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Induction of immune response and safety profiles in rhesus macaques immunized with a DNA vaccine expressing human prostate specific antigen

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Prostate specific antigen (PSA) is a widely used marker for prostate cancer, which is secreted by normal prostate cells at low levels, but is produced more substantially by cancer cells. We have previously reported on the use of a DNA vaccine construct that encodes for human PSA gene to elicit host immune responses against cells producing PSA. DNA immunization strategy delivers DNA constructs encoding for a specific immunogen into the host, who becomes the in vivo protein source for the production of antigen. This antigen then is the focus of the resulting immune response. In this study, we examine the induction of immune responses and safety profiles in rhesus macaques immunized with DNA-based PSA vaccine. We observed induction of PSA-specific humoral response as well as positive PSA-specific lymphoproliferative (LPA) response in the vaccinated macaques. We also observed that the stimulated T cells from the PSA-immunized rhesus macaques produced higher levels of Th1 type cytokine IFN-gamma than the control vector immunized animals. On the other hand, DNA immunization did not result in any adverse effects in the immunized macaques, as indicated by complete blood counts, leukocyte differentials and hepatic and renal chemistries. The macaques appeared healthy, without any physical signs of toxicity throughout the observation period. In addition, we did not observe any adverse effect on the vaccination site. The apparent safety and immunogenecity of DNA immunization in this study suggest that further evaluation of this vaccination strategy is warranted.
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Induction of immune responses and safety pro®les in rhesus macaques
immunized with a DNA vaccine expressing human prostate speci®c antigen
J Joseph Kim
1,2
, Joo-Sung Yang
2
, Liesl K Nottingham
2
, Waixing Tang
3
, Kesen Dang
2
,
Kelledy H Manson
4
, Michael S Wyand
4
, Darren M Wilson
2
and David B Weiner*
,2,5
1
Viral Genomix, Inc., Philadelphia, Pennsylvania, PA 19104, USA;
2
Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania, PA 19104, USA;
3
Institute of Human Gene Therapy, Philadelphia,
Pennsylvania, PA 19104, USA;
4
Primedica Mason Labs, Worcester, Massachusetts, USA;
5
The Wistar Institute, Philadelphia,
Pennsylvania, PA 19104, USA
Prostate speci®c antigen (PSA) is a widely used marker
for prostate cancer, which is secreted by normal prostate
cells at low levels, but is produced more substantially by
cancer cells. We have previously reported on the use of a
DNA vaccine construct that encodes for human PSA
gene to elicit host immune responses against cells
producing PSA. DNA immunization strategy delivers
DNA constructs encoding for a speci®c immunogen into
the host, who becomes the in vivo protein source for the
production of antigen. This antigen then is the focus of
the resulting immune response. In this study, we examine
the induction of immune responses and safety pro®les in
rhesus macaques immunized with DNA-based PSA
vaccine. We observed induction of PSA-speci®c humoral
response as well as positive PSA-speci®c lymphoproli-
ferative (LPA) response in the vaccinated macaques. We
also observed that the stimulated T cells from the PSA-
immunized rhesus macaques produced higher levels of
Th1 type cytokine IFN-gthan the control vector
immunized animals. On the other hand, DNA immuniza-
tion did not result in any adverse eects in the
immunized macaques, as indicated by complete blood
counts, leukocyte dierentials and hepatic and renal
chemistries. The macaques appeared healthy, without
any physical signs of toxicity throughout the observation
period. In addition, we did not observe any adverse eect
on the vaccination site. The apparent safety and
immunogenecity of DNA immunization in this study
suggest that further evaluation of this vaccination
strategy is warranted. Oncogene (2001) 20, 4497 ±
4506.
Keywords: prostate speci®c antigen; DNA vaccines;
prostate cancer; immune responses; rhesus macaques
Introduction
Prostate cancer is the most common malignancy in
American men and is the second leading cause of cancer
related death in the male population (Boring et al, 1994).
It is estimated that 184 500 men were diagnosed with
prostate cancer and 39 200 men died of the disease in
1998 in the United States (Garnick and Fair, 1998).
Present treatment for prostate cancer includes radical
prostectomy, radiation therapy or hormonal therapy.
Even though traditional surgical androgen deprivation
has been largely replaced by hormonal therapy, no
systemic therapy has clearly improved the hormone
refractory disease. Even early treatment through surgery
or radiation therapy does not always achieve complete
eradication of the tumor and can lead to unwanted side
eects such as impotence and urinary incontinence
(D'Amico et al., 1998; O'Donnell and Finan, 1989;
Zagars and Pollack, 1995).
Prostate speci®c antigen (PSA) is a 240 amino acid
member of the glandular kallikrein gene family (Wang
et al., 1982; Watt et al., 1986). PSA is a serine protease
secreted by both normal and transformed epithelial
cells of the prostate gland (Wang et al., 1982). PSA can
be detected in the sera of healthy males at low levels
without clinical evidence of prostate cancer. However,
PSA is secreted more substantially by cancer cells
(Wang et al., 1982; Stenman et al., 1999). Since PSA
expression appears to be limited to prostate cells, it is
now the most widely used marker for prostate cancer
(Godley, 1999; Labrie et al., 1992). Furthermore, the
tissue speci®city of PSA makes it a potential target for
the development of immunotherapies against prostate
cancer (Armbruster, 1993; Hodge et al., 1995).
However, the lack of strong surface expressed PSA
makes targeting these antigen expressing cells complex.
We have previously reported on the use of DNA
immunization in mice to elicit host immune responses
against cells producing PSA (Kim et al., 1998b).
