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Enhancement of Sindbis Virus
Self-Replicating RNA Vaccine Potency by
Linkage of Mycobacterium tuberculosis Heat
Shock Protein 70 Gene to an Antigen Gene
Wen-Fang Cheng, Chien-Fu Hung, Chee-Yin Chai, Keng-Fu
Hsu, Liangmai He, Charles M. Rice, Morris Ling and T.-C.
Wu
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 166:6218-6226; ;
doi: 10.4049/jimmunol.166.10.6218
http://www.jimmunol.org/content/166/10/6218
Enhancement of Sindbis Virus Self-Replicating RNA Vaccine
Potency by Linkage of Mycobacterium tuberculosis Heat Shock
Protein 70 Gene to an Antigen Gene1
Wen-Fang Cheng,2*¶ Chien-Fu Hung,2* Chee-Yin Chai,*储 Keng-Fu Hsu,*# Liangmai He,*
Charles M. Rice,** Morris Ling,* and T.-C. Wu3*†‡§
NA replicon vaccines can be derived from ␣ virus vectors, such as Sindbis virus (1), Semliki Forest virus (2, 3),
or Venezuelan equine encephalitis virus (4) vectors.
These vaccines are self-replicating and self-limiting and may be
administered as either RNA or DNA, which is then transcribed into
RNA replicons in transfected cells or in vivo (5, 6). Self-replicating RNA is capable of replicating in a diverse range of cell types
and allows the expression of the Ag of interest at high levels (7).
Additionally, self-replicating RNA eventually causes lysis of
transfected cells (8). These vectors therefore do not raise the concern associated with naked DNA vaccines of integration into the
host genome. This is particularly important for vaccine development targeting proteins that are potentially oncogenic, such as the
human papillomavirus (HPV)4 E6 and E7 proteins.
R
Departments of *Pathology, †Obstetrics and Gynecology, ‡Molecular Microbiology
and Immunology, and §Oncology, Johns Hopkins Medical Institutions, Baltimore,
MD 21205; ¶Department of Obstetrics and Gynecology, National Taiwan University
Hospital, National Taiwan University, Taipei, Taiwan; 储Department of Pathology,
School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; #Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, Taiwan; and **Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63130
Received for publication May 10, 2000. Accepted for publication March 1, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants U19 CA72108-02 and RO1 CA72631-01, Cancer Research Institutes, American Cancer Society, and the Alexander and Margaret
Stewart Trust grant.
2
W.-F.C. and C.-F.H. contributed equally to this paper.
3
Address correspondence and reprint requests to Dr. T.-C. Wu, Department of Pathology,
Johns Hopkins University School of Medicine, Ross Research Building, Room 659, 720
Rutland Avenue, Baltimore, MD 21205. E-mail address: [email protected]
4
Abbreviations used in this paper: HPV, human papillomavirus; HSP, heat shock
protein; DCs, dendritic cells; GFP, green-fluorescent protein; BHK, baby hamster
kidney; ␤-gal, ␤-galactosidase; LDH, lactate dehydrogenase.
Copyright © 2001 by The American Association of Immunologists
In the past few years, studies have demonstrated that immunization with heat shock protein (HSP) complexes isolated from tumor or virus-infected cells are able to induce potent antitumor (9)
or antiviral immunity (10). Immunogenic HSP-peptide complexes
can also be reconstituted in vitro by mixing the peptides with HSPs
(11), and HSP-based protein vaccines can also be administered by
fusing Ags to HSPs (12). We have recently demonstrated that linkage of HPV-16 E7 Ag to Mycobacterium tuberculosis HSP70
leads to the enhancement of DNA vaccine potency (13, 14). These
investigations have made HSPs attractive for use in immunotherapy. The HSP vaccines that have been tested thus far have been in
the form of peptide/protein-based vaccines or DNA-based vaccines. To date, HSPs have not been applied in the form of selfreplicating RNA vaccines.
In this study, we chose HPV-16 E7 as a model Ag for vaccine
development because HPVs, particularly HPV-16, are associated
with most cervical cancers. HPV oncogenic proteins, E6 and E7,
are coexpressed in most HPV-containing cervical cancers and are
important in the induction and maintenance of cellular transformation. Therefore, vaccines targeting E6 or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical
malignancies (for review, see Refs. 15 and 16). HPV-16 E7 is a
well-characterized cytoplasmic/nuclear protein that is more conserved than E6 in HPV-associated cancer cells and has been applied in a variety of HPV vaccines. We therefore chose to use
HPV-16 E7 as our model Ag.
