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Wistar Institute, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently reported that an E1-deleted adenoviral recombinant derived from the human strain 5 expressing the rabies virus glycoprotein induces a strong secretory Ab response in the intestines and genital tract after i.n. immunization (4). Adenoviral recombinants are replication defective and thus exceptionally safe. Furthermore, they show high efficacy in inducing B, Th, and cytolytic T cell responses to the encoded transgene product (5, 6, 7). Nevertheless, one potential drawback for the use of human adenoviral recombinants as vaccine carriers in humans is the preexisting immunity to this common pathogen, which will interfere with the induction of an immune response to the transgene product.
DNA vaccines, one of the latest advances in vaccinology, are bacterial vectors that carry the gene of a pathogen under the control of a strong constitutively active promoter such as the one derived from CMV (8, 9). Inoculation of DNA vaccines into muscle or skin by a syringe or a propulsion device such as a gene gun has been shown to result in the uptake of DNA into cells followed by transcription and translation of the pathogen’s gene, resulting in an immune response composed of Abs, Th cells, and cytolytic T cells. Although some groups reported induction of mucosal immunity upon application of DNA vaccines to mucosal surfaces (10, 11, 12, 13), such responses either were rather low or required the addition of adjuvants or other specific formulations. DNA vaccines were shown in several systems to prime the immune system to a subsequent booster immunization with a traditional vaccine (14, 15). In these reports, the DNA vaccines as well as the Ags used for booster immunization were applied systemically. Considering the significant cross-talk between the central and the mucosal immune systems, we tested whether DNA vaccines given systemically could affect the vaginal Ab response to a subsequent i.n. inoculation of an adenoviral recombinant expressing the same transgene product. In this article we show that DNA vaccines to rabies virus given i.m. or i.d., or applied to mucosal surfaces induce strong systemic Ab responses but elicit only borderline responses at the genital mucosa. Nevertheless, after a second i.n. immunization with an E1-deleted adenoviral recombinant to rabies virus, mice primed with the DNA vaccines developed augmented and prolonged vaginal Ab responses. The responses could further be enhanced by using Th2 cytokines such as IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female 6–10-wk-old C3H/He mice purchased from The Jackson Laboratory (Bar Harbor, ME) were kept in the Animal Facility of The Wistar Institute. They were used between 2 and 3 mo of age.
Viruses
Rabies virus of the Evelyn Rokitniki Abelseth (ERA)4 strain was propagated and titrated on baby hamster kidney (BHK)-21 cells and inactivated by ß-propionolactone treatment (16). The E1-deleted adenovirus recombinant expressing the rabies virus glycoprotein of the ERA strain (Adrab.gp) was grown on E1-transfected 293 cells (7). An E1-deleted adenoviral recombinant expressing E7 of human papilloma virus type 16 (AdE7) was generated using previously described methods (7). The vaccinia virus recombinant expressing the glycoprotein of rabies virus strain ERA was propagated and titrated on HeLa cells (17). Purified adenovirus human strain 5 was purchased from the Vector Core Facility of the Gene Therapy Group of the University of Pennsylvania.
Mammalian cell lines
BHK-21, HeLa, and 293 cells were grown in DMEM supplemented with 10% FBS and antibiotics.
Bacterial cell lines
Escherichia coli DH5
cells were grown in
Luria-Brtani (LB) broth. Transformed cells were propagated in LB broth
supplemented with ampicillin.
