb c
mice with the HLA-A*0201 construct elicited CD4+ and CD8+ T cell proliferative responses, IgG
specific antibodies in mice and in human T cell proliferation and APOBEC3G mRNA. These
adaptive and innate immune responses induced by a novel recombinant HLA construct in
human cells and mice suggest their application as a potential vaccine candidate against HIV
infection.
Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Cloning and production of HLA-A*0201 heavy chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Cloning and production of human β2 microglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Assembling the HLA complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Construction of expression vectors and production of biotinylated C-terminal peptide of M. tuberculosis HSP70 (HSP70359–608)
2.5.
Preparation of the Dextramer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.
Immunization of mice with the Dextramer and HIVgp120 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.
Mouse T cell proliferative assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8.
Antibody analysis by ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9.
Human T cell proliferative assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.10. APOBEC3G mRNA assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: A3G, APOBEC3G;CMV, cytomegalovirus;DC, dendritic cells;FITC, fluorescein isothiocyanate;HIV, human immunodeficiency virus;HLA, human
leukocyte antigens;SIV, simian immunodeficiency virus;HSP, heat shock protein;TH1, T helper 1.
⁎ Corresponding author. Kings College London, Mucosal Immunology Unit, Tower Wing Floor 28, Guy's Hospital, London SE1 9RT, England, United Kingdom.
Tel.: +44 207188 3072; fax: +44 207188 4375.
E-mail address: [email protected] (T. Lehner).
0022-1759/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2010.05.008
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J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
3.
4.
Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Stimulation of human T cell proliferation with the Dextramer .
4.2.
Stimulation of A3GmRNA. . . . . . . . . . . . . . . . . .
4.3.
Splenic T cell proliferative responses in mice immunized with
4.4.
IgG antibodies to HIVgp120 and to HLA-A*02 Dextramer . . .
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Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Immunization of macaques with SIV grown in human CD4+
T cells (Desrosiers et al., 1989; Murphey-Corb et al., 1989; Stott
et al., 1990; Carlson et al., 1990; Hunsmann et al., 1995;
Biberfeld and Putkonen, 1995) or with the cells alone (Stott,
1991; Stott et al., 1994; Arthur et al., 1995; Chan et al., 1995)
yielded about 85% and 55% protection against SIV infection,
respectively. The protection was dependent on HLA antigens
acquired by the virions in the process of budding through the
human CD4+ T cell membrane in which the SIV was grown.
Despite the reproducibility of preventing SIV infection in
macaques, this approach was abandoned largely because of
the potential adverse effects of immunization with HLA+ cells,
and using CD4+ T cell lines propagated with oncogenic viruses.
There is also evidence in humans that alloimmune
responses can prevent HIV-1 infection. This was demonstrated in vitro by inducing cytotoxic lymphocytes and soluble
factors (Shearer et al., 1993). Systemic in vivo alloimmunization of women revealed that HIV-1 replication in CD4+ T cells
ex vivo is inhibited (Wang et al., 1999). Epidemiological
evidence suggests that transmission of HIV from mother to
baby occurs more frequently among uniparous women and
mother–child HLA-class I concordance increases perinatal
HIV-1 transmission (McDonald et al., 1998). Furthermore,
sera from multiparous women may contain alloantibodies
and CCR5 antibodies which inhibit ex vivo HIV-1 replication
(Wang et al., 2002a). Indeed, alloimmunization has been
proposed as a strategy for inducing immune protection
against HIV infection (Lehner et al., 2000).
The objective of this study was to evaluate the immunogenic potential of recombinant HLA-class I antigen (A*0201)
linked by the biotin–streptavidin method to dextran (Dextramer) and to use HSP70 as an adjuvant to elicit immune
responses. Immunization of Balb c mice with the HLA-A*0201
Dextramer construct elicited an increase in both CD4+ and
CD8+ T cell proliferation and IgG antibodies to the HLAA*0201 dextramer. Human CD4+ and CD8+ T cell proliferative responses and an innate immune response were induced
in vitro by the Dextramer stimulating upregulation of
APOBEC3G mRNA in human CD4+ T cells.
