RESEARCH

Complexes of Streptavidin-Fused Antigens with BiotinylatedAntibodies Targeting Receptors on Dendritic Cell Surface:A Novel Tool for Induction of Specific T-Cell Immune Responses

Ondrej Stanek • Irena Linhartova •

Laleh Majlessi • Claude Leclerc • Peter Sebo

Published online: 18 October 2011

� Springer Science+Business Media, LLC 2011

Abstract The choice of tools that enable efficient tar-

geting of exogenous antigens (Ag) for processing and

presentation by professional Ag-presenting cells (APC)

remains limited. This represents, indeed, a bottleneck in

development of vaccines inducing specific T-cell respon-

ses. Here, we describe a novel strategy of Ag delivery into

APCs. The Ag of choice is fused to the N- or C-terminus of

streptavidin (SA) and tetrameric Ag–SA or SA–Ag fusion

proteins are produced in E. coli and purified by 2-Imino-

biotin-Agarose affinity chromatography. Alternatively,

Ag–SA proteins are purified from urea extracts of E. coli

inclusion bodies and refolded in vitro into functional tet-

ramers. Complexes with biotinylated antibodies targeting

cell surface receptors are formed and used to deliver the

Ags of choice for processing and presentation by APCs and

induction of Ag-specific CD4? and CD8? T-cell responses

in vitro and in vivo.

Keywords Streptavidin � Antigen delivery �Biotinylated antibody � T-cell response � Dendritic cell �Receptor targeting

Introduction

For many vaccine and diagnostic applications, it is

important to specifically stimulate Ag-specific CD4? or

CD8? T-cell immune responses. This requires delivery of

the Ags of choice into professional Ag-presenting cells

(APC), which are equipped for Ag processing by endo-

somal proteases or cytosolic proteasome, and can load the

processed antigenic peptides onto class I and class II MHC

glycoproteins, in order to present them on cell surface to

specific T-lymphocytes. In the presence of co-stimulatory

molecules, immunological synapses can then form between

the peptide-loaded MHC molecules of APCs and the cog-

nate Ag-specific T-cell receptors (TCR), resulting in spe-

cific T-cell activation.

Over the past three decades, various strategies enabling

delivery of Ags for presentation by APCs have been

designed and used with varying degree of success [1]. An

intensely explored line of approaches, used particularly to

induce MHC-I-restricted CD8? T-cell responses, employs

DNA vaccines or live attenuated Ag vectors derived from

intracellular parasites, bacteria, or viruses [2, 3]. This

approach relies on intracellular expression of Ag from

transfected DNA or its endogenous production by live

intracellular microorganisms inside APCs and subsequent

presentation in vivo. Another line of strategies for Ag

delivery into APCs comprises chemically defined formula-

tions of peptides with liposomes, or cell-penetrating agents

[4, 5]. A third line of Ag delivery strategies then consists in

the use of cell invasive protein carriers, such as those derived

Claude Leclerc, Laleh Majlessi and Peter Sebo are co-senior authors.

O. Stanek � I. Linhartova � P. Sebo (&)

Laboratory of Molecular Biology of Bacterial Pathogens,

Institute of Microbiology of the ASCR, v. v. i., Videnska 1083,

142 20 Prague, Czech Republic

e-mail: sebo@biomed.cas.cz

O. Stanek

Institute of Chemical Technology, Prague, Czech Republic

L. Majlessi � C. Leclerc

Institut Pasteur, Unite de Regulation Immunitaire et

Vaccinologie, 25-28 Rue du Dr Roux, 75724 Paris, France

L. Majlessi � C. Leclerc

INSERM U1041, 75015 Paris, France

123

Mol Biotechnol (2012) 51:221–232

DOI 10.1007/s12033-011-9459-6

from the TAT protein, or of various specific receptor-bind-

ing proteins that selectively target APCs [6–15].

Recently, an alternative approach was developed that

employs APC targeting by monoclonal antibodies (mAb)

selectively recognizing receptors on the surface of dendritic

cells (DC), e.g., CD205, CD207 (Langerin) mannose

receptors, the b2 integrin CD11c/CD18, or the C-type lectin

receptor Clec9A, respectively. The Ags were chemically

coupled or genetically fused to such antibodies, in order to

enable their selective delivery into APCs. This strategy was

successfully applied for efficient induction of Ag-specific

CD4? and CD8? T-cell responses [16–21]. A recently

employed alternative approach consists in the use of recep-

tor-specific single chain antibody fragments (scFv) that are

fused at the N- or C-termini of SA and enable binding and

delivery of biotinylated cargo Ags into APCs [22–26].

All these strategies, however, suffer of the tediousness

of construction of Ag delivery tools and lack the flexibility

of receptor choice, particularly when the need appears to

rapidly screen for the most suitable receptor on APCs. Such

screening and examination of alternative uptake and traf-

ficking pathways for Ag delivery into APCs may, indeed,

be particularly useful to undertake when testing of various

routes of Ag administration (e.g., mucosal, intradermal,

subcutaneous, or intraperitoneal) is appropriate for induc-

tion of an optimal immune response. Moreover, the pos-

sibility to rapidly screen and identify the optimal APC

subpopulation and/or surface receptor to be targeted in

different tissues, or epithelial surfaces, may be helpful in

achieving optimal presentation of the given Ag to T-cells

in a particular location of the body.

Here, we describe a novel Ag delivery approach in

which the Ags are genetically fused to N- or C-termini of

SA. This allows combinatorial complexing of the given

tetrameric Ag–SA or SA–Ag fusion protein with a broad

range of commercially available or purpose-prepared bio-

tinylated antibodies, recognizing receptors of choice on

different APC subsets. This generates an inherent flexibil-

ity in the choice of receptor, APC subtype, and uptake

pathway into which the Ag will be directed.

The proof of concept and usefulness of the developed

tools for induction of specific T-cell responses in vitro and

in vivo is documented here, using ovalbumin as model Ag

and CFP-10 and ESAT-6 as two immunodominant myco-

bacterial Ags.

