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,
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Centre for Gene Therapeutics, Departments of
*
Pathology and Molecular Medicine and
Biology, McMaster University, Hamilton, Ontario, Canada;
Loeb Health Research Institute at the Ottawa Hospital and Faculties of Health Sciences and Medicine, University of Ottawa, Ottawa, Ontario, Canada; and
Coley Pharmaceuticals Group, Wellesley, MA 02481
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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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The administration of Ag to mucosal surfaces is possibly the best
method of inducing mucosal immune responses at distant as well as local
sites (1, 2, 3). However, one of the major impediments to
implementation of mucosal vaccines has been the lack of effective yet
safe mucosal adjuvants. A new class of adjuvant is CpG DNA, which
contains unmethylated CpG dideoxynucleotides in particular base
contexts (CpG motifs) and which is most often given in the form of
synthetic oligodeoxynucleotides (CpG
ODN)3
(11). CpG ODN protects B cells from apoptosis and triggers
them to proliferate and secrete Ig and cytokines (IL-6 and IL-12)
(11, 12, 13, 14). In addition, CpG ODN directly activates
monocytes, macrophages, and dendritic cells to secrete IFN-
,
IL-6, IL-12, GM-CSF, chemokines, and TNF- (14, 15).
These cytokines stimulate NK cells to secrete IFN-
and have
increased lytic activity (14, 16, 17). CpG ODN also
enhances expression of class II MHC and B7 costimulatory molecules
(18, 19). Overall, CpG induces a Th1-biased cytokine
profile with a predominance of high IgG2a titers in serum and CTL
(20, 21, 22). In contrast, ODN that do not contain CpG motifs
(non-CpG ODN) do not have stimulatory effects in vitro or following
parenteral administration (19). Recently, several studies
showed that intranasal (i.n.) delivery of CpG ODN as an adjuvant
results in strong systemic and mucosal immune responses to
coadministered Ags including hepatitis B surface Ag (23, 24), -galactosidase (25), and whole killed
influenza virus (26). Although non-CpG ODN had an adjuvant
effect at mucosal surfaces, this was weaker than with CpG ODN and was
not Th1-biased (27).
Here, we have evaluated the ability of CpG ODN to act as a mucosal adjuvant for immunization against the sexually transmitted HSV-2. Mice were immunized i.n. with recombinant HSV-1 gB (rgB) plus CpG ODN, and immunity was evaluated in the female genital tract and compared with immunization with non-CpG ODN, rgB alone, or recombinant human adenovirus type 5 expressing HSV gB (AdgB).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred, female C57BL/6 mice (Charles River Canada, St. Constant, Quebec, Canada), 6–8 wk old, were used for these studies. Mouse colonies were maintained on a 12-h light/dark cycle.
Vaccine preparations and cell culture
Vero, 293, MC57, and LTA cells were grown in -MEM (Life
Technologies, Burlington, Ontario, Canada) supplemented with 10% FCS
(Life Technologies) and 1% penicillin-streptomycin and
L-glutamine (Life Technologies). The production of AdgB has
been previously described (28) and used as a mucosal
vaccine against herpes infections (29, 30, 31, 32, 33, 34). Mice were
immunized with 2–5 x 108 PFU of AdgB.
HSV-2 strain 333 was propagated and titered on Vero cells. The CpG ODN
(5'-TCCATGACGTTCCTGACGTT-3') and non-CpG control ODN
(5'-TCCAGGACTTCTCTCAGGTT-3') (Coley Pharmaceutical Group,
Wellesley, MA) were used at 10 µg/immunization. Both ODN had a
nuclease-resistant phosphorothioate backbone and have been previously
used for both parenteral (19) and mucosal immunizations
(23). Recombinant glycoprotein B of HSV-1 (Chiron,
Emeryville, CA) was used at 10 µg/immunization.
Immunizations
All groups of mice were immunized i.n. with vaccine plus PBS in a total volume of 15 µl, as previously described (34). In short, each mouse was halothane anesthetized and held inverted with nose down until droplets of vaccine that were applied to the external nares were completely inhaled. All mice were immunized twice with a 2-wk interval between.