2. Materials and methods
2.1. Cloning and production of HLA-A*0201 heavy chain
The human MHC-class 1 HLA-A*0201 coding sequence
minus the signal peptide and transmembrane regions was
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HLA-A*02
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HIVgp120
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Dextramer construct
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obtained from GenBank (acc # M84379) by backtranslation.
Codon usage was optimized to E. coli using www.entelechon.
com before the gene was synthesized by PCR using 10
overlapping DNA primers (DNA Technology, Denmark) and
KOD polymerase (EMD Chemicals, Novagen). The sequence
was verified by repeated DNA sequencing (MWG Biotech,
Ebersberg, Germany) and base errors introduced by the PCR
were corrected using Quick Change multi site-directed
mutagenesis kit (Stratagene, La Jolla, CA) before cloning in
pGarboczi (Garboczi et al., 1992).
Recombinant HLA-A*0201 was produced by E. coli batch
fermentation. Bacteria were harvested by centrifugation and
resuspended in ice cold buffer (50 mM Tris–HCl pH 8.3, and
150 mM NaCl). Proteinase inhibitor AEBSF was added to a
final concentration of 0.5 mM before lyses of the cells by cell
disruption. Inclusion bodies were isolated from the cell lysate
by centrifugation (20 g rpm/4 °C) and washed thoroughly
three times in ice cold buffer (2 M urea, 2% Triton X-100,
0.5 M NaCl, and 20 mM Tris–HCl pH 8.0). After the final wash
the inclusion body pellet was resuspended 8 M Urea, 150 mM
NaCl, and 20 mM Tris–HCl pH 8.0). Undissolved material was
removed by centrifugation and the supernatant was filtered
through 0.2 μm filter before loading on to an ion exchange
column (Q Sepharose fast flow). The HLA heavy chain was
eluted by a 0–100% gradient of 8 M Urea, 500 mM NaCl, and
20 mM Tris–HCl pH 8.0. Relevant fractions were identified by
SDS-PAGE and concentrated.
2.2. Cloning and production of human β2 microglobulin
Cloned human β2 microglobulin (GenBank acc #
CAG33347.1) was a kind gift from L. Østergaard (Danish Cancer
Society). Recombinant β2M was produced by E. coli batch
fermentation. Bacteria were harvested by centrifugation and
resuspended in ice cold buffer (50 mM Tris–HCl pH 8.3, and
150 mM NaCl). Proteinase inhibitor AEBSF (Sigma, St. Louis)
was added to a final concentration of 0.5 mM before lyses of the
cells by cell disruption. Inclusion bodies were isolated as
described above for the heavy chain. Undissolved material was
removed by centrifugation and the supernatant was filtered
through 0.2 μm filter before loading on to an IMAC column. The
β2M preparation was then washed in 125 mM NaCl, and
20 mM Tris–HCl pH 8.0 before elution by an imidazole gradient
(0–100%, 500 mM imidazole, 125 mM NaCl, and 20 mM Tris–
HCl pH 8.0). The relevant β2M fractions were identified by SDSPAGE, sterile filtered and concentrated before gel filtration
(column Hi-Load 26/60 Superdex 75) was performed to isolate
the monomeric β2M from dimeric and multimeric forms.
J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
3
Fig. 1. (A) Elution profile of folded MHC complex HLA-A*0201(NLVPMVATV). Absorbance at 280 nm (solid line), conductivity (dotted line). 2.5 ml fractions are
indicated on the X-axis. Fractions 86–89 were isolated. (B) SDS-PAGE of the folded and purified HLA-A*0201 peptide complex used for vaccine preparation.
(C) SDS gel of HSP70359–609 and anti-his blot with mouse IgG HRP *Pep3 ± HSP359–608.