Speci®cally, a DNA vaccine construct which encodes
for human PSA gene was cloned into expression
vectors. Then the mice were immunized intramuscu-
larly with these DNA vaccines and the in vivo immune
responses were examined. Injection of the PSA plasmid
Oncogene (2001) 20, 4497 ± 4506
ã
2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
www.nature.com/onc
*Correspondence: DB Weiner, Department of Pathology and
Laboratory Medicine, University of Pennsylvania, 505 Stellar-
Chance, 422 Curie Blvd., Philadelphia, PA 19104, USA;
E-mail: dbweiner@mail.med.upenn.edu
Received 23 January 2001; revised 9 April 2001; accepted 12 April
2001
induced a strong and persistent antibody response
against PSA following immunization. In addition,
DNA immunization induced signi®cant PSA-speci®c
T helper cell proliferative as well as CD8
+
T cell-
restricted cytotoxic T lymphocyte responses in mice.
The CTL response allows for destruction of PSA-
expressing cells without the requirement for strong
surface expression. These results demonstrate the
potential utility of targeting PSA as a testable
immunotherapy against prostate cancer.
Important for the ultimate use of DNA vaccine
technology in humans is that results originally
observed in mouse systems translate to animal models
more similar to the clinical setting. Previously, it has
been reported that primates may have a limited ability
to produce DNA vaccine-encoded proteins through
direct genetic inoculation into muscle (Jiao et al.,
1992). More speci®cally, it has been reported that
DNA immunizations alone in primates induce weak
immune response (Letvin et al., 1997). These observa-
tions suggest reduced immunogenecity of DNA
vaccines in hon-human primates, potentially limiting
their utility. Because of the high degree of similarity
between the rhesus macaques and human prostate
gland and PSA (94%), the rhesus macaque model is
well suited to accurately assess the eects of PSA
vaccine.
In this study, we report on the ®rst examination of
immune responses and safety in rhesus macaques
immunized with DNA-based PSA vaccine. We ob-
served induction of PSA-speci®c immune responses in
rhesus macaques without any signs of adverse reaction
to the vaccination.
Results
Vector construction and in vitro expression
DNA vaccine constructs expressing human PSA
(pCPSA) was cloned into an expression vector under
control of a CMV promoter as previously described
(MacGregor et al., 1998). The vector has a Kanamycin
resistance gene as a selectable marker. Similar DNA
backbones have been previously evaluated in hundreds
of humans without any major side eect (MacGregor
et al., 1998). To con®rm the expression of pCPSA in a
monkey cell line, we transfected COS cells in vitro
using a method previously described. Three days
following transfection, the transfected cells were
stained with a-PSA antibody and observed under
microscope. As shown in Figure 1b, we observed an
expression of PSA in COS cells transfected with
pCPSA. Untransfected control cells did not show
expression of PSA (Figure 1a).
DNA immunization induces PSA-specific antibody
responses in rhesus macaques
We investigated whether the induction of PSA-speci®c
immune responses observed in mice through DNA
immunization could also be achieved in rhesus
macaques. We immunized four rhesus macaques (two
male and two female) intramuscularly with 500 mgof
pCPSA DNA vaccine construct at weeks 0, 4, and 10.
The sera samples were collected at various time points
and were assayed for the induction of PSA-speci®c IgG
antibodies. We observed that DNA immunization can
elicit PSA-speci®c antibody responses in two of four
animals (50%) (Figure 2a). The PSA-speci®c humoral
response was detected in the vaccinated macaques as
early as 6 weeks post-immunization and remained
persistent in these responders for at least 14 weeks. It is
interesting to note that 50% of female macaques,
which do not produce PSA, responded positively to
pCPSA vaccination. It is also interesting that the same
percentage of male macaques (which already produce
their own macaque PSA) displayed PSA-speci®c
humoral responses. Moreover, a booster eect was
observed in both responders after the third injection at
week 10.
Furthermore, these antigen-reactive sera from the
immunized monkeys were PSA-positive by Western
blot analysis. As shown in Figure 2b, the sera collected
from monkeys (MK1 and MK4) at 20 weeks post-
immunization were analysed by Western blot analysis.
The sera of control monkeys did not show reactivity.
Figure 1 Construction and in vitro expression of pCPSA. The
complete coding sequence of human PSA was cloned into a
clinical expression vector under control of a CMV promoter and
a Kanamycin resistance gene as a selectable marker. The proper
expression of PSA protein was assayed by transfecting the pCPSA
plasmids into COS cells. The control untransfected cells (a) and
PSA-transfected cells (b) were stained with a-PSA antibodies
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
JJ Kim et al
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Oncogene
In contrast, the sera collected from animals immunized
with pCPSA showed distinct PSA band at the
molecular weight of 30 kD. This ®nding illustrates
the strength of the humoral response in these animals.
DNA immunization induces PSA-specific cellular
responses in rhesus macaques
The eect of DNA immunization on antigen-speci®c
lymphoproliferative responses was examined in maca-
ques immunized as described above. As shown on
Figure 3, we observed positive PSA-speci®c lympho-
proliferative (LPA) responses in three out of four
animals (SI42), and signi®cant LPA responses (SI44)
were induced in 50% of animals. It was interesting to
note that macaques MK2 and MK3 displayed higher
levels of lymphoproliferative response while MK1 and
MK4 had higher levels of humoral response. Addi-
tional studies with more macaques would be needed to
determine the signi®cance of such immune response
polarity.
Given these immune responses following the DNA
immunization in macaques, the eects of increase in
vaccine dosage and frequency of administration on the
induction of PSA-speci®c LPA responses were investi-
gated. Two additional rhesus macaques (MK5 and
MK6) were immunized intramuscularly with 1 mg of
pCPSA DNA vaccine construct at weeks 0, 4, 10 and
14 (4 mg total doses). One control animal was injected
with control vector pCDNA3 with identical regimen.
Induction of PSA-speci®c LPA responses were ana-
lysed in these animals. As shown in Figure 4, four
immunizations with 1 mg vaccine dosage resulted in a
signi®cant induction of PSA-speci®c LPA responses in
the PSA vaccine immunized macaques (MK5 and
MK6). These results demonstrate that more frequent
immunizations with a higher dose of vaccine can
signi®cantly enhance the level of LPA responses. In
addition, a booster eect was observed following the
fourth injections.
