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*
Department of Immunobiology, Guy’s Kings College and St. Thomas’ Medical and Dental Schools, London, United Kingdom; and
Vical, Inc., San Diego, CA 92121
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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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Recently, several groups have reported the ability of DNA expression vectors encoding foreign proteins to elicit both cellular and humoral immune responses to the encoded protein following immunization by the i.m. (14, 15, 16, 17, 18) or the intradermal (19, 20) route. Protection against systemic infection in several murine and non-human primate models by this technology (16, 18, 21, 22) suggests that this novel approach may also elicit protective mucosal immunity, provided the plasmid DNA is targeted to the specialized inductive sites of the mucosal immune system. Such sites may include the organized lymphoid tissues associated with the gut, rectum, bronchus, and nasopharynx or the draining LNs (23, 24, 25). Ags presented at these sites induce the priming and homing of IgA-committed B cells and effector T cells to a variety of mucosal tissues (25, 26). Such an approach has the advantage of potentially achieving both systemic immunity and secretion of Ag-specific IgA at distant mucosal sites.
The nasal mucosa presents an attractive, noninvasive route for the delivery of DNA vaccines. Based on the concept of a common mucosal immune system (26, 27), we have addressed whether genetic immunization by the intranasal route will elicit both systemic and secretory Ab responses at the cervical-vaginal and rectal mucosa and MHC-restricted CTL in the regional LNs and spleen. To evaluate the feasibility of this approach for the development of vaccines against sexually transmitted pathogens, we used firefly luciferase (Photinus pyralis) as a model Ag to investigate 1) the efficiency of cationic lipids to augment gene expression and indirectly influence Ag presentation at the nasal mucosa, 2) the distribution and duration of plasmid DNA and mRNA following vaccination, and 3) the efficiency of cationic lipid-mediated genetic immunization to induce systemic and mucosal immunity in the female genital tract.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Firefly luciferase cDNA was subcloned from plasmid pGEM-luc
(Promega, Madison, WI) into the BglII site of the eukaryotic
expression vector VIJ (28). This vector consists of a pUC-19 backbone
with the CMV IEI enhancer, promoter and intron A transcription
regulatory elements, a bovine growth hormone polyadenylation sequence,
and an ampicillin resistance gene (28). Plasmid VIJ-luc was transformed
into Escherichia coli DH5
and purified by alkaline lysis
and double cesium chloride gradient ultracentrifugation (29) followed
by ethanol precipitation. Plasmid DNA was resuspended in pyrogen-free
water. The spectrophotometric
A260/A280 ratios were
between 1.7 and 2.0. The endotoxin content was <5 ng/mg of plasmid DNA
determined by the Limulus amebocyte lysate assay (Sigma, St.
Louis, MO).
Plasmid DNA-lipid formulation
To enhance the efficiency of plasmid DNA uptake by nasal epithelium, plasmid DNA was complexed with DMRIE/DOPE ((+)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine) provided by our colleagues at Vical (San Diego, CA). Characterization of this lipid has been reported previously (30). The lipid formulation was prepared by dissolving DMRIE (30) and DOPE into chloroform and was lyophilized by rotary evaporation and overnight evacuation (31). All plasmid DNAs were complexed by incorporating the DNA into the lipid using a 1/1 molar ratio of DMRIE/DOPE at a DNA/lipid ratio of 5/1 (w/w) based on the mass of DMRIE in the liposome complex. The final plasmid DNA concentration was 2.5 mg/ml.
Immunization
Seven- to eight-week-old female BALB/c mice (Harlan Olac, Bichester, U.K.) received 20 µl of plasmid DNA-lipid complexes containing 50 µg of plasmid DNA to the nares under general anesthesia (Hypnovel, Roche, Welwyn Garden City, U.K.; and Hypnorm, Janssen, Beerse, Belgium). In certain experiments, the quadriceps muscles of anesthetized mice were injected with 50 µg of DNA in saline. Animal care throughout the study was in accordance with U.K. Home Office guidelines.
