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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3172-3176
Gene Transfer in Dendritic Cells, Induced by Oral DNA Vaccination
With Salmonella typhimurium, Results in Protective
Immunity Against a Murine Fibrosarcoma
By
Paola Paglia,
Eva Medina,
Ivano Arioli,
Carlos A. Guzman, and
Mario P. Colombo
From the Division of Experimental Oncology D, Istituto Nazionale per
Lo Studio e la Cura dei Tumori, Milano, Italy; and the Division of
Microbiology, GBF-National Research Centre for Biotechnology,
Braunschweig, Germany.
 |
ABSTRACT |
A live attenuated AroA auxotrophic mutant of
Salmonella typhimurium (SL7207) has been used as carrier for
the pCMV vector that contains the -galactosidase (-gal) gene
under the control of the immediate early promoter of
Cytomegalovirus (CMV). We tested whether orally administered
bacterial carrier could enter and deliver the transgene to
antigen-presenting cells (APCs) through the natural enteric route of
infection and whether -gal expression could generate a protective
response against an aggressive murine fibrosarcoma transduced with the
-gal gene (F1.A11) that behaves operationally as a tumor-associated
antigen. After three courses, at 15-day intervals, mice developed both
cell-mediated and systemic humoral responses to -gal. Mice
vaccinated with the Salmonella harboring pCMV, but not with
plasmid-less carrier, showed resistance to a challenge with F1.A11
cells. These experiments suggest that Salmonella-based DNA
immunization allows us to specifically target antigen expression in
vivo to APCs. To prove that the transgene is actually expressed by APCs
as a function of an eukaryotic promoter, the green fluorescent protein
(GFP) was placed under the control of either the eukariotic CMV or a
prokaryotic promoter. Using cytofluorometric analysis, GFP was detected
only in splenocytes of mice receiving a Salmonella carrier harboring
GFP under the CMV promoter. These results indicate that transgene
expression occurs because of a Salmonella-mediated gene
transfer to eukaryotic cells. Finally, approximately 19% of the
splenocytes expressed GFP. Among them, F4/80+ macrophages
and CD11cbright dendritic cells (DCs) were scored as
positive for GFP expression. Extensive work has been performed trying
to optimize the way to transfect DCs, ex vivo, with genes coding for
relevant antigens. We show here, for the first time, that DCs can be
directly and specifically transduced in vivo such to induce DNA
vaccination against tumors.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
CANCER VACCINES are proposed for
management of patients with minimal residual disease or with high risk
of tumor recurrence, an approach that could also be extended to the
treatment of individuals with genetic predisposition to
cancer.1 Compared with conventional therapies, oral
vaccination is particularly suitable for this category of patients
because of its noninvasive nature and minimal associated distress or
side effects.2 The primary goal of a vaccine is to target
the immunizing antigen(s) to appropriate bone marrow-derived
antigen-presenting cells (APCs), especially dendritic cells (DCs),
which are instrumental for the activation of virgin T
lymphocytes.3
Naked DNA-based vaccines, administered by intradermal or intramuscular
injection, have emerged as a promising approach to control infectious
diseases and, to some extent, tumors.4 The mechanism
inducing host immune response after DNA immunization is not yet fully
elucidated. Several recent reports point to APCs, rather than to
myocytes or other somatic cells, as responsible for antigen processing
and presentation after DNA injection in vivo.5-7 Because of
the low number of APCs present in muscle, especially in the absence of
inflammatory stimuli, immune response induced by intramuscular
injection of plasmid DNA seems to be limited to very potent antigens.
This makes highly desirable the ability to deliver the
antigen-encoding DNA specifically to APC at inductive sites of immune
responses and possibly within a context able to signal the immune
system with the presence of an existing danger.
Attenuated bacterial carrier strains have been recently used as a DNA
delivery system in vitro8-10 and in vivo.11 We
report here that, using Salmonella typhimurium as an oral
carrier for genetic immunization, effective protection against tumor
development can be achieved as a function of in vivo transduction of
APCs, including DCs.
| |
MATERIALS AND METHODS |
Mice.
Female BALB/c (H-2d), 6 to 12 weeks of age, were purchased
from Charles River (Calco, Italy) and maintained at the Istituto Nazionale Tumori under standard conditions according to Institutional Guidelines. This study was approved by the Institutional Ethic Committee for animal use in experimental research.
Cell lines and peptides.