Collection of fluids and estrous staging
Serum was collected from each mouse 2 wk following a second immunization. Vaginal washes for estrous staging and Ab determinations were collected daily by pipetting 30 µl of PBS into and out of the vagina six to eight times as previously described (33, 34). The staging of the estrous cycle (estrus, metestrus, diestrus, or proestrus) for each mouse was based on a smear from these washings (35). Smears were stained with Diff-Quik (Baxter Scientific Products, Miami, FL) and staged by examining the cells present in the smears.
Ab determination
HSV-1 gB-specific Ab titers were determined by ELISA as previously described (34) using the ELISA amplification system (EAS; Life Technologies). We have previously shown that IgA and IgG Ab levels fluctuate in an inverse manner over the course of the estrous cycle such that at estrus IgA levels are relatively higher than at diestrus and, conversely, IgG levels are relatively higher at diestrus. As a result, to correctly measure maximal IgA levels, four estrus stages per animal were pooled together for IgA Ab determinations (33). Similarly, vaginal washes from four diestrus stages were pooled for IgG Ab determinations. End point Ab titers were determined and are expressed as the geometric mean titer ± SEM and represent the inverse dilution of the sample at which two times the background absorbance of serum or lavage fluid from control mice was reached.
Spot-forming cell enumeration by enzyme-linked immunospot (ELISPOT) assay
Single cell suspensions from the genital tract were prepared as
previously described (34). Briefly, the vagina, cervix,
and uterine horns were removed from groups of mice, minced, and then
digested with an enzyme solution containing collagenase (0.5 U/ml;
Boehringer Mannheim, Indianapolis, IN), Dispase II (1.2 U/ml;
Boehringer Mannheim), and DNase (5 U/ml; Calbiochem, La Jolla, CA). Two
successive digests were performed for 1 h at 37°C. Lymphocytes
were isolated by passing the cell fractions over Ficoll gradients and
then culturing for 2 h to remove adherent epithelial and
fibroblast cells. Ninety-six-well nitrocellulose plates (Millipore,
Bedford, MA) were coated with 5 µg/ml rgB or anti-IFN-
(PharMingen, Mississauga, Ontario, Canada) Ab in PBS and incubated
overnight at 4°C. Plates were blocked with 10 mg/ml BSA in PBS, pH
7.4. Serially diluted single cell suspensions of genital tract digests
plus supplemented RPMI 1640 medium (10% FCS) were plated at 37°C for
48 h. For enumeration of HSV-specific CTL, the gB CTL peptide
(SSIEFARL) was added to induce production of IFN-. The number of
rgB-specific CTL or rgB-specific IgA or IgG Ab-secreting cells (ASC) in
genital tract digests was determined by the addition of biotinylated
anti-IFN- (PharMingen) or goat anti-mouse IgA or IgG
(Southern Biotechnology Associates, Birmingham, AL) Abs, respectively,
and developed by avidin-peroxidase substrate containing
H2O2 and
3-amino-9-ethylcarboazole (AEC) in acetate buffer as previously
described (34).
CTL activity and enumeration of intracellular IFN-+
CD8+ T cells
Splenocytes and iliac lymph node (ILN) cells isolated from
individual mice were tested for CTL activity against HSV-2-infected
targets as previously described (32). Briefly, splenocytes
were cultured for 5 days with -irradiated, AdgB-infected, syngeneic
MC57 cells at a stimulator-to-responder ratio of 1:166 in RPMI 1640
medium (10% FCS). Lymphocytes from ILN, which drain the genital tract,
were harvested 48–60 h following intravaginal (IVAG) HSV-2 infection
and cultured in vitro for 3 days. Spleen or lymph node effector cells
were then incubated with target cells in a 51Cr
release assay as previously described (32). MC57
(H-2b) or LTA (H-2k) target
cells were cultured with gB CTL peptide or infected with HSV-2 for
6 h before incubation with effector cells. The cytotoxic activity
was calculated using the following formula: % specific lysis =
(test counts - spontaneous counts)/(max counts -
spontaneous counts). For intracellular IFN- staining, ILN cells were
cultured for 5 h with gB CTL epitope as above in the presence of
Golgi plug (PharMingen) to block protein export and thereby increase
intracellular levels of IFN-. Cells were then fixed and
permeabilized with Perm/fix solution (PharMingen) and stained with anti
IFN--FITC and anti-CD8-PE before FACS analysis.