2.3. Assembling the HLA complex
The HLA-class 1 complex is a trimeric complex consisting
of the HLA heavy chain, β2M, and a peptide with an amino
acid sequence corresponding to the binding motif of the HLA
molecule. The peptide used was the CMV epitope
NLVPMVATV and N95% pure peptide was obtained from
Neosystems (France). Assembly of the complex was performed at 10 °C by continuous mixing in refolding buffer,
adjusted to 0.1 mM DTT, and containing the protease
inhibitors, PMSF 1 mM, Pepstatin A (20 μg/ml) and Leupeptin
(20 μg/ml). The folded MHC complex was biotinylated using
d-biotin, Bir-A enzyme in the presence of Pepstatin A and
Leupeptin. The resulting folded and biotinylated HLA complex was isolated from excess biotin, β2M and aggregated
heavy chain by gel filtration on Superdex using 50 mM NaCl,
and 20 mM Tris pH 8.0 as elution buffer. The elution profile
(Fig. 1A) clearly distinguishes the folded MHC complex. SDSPAGE of MHC complex elution peak shows the HLA heavy
chain and β2M bands. The bound peptide is too small to be
seen on the gel (Fig. 1B). Correct assembly was verified by a
conformational ELISA using the conformational sensitive
mouse anti-HLA ABC monoclonal W6/32 antibody (Dako
MO736) as primary catchment antibody. Maxisorb ELISA
plates (Nunc, Denmark) were coated with 5 μg/ml of W6/32
overnight, at 2–4 °C and blocked using 1% skimmed milk
powder. The folded MHC was diluted to approx 0.3 mg/ml
before application in two-fold serial dilutions. As positive
control the human myeloid cell line KG-1 was used. Negative
control was the THT ELISA assay buffer (100 mM NaCl, 50 mM
Tris pH 7.2, and 0.01% Bronidox). Secondary rabbit anti-β2M
antibody (Dako P0174) was used to detect binding of the HLA
complex. Amplification was performed using HRP conjugated
polyclonal goat-anti-rabbit immunoglobulin. The ELISA assay
(Table 1) confirms correct folding of the MHC complex with
an absorbance value for the undiluted complex in five-fold
excess of the positive control cell absorbance. The complex
stays together as the tri-molecular complex with the peptide
bound in the MHC grove is inherently stable, which is
enhanced by conjugation to the dextran backbone (Zhu
et al., 2010).
Table 1
MHC ELISA folding assay. Two-fold serial dilution of folded MHC was applied
in duplicate. 1×THT buffer shows background staining. The positive control
was of MHC-class 1 expressing KG-1 human myeloma cells.
Sample
HLA-A*0201(NLVPMVATV)
Undiluted
1:2
1:4
1:8
1:16
1:32
1:64
Controls
0.593
0.510
0.476
0.249
0.272
0.170
0.108
1×THT
0.041
0.513
0.554
0.453
0.405
0.303
0.184
0.118
Positive control
0.110
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J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
2.4. Construction of expression vectors and production
of biotinylated C-terminal peptide of M. tuberculosis
HSP70 (HSP70359–608)
known number of molecules of SA was mixed with a known
number of molecules of dextran in the conjugation process.
The conjugate was then purified on a Superdex 200, Hi-Load
16/60 and the fractions were collected. All SA-coated dextran
was isolated and the bound and unbound amount was
measured. The percentage of bound SA was then determined
and the average number of SA per dextran was calculated. The
number of MHC was then determined by the available dextran
backbone which on average showed 8 streptavidin binding
sites per dextran molecule. For each SA site a maximum of
three biotinylated MHC molecules were bound due to steric
constraint. Thus, 24 MHC molecules was the optimum number
per dextran. For the multimeric HLA/HSP70 Dextramer
equimolar amounts of HLA and HSP70 was attached resulting
in a molar ratio of 12/12/1 HLA complex/HSP70/dextran. The
attachment of HLA complexes and HSP70 to the dextran
backbone was verified by solid-phase ELISA using again the
conformational sensitive mouse anti-HLA ABC monoclonal
W6/32 (Dako MO736) described above or anti-HSP70 as
catchment antibody. In both cases secondary rabbit anti FITC
(Dako P5100) was used to detect binding of the dextramer
construct. Amplification was performed using HRP conjugated
polyclonal goat-anti-rabbit immunoglobulin. The recombinant HIVgp120 (IIIB, EVA607) (NIBSC, Potters Bar, UK) was
conjugated to HSP70 using N-succinimidyl(3[2-pyridyl]-ditio
(SPDP) as previously described (Bogers et al., 2004).