The clone F1.A11 (H-2d) expressing -galactosidase
(-gal) was obtained by transduction of spontaneously transformed
BALB/c fibroblast cell line F1 with the LBSN retroviral
vector.12 These cells express major histocompatibility
complex-I (MHC-I) molecules but not MHC-II and are highly
tumorigenic. P13.1 (H-2d) expressing -gal and its
parental P815 cell lines were used in the cytotoxicity measurement. The
GP1 peptide, encompassing the immunodominant H-2Ld
restricted -gal epitope, was used for in vitro cytotoxic T
lymphocytes (CTL) restimulation.13
Bacterial strains, plasmids, and media.
The auxotrophic S typhimurium AroA strain SL7207
(S typhimurium 2337-65 derivative hisG46, DEL407
[aroA::Tn10{Tc-s}]) was kindly provided by B.A.D. Stocker
(Stanford University, School of Medicine, Stanford, CA). Bacterial
strains were routinely grown at 37°C in LB broth or agar (Sigma, St
Louis, MO), supplemented with 100 µg/mL of ampicillin
when required. The prokaryotic and eukaryotic green fluorescent protein
(GFP) expression vectors pASV214 and pEGFP-C2
(Clontech, Palo Alto, CA) were used to characterize gene
transfer in vivo, whereas the plasmid pCMV (Clontech) was used for
genetic immunization in tumor protection studies. Listeria
monocytogenes vaccine strain  mpl2pGKV20 and its
preparation have been previously described.12
Immunization protocols.
For vaccination, bacteria were grown overnight until they reached
mid-log phase. They were then harvested by centrifugation (3,000g) and resuspended in 5% sodium bicarbonate buffer. Mice were immunized four times at 15-day intervals by gently feeding them
with the bacterial suspension (5 × 108 colony-forming
units [CFU]/mouse) in a volume of approximately 30 µL.
Control mice received the plasmid-less SL7207 strain or buffer only as
sham vaccine.
Measurement of antigen-specific antibodies and Ig isotype assay.
Sera samples were taken 8 days after the second immunization and tested
for their reactivity to -gal in an antigen capture enzyme-linked
immunosorbent assay (ELISA) that can detect IgG levels down to 10 ng/mL, as previously described.12 Sera were also tested on
ovalbumin coated plates (10 µg/mL) as negative control.
Analysis of cytotoxic T-cell activity.
At days 15, 30, and 45 after primary immunization, spleens were removed
from 3 immunized mice and pooled in a single-cell suspension prepared
by mechanical dissociation. Splenocytes were restimulated at 5 × 106 cells with the synthetic peptide GP1 (1 µmol/L) in 1.5 mL medium final volume in 24-well plates. After 5 to 7 days, viable cells were harvested and tested in a
51Cr-release assay for their ability to lyse the
-gal-expressing tumor cell line P13.1; the P815 parental cell line
was used as a negative target.
Cytokines assay.
Cytokines were assayed in supernatants of nylon wool-purified T
lymphocytes (1 × 104/well) cultured 24 to 48 hours in
the presence of immobilized anti-CD3 antibody (clone 145-2C11; 20 µg/mL) in 96-well flat-bottomed plates. Cytokines were measured by
sandwich ELISA using monoclonal antibody (MoAb) pairs
BVDA-1D11/BVD6-24G2 and R4-6A2/XMG1.2 (Pharmingen, San Diego,
CA) for interleukin-4 (IL-4) and interferon- (IFN-) determination, respectively.
In vivo protection studies.
Two weeks after the last immunization, animals received in the left
rear flank a subcutaneous challenge of F1.A11 living cells (104 cells/mouse). Mice were inspected for tumor growth and
size every second day. Tumor growth was measured using calipers and was
recorded as the narrowest and longest surface length. Tumor size (in
square millimeters) was calculated as the product of the mean of these two lengths per animal averaged over the total number of animals in the
group. Statistical differences in tumor size were calculated using the
Student's t-test. The differences in tumor take were evaluated
by  2 test. Mice that were tumor free about 40 days after
first challenge received a second contralateral challenge and were
further inspected for tumor growth.
Characterization of in vivo gene transfer.