In our current study, we investigated whether genes linking
HSP70 to full-length E7 can enhance the potency of self-replicating Sindbis RNA vaccines. We showed that a Sindbis RNA vaccine linking HSP70 to E7 significantly increased expansion and
activation of E7-specific CD8⫹ T cells and NK cells, bypassing the
CD4 arm and resulting in potent antitumor immunity against E7expressing tumors. We also found that the Sindbis E7/HSP70 RNA
vaccine could induce apoptotic death of transfected cells. Our in
0022-1767/01/$02.00
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Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern
of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to
Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus
self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene
generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the
wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can
be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can
wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into
one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8ⴙ T cells.
These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines. The
Journal of Immunology, 2001, 166: 6218 – 6226.
The Journal of Immunology
vitro studies indicated that E7 Ag from E7/HSP70 RNA replicontransfected cells can be processed by bone marrow-derived dendritic cells (DCs) and presented more efficiently through the MHC
class I pathway than wild-type E7 RNA replicon-transfected cells.
Our study may have important implications for vaccine
development.
Materials and Methods
Plasmid DNA constructs and preparation
In vitro RNA preparation
The generation of RNA transcripts from SINrep5-HSP70, SINrep5-E7,
SINrep5-E7/GFP, SINrep5-E7/HSP70, and SINrep5 was performed using
the protocol as previously described (18). Briefly, SpeI was used to linearize DNA templates for the synthesis of RNA replicons from SINrep5HSP70, SINrep5-E7, SINrep5-E7/HSP70, SINrep5-E7/GFP, and SINrep5
constructs. RNA vaccines were transcribed in vitro and capped using SP6
RNA polymerase and capping analogue from the in vitro transcription kit
(Life Technologies, Gaithersburg, MD) according to vendor’s manual. After synthesis, DNA was removed by digestion with DNase I. Synthesized
RNA was quantified and analyzed using denaturing formaldehyde agarose
gels (19). The purified RNA was divided into aliquots to be used for vaccination in animals and for transfection of a baby hamster kidney (BHK21)
cell line. The protein expression of the transcripts was assessed by transfection of the RNA into BHK21 cells using electroporation.
Cell lines
BHK21 cells were obtained from the American Type Culture Collection
(Manassas, VA) and grown in Glasgow MEM supplemented with 5% FBS,
10% tryptose phosphate broth, 2 mM glutamine, and antibiotics. Cells were
kept at 37°C in a humidified 5% CO2 atmosphere and were passaged every
2 days. The production and maintenance of TC-1, an HPV-16 E7-expressing tumor cell line, have been described previously (20). On the day of
tumor challenge, TC-1 cells were harvested by trypsinization, washed
twice with 1⫻ HBSS, and finally resuspended in 1⫻ HBSS to the designated concentration for injection.
CTL assays
Cytolysis was determined by quantitative measurements of lactate dehydrogenase (LDH) using CytoTox96 nonradioactive cytotoxicity assay kits
(Promega, Madison, WI) according to the manufacturer’s protocol. Briefly,
splenocytes were harvested and pooled 2 wk after RNA vaccination. Five
mice were used for each vaccinated group. Splenocytes were cultured with
1 ␮g/ml E7 peptide (aa 49 –57, RAHYNIVTF) in a total volume of 2 ml
RPMI 1640, supplemented with 10% (v/v) FBS, 50 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 2 mM nonessential amino acids in a 24-well tissue culture plate for 6 days as effector
cells. For controls, mAb GK1.5 (21) was used for CD4 blocking, and mAb
2.43 (22) was used for CD8 blocking. mAb 2.43 or mAb GK1.5 was added
to the cultured splenocytes at a concentration of 50 ␮g/ml on day 5 and
incubated overnight. CTL assays were performed on day 6. TC-1 tumor
cells were used as target cells. The TC-1 cells mixed with splenocytes at
various E:T ratios. After 5 h incubation at 37°C, 50 ␮l of the cultured
medium were collected to assess the amount of LDH in the cultured medium according to the manufacturer’s protocol. The percentage of lysis was
calculated from the equation: 100 ⫻ [(A ⫺ B)/(C ⫺ D)], in which A is the
reading of experimental-effector signal value, B is the effector spontaneous
background signal value, C is maximum signal value from target cells, D
is the target spontaneous background signal value.
ELISA
For the determination of IFN-␥ in the supernatant of cultured splenocytes,
splenocytes were harvested 2 wk after vaccination and cultured with 1
␮g/ml E7 peptide (aa 49 –57) containing the MHC class I epitope (RA
HYNIVTF) (23) or 10 ␮g/ml E7 peptide (aa 30 – 67) containing the class
II epitope (DSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRL)
(24) in a total volume of 2 ml RPMI 1640, supplemented with 10% (v/v)
FBS, 50 U/ml penicillin and streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 2 mM nonessential amino acids in a 24-well tissue
culture plate for 6 days. As a control, mAb 2.43 (22) was used for CD8
blocking. mAb 2.43 (50 ␮g/ml) was added to the cultured splenocytes
during the incubation period. The supernatants were harvested and assayed
for the presence of IFN-␥ using ELISA kits (Endogen, Woburn, MA) according to the manufacturer’s protocol. Splenocytes from mice vaccinated
with Sig/E7/LAMP-1 RNA vaccine (18) were pulsed with E7 peptide and
used as a positive control for the ELISA.