To enable in vivo biotinylation of the C-terminal peptide of
M. tuberculosis HSP70 (aa 359–608) was subcloned. A primer
of 76 bases for PCR was designed that allows fusion of the
recognition site for in vivo biotinylation in E. coli to the Cterminal end of HSP70(359–608). Together with a forward
cloning primer the HSP70(359–608) fusion gene was amplified
and after restriction with NdeI and XhoI inserted into the
expression vector pET26. The resulting plasmid pLEXWO1674 was transformed into E. coli BL21 (DE3). The insert of
plasmid pLEXWO167-4 was confirmed by sequencing.
For rapid purification metal chelate chromatography
biotinylated HSP70(359–609) was fused to a 6-fold N-terminal
histidine tag. HSP70(359–609) gene was excised from plasmid
pLEXWO167-4 using restriction endonucleases NdeI and XhoI
and inserted into pET28 which provides the sequence for the
N-terminal histidine tag. The resulting plasmid pLEXWO1754 was transformed into E. coli BL21 (DE3). This strain was
used for production of biomass of biotinylated HSP70(359–608)
with N-terminal histidine tag.
The cell mass was suspended in 20 mM tris, 100 mM NaCl,
and 10 mM imidazole, pH 8.0 and then disrupted by homogenization and sonication at 4 °C. After centrifugation the clear
supernatant was applied on Ni-NTA resin (Qiagen, Hilden).
After washing the bound protein was eluted in a linear gradient
from 20 mM tris, 100 mM NaCl, and 10 mM imidazole, pH 8.0 to
20 mM tris, 100 mM NaCl, and 500 mM imidazole, pH 8.0.
Fractions containing the target protein were pooled, diluted
tenfold with 20 mM tris, pH 8.0 and applied on a Q Sepharose
High Perfomance resin (GE Healthcare). After washing the
protein was eluted in a linear gradient from 20 mM tris, pH 8.0
to 20 mM tris, and 1 M NaCl, pH 8.0. Fractions containing the
pure target protein were pooled and underwent the final buffer
exchange step. The protein was applied on a Sephadex G25 fine
resin (GE Healthcare) and eluted with PBS, pH 7.4. The purified
N-his HSP359–608-bio was subjected to final quality control and
a Western blot is shown in Fig. 1C. The protein pool was filter
sterilized through 0.2 μM PES filters, aliquoted and stored
frozen at −26 to −28 °C.
Animals were housed according to UK Home Office guidelines for animal experimentation. Three groups of 6 mice/group
of 6 week old Balb C mice were immunized subcutaneously in
the base of the tail with 10 or 20 μg/mouse at day 0 and 21, and
an unimmunized control group presented in Fig. 4C. Group 1
received 10 μg of Dextramer-HSP70 mixed with 20 μg of
HIVgp120 chemically conjugated to HSP70; Group 2 received
20 μg of HIVgp120 conjugated to HSP70; and Group 3 received
10 μg of dextran per mouse. Blood samples were taken from the
tail before and 10 days after the second immunization. The
animals were killed by cervical dislocation 10 days after the
second immunization. Spleens were removed for T cell
proliferative studies.
2.5. Preparation of the Dextramer
2.7. Mouse T cell proliferative assay
The dextramer backbone consisting of a 270 kDa dextran
(Pharmacosmos, Denmark) was activated by divinyl sulfone
acid by incubation of 1 g dextran and 5 g divinyl sulfone acid
in 0.2 M Na2CO3 for 20 min with heavy stirring. Reaction was
stopped by concentrated acetic acid and then thoroughly
dialyzed against water (Lihme and Boenisch, 1994). Strepavidin in heavy excess was reacted with the activated dextran
in the presence of a minor amount of FITC for 3 h resulting in
8% incorporation of streptavidin. Biotinylated HLA complex
alone or together with biotinylated HSP70 molecules was
bound to a divinyl sulfone acid activated Streptavidin coated
270 kDa dextran-FITC backbone (Lihme and Boenisch, 1994).