Mice were fed with SL7207 harboring either pASV2 or pEGFP-C2 vectors
that contain the GFP gene under the control of prokaryotic and
eukaryotic promoters, respectively, following the above-mentioned vaccination protocol. GFP-expressing cells in spleen suspensions were
detected by flow cytometry. The phenotype of the GFP-positive cells
(FL1+) was determined by double-fluorescence analysis after
staining with biotinylated anti-CD11c (clone N418), anti-F4/80 (clone
F4/80), anti-B220 (clone RA3), anti-Thy1.2 (clone B5.5), and
appropriate isotype-matched negative controls (Pharmigen) in the
presence of Fc-blocking reagent (clone 24G2; Pharmingen) and using
phycoerythrin (PE)-Streptavidin (Pharmingen) as
second-step reagent.
| |
RESULTS |
It has been recently shown that S typhimurium carrier strain
harboring eukaryotic expression vectors can mediate gene transfer in
vivo and trigger both cellular and humoral immune responses against the
encoded antigens.11
To assess the potential of oral genetic immunization to control tumor
development, we had used a tumor model based on a highly aggressive
murine fibrosarcoma transduced with the -gal gene (F1.A11 cells),
which has been shown to behave operationally as a tumor-associated
antigen (TAA).13,15 The attenuated S typhimurium AroA SL7207 strain harboring the pCMV vector that
contains -gal gene under the control of the immediate early promoter
of Cytomegalovirus (CMV) has been used.11 After a
primary immunization per oral, mice received three boosts
at 15-day intervals via the same route. In vitro infection with this
strain resulted in an efficient expression of -gal by spleen, bone
marrow, and peritoneal macrophages (not shown). In vivo vaccination
elicited a tumor-specific T-cell-mediated response: a TAA-specific
cytotoxic response was detectable within 3 weeks after the primary
immunization and steadily increased during the course of the treatment
(Fig 1A). Additionally, a systemic humoral
response was measurable as the presence of specific anti-TAA antibodies
in the sera of immunized mice (Fig 1B). To determine the subclass
distribution of the anti--gal IgG, serum samples were analyzed for
levels of IgG1, 2a, 2b, and 3, resulting in a predominant IgG2a isotype
(not shown). In addition, cytokines secreted by T lymphocytes from
vaccinated mice were measured by ELISA after stimulation with
immobilized anti-CD3 antibody. Mice receiving SL7207 (pCMV) vaccine
display a typical Th1 profile characterized by significant production
of IFN- and negligible IL-4 release; interestingly, lymphocytes from
mice receiving the plasmid-less carrier displayed the same cytokine
profile, which most likely contributes to the explanation
of the adjuvant effect of the carrier per se
(Fig 2). These results confirm that DNA encoding the TAA has been delivered by the carrier to host cells and
that it has been properly transcribed, translated, and presented to the
elements of immune system, resulting in type-1 cellular and humoral
immune responses.
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| Fig 1.
Immune response elicited by S
typhimurium-mediated DNA immunization. (A) CTL-mediated recognition
of -gal-expressing tumor target cells. Splenocyte cells from mice
vaccinated with SL7207 (pCMV;  ) or SL7207 ( ) at the indicated
time points (arrows) were restimulated in vitro for 5 days in presence
of GP1 peptide. After culture, lymphocytes were tested in a
51Cr-release assay at a 50:1 E/T ratio using P13.1 as
target. Results are the mean of triplicates; the percentage of
nonspecific lysis of P815 cells has been subtracted.
(B) Serum IgG levels were measured in antigen-capture
ELISA: () -gal-reactive IgG; ( ) ovalbumin-reactive IgG.
Results are from sera pools of 3 mice each.
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| Fig 2.
Profiles of cytokines released by anti-CD3-stimulated T
lymphocytes from mice receiving Salmonella-based vaccines.
Spleen T lymphocytes were isolated and nylon-wool purified 45 days
after primary vaccine administration and plated in the presence of
immobilized anti-CD3 MoAb for 24 or 48 hours, respectively, to measure
IFN- and IL-4 release. Cytokine levels were determined by sandwich
ELISA.
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We analyzed the efficacy of the immune response elicited after
Salmonella-mediated DNA-transfer to prevent tumor take. Mice immunized with the strain SL7207 (pCMV) or plasmid-less carrier as
control were challenged subcutaneously with F1.A11 cells
(104/mouse). About 80% of the animals vaccinated with the
plasmid-less carrier developed tumors (Fig
3A), whereas tumor take was significantly reduced (P < .01)
in the animals receiving the recombinant carrier. Within this group,
the few animals that developed tumors had significantly reduced tumor
size and prolonged survival compared with controls (Fig 3B).
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| Fig 3.
Protective memory response against tumor challenge in
mice receiving S typhimurium-mediated DNA oral immunization.
(A) Tumor-take curves. After immunization with SL7207 (pCMV;
squares) or SL7207 (triangles), mice received a first subcutaneous
challenge with F1A11 tumor cells (solid symbols); after 40 days,
tumor-free mice received a second contralateral challenge (open
symbols). Tumor growth was inspected every other day by palpation. Each
group included 15 mice. (B) Tumor size (in square millimeters)
progression in immunized mice. Statistically significant differences
are indicated: P < .01 (**).