Cytokines play a key role in directing and targeting
immune cells during the development of the immune
response. For instance, IFN-gis produced by Th1 and
CD8
+
T cells and is intricately involved in the
regulation or development of anti-viral T cell-mediated
immune responses (Clerici et al., 1993; Rosenberg et
Figure 2 Induction of PSA-speci®c antibody responses in rhesus macaques. (a) Four rhesus macaques (two male and two female)
were immunized intramuscularly with 500 mg of pCPSA DNA vaccine construct at weeks 0, 4, and 10 (as indicated by the arrows).
One control animal was injected with control vector pCDNA3 with identical regimen. The sera samples were collected and were
assayed for the induction of PSA-speci®c antibodies. At 1 : 128 dilution, PSA-speci®c antibody response was detected as early as
week 6 in 50% of pCPSA immunized animals. The antigen-speci®c antibody response remained persistent in these responders. (b)
Induction of PSA-speci®c Western reactivity in immunized macaques. The sera collected from monkeys MK1 (lane 1) and MK4
(lane 2) at 20 weeks post-immunization were analysed by Western bot analysis against recombinant PSA protein. The PSA band at
the molecular weight of 30 kD is indicated by the arrow. The sera from the control macaque (lane 3) was negative, while both
positive control lanes, the monoclonal PSA Ab (lane 4) and the Rabbit polyclonal anti-PSA Ab (lane 5), were positive
Oncogene
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
JJ Kim et al
4499
al., 1997). In contrast, IL-4 plays a dominant role in B
cell-mediated immune responses (Seder and Paul,
1994). Thus, analysis of these cytokines secreted by
stimulated T cells may be important in elucidating the
extent of cell-mediated responses following immuniza-
tion (Lekutis et al., 1997). As shown in Figure 5, the
stimulated T cells from the PSA-immunized rhesus
macaques (MK5 and MK6) produced higher levels of
IFN-gthan the control vector immunized animals. On
the other hand, the level of IL-4 produced by either
group was similar. These results support that the
responses in immunized macaques displayed a strong
Th1 phenotype with potent induction of IFN-g. Th1
phenotypes are a hallmark of this vaccine approach in
mice (Kim et al., 1998a) and in humans (Boyer et al.,
1999).
Physical consequences of immunization
Since the ultimate goal of vaccine or therapeutic
strategies requires understanding of the consequences
of a particular immunization strategy, the eects of
PSA vaccine were examined in these primates. The
macaques were tested for complete blood counts,
leukocyte dierentials and hepatic and renal chemis-
tries. As shown in Table 1, the complete blood counts
remained within the normal limits throughout the
observation periods. In addition, hepatic and renal
functional measurements did not indicate any adverse
reaction to immunization (Table 2). We also examined
the vaccine injection sites at speci®c times following
immunization and did not observe any signi®cant
irritation on the vaccination site (data not shown).
These results are consistent with the safety observation
we had previously made in humans receiving vaccina-
tion with DNA constructs encoding for HIV-1 antigens
(MacGregor et al., 1998).
We also examined the eect of immunization on the
prostate of the immunized animals (males only) 24
weeks post-last immunization. We prepared frozen
section slides of the prostate tissue received and stained
them with hematoxylin and eosin (H&E) stain. As
shown in Figure 6, we observed some evidence of
in®ltrating cells in the prostate tissue, while suggesting
a direct link between this in®ltration and the vaccine
protocol will require more study. Overall, it is
Figure 3 Induction of PSA-speci®c lymphoproliferative responses in rhesus macaques. Induction of lymphoproliferative responses
against PSA proteins were examined in animals immunized as described in Figure 2. PSA-speci®c lymphoproliferative responses
were observed in three out of four animals (SI42), and 50% of animals displayed signi®cant lymphoproliferative responses (SI44)
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
JJ Kim et al
4500
Oncogene
interesting to note that no adverse symptoms were
noted during the routine and clinical examinations of
these macaques throughout the study period (Tables 1
and 2). The clinical signi®cance of the prostate tissue
in®ltration observed here is currently not clear.
Discussion
Immune-based therapies are promising strategies to
treat cancer. Several immunotherapeutics against
cancer have already been investigated (Small, 1999;
Tuting et al., 1997). Examples include the injection of
live, irradiated, and autologous tumor cells transfected
with cytokines (Sandra, 1994; Vieweg et al., 1994). In
addition, a recombinant vaccinia virus engineered to
express tumor speci®c antigens and autologous den-
dritic cells loaded with peptide sequences of cancer
antigens have been investigated as possible vaccines
(Hodge et al., 1995; Lotze et al., 1997; Malkowicz and
Johnson, 1998; Murphy et al., 1996; Porgador et al.,
1996; Tjoa et al., 1996).
DNA vaccination is an important candidate for
potential immunotherapy against cancer. Extensive
experiments have shown that the DNA vaccines' ability
to elicit humoral and cellular responses in vivo in a safe
and well-tolerated manner in various model systems,
including rodents and non-human primates, is now
being explored in humans. The ®rst DNA vaccine
studies to enter the clinic were DNA vaccines encoding
for HIV-1 MN envelope (MacGregor et al., 1998).