Plasmid vectors
The pSG5rab.gp vector expressing the glycoprotein of the ERA
strain of rabies virus under the control of the SV40 promoter has been
described previously (18). The pVR1012 vector (provided by Vical,
San Diego, CA) was first modified by replacing the kanamycin resistance
gene with the ampicillin resistance gene obtained from the pUC19
vector. The pVRamp.rab.gp vector was constructed by cloning the
rabies virus glycoprotein gene into the multicloning site
downstream of the CMV promoter. The resulting vector was
subsequently analyzed by restriction enzyme digest and upon transient
transfection of BHK-21 cells for appropriate expression of the viral
glycoprotein using a specific mAb in an indirect immunofluorescence
assay followed by analysis in a FACS (18). The mouse IL-4 cDNA and the
ß-galactosidase (lacZ) coding sequence were cloned into the pSG5
vector using standard molecular techniques to generate the pSG5IL-4 and
pSG5lacZ vectors. The mouse IL-5 cDNA was cloned into the pcDNA3
vector. The ampicillin-resistant pVR1012.2 vector, the pSG5 vector
without insert, or the pSG5lacZ vector were used as control vectors
throughout these studies. A list of the different plasmids as well as
the viral recombinants used in these studies are shown in Table I
. Vectors were expanded in DH5 and
purified using the Mega Purification Kit (Promega, Madison, WI)
according to the manufacturer’s specification. DNA was quantitated by
electrophoresis on agarose gel against a known standard.
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Groups of five female mice were injected with 50 µg of vector diluted in 50 µl of either saline or water into both quadriceps muscles, dividing the dose equally to reduce variability. Injections were given i.d. at two sites into the skin of the lower back. Inoculation by the i.t. route was conducted in anesthetized mice after surgical exposure of the trachea. Adenovirus was either given s.c. in 200 µl of saline into the flank or i.n. by depositing 50 µl of saline containing varied amounts of virus onto both nostrils.
Preparation of samples
Serum was prepared from blood harvested by retroorbital puncture. For all titrations, sera were tested at serial dilutions starting with a 1:200 dilution. For isotype determination, sera were tested at a 1:800 dilution. Vaginal lavage was obtained by rinsing the vaginal cavity three times with 50 µl of sterile saline. Debris was removed by centrifugation. Vaginal lavage was in most experiments tested immediately after harvesting starting for titrations with a 1:2 dilution of the lavage fluid (total of 150 µl/mouse). A 1:8 dilution of the lavage fluids was used for isotype testing. In some of the initial experiments, total Ig was determined in several different pooled lavage fluids with an ELISA using microtiter plates coated with a rabbit anti-mouse Ig to assess potential variability in the ratio of harvested lavage fluid to washing fluid. Using 4–5 mice/group, the amount of variability of total Ig in vaginal lavage fluids of different groups was negligible (data not shown).
Enzyme-linked immunoadsorbent assay
Sera and vaginal lavage samples were tested for Abs to rabies virus on microtiter plate wells coated with purified ß-propionolactone-inactivated ERA virus or adenovirus virus as described (7, 19). The x-axis units of the different graphs showing titrations reflect the relative dilution of samples on a logarithmic scale. Sera were tested in serial 2-fold dilutions starting with a dilution of 1:200; vaginal lavages, also tested at serial 2-fold dilutions, were started at a dilution of 1:2. Isotypes of specific Abs were determined using the Calbiochem Hybridoma Isotyping Kit (Calbiochem, La Jolla, CA) as described (19).
Statistical analysis
Data were analyzed by Student’s t test. Samples with p value <0.05 were considered to show a significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first set of experiments was designed to compare the ability
of DNA vaccines with that of traditional vaccines to prime the Ab
response to a subsequent booster immunization with an E1-deleted
adenoviral recombinant. Groups of five C3H/He mice were inoculated with
104 PFU of Adrab.gp virus, 106 PFU of vaccinia
rabies glycoprotein (VRG) virus, both given s.c., or 50 µg of
pSG5rab.gp vector or pSG5lacZ vector as a control, both given i.m. Mice
were bled 10 wk later and tested for Abs to rabies virus (Fig. 1). The Ab response to rabies virus was
statistically significantly highest in mice immunized with a low dose
of the adenoviral recombinant that we had described previously to be an
exceedingly potent vaccine (7). The DNA vaccine and the VRG recombinant
induced comparable Ab titers, while Ab titers in sera of mice injected
with the lacZ-expressing vector were identical with those of sera from
naive mice. All groups of mice were boosted the day after harvesting
the serum with 104 PFU of Adrab.gp virus given s.c. Blood
was collected 5 and 10 days later, and again serum Ab titers to rabies
were determined by an ELISA. As shown in Fig. 1, 5 days after booster
immunization the adenoviral recombinant given twice resulted in the
highest Ab titers followed by titers of sera from mice primed with the
DNA vaccine. Mice primed with the vaccinia recombinant showed at this
early time point only a small increase in titers, while Abs in the
pSG5lacZ-primed control mice were still below the level of
detectability. Titers in mice immunized with the Adrab.gp recombinant
remained stable 10 days after priming, while titers in VRG virus or
pSG5rab.gp vector-primed mice increased further. At day 10, there was
no statistically significant difference in Ab titers to rabies virus
between any of the three groups of mice primed with either of the
recombinant vaccines or the DNA vaccine. Titers of control mice primed
with the pSG5lacZ vector remained well below those observed in
Ag-primed mice. We next determined the isotype profile of mice primed
with the different constructs and then boosted with the adenoviral
recombinant. In all three groups, as well as in the control group
primed with the pSG5lacZ construct, isotypes of Abs to rabies virus
were mixed, i.e., composed of IgG1, IgG2a, and IgG2b with IgG2a being
predominant (data not shown). These data confirm that DNA vaccines are
highly suited to prime the immune system to a systemic booster
immunization with a potent viral recombinant vaccine.
