Enhancing Efficacy of Recombinant Anticancer
Vaccines With Prime/Boost Regimens That Use Two
Different Vectors
Kari R. Irvine, Ronald S. Chamberlain, Eliza P. Shulman,
Deborah R. Surman, Steven A. Rosenberg, Nicholas P. Restifo*
The identification of tumor-associated antigens has enabled
the development of recombinant and synthetic anticancer vaccines for use in clinical trials (1,2). A number of different recombinant and synthetic vectors, including recombinant vaccinia virus (rVV), recombinant fowlpox virus (rFPV), and
recombinant DNA plasmids (pDNA), developed to express large
quantities of the tumor-associated antigens have been demonstrated to effectively elicit potent humoral (antibody) and cellmediated immune (e.g., cytotoxic T lymphcytes [CTLs]) responses in experimental murine model antigen systems (3–5).
Most significantly, these approaches have been shown to have
therapeutic efficacy in the treatment of tumor-bearing mice (3–
5). Several of these vaccination strategies are now being tested
in clinical trials on patients with metastatic cancer at the National Cancer Institute and elsewhere.
The induction of an effective immune response against cancer
antigens may require repetitive immunizations, since the aim of
these experimental vaccines is the treatment of established tumors. Few studies have examined the therapeutic effect on tumor growth of repetitive boosting with recombinant vectors. It
has been reported, however, that humoral and cellular immune
responses are considerably reduced against the heterologous
protein products generated by the rVV in individuals who have
had previous exposure to the virus (6,7). Preimmunization of
mice with a wild-type vaccinia virus followed by a recombinant
version of the same virus has been shown to preclude the induction of immunity to the recombinant antigen for the natural
lifespan of the animal (8). While these studies were limited to
recombinant vaccinia vaccines, many recombinant vectors including fowlpox, influenza, adenovirus, and bacteria are also
likely to induce anamnestic responses to the vector following
repeated boosting.
In this study, we compared different vaccination strategies of
priming and boosting either with a single vector (homologous
boosting) or with two different vectors (heterologous boosting).
We analyzed the generation of antigen-specific secondary CTLs,
antibody responses, and most importantly, the elicitation of
therapeutic antitumor immunity to develop optimal strategies for
immunization against cancer.
Materials and Methods
Tumor cell lines. CT26.WT is a clone of the N-nitroso-N-methylurethaneinduced (H-2d), undifferentiated colon carcinoma of BALB/c murine origin.
After transduction with a retrovirus encoding the LacZ gene, CT26.WT was
subcloned to generate the b-galactosidase (b-gal)-expressing cell line
CT26.CL25 (4). Cell lines were maintained in complete medium consisting of
RPMI-1640 medium, 10% heat-inactivated fetal calf serum (Biofluids, Rockville, MD), 0.03% L-glutamine, 100 mg/mL streptomycin, 100 U/mL penicillin,
and 50 mg/mL gentamicin sulfate (National Institutes of Health Media Center).
CT26.CL25 was maintained in medium containing 400 or 800 mg/mL G418
(Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD).
Mice. Female BALB/c mice, 6–10 weeks old, were obtained from the Animal
Production Colonies, National Cancer Institute-Frederick Cancer Research and
*Affiliation of authors: Surgery Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD.
Correspondence to: Nicholas P. Restifo, M.D., National Institutes of Health,
Bldg. 10, Rm. 2B54, Bethesda, MD 20892–1502.
See ‘‘Notes’’ following ‘‘References.’’
© Oxford University Press
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
ARTICLES 1595
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
Background: The identification of tumor-associated antigens
and the cloning of DNA sequences encoding them have enabled the development of anticancer vaccines. Such vaccines
target tumors by stimulating an immune response against
the antigens. One method of vaccination involves the delivery of antigen-encoding DNA sequences, and a number of
recombinant vectors have been used for this purpose. To
optimize the efficacy of recombinant vaccines, we compared
primary and booster treatment regimens that used a single
vector (i.e., homologous boosting) with regimens that used
two different vectors (i.e., heterologous boosting). Methods:
Pulmonary tumors (experimental metastases) were induced
in BALB/c mice inoculated with CT26.CL25 murine colon
carcinoma cells, which express recombinant bacterial bgalactosidase (the model antigen). Protocols for subsequent
vaccination used three vectors that encoded b-galactosidase—vaccinia (cowpox) virus, fowlpox virus, naked
bacterial plasmid DNA. Mouse survival was evaluated in
conjunction with antibody and cytotoxic T-lymphocyte responses to b-galactosidase. Results: Heterologous boosting
resulted in significantly longer mouse survival than homologous boosting (all P<.00001, two-sided). Potent antigenspecific cytotoxic T lymphocytes were generated following
heterologous boosting with poxvirus vectors. This response
was not observed with any of the homologous boosting regimens. Mice primed with recombinant poxvirus vectors generated highly specific antibodies against viral proteins. Conclusions: The poor efficacy of homologous boosting regimens
with viral vectors was probably a consequence of the induction of a strong antiviral antibody response. Heterologous
boosting augmented antitumor immunity by generating a
strong antigen-specific cytotoxic T-lymphocyte response.