Fifteen healthy HIV-1 sero-positive volunteers in the
trial received three injections each separated by 10
weeks with escalating dosage (three dosage groups of
®ve subjects) of envelope vaccine. Preliminary results
reveal no signi®cant clinical or laboratory adverse
eects measured in all three dosage groups (30, 100,
300 mg). More importantly, the immunized individuals
developed an increase in antibody responses to
envelope proteins and peptides after receiving the
100 mg dose. Some increases in cellular responses
including the lymphoproliferative and CTL responses
as well as b-chemokine expression were also observed
(MacGregor et al., 1998). In addition, phase I trials
evaluating a gag/pol construct as a therapeutic vaccine
as well as a prophylactic DNA vaccine study for HIV
has been undertaken. In another clinical study, the
healthy volunteers who were immunized with DNA
vaccines encoding for malaria proteins developed CTL
responses against the target cells prepared with malaria
peptides (Wang et al., 1998).
A major focus of developing DNA vaccines against
cancer has been the use of tumor associated antigens.
These are proteins produced by tumor cells, which can
be presented on the cell surface in the context of major
histocompatibility complexes (Kelley and Cole, 1998).
Figure 4 Induction of PSA-speci®c lymphoproliferative responses in rhesus macaques. Two rhesus macaques (MK5 and MK6)
were immunized intramuscularly with 1 mg of pCPSA DNA vaccine construct at weeks 0, 4, 10 and 14 (as indicated by the arrows).
One control animal was injected with control vector pCDNA3 with identical regimen. Induction of PSA-speci®c lymphoproliferative
responses were analysed in these animals
Oncogene
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
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Recently, the antigens such as the carcinoembryonic
antigen (CEA) and PSA have been the targets of
immunotherapy of cancer. DNA vaccine encoding for
human CEA was characterized in a murine model, and
CEA speci®c humoral and cellular responses were
detected in the immunized mice (Conry et al., 1994).
The CEA DNA vaccine was also characterized in a
canine model, where sera obtained from dogs injected
intramuscularly with the construct demonstrated an
increase in antibody levels (Smith et al., 1998). The
CEA DNA vaccine was also examined in the pig-tail
macaques, and this strategy is currently being
investigated in humans (Conry et al., 1998).
We have previously reported on the use of DNA
vaccine which encodes for human PSA to induce
strong and persistent antibody responses against PSA
for at least 180 days following immunization (Kim et
al., 1998b). In addition, signi®cant CD4
+
T helper cell
as well as CD8
+
CTL responses were observed. These
results demonstrate the potential utility of targeting
PSA as a testable immunotherapy against prostate
cancer.
In another study, recombinant vaccinia virus
vaccines expressing human PSA (rV-PSA) were studied
in rodent as well as in non-human primate models
(Hodge et al., 1995). Hodge et al. (1995) investigated
the immunological eects of a recombinant vaccinia
virus expressing human PSA (rV-PSA) in rhesus
macaques and observed that the rhesus monkeys
exhibited little vaccine toxicity other than the low-
grade fever expected with vaccinia infection. In
addition, the macaques receiving the high dose rV-
PSA vaccination exhibited cellular immune responses
speci®c to PSA. This vaccinia virus-based PSA vaccine
has also been tested in Phase I trial (Eder et al., 2001).
In this report, we extend this strategy to investigate
for immunology and safety in macaques. Because of
the high degree of similarity between the rhesus and
human prostate gland and PSA (94%), this animal
model was well suited to accurately assess the eects of
PSA vaccine. We immunized four rhesus macaques
(two male and two female) intramuscularly with 500 mg
of pCPSA DNA vaccine construct at weeks 0, 4, and
10. The PSA-speci®c IgG response was detected in the
vaccinated macaques as early as 6 weeks post-
immunization and remained persistent in these respon-
ders (50%) through later time points. It is of interest
Figure 5 Expression of cytokines by stimulated T cells. Super-
natants from PSA-speci®c lymphoproliferative assay described in
Figure 3 were collected and tested for cytokine pro®le using
ELISA kits for IFN-g(a) and IL-4 (b)
Table 1 Summary of eects on rhesus macaques
PSA-immunized (n=4) Control-immunized (n=3) Normal
Week 10 Week 14 Week 34 Week 10 Week 14 Week 34 range
WBC (610
3
) 6.1 (+1.5) 6.6 (+3.1) 6.5 (+2.0) 6.5 (+0.7) 5.6 (+1.7) 8.7 (+1.8) (1.4 ± 7.6)
RBC (610
6
) 5.4 (+0.2) 5.4 (+0.3) 5.6 (+0.2) 5.4 (+0.1) 5.5 (+0.2) 5.6 (+0.2) (4.5 ± 6.9)
HGB (gm/dl) 12.7 (+0.7) 12.7 (+0.5) 13.2 (+0.5) 12.0 (+0.4) 12.2 (+0.5) 12.5 (+0.5) (10.6 ± 15.6)
HCT (%) 39.1 (+2.1) 39.2 (+1.0) 40.6 (+1.1) 27.9 (+18.8) 39.0 (+1.4) 39.6 (+1.5) (35.0 ± 48.2)
MCV (fl) 73 (+3.7) 73.5 (+3.7) 73.0 (+3.7) 71.7 (+0.6) 71.3 (+0.6) 71.3 (+0.6) (66.0 ± 80.2)
MCH (pg) 23.7 (+1.1) 23.7 (+1.4) 23.6 (+1.1) 22.4 (+0.3) 22.3 (+0.2) 22.4 (+0.1) (21.0 ± 25.0)
MCHC (g/dl) 32.5 (+0.5) 32.3 (+0.4) 32.5 (+1.3) 31.3 (+0.5) 31.2 (+0.4) 31.5 (+0.2) (29.2 ± 33.8)
Poly (%) 55.5 (+13.6) 57.0 (+18.7) 48.5 (+13.5) 45.3 (+9.0) 50.7 (+13.1) 53.7 (+11.6) (14.2 ± 87.8)
Abs Poly 3.5 (+1.5) 4.0 (+3.0) 3.0 (+0.9) 2.9 (+0.7) 3.0 (+1.5) 4.6 (+0.9) (0 ± 12.4)
Lymph (%) 42.5 (+14.3) 41.3 (+18.2) 48.3 (+16.2) 54.0 (+8.7) 49.0 (+13.5) 46.0 (+12.1) (11.0 ± 83.0)
Abs Lymph 2.5 (+0.7) 2.4 (+1.3) 3.3 (+1.7) 3.5 (+0.6) 2.6 (+0.1) 4.1 (+1.6) (0.2 ± 8.1)
PLT (610
3
) 366.0 (+115.2) 396.3 (+108.0) 380.0 (+79.4) 378.3 (+32.5) 381.0 (+48.5) 346.3 (+31.1) (181.8 ± 661.8)
Total leukocyte count (WBC); Erythrocyte count (RBC); Hemoglobin concentration (HGB); Hematocrit value (HCT); Mean corpuscular
volume (MCV); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin concentration (MCHC); Platelet count (PLT)
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
JJ Kim et al
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Oncogene
that the frequency and the degree of PSA-speci®c IgG
responses induced from DNA immunization were more
potent than those induced from the high dose
administration of rV-PSA in rhesus macaques (Hodge
et al., 1995).