Materials and Methods

Recombinant Antigens Fused to Streptavidin

The codon-optimized synthetic gene encoding residues

13–139 of natural core of SA from Streptomyces avidinii

[27] was purchased (GenScript, Piscataway, NJ, USA) and

inserted as an NcoI-HindIII fragment into the pET28b

expression vector (Novagen, Darmstadt, Germany). Syn-

thetic oligonucleotides introducing multiple cloning sites

for in-frame fusion of Ag-coding sequences were next

inserted into the restored NcoI and HindIII sites, as

depicted in detail in Fig. 1, to yield the basic cloning and

expression vector pET28-SAmcs.

The genes encoding for mycobacterial 10-kDa Culture

Filtrate Protein Rv3874 (CFP-10) [28, 29] and 6-kDa Early

Secreted Antigenic Target Rv3875 (ESAT-6) [30] were

PCR amplified on the templates of genomic DNA of

Mycobacterium tuberculosis H37Rv. The amplified open

reading frames were cloned as NcoI and BamHI fragments

into pET28-SAmcs to yield the plasmids pET28b-CFP-10-

SA and pET28b-ESAT-6-SA, respectively. The pET28b-

CFP-10/ESAT-6-SA plasmid for co-expression of the

CFP-10 chaperone together with ESAT-6-SA fusion protein

was constructed as depicted in Fig. 1a. Briefly, the Rv3875

open reading frame was fused in frame to the 50 end of the

open reading frame encoding the SA core on the pET28-

SAmcs. Next an Rv3874 expression cassette was added, so

that from the same T7 promotor the Rv3874 and Rv3875

genes were expressed as a bicistronic mRNA (Fig. 1a).

The portions of the chicken ovalbumin gene (ova) were

amplified using primers listed in Table 1 employing the

ova cDNA cloned in a bacterial vector. The OVA Ag

(OT-II) epitope-coding sequence was inserted as a pair of

synthetic oligonucleotides (Table 1). The PCR-amplified

fragments and oligonucleotide pairs were fused in frame to

SA core as depicted in Fig. 1a. Sequences of all constructs

were confirmed and will be provided upon request.

Expression and Purification of Ag–SA Tetramers

and Monomers

Plasmids derived from pET28-SAmcs were transformed

into E coli Artic Express DE3 cells (Stratagene, Santa

Clara, CA, USA) for IPTG inducible production of Ags

fused to SA. Cultures of transformants were grown in LB

medium containing 60 lg/mL of kanamycin and 20 lg/mL

of gentamicin at 28 �C until optical density at 600 nm

of 0.8 was reached. Expression of the SA alone or of

SA-fused Ag was induced by addition of IPTG to a final

concentration of 0.5 mM and the growth temperature was

rapidly decreased to 10 �C. The cells were harvested 24 h

later, washed in 50 mM CH3COONH4 buffered to pH 9 by

25% NH3�H2O (AC buffer) and stored frozen at -20 �C.

Bacterial pellets were resuspended in AC buffer and

lyzed by ultrasonic disruption. Some of the fusion proteins

were purified directly from soluble cytosolic extracts, while

other SA fusion tetramers were solubilized from cell debris

222 Mol Biotechnol (2012) 51:221–232

123

by extraction with 2 M Urea in AC buffer without pro-

voking tetramer dissociation. Alternatively, Ags fused to

SA were also produced as insoluble monomers and

extracted from inclusion bodies with 8 M urea.

Extracts containing tetrameric forms of the Ag–SA

fusions were loaded onto 2-Iminobiotin-Agarose columns

(Sigma-Aldrich, St. Louis, MO, USA) equilibrated in

50 mM AC buffer (pH 9) and supplemented with 0.5 M

NaCl. The columns were washed with several bed volumes

of equilibration buffer, followed by extensive washing with

0.1 M acetic acid pH 2.9, 0.5 M NaCl. Finally, protein

elution was achieved with 0.1 M acetic acid pH 2.9 without

salt and fractions of eluted protein were immediately

neutralized and buffered to pH 9 by addition of 1/50 of

fraction volume of 25% solution of NH3�H2O.

The Ag–SA fusion proteins were concentrated by ultra-

filtration and the residual lipopolysacharide (endotoxin) was

removed by passage through EndoTrap columns (Profos,

Regensburg, Germany). This procedure allowed to reduce

endotoxin levels below 50 EU/mg of protein, as assessed by

the chromogenic LAL test assay kit (Lonza, Walkersville,

MD, USA). Formation of Ag–SA tetramers was controlled

using Tris-Tricine SDS-PAGE gels (15%) and the capacity

to bind biotin was controlled in Western blots by detection

of biotinylated marker proteins by Ag–SA fusions, which

were next detected by a sandwich of Ag-specific polyclonal

sera and anti-rabbit-peroxidase conjugate.

The insoluble OVA–SA protein was extracted with 8 M

urea from bacterial cell debris and loaded onto a DEAE-

Sepharose column equilibrated in 50 mM ammonium

pET28b-CFP-10/ESAT-6-SA

HindIII

stop codon start codon

NcoI

NheI

BamHI

EcoRI

PstI

XhoISpeI

SacI

pT7

RBS

pT7

RBS

NcoI HindIII

stop codonstart codon

pT7

RBS

NcoI HindIII

cfp-10 sa

stop codonstart codon

pT7

RBS

NcoI HindIII

esat-6 sa

stop codonstart codon

pT7

RBS

stop codon

NcoI BamHI BglII HindIII

RBScfp-10 esat-6 sa

stop codonstart codon start codon

RBSpT7Ribosome binding site pT7 promoter cleavage siteRBS

5’-terminal sequence

3’-terminal sequence

MAKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKI

KVYLPRMKMEEKYNLTSVEFM- -

LETSAESLKISQAVHAAHAEINEAGREVEFTVKL

ACC ATG GCT AGC GGA TCC CTG CAG GAA TTC ATG GAAThr Met Ala Ser Gly Ser Leu Gln Glu Phe Met Glu

NcoINheI

BamHI EcoRIPstI

SA (13-139 AA)

B

ACT AGT GAG CTC AAG CTT TAA CTC GAG CAC CAC CAC CAC CAC CAC TGAThr Ser Glu Leu Lys Leu stop Leu Glu His His His His His His stop

SpeI HindIII XhoIPstI

pET28b-SAmcs

pET28b-OVA-SA

pET28b-CFP-10-SA

pET28b-ESAT-6-SA

A

OVA228-296 OVA317-341sa

sa

Fig. 1 Schematic depiction of

constructs used for Ag–SA

fusion production. a Scheme of

the pET28b-SAmcs construct

bearing the codon-optimized

open reading frame (ORF)

encoding residues 13–139 of SA

(GenBank: CAA00084.1). The

50 and 30 ends of the ORF were

modified by insertion of

multiple restriction sites for

in-frame insertion of sequences

encoding the Ags of interest.

b DNA fragments encoding

residues 228–296 and 317–341

of chicken ovalbumin (OVA)

were fused in frame to 50- and

30- ends of the SA core-

encoding ORF, respectively.