These data suggest that heterologous boosting strategies may
be useful in increasing the efficacy of recombinant DNA
anticancer vaccines that have now entered clinical trials. [J
Natl Cancer Inst 1997;89:1595–1601]
1596 ARTICLES
ELISA. Serum from immunized mice was collected 21 days after the primary
immunization and 8 days after the final boost to be analyzed for the presence of
antibodies against b-gal protein, wild-type vaccinia virus, or wild-type fowlpox
virus by ELISA, as previously described (3). Specifically, microtiter plates were
either dried down overnight at 37 °C in a nonhumidified incubator with 200
ng/50 mL per well of purified b-gal protein (Sigma Chemical Co., St. Louis,
MO) or coated with either wild-type vaccinia virus (5 × 105 PFU/50 mL per well)
or wild-type fowlpox virus (5 × 105 PFU/50 mL per well) at 4 °C overnight.
Incubation of 5% bovine serum albumin (BSA) in phosphate-buffered saline
(PBS) on each well for 1 hour to prevent nonspecific antibody binding was
followed by a second 1-hour incubation with 50 mL of fivefold dilutions (starting
at 1:100) of test sera. After being washed with 1% BSA in PBS, horseradish
peroxidase-conjugated sheep anti-mouse immunoglobulin G (IgG) F(ab8)2 fragments (at 1:3000 dilution in 1% BSA in PBS) (Amersham International, Amersham, U.K.) were added for 1 hour at 37 °C to detect antibodies immobilized
on the wells. The resulting complex was detected by the chromogen ophenylenediamine (Sigma Chemical Co.). Absorbance was read on a Titertek
Multiskan Plus Reader (Flow Laboratories, McLean, VA) with the use of a
490-nm filter.
Western blot analysis. Mouse antiserum obtained 21 days after the primary
immunization was tested in a western blot for reactivity against b-gal protein,
wild-type vaccinia virus, and wild-type fowlpox virus. To this end, 5 mg of b-gal
protein, 6.6 × 106 PFU of wild-type vaccinia virus, and 2 × 107 PFU of wild-type
fowlpox virus were dissolved in sodium dodecyl sulfate (SDS)–polyacrylamide
gel electrophoresis sample buffer, boiled for 5 minutes, and subjected to electrophoresis by use of a 6%–18% linear gradient SDS–polyacrylamide gel. After
electrophoresis, proteins were transferred for 2 hours onto nitrocellulose paper
(0.45-mm pore size) by use of transfer buffer at room temperature at 25 V. The
blots were then incubated in PBS containing 5% nonfat dry milk for 1 hour at
room temperature. Ten milliliters of a 1:200 dilution of antiserum in PBS with
2% nonfat dry milk was added to each nitrocellulose strip, and the strips were
incubated for 2 hours at room temperature with gentle agitation. After the blots
were washed with PBS containing 0.05% Tween-20, they were incubated with
horseradish peroxidase-conjugated sheep anti-mouse IgG F(ab8)2 fragments (1:
1000 dilution in 1% BSA in PBS) to visualize antibody binding. Bound immunoglobulin was then detected by the incubation of the blots for approximately 3
minutes in 3,38-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) dissolved in distilled water. The reaction was stopped by washing the nitrocellulose
blots for 5 minutes with distilled water.
Results
Prolongation of Survival of Tumor-Bearing Mice by
Boosting With Heterologous Vectors
To compare the effect of repetitive immunization of the recombinant vaccine vectors on tumor growth, long-term survival
studies were performed (Fig. 1). BALB/c mice challenged intravenously with CT26.CL25 (b-gal+) tumor cells were immunized 3 days later with either no immunogen, pDNA (pCMV/
b-gal), rVV expressing b-gal (VJS6), or rFPV expressing b-gal
(rFPV.bg40k). Seventeen days after tumor inoculation, each
group of primed mice was divided into four groups and boosted
with each immunogen to compare all possible heterologous and
homologous immunization strategies.
Mice immunized 17 days after tumor administration with
either pDNA (pCMV/b-gal) or rFPV (rFPV.bg40k) had a slight
increase in survival compared with that of nonimmunized mice
(Fig. 1, A). Two immunizations with rVV (VJS6) significantly
prolonged survival compared with that of unvaccinated mice,
but this result was not statistically different from the result obtained with one immunization 3 days after tumor challenge (Fig.
1, B). Mice that received a boost with a heterologous recombinant viral vector, rFPV, after an initial immunization with rVV
had a longer survival time than mice that received the homologous prime and boost with rVV (P<.00001; Fig. 1, B). Indeed,
50% of the heterologously boosted mice survived longer than 60
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
Development Center, National Institutes of Health (Frederick, MD). The mice
received care in accordance with the guidelines set forth by the animal research
advisory committee of the National Institutes of Health.
Peptides. The synthetic peptide TPHPARIGL, representing the naturally processed H-2 Ld- restricted epitope spanning 876–884 of b-gal, was synthesized by
Peptide Technologies (Washington, DC) to a purity of greater than 99%, as
assessed by high-performance liquid chromatography and amino acid analysis.