Furthermore, we observed positive PSA-speci®c LPA
responses in three out of four animals. The potency of
PSA-speci®c LPA responses induced from DNA
immunization was similar to that from the high dose
administration of rV-PSA in rhesus macaques (Hodge
et al., 1995). The positive immune response rate may
be dose-dependent since two additional rhesus maca-
ques (MK5 and MK6) immunized with a higher 1 mg
dose of pCPSA DNA vaccine construct at weeks 0, 4,
10 and 14 resulted in 100% positive immune response
rate. In addition, more frequent immunization with
higher dose of vaccine seemed to enhance the level of
LPA responses, and a booster eect was observed
following the third and fourth injections. Moreover, we
observed that the stimulated T cells from the PSA-
immunized rhesus macaques produced higher levels of
Th1 type cytokine IFN-gthan the control vector
immunized animals.
While the pCPSA immunizations induced humoral
and cellular immune responses, the injections did not
result in speci®c adverse eects in the immunized
macaques, as indicated by complete blood counts,
leukocyte dierentials and hepatic and renal chemis-
tries. The macaques appeared healthy, without any
physical signs of toxicity throughout the observation
period. In addition, we did not observe any adverse
eect on the vaccination site from the immunization
(data not shown). We did observe some evidence of
in®ltrating cells in the prostate tissue almost 6 months
from the last immunization. Further studies are needed
to elucidate the clinical signi®cance of the prostate
tissue in®ltration.
These safety observations are consistent with the
results observed in humans receiving vaccination with
DNA constructs encoding for HIV-1 antigens (Mac-
Gregor et al., 1998). Overall the vaccine recipients
received the vaccines in a well tolerated manner, with
no signi®cant clinical or laboratory adverse eects
measured in all dosage groups (30, 100, 300 mg per
dose). In addition, these observations are similar to the
results observed in a study on the immunization of
Table 2 Serum chemistry summary
PSA-immunized (n=3) Control-immunized (n=3)
Week 39 Week 41 Week 39 Week 41 Normal range
Glucose (mg/dl) 59.0 (+6.2) 59.3 (+4.6) 63.3 (+2.3) 56.7 (+6.4) (28.1 ± 123.2)
BUN (mg/dl) 19.0 (+1.0) 187 (+0.6) 20.7 (+3.1) 19.0 (+2.6) (10.4 ± 28.9)
Creatinine (mg/dl) 1.0 (+0.2) 0.9 (+0.1) 0.8 (+0.0) 0.8 (+0.0) (0.6± 1.4)
Total protein (g/dl) 7.6 (+0.5) 7.4 (+0.4) 7.0 (+0.2) 6.8 (+0.2) (6.7 ± 8.4)
Albumin (g/dl) 4.4 (+0.4) 4.3 (+0.3) 4.3 (+0.3) 4.2 (+0.3) (3.1 ± 5.4)
Globulin (g/dl) 3.1 (+0.4) 3.1 (+0.6) 2.7 (+0.2) 2.6 (+0.2) (2.0± 4.4)
A/G ratio 1.4 (+0.2) 1.4 (+0.3) 1.6 (+0.2) 1.6 (+0.2) (0.7 ± 2.0)
Calcium (mg/dl) 9.5 (+0.6) 9.2 (+0.5) 9.4 (+0.3) 9.3 (+0.2) (8.8 ± 10.7)
Phosphorus (mg/dl) 5.3 (+1.2) 5.5 (+1.2) 6.2 (+0.9) 6.9 (+0.5) (4.3± 7.9)
Total bilirubin (mg/dl) 0.1 (+0.1) 0.2 (+0.1) 0.1 (+0.0) 0.2 (+0.1) (0.0 ± 0.6)
ALKP (u/l) 315.0 (+281.4) 307.3 (+264.2) 675.0 (+151.6) 715.0 (+164.9) (120.5 ± 1053.5)
ALT (u/l) 54.0 (+33.6) 41.0 (+28.2) 26.3 (+7.5) 23.2 (+4.2) (4.8 ± 75.7)
AST (u/l) 44.7 (+11.7) 39.3 (+11.6) 30.7 (+4.7) 33.0 (+6.1) (7.4 ± 91.7)
Sodium (mEq/l) 143.0 (+1.7) 145.0 (+1.7) 145.0 (+1.0) 145.0 (+1.0) (143.3 ± 152.9)
Potassium (mEq/l) 3.6 (+0.5) 3.4 (+0.2) 3.2 (+0.3) 3.4 (+0.1) (2.9 ± 5.6)
Chloride (mEq/l) 106.7 (+0.6) 108.3 (+0.6) 106.7 (+2.5) 108.7 (+2.5) (102.6 ± 113.3)
Triglycerides (mg/dl) 44.0 (+11.3) 39.7 (+13.0) 54.7 (+7.6) 46.0 (+6.0) (0.0 ± 126.4)
Cholesterol (mg/dl) 147.3 (+13.9) 144.3 (+15.5) 158 (+11.4) 148.7 (+8.5) (91.5 ± 212.3)
GGT (u/l) 82.0 (+4.4) 80.7 (+6.4) 82.7 (+13.1) 87.7 (+19.0) (24.4 ± 108.9)
Blood urea nitrogen (BUN); Alkaline phosphatase (ALKP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST)
Figure 6 Immunohistochemical analysis of prostate tissues collected from immunized macaques. At week 34, frozen section slides
were prepared from the prostate tissues from pCPSA immunized macaques (aand b) and control macaque (c) were stained with
hematoxylin and eosin (H&E) stain
Oncogene
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
JJ Kim et al
4503
recombinant vaccinia virus vaccines expressing human
PSA in rhesus macaques (Hodge et al., 1995). Hodge et
al. (1995) reported that PSA-vaccinia immunized
rhesus macaques exhibited little vaccine toxicity other
than the low-grade fever expected with wild-type
control vaccinia infection.