Measurement of luciferase expression
At varying times following intranasal immunization, animals were euthanized with an overdose of anesthesia (Hypnovel and Hypnorm). Nasal tissue was excised, weighed, and snap-frozen on dry ice. Frozen tissues were homogenized in 100 µl of lysis buffer (Promega, Cell Culture Lysis Reagent) and centrifuged for 3 min at 10,000 x g, and 20 µl of the supernatant was assayed for luciferase enzyme activity by chemiluminescence assay (Promega Luciferase Assay System) using a Turner TD-20e luminometer. The luciferase content of samples was calculated from relative light units using a standard curve of purified firefly luciferase (Sigma). The total protein concentration of each nasal extract was determined by the Bio-Rad DC protein assay (Richmond, CA). Luciferase values were expressed as picograms per milligram of total protein extract.
In situ hybridization
In situ hybridization was performed on snap-frozen unfixed nasal tissue as described previously (32). A fluorescein-labeled antisense luciferase RNA was synthesized in vitro by run-off transcription from plasmid pGEM-luc DNA (Promega), incorporating fluorescein-12-UTP. Frozen sections were dehydrated in alcohol, and 30 ng of the antisense probe in hybridization buffer was added to each slide (hybridization buffer is 1x Denhardt’s solution, 4x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate), 100 µg/ml polyadenylitic acid, 10% dextran sulfate, 50% deionized formamide, and 100 µg/ml denatured herring sperm DNA). Slides were incubated overnight at 55°C and washed for 30 min at room temperature with 0.1x SSC followed by 30 min at 65°C with 0.5x SSC, for 10 min at room temperature with 0.5x SSC, and for 5 min at room temperature with 0.1 M Tris (pH 7.5) containing 0.4 M NaCl. Sections were incubated for 30 min at room temperature with 0.5% Amersham blocking agent (RPN 3023, Amersham, Aylesbury, U.K.) in 0.1 M Tris (pH 7.5) containing 0.4 M NaCl, followed by overnight incubation with an alkaline phosphatase-labeled anti-fluorescein Ab (Amersham). Hybridization signal was detected by incubation with 5-bromo-4-chloride-indolyl-phosphate and 4-nitro blue tetrazolium chloride (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions.
PCR
Individual tissues isolated with separate sterile instruments (as a precaution to prevent cross-contamination) were snap-frozen and stored at -70°C. DNA was extracted from minced tissues in 1.2 ml of lysis buffer (100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8), 0.5% SDS, and 0.1 mg of proteinase K/100 mg tissue) using standard techniques (28). PCR was performed in a 50-µl reaction volume containing 0.1 µg of DNA, 10 mM Tris-HCl (pH 8.3) 1.5 mM MgCl2, 50 mM KCl (pH 8.3), 2 mM dNTPs, 0.75 ng of each primer, and 2 U of Taq DNA polymerase (Boehringer Mannheim). The oligonucleotide primers used were; luciferase, 5'-TCTGACGCAGGCAGTTCTATG-3' (sense) and 5'-ACTACAAGTGGAGCTATACAC-3' (antisense) amplifying a 520-bp fragment; and glyceraldehyde-3-phosphate dehydrogenase, 5'-TCATGACCACAGTCCATGCCATCAC-3' (sense) and 5'-GACCATACTGTTACTTATGCCGATG-3' (antisense) amplifying a 436-bp fragment. The samples were processed on a Biometra Trio-Thermoblock thermal cycler (Tampa, FL) programmed for one cycle at 96°C for 2 min and 40 cycles each of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min. Ten-microliter aliquots of the PCR products were analyzed on a 1.5% agarose gel and visualized with ethidium bromide.
RT-PCR
Total RNA was extracted from frozen minced tissues using 1.0 ml of a guanidinium isothiocyanate/phenol-based monophasic extraction solution (TRIzol, Life Technologies, Grand Island, NY) as lysis buffer. After incubation for 5 min at room temperature, 0.2 ml of chloroform was added, and samples were vortexed and incubated 3 min at room temperature. The aqueous phase, separated by centrifugation for 15 min at 12,000 x g, was transferred to a new tube and combined with 0.5 ml of isopropyl alcohol. RNA was recovered by centrifugation (15 min at 12,000 x g), and following washing in 75% ethanol, the pellet was resuspended in diethylpyrocarbonate-treated water. To remove any possible contaminating plasmid DNA, RNA samples were incubated with 2.5 U of RNase-free DNase (60031, Stratagene, La Jolla, CA) for 1.5 h at 37°C, followed by phenol/chloroform extraction and ethanol precipitation. Five micrograms of total RNA was reverse transcribed using the Stratagene RT-PCR kit. As a control, reverse transcriptase was omitted from the reaction with a second aliquot of RNA. A 5-µl aliquot of each preparation (with or without reverse transcriptase) was then amplified using the oligonucleotide primers and PCR protocols described above and was analyzed by agarose gel electrophoresis.