Plasmid preparations and delivery of DNA by use of a gene gun. A
plasmid encoding the Escherichia coli lacZ gene under the control of the human
cytomegalovirus (CMV) intermediate-early promoter, designated pCMV/b-gal,
was provided by J. Haynes (Agracetus, Middleton, WI). Closed, circular plasmid
DNA was isolated with the use of Wizard Maxipreps DNA purification kits
(Promega Corp., Madison, WI). Plasmid DNA and gold were coprecipitated by
the addition of 200 mL of 2.5 M CaCl2 during vortex mixing as previously
described (9). DNA-coated gold particles were delivered into the abdominal
epidermis by use of the hand-held helium-driven Accell gene delivery system
(provided by Geniva, Middleton, WI). Each animal received 10 nonoverlapping
deliveries per immunization at a pressure of 400 psi of helium.
Recombinant viruses. The rVV vaccine VJS6 was engineered such that the
E. coli lacZ gene encoding for b-gal was under the control of the early/late
vaccinia virus 7.5K promoter from plasmid pSC65 (5). The recombinant stocks
were initially propagated in the BSC-1 monkey kidney cell line to create a crude
lysate, which was then further purified over a sucrose cushion (10). The rFPV
vaccine used in these studies (rFPV.bg40k) contains the E. coli lacZ gene under
control of the vaccinia virus 40K promoter inserted into the BamHI region of the
fowlpox virus genome as previously described (Therion Biologics Corp., Cambridge, MA) (4).
In vivo treatment studies. BALB/c mice were challenged intravenously with
105 CT26.CL25 tumor cells to establish pulmonary metastases (11). Three days
later, four groups of mice (37–40 mice per group) were primed with either no
immunogen, 10 mg of pCMV/b-gal administered intradermally with the gene
gun, 107 plaque-forming units (PFU) of rVV-b-gal VJS6 administered intravenously, or 107 PFU of rFPV.bg40k administered intravenously. Fourteen days
after tumor inoculation, each group was divided into four groups of nine to 10
mice, each of which was subsequently boosted with the same amount of either
no immunogen, pCMV/b-gal DNA, rVV-b-gal, or rFPV.bg40k to compare the
16 possible heterologous and homologous immunization strategies. These mice
were followed for long-term survival.
Statistical analysis. Kaplan–Meier plots were generated, and the Mantel–
Haenszel test was used to compare the survival of mice belonging to the different
treatment groups; in each case, an adjustment was made for the number of tests
accomplished. This experiment was performed two times with similar results.
The P values stated are derived from two-sided tests.
Generation of CTL and antibody responses. BALB/c mice (eight mice per
group) were vaccinated with either no immunogen, 10 mg of DNA encoding
b-gal intradermally with the gene gun, 107 PFU of rVV (VJS6) intravenously, or
107 PFU of rFPV.bg40k intravenously. Twenty-one days later, each group of
primed mice was divided into four groups of two mice each and were boosted
with each immunogen to compare all heterologous and homologous possibilities.
For the determination of the optimal kinetics of an in vivo secondary CTL
response, mice were killed on days 2, 4, 6, or 8 after the second vaccination, at
which time their spleens were removed and CTL lytic reactivity was assessed
against b-gal-expressing tumor cells without an in vitro stimulation step in a
standard 6-hour 51Cr release assay described below (data not shown). For all
other experiments, mice were killed at the optimal time point, i.e., 4 days following the second vaccination, and in vivo CTL lytic reactivity was assessed.
Pooled serum (from two mice per group) was also taken 8 days after the boost
to evaluate antibody reactivity to b-gal protein via an enzyme-linked immunosorbent assay (ELISA).
51
Cr release assay. Six-hour 51Cr release assays were performed as previously described (12). Briefly, for the radioactive labeling of the target cells, 2 ×
106 of either CT26.WT cells, CT26.CL25 cells, or CT26.WT cells pulsed with
b-gal peptide were incubated with 200 mCi of the g emitter Na51CrO4 in 0.2 mL
of complete medium for 90 minutes. Peptide-pulsed CT26.WT cells were
incubated with 1 mg/mL (approximately 1 mM) antigenic b-gal peptide
(TPHPARIGL) during 51Cr labeling as previously described (3–5). Target cells
were then mixed with effector cells for 6 hours at 37 °C at the effector-to-target
cell ratios indicated (100:1, 50:1, 25:1, etc.). The amount of 51Cr released was
determined by gamma counting, and the percentage of specific lysis was calculated as follows: percent specific lysis 4 [(experimental cpm − spontaneous
cpm)/(maximal cpm − spontaneous cpm)] × 100.
creased the lifespan of mice primed with either rVV or rFPV
compared with the lifespan of mice immunized two times with
pDNA (P 4 .0026 and P 4 .0046 for rVV and rFPV priming,
respectively) (Fig. 1, E). No statistically significant difference in
survival was observed between mice primed with either rVV or
rFPV and boosted with pDNA and the groups of mice that
received a homologous prime and boost of either rFPV or rVV
(Fig. 1, D and E). Considered together, these data suggested that
immunizing and boosting with two different vectors expressing
the same tumor-associated antigen prolonged survival of tumorbearing mice more efficiently than multiple immunizations with
the same vector.