This study utilizes a relevant pre-clinical model to
evaluate the pCPSA vaccine immunogenecity and
safety and extends the immunogenecity ®ndings
observed from the rodent studies. Due to 94%
similarity between the rhesus macaques and human
PSA protein, the rhesus macaque model was well
suited to assess the eects of PSA vaccine. We
observed that the immunization of rhesus macaques
with pCPSA vaccine induced potent humoral cellular
immune responses without any measurable adverse
eects. The apparent safety and immunogenecity of
the vaccine, especially the induction of potent
cellular immune responses support the potential
utility of this vaccine strategy. Because human and
rhesus PSA do not share 100% homology, the
ultimate test of this strategy rests with appropriate
studies in the clinic.
Materials and methods
Construction and expression of pCPSA DNA constructs
DNA vaccine cassettes expressing PSA were constructed by
cloning the complete coding sequence of PSA into clinical
expression vector under the control of CMV promoter. The
expression of PSA by the pCPSA plasmids was assayed by
transfecting them into COS cells using the calcium phosphate
transfection method. Brie¯y, monkey COS cells line cells
(1610
6
) grown in DMEM (Gibco ± BRL, Grand Island, NY,
USA) supplemented with 10% FBS, 100 U/ml penicillin and
100 mg/ml streptomycin were plated on 60 mm plates for 24 h
prior to being transfected with 10 mg of pCPSA plasmid
DNA. The cells were harvested at 5 days post-transfection
and were washed twice with PBS (Gibco ± BRL) and ®xed
with 4% paraformaldehyde solution. These ®xed cell
suspensions were dropped onto a slide and air-dried for at
least 2 h. The cells were dehydrated by immersing the slides
in solutions of 50, 70, 90, then 98% of EtOH for 3 min in
each solution, and then air-dried again for 20 min. The cells
were blocked with 3% goat serum (Gibco ± BRL) in PBS for
20 min, and then incubated with 1 : 250 dilution of rabbit
anti-PSA primary antibody (Fitzgerald Industries Interna-
tional, Inc. Concord, MA, USA) for 1 h at room
temperature. After washing with PBS for 5 min, the cells
were incubated with biotinylated anti-rabbit IgG (Sigma
Chemical Co, St. Louis, MO, USA) in a 1 : 500 dilution for
45 min at room temperature. After washing, the slides were
incubated with Avidine and then Biotinylated Alkaline
Phosphatase in 1 : 500 dilutions for 30 min each, and the
color was developed by adding substrate nitro-blue tetra-
zolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP).
The slides were viewed with a Nikon OPTIPHOT ¯uorescing
microscope (Nikon Inc., Tokyo, Japan) using a 2006
objective (Nikon Fluo 40X Ph3D2). Slide photographs were
obtained using a Nikon camera FX35DX with exposure
control by Nikon UFX-II and Kodak Ektachrome 160T slide
®lm.
DNA inoculation of animals
Rhesus macaques (Macaca mulatta) were individually housed
at the Primedica Mason Labs (Worcester, MA, USA). All
animal care and use procedures conformed to the revised
Public Health Service Policy on Humane Care and Use of
Laboratory Animals. Animals were anesthetized with keta-
mine HCL for all technical procedures. Macaques were
immunized intramuscularly (IM) in the quadriceps with DNA
preparations formulated in PBS and 0.25% bupivacaine-HCl
on multiple occasions.
ELISA
Serum antibody reactivity puri®ed PSA protein was analysed
by ELISA as previously described (Boyer et al., 1997).
Brie¯y, recombinant PSA protein (Fitzgerald Industries) was
resuspended in PBS to a concentration of 0.5 mg/ml. Fifty ml
(25 ng) of each protein preparation was incubated in each of
the ELISA wells overnight at 48C. Plates were then rinsed
with washing buer (0.45% NaCl in deionized water
containing 0.05% Tween-20) and blocked with blocking
buer (5% non-fat dry milk in PBS with 1% BSA and 0.05%
Tween-20) for 2 h at 378C. Serum samples were then diluted
in dilution buer (5% non-fat dry milk in PBS with 0.05%
Tween-20) at the appropriate dilutions and incubated in
duplicate or triplicate in recombinant protein coated wells for
1 h at 378C, washed and then incubated for 1 h at 378C with
a goat anti-human Ig-horseradish peroxidase conjugate
(Sigma Chemical Co) diluted in dilution buer at the
concentration suggested by the manufacturer. After extensive
washing the plates were developed with 3,3',5,5'-tetramethyl-
benzidine dihydrochloride (TMB) substrate (100 mg/ml), the
reaction was stopped with 2 N H
2
SO
4
and color development
was quantitated at 450 nm. BSA coated wells were used as
negative binding control wells in these assays. Speci®c
binding (absorbance at 450 nm) was calculated by subtracting
A
450
values from sera samples bound to BSA (that is,
control) from A
450
values from sera samples bound to gp120;
that is, experimental wells (A
450
experimental7A
450
control).