For the present study a Dextramer with the molar ratio of 24
HLA molecules to 1 dextran was used. The number of
streptavidin (SA) per dextran molecule was determined
after the conjugation of SA to the activated dextran. A
The CFSE (carboxyfluorescein diacetate succinimidyl ester)
cell labelling method of dye dilution, which is halved with each
cell division has been used for both the murine and human
lymphocyte proliferation assays. Mouse spleens were collected
and teased apart to release splenocytes. The cell suspension was
filtered through BD Falcon 100 μm Nylon cell strainer (BD
Biosciences) to remove the remaining tissues. The cells were then
washed and resuspended in RPMI 1640 medium supplemented
with 10% FCS, 100 μg/ml of penicillin and streptomycin, and
2 mM glutamine. 20×107 of the splenocytes were washed once
with PBS containing 1% BSA and cell pellets were resuspended in
1 ml of PBS and labelled with CFSE (Molecular Probes, Invitrogen) according to the manufacturer's instruction. CFSE-labelled
cells were washed twice, resuspended in the culture medium and
3×105 cells in 100 μl were plated into 96 well plates. Two to
three concentrations of HSP70, HLA-dextramer, 5 μg/ml of Con A
2.6. Immunization of mice with the Dextramer and HIVgp120
J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
as positive control and 10 μg/ml of BSA as negative control were
added to the cultures. After 7 days of incubation, proliferation
was assayed by flow cytometry (BD Canto II) and proliferation
was expressed as percent proliferated cells of the total
population. Proliferated CD4+ and CD8+ T cells were assayed
by labelling cells with PE conjugated antibodies to murine CD4
and CD8 (Immunotool, UK).
5
2.8. Antibody analysis by ELISA
A solid-phase enzyme-linked immunosorbent assay
(ELISA) method was used for the detection of serum IgG
antibodies to the Dextramer and HIVgp120. Microtitre plates
(Dynatech M 129B, Billingshurst, Kent, U.K.) were coated
with 1 μg/ml of Dextramer or 1 μg/ml HIVgp120 (100 μl per
Fig. 2. (A) Stimulation of human PBMC, CD4+ and CD8+ human T cell proliferation stimulated by HLA-A*02 Dextramers, which was compared with irradiated allogeneic
cells and presented as mean (±sem) (n= 3). (B) Gating of the lymphocytes examined (P1) and double staining for CD4+ and CD8+ T cells. (C) Representative flow
cytometry of proliferation of CD4+ and (D) CD8+ T cells in response to the Dextramer without or with HSP70, dextran, HSP70 alone or allogeneic stimulation.
6
J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
well) and then incubated overnight at 4 °C. The plates were
washed ×3 in PBS (pH 7.4) and blocked by adding 200 μl of
PBS/BSA/T20 at 37 °C for 1 h. Dilutions of samples in
duplicates starting from 1:50 to 1:64,000 (100 μl per well)
were added to the plates and incubated at 37 °C for 2 h after
which the plates were washed (3×) with PBS. 100 μl/well
goat-anti-mouse IgG alkaline phosphatase conjugate (1:2000
Sigma) in PBS/BSA/T20 was added, the plates were incubated
for 2 h at 37 °C and then washed twice in PBS and once in
distilled water. One tablet of p-nitrophenol was dissolved in
5 ml of diethanolamine buffer and 100 μl was added to each
well. When the colour had developed the reaction was
stopped by the addition of 50 μl per well of 3 M sodium
hydroxide and read at 405 nm in an ELISA reader (Anthos
2001 Labtec International UK).
2.9. Human T cell proliferative assay
Dextran, Dextramer, Dextramer linked to HSP70, each at
10 μg per ml, or 104 irradiated allogeneic monocytes were
incubated with 105 CFSE-labelled human peripheral blood
mononuclear cells (PBMC) for seven days, avoiding HLAA*0201 cells. The cells were stained with PE anti-CD8 and
APC-Cy7 anti-CD4 and analysed by flow cytometry using a BD
FACSCanto II flow cytometer and FACSDiva software. Proliferation was determined by measuring the percentage of
PBMC, CD4 T cells, or CD8 T cells with reduced CFSE content.