Collection of fluids
Serum was obtained from tail vein blood or from blood collected from the subclavian veins when mice were euthanized. Vaginal and rectal fluids were obtained by introducing 50 µl of PBS with a pipette and withdrawing it five times. Vaginal and rectal fluids were collected twice over 72 h and pooled from individual mice. The fluids were stored at -20°C and centrifuged before assay.
ELISA for luciferase Ab
Firefly luciferase (Sigma) in 0.1 M carbonate-bicarbonate buffer, pH 9.6, was absorbed onto microtiter plates (Immulon 4, Dynatech, Chantilly, VA) overnight at 4°C. Plates were washed twice with PBS containing 0.05% Tween-20 (Merck Chemicals, Poole, U.K.) and blocked with 3% BSA (Sigma) for 2 h at room temperature. Plates were washed and incubated with 50 µl of serial dilutions of serum or vaginal or rectal fluids (in duplicate) for 2 h at room temperature. Plates were then washed and incubated for 2 h at room temperature with 100 µl of affinity-purified goat anti-mouse IgA or IgG conjugated to alkaline phosphatase (Sigma). Ab binding was detected by addition of p-nitrophenyl-phosphate disodium substrate (Sigma) and was measured by the OD emitted at 405 nm on a Bio-Rad model 2550 ELISA plate reader. The results were expressed as the lowest dilution giving an OD of 3 SD above the mean of the background sample for all serum samples and rectal and vaginal washings.
Measurement of sIgA
To detect the secretory component associated with luciferase-specific IgA, vaginal fluids were pooled separately (five per group) from mice immunized with plasmid DNA-cytofectin complexes containing either V1J-Luc or the empty V1J vector. Fluids were clarified by centrifugation at 10,000 x g and concentrated 10-fold with Centricon-30 microconcentration filters (Amicon, Beverley, MA). Serial dilutions of vaginal fluids were incubated with luciferase Ag-coated ELISA plates (as described above). Plates were then washed and incubated for 2 h at room temperature with 100 µl of a goat antiserum to rat secretory component that is cross-reactive with mouse secretory component (33). This antiserum has been repeatedly absorbed with rat serum and shows no binding to rat or mouse IgA (33). After washing, plates were incubated with 100 µl of affinity-purified sheep anti-goat IgG conjugated to alkaline phosphatase (Sigma), and specific binding was detected by addition of p-nitrophenyl-phosphate disodium substrate as indicated above.
Measurement of CTL activity
Mononuclear cells isolated from the spleen or cervical or iliac LNs were resuspended in RPMI supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (Sigma), and 5 x 10-5 M 2-ME (Eastman Kodak, Rochester NY). Responder cells (4 x 107) were cocultured with 4 x 106 syngeneic UV and gamma-irradiated (3000 rad) spleen cells infected 16 h previously with a recombinant Adeno 5 virus expressing luciferase (Ad5-Luc 3) (34). Effector cells were harvested 5 days following in vitro culture and washed. Serial dilutions of effector cells were tested in a standard 51Cr release assay (35) with 5 x 103 51Cr-labeled target cells. These included P815, a H-2d cell line either uninfected or infected 16 h previously with 5 plaque-forming units/cell vaccinia virus expressing luciferase (VVLuc) (36) or vaccinia virus expressing SIV gag p55 (VVgag; provided by M. Mackett, Manchester, U.K.) and RMA, a H-2b target cell line infected with 5 plaque-forming units/cell VV Luc. Cytotoxic activity was calculated as the percent specific lysis from the formula (experimental release - spontaneous release)/(total release - spontaneous release) x 100. Total release was determined by incubation of targets with 5% Triton X-100 containing 2% SDS.