For the determination of E7 protein from BHK21 cells transfected with
SINrep5-E7 or -E7/HSP70 RNA, an indirect ELISA method was used as
described previously (18). The ELISA plate was read with a standard
ELISA reader at 450 nm.
In vivo tumor protection experiments
For the tumor protection experiment, mice (5 per group) were immunized
i.m. with 10 ␮g/mouse SINrep5 RNA, SINrep5-HSP70, SINrep5-E7, SINrep5-E7 mixed with SINrep5-HSP70, SINrep5-E7/GFP, or 0.1, 1, or 10
␮g/mouse SINrep5-E7/HSP70 RNA. Fourteen days after immunization,
mice were injected i.v. with 1 ⫻ 104 cells/mouse TC-1 tumor cells in the
tail vein. Three weeks after tumor challenge, mice were euthanized. The
number of tumor nodules on the lung surface in each mouse was evaluated
and counted by experimenters blinded to sample identity.
In vivo Ab depletion experiments
Female C57BL/6 mice (6 to 8 wk old) from the National Cancer Institute
(Frederick, MD) were purchased and kept in the oncology animal facility
of The Johns Hopkins Hospital (Baltimore, MD). All animal procedures
were performed according to approved protocols and in accordance with
recommendations for the proper use and care of laboratory animals.
The procedure for in vivo Ab depletion has been described previously (18,
25). In brief, mice were vaccinated with 1 ␮g/mouse self-replicating SINrep5-E7/HSP70 RNA i.m. and challenged with 1⫻ 104 cells/mouse TC-1
tumor cells via tail vein injection. Depletions were started 1 wk before
tumor challenge. Isotype IgG2a Ab (PharMingen, San Diego, CA) was
used as a nonspecific control. mAb GK1.5 (21) was used for CD4 depletion, mAb 2.43 (22) was used for CD8 depletion, and mAb PK136 (26) was
used for NK1.1 depletion. Flow cytometry analysis revealed that ⬎95% of
the appropriate lymphocytes subset were depleted with a normal level of
other subsets. Depletion was terminated on day 21 after tumor challenge.
RNA vaccination
Cell surface marker staining and flow cytometry analysis
All SINrep5 RNA vaccines were generated using in vitro transcription as
described above. RNA concentration was determined by OD at 260 nm.
The integrity and quantity of RNA transcripts were further checked using
denaturing gel electrophoresis. Mice were vaccinated i.m. with 10 ␮g/
mouse SINrep5-HSP70, SINrep5-E7, SINrep5-E7 mixed with SINrep5HSP70, SINrep5-E7/GFP, or SINrep5 RNA vaccines in the right hind leg
while SINrep5-E7/HSP70 was administered in doses of 0.1, 1, and 10
␮g/mouse.
Splenocytes removed from naive or vaccinated groups of mice were immediately treated with cell surface marker staining using a previously described protocol (18). Cells were then washed once in FACScan buffer and
stained with PE-conjugated monoclonal rat anti-mouse NK1.1 Ab and
FITC-conjugated monoclonal rat anti-mouse CD3 Ab (PharMingen). The
population of NK cells was NK1.1⫹ and CD3⫺. The percentage of NK
cells in mice immunized with various self-replicating RNA vaccines was
analyzed using flow cytometry.
Mice
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The generation of pcDNA3-HSP70, pcDNA3-E7, and pcDNA3-E7/HSP70
has been described previously (13). For the generation of pcDNA3-GFP, a
DNA fragment encoding the green-fluorescent protein (GFP) was first amplified with PCR using pEGFPN1 (Clontech, Palo Alto, CA) as a template
and a set of primers: 5⬘-atcggatccatggtgagcaagggcgaggag-3⬘ and 5⬘-gg
gaagctttacttgtacagctcgtccatg-3⬘. The amplified product was further cloned
into the BamHI/HindIII cloning sites of pcDNA3 vector. For the generation
of pDNA3-E7/GFP, a DNA fragment encoding HPV-16 E7 was first amplified with PCR using pcDNA3-E7 as a template and a set of primers:
5⬘-ggggaattcatgcatggagatacaccta-3⬘ and 5⬘-ggtggatccttgagaacagatgg-3⬘.