In Vivo Secondary CTL Responses Induced in Mice
Immunized and Boosted With Different Vectors
Expressing the Same Tumor-Associated Antigen
To determine the effect of the different immunization schema
on the induction of an antigen-specific CTL response, we im-
Fig. 1. Immunizing and boosting with different recombinant vectors prolongs the survival of tumor-bearing animals. BALB/c mice were challenged intravenously with 105 CT26.CL25 tumor cells to establish pulmonary metastases (12). Three days later, groups of mice (37–40 animals per
group in each experiment) were primed with either A) no immunogen
(None), B) 107 plaque-forming units (PFU) of recombinant vaccinia virus
VJS6 (rVV) expressing b-galactosidase (b-gal) intravenously, C) 107 PFU
of recombinant fowlpox virus (rFPV.bg40k) expressing b-gal (rFPV) intravenously, D) 10 mg of pCMV/b-gal (recombinant DNA plasmids
[pDNA]) intradermally with the gene gun. Fourteen days after tumor inoculation, each group of mice was divided into four groups of nine to 10
mice each; mice in each of these groups received a second immunization
(boost) with either no immunogen, pDNA, rVV, or rFPV to compare all
possible heterologous and homologous immunization strategies. In panel
E, mice were administered either no treatment, rVV, rFPV, or pDNA 3
days after tumor inoculation and then boosted with pDNA 14 days later.
The control group that received no treatment is shown in all panels as
None-None. This figure represents pooled data from two experiments performed identically. Mice initially immunized with rVV then received a
boosting vaccination with either the rFPV (B) or pDNA (E); each of these
groups of mice exhibited prolonged survival compared with the survival
among the control, unvaccinated group (P<.00001). Mice initially immunized with an rFPV then received a boosting vaccination with either rVV
(C) or pDNA (E); each of these groups exhibited prolonged survival compared with that of the control, unvaccinated group (P<.00001). Mice that
were initially immunized with pDNA and then received a boosting vaccination with either rVV or rFPV exhibited prolonged survival compared
with that of the untreated group (P<.00001 for both) (panel D). All P
values are two-sided.
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
ARTICLES 1597
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
days. A similar pattern was observed for rFPV immunization
(Fig. 1, C). Whereas one immunization with rFPV did not significantly enhance survival compared with that of control mice,
a prolongation of survival was seen with two sequential immunizations of rFPV (P<.0001). However, mice administered rFPV
and boosted with the heterologous vector rVV had a significant
increase in survival compared with that of the mice that received
two doses of rFPV (P<.00001); 80% of the mice that received
the heterologous combination survived for greater than 60 days
(Fig. 1, C).
For pDNA immunization, a small but significant increase in
survival was observed in the group of mice that received a prime
and a boost with pDNA (P 4 .0026) (Fig. 1, D). Boosting
pDNA immunization with either heterologous viral vector, rVV
or rFPV, significantly extended longevity compared with the
lifespan of the untreated group (P<.0001) or with the results
obtained with a prime and boost of pDNA (P 4 .0002 and P 4
.0028, for boosting pDNA immunization with rVV and rFPV,
respectively) (Fig. 1, D). Conversely, boosting with pDNA in-
Augmented Anti-b-gal Antibody Responses Elicited After
a Boost With Any Combination of pDNA, rVV, or rFPV
To study antigen-specific humoral immunity by use of the
different combinations of the rDNA, rVV, and rFPV vaccines,
we tested serum samples, harvested 8 days after the boost, by
ELISA for antibody reactivity against b-gal protein. b-galspecific antibody titers were increased following a primary immunization with rVV with boosts of either pDNA, rVV, or
rFPV. (Titers increased from 1:50 with no boost to 1:250 for
each group [Fig. 3, A].) After rFPV immunization, b-gal titers
were also dramatically boosted by a second immunization with
either pDNA (titer 4 1:6250), rVV (titer 4 1:3000), or rFPV
(titer 4 1:1500). The b-gal-specific antibody titers were also
boosted when either pDNA, rVV, or rFPV was administered as
a boost following pDNA priming; these titers ranged from 1:200
to 1:2500 for each (Fig. 3, A). Mice that were primed with rVV
demonstrated lower titers against b-gal after the boost than those
that were primed with either pDNA or the rFPV. An enhancement of the anti-b-gal antibody response was observed regardless of boosting with either a homologous vector or a heterologous vector expressing the same tumor-associated antigen.
Induction of Vector-Specific, High-Titered Antibodies
After a Single Immunization With Either rVV or rFPV
To characterize vector-specific humoral immunity induced by
immunization with either of the pDNA, rVV, or rFPV vaccines,
we tested serum samples harvested 21 days after the primary
immunization by ELISA and western blot for antibody reactivity
against wild-type vaccinia virus or wild-type fowlpox virus
(Figs. 3, B, and 4).