Statistics
To determine the significance of differences in the concentration of luciferase expressed in nasal extracts following immunization with naked plasmid DNA and plasmid DNA-cytofectin complexes, an analysis of variance was used. For all other statistical analyses, Student’s t test was used. A p value of 0.05 or less was considered significant in both tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nasal epithelium and associated secretions present a
formidable physical and chemical barrier to substances entering the
respiratory tract (37). Such barriers may preclude the efficacy of
nasal immunization with nucleic acid. Therefore, we first addressed the
efficiency of DNA uptake and protein expression by intranasal
immunization. In view of the reported inefficiency of naked DNA uptake,
particularly by tissues other than cardiac and skeletal muscle (38) or
skin, we evaluated protein expression following cytofectin-mediated
plasmid DNA delivery using DMRIE/DOPE compared with naked DNA alone.
Plasmid DNA-cytofectin complexes containing the firefly luciferase
expression vector VIJ-Luc or the empty plasmid V1J were administered
topically to the nares of BALB/c mice. Three days following
instillation, extracts of nasal epithelium isolated from recipients of
V1J-Luc cytofectin complexes showed a 30-fold increase in luciferase
protein compared with recipients of naked plasmid DNA (Fig. 1
A; p <
0.001). Luciferase expression persisted for at least 28 days following
transfection with plasmid DNA-cytofectin complexes containing V1J-Luc.
Although the mean concentration of luciferase declined by day 7 to 33%
of the day 3 value (found to be the peak, relative to day 1 or 2;
L. S. Klavinskis et al., unpublished observations), expression was
maintained at a residual level corresponding to 4.5% of the day 3
value on day 28 (Fig. 1A). An analysis of variance of the
concentration of luciferase expressed in nasal tissue at each time
point indicated a significant increase in expression by administering
plasmid DNA complexed with DMRIE/DOPE compared with naked DNA alone
(F(1, 24) = 124.59; p < 0.001).
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B). No signal was detected in sequential tissue sections
treated with RNase and hybridized with the luciferase probe,
demonstrating that the hybridization signal was specific to the RNA
transcripts (Fig. 1C). Similarly, no hybridization signal
was detected from mice transfected with plasmid DNA-cytofectin
complexes containing the empty plasmid, V1J, demonstrating specificity
to luciferase (Fig. 1D). Distribution and duration of plasmid DNA and mRNA
Since nasal instillation with a DNA vaccine could potentially
target and prime the organized mucosal associated lymphoid tissues of
the upper and lower respiratory tract, we used a PCR-based approach to
determine the location of plasmid DNA and luciferase mRNA following
intranasal administration. Using luciferase-specific primers, amplified
Luc DNA PCR product was detected in isolated tissues throughout the
respiratory tract (nasopharynx, trachea, and lung) and also the gut,
spleen, and cervical and mesenteric LNs (Fig. 2a) at 3 days following
intranasal inoculation. The absence of PCR product from the brain,
liver, quadriceps muscle, and ovary (Fig. 2a) suggested that
the distribution of plasmid DNA was not random, but was restricted to
the respiratory and gastrointestinal tract as well as the spleen. The
strongest PCR signal was consistently observed for nasal tissue, lung,
and cervical LNs. RT-PCR analysis of RNA isolated on day 3 demonstrated
a pattern of luciferase mRNA tissue expression that was the same as
that seen for plasmid DNA (Fig. 2b). Southern blot analysis
of the RT-PCR products confirmed that the PCR product was the
luciferase sequence (not shown). No signal was obtained from tissues
isolated from animals administered plasmid DNA-cytofectin complexes
containing the empty vector DNA (Fig. 2c). Thus, intranasal
immunization is associated with DNA delivery to mucosal surfaces
throughout the respiratory and gastrointestinal tract. Preliminary data
from PCR studies indicate that intranasal immunization is associated
with transfection of dendritic cells in the organized lymphoid tissue
of the nasal cavity and the draining LNs (C. Barnfield and L. S.
Klavinskis, unpublished observations).
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We next investigated whether intranasal immunization with
either naked plasmid DNA or plasmid DNA complexed with DMRIE/DOPE would
elicit luciferase-specific IgA and IgG Abs in serum and vaginal and
rectal fluids. Four weeks after the primary immunization, mice
immunized with the naked plasmid V1J-Luc showed negligible serum
anti-luciferase IgG or IgA Ab by ELISA; the mean titers were 72 and
24 above the preimmunization value (Fig. 3, a and b). In
contrast, all 10 mice immunized with V1J-Luc cytofectin complexes
demonstrated serum anti-luciferase IgG and IgA Ab, with mean titers
of 3061 and 576, respectively (Fig. 3, a and b).