The N-terminal portion of the

OVA-SA protein bears the

H-2 Kb-restricted OVA257–264

MHC class I epitope

(underlined italics) and the

I-Ab-restricted MHC class II

epitope OVA258–276 (bold). The

C-terminal sequence comprises

an H-2b-restricted OVA323–339

epitope (italics, bold), which is

recognized by transgenic TCR

from OT-II mice

Mol Biotechnol (2012) 51:221–232 223

123

acetate buffer (pH 9) containing 8 M urea, without salt.

The column was washed with loading buffer supplemented

with 50 mM NaCl and the monomeric denatured OVA–SA

protein was eluted in buffer containing 0.1 M NaCl (pH 9).

The protein sample was diluted 1:4 with ice-cold AC buffer

without urea and loaded onto a Phenyl–Sepharose column.

The column was washed extensively with ten bed volumes

of AC buffer and endotoxin was removed by further

washing with ten bed volumes of 50 mM AC buffer con-

taining 60% isopropanol. This combined washing cycle

was repeated three times, before the protein was eluted in

50 mM ammonium acetate buffer, 8 M urea, 0.1 M NaCl,

pH 9. Tetramers of the refolded OVA–SA were formed by

dilution of the urea-containing protein solution 1:100 into a

buffer solution containing the biotinylated antibody.

Biotinylated Antibodies and Complex Formation

Monoclonal antibodies specific to CD11b (clone M1/

70.15.11.5.HL, rat IgG2b, ATTC-TIB-12), CD11c (clone

N418, Armenian hamster IgG, ATTC-HB-224) or to MHC-

II (I-A/I-E) (clone M5/114.15.2, rat IgG2b) or the control

Ig (clone R187, rat IgG, ATCC-CRL-1912) were prepared

from supernatants of B-cell hybridomas, cultured in serum-

free, synthetic HL-1 medium (Lonza BioWhittaker,

Walkersville, MD) complemented with 2 mM L-glutamax,

5 9 10-5 M ß-mercapto-ethanol, 100 IU/mL penicillin

and 100 lg/mL streptomycin. Antibodies were precipitated

from supernatants at 4 �C with endotoxin-free (NH4)2SO4

at a 50% final concentration and dialyzed extensively

against PBS. Antibodies were biotinylated (*2:1 ratio)

using the EZ-Link Sulfo-NHS-LC kit (Pierce, Rockford,

IL), free biotin was removed by dialysis and the antibody

solutions in PBS were sterilized by filtration through

0.2 lm filters.

Biotinylated anti-CD11c (clone N418) was from, eBio-

science (San Diego, CA) and anti-CD206 (clone MR5D3)

was from BioLegend (San Diego, CA), respectively.

Complexes of the biotinylated antibodies and tetrameric

SA–Ag fusions were allowed to form on ice for 2 h before

use.

Cell Lines

The CD8? T-cell hybridoma B3Z, recognizing OVA257–264

presented on H-2 Kb molecules [31], was a generous gift of

Darren E. Higgins (Harvard Medical School, Boston, USA).

MF2.2D9 CD4? T-cell hybridoma recognizing OVA258–276

in the context of I-Ab molecules [32] was kindly provided by

Kenneth L. Rock (University of Massachusetts, USA). T-cell

lines were incubated in RPMI 1640 medium supplemented

with 10% FCS (Life Technologies, Carlsbad, CA, USA),

Table 1 List of PCR primers and oligonucleotides used in cloning

Primer/oligonucleotide Sequence Restriction site Gene designation

CFP-10-I ATTACCATGGCAGAGATGAAGACC NcoI CFP-10-SA

CFP-10-II ATTCCCCATGGAGAAGCCCATTTGCGAGGA NcoI

ESAT-6-I ATTACCATGACAGAGCAGCAGTGG NcoI ESAT-6-SA

ESAT-6-II ATTTTCCATGGATGCGAACATCCCAGTGAC NcoI

CFP-10 coex.I ATTACCATGGCAGAGATGAAGACC NcoI co-expression of

CFP-10 and ESAT-6CFP-10 coex.II TAAGGATCCTCTTTTCGTAATAGCGGGTC BamHI

ESAT-6 coex.I TTAAGATCTAGGAGATATACCATGACTGTA BglII

ESAT-6 coex.II TAAAAAGCTTCTATGCGAACATCCCAGTGAC HindIII

MCSa-N-ter.I CATGGCTAGCGGATCCCTGCAGG NcoI Ag fusion site on the

N-terminus of SAMCSa-N-ter.II AATTCCTGCAGGGATCCGCTAGC EcoRI

MCSa-C-ter.I TCGAAACTAGTGAGCTCAAGCTTTAACTCGAGA XhoI Ag fusion site on the

C-terminus of SAMCSa-C-ter.II AGCTTCTCGAGTTAAAGCTTGAGCTCACTAGTT HindIII

OVA-I CTACCATGGCTAAGATCCTGGAGCTTCCAT NcoI OVA (aa 228–296)

OVA-II TACGAATTCGACAGATGTGAGGTTGTATT EcoRI

OTII-I CTAGTGCTGAATCTCTGAAAATCTCTCAGGCTGT

TCACGCTGCTCACGCTGAAATCAACGAAGCTGG

TCGTGAAGTTGAATTTACCGTAA

SpeI OT-II epitope OVA

(aa 323–339)

OTII-II AGCTTTACGGTAAATTCAACTTCACGACCAGCTTC

GTTGATTTCAGCGTGAGCAGCGTGAACAGCCTGA

GAGATTTTCAGAGATTCAGCA

HindIII

a MCS, multiple cloning site

224 Mol Biotechnol (2012) 51:221–232

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0.1 mg/mL streptomycin, 1000 U/mL penicillin and

0.25 lg/mL amphotericin (Sigma–Aldrich, St. Louis, MO,

USA), 50 lM 2-mercaptoethanol, 1% non-essential amino

acids (Biochrom, Berlin, Germany), 1 mM sodium pyruvate,

2 mM glutamine and 6.5 g/L glucose.