High titers of anti-vaccinia virus antibody were seen in the
serum from mice primed with rVV (titer 4 1:31 250), but not in
the serum from mice immunized with pDNA or rFPV (Fig. 3,
B). Western blot analysis demonstrated that immunization with
rVV induced antibodies against both a single band of b-gal
protein (Fig. 4, lane 4) and multiple bands of wild-type vaccinia
virus (Fig. 4, lane 5), but no reactivity was observed against
wild-type fowlpox virus (Fig. 4, lane 6). Similarly, titers of
anti-fowlpox virus antibodies were found only in the sera of
mice primed with rFPV (titer 4 1:1250; Fig. 3, B). Western blot
analysis showed that vaccination with rFPV induced antibodies
that recognized b-gal protein (Fig. 4, lane 7), 14–20 bands of
wild-type fowlpox virus (Fig. 4, lane 9), and no bands of wildtype vaccinia virus (Fig. 4, lane 8). The antibodies induced by
immunization with pDNA did not react with either wild-type
vaccinia virus or wild-type fowlpox virus, but they did recognize
b-gal protein both by ELISA and by western blot analysis (Figs.
3, B, and 4). These data show that high titers of vector-specific
antibodies were induced by immunization with either vaccinia
virus or fowlpox viruses.
Altogether, vaccination strategies using different recombinant vectors expressing the same tumor-associated antigen resulted in no cross-reactive antibodies among vectors, enhanced
Fig. 2. In vivo secondary cytotoxic T-lymphocyte (CTL) responses in
mice immunized with different homologous and heterologous vaccination regimens. Previously untreated BALB/c mice (eight per group)
were primed with either no immunogen (None), 107 plaque-forming
units (PFU) of VJS6 (rVV) expressing b-galactosidase (b-gal) intravenously, 107 PFU of rFPV.bg40k (rFPV) intravenously, or 10 mg of
pCMV/b-gal (pDNA) intradermally with the gene gun. Twenty-one
days later, each group of mice was divided into four groups and boosted
with the same amount of either no immunogen, rVV, rFPV, or pDNA
to compare heterologous and homologous vaccination regimens. Mice
were killed 4 days after the boost, and a standard 6-hour 51Cr release
assay was performed immediately to assess secondary in vivo CTL
responses against b-gal as described in the ‘‘Materials and Methods’’
section at effector-to-target cell (E:T) ratio of 100:1 and after twofold
dilutions of the effectors to give E:T ratios of 50:1, 25:1, etc. CT26.WT
(b-gal−, s) and CT26.CL25 (b-gal+, ●) served as targets. This experiment was repeated seven times with similar results.
1598 ARTICLES
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
munized the mice with the different heterologous and homologous combinations of the pDNA, rVV, and rFPV vaccines as
described in the ‘‘Materials and Methods’’ section (Fig. 2).
Unprimed mice administered rVV or rFPV and tested for
CTL reactivity 4 days later failed to induce a lytic response
against either CT26.CL25 (b-gal+) or CT26.WT (b-gal−). Mice
primed with either rVV or rFPV and tested 21 days later did not
elicit b-gal-specific CTLs (data not shown). No CTL activity
was observed when mice were immunized and boosted with the
same vector, either rVV or rFPV (Fig. 2). However, boosting the
rVV-primed mice with a different vector, rFPV, induced antigen-specific CTLs (Fig. 2). Mice primed and boosted with rFPV
also did not induce anti-b-gal CTLs. However, rFPV-primed
mice boosted with the heterologous vector rVV elicited antigenspecific CTLs (Fig. 2). Mice primed with pDNA induced b-galspecific CTLs only when they were boosted with either rVV or
rFPV (Fig. 2). The order of this immunization appeared to be
important because, when either an rVV or an rFPV immunization was followed by a booster with pDNA, no lytic activity was
observed (data not shown). These studies suggest that repetitive
vaccination with the same vector does not promote the expansion of antigen-specific CTLs. However, the immunization strategy using two different recombinant vectors expressing the same
antigen does induce enhanced lytic activity.
CTL responses, and prolonged survival of tumor-bearing mice.
Thus, this strategy of immunizing and boosting with alternating
recombinant vectors may represent a more potent means of enhancing an immune response against a desired antigen than repetitive immunizations with the same vector.
Discussion
Exposure of the immune system to an antigen results in ‘‘immunological memory,’’ which in turn enhances the body’s ability to respond to a repeat exposure to the same antigen. The
rationale to employ prime/boost regimens is based on observations of secondary immune responses, which are faster and
larger than the first (primary) immune response (13,14). Specifically, a priming dose of an antigen results in the activation
and expansion of clonotypes capable of recognizing a particular
peptide antigen presented in the context of its restricting major
histocompatibility complex (MHC) molecule. It has been hy-
Fig. 4. Western blot of purified b-galactosidase (b-gal) protein, wild-type vaccinia virus, and wild-type fowlpox virus with the use of serum samples from
mice immunized with recombinant vaccinia virus (rVV), recombinant fowlpox
virus (rFPV), and recombinant DNA plasmids (pDNA). Mice were immunized
one time with either 10 mg of pCMV/b-gal (pDNA) intradermally with the gene
gun (left panel), 107 plaque-forming units (PFU) of VJS6 (rVV) intravenously
(middle panel), or 107 PFU of rFPV.bg40k (rFPV) intravenously (right panel).