IVAG HSV-2 challenge
Three weeks after the second immunization, mice were injected s.c. with 2 mg of progesterone/mouse (Depo-Provera; Upjohn, Don Mills, Ontario, Canada). Five days later mice were anesthetized using halothane, swabbed IVAG with a cotton applicator, placed on their backs, and infected IVAG for 1 h with 10 µl of HSV-2 while being maintained under anesthetic. Mice were IVAG washed daily by pipetting 2 x 30 µl of PBS into and out of the vagina six to eight times. Viral titers in IVAG washes were determined by plaque assay on Vero cell monolayers. Genital pathology was monitored daily following HSV-2 challenge, and scoring was performed blinded. Pathology was scored on a five-point scale: 0, no apparent infection; 1, slight redness of external vagina; 2, redness and swelling of external vagina; 3, severe redness and swelling of external vaginal and surrounding tissue; 4, genital ulceration with severe redness, swelling, and hair loss of genital and surrounding tissue; 5, severe genital ulceration extending to surrounding tissue. Mice were sacrificed upon reaching stage 5.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The levels of specific anti-gB Abs induced following i.n.
immunization with rgB alone or mixed with CpG or control non-CpG ODN
were evaluated in serum and genital tract washes. Additionally, mice
were immunized with AdgB, which we previously showed induced strong
mucosal immunity and protection (2), were evaluated. Fig. 1
shows that only AdgB or CpG ODN plus
rgB immunization induced significantly greater levels of serum
anti-gB IgA and IgG (p 
0.05) than in
mice immunized with rgB alone. Furthermore, control mice immunized i.n.
with CpG ODN alone did not generate any detectable specific serum
anti-gB IgG or IgA Abs (Fig. 1).
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). IgA but not IgG levels were also
significantly higher (p 0.001) in rgB plus
CpG ODN-immunized mice compared with mice immunized with rgB alone, and
several animals had IgA titers a log higher than in any other group
including AdgB. Control mice immunized with CpG ODN alone did not have
any detectable specific IgG or IgA Abs in their genital washes (Fig. 1). These results demonstrate the ability of i.n. administered CpG ODN
to act as a mucosal adjuvant for the induction of specific mucosal Ab
responses in distant mucosal tissues. Effects of the estrous cycle on gB-specific Ab levels
The levels of specific and total Abs in the murine genital tract
have been shown to rise and fall with the stages of the estrous cycle
(33, 36). Here, the ratios of IgA to IgG Abs over one
estrous cycle were highest during estrus and lowest during diestrus
(Fig. 2), consistent with our previous
results (33). Interestingly, although ratios in all groups
of immunized mice displayed this same profile, the ratios were up to a
log higher in CpG ODN plus rgB-immunized mice, reflecting the overall
higher levels of anti-gB IgA found in these animals. As a result,
levels of specific IgA were often higher during diestrus in the rgB
plus CpG-immunized mice when compared with estrus levels in mice from
other groups.
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Next we addressed whether i.n. immunization with CpG ODN plus rgB
induced ASC in the genital tract. Four weeks following immunization,
mice were IVAG challenged with HSV-2, and the ASC responses were
assessed in genital tissues. As early as 2 days after challenge, the
numbers of IgA ASC specific for gB were 3-fold and more than a log
higher in rgB plus CpG ODN-immunized mice than in mice immunized with
rgB plus non-CpG ODN or rgB alone, respectively (Fig. 3). By 6 days post IVAG challenge,
numbers of IgA ASC had increased dramatically (over 1600) in CpG plus
rgB-immunized mice, but not in any of the other groups of mice (Fig. 3). In contrast to these responses, at days 2 or 6 post IVAG challenge
the IgG ASC responses in CpG ODN plus rgB-immunized mice were no
different or lower than those of mice immunized with non-CpG ODN plus
rgB or AdgB. Interestingly, although initial numbers of IgG ASC were
extremely high, they decreased by over a log from days 2 to 6 in all
groups. Thus, i.n. CpG ODN plus rgB immunization preferentially induced
and maintained large numbers of gB IgA ASC capable of homing to the
genital tract in response to local Ag challenge.