2.10. APOBEC3G mRNA assay
A3G mRNA expression by 1 × 106 CD4+ MACS isolated cells
was measured by real-time PCR following an 18 hour stimu-
lation with medium alone or different dilutions (1:5, 1:10 or
1:20) of either dextramer, dextran, dextramer linked to
HSP70(359–608) or HSP70(359–608) alone as previously described
(Pido-Lopez et al., 2007). The 18 hour stimulation period was
determined after undertaking a time-course study. In addition
A3G mRNA was measured following allogeneic stimulation of
1 × 106 CD4+ cells with HLA mismatched 1 × 106 C8166 T cells.
A3G was calculated as fold increase in relation to unstimulated
(medium alone) A3G levels.
3. Theory
The rationale of linking recombinant HLA-class I molecules
and HSP70 to a dextran backbone is to enable stable attachment
of a number of protein molecules in proximity by using the
biotin–streptavidin technique. The linked construct may be
more effective in inducing innate and adaptive immune
responses by two or more antigens than when used separately.
4. Results
4.1. Stimulation of human T cell proliferation with the Dextramer
Human CD4+ T cell proliferation stimulated in vitro by the
Dextramer construct was significantly increased from mean
(±sem) 3.4(±1.6) to 7.7(±4.5)% and further enhanced
when linked to HSP70 (11.0 ± 2.6%), which is not significantly
different from proliferation in response to irradiated allogeneic cells (18.4 ± 2.2%; Fig. 2A). PBMC yielded similar results.
However, human CD8+ T cell proliferation stimulated in vitro
by the Dextramer was lower (4.6 ± 3.4%) than that of CD4+ T
cells and when the Dextramer was linked to HSP70 it failed to
Fig. 3. Innate anti-HIV factor — APOBEC3G mRNA elicited by stimulating human CD4+ T cells with the Dextramer (HLA-A*0201) alone or linked with HSP70,
Dextran, HSP70 or allogeneic human CD4+ T cells (n = 8).
J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
enhance proliferation to the same level as that stimulated
with allogeneic cells (Fig. 2A). Representative flow cytometry
is illustrated in Fig. 2B. The positive control, concanavalin A
was consistently high (71.2–96.4%; data not presented) and
the negative control, without any stimulant was low (1.7–
14.8) and subtracted from the above values.
4.2. Stimulation of A3GmRNA
Stimulation of human CD4+ T cells with the Dextramer
preparations clearly demonstrated a significant increase in
A3G mRNA expression induced by the Dextramer linked to
HSP70 at 1:5, 1:10 and 1:20 dilution (Fig. 3), compared with
Dextramer alone (p = 0.02). However, stimulation of CD4+ T
cells with Dextran alone failed to increase A3G mRNA,
whereas HSP70 alone induced a 4.3 ± 0.7-fold increase in
A3G mRNA. Allogeneic stimulation with irradiated CD4+ T
cells (C8166 cell line) induced 10.7(±2.7)-fold increase in
mRNA, which compares favourably with 9.0-10.5 fold
increase with the Dextramer linked to HSP70 (Fig. 3).
4.3. Splenic T cell proliferative responses in mice immunized
with the HLA-A*02 and HIVgp120 Dextramer construct
Immunization with Dextramer linked to HSP70 yielded the
highest CD4+ T cell proliferative response to the Dextramer
7
(27.9%) but also high responses when stimulated with
Dextran (16.1%) or HSP70 (19.9%) (Fig. 4A). Immunization
with Dextramer alone also yielded a high response to the
Dextramer (21%) and Dextran (13.2%) but no response to
HSP70. CD8+ T cell proliferation was also maximal when
immunized with the Dextramer linked to HSP70 (27.9%), with
no increase in the CD8+ T cells of Dextramer immunized mice
(Fig. 4B). Thus, Dextramer linked with HSP70 resulted in the
highest CD4+ and CD8+ T cell proliferative responses to the
Dextramer which, however, also yielded significant responses
to Dextran and HSP70. Immunization with HIVgp120 linked to
HSP70 elicited 5.4(±2.7)% CD4+ T cell proliferation (Fig. 4C),
which was greatly enhanced when mixed with DextramerHSP70 (14.7 ± 7.0). However, the possibility that some of
the proliferative response may have been due to the
immunogenicity of streptavidin cannot be excluded, though
the low CD8+ T cell proliferation with Dextramer alone makes
this less likely (Fig. 4A). Negligible Dextramer or HIVgp120
stimulated proliferation was found in the Dextran immunized
or unimmunized control animals (Fig. 4A,B).