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In the next set of experiments we tested whether DNA vaccines
could induce mucosal immunity. Groups of 5 C3H/He mice were inoculated
i.n., i.t., or i.m. with 50 µg of the pSG5rab.gp vector. Serum Ab
titers determined 4 and 6 wk later showed that i.m. vaccination
resulted in the highest Ab titers followed by i.t. immunization,
whereas titers upon i.n. inoculation were only borderline positive,
suggesting that vector DNA was preferentially taken up by the lower
rather than the upper airways. (Fig. 2
shows data for the sera harvested after 6 wk; data for i.n.
immunization are not shown.) Ab titers in vaginal lavage were
determined 6 weeks after vaccination. Mice immunized i.t. had no
detectable Abs to rabies virus in their vaginal secretions while low
but statistically significant titers could be detected in i.m.
immunized mice. We reported previously that an E1-deleted adenoviral
recombinant to rabies virus given i.n. induces a strong vaginal Ab
response (4). To test whether mice primed with a DNA vaccine to rabies
virus either systemically by i.m. injection, or through mucosal
surfaces by i.t. application, showed an enhanced vaginal Ab response
upon i.n. booster immunization with the Adrab.gp recombinant, DNA
vaccine-primed as well as naive control mice were booster with 2
x 106 PFU of the Adrab.gp recombinant given i.n. Sera and
vaginal lavage were harvested 4 wk later and tested for Abs to rabies
virus. As shown in Fig. 2, priming with the pSG5rab.gp vector enhanced
Ab titers in both serum and vaginal lavage. Both routes of DNA vaccine
priming, i.e., i.m. or i.t., were equally effective. To determine
whether the different routes of priming had favored the development of
distinct Ab isotypes, the isotype profiles of Abs to rabies virus in
sera and vaginal lavage were determined. As shown in Fig. 3, the response in sera upon DNA vaccine
priming by either route was clearly predominated by IgG2a. In vaginal
lavage, primed as well as unprimed mice developed comparable levels of
IgA, the difference in titers (Fig. 2) was mainly a reflection of
levels of IgG2a which were significantly higher in DNA vaccine-primed
mice (Fig. 3). These data show that DNA priming can augment the vaginal
Ab response upon booster immunization with an adenoviral recombinant
given i.n., mainly by enhancing IgG2a titers. Systemic priming is
equally effective to prime through the lower airways.
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). After booster immunization with the
Adrab.gp recombinant given i.n., vaginal lavage titers in primed mice
increased rapidly by day 5 when titers in control mice were still
undetectable. A further increase was observed by day 10 when titers in
mice primed with the empty vector before the booster immunization with
the Adrab.gp recombinant started to become positive. At later time
points, i.e., 24 days and 3 mo after booster immunization, control mice
had substantial levels of Abs to rabies virus in their vaginal lavage;
nevertheless, titers of DNA vaccine-primed mice remained clearly
superior. This was even more pronounced after 4 mo when titers in
vaginal lavage of control mice had declined to levels below
detectability, while titers of DNA vaccine-primed mice were still
readily measurable (Fig. 4).