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Ratios of gB-specific IgG subclass Abs in serum were examined as
an indication of the phenotype of the immune response induced by CpG
ODN plus rgB immunization (Fig. 4).
Recombinant gB-specific IgG2a-to-IgG1 Ab ratios were found to be
significantly greater (p 0.001) in the CpG
ODN plus rgB (3.4 ± 1.9 vs 0.5 ± 0.27 for gB/ODN; 0.19
± 0.087 for gB alone; and 0.9 ± 0.13 for AdgB)-immunized group
than in any other group and always >1. In contrast, with the exception
of a single animal, mice receiving rgB alone or rgB plus control
non-CpG ODN had ratios below 1. These observations demonstrate that an
IgG2a-dominated Ab response was seen in the serum of mice immunized
i.n. with AdgB and especially in mice immunized i.n. with CpG ODN
plus rgB.
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The induction of Th1 responses, and predominantly CTL, is
considered an integral component of a successful HSV vaccine. In two
separate experiments, we examined whether CpG ODN plus rgB-immunized
mice contained any CTL against HSV in their spleens. Results from one
experiment are shown in Fig. 5, where it
is apparent that AdgB immunization resulted in elevated levels of
HSV-specific CTL. Together with the second experiment, eight of
eight AdgB-immunized mice were found to contain high levels of splenic
CTL specific for HSV. In contrast, lower levels of HSV-specific
CTL-mediated killing were observed in mice immunized with rgB plus ODN
with or without CpG (Fig. 5). Indeed, only three of eight and two of
eight mice immunized with rgB plus either CpG ODN or non-CpG ODN,
respectively, had anti-HSV CTL activity in their spleens.
HSV-specific CTL were not observed in any animals immunized with rgB
alone (Fig. 5) or in control mice immunized with CpG ODN alone (data
not shown).
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A).
Interestingly, only rgB plus CpG and AdgB groups were significantly
higher than rgB alone (p 0.005). When
CD8+ T cells (gated for CD8) from the ILN were
examined by FACS for IFN- production following in vitro gB-peptide
stimulation all three groups of mice had significantly higher
(p 0.05) percentages of IFN--positive
cells compared with mice immunized with rgB alone (Fig. 6B).
In addition to the lymph nodes, the genital tissues were examined
for CTL activity by determining the number of IFN--positive cells in
response to peptide stimulation by ELISPOT (Fig. 6C)
and intracellular IFN- staining (data not shown). From these
experiments it was clear that mice immunized with AdgB or rgB plus
CpG/ODN or rgB plus non-CpG ODN developed CTL rapidly in the genital
tissues following IVAG infection.
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To evaluate the level of protection mediated by i.n. immunization
with CpG ODN plus rgB, mice were challenged IVAG with HSV-2. From both
low and high dose challenge experiments it was clear that only mice
immunized with CpG ODN plus rgB or AdgB were protected from overt signs
of disease (Table I). In addition,
overall severity scores were significantly lower in these two groups of
mice than those for any of the other groups (Table I; p
0.05). Actual survival from low dose challenge was also
predominantly observed in rgB plus CpG ODN- and AdgB-immunized mice.
However, high dose challenge overcame this protection, especially in
AdgB-immunized mice.