Western blot analysis
Three mg of PSA protein was resolved on a 10% SDS ±
PAGE prep gel and the protein was transferred to PVDF
Immobilon
2
-P membrane (Millipore, Bedford, MA, USA).
The membrane was cut into strips and each strip was
incubated with PSA immunized monkey sera, mouse
monoclonal PSA Ab-3 (ER-PR8+A67-B/E3) (NeoMarkers
Inc., Union City, CA, USA), or rabbit anti-PSA primary Ab
in 1 : 250 dilution. After incubation with HRP conjugated
secondary Abs, the presence of Abs was con®rmed by light
emitting non-radioactive reagent ECLTM Western blotting
(Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).
Lymphoproliferative assay
Peripheral blood lymphocytes were prepared as previously
described (Boyer, 1997). The isolated cell suspensions were
resuspended to a concentration of 5610
5
cells/ml in a media
consisting of RPMI 1640 (Gibco ± BRL) with 10% fetal calf
serum (Gibco ± BRL). A 100 ml aliquot containing 5610
5
cells was immediately added to each well of a 96-well
microtiter round bottom plate. Recombinant PSA protein
(Fitzgerald Industries) at the ®nal concentrations of 5 and
1mg/ml were added to wells in triplicate. The cells were
incubated at 378Cin5%CO
2
for 3 days. One mCi of tritiated
Immunology and safety in rhesus macaques immunized with a PSA DNA vaccine
JJ Kim et al
4504
Oncogene
thymidine was added to each well and the cells were
incubated for 12 ± 18 h at 378C. The plates were harvested
and the amount of incorporated tritiated thymidine was
measured in a Beta Plate reader (Wallac, Turku, Finland).
Stimulation Index was determined from the formula:
Stimulation Index SIexperimental count=spontaneous count
Spontaneous count wells (media only) include 10% fetal calf
serum. To assure that cells were healthy, Concanavalin A
(Sigma) was used as a polyclonal stimulator positive control.
The data was analysed statistically using a paired Student
t-test.
Cytokine expression analysis
Supernatants from PSA-speci®c lymphoproliferative assay
were collected and tested for cytokine pro®le using ELISA
kits for IFN-g(Figure 5a) and IL-4 (Figure 5b) (Biosource
International Inc., Camarillo, CA, USA). For each sample at
each time point, 100 ml of supernatant were tested in
triplicate wells. Each well value was used to derive the
average and the standard deviation values. The Pvalues were
calculated using the Student t-test.
Immunohistochemical analysis of prostate from immunized
macaques
Frozen section slides were prepared from the prostate tissues
from pCPSA immunized macaques. Four micron frozen
sections were made using a Leica 1800 cryostat (Leica Inc.,
Deer®eld, IL, USA). To detect the presence of lymphocytes
in muscle, the slides were stained with hematoxylin and eosin
(H&E) stain (Vector Labs). The slides were viewed with a
Nikon OPTIPHOT ¯uorescing microscope (Nikon Inc.,
Tokyo, Japan) using a 406objective (Nikon Fluo
406Ph3D2). Slide photographs were obtained using a Nikon
camera FX35DX with exposure control by Nikon UFX-II
and Kodak Ektachrome 160T slide ®lm.
Acknowledgments
This work was supported in part by grants from NIH to
DB Weiner.
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Oncogene
... Also the safety of DNA vaccines has been established in various trials in several species, including humans J. Vet. Adv., 2012, 2(4):139-148 (Bagarazzi et al., 1998;Kim et al., 2001). Since its invention, a variety of DNA vaccines have undergone clinical trials in veterinary practice (Babiuk et al., 1998;Dunham, 2002;Oshop et al., 2002). ...
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Article
The causes of prostate cancer reflect a complex interaction between environmental and genetic factors. Improvement in screening has reduced the incidence of prostate cancer, and risk assessment schemata have enhanced therapy, both for localized disease and for locally recurrent prostate cancer. The use of hormone therapy has been further evaluated, as primary therapy for locally advanced cancers, for lymph node–positive cancers, and for de novo metastatic cancer. Modest inroads have been made in the treatment and understanding of androgen-independent prostate cancer. Advances have been made in the understanding of the risk factors, genetic and environmental, associated with the development and progression of prostate cancer; in screening; and in optimizing therapy for localized, locally recurrent, and advanced disease. This article reviews the most salient observations reported between November 1, 1997 and October 31, 1998.
Article
BACKGROUND In this study, the authors evaluated whether a clinically relevant stratification of prostate specific antigen (PSA) failure free survival (bNED) after definitive local therapy could be made for patients with prostate carcinoma clinically classified as T1 or T2 and pretreatment PSA levels of 4-20 ng/mL.METHODS Multivariate Cox regression analysis and Kaplan-Meier analysis were performed for clinically localized prostate carcinoma patients who presented with PSA levels of 4-20 ng/mL. Three hundred forty-eight of the patients were managed definitively with conventional external beam radiation therapy (median dose, 67 gray), whereas 547 of the patients were managed definitively with a radical retropubic prostatectomy. The outcome tested was time to posttreatment PSA failure. The clinical predictors evaluated included the standard paradigm (PSA, biopsy Gleason score, and clinical stage); type of local therapy; and a newly defined factor, the calculated prostate cancer volume (cVCa).RESULTSTime to posttreatment PSA failure was equivalent (P = 0.52) independent of the type of local therapy. The cVCa (P < 0.0001), pretreatment PSA (P = 0.003), and clinical classification of T2c (P = 0.04) remained significant predictors of time to posttreatment PSA failure in multivariate analysis.CONCLUSIONS The staging system described herein, which is based on cVCa and PSA, may optimize patient selection for definitive local therapy and entry onto randomized clinical trials examining the use of adjuvant hormonal or chemotherapy in patients with clinically localized disease who present with PSA levels of 4-20 ng/mL. Validation of this staging system by other investigators is currently underway. Cancer 1998;82:334-41. © 1998 American Cancer Society.