4.4. IgG antibodies to HIVgp120 and to HLA-A*02 Dextramer
High IgG antibody titres were elicited to HIVgp120 in mice
immunized with the Dextramer, linked to HSP70 and mixed
with HIVgp120-HSP70 (9600 ± 1848) (Fig. 4C). A low antibody
Fig. 4. (A) CD4+ and (B) CD8+ T cell proliferative responses to dextran, Dextramer (HLA-A*0201) and HSP70 in Balb c mice immunized with the Dextramer (HLAA*0201) and covalently linked with HSP70. (C) CD4+ T cell proliferation stimulated by HIVgp120, antibodies to HIVgp120 and the Dextramer (HLA-A*02) were
measured by ELISA and the results were expressed as mean (± sem); n = 4–5).
8
J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
titre to HIVgp120 was also elicited by HIVgp120-HSP70 (350±
38). However, antibodies to HLA-A*02 were recorded only in
group 1 mice immunized with Dextramer-HSP70 mixed with
HIVgp120-HSP70 (1600 ± 566; Fig. 4C). This is consistent with
the highest level of CD4+ T cell proliferative responses (Fig. 4C).
As with T cell proliferative studies, we cannot exclude the
possibility that some of the antibodies to Dextramer may have
been induced by streptavidin.
5. Discussion
Conventional alloimmunization with cells elicits not only
immune responses to the foreign HLA molecules but also a
variety of cytokines and chemokines, which may boost the
immune response. In principle it was assumed that the
response to recombinant alloantigens would prove to be
inferior to the corresponding cell-induced alloimmune
responses, especially as the cells express HLA-A, B, CW, DR
and DQ molecules, in contrast to the single recombinant HLAclass I A*0201 molecule. Surprisingly, the proliferative human
CD4+ and CD8+ T cell responses stimulated in vitro by the
recombinant HLA antigen was comparable to allostimulated
cells. In addition to the specific alloimmune response,
significant T cell proliferation was induced to dextran and
HSP70 to which the HLA antigens were linked. Microbial
HSP70 was used as an adjuvant as there is a great deal of
evidence that it is capable of internalizing exogenous antigen
not only by the MHC-class 2 but also the class I (crosspresentation) pathway (Castellino et al., 2000). It stimulates
production of 3 CC chemokines (CCL-3, CCL-4 and CCL-5), of
which CCL5 is a potent chemoattractant of monocytes, CD4
cells and activated CD8 cells (Schall et al., 1990; Murphy et al.,
1994; Meurer et al., 1993; Kim et al., 1998). CCL3 and CCL4
attract CD4+ T- and B-cells (Schall et al., 1993) and all three
chemokines attract immature DC (Dieu et al., 1998). HSP70
also stimulates the production of a number of cytokines (IL12, TNF-α, IL-1β and IL-6) (Wang et al., 2002b; MacAry et al.,
2004); IL-12 is one of the most potent cytokines inducing TH1 polarization (Trichieri, 1994) and the C-terminal portion of
the HSP70-linked peptide elicits high serum IgG2a and IgG3
subclasses of antibodies, consistent with Th1-polarizing
response (Wang et al., 2002b) and induces maturation of DC.
Alloimmunization with human cells would have to utilize
cell lines, which raises the problem of removing oncogenic
viruses commonly used for the production of cell lines.
Recombinant alloantigens would bypass this problem and
restrict the immune response to a single antigen, quite apart
from the superior quality control over the production of a
recombinant alloantigen, compared with cell lines.