The amplified product was further cloned into the EcoRI/BamHI cloning
sites of pcDNA3-GFP. The Sindbis virus RNA replicon vector, SINrep5
(17), and SINrep5-E7 (18) have been described previously. For the generation of SINrep5-HSP70 and SINrep5-E7/HSP70, DNA fragments encoding M. tuberculosis HSP70 and chimeric E7/HSP70 were isolated from
pcDNA3-HSP70 and pcDNA3-E7/HSP70, respectively, and further cloned
into the corresponding XbaI and PmeI sites of the SINrep5 vector to generate SINrep5-HSP70 and SINrep5-E7/HSP70 constructs. For the generation of SINrep5-E7/GFP, DNA encoding E7/GFP was isolated from
pcDNA3-E7/GFP and further cloned into XbaI/PmeI sites of SINrep5. The
accuracy of these constructs was confirmed by DNA sequencing.
6219
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SELF-REPLICATING E7/HSP70 RNA VACCINE FOR E7-EXPRESSING TUMORS
FIGURE 1. Schematic diagram of the SINrep5 self-replicating RNA
transcripts. A methylated m7Guo (M7G) “cap” is located at the 5⬘-end of
the mRNA, followed by a sequence responsible for the self-replication
(replicase), the gene of interest (i.e., E7, HSP70, E7/GFP, or E7/HSP70),
and a polyadenylated tail (AAAA).
In vitro cell death analysis
CTL assay using DCs pulsed with BHK21 cells transfected with
various RNA transcripts
We performed CTL assays using DCs pulsed with BHK21 cells transfected
with various RNA transcripts using a protocol similar to what has been
described by Albert et al. (28, 29) with modifications. DCs were generated
by culture of bone marrow cells in the presence of GM-CSF as described
previously (18). BHK21 cells (1 ⫻ 107) were transfected with 4 ␮g various
Results
Construction and characterization of self-replicating RNA
constructs
Generation of plasmid DNA constructs and subsequent preparation
of self-replicating SINrep5 RNA constructs was performed as described in Materials and Methods. The SINrep5 vector contains
the genes encoding Sindbis virus RNA replicase and the SP6 promoter (17). A schematic diagram of SINrep5, SINrep5-HSP70,
SINrep5-E7, SINrep5-E7/GFP, and SINrep5-E7/HSP70 RNA
transcripts using SP6 RNA polymerase is shown in Fig. 1. An
ELISA was performed to demonstrate E7 protein expression in
BHK21 cells transfected with various self-replicating RNA constructs. SINrep5-E7 and SINrep5-E7/HSP70 expressed comparable amounts of E7 protein (data not shown).
Vaccination with self-replicating SINrep5-E7/HSP70 RNA
enhances an E7-specific cytotoxic immune response
CD8⫹ T lymphocytes are one of the most crucial effectors for
inducing antitumor immunity. To determine the quantity of E7specific CD8⫹ T cell responses generated by the SINrep5-E7/
FIGURE 2. CTL assays. A, Mice (5 per group) were immunized with various RNA vaccines via i.m. injection. Splenocytes from mice were pooled 14
days after vaccination. To perform the cytotoxicity assay, splenocytes were cultured with E7 peptide (aa 49 –57, RAHYNIVTF) containing MHC class I
epitope for 6 days and used as effector cells. TC-1 tumor cells served as target cells. The TC-1 cells mixed with splenocytes at various E:T ratios. Cytolysis
was determined by quantitative measurements of LDH. The self-replicating RNA E7/HSP70 vaccine generated a significantly higher percentage of specific
lysis than did the other RNA vaccines (p ⬍ 0.001). B, CTL assays using splenocytes from SINrep5-E7/HSP70-vaccinated mice with blocking of subsets
of lymphocytes. CTL assays with CD4 blocking (䡺), CD8 blocking (E), or without blocking (f). CD8 blocking led to a significant loss of CTL activity.
Error bars apply to three samples from each vaccination group. CTL assays shown here are from one representative experiment of two performed.
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BHK21 cells (1 ⫻ 107) were transfected with 4 ␮g SINrep5, SINrep5-E7,
SINrep5-HSP70, or SINrep5-E7/HSP70 RNA transcripts. We used cells
transfected with SINrep5-␤-gal to determine transfection efficiency. The
SINrep5-␤-gal transfected cells were fixed and stained for lacZ expression
using 5-bromo-4-chloro-3-indolyl-D-galactoside (27). In general, the transfection efficiency in our electroporation was consistent and measured to be
⬃30%. Unmodified BHK21 cells and electroporated BHK 21 cells without
RNA were used as controls. BHK21 cells were collected and assessed
every 24 h, until h 72. The percentage of apoptotic BHK21 cells was
analyzed using annexin V apoptosis detection kits (PharMingen) according
to the manufacturer’s protocol, followed by flow cytometry analysis. The
percentage of apoptotic cells was corrected for transfection efficiency.
self-replicating SINrep5 RNA constructs via electroporation as described
above. BHK21 cells were collected 16 –20 h after electroporation. The
levels of E7 protein expression in BHK21 cells transfected with SINrep5-E7 or SINrep5-E7/HSP70 RNA transcripts were determined by
ELISA as described above. Transfected BHK21 cells (3 ⫻ 105) were then
coincubated with 1 ⫻ 105 bone marrow-derived DCs at 37°C for 48 h.