Generation of Mouse Bone Marrow-Derived Dendritic

Cells

Bone marrow-derived dendritic cells (BM-DC) were gen-

erated according to Lutz et al. [33]. Briefly, mouse tibias

and femurs were flushed with ice-cold PBS and the

obtained cells were collected for 5 min at 10009g, resus-

pended in RPMI 1640 medium, supplemented with 10%

FCS (Life Technologies, Carlsbad, CA, USA), 0.1 mg/mL

streptomycin, 1000 U/mL penicillin and 0.25 lg/mL

amphotericin (Sigma–Aldrich, St. Louis, MO, USA),

50 lM 2-mercaptoethanol, 1% non-essential amino acids

(Biochrom, Berlin, Germany), 1 mM sodium pyruvate,

2 mM glutamine and 20 ng/mL Granulocyte–Macrophage

Colony-Stimulating Factor (GM-CSF). Bone-marrow

hematopoietic precursors were seeded at 2 9 106 cells per

100 mm non-treated cell culture plates in 10 mL of con-

ditioned medium and incubated at 37 �C in a 5% CO2

humidified atmosphere. On days 3, 6 and 8 one half of the

medium was replaced. At day 8 or 9 the percentage of

CD11c? cells was higher than 70–80% and the percentage

of CD11b? cells exceeded 90%, as determined by cyto-

fluorometric analysis.

In Vitro Antigen Presentation Assay

1 9 105 BM-DC per well were seeded in 96-well plates in

triplicates and incubated with the mixture of Ag–SA tet-

ramers, with or without the appropriate biotinylated anti-

body. In parallel, BM-DC were incubated with free soluble

ovalbumin (OVA; albumin from chicken egg white from

Sigma–Aldrich, St. Louis, MO, USA), or free MHC-II-

restricted IINFEKLTEWTSSNVMEER (Vidia, Vestec,

Czech Republic) or MHC-I-restricted SIINFEKL peptides

(Sigma-Aldrich, St. Louis, MO, USA), used as controls.

After 3 h incubation, the medium was discarded, cells were

washed and 1 9 105 MF2.2D9 (CD4?) or B3Z (CD8?)

T-hybridoma cells were added per well for additional 16 h.

Cultures were frozen for at least 2 h at -80 �C and the

concentration of IL-2 was determined by a sandwich

ELISA using paired rat-anti-mouse IL-2 capture antibody

(JES6-1A12) and biotinylated rat-anti-mouse IL-2 detec-

tion antibody (JES6-5H4) from BD Pharmingen (Exbio,

Vestec, Czech Republic). Stimulation of the B3Z (CD8?)

T-hybridoma was measured as ß-galactosidase activity

accumulated in B3Z cells upon coculture with DC pre-

senting OVA [34].

Mice, Immunization, Detection of T-Cell Responses

Ten to twelve weeks-old female C57BL/6 mice (Charles

Rivers, Saint Germain sur l’Arbresle, France) were

immunized by single i.v. injections of OVA-SA at the

indicated dose, complexed with biotinylated anti-CD11b or

anti-CD11c mAb or with a biotinylated control Ig, in the

presence of 25 lg/mouse of poly I:C. At the indicated time

point post immunization, IFN-c T-cell responses were

analyzed subsequent to in vitro stimulation of total

splenocytes with appropriate proteins or antigenic peptides,

in complete HL-1 medium. At 72 h post-incubation, IFN-cwas quantified in culture supernatants by ELISA, as pre-

viously described [35].

Results

Production of Antigen-Streptavidin Fused Proteins

Previously, fusion proteins to full-length SA were found to

be proteolytically cleaved in E. coli within the N- and

C-termini sequences of SA [27, 36, 37]. Therefore, we used

only the core sequence of natural SA, corresponding to its

residues 13–139. The synthetic gene with codons opti-

mized for protein production in E. coli was cloned into the

pET28b expression vector and its 50 and 30 terminal por-

tions were modified by introduction of restriction sites, to

allow in-frame fusion of open reading frames encoding Ags

of choice (Fig. 1a). Initially, we constructed fusions of SA

with only the short OVA peptides attached, which corre-

sponded to immunodominant epitopes of ovalbumin.

These, however, turned out to be too unstable and failed to

yield soluble Ag–SA tetramers in E. coli. Therefore, a

larger construct was prepared, in which an ovalbumin

portion comprising residues 228–296 and harboring the

MHC-I-restricted epitope SIINFEKL was fused to the

N-terminus of SA. Next, the ovalbumin segment com-

prising residues 317–341 and containing the MHC-II-

restricted OT-II epitope was attached at the C-terminus of

SA, to yield the construct called OVA–SA (Fig. 1b).

in E coli grown at 37 �C the OVA-SA protein could

only be produced in form of inclusion bodies and its sub-

sequent refolding from urea extracts into tetramers was

inefficient (Table 2). To circumvent the problem, we used

a low culture temperature (10–12 �C) and the E. coli Artic

Express DE3 strain, in which co-expression of the cold-

shock chaperons Cpn10 and Cpn60 allows stabilization of

produced proteins. This allowed production of the OVA–

SA protein in form of soluble tetramers in the cytosolic

fraction of E. coli.