Analysis of the Ag-specific serum IgG isotype from these mice indicated
a 1.6-fold increase in IgG1 Ab relative to IgG2a, which may represent a
mixed Th1/Th2 response (data not shown). To determine whether
intranasal immunization with plasmid DNA also stimulated a mucosal IgA
response in the female genital tract and rectum, we analyzed rectal and
vaginal fluids for luciferase Ab. An Ag-specific IgA response in rectal
and vaginal fluids could only be detected in plasmid
DNA-lipid-immunized mice (Fig. 3, c and d). A
second immunization via the i.m. route with the naked V1J-Luc plasmid
still failed to elicit detectable luciferase-specific IgA Ab in rectal
or vaginal fluids (data not shown). Thus, cytofectins were required for
successful immunization with DNA by the intranasal route.
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Detection of luciferase IgA in vaginal or rectal
fluids could infer transudation from serum, perhaps through trauma or
abrasion through sampling. To exclude this possibility, we addressed
whether systemic immunization with naked V1J-Luc in saline into the
quadriceps muscle would elicit a detectable luciferase IgA response in
vaginal fluids. As indicated in Fig. 4a, i.m. delivery of plasmid
DNA-lipid complexes did not induce luciferase-specific IgA in vaginal
fluid, although this route induced a high titered IgG response in
serum. For example, at 8 wk postimmunization, the reciprocal Ab titer
was 2176 (Fig. 4a). The ratio of luciferase IgG to IgA Ab
titers in the vaginal fluids of intranasal immunized mice was
significantly lower than that in serum (0.10 and 5.3, respectively;
p < 0.05, by paired Student’s t test). To
further investigate whether luciferase IgA detected in vaginal fluid
was mainly of the secretory type, we analyzed pooled vaginal fluids by
ELISA using luciferase as the target Ag and antisera to the secretory
component as the detection component of the assay. The
anti-secretory component antisera used in these assays does not
bind murine serum IgA (33). Vaginal fluid from mice immunized with
plasmid DNA-cytofectin complexes containing V1J-Luc, but not the empty
vector, demonstrated that significant titers of luciferase IgA Ab
associated with secretory component were induced (Fig. 4b).
These results clearly support the conclusion that cytofectin-mediated
intranasal immunization elicits an Ag-specific vaginal sIgA
response.
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An ideal vaccine capable of preventing transmission or
dissemination of sexually acquired pathogens may rely on the ability of
the vaccine to elicit Ag-specific CTL in genital/rectal tissues and
draining lymph nodes. We therefore examined whether intranasal
immunization with plasmid DNA-cytofectin complexes would elicit CTL in
the spleen and iliac lymph nodes draining the genito-rectal mucosa.
Splenocytes from H-2d mice immunized intranasally with
VIJ-Luc DMRIE/DOPE complexes demonstrated an MHC-restricted
luciferase-specific CTL response following in vitro expansion (Fig. 5a). This response was
specific to immunization with DNA encoding luciferase, since mice
immunized with cytofectin complexes containing only the empty V1J
vector failed to show specific recognition and lysis of
luciferase-expressing target cells (Fig. 5b). Immunization
with DNA cytofectin complexes encoding luciferase elicited a stronger
CTL response in the spleen than immunization with naked DNA encoding
luciferase (62 and 21% specific lysis, respectively, at an E:T cell
ratio of 50:1; data not shown). Mononuclear cells isolated from the
cervical (Fig. 5c) and iliac (Fig. 5d) LNs
elicited a vigorous MHC-restricted luciferase-specific CTL response.
Thus, intranasal immunization with plasmid DNA-lipid complexes induces
CTL in LNs draining the nasopharynx and iliac LNs draining the
genito-rectal mucosa.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results show that the delivery vehicle and route of immunization
are critical factors governing the efficiency with which plasmid DNA
can elicit specific vaginal and rectal sIgA. The absence of detectable
Ag-specific IgA in vaginal and rectal fluids after i.m. immunization
with plasmid DNA (Fig. 4) is consistent with a preferential requirement
to target mucosa-associated lymphoid tissues to initiate immune
responses in the vagina. Intranasal immunization with a plasmid
DNA-lipid complex was shown to elicit vaginal IgA Ab that was
predominantly of the secretory form, as indicated by the presence of
secretory component associated with specific IgA (Fig. 4b).