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and Fig. 7). These results suggest that mucosal
immunization with rgB plus CpG ODN but not non-CpG ODN induced an
immune response that is critical for protection from genital HSV-2
challenge. In addition, over the first 2 days after IVAG challenge the
levels of virus recovered from mice immunized with rgB plus CpG ODN
were up to a log lower than in other groups of mice including those
immunized with AdgB (Fig. 7). The ability of rgB plus CpG ODN to
achieve such a significant reduction in viral titer is a major
achievement with regard to control of sexually transmitted virus
infections.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intranasal administration of CpG ODN plus rgB induced high levels of specific IgG and IgA in the serum and genital tract that were significantly greater than mice immunized with rgB alone. Furthermore, i.n. immunization with CpG ODN alone did not induce detectable gB-specific Abs in serum or genital washes. We previously demonstrated that the levels of specific and total Abs in the murine genital tract rise and fall with the stages of the estrous cycle (33). These observations have been confirmed by others (36) and indicate that to properly assess local Ab levels, daily IVAG washes need to be taken from individual mice and the stage of the estrous cycle determined. Following this protocol, and in agreement with our previous findings, the ratios of Ag-specific IgA to IgG Abs were highest during estrus and lowest during diestrus. Interestingly, although all groups of immunized mice displayed this profile, the ratios were up to a log higher in CpG ODN plus rgB-immunized mice, reflecting the overall higher levels of anti-HSVgB IgA in the genital tract of these animals. Importantly, the levels of specific IgA were often higher during diestrus in the rgB plus CpG ODN-immunized mice when compared with estrus levels in mice from other groups. These results indicate that use of CpG ODN as a mucosal adjuvant may induce sufficient levels of specific IgA in the genital tract to protect from infection throughout the murine reproductive cycle. Because similar fluctuations in Ab levels have been associated with the menstrual cycle (37, 38, 39), these results may also extend to primates and humans.
To evaluate whether the dramatically elevated levels of IgA in the
genital washes of CpG ODN-immunized mice were associated with a local
specific B cell immune response, the number of HSVgB-specific ASC
in the genital tract were determined. As early as 2 days following IVAG
challenge with HSV-2, IgA ASC were observed in the genital tracts of
all immunized mice. The numbers of IgA ASC, though, were 3-fold and
more than a log higher in rgB plus CpG ODN-immunized mice than in mice
immunized with rgB plus non-CpG ODN or rgB alone, respectively.
Interestingly, 2 days after IVAG challenge, mice immunized with AdgB
had comparable numbers of IgA ASC in the genital tract as CpG
ODN-immunized mice but by 6 days after challenge the numbers of IgA ASC
had increased dramatically (over 1600) in the CpG-immunized mice but
not in any other group. Thus, IgA ASC dramatically increased in number
over time following local viral challenge in rgB plus CpG ODN-immunized
mice. The high and sustained number of specific IgA ASC in the genital
tract following infection strongly suggests a local component to the
elevated levels of IgA observed in genital washes throughout the
reproductive cycle. The sustained IgA ASC response may be due to the
ability of CpG ODN to directly activate B cells, macrophages, and
dendritic cells (11, 12, 14) to secrete Th1-like cytokines
such as IFN- and IL-12, express costimulatory molecules, and
increase Ag presentation (14, 15, 18, 19). In addition, B
cell activation by CpG shows strong synergy with signaling through the
specific B cell receptor (21) and promotes
anti-apoptotic activities (12). Thus, the significant
increase and maintenance of Ag-specific IgA ASC following recombinant
viral protein plus CpG ODN immunization may reflect the protection of
activated B cells from apoptosis or, alternatively, a
cytokine/chemokine milieu that induces a population of B cells that can
participate in local mucosal immune responses to infection.
Our results also confirm that i.n. immunization with CpG ODN plus rgB, but not non-CpG ODN, induced an IgG2a-dominant Ab response in serum. HSVgB-specific IgG2a-to-IgG1 Ab ratios were significantly greater in the CpG ODN plus rgB-immunized group than in any other group. Similarly, an IgG2a-dominated response was seen in AdgB-immunized mice. IgG2a-dominant responses are typically associated with a Th1 T cell response, and CpG ODN has been shown to predominantly induce Th1 cytokines (20, 21, 22).