The ESAT-6-SA protein could be produced to high

levels in form of inclusion bodies in E. coli BL21 kDE3 at

Mol Biotechnol (2012) 51:221–232 225

123

20 �C, while the yields of soluble tetrameric ESAT-6-SA

produced in E. coli Artic express cells at 10–12 �C

(Table 2) were quite low (data not shown). Since ESAT-6

is naturally produced and secreted in complex with its

chaperon CFP-10, and this is soluble when expressed in

E. coli [38, 39], the CFP-10 chaperone was produced in the

same cells together with ESAT-6-SA using the pET28b-

CFP-10/ESAT-6-SA plasmid (Fig. 1a). Co-expression of

this chaperone then allowed high level production of sol-

uble tetrameric CFP-10/ESAT-6-SA complexes also in

E. coli BL21 kDE3 at 20 �C (Table 2).

Soluble tetrameric Ag–SA complexes were next purified

from cell lyzates by affinity chromatography on 2-Imino-

biotin-Agarose. The critical step of this procedure appeared

to be the decrease of buffer pH during replacement of the

loading solution (pH 9) by the buffer used for protein

elution (pH 2.9). Indeed, the use of sodium acetate pH 4.0

for protein elution according to recommendation of the

supplier of the 2-Iminobiotin-Agarose resin reproducibly

resulted in precipitation of Ag–SA in the eluate. The

problem could, however, be solved by introducing a wash

of the resin-bound protein with 0.5 M NaCl in 0.1 M acetic

acid at pH 2.9. This allowed to obtain soluble Ag–SA

tetramers upon subsequent elution from the resin in 0.1 M

acetic acid pH 2.9 without salt (Table 3 and Fig. 2a).

All produced Ag–SA fusions proteins could be purified

as stable and soluble tetrameric proteins (Table 2, Fig. 2b)

that had to be heat-denatured in SDS-PAGE loading buffer

in order to dissociate into monomers (Fig. 2b). Alterna-

tively, insoluble monomers of OVA–SA were also

extracted in buffers containing 8 M urea from inclusion

bodies produced at 37 �C and the protein was purified by a

combination of ion exchange and hydrophobic chroma-

tography (Fig. 2c). Since attempts to refold denatured

OVA–SA into soluble tetramers in the absence of biotin

failed, the purified monomeric OVA–SA protein was

refolded into functional tetramers on the template of bio-

tinylated antibodies (see below).

Table 2 Solubility of the Ag–SA proteins produced in E. coli at different temperatures

Protein solubilitya

E. coli BL21 kDE3 E. coli Artic Express DE3

37 �C 30 �C 25 �C 20 �C 10 �C (%)

SA I.B.b I.B. I.B. 60% 100

CFP-10-SA I.B. I.B. 60% 90% 100

ESAT-6-SA I.B. I.B. I.B. I.B. 90

CFP-10/ESAT-SA I.B. I.B. I.B. 60% 100

OVA–SA I.B. I.B. I.B. I.B. 30

a % of the produced protein recovered in the cytosolic fraction. The ratio was deduced from scans of Tris-Tricine SDS-PAGE gel separations of

cytosolic fractions and urea extracts of particulate cellular debrisb I.B. insoluble protein, expression into inclusion bodies

Table 3 Solubility of Ag–SA tetramers eluted from 2-Iminobiotin agarose columns

% Aggregation

Protein Elution buffera

0.1 M CH3COOH/

CH3COONa, pH 4 (%)

0.1 M CH3COOH,

pH 2.9 (%)

0.1 M CH3COOH/

0.5 M NaCl, pH 2.9

Wash at pH 2.9 with

0.5 M NaCl followed by 0.1 M

CH3COOH, pH 2.9 (%)

SA 50 40 0% 0

CFP-10-SA 20 10 No elution 0

ESAT-6-SA 60 30 No elution 0

CFP-10/ESAT-6-SA 20 10 No elution 0

OVA–SA 70 50 No elution 10

Protein samples were loaded onto 2-iminobiotin agarose columns in 50 mM CH3COONH4, 0.5 M NaCl pH 9 and washed in the loading buffer.

The proteins were eluted in the indicated buffersa Protein precipitates were collected by centrifugation. Supernatants were recovered and the precipitates were dissolved in 8 M urea to the initial

volume of the individual samples. Equal volumes of supernatants and dissolved precipitate samples were separated on 15% Tris-Tricine SDS-

PAGE gels and the percentage of precipitated protein was determined by densitometry of Coomassie-stained gels

226 Mol Biotechnol (2012) 51:221–232

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In Vitro Presentation of Streptavidin-Fused Antigen

We next evaluated in vitro the efficiency of Ag delivery to

DC by pre-formed complexes of Ag–SA tetramers with

biotinylated targeting antibodies. Purified 100 kDa OVA–

SA tetramers were respectively mixed at 1:1, 1:2 or 2:1

molar ratios with biotinylated mAbs specific to the b2

integrin subunit CD11c, or to the mannose receptor CD206.

Alternatively, urea-extracted OVA–SA monomers purified

under denaturing conditions (in 8 M urea) were refolded by

direct dilution into solution of the anti-CD11c antibody.

BM-DCs (H-2b) were incubated for 3 h with various con-

centrations of such tetramer:antibody complexes, before

being extensively washed, to remove unbound Ag, and

co-incubated with MHC-II-restricted and OVA-specific

MF2.2D9 CD4? T hybridoma cells. In this protocol, if the

OVA Ag is delivered to BM-DC via the biotinylated

antibody and is endocytosed, processed, loaded onto MHC-

II molecules and presented on the surface of BM-DC,

respectively, the TCR of the OVA-specific T-cell hybrid-

oma is triggered. This leads to IL-2 production proportional

to Ag processing and presentation by BM-DCs.

As shown in Fig. 3, at all three tested tetramer:antibody

molar ratios, an efficient processing and presentation of

OVA was detected for the complexes of OVA–SA tetra-

mers present at subnanomolar concentrations, when the

biotinylated anti-CD11c mAb was used for targeting.

Interestingly, a comparably efficient OVA delivery was

observed also when the denatured OVA–SA protein was

refolded from 8 M urea-containing stocks by a direct

[100-fold dilution into solution of the biotinylated tar-

geting antibody (Fig. 3, OVA–SA urea ? anti-CD11c

lines). In contrast, free OVA–SA (not complexed to a

targeting antibody) was barely presented by BM-DCs even

at the highest input concentrations, likely due to a low level

of receptor independent uptake of the OVA–SA antigen by

macropinocytosis. The biotinylated anti-CD206 antibody

mediated only a weak presentation of OVA–SA (Fig. 3a),

in line with the poor expression of the CD206 receptor on

the surface of BM-DCs (data not shown). Finally, the

ESAT-6-SA Ag, used as negative control, did not induce

any stimulation of MF2.2D9 T-cells, thus documenting the

specificity of the OVA presentation assay. These results

demonstrate that when OVA–SA was targeted to DC via

the b2 integrin CD11c/CD18, expressed on DC surface, the

Ag was able to efficiently gain access to the MHC-II

pathway of DCs in order to be presented to specific CD4?