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Also, recombinant Listeria carriers expressing the target
antigen as a bacterial protein have been previously tested for oral vaccination in animal tumor models.12,16 We compared the
efficacy of Salmonella-based DNA vaccine with that of
Listeria-derived protein vaccine (mpl2pGK20)
previously described. Salmonella-based vaccine seems more
efficient, although moderately, than Listeria mpl2pGK20 in
preventing the take of F1.A11 fibrosarcoma
(Fig 4). However, more
extensive comparison between DNA and protein-based oral vaccination
mediated by the same bacterial carrier are necessary to raise a
conclusion.
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| Fig 4.
Comparison of the efficacy of Salmonella- and
Listeria-based vaccines in preventing the growth of murine
fibrosarcoma F1.A11. After immunization with Salmonella SL7207
(pCMV; ), Listeria  mpl2pGK20 ( ), and saline
only ( ) mice received a subcutaneous challenge with
F1A11 tumor cells. Tumor growth was inspected every other day by
palpation. Each group included 15 mice.
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Long-lasting immune memory is a requisite for an effective antitumor
response and metastases control.12 Therefore, the ability of Salmonella-based vaccine to trigger a persistent antitumor immunity was further evaluated. Mice that remained disease-free after
the first challenge were subjected to a second contralateral challenge
with F1.A11 cells. Animals immunized with SL7207 (pCMV) completely
rejected the second challenge (Fig 3A,  ). In contrast, the few mice
(20%) that remained tumor free in the group receiving the plasmid-less
carrier quickly developed tumors (Fig 3A,  ), likely due to their
lack of TAA-specific immune response (Fig 1A and B).
To determine whether APCs express the transgene delivered by the orally
administered Salmonella carrier, mice were fed with the SL7207
strain harboring an expression vector encoding the GFP under the
control of either an eukaryotic (pEGFP-C2) or a prokaryotic (pASV2)
promoter. Using cytofluorometric analysis, GFP was detectable in spleen
cells of mice treated with SL7207 (pEGFP-C2) starting on day 28 after
the first vaccine administration (Fig 5B).
In contrast, GFP was not detectable in spleen cells of animals
receiving the Salmonella containing the constitutive expression
system (pASV2), which directs GFP synthesis only within the carrier
(Fig 5A, D, and F). GFP expression by pASV2 was undetectable even at
earlier time points, immediately after vaccination (8 to 24 hours; data
not shown), likely because of the rapid in vivo clearance of the
bacterial carrier. These results confirmed that antigen can be
expressed in vivo, at the systemic level, by eukaryotic cells after
Salmonella-mediated gene transfer. The phenotype of the
splenocytes subpopulation(s) expressing GFP was characterized with
MoAbs recognizing DC, macrophage, or lymphocyte subsets. Approximately
19% of the splenocytes expressed GFP (Fig 5B). Among them, about 50%
of the CD11cbright DCs (Fig 5C) and 30% of
F4/80+ macrophages (Fig 5E) were scored as positive for GFP
expression. No B220+ or Thy1.2+ lymphocytes
were scored positive for GFP expression (Fig 5G and H). These results
confirm that Salmonella-mediated transgene expression occurs
within APCs. This expression is long-lasting, being detectable up to 7 days after the last vaccine administration, and it is detectable within
secondary lymphoid organs. GFP expression by DCs points to the
potential of a vaccination approach that results in DC-mediated
delivery of transfected antigens to the T-cell-dependent areas of
secondary lymphoid organs.
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| Fig 5.
Characterization of in vivo S
typhimurium-mediated DNA gene transfer to APCs in orally vaccinated
mice. Expression of GFP, naturally emitting green fluorescence, was
determined by flow cytometry in total spleen cells from mice receiving
the S typhimurium SL7207 strain carrying either the expression
vector pASV2 (A, D, and F) or pEGFP-C2 (B, C, E, G, and H), which
contain the GFP coding gene under the control of either a constitutive
prokaryotic or eukaryotic promoter, respectively. Spleen cell subsets
expressing GFP were identified by two-color fluorocytometric analysis
after staining with biotinylated anti-CD11c, anti-F4/80, anti-B220, and
anti-Thy1.2 followed by PE-Streptavidin. (C) and (D) display GFP
expression by cells gated as positive for CD11c marker; (E) and (F)
display GFP expression by cells gated as positive for F4/80 marker; (G)
and (H) display GFP expression by cells gated as positive for B220 and
Thy1.2, respectively. Control samples stained with appropriated
biotin-conjugated isotype-matched negative control MoAbs followed by
PE-Streptavidin were included for gate setting.