These DCs were used as target cells and Db-restricted E7-specific CD8⫹ T
cells (30) were used as effector cells. CTL assays were performed with
effector cells and targets cells (1 ⫻ 104/well) mixed together at various E:T
ratios (1:1, 3:1, and 9:1) in a final volume of 200 ␮l. After 5 h incubation
at 37°C, 50 ␮l of the cultured medium were collected to assess the amount
of LDH in the cultured medium as described above. DCs coincubated with
untransfected BHK21 cells, transfected BHK21 cells alone, untreated DCs
alone, and CD8⫹ T cell line alone were included as negative controls.
The Journal of Immunology
Vaccination with self-replicating SINrep5-E7/HSP70 RNA does
not enhance IFN-␥-secreting E7-specific CD4⫹ T cell activity or
anti-E7 Ab titers
To assess the E7-specific CD4⫹ T cell immune responses generated by self-replicating SINrep5-E7/HSP70 RNA, an ELISA was
performed to determine the concentration of IFN-␥ in the supernatant of cultured splenocytes. Splenocytes obtained from mice
vaccinated with various self-replicating SINrep5 RNA vaccines
were cultured in vitro with E7 peptide (aa 30 – 67) containing the
MHC class II epitope (24) for 6 days. As a negative control, an
ELISA was also performed without peptide. In addition, we used
splenocytes from Sig/E7/LAMP-1 RNA-vaccinated mice (18)
pulsed with E7 peptide as a positive control for the ELISA to
ensure the success of this assay. We have previously shown that
mice vaccinated with Sig/E7/LAMP-1 RNA vaccine generated a
significant increase of IFN-␥-secreting CD4⫹ E7-specific T cells
(18). As shown in Fig. 4, splenocytes from mice vaccinated with
SINrep5-E7/HSP70 RNA did not generate a significant increase in
IFN-␥ concentration compared with splenocytes from mice vaccinated with other SINrep5 RNA vaccines. These results suggested
that fusion of HSP70 to E7 did not significantly enhance IFN-␥secreting E7-specific CD4⫹ T cell activity.
The quantity of anti-HPV-16 E7 Abs in the sera of the vaccinated mice was determined using a direct ELISA 2 wk after vaccination. Mice vaccinated with SINrep5-E7/HSP70 did not exhibit
higher titers of E7-specific Abs in the sera of mice than mice vaccinated with the other RNA vaccine constructs (data not shown).
Vaccination with self-replicating SINrep5-E7/HSP70 RNA
protects mice against the growth of TC-1 tumors
To determine whether vaccination with the self-replicating SINrep5-E7/HSP70 RNA could protect mice against E7-expressing
FIGURE 3. ELISA for IFN-␥ secreted by E7-specific CD8⫹ T cells.
Mice were immunized with various self-replicating RNA vaccines via i.m.
injection. Splenocytes were collected 14 days after vaccination. Splenocytes obtained from mice vaccinated with various self-replicating SINrep5
RNA vaccines were cultured in vitro with E7 peptide (aa 49 –57, RAHYNIVTF) containing the MHC class I epitope or without any peptide for
6 days. The supernatants of the culture medium were collected to detect
IFN-␥ concentration using an ELISA. Splenocytes from mice vaccinated
with E7/HSP70 RNA secreted the highest concentration of IFN- ␥ compared with the other RNA vaccines (p ⬍ 0.001, one-way ANOVA). Blocking with CD8-specific Ab (mAb 2.43) (22) led to a significant decrease in
IFN-␥ concentration. Results from the ELISA are from one representative
experiment of three performed.
FIGURE 4. ELISA for IFN-␥ secreted by E7-specific CD4⫹ T cells.
Splenocytes from mice vaccinated with various self-replicating RNA vaccines were cultured in vitro with E7 peptide containing the MHC class II
epitope (aa 30 – 67, DSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKC
DSTLRL) or no peptide (control). The supernatant of the culture medium
was collected to detect IFN-␥ concentration using an ELISA. We used
IFN-␥-secreting splenocytes from Sig/E7/LAMP-1 RNA-vaccinated mice
(18) as a positive control for CD4⫹ IFN-␥-secreting T cells to ensure the
success of this assay. Results from the ELISA are from one representative
experiment of three performed.