T-cells.

1 2 3 4Mr

1 2 3 4 5 6Mr

25 kDa

100 kDa

25 kDa

100 kDah h h h

25 kDa

100 kDa

monomer

tetramer

Mr

monomer

tetramer

A B

C

SAT

SAT

Fig. 2 Purification of fused Ag-SA. a Fractions from isolation of the

soluble OVA–SA tetramer. Lane 1 2 M urea extract of cell debris,

lane 2 2-Iminobiotin-Agarose column flow through, lane 3 eluted

tetrameric OVA–SA, lane 4: monomeric OVA–SA obtained by

tetramer denaturation for 5 min at 100 �C. b Purified tetramers of the

Ag–SA fusion proteins. 5 lg of the Ag–SA tetramers eluted from the

2-Iminobiotin-Agarose column were loaded on the gel as such, or

upon denaturation by sample heating at 100 �C for 5 min. All samples

were separated on 15% Tris-Tricine SDS-PAGE gels and the proteins

were visualized by Coomassie blue staining. h heat tetramer

denaturation for 5 min at 100 �C. c Isolation of Ag–SA monomers

from 8 M urea extracts of inclusion bodies. Lane 1 extract of cell

debris in 8 M urea, lane 2 DEAE Sepharose column flow through,

lane 3 column wash, lane 4 eluted Ag–SA fraction before loading on

a Phenyl Sepharose column, lane 5 flow through of Ag–SA (diluted to

2 M urea) loaded on the Phenyl Sepharose column, lane 6 elution

from Phenyl Sepharose. Mr molecular weight markers

Mol Biotechnol (2012) 51:221–232 227

123

Induction of Ag-Specific T-Cell Responses

by In Vivo DC Targeting

We next sought to evaluate the potential of Ag–SA com-

plexes with biotinylated mAbs specific for DC subsets to

induce Ag-specific T-cell responses in vivo. To this end,

we immunized C57BL/6 mice by i.v. injection of OVA–SA

in complex with biotinylated anti-CD11c mAb (4:1), or

with a biotinylated control IgG, using poly I:C as adjuvant.

The mice that received 10 lg of OVA–SA in complex with

biotinylated anti-CD11c mAb displayed OVA-specific

T-cell responses on day 10 after immunization, as judged

by strong IFN-c production of their splenocytes stimulated

in vitro with soluble OVA protein (Fig. 4, left panel black

bars). No such splenocyte responses were detected in

control mice injected with complexes of OVA–SA to

biotinylated control IgG (Fig. 4 left, empty bars), or after in

vitro restimulation of splenocytes from immunized mice

with the control protein MalE (Fig. 4 right panel). In one

out of three immunized mice a weak anti-SA T-cell

response was detected (data not shown).

OVA-SA nM

A B

IL-2

(O

D49

2nm

)

COVA-SA + anti-CD11c

OVA-SA urea + anti-CD11c

ESAT-6-SA + anti-CD11c

OVA-SA + anti-CD206

OVA-SA w/o antibody

ESAT-6-SA

Ag-SA/antibodymolar ratio = 1:1

0

0.1

0.2

0.3

0.4

0.5

0.6

10-3 10-2 10-1 100

OVA-SA nM

IL-2

(O

D49

2nm

)IL

-2 (

OD

492n

m)

Ag-SA/antibodymolar ratio = 2:1

0

0.1

0.2

0.3

0.4

0.5

0.6

10-3 10-2 10-1 100

Ag-SA/antibodymolar ratio = 1:2

0

0.1

0.2

0.3

0.4

0.5

0.6

10-3 10-2 10-1 100

OVA-SA nM

Fig. 3 Biotinylated anti-CD11c mAb delivers the OVA–SA protein

for presentation of the OVA258–276 epitope on MHC class II

molecules in vitro. Soluble tetramers of Ag–SA (OVA–SA, ESAT-

6-SA), or the denatured monomers (OVA–SA urea) refolded by

[100-fold dilution into solution of biotinylated anti-CD11c mAb in

PBS with 1% BSA, were complexed with the biotinylated anti-CD11c

mAb at a molar ratio of mAb:Ag–SA = 2:1 (a), 1:2 (b) and 1:1 (c),

respectively. The mAb:Ag–SA complexes were then incubated at

indicated concentrations with 105 BM-DCs in 200 lL of culture

medium at 37 �C under 5% CO2 atmosphere for 3 h, before the

medium was discarded and 105 MF2.2D9 CD4? T-cell hybridoma

cells were added for additional 16 h of co-incubation with BM-DCs.

Cultures were frozen at -80 �C for 2 h before released IL-2 was

determined by ELISA. The experiment was reproduced three times

and a representative result is shown

Ag (µg/ml)

OVA MalE

Ag (µg/ml)

0.1 1 1000

1

2

3

4

5

6

7biot-anti-CD11cbiot-Ctrl IgG

0.1 1 1000

1

2

3

4

5

6

7

Fig. 4 In vivo induction of OVA-specific T-cell response by delivery

of OVA–SA to CD11c? cells. Female C57BL/6 mice (three per

group) were immunized by a single i.v. injection of 13 lg (500 pmol/

mouse) of OVA–SA, complexed at a molar ratio of 4:1, with

biotinylated anti-CD11c antibody, or a biotinylated control IgG, in the

presence of 25 lg/mouse of poly I:C. Ten days after immunization,

the T-cell response of individual mice was analyzed by ELISA as the

release of IFN-c upon in vitro stimulation of splenocytes with various

concentrations of soluble OVA protein, or of the unrelated MalE

control. Individual bars correspond to individual mice, where full

bars are for results obtained with complexes to biotinylated anti-

CD11c and empty bars are results obtained with biotinylated control

IgG

228 Mol Biotechnol (2012) 51:221–232

123

Furthermore, as shown in Fig. 5, OVA-specific CD8?