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| |
DISCUSSION |
We have shown that Salmonella-based DNA immunization allows us
to specifically target antigen expression in vivo to APCs, thus
inducing MHC-I- and MHC-II-restricted antitumor immune responses. Vaccine-mediated induction of TAA-specific immune response, although required, could be insufficient to reject a tumor in absence of appropriate inflammatory costimuli, because such costimulation may
avoid tolerance or ignorance of the antigen.17 The
bacterial carrier may function as a natural adjuvant, because bacteria
are known to induce release of tumor necrosis factor- (TNF-),
IFN-, IL-12, and other proinflammatory mediators18 that
enhance early innate immunity and thereafter create an inflammatory
context that likely favors DC maturation to antigen-presenting
function.3 Therefore, Salmonella not only carries
the eukaryotic expression cassette, but also may act as natural
adjuvant.
After DNA immunization, muscle cells receiving the naked DNA injection
express the antigen that is likely captured, processed, and presented
by APCs as a consequence of cross-priming.7,19,20 Few APCs
may also be directly transduced by injected DNA.21 Antigen
presentation by somatic cells is generally inefficient because of the
lack of either MHC-II or costimulatory molecules; even resting DCs are
a priori poor APCs and require additional signals such as tissue injury
or infections to complete their maturation and activate their
functions. Therefore, the efficacy of naked DNA vaccination largely
depends on the presence of either immunostimulatory motifs within
plasmid DNA22 or coinjected DNA coding for inflammatory
cytokines15,23 or on drug-mediated tissue
injury,24 all of which induce local inflammation and
recruitment of APCs. In mice vaccinated with Salmonella-based
carriers, gene transfer would preferentially occur in phagocytic cells,
which are the main target for bacterial infection in
vivo.25 The protein(s) encoded by the transgene would then
be released within the APCs and delivered to the antigen presentation
pathways. Stimulation of APCs by bacterial compounds would further
promote their functional maturation and migration towards inductive
sites of lymphoid organs tuning up the immune response.17
Recombinant vaccines that involve the use of engineered bacteria have
been approached. Listeria monocytogenes displays
characteristics that made it a promising cancer vaccine
vector.12,16,26 Antigens have been expressed in
Listeria after different strategies to get intracellular
protein, protein fused with listeriolisin O, or fusion protein to be
retained on the bacterial surface or exported outside the
carrier. However, the use of bacterial carrier for transfer of genetic material has never been approached before in vivo,
despite evidence that indicates that bacteria can mediate DNA transfer
to mammalian cells in vitro.8-10 The possibility of
macromolecule delivery in vivo to specific tissues, cells, or cellular
compartments remains one of the major challenges that biotechnology
should face. Bacterial carriers might offer the possibility of
targeting and expressing in vivo human proteins other than small viral
or bacterial protein into phagocytic cells. In aiming at this
goal, some factors should be considered: (1) the
molecular size and the posttranscriptional modifications of the
protein; (2) the possible toxicity of the protein for the carrier; and
(3) the possibility that the competition between the
synthesis of heterologous and bacterial proteins may influence the
performance of the carrier by affecting its viability and invasiveness.
Extensive work has been performed to transfect DCs in vitro with genes
coding for relevant antigens for immunotherapy of tumors.27
We demonstrate, for the first time to our knowledge, that S
typhimurium, used as a DNA-delivery system, induces antigen expression in vivo not only in macrophages, but also in DCs. This finding underscores the possibility of loading DCs without need for ex
vivo manipulations. We cannot exclude the possibility that cells other
than APCs expressing the antigen may also contribute to the overall
vaccine efficacy. Finally, S typhimurium-based carriers may
offer the opportunity to gene-target monocyte/macrophages to correct
their genetic defects.
| |
FOOTNOTES |
Submitted April 14, 1998;
accepted June 24, 1998.
Supported by Telethon-Italy (Grant No. A102), AIRC, and CNR.
Address reprint requests to Mario P. Colombo, PhD,
Division of Experimental Oncology D, Istituto Nazionale per Lo Studio e la Cura dei Tumori, Via Venezian 1, I-20133 Milano, Italy; e-mail: mcolombo{at}istitutotumori.mi.it.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank B.A.D. Stocker for kindly providing us with the
SL7297 strain, Prof K.N. Timmis for valuable discussion, and Ralph
Steinman, B. Perussia, and G. Parmiani for critical reading.
| |
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Abstract
Introduction
Abstract