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HSP70 RNA vaccine, CTL assays were performed. Splenocytes
from vaccinated mice were cultured with E7 peptide (aa 49 –57)
containing the MHC class I epitope and served as effector cells.
TC-1 tumor cells were used as target cells. As shown in Fig. 2A,
vaccination with SINrep5-E7/HSP70 generated a significantly
higher percentage of specific lysis compared with vaccination with
other SINrep5 RNA vaccines ( p ⬍ 0.001, one-way ANOVA).
Furthermore, CTL activity appeared to be CD8 specific because
blocking with CD8-specific Ab led to a significant loss of specific
lysis (Fig. 2B). Interestingly, vaccination with SINrep5-E7 RNA
resulted in a percentage of specific lysis only slightly higher than
background. This finding was consistent with our previous observation that vaccination with naked wild-type E7 DNA (13) or SINrep5-E7 RNA (18, 25) did not generate strong E7-specific CD8⫹
T cell immune responses, suggesting that wild-type E7 is a
weak Ag.
We also performed an ELISA to determine the concentration of
IFN-␥ in the supernatant of cultured splenocytes. As shown in Fig.
3, splenocytes from mice vaccinated with SINrep5-E7/HSP70
RNA secreted the highest concentration of IFN-␥ compared with
splenocytes from mice vaccinated with other SINrep5 RNA vaccines ( p ⬍ 0.001, one-way ANOVA). The increase of IFN-␥ secretion is likely due to CD8-specific T cells because blocking with
CD8-specific Ab during the in vitro peptide stimulation led to a
significant decrease in IFN-␥ concentration (Fig. 3). These results
also indicated that fusion of HSP70 to E7 significantly enhanced
IFN-␥-secreting E7-specific CD8⫹ T cell activity.
6221
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SELF-REPLICATING E7/HSP70 RNA VACCINE FOR E7-EXPRESSING TUMORS
tumors, an in vivo tumor protection experiment was performed as
described in Materials and Methods. As shown in Fig. 5A, fewer
pulmonary tumor nodules were identified in mice vaccinated with
the self-replicating E7/HSP70 RNA vaccines (0.1, 1, and 10 ␮g)
compared with mice vaccinated with the other RNA vaccines ( p ⬍
0.001, one-way ANOVA). Representative gross photographs of
the lung tumors are shown in Fig. 5B. Our results demonstrated
that self-replicating SINrep5-E7/HSP70 RNA vaccines protected
mice from i.v. tumor challenge even at the low dosage of 0.1 ␮g,
whereas mice vaccinated with 10 ␮g of the other SINrep5 RNA
vaccines developed numerous lung nodules from TC-1 tumor challenge. Our data also showed that linkage of E7 to an irrelevant
protein such as GFP does not generate a significant antitumor effect and that the enhancement of antitumor effect by HSP70 requires physical linkage of HSP70 to E7.
CD8⫹ T cells and NK cells are important for the antitumor
effect generated by vaccination with SINrep5-E7/HSP70 RNA
vaccines
To determine the subset of lymphocytes that are important for
protection against E7-expressing tumor cells, we performed in
vivo Ab depletion experiments. Ab depletion was initiated 1 wk
before tumor challenge and terminated on day 21 after tumor
challenge. As shown in Fig. 6, the mean number of pulmonary
nodules from mice depleted of CD8⫹ T cells or NK1.1 cells was
significantly higher than that observed in mice treated with the
control IgG2a isotype Ab. Similar results were observed in mice
treated with control IgG2a isotype Ab and in mice without Ab
depletion. Furthermore, depletion of NK1.1 cells resulted in a
higher mean number of tumor lung nodules than did CD8⫹
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FIGURE 5. Tumor protection experiments using various SINrep5 self-replicating RNA vaccines. Mice were immunized with various SINrep5 selfreplicating RNA vaccines and challenged with TC-1 tumor cells as described in Materials and Methods. A, The mean number of tumor nodules on the lung
surface of the vaccinated mice was used as a measurement of the effectiveness of the various self-replicating RNA vaccines in controlling HPV-16
E7-expressing tumor growth. There were fewer mean pulmonary nodules in mice vaccinated with self-replicating E7/HSP70 RNA vaccines (0.1, 1, and
10 ␮g) than in mice vaccinated with the other RNA vaccines (10 ␮g; p ⬍ 0.001, one-way ANOVA). The tumor protection experiments were repeated three
times, yielding similar results. B, Representative gross pictures of the lung tumors in each vaccinated group. There are multiple grossly visible lung tumors
in unvaccinated control mice and mice vaccinated with SINrep5 or SINrep5-E7 RNA vaccines. Lung tumors in the SINrep5-E7/HSP70 RNA-vaccinated
group cannot be seen at the magnification provided in this figure.