T-cell responses were detected in C57BL/6 mice ten days

after immunization with OVA–SA in complex to anti-

CD11b mAb. Splenocytes from such immunized mice

produced high levels of IFN-c in response to stimulation in

vitro with the MHC I-restricted OVA257–264 peptide

SIINFEKL, but not upon stimulation with a control peptide

(Fig. 5, Ctrl pep). In turn, mice injected with OVA–SA

complexed to control IgG did not mount any SIINFEKL-

specific T-cell responses (Fig. 5, biot-Ctrl Ig). Altogether,

these in vivo results provide the proof of concept for the

developed technology for induction of Ag-specific T-cell

responses.

Extension of the Delivery Technology to Other

Antigens and Receptors

To extend this Ag delivery strategy from the OVA model

to Ags with diagnostic or vaccinal potential, we examined

in vitro delivery and presentation of the mycobacterial Ag

ESAT-6. To do so, complexes of CFP-10/ESAT-6-SA Ag

(c.f. Fig. 1a) with biotinylated mAbs bound to MHC-II,

CD11b or CD11c molecules were assembled on the surface

of BM-DC (H-2b) at 4�C and upon extensive washing, the

cells were co-incubated at 37�C with the ESAT-6:1–20-

specific (MHC-II-restricted) CD4? T-cell hybridoma NB11

[40]. As shown in Fig. 6, targeting of any of the three

surface molecules enabled delivery of the CFP-10/ESAT-6

Ag complex for processing and presentation of the

immunodominant ESAT-6:1–20 epitope to the NB11

T-cells in a highly specific and efficient manner. In

contrast, equal amounts of CFP-10/ESAT-6 complexed to

biotinylated control IgG yielded only a weak Ag presen-

tation response and no stimulation of the T-hybridoma cells

was observed with SA control Ag alone. These results,

hence, demonstrate the feasibility of targeted delivery to

DCs of mycobacterial Ags, which are of high interest for

use in vaccines and as tools for in vitro diagnostics.

Discussion

We describe here, the design and use of a novel tool for Ag

delivery into dendritic cells that is based on Ag targeting

through antibodies specific for selected receptors on the

surface of DCs. The system employs Ag–SA fusion pro-

teins that are complexed with biotinylated targeting anti-

bodies, taking advantage of the very high affinity binding

of biotin to tetrameric SA (Kd * 10-15 M).

We first focused on definition of conditions under which

the Ag–SA fusion proteins would be produced as soluble

tetramers into the cytosolic fraction of E. coli. Solubility of

different fused Ag–SA upon expression in E. coli is likely

to vary and finding of optimal conditions for refolding of

individual Ag–SA proteins into soluble Ag–SA tetramers

in vitro may be difficult. With the OVA and ESAT-6 Ags

tested here, the soluble Ag–SA tetramers could be pro-

duced at low growth temperatures (10–12 �C) using the

E. coli Arctic express strain producing cold shock-induced

chaperons Cpn10 and Cpn60 that can assist folding of

SIINFEKL Ctrl pep SIINFEKL Ctrl pep

biot-anti-CD11b biot-Ctrl IgG

0

3

6

9

12

15

18

Fig. 5 Immunization by OVA–SA complexed with biotinylated anti-

CD11b mAb induces OVA-specific CD8? T-cell responses in vivo.

Female C57BL/6 mice (2 per group) were immunized by a single i.v.injection of 1.3 lg (50 pmol/mouse) of complexed at a molar ratio of

2:1 to biotinylated anti-CD11b mAb (left), or to biotinylated control

IgG (right), in the presence of 25 lg/mouse of poly I:C. 10 days after

immunization, the T-cell response of individual mice was analyzed by

ELISA as the release of IFN-c upon in vitro stimulation of

splenocytes with the OVA257–264 SIINFEKL peptide, as compared

to a control peptide. Mean ± SD are calculated for two individuals of

each experimental group. Results are representative of two indepen-

dent experiments

biot-Ctrl IgG biot-anti-MHC-II biot-anti-CD11b biot-anti-CD11c0

5

10

15

20Ctrl-SA

IL-2

(ng

/ml)

CFP-10/ESAT-6-SA

Fig. 6 Targeting of three different surface markers of BM-DCs by

biotinylated antibodies enables delivery of the CFP-10/ESAT-6-SA

fusion protein into the MHC-II presentation pathway. BM-DC from

C57BL/6 (H-2b) mice were incubated at 4 �C with biotinylated

control IgG or the indicated biotinylated mAbs specific to MHC-II,

CD11b or CD11c molecules. Cells were washed and incubated at

4 �C for 1 h with 1 lg/mL of mock SA (Ctrl-SA), or with the CFP-

10/ESAT-6-SA fusion protein. Cells were washed and co-cultured

with anti-ESAT-6:1-20, I-Ab-restricted NB11 T-cell hybridoma. The

presentation of the immunodominant ESAT-6:1-20 epitope, retricted

by I-Ab by the NB11 T-cell hybridoma was assessed by ELISA for

released IL-2 in the co-culture supernatants after 24 h, which is

proportional to the intensity of the Ag presentation and thereby the

TCR triggering. Mean ± SD of culture duplicates are shown and

results are representative of two independent experiments

Mol Biotechnol (2012) 51:221–232 229

123

poorly soluble proteins. Moreover, for the ESAT-6-SA

fusion the co-expression of the ESAT-specific chaperone

CFP-10 [28] allowed expression of soluble and tetrameric

CFP-10/ESAT-6-SA complexes also at increased temper-

atures (20 �C), using the more common E. coli strain

BL21(kDE3). This allowed, indeed, to substantially

increase the yields of the produced protein (not shown).

Surprisingly, we also found that the OVA–SA protein,

which was produced at high levels into inclusion bodies in

E. coli at standard growth conditions (37 �C), could still be

quite efficiently targeted into BM-DCs for processing and

presentation upon simple refolding by dilution-out from

8 M urea [100-fold into solution of the biotinylated tar-

geting antibody. Our ongoing experiments with different

Ag–SA fusions now, indeed, suggest that in many cases it

may be easier to obtain the Ag–SA at higher purity and

higher amounts, when produced into inclusion bodies. This

option, hence, deserves further testing with other ‘difficult’

Ag–SA proteins, as it would represent a very cost-effective

method of preparation of high amounts of Ag–SA protein

complexes with biotinylated mAbs.