Studies concerned with the induction of anti-HSV CTL demonstrated
that although AdgB induced high levels of specific CTL in the spleens
of all immunized mice, CpG ODN plus rgB induced moderate CTL levels
that were undetectable in some animals. Indeed, comparable levels of
HSV-specific CTL-mediated killing were observed in mice immunized with
rgB plus ODN with or without CpG. In contrast to splenic CTL, CpG ODN
induced high levels of CTL in the ILN that drain the genital tract
shortly following IVAG HSV-2 infection. Indeed, the high levels of
specific CTL were confirmed in studies that examined the numbers of
CD8+ T cells in the ILN that contained IFN- in
response to gB epitope exposure. Previously we demonstrated that
mucosal but not systemic immunization with AdgB led to
compartmentalization of anti-viral CTL to mucosal-associated lymph
nodes over time (32). Although it has been shown that
systemic administration of CpG ODN induces potent CTL responses in the
spleen, it seems clear from our results that mucosal administration of
CpG ODN promotes a more localized and compartmentalized CTL response in
the genital tract.
Following both low and high dose IVAG challenge with HSV-2, only mice immunized with CpG ODN plus rgB or AdgB were protected from overt signs of disease, had significantly lower severity scores, and survived low dose virus challenge. Interestingly, mice immunized with rgB plus non-CpG ODN induced relatively high levels of anti-gB IgG and IgA and comparable levels of anti-HSV CTL in the spleen and genital tract as CpG ODN-immunized mice but were not protected against IVAG challenge with HSV-2. These results suggest that mucosal immunization with rgB plus CpG ODN but not non-CpG ODN induced an immune response that is critical for protection from genital challenge. This could also relate to the observation that non-CpG ODN did not induce an IgG2a-dominant response and thus may not have induced a Th1-like response. In addition, over the first 2 days after IVAG challenge the levels of virus recovered from mice immunized with rgB plus CpG ODN were up to a log lower than in other groups of mice, including those immunized with AdgB. The ability of recombinant viral protein plus CpG ODN to achieve such a significant reduction in the titer of virus in the genital tract is a major achievement with regard to control of sexually transmitted virus infections. The lower levels of free virus in the genital tracts of rgB plus CpG ODN-immunized mice may reflect neutralization of the initial infectious load. Alternatively, virus levels may have appeared lower due to Abs in the genital tract that could have neutralized virus being shed into IVAG washes. Nevertheless, these results are the first to show that mucosal (i.n.) immunization with CpG ODN plus Ag induced significant specific IgA and anti-HSV CTL in the genital tract that remained high throughout the estrous cycle and protected mice from challenge in the genital tract. The sustained presence of specific IgA Abs in the genital tract and the induction of localized HSV-specific CTL might also serve to reduce the transmission of virus from infected to uninfected individuals via sexual transmission.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenneth L. Rosenthal, Department of Pathology and Molecular Medicine, McMaster University Health Sciences Center Room 4H17, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
3 Abbreviations used in this paper: ODN, oligodeoxynucleotide(s); non-CpG ODN, ODN that do not contain CpG motifs; AdgB, recombinant human adenovirus type 5 expressing HSV glycoprotein B; ASC, Ab-secreting cell(s); ILN, iliac lymph node(s); i.n., intranasal(ly); gB, glycoprotein B; rgB, recombinant HSV-1 gB; IVAG, intravaginal(ly); ELISPOT, enzyme-linked immunospot.