Twenty-one days later, serum was harvested and tested at a 1:200 dilution by
western blot against nitrocellulose blots of 5 mg of b-gal protein (lanes 1, 4, and
7), 6.6 × 106 PFU of wild-type vaccinia virus (lanes 2, 5, and 8), or 2 × 107 PFU
of wild-type fowlpox virus (lanes 3, 6, and 9) as described in the ‘‘Materials and
Methods’’ section. The blots were then washed and incubated with horseradish
peroxidase-conjugated sheep anti-mouse immunoglobulin G F(ab8)2 fragments
(1:1000 diluted in 1× phosphate-buffered saline with 1% bovine serum albumin)
to visualize antibody binding. Bound immunoglobulin was then detected by
incubating the blots for approximately 3 minutes in 3,38-diaminobenzidine tetrahydrochloride dissolved in distilled water. The reaction was stopped by washing the nitrocellulose blots for 5 minutes with distilled water. The molecular
weight markers Mr(K) are shown on the right of the figure.
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
ARTICLES 1599
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
Fig. 3. Antibody reactivity of sera from mice immunized with different homologous and heterologous vaccination regimens. Previously untreated BALB/c mice
were primed with either no immunogen (None), 10 mg of b-galactosidase (b-gal)
DNA (pDNA) intradermally with the gene gun, 107 plaque-forming units (PFU)
of VJS6 (rVV) expressing b-gal intravenously, or 107 PFU of rFPV.bg40k
(rFPV) intravenously. Twenty-one days later, each group of mice (two mice per
group) was boosted with the same amount of each immunogen to compare all
heterologous and homologous immunization regimens. A) Sera harvested 8 days
after the boost were assayed by enzyme-linked immunosorbent assay (ELISA)
for antibody reactivity against b-gal protein as described in the ‘‘Materials and
Methods’’ section. B) Sera from mice harvested the day of the boost (21 days
after the initial priming immunization) were tested by ELISA for antibody reactivity against wild-type vaccinia virus (left panel) or wild-type fowlpox virus
(right panel) as described in the ‘‘Materials and Methods’’ section. Serum titers
to either b-gal protein, wild-type vaccinia virus, or wild-type fowlpox virus were
calculated with the use of the dilution observed at an optical density of 0.3.
pothesized that secondary immune responses are more vigorous
than primary immune responses not only because of an increased number of antigen-specific precursor T cells but also
because of a ‘‘memory’’ state of T-lymphocyte activation. Although the mechanisms of T-cell memory are still the subject of
some debate (i.e., whether there is a persistence of antigen or a
persistence of particular T cells), this memory can last for the
lifetime of the vaccinated animal and results in a rapid expansion
of memory T lymphocytes upon re-exposure to relatively lower
concentrations of the relevant antigen (15,16).
To our knowledge, this study represents the first report to
suggest that immunizing and boosting with two different recombinant vectors expressing the same tumor-associated antigen
may be better than repeated vaccinations with the same recombinant vector for the active treatment of malignant disease. We
selected rVV, rFPV, and pDNA as vectors for this study because
they deliver antigen intracellularly to mediate powerful CTL
responses and antitumor immunity (3–5,17–19).
Whereas boosting with the identical recombinant vectors augmented antibody reactivity against b-gal, immunizing and boosting with the same immunogen failed to elicit secondary CD8+
T-lymphocyte responses, as shown by lysis of antigenexpressing tumor cells (Fig. 2). These data may be a result of an
increase in precursor CTL frequency following the boosting immunization that is below the level of detection of our in vivo
primary CTL assay. However, Murata et al. (20) also have recently demonstrated that priming and boosting with the same
1600 ARTICLES
reasons. DNA vaccination may not produce the quantity of the
antigenic protein required to elicit therapeutic immune responses
in an active treatment regimen. By contrast, viral vectors produce large quantities of antigen that persist for long periods. In
addition, viruses express many immunogenic proteins that may
serve as adjuvants that boost immunity against the recombinant
protein. Our results demonstrated, however, that DNA could be
utilized both as a priming or as a boosting agent to prolong the
survival of tumor-bearing mice.
These data help to establish a strategy of immunizing and
boosting with alternating recombinant vectors that could be useful in the clinical use of recombinant anticancer vaccines. However, unlike many of the recently identified tumor-associated
antigens cloned from melanoma that are nonmutated differentiation antigens, b-gal is a foreign molecule to a mouse, and the
responses induced by these recombinant constructs may not be
the same as those responses induced by self antigens. Future
studies will be aimed at generating immunity against self molecules with the use of the strategy of immunization with heterologous recombinant vectors encoding self antigens, such as the
murine homologues of the human melanoma antigens. Clinical
trials applying the use of alternating recombinant vectors are
also currently ongoing in the National Cancer Institute in patients with metastatic melanoma. This strategy represents a
novel approach to enhance the potency of therapeutic recombinant vectors for the treatment of established disease in vivo.