Interestingly, direct immunization of the vagina with naked plasmid DNA
does not appear to be particularly effective at inducing sIgA in the
genital tract (43), consistent with the results of studies using
recombinant Ags (44, 45). This difference in the efficiency of priming
for genital responses by plasmid DNA may reflect the lack of
Ag-sampling M cells and functional mucosa-associated lymphoid tissue
able to initiate immune responses in the vagina (46). The barrier
function of the cervico-vaginal epithelium at estrus (46, 47) and the
requirement for delivery vehicles such as DMRIE/DOPE to penetrate the
mucosal epithelium and enhance gene expression may also play a role.
Complexing plasmid DNA with cytofectins for nasal administration
was found to be essential to elicit a specific vaginal IgA response.
The cytofectin also increased the specific serum IgA and IgG Ab titers
(Fig. 3a). We propose that these effects may be due to an
increase in Ag presentation of the DNA-encoded protein within the
mucosal associated lymphoid tissue of the respiratory tract and
the draining LNs. In support of this, we found that complexing plasmid
DNA with DMRIE/DOPE increased the expression of the encoded
protein by 30-fold (Fig. 1) in nasal tissue compared with naked DNA,
consistent with previous reports of the delivery of plasmid DNA-encoded
protein to the lung (48, 49, 50). The level of gene expression achieved at
mucosal inductive sites may account in part for the magnitude of the
sIgA and serum Ab responses achieved. This would explain the absence of
specific sIgA observed by Ban and colleagues following nasal delivery
of plasmid DNA complexed with DOTAP/DOPE (51), which exhibits lower
transfection activity in the respiratory tract (48). Cytofectins may
also provide a secondary role as an adjuvant, facilitating uptake of
plasmid DNA by APCs or creating inflammation. Conventional liposomes
based on phospholipids and cholesterol are reported to accumulate in
the Peyer’s patches following oral administration (52) and have been
shown to induce elevated cellular and humoral immune responses to
protein Ags (53) and plasmid DNA-encoded proteins (54, 55). Other
adjuvants, including cholera toxin, have been used to promote mucosal
immunity (2). However, we found no difference in gene expression in
nasal tissue, serum Ab, or vaginal IgA Ab titer following
coadministration of cytofectin-DNA complexes with cholera toxin,
suggesting the absence of any synergistic effect (unpublished
observations). In contrast, an adjuvant effect of cholera toxin in
eliciting mucosal immunity with naked DNA has been reported (56). The
basis for this difference requires further investigation.
It is currently thought that vaccines effective against sexually
transmitted diseases may need to elicit a series of immunological
barriers to limit dissemination of pathogens from the mucosal
epithelium (57). Although a protective role for vaginal IgA in
preventing HIV infection has been highlighted from a cohort of exposed
seronegative individuals (39), and neutralization of HIV has been
achieved with secretory anti-HIV Ab (1), development of successful
vaccines against intracellular pathogens such as HIV, HSV, and
chlamydia trachomatis may also need to elicit CTL or delayed-type
hypersensitivity responses in the genital tissues and draining LNs. Our
results indicate that intranasal immunization with plasmid DNA-lipid
complexes stimulates the induction of MHC class I-restricted CTL in the
spleen and cervical LNs draining the respiratory tract (Fig. 5, a and c). Moreover, this is the first report to
demonstrate that intranasal administered plasmid DNA can prime for
mucosal CTL that recirculate and localize in the iliac LNs (Fig. 5d). In this murine system we are unable to address whether
the elicited CTL also home to the vaginal mucosa. Previous studies
suggest that primed CD8+ T cells from the iliac LNs migrate
to the vaginal mucosa following local Ag stimulation (8) and that
virus-specific CTL localizing in the genital LNs are effective in
clearance of virus in the vagina (6). Thus, cytofectin-mediated
intranasal immunization with plasmid DNA may provide a safe,
noninvasive and noninfectious, generic strategy to elicit mucosal T
cells contributing an effective barrier to dissemination of pathogens
from the vaginal epithelium and submucosa.