The Journal of Immunology
6223
Self-replicating RNA vaccines induce apoptosis
FIGURE 6. In vivo Ab depletion experiments to determine the effect of
lymphocyte subsets on the potency of self-replicating SINrep5-E7/HSP70
RNA vaccine. Mice were immunized with 1 ␮g/mouse self-replicating
SINrep5-E7/HSP70 RNA via i.m. injection. Two weeks after vaccination,
mice were challenged with 1 ⫻ 104 TC-1 cells/mouse i.v. via tail vein.
Depletions were initiated 1 wk before tumor challenge and lasted for 28
days. Three weeks after tumor challenge, the mice were sacrificed. The
number of pulmonary tumor nodules in mice depleted of CD8⫹ T cells and
NK1.1 cells was significantly higher than those in mice depleted of CD4⫹
T cells or receiving control IgG2a isotype Ab.
Self-replicating RNA vaccines have been shown to induce apoptotic changes after uptake by cells (3). We therefore evaluated the
percentage of apoptotic cells in BHK21 cells transfected with various RNA vaccines. Because transfection efficiency was only 30%,
the percentages of apoptotic BHK21 cells were corrected for transfection efficiency. As shown in Fig. 8, all of the BHK21 cells
transfected with various RNA vaccines generated a higher percentage of apoptosis compared with the two control groups (untransfected or electroporated without RNA vaccines). We observed no
FIGURE 7. Flow cytometry analysis of NK cells in mice immunized with various self-replicating SINrep5 RNA vaccines. Mice were immunized, and
splenocytes were collected as described in Materials and Methods. Splenocytes were stained for CD3 and NK1.1 immediately without any stimulation. A,
Flow cytometry analysis to demonstrate the number of NK cells in mice immunized with various self-replicating RNA vaccines. The percentage of NK
cells in splenocytes is indicated in the upper left corner. B, Histogram demonstrating the percentages of NK cells in vaccinated mice. The percentage of
NK cells from the splenocytes of mice immunized with self-replicating RNA vaccines was higher than those in mice receiving no immunization. There
was no significant difference in the percentage of NK cells generated by the various self-replicating RNA vaccines. Data from the NK cell surface marker
staining shown here are from one representative experiment of two performed.
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depletion. In comparison, the mean number of pulmonary nodules from mice depleted of CD4⫹ T cells resembled results
obtained from the mice receiving IgG2a isotype Ab, indicating
that CD4⫹ T cells were not critical in generating this effect.
These results suggest that both CD8⫹ T cells and NK cells were
essential for the Ag-specific antitumor immunity generated by
SINrep5-E7/HSP70 RNA vaccine. CD8⫹ T cells and NK cells
have also been found to be important in generating an antitumor
effect in mice treated with autologous tumor-derived heat shock
protein preparations (9).
To investigate whether NK cells were significantly expanded in
mice vaccinated with various RNA vaccines, we performed flow
cytometry analysis of CD3⫺NK1.1⫹ cells. We found that the presence of NK cells was markedly increased in all constructs (E7/
HSP70, E7, HSP70, and control plasmid) relative to naive mice,
indicating that the expansion of NK cells is not limited to the
E7/HSP70 RNA vaccine (Fig. 7). Because our data indicated that
the various RNA replicon-based vaccines each produced a similar
number of NK cells, NK cells alone are probably not sufficient to
account for the antitumor effect generated by the E7/HSP70 RNA
vaccine.
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SELF-REPLICATING E7/HSP70 RNA VACCINE FOR E7-EXPRESSING TUMORS
significant difference in apoptotic changes induced by the different
RNA constructs. Furthermore, we found that there was a steady
decline in apoptosis of BHK21 cells from 24 to 72 h after transfection (with SIN-E7/HSP70: 70.3 ⫾ 3.6% for 24 h, 49.3 ⫾ 4.2%
for 48 h, 18.0 ⫾ 3.1% for 72 h; p ⬍ 0.001, one-way ANOVA).
These data indicate that cells transfected with self-replicating RNA
vaccines undergo apoptotic changes.