Received for publication August 17, 2000. Accepted for publication December 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 93:2879. production by stimulation of interleukin-12 and tumor necrosis factor-. Cell. Immunol. 167:72.[Medline]
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S.-M. Kang, L. Guo, Q. Yao, I. Skountzou, and R. W. Compans Intranasal Immunization with Inactivated Influenza Virus Enhances Immune Responses to Coadministered Simian-Human Immunodeficiency Virus-Like Particle Antigens J. Virol., September 15, 2004; 78(18): 9624 - 9632. [Abstract] [Full Text] [PDF]
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L. Aurelian Herpes Simplex Virus Type 2 Vaccines: New Ground for Optimism? Clin. Vaccine Immunol., May 1, 2004; 11(3): 437 - 445. [Abstract] [Full Text] [PDF]
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B. Garulli, Y. Kawaoka, and M. R. Castrucci Mucosal and Systemic Immune Responses to a Human Immunodeficiency Virus Type 1 Epitope Induced upon Vaginal Infection with a Recombinant Influenza A Virus J. Virol., January 15, 2004; 78(2): 1020 - 1025. [Abstract] [Full Text] [PDF]
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G. A. Prince, J. J. Mond, D. D. Porter, K. C. Yim, S. J. Lan, and D. M. Klinman Immunoprotective Activity and Safety of a Respiratory Syncytial Virus Vaccine: Mucosal Delivery of Fusion Glycoprotein with a CpG Oligodeoxynucleotide Adjuvant J. Virol., December 15, 2003; 77(24): 13156 - 13160. [Abstract] [Full Text] [PDF]
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F. N. Toka, M. Gierynska, and B. T. Rouse Codelivery of CCR7 Ligands as Molecular Adjuvants Enhances the Protective Immune Response against Herpes Simplex Virus Type 1 J. Virol., December 1, 2003; 77(23): 12742 - 12752. [Abstract] [Full Text] [PDF]
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A. A. Ashkar, S. Bauer, W. J. Mitchell, J. Vieira, and K. L. Rosenthal Local Delivery of CpG Oligodeoxynucleotides Induces Rapid Changes in the Genital Mucosa and Inhibits Replication, but Not Entry, of Herpes Simplex Virus Type 2 J. Virol., August 15, 2003; 77(16): 8948 - 8956. [Abstract] [Full Text] [PDF]
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C. Kaushic, A. A. Ashkar, L. A. Reid, and K. L. Rosenthal Progesterone Increases Susceptibility and Decreases Immune Responses to Genital Herpes Infection J. Virol., April 15, 2003; 77(8): 4558 - 4565. [Abstract] [Full Text] [PDF]
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S.-M. Kang and R. W. Compans Enhancement of Mucosal Immunization with Virus-Like Particles of Simian Immunodeficiency Virus J. Virol., March 15, 2003; 77(6): 3615 - 3623. [Abstract] [Full Text] [PDF]
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 D. M. Koelle and L. Corey Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research Clin. Microbiol. Rev., January 1, 2003; 16(1): 96 - 113. [Abstract] [Full Text] [PDF]
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W. Jiang, H. J. Baker, and B. F. Smith Mucosal Immunization with Helicobacter, CpG DNA, and Cholera Toxin Is Protective Infect. Immun., January 1, 2003; 71(1): 40 - 46. [Abstract] [Full Text] [PDF]
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 T.-Y. Kim, H.-J. Myoung, J.-H. Kim, I.-S. Moon, T.-G. Kim, W.-S. Ahn, and J.-I. Sin Both E7 and CpG-Oligodeoxynucleotide Are Required for Protective Immunity against Challenge with Human Papillomavirus 16 (E6/E7) Immortalized Tumor Cells: Involvement of CD4+ and CD8+ T Cells in Protection Cancer Res., December 15, 2002; 62(24): 7234 - 7240. [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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R. B. Pyles, D. Higgins, C. Chalk, A. Zalar, J. Eiden, C. Brown, G. Van Nest, and L. R. Stanberry Use of Immunostimulatory Sequence-Containing Oligonucleotides as Topical Therapy for Genital Herpes Simplex Virus Type 2 Infection J. Virol., October 11, 2002; 76(22): 11387 - 11396. [Abstract] [Full Text] [PDF]
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 M R Wills, A J Carmichael, and J G P Sissons Vaccines against persistent DNA virus infections Br. Med. Bull., July 1, 2002; 62(1): 125 - 138. [Abstract] [Full Text] [PDF]
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 J. N. Kline, K. Kitagaki, T. R. Businga, and V. V. Jain Treatment of established asthma in a murine model using CpG oligodeoxynucleotides Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L170 - L179. [Abstract] [Full Text] [PDF]
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M. Gierynska, U. Kumaraguru, S.-K. Eo, S. Lee, A. Krieg, and B. T. Rouse Induction of CD8 T-Cell-Specific Systemic and Mucosal Immunity against Herpes Simplex Virus with CpG-Peptide Complexes J. Virol., June 5, 2002; 76(13): 6568 - 6576. [Abstract] [Full Text] [PDF]
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