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). Mucosal B cell response upon i.m.or i.d. priming with a DNA vaccine
DNA immunization given i.m. results in a preferential Th1 response
while, as was shown in some systems (20), i.d. injection favors
activation of a Th2 response which is required to induce activation of
IgA-secreting B cells. We therefore next compared the effect of
i.d. vs i.m. priming with the pSG5rab.gp vector on the mucosal Ab
response to i.n. booster immunization with the Adrab.gp vaccine. Groups
of 5 C3H/He mice were inoculated either i.m. or i.d. with 50 µg
of pSG5rab.gp or pSG5 vector. Vaginal lavage and sera were tested 8 wk
later for Abs to rabies virus. While Ab titers in sera were
substantial, with those achieved upon i.d. immunization being higher
(data not shown), titers in vaginal lavage were comparable upon both
routes of immunization. Mice were then boosted i.n. with 2 x
106 PFU of the adenoviral recombinant and serum and vaginal
lavage titers were measured 5 days as well as 4 wk later. Mice primed
with the Ag-expressing vector exhibited clearly higher titers in sera
(data not shown) and vaginal lavage compared with those inoculated with
the control construct (data for vaginal lavage shown in Fig. 6). In this experiment, i.d. DNA vaccine
priming resulted at both time points in slightly superior titers in
sera and vaginal lavage compared with i.m. priming. This pattern was
variable, and in subsequent experiments the difference in titers in
either fluid upon i.m. or i.d. immunization was less pronounced or even
reversed. Mice immunized i.d. with the control vector developed
reproducibly very poor Ab responses compared with mice immunized i.m.
with the same construct. We assume that this is in part a reflection of
the immunomodulatory activity of bacterial DNA, which might have
different effects depending on the anatomic site of vaccination.
The isotypes of vaginal Abs to rabies virus tested after 5 days
and 4 wk showed that i.m. DNA priming resulted in a preferential
increase of IgG2a while i.d. priming strongly augmented an IgA response
at the early time point that by 4 wk was in part replaced by IgG1 and
IgG2a. This difference in isotype distribution was observed in all
experiments.
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The effect of DNA vaccine priming on the Ab response to the
adenoviral Ags of the Adrab.gp vaccine was investigated. Groups of
C3H/He mice were primed with 50 µg of the pSG5rab.gp vaccine and
boosted with the Adrab.gp construct given i.n. 6 wk later. Before the
booster immunization, DNA-vaccinated mice still had serum Abs and low
but detectable vaginal lavage Abs to rabies virus. Sera and vaginal
lavage were tested 1 wk after the adenoviral boost for Abs to rabies
virus and for Abs to adenovirus. As expected, pSG5rab.gp-primed mice
developed enhanced serum and vaginal lavage Ab titers upon booster
immunization when compared with control mice. The magnitude of titers
to the adenoviral Ags was reversed; mice that had been primed with the
DNA vaccine to rabies virus developed only marginal serum and vaginal
lavage titers to adenoviral Ags, while unprimed Adrab.gp-immunized mice
developed substantial Ab titers at both sites, indicating that the
preexisting immune response to the plasmid-encoded Ag had actively
interfered with the Ab response to the vaccine carrier (Fig. 7).
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Although the E1-deleted adenoviral recombinant to rabies virus is
an exceedingly potent vaccine in rodents, humans will most likely show
reduced efficacy due to preexisting immunity to wild-type adenovirus
human strain 5, which forms the basis of the Adrab.gp recombinant. To
test whether DNA vaccines could augment the Ab response to a transgene
product expressed by an adenoviral recombinant in mice with preexisting
immunity to the vaccine carrier, groups of 5 C3H/He mice were immunized
with 2 x 106 PFU of the AdE7 recombinant given i.n.
Some of these mice were boosted 1 month later with 50 µg of
pSG5rab.gp vector or pSG5 vector given i.m. After 2 mo, mice were
boosted i.n. with 2 x 106 PFU of Adrab.gp virus.