An important issue that still needs to be resolved is the underlying
mechanism by which intranasal delivery of plasmid DNA elicits both
systemic and vaginal immune responses. Following intranasal
administration, we demonstrated distribution of plasmid DNA and also
the encoded transcripts throughout the respiratory tract, in agreement
with previous observations (48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58). Expression was also detected in
the gut, spleen, and cervical and mesenteric LNs, but not in the liver,
brain, or ovaries (Fig. 2). It is not inconceivable that plasmid DNA is
swallowed following nasal delivery, accounting for expression in the
gut. Preliminary evidence indicates that enteric delivery of plasmid
DNA-cytofectin complexes also elicits mucosal immunity to the encoded
Ag (C. Barnfield and L. S. Klavinskis, unpublished observations).
With the exception of the liver, the distribution of plasmid DNA and
encoded transcripts is redolent of the dissemination of
phosholipid-based liposomes to reticuloendothelial tissues (58),
although the plasmid DNA-DMRIE/DOPE complex is not a classical liposome
(30). It is possible that in addition to direct transfection of the
nasal epithelium (demonstrated by in situ hybridization; Fig. 1b), plasmid DNA-lipid complexes are endocytosed by M cells
in the follicle-associated epithelium and transported intact to the
underlying lamina propria. Preliminary data indicate that dendritic
cells are transfected in both nasal tissue and cervical LNs (C.
Barnfield and L. S. Klavinskis, unpublished observations).
However, it is currently unclear whether free plasmid DNA-cytofectin
complexes drain via lymph to the regional LNs or whether dendritic
cells or macrophages that are intimately associated with the
basolateral surface of M cells (59) are directly transfected and
migrate to the regional LNs. Conceivably, both mechanisms are operable,
and this is under investigation. Alternatively, from the highly
vascularized tissue of the nasopharynx, plasmid DNA complexes may
directly enter the circulation and disseminate to the spleen, priming
for the systemic immune responses reported herein.
The persistence of the encoded protein in nasal tissue to residual levels by day 28 and the short term expression of transcripts in the lung are consistent with previous observations addressing cytofectin-mediated gene delivery to the respiratory tract (48, 49, 50, 51). While short term expression may be undesirable for gene replacement therapy, in the context of mucosal vaccination such short term expression may be an attribute to prevent the induction of systemic tolerance reported previously with repeated exposure of Ag (60). Whether low levels of Ag and specific transcripts are expressed below the detection of our assays is unclear.
In conclusion, this study demonstrates that intranasal immunization with plasmid DNA-lipid complexes is effective in eliciting Ag-specific CTL and sIgA in the genital and rectal tracts, with specific IgA and IgG Abs and CTL also induced in the systemic compartment. A vaccine capable of stimulating these responses may prove effective in the prevention of sexually transmitted disease, consistent with the current hypothesis that a succession of cellular and humoral immune barriers from the genital mucosa may be required (57). Studies are currently in progress to determine the efficacy of this simple, noninvasive plasmid DNA immunization strategy in preventing genito-rectal infection of macaques with SIV.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda Klavinskis, Department of Immunobiology, New Guy’s House Floor 3, Guy’s Hospital, GKT, St. Thomas St., London, SE1 9RT, U.K. E-mail address:
3 Abbreviations used in this paper: HSV, herpes simplex virus; LN, lymph node; sIgA, secretory immunoglobulin A; DMRIE/DOPE, (+)-N-(2-hydroxyethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide and 1,2-dioleyl-sn-glycero- 3-phosphoethanolamine.