In vitro allogeneic stimulation of human CD4+ and CD8+ T
cell proliferation was followed by in vivo immunization of Balb c
mice. Significant CD4+ and CD8+ T cell proliferative responses
were elicited by the Dextramer. Surprisingly, both human and
murine CD4+ T cell proliferative responses were elicited with
the HLA-class I antigen, which differs from the conventional
CD8+ T cell response and will need to be further investigated.
Significantly raised IgG antibody titres to HLA-A*02 were found
in the sera from the Dextramer immunized mice, but the former
gave a smaller standard error of the mean, suggesting greater
consistency of response. These results suggest that recombinant alloantigens might prove to be useful reagents in
immunotherapy. A critical issue in utilizing this vaccination
strategy is to alloimmunize the individual subject. With the
help of Dr. R. Vaughan we have calculated from the frequency of
the alleles found in different populations that to cover over 90%
of the Caucasian, African and Chinese populations may require
four HLA-class I (HLA-A*0101, *0201, *0301 and *1101) and one
HLA-class II (DRB1*04) alleles. However, the potential application of alloimmunization in prevention of HIV-1 infection or the
treatment of tumours may cause future problems if an organ
transplant was required. Recently developed immunosuppressive agents may deal with these, but the risk: benefit ratio will
no doubt have to be considered. Furthermore, the vaccine
needs to be affordable, especially with a multiple allele
constructs, and this may be resolved if the vaccine was to be
produced on an industrial scale.
Immunization with the Dextramer mixed with HIVgp120HSP70, also yielded higher CD4+ T cell proliferation to
HIVgp120 than immunization with HIVgp120-HSP70 alone.
This is consistent with the Dextramer mixed with HIVgp120HSP70 enhancing CD4+ T cell proliferative response and B cell
antibody response. Thus, alloimmunization with the HLAclass I mixed with HIVgp120-HSP70 yields high CD4+ T cell
responses and antibodies to both HLA-I and HIVgp120. It will
be of interest to follow up systemic with mucosal immunization, as in both heterosexual and homosexual HIV infection
the virus is transmitted by the mucosal route.
Allostimulation induces the innate anti-viral A3G factor
(Pido-Lopez et al., 2009) and this lead us to examine human
CD4+ T cells to find out if stimulation with the recombinant
HLA-class I antigen will induce A3G mRNA. Indeed, significant
A3G mRNA resulted if human CD4+ T cells were stimulated
with the HLA-A*0201 Dextramer, with a maximum reached
when linked to HSP70. A comparative assay of the dextramer
with allogeneic irradiated cells suggests that they are equally
potent in stimulating A3G mRNA production.
Thus, allostimulation in vitro with the recombinant HLAclass I construct elicits adaptive immune T cell proliferative
responses in human CD4+ and CD8+ T cells and innate A3G
mRNA expression in CD4+ T cells. HLA immunization in vivo
in mice induces CD4+ and CD8+ T cell proliferative responses
and IgG antibodies to the alloantigen. Early production of A3G
may be important in HIV-1 vaccination, as the innate
response is required to prevent early infection and destruction of CD4+ CCR5+ T cells within 2 weeks of the onset of
infection. Indeed, single alloimmunization of women elicited
significant A3G expression in their PBMC and inhibited ex vivo
HIV replication (Wang et al., 1999; Pido-Lopez et al., 2009).
6. Conclusions
A novel recombinant HLA-class I and HSP70 molecules
were linked to dextran backbones using the biotin–avidin
method. Stimulation of human PBMC in vitro with this
construct induced CD4+ and CD8+ T cell proliferation and
the innate anti-viral factor APOBEC3G. Immunization of Balb c
mice with the HLA-A*0201 Dextramer also elicited an
increase in CD4+ and CD8+ T cell proliferation and IgG
antibodies to the HLA-A*0201. The recombinant HLA-class Idextran-HSP70 construct compared favourably with the
immune responses elicited by native allogeneic stimulation.
The application of this construct as a potential vaccine
J. Schøller et al. / Journal of Immunological Methods 360 (2010) 1–9
candidate will be studied in preventing SHIV infection in
macaques.
Acknowledgements
This investigation was partly supported by a grant from
The Bill and Melinda Gates Foundation (number 38608). We
thank Jann Kyhn for excellent technical assistance in
preparation of the Dextramers.
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