Naive control mice were immunized i.n. at the same time with Adrab.gp
virus. Sera and vaginal lavage were harvested 10 days later and tested
for Abs to rabies virus. As shown in Fig. 8, the Ab response to rabies virus as
expressed by the adenoviral recombinant was reduced in sera of
adenovirus preimmune mice, while no difference was observed in vaginal
lavage. In both fluids, DNA vaccine priming strongly augmented the Ab
response above that seen in naive mice vaccinated with the Adrab.gp
recombinant only, indicating that this combination vaccine regimen was
suited to overcome the impairment of the Ab response to the rabies
virus Ag due to preexisting immunity to the vaccine carrier.
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The immune response to a DNA vaccine can readily be enhanced by
the use of genetic adjuvants in the form of plasmid vectors expressing
mouse cytokines given concomitantly with the DNA vaccine (21, 22). In
the next set of experiments, we tested the effect of IL-4 and IL-5, two
cytokines known to promote mucosal IgA responses, on the Ab response
upon DNA vaccine priming followed by i.n. booster immunization with the
adenoviral recombinant. Mice vaccinated with the DNA vaccine in the
presence of IL-4 had increased serum Ab titers before booster
immunization, IL-4 had no such effect on titers in vaginal lavage (data
not shown). IL-5 in combination with the pSG5rab.gp vector did not
increase serum Ab titers but enhanced vaginal lavage titers tested
before boosting. Mice were boosted 6–8 wk later with the Adrab.gp
recombinant given i.n. Adrab.gp was used at 2 x 106
PFU in the initial experiments. The IL-5-encoding plasmid, as shown in
Fig. 9, markedly increased Ab titers in
vaginal lavage, serum Ab titers were not enhanced. IL-5 augmented
mainly Th2-type isotypes such as IgA and IgG1 in vaginal lavage. IL-5
also caused a slight but statistically significant increase in
secretion of IgG2a, an isotype associated with Th1 responses. IL-5
given without the DNA vaccine had only a marginal effect on Ab titers
in vaginal lavage after booster immunization with the Adrab.gp
recombinant. IL-4 also augmented Ab titers in vaginal lavage following
a booster immunization with 2 x 106 PFU of Adrab.gp
virus (data not shown). Nevertheless, the effect was not as striking as
that observed with IL-5. Upon using a lowered dose of Adrab.gp virus
given at 5 x 103 PFU i.n., the IL-4-encoding plasmid
given concomitantly with the DNA vaccine strongly increased the vaginal
Ab response (Fig. 10). The IL-4 vector
given alone, i.e., without the pSG5rab.gp vector, had no such effect
(data not shown). In contrast to IL-5, IL-4 mainly increased
Th1-related Abs of the IgG2a isotype in vaginal lavage.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The comparatively low immunogenicity of DNA vaccines can be reversed by using DNA vaccines for priming followed by a traditional, more potent vaccine for booster immunization (14, 15). We tested in the experiments detailed here whether DNA vaccines, which are known to prime the systemic immune response, were also able to augment the mucosal immune response upon i.n. booster immunization with a replication-defective E1-deleted adenoviral recombinant.
E1-deleted adenoviral recombinants of the human strain 5 were initially developed for gene therapy, especially for the treatment of lung disorders such as cystic fibrosis (27). Such recombinants expressing reporter proteins were found, during preclinical trials, to infect cells of the airways efficiently, leading to high levels of transgene expression (28). Side effects but for a mild inflammatory reaction were not observed (29) even upon application of the high doses of recombinant virus needed to achieve eventually therapeutic levels of the missing protein. Nevertheless, expression of the transgene product was transient (6, 27). In immunocompetent individuals, the E1-deleted adenoviral recombinants induced cytolytic T cells to the structural proteins of adenovirus as well as to the transgene product, which rapidly eliminated the infected cells. A concomitantly induced CD4+ T cell response sponsored activation of B cells including those secreting neutralizing Abs to adenovirus. The Abs reduced uptake of the adenoviral gene therapy vehicle upon readministration. Although the strong immune response to the adenoviral recombinant restricted their use for permanent replacement of missing or faulty genes, it suggested their suitability as vaccine carriers. We previously described that the E1-deleted adenoviral recombinant expressing the glycoprotein of rabies virus induces a potent rabies virus-neutralizing Ab response after s.c. inoculation. The efficacy of the replication-defective adenoviral recombinant exceeded that of a vaccinia recombinant expressing the same Ag of rabies virus by a factor of at least 100; while 104 PFU of the adenoviral recombinant induced complete protection to challenge with rabies, the vaccinia recombinant used at 106 PFU induced protection in less than one-half of the animals. Adenoviral recombinants also have the advantage of inducing both central and mucosal immunity upon i.n. application.