References
(1) Boon T, Gajewski TF, Coulie PG. From defined human tumor antigens to
effective immunization? Immunol Today 1995;16:334–6.
(2) Rosenberg SA. Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens. J Natl Cancer
Inst 1996;88:1635–44.
(3) Irvine KR, Rao JB, Rosenberg SA, Restifo NP. Cytokine enhancement of
DNA immunization leads to effective treatment of established pulmonary
metastases. J Immunol 1996;156:238–45.
(4) Wang M, Bronte V, Chen PW, Gritz L, Panicali D, Rosenberg SA, et al.
Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen. J Immunol 1995;
154:4685–92.
(5) Bronte V, Tsung K, Rao JB, Chen PW, Wang M, Rosenberg SA, et al. IL-2
enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases. J Immunol 1995;154:5282–92.
(6) Cooney EL, Collier AC, Greenberg PD, Coombs RW, Zarling J, Arditti
DE, et al. Safety of and immunological response to a recombinant vaccinia
virus vaccine expressing HIV envelope glycoprotein. Lancet 1991;337:
567–72.
(7) Graham BS, Belshe RB, Clements ML, Dolin R, Corey L, Wright PF, et al.
Vaccination of vaccinia-naive adults with human immunodeficiency virus
type 1 gp160 recombinant vaccinia virus in a blinded, controlled, randomized clinical trial. The AIDS Vaccine Clinical Trials Network. J Infect Dis
1992;166:244–52.
(8) Kundig TM, Kalberer CP, Hengartner H, Zinkernagel RM. Vaccination
with two different vaccinia recombinant viruses: long-term inhibition of
secondary vaccination. Vaccine 1993;11:1154–8.
(9) Fuller DH, Haynes JR. A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice
receiving a DNA-based glycoprotein 120 vaccine. AIDS Res Hum Retroviruses 1994;10:1433–41.
(10) Earl PL, Moss B. Expression of proteins in mammalian cells using vaccinia
viral vectors. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman
JG, South JA, et al., editors. Current protocols in molecular biology. New
York: Green Publishing Associates, 1994:16.15.1–16.18.10.
(11) Rao JB, Chamberlain RS, Bronte V, Carroll MW, Irvine KR, Moss B, et al.
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
recombinant virus did not result in the expansion of the secondary CD8+ T-cell population specific for the heterologous antigen. In the present study, a strong secondary b-gal-specific lytic
response was observed when mice were immunized and boosted
in any order with the two different recombinant poxvirus vectors
expressing the same antigen. In agreement with these findings,
Murata et al. (20) demonstrated a 20- to 30-fold expansion in the
number of antigen-specific CD8+ precursor T cells in mice
primed with a recombinant influenza virus and boosted with a
recombinant vaccinia virus expressing the same MHC class Irestricted epitope. Similarly, Hodge et al. (21) showed that splenocytes derived from mice primed with rVV expressing the
tumor-associated antigen, carcinoembryonic antigen (CEA), and
boosted with the recombinant avian poxvirus ALVAC-CEA
demonstrated a fourfold increase in CEA-specific T-cell proliferation as compared with mice primed and boosted with rVV
expressing CEA alone.
Pre-existing neutralizing antibodies against the viral structural proteins may reduce the CTL responses against tumorassociated antigens and antitumor immunity by rapidly neutralizing the boosting recombinant viral infection and thus reducing
the expansion of precursor T cells against the tumor-associated
antigen (6–8). In accordance with these studies, we observed
high titers of specific anti-vector antibodies after one immunization with the recombinant viruses. Antiviral CTLs may also
play a role in the elimination of recombinant virus and thus the
reduced tumor-associated antigen-specific secondary CTL and
antitumor responses (22). The limited immunogenicity to the
tumor-associated antigen observed after a boost with the same
viral vector may also be due to a specific suppression of responses against weaker determinants by stronger or immunodominant determinants (23–25). Vaccinia virus and fowlpox virus both produce more than 200 proteins responsible for their
structure, transcription, and replication that could function as
potential immunodominant epitopes. The abundant expression
of vaccinia virus structural proteins by the replicating rVV could
also account for the weak induction of anti-b-gal antibodies
(Fig. 3, A).
Although some of the proteins expressed by fowlpox virus
and vaccinia virus share homology, it is not known whether the
subset of proteins that shares homology also shares immunogenic determinants (26,27). Our studies demonstrated that little
or no antibody cross-reactivity exists between rVV and rFPV
(Figs. 3, B, and 4). This lack of cross-reactivity between vaccinia
virus and fowlpox virus may contribute to the enhancement of
CTL expansion and antitumor immunity following a boost with
a heterologous vector.