Received for publication June 17, 1998. Accepted for publication August 31, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. M. Gherardi, J. L. Najera, E. Perez-Jimenez, S. Guerra, A. Garcia-Sastre, and M. Esteban Prime-Boost Immunization Schedules Based on Influenza Virus and Vaccinia Virus Vectors Potentiate Cellular Immune Responses against Human Immunodeficiency Virus Env Protein Systemically and in the Genitorectal Draining Lymph Nodes J. Virol., June 15, 2003; 77(12): 7048 - 7057. [Abstract] [Full Text] [PDF]
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 K. L. Csencsits and D. W. Pascual Absence of L-Selectin Delays Mucosal B Cell Responses in Nonintestinal Effector Tissues J. Immunol., November 15, 2002; 169(10): 5649 - 5659. [Abstract] [Full Text] [PDF]
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 M.W. Fan, Z. Bian, Z.X. Peng, Y. Zhong, Z. Chen, B. Peng, and R. Jia A DNA Vaccine Encoding a Cell-surface Protein Antigen of Streptococcus mutans Protects Gnotobiotic Rats from Caries Journal of Dental Research, November 1, 2002; 81(11): 784 - 787. [Abstract] [Full Text] [PDF]
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L. Stevceva, X. Alvarez, A. A. Lackner, E. Tryniszewska, B. Kelsall, J. Nacsa, J. Tartaglia, W. Strober, and G. Franchini Both Mucosal and Systemic Routes of Immunization with the Live, Attenuated NYVAC/Simian Immunodeficiency Virus SIVgpe Recombinant Vaccine Result in Gag-Specific CD8+ T-Cell Responses in Mucosal Tissues of Macaques J. Virol., October 11, 2002; 76(22): 11659 - 11676. [Abstract] [Full Text] [PDF]
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 B. Guy Evaluation of Events Occurring at Mucosal Surfaces: Techniques Used To Collect and Analyze Mucosal Secretions and Cells Clin. Vaccine Immunol., July 1, 2002; 9(4): 753 - 762. [Full Text] [PDF]
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 G. P. Priebe, M. M. Brinig, K. Hatano, M. Grout, F. T. Coleman, G. B. Pier, and J. B. Goldberg Construction and Characterization of a Live, Attenuated aroA Deletion Mutant of Pseudomonas aeruginosa as a Candidate Intranasal Vaccine Infect. Immun., March 1, 2002; 70(3): 1507 - 1517. [Abstract] [Full Text] [PDF]
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B. Ferko, J. Stasakova, S. Sereinig, J. Romanova, D. Katinger, B. Niebler, H. Katinger, and A. Egorov Hyperattenuated Recombinant Influenza A Virus Nonstructural-Protein-Encoding Vectors Induce Human Immunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal Immune Responses in Mice J. Virol., October 1, 2001; 75(19): 8899 - 8908. [Abstract] [Full Text] [PDF]
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L. M. Hodge, M. Marinaro, H. P. Jones, J. R. McGhee, H. Kiyono, and J. W. Simecka Immunoglobulin A (IgA) Responses and IgE-Associated Inflammation along the Respiratory Tract after Mucosal but Not Systemic Immunization Infect. Immun., April 1, 2001; 69(4): 2328 - 2338. [Abstract] [Full Text] [PDF]
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D. E. Hassett, M. K. Slifka, J. Zhang, and J. L. Whitton Direct Ex Vivo Kinetic and Phenotypic Analyses of CD8+ T-Cell Responses Induced by DNA Immunization J. Virol., September 15, 2000; 74(18): 8286 - 8291. [Abstract] [Full Text]
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 P. J. Delves and I. M. Roitt The Immune System- Second of Two Parts N. Engl. J. Med., July 13, 2000; 343(2): 108 - 117. [Full Text] [PDF]
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B. I. Loehr, P. Willson, L. A. Babiuk, and S. van Drunen Littel-van den Hurk Gene Gun-Mediated DNA Immunization Primes Development of Mucosal Immunity against Bovine Herpesvirus 1 in Cattle J. Virol., July 1, 2000; 74(13): 6077 - 6086. [Abstract] [Full Text]
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C. Dupuy, D. Buzoni-Gatel, A. Touze, D. Bout, and P. Coursaget Nasal Immunization of Mice with Human Papillomavirus Type 16 (HPV-16) Virus-Like Particles or with the HPV-16 L1 Gene Elicits Specific Cytotoxic T Lymphocytes in Vaginal Draining Lymph Nodes J. Virol., November 1, 1999; 73(11): 9063 - 9071. [Abstract] [Full Text] [PDF]
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S. Chun, M. Daheshia, S. Lee, S. K. Eo, and B. T. Rouse Distribution Fate and Mechanism of Immune Modulation Following Mucosal Delivery of Plasmid DNA Encoding IL-10 J. Immunol., September 1, 1999; 163(5): 2393 - 2402. [Abstract] [Full Text] [PDF]
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)
are shown for mice immunized with plasmid DNA-lipid containing V1J-Luc
or the empty V1J vector only (
) as described in Materials and
Methods.