Enhanced presentation of E7 through the MHC class I pathway
in DCs pulsed with cells transfected with SINrep5-E7/HSP70
RNA
A potential mechanism for E7-specific CD8⫹ T cell immune activity in vivo is the presentation of E7 through the MHC class I
pathway in DCs after uptake of E7 from RNA replicon-transfected
cells. We used bone marrow-derived DCs coincubated with transfected BHK21 cells as target cells and E7-specific CD8⫹ T cells
(30) as effector cells. CTL assays were performed with various E:T
ratios. As shown in Fig. 9, DCs coincubated with BHK21 cells
transfected with SINrep5-E7/HSP70 RNA generated significantly
higher percentages of specific lysis compared with DCs coincubated with BHK21cells transfected with SINrep5-E7 RNA ( p ⬍
0.001). These results suggested that DCs pulsed with cells expressing E7/HSP70 fusion protein presented E7 Ag through the MHC
class I pathway more efficiently than DCs pulsed with cells expressing wild-type E7 protein.
Discussion
In this study, we observed that the linkage of M. tuberculosis
HSP70 to E7 Ag in a Sindbis virus RNA vector led to enhance-
FIGURE 9. CTL assays to demonstrate enhanced MHC class I presentation of E7 in bone marrow-derived DCs pulsed with BHK21 cells transfected by SINrep5-E7/HSP70 RNA. Bone marrow-derived DCs were
cocultured with various RNA-transfected BHK21 cells and used as target
cells. Db-restricted E7-specific CD8⫹ T cells were used as effector cells
(30). Cytolysis was determined by quantitative measurements of LDH as
described in Materials and Methods. The self-replicating E7/HSP70 RNA
vaccines generated significantly higher percentages of specific lysis (E:T
ratios, 3:1 and 9:1) compared with the other RNA vaccines (p ⬍ 0.001).
CTL assays shown here are from one representative experiment of two
performed.
ment of E7-specific CD8⫹ T cell-mediated immunity and an
impressive antitumor effect. Similarly, we previously demonstrated that E7/HSP70 generated potent immunological and antitumor effects in a naked conventional DNA vector (13) and in
a DNA-based self-replicating RNA replicon vector, also known
as “suicidal DNA” (14). Our data are consistent with prior reports using HIV-1 p24 (12), OVA (31), or influenza nucleoprotein (32) as model Ags linked to HSP70. Fusion to HSP70 increased the immunogenicity of these Ags. Although chimeric
HSP70 fusions have been shown to work well in these models,
at least one study found that gp96, another member of heat
shock proteins, fused with ␤-gal did not lead to activation of
␤-gal-specific T cells in vivo (33). Thus, the strategy of linking
HSP to Ag to enhance vaccine potency may depend on the type
of HSP used for the linkage.
We observed that SINrep5-E7/HSP70 significantly enhanced
E7-specific CD8⫹ T cell responses compared with SINrep5-E7
RNA vaccines in vivo. It seems unlikely that the observed enhancement of E7-specific CD8⫹ T cell responses in vivo occurs
through improvement of direct MHC class I presentation of E7
to CTLs by cells expressing E7/HSP70, a process known as
“direct priming.” Intramuscular delivery of RNA replicons
probably delivers RNA into muscle cells, which are not ideal
professional APCs because they lack costimulatory molecules
that are important for efficient activation of T cells. Even if the
various SINrep5 constructs are delivered to cells other than
muscle cells, self-replicating RNA eventually induces apoptosis
(8). The initially transfected cells are thus unlikely to directly
present Ag in an efficient manner.
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FIGURE 8. Flow cytometry analysis to determine apoptotic death of
host cells induced by self-replicating RNA vaccines. BHK21 cells transfected with various RNA vaccines were stained with annexin V-FITC and
propidium iodide followed by flow cytometry analysis. Unmodified
BHK21 cells and electroporated BHK 21 cells without RNA were used as
controls. The percentages of apoptotic cells revealed a statistical decline
when transfected with SINrep5 RNA 24 –72 h after electroporation (representative with SIN-E7/HSP70: 70.3 ⫾ 3.6% for 24 h, 49.3 ⫾ 4.2% for
48 h, 18.0 ⫾ 3.1% for 72 h; p ⬍ 0.001, one-way ANOVA). No statistical
difference could be found in the percentages of apoptotic cells transfected
with various SINrep5 RNA vaccines. This experiment was repeated twice,
yielding similar results.
The Journal of Immunology
In summary, our results revealed that fusion of the gene encoding M. tuberculosis HSP70 to HPV-16 E7 gene in RNA replicon
can generate significant E7-specific CD8⫹ T cell-mediated immune responses and antitumor effects against HPV-16 E7-expressing murine tumors. Our study also indicated that fusion of HSP70
to an Ag gene may greatly enhance the potency of RNA repliconbased vaccines and can potentially be applied to other cancer systems with known tumor-specific Ags or other infectious diseases.
Acknowledgments
We thank Drs. Drew M. Pardoll, Robert J. Kurman, Chang-Yao Hsieh, and
Keertie Shah for helpful discussions. We thank Drs. Ralph Hruban and
Richard Roden for critical review of the manuscript.
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