The mucosal immune system differs from the systemic immune system with regard to a number of parameters; nevertheless, there appears to be substantial cross-communication between those two systems. Systemic immunization commonly results in low but measurable immune responses at mucosal surfaces while vice versa mucosal immunization causes activation of central immune responses. Furthermore, previous studies have shown that s.c. priming with a protein-based vaccine to influenza virus followed by a systemic booster immunization with the same vaccine augmented the local mucosal immune response (30). Vice versa mucosal priming with a vaccine to HIV has been shown to augment the mucosal immune response to an i.d. booster immunization with the same recombinant (31). Although it is currently unclear whether the two immune systems communicate by lymphocyte trafficking or by migration of APC, these data nevertheless strongly suggest that prime boost regimens using alternating central and mucosal routes of immunization might enhance the immune response at both sites.
We initially tested the effect of i.m., i.t., and i.d DNA vaccine
priming on the vaginal Ab response to an i.n. booster immunization with
the Adrab.gp recombinant to establish whether any of these routes was
superior in priming vaginal Ab secretion. The i.t. immunization was
tested as a mucosal immunization route. Immunization by the i.d. route
was explored, because in some systems this route was shown to
generate a preferential Th2 response compared with i.m. DNA
vaccine immunization (20), which due to the IL-12 and IFN-
induction
by the CpG sequences of the vector backbone causes a strongly biased
Th1 response (25). Furthermore, T cells residing in the skin show
phenotypic similarities with T cells in the vaginal epithelium (32),
suggesting not only common ancestors but potentially also
interchangeable homing patterns. All three routes of DNA vaccination
induced vaginal Ab responses upon i.n. Adrab.gp booster immunization
that were comparable in magnitude; the only reproducible difference was
that i.d. priming induced higher IgA responses compared with i.m. or
i.t. priming which is consistent with the notion that this route of DNA
vaccination sponsors Th2 type immunity. The route chosen for all of the
subsequent experiments, i.m. DNA vaccine priming, not only enhanced the
vaginal Ab response following i.n. Adrab.gp booster immunization but
also extended the longevity of the vaginal Ab response.
Although our data show that the systemic and mucosal Ab response in experimental animals to a single i.n. immunization with the Adrab.gp recombinant is not only substantial but also long-lasting, in humans, the natural host for wild-type adenovirus human strain 5, preexisting Abs to structural proteins of the virus that can be demonstrated in >95% of the population are likely to affect the efficacy of the vaccine. We had shown previously that this could be counterbalanced by increasing the vaccine dose, an approach that might not be practical for clinical use as this would augment the cost of the vaccine and enhance the risk of side effects. The use of DNA vaccine priming also rescued the Ab response to the rabies virus glycoprotein expressed by the Adrab.gp recombinant in mice preimmune to adenoviral Ags, suggesting that such a vaccine regimen might be highly suitable for use in humans with previous exposure to wild-type adenovirus. Conversely, DNA vaccine priming reduced the Ab response to the adenoviral Ags after i.n. booster immunization with the Adrab.gp recombinant. This phenomenon, which we cannot yet fully explain, might reflect cytokine competition between already activated B cells to rabies virus and naive B cells to the adenoviral Ags. Regardless of the mechanisms, this observed reduction in the Ab response to the vaccine carrier is very encouraging in that it suggests that using a combination of DNA vaccine priming before a booster immunization with a viral recombinant vaccine might allow for repeated use of the recombinant. Nevertheless, these experiments remain to be extended to additional recombinant vaccines based on carriers other than adenovirus to ensure that DNA vaccine priming in general impairs the B cell response to the Ags of the recombinant vaccine.