Another critical step of the Ag delivery technology

presented here was the purification of tetrameric Ag–SA

fusion proteins on 2-Iminobiotin-Agarose columns. The

procedures recommended by the supplier of the resin

yielded aggregated protein. This was most likely due to

precipitation in the course of transition through the iso-

electric point, during the sharp pH change, when the

loading buffer (pH 9) was directly replaced by the elution

buffer (pH 2.9). This problem was solved by inclusion of a

washing step at pH 2.9, at which elution of the protein was

prevented by addition of 0.5 M NaCl that stabilized the

binding interaction of SA with 2-iminobiotin. Crossing of

the isoelectric point of the fusion protein while this

remained bound to the resin prevented its precipitation.

Subsequent elution with buffer without salt then yielded

soluble tetramers that resisted subsequent neutralization of

the 0.1 M acetic acid elution buffer. This procedure was,

indeed, successfully applied also for purification of several

additional mycobacterial Ag–SA fusion proteins used for in

vivo immunization studies (Dong et al., manuscript in

preparation).

In the used in vitro system, only presentation of OVA on

the MHC II molecules was observed and its MHC-I-

restricted presentation was inefficient. It is likely that upon

endocytosis of the OVA–SA:mAb complexes by BM-DCs

in vitro the MHC I cross-presentation was hampered by

destruction of the OVA257–264 MHC-I epitope (SIINFEKL)

due to excision of the overlapping MHC-II epitope. Nev-

ertheless, good MHC I restricted presentation of the

OVA257–264 epitope was observed in vivo upon adminis-

tration of the OVA–SA fusion in complex with biotinylated

anti-CD11b mAb. It remains, hence, to be clarified if the

processing of the fusion protein followed a different path

when taken up by circulating APCs in the blood. Indeed,

we demonstrated that in vitro formed complexes between

the Ag–SA tetramers and the biotinylated antibodies

against CD11b or CD11c subunits of the b2 integrins, the

MHC-II molecules, and to a lesser extent also to the CD206

mannose receptor, respectively, could all deliver these

immunogens to the BM-DC. Upon endocytosis, these

immunogens gained access into MHC-II processing path-

way and were efficiently presented in the context of MHC-

II molecules to specific T-cells. Moreover, immunization

with OVA–SA complexed to biotinylated anti-CD11b or

anti-CD11c antibodies allowed also induction of strong

OVA-specific CD4? or CD8? T-cell responses in vivo.

These observations demonstrate the feasibility of using this

strategy of APC-specific Ag targeting and pave the way to

development of immunization tools for antibody-mediated

Ag delivery to specific DC subsets.

Compared to direct fusions of Ags to the targeting

antibody [16–21], to the SA-antibody fusions, or to the use

of biotinylated Ag complexes [22–26], respectively, the

here-described Ag fusions to SA would potentially present

several advantages. The most important, perhaps, is the

flexibility, where one Ag–SA protein can be combined with

numerous different biotinylated targeting antibodies in the

search for mAbs targeting appropriate cell surface recep-

tors, or recognizing the most appropriate APC subsets.

Furthermore, due to the tetrameric nature of the Ag–SA

fusion protein, a two to four-fold higher Ag dose is

delivered per antibody molecule.

The here-described strategy is likely to allow also vac-

cine delivery through alternative routes, such as intrana-

sally, to direct Ags of interest to the airway DC subsets, or

intradermally to dermal DCs. For instance, delivery of

protective immunogens to dermal DC subsets by Ag–SA

complexes with mAbs to CD207 (Langerin) certainly

deserves exploration. This is likely to benefit from the use

of recently developed transdermal delivery systems capa-

ble to increase Ag permeation in skin by the use of

chemical enhancers, ultrasound, iontophoresis, or micro-

needles [41–43].

Besides of appropriate Ag delivery and processing/pre-

sentation by the right APC, the presence of a second signal

that activates the APC appears to be primordial for

inducing T-cell responses. However, direct cross-linking of

b2-integrins by specific mAbs has not been reported to

provide appropriate DC activation signals. To circumvent

this problem in the present study, and to evaluate the fea-

sibility of in vivo triggering of specific T-cell responses by

the use of OVA–SA fusion complexed to biot-mAbs, we

have systematically used Poly I:C as adjuvant. This syn-

thetic analog of dsRNA was, indeed, shown to interact with

endosomal TLR3 or with cytosolic dsRNA sensors, the

230 Mol Biotechnol (2012) 51:221–232

123

Retinoic acid-Inducible Gene-I (RIG-I) or Melanoma Dif-

ferentiation-Associated gene-5 (MDA5) RNA helicases,

the capacity of which to promote induction of Th1 and

CD8? CTL responses has been largely documented [44].

It should be stressed that the biot-mAbs used here for

Ag–SA delivery were derived from rat or hamster. For a

future use of this Ag delivery system in human vaccination,

however, humanized antibodies will have to be used, so as

to avoid induction of immune responses against xenoge-

neic determinants of the targeting antibodies.

Further, it has to be taken in account that diverse

parameters may influence in vivo endocytosis of Ag,

including immunosuppressive agents and regulatory T cells

(Treg). In vitro treatment of mouse BM-DC with the

immunosuppressive macrolide rapamycin can, indeed,

decrease macropinocytosis and mannose receptor-mediated

endocytosis, albeit it does not totally inhibit endocytic

capacities of BM-DC [45]. Therefore, in the process of

extension of the developed strategy for vaccine delivery

into human DC, the status of the immune system of the

vaccinated individuals will have to be taken in consider-

ation. The Treg cells have also been observed to reduce the

expression of endocytic receptors on human DC in vitro in

certain conditions [46]. However, our preliminary results

suggest that Ag targeting to different DC subsets in vivo,

through diverse endocytic C-type lectins or b2-integrins, is

readily feasible in mice having an intact Treg compartment

(data not shown).

The results presented here, hence, open the way to use of

the developed antigen delivery tools in vaccination, for the

purpose of de novo induction of Ag-specific T-cell immune

responses, as well as for in vitro diagnostic applications,

where specific antigen delivery for presentation by APC is

required for stimulation of memory T-cells in recall

response assays.

Acknowledgments This work was supported by grants from the

Ligue Nationale Contre le Cancer (Equipe Labellisee 2011) and

ANR Emergence (ANR-2010-EMMA-008-01) to C.L., the grants

KAN200520702, 310/08/0447 and 2B06161 to P.S., and the Research

Plan AV0Z50200510.

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