Article
Prostate-specific antigen (PSA) is. a serine protease secreted by prostatic epithelial cells and is widely used as a marker for prostate cancer. The tissue specificity of PSA makes it a potential target for active specific immunotherapy, especially in prostate cancer patients who have undergone prostatectomy and in whom the only PSA-expressing tissue in the body resides in metastatic deposits. We report here the cloning, construction and immunological consequences of immunization of rhesus monkeys with a recombinant vaccinia virus expressing human PSA (designated rV-PSA). The prostate gland of the rhesus is structurally and functionally similar to the human prostate. While rodent and other mammalian species do not share homology with human PSA, there is 94% homology between the amino acid sequences of rhesus and human PSA. Immunization of rhesus monkeys with wild-type vaccinia virus or rV-PSA elicited the usual low-grade constitutional symptoms of vaccinia virus infection. There was no evidence of any adverse effects in any immunized monkeys. A short-lived PSA-specific IgM antibody response was noted in all rV-PSA immunized monkeys regardless of dose level. All monkeys receiving the 108pfu dose of rV-PSA demonstrated PSA-specific T-cell responses that were maintained up to 270 days. No differences in anti-PSA immune responses or toxicity were observed in animals that received prostatectomy prior to immunization. Our results thus demonstrate the safety and immunogenicity of rV-PSA in a non-human primate and have implications for potential specific immunotherapy protocols using PSA as a target.
Article
Dendritic cells (DCs) are “professional” antigen-presenting cells capable of stimulating T-cell proliferation and cytotoxicity when loaded with and presenting specific antigens, including tumor antigens. We demonstrated the stimulation of an autologous cytotoxic T-cell response elicited by DC loaded with autologous tumor cell lysate derived from primary prostate tumor. A candidate tumor antigen is prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer patients. We identified a HLA-A2 motif in PSMA, isolated patient DC, loaded peptide into DC, and stimulated autologous T cells to proliferate. The ability to use DC for presentation of either tumor or peptide antigen in an HLA-restricted fashion in order to stimulate T-cell proliferation and cytotoxicity demonstrates the potential of this technology for development of a prostate cancer vaccine. © 1996 Wiley-Liss, Inc.
Article
A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts ≥500/μl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 μg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-μg dose group also developed increased MIP-1α levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 μg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.
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Prostate specific antigen (PSA) is serine protease produced at high concentrations by normal and malignant prostatic epithelium. It is mainly secreted into seminal fluid, where it digests the gel forming after ejaculation. Only minor amounts of PSA leak out into circulation from the normal prostate, but the release of PSA is increased in prostatic disease. Thus PSA is a sensitive serum marker for prostate cancer but its specificity is limited by a high frequency of falsely elevated values in men with benign prostatic hyperplasia (BPH). Approximately two-thirds of all elevated values (>4 μg/l) in men over 50 years of age are due to BPH. In serum, most of the PSA immunoreactivity consists of a complex between PSA and α1-antichymotrypsin (PSA-ACT) whereas approximately 5–40% are free. The proportion of PSA-ACT is larger and the free fraction is smaller in prostate cancer than in benign prostatic hyperplasia (BPH). Determination of the proportion of free PSA has become widely used to improve the cancer specificity of PSA especially in men with PSA values in the `grey zone' (4–10 μg/l). PSA also occurs in complexes with other protease inhibitors and determination of these and other markers may further improve the diagnostic accuracy for prostate cancer. Interpretation of the results for many different markers is complicated, but this can be simplified by using statistical methods. The diagnostic accuracy can be further improved by using logistic regression or neural networks to estimate the combined impact of marker results and other findings like digital rectal examination (DRE), transrectal ultrasound (TRUS) and heredity.
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The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases of the kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the charge-relay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and--to a lesser extent--gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the alpha-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide yielded a specific activity of 9.21 microM per min per mg of PA and Km and kcat values of 15.3 mM and 0.075s-1, respectively. "Trypsin-like" activity of PA was also detected with N alpha-benzoyl-DL-arginine p-nitroanilide and gave a specific activity of 1.98 microM per min per mg of PA. Protease inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl ketone, aprotinin, leupeptin, soybean trypsin inhibitor as well as Zn2+ and spermidine were effective inhibitors of PA enzymatic activity.
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Urinary incontinence after radical prostatectomy is a difficult postoperative problem and often is a major consideration in the selection of therapy for clinically localized disease. The occurrence of incontinence is unpredictable and the relationship of incontinence to operative technique is unclear. We compared urinary continence in 68 consecutive patients undergoing radical prostatectomy. In 34 patients nonnerve-sparing radical prostatectomy was performed and in 34 subsequent patients a nerve-sparing operation was done. Patient age, Gleason score and stage of the tumor, and operative time were not significantly different between the groups. In the nonnerve-sparing operated group there were 4 patients (12%) with total and 6 (18%) with stress incontinence requiring absorbent pads, compared to 0 and 2 (6%), respectively, in the nerve-sparing group. The postoperative functional urethral length in the nonnerve-sparing group was 1.9 +/- 0.6 cm. (standard deviation) and in the nerve-sparing group it was 2.3 +/- 0.5 cm., which was significantly different (p less than 0.05). The peak resting urethral pressure of the nonnerve-sparing group was 35.4 +/- 14.2 cm. water and in the nerve-sparing group it was 46.5 +/- 12.3 cm. water, which also was significantly different (p less than 0.05). The study indicates that preservation of the pelvic nerves during radical prostatectomy has a major role in the functional preservation of urinary continence.