Vaccination with recombinant plasmid DNA is accomplished
by the expression of plasmid DNA encoding a single antigen of
interest preceded by a eukaryotic promoter and therefore shares
no epitopes in common with either viral vector (3,28–30). In the
case of immunization with pCMV/b-gal, we did not observe
antibodies cross-reactive with either rVV or rFPV. The use of
plasmid DNA may eliminate problems of memory responses to
vector-related proteins, although humoral responses to DNA in
this setting have not been adequately studied. Despite this characteristic, DNA boosted by DNA was not as effective as a DNA
prime boosted by either of the recombinant viral vectors. The
poorer efficacy of DNA vaccination may be due to a number of
(12)
(13)
(14)
(15)
(16)
(17)
(19)
(20)
(21)
(22)
(23) Deng Y, Yewdell JW, Eisenlohr LC, Bennink JR. MHC affinity, peptide
liberation, T cell repertoire, and immunodominance all contribute to the
paucity of MHC class I-restricted peptides recognized by antiviral CTL. J
Immunol 1997;158:1507–15.
(24) Oldstone MB, Lewicki H, Borrow P, Hudrisier D, Gairin J. Discriminated
selection among viral peptides with the appropriate anchor residues: implication for the size of the cytotoxic T-lymphocyte repertoire and control
of viral infection. J Virol 1995;69:7423–9.
(25) Mylin LM, Bonneau RH, Lippolis JD, Tevethia SS. Hierarchy among
multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian
virus 40 T antigen. J Virol 1995;69:6665–77.
(26) Skinner MA, Moore JB, Binns MM, Smith GL, Boursnell ME. Deletion of
fowlpox virus homologues of vaccinia virus genes between the 3 betahydroxysteroid dehydrogenase (A44L) and DNA ligase (A50R) genes. J
Gen Virol 1994;75:2495–8.
(27) Tartaglia J, Winslow J, Goebel S, Johnson GP, Taylor J, Paoletti E. Nucleotide sequence analysis of a 10.5 kbp HindIII fragment of fowlpox virus:
relatedness to the central portion of the vaccinia virus HindIII D region. J
Gen Virol 1990;71:1517–24.
(28) Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al.
Direct gene transfer into mouse muscle in vivo. Science 1990;247:1465–8.
(29) Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et
al. Heterologous protection against influenza by injection of DNA encoding
a viral protein. Science 1993;259:1745–9.
(30) Pertmer TM, Eisenbraun MD, McCabe D, Prayaga SK, Fuller DH, Haynes
JR. Gene gun-based nucleic acid immunization: elicitation of humoral and
cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine 1995;13:1427–30.
Notes
The first two authors contributed equally to this work.
Present address: R. S. Chamberlain, Department of Surgery, The George
Washington University, Washington, DC.
Manuscript received April 24, 1997; revised July 30, 1997; accepted September 3, 1997.
Journal of the National Cancer Institute, Vol. 89, No. 21, November 5, 1997
ARTICLES 1601
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on March 4, 2014
(18)
IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor
vaccines: enhancement by simultaneous B7-1 expression. J Immunol 1996;
156:3357–65.
Restifo NP, Esquivel F, Kawakami Y, Yewdell JW, Mule JJ, Rosenberg
SA, et al. Identification of human cancers deficient in antigen processing.
J Exp Med 1993;177:265–72.
Zinkernagel RM, Bachmann MF, Kundig TM, Oehen S, Pirchet H, Hengartner H. On immunological memory. Annu Rev Immunol 1996;14:
333–67.
Bruno L, Kirberg J, von Boehmer H. On the cellular basis of immunological T cell memory. Immunity 1995;2:37–43.
Gray D, Matzinger P. T cell memory is short-lived in the absence of
antigen. J Exp Med 1991;174:969–74.
Kundig TM, Bachmann MF, Ohashi PS, Pircher H, Hengartner H, Zinkernagel RM. On T cell memory: arguments for antigen dependence. Immunol
Rev 1996;150:63–90.
Conry RM, LoBuglio AF, Kantor J, Schlom J, Loechel F, Moore SE, et al.
Immune response to a carcinoembryonic antigen polynucleotide vaccine.
Cancer Res 1994;54:1164–8.
Kantor J, Irvine K, Abrams S, Kaufman H, DiPietro J, Schlom J. Antitumor
activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine. J Natl Cancer Inst 1992;84:1084–91.
Estin CD, Stevenson US, Plowman GD, Hu SL, Sridhar P, Hellstrom I, et
al. Recombinant vaccinia virus against the human melanoma antigen p97
for use in immunotherapy. Proc Natl Acad Sci U S A 1988;85:1052–6.
Murata K, Garcia-Sastre A, Tsuji M, Rodrigues M, Rodriguez D, Rodriguez JR, et al. Characterization of in vivo primary and secondary CD8+
T cell responses induced by recombinant influenza and vaccinia viruses.
Cell Immunol 1996;173:96–107.
Hodge JW, McLaughlin JP, Kantor JA, Schlom J. Diversified prime and
boost protocols using recombinant vaccinia virus and recombinant nonreplicating avian pox virus to enhance T-cell immunity and antitumor responses. Vaccine 1997;15:759–68.
Battegay M, Moskophidis D, Waldner H, Brundler MA, Fung-Leung WP,
Mak TW, et al. Impairment and delay of neutralizing antiviral antibody
responses by virus-specific cytotoxic T cells [published erratum appears in
J Immunol 1994;152:1635]. J Immunol 1993;151:5408–15.