In an attempt to further augment the vaginal Ab response, we tested two Th2 cytokines, i.e., IL-4 and IL-5 which both promote stimulation of IgA-secreting B cells, as genetic adjuvants given concomitantly with the DNA vaccine. Numerous studies have shown that most cytokines expressed by plasmid vectors enhance or modulate the systemic immune response to a DNA vaccine (33). We had reported previously that IL-4 augmented the central B and T cell response to a DNA vaccine given i.m. IL-4 did not switch the immune response toward the Th2 pathway but rather augmented Th1-linked immunity including cytolytic T cells and B cells secreting IgG2a Abs (21). At the mucosa, the effect of IL-4 given with the DNA vaccine priming could best be demonstrated upon lowering the dose of the Adrab.gp recombinant used for i.n. booster immunization. As we had observed previously, IL-4 augmented secretion of IgG2a, a Th1-type isotype, rather than sponsoring Th2-related isotypes. IL-5 was more efficacious as a mucosal genetic adjuvant and strongly augmented vaginal Ab secretions even before boost or upon the use of high doses of the Adrab.gp vaccine for reimmunization. In contrast to IL-4, IL-5 favored stimulation of Th2 type Ab isotypes such as IgA and IgG1.
In summary, our data, using a simple and well-defined viral test system in mice, show that DNA vaccines given i.m. can prime a vaginal Ab response including secretion of specific IgA to i.n. immunization with an adenoviral recombinant expressing the same Ag. The vaginal Ab response could further be augmented by using IL-4 and especially IL-5 as genetic adjuvants concomitantly with the DNA vaccine. These results demonstrate that such a vaccine regimen, which does not require the use of toxic adjuvants, might be suitable for prevention of diseases, such as sexually transmitted diseases, where the pathogens infect through mucosal surfaces.
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Acknowledgments |
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Footnotes |
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2 Current address: Imperial Cancer Research Fund Molecular Oncology Unit, Imperial College School of Medicine Cyclotron Building, Floor 8, Hammersmith Hospital, Du Cane Road, London W12 OHS, U.K.
3 Address correspondence and reprint requests to Dr. Hildegund C. J. Ertl, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
4 Abbreviations used in this paper: ERA, Evelyn Rokitniki Abelseth; BHK, baby hamster kidney; Adrab.gp, glycoprotein of the ERA strain; LB, Luria-Bertani; VRG, vaccinia rabies glycoprotein.
Received for publication December 28, 1998. Accepted for publication March 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 93:2879.
T cell receptor repertoire of murine T cells associated with the vaginal epithelium. J. Immunol. 147:1773.[Abstract]
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Z. Xiang, G. Gao, A. Reyes-Sandoval, C. J. Cohen, Y. Li, J. M. Bergelson, J. M. Wilson, and H. C. J. Ertl Novel, Chimpanzee Serotype 68-Based Adenoviral Vaccine Carrier for Induction of Antibodies to a Transgene Product J. Virol., February 22, 2002; 76(6): 2667 - 2675. [Abstract] [Full Text] [PDF]
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 C. J. Cohen, Z. Q. Xiang, G.-P. Gao, H. C. J. Ertl, J. M. Wilson, and J. M. Bergelson Chimpanzee adenovirus CV-68 adapted as a gene delivery vector interacts with the coxsackievirus and adenovirus receptor J. Gen. Virol., January 1, 2002; 83(1): 151 - 155. [Abstract] [Full Text] [PDF]
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). All mice were
boosted with 104 PFU of Adrab.gp virus given s.c. Mice were
bled 5 (
) and 10 (
) days later. Normal mouse serum was used as a
control (x).
, pVRamp prime +
Adrab.gp boost; 
, Adrab.gp
virus only. Mice were retested 4 wk after booster immunization with the
Adrab.gp virus (bottom).
, Adrab.gp
virus only; 
, pSG5rab.gp priming given i.d.; 
, 
, pSG5 i.d. + Adrab.gp; 
,
, pSG5 i.m. +
Adrab.gp; x, 
