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Departments of
*
Medicine and
Pathology, University of California at San Diego, La Jolla, CA 92093; and
Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines, which are known
to inhibit the use of the CCR5 coreceptor by HIV; 4) CTL activity; 5)
mucosal immune responses; and 6) CD8 T cell responses that were
independent of CD4 T cell help. Based on these results, ISS-based
immunization holds promise for the development of an effective
preventive and therapeutic HIV vaccine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial DNA is also a potent activator of innate immunity, stimulating APCs to produce cytokines (6, 7, 8), up-regulate MHC and costimulatory molecules (9, 10, 11), and induce cross-presentation of soluble Ag on MHC class I (12). This immunostimulatory activity of bacterial DNA is dependent on immunostimulatory DNA sequences (ISS). ISS are structurally defined by their content of CpG motifs (5'-purine-purine-CpG-pyrimidine-pyrimidine-3') (7). In vertebrate genomes, there is a diminished frequency of CpG motifs as well as inactivation of them by cytosine methylation (7, 13). Based on their ability to activate innate immunity and their structural differences from vertebrate DNA, ISS contained in bacterial DNA fulfill the definition for PAMPs. The cellular recognition systems for bacterial DNA are just beginning to be elucidated. The potent induction of type I IFNs by ISS and dsRNA but not by LPS (14, 15) suggests that ISS-induced signaling is more analogous to the dsRNA/PKR signaling system than to the LPS recognition system. Recently published work suggests that Toll-like receptor 9 and the intracellular DNA-PK catalytic subunit are essential for ISS-induced activation of innate immunity (16, 17).
Recent studies have emphasized the central role of dendritic cells in initiating adaptive immune responses after exposure to PAMPs in their microenvironment (18). Because the response of cells of the innate immune system, such as dendritic cells, to PAMPs is deeply rooted in evolution, it is likely that PAMPs act as natural adjuvants for the induction of protective immune responses against the pathogens that contain them. This suggests an intriguing strategy for vaccine design: combining a suitable PAMP with pathogen-derived Ags to create better immunogens.
The potent immunostimulatory properties of ISS led us to hypothesize
that they could be used as a PAMP candidate for HIV vaccine
development. The ability to produce large quantities of synthetic
oligodeoxynucleotides (ODNs) containing ISS, and the relatively low
toxicity of these synthetic ODNs when compared with other PAMPs such as
LPS, makes ISS an especially attractive reagent for future clinical
use. In the murine studies presented here, we evaluated the immune
response elicited by a model HIV-derived Ag (gp120) coadministered
with, or directly conjugated to, ISS. We show that these ISS-based
immunization schemes elicit potent Ag-specific immune responses,
including Ab, Th1 cytokines, CCR5-specific -chemokines, and CTL
activity. These immune responses can be induced systemically, as well
as at mucosal sites. Furthermore, ISS-based vaccination elicits these
cytokine, chemokine, and CTL responses from CD8 T cells independently
of CD4 T cell help. Our findings suggest that ISS-based immunization
might also elicit the robust and multifaceted immune responses that
will be required for protection against HIV infection and for
vaccination in the setting of CD4 T cell deficiency, as seen in AIDS
patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HIV gp120 protein from HIV-1LAI expressed
in Chinese hamster ovary cells was obtained from Quality Biological
(Gaithersburg, MD). ISS and mutated phosphorothioate
oligodeoxynucleotides (mODN) were purchased from Trilink
Biotechnologies (San Diego, CA). The sequence of the ISS used in these
studies is 5'-TGACTGTGAACGTTCGAGATGA-3'. The mODN has the
sequence 5'-TGACTGTGAACCTTAGAGATGA-3'. gp120:ISS and
gp120:mODN conjugates were produced in a three-step process as
previously described (12, 19). Introduction of maleimido
groups onto gp120 molecules was achieved by incubation with a 20 M
excess of
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Pierce Chemicals, Rockford, IL) for 2 h followed by purification
on an NAP-25 column (Amersham Pharmacia, Uppsala, Sweden). 5'
activation of oligodeoxynucleotides was conducted by incubation with
0.2 M tricarboxyethylphosphine (Pierce Chemicals), and activated ODNs
were subsequently purified on an NAP-10 column. Maleimido-modified
gp120 and thiol-activated ODNs were then incubated together overnight,
and free ODNs were removed by filtration using an Amicon 50 spin column
(Amicon, Beverly, MA). The conjugate was analyzed by SDS-PAGE. After
electrophoresis, the samples were transferred onto nitrocellulose
membranes and visualized by chemiluminescent detection of
anti-gp120 Ab (Western blotting), autoradiography after
hybridization with complementary
[
-32P]ATP-labeled oligodeoxynucleotides
(Southwestern blotting), or UV visualization of shadowing of the
oligodeoxynucleotides on TLC membranes (UV shadowing).
Immunization protocols
Female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) age 4–6 wk were immunized with gp120 (10 µg) alone or with ISS (50 µg) or mODN (50 µg). Alternatively, mice were vaccinated with gp120:ISS or gp120:mODN conjugate (10 µg based on gp120 content). For intradermal (i.d.) immunization, reagents were delivered in 50 µl saline by injection into the base of the tail. For intranasal (i.n.) immunization, reagents were applied topically in 30 µl saline divided equally and delivered to each nare of lightly anesthetized mice. Immunizations were delivered on three occasions spaced 2 wk apart. For CD4 T cell depletion, mice received 1 mg GK 1.5 mAb (Bio Express, West Lebanon, NH) i.p. on three occasions, 4 wk apart. With the use of flow cytometry, we determined that mice receiving GK 1.5 mAb had <1% of the peripheral blood and splenic CD4 T cell counts of untreated mice throughout the course of these experiments. All animal procedures followed the University of California, San Diego, animal care guidelines.
Sample collection and processing
Serum was obtained by serial retro-orbital bleedings until mice were sacrificed at week 12. Vaginal washes were obtained during week 12 by lavage with 50 µl PBS. Samples were spun to remove cellular debris and frozen at -70°C until the IgA assay was performed. Feces were collected at week 12, and IgA was extracted by routine methods. Briefly, three to six pieces of freshly voided feces were collected and subsequently dried in a Speed Vac concentrator. After drying, net dry weights were recorded, and the material was resuspended in PBS with 5% nonfat dry milk and protease inhibitors at a ratio of 20 µl/mg feces to standardize for variability in the amount of fecal material collected. The solid matter was resuspended by vortexing for 12 h followed by centrifugation at 16,000 x g for 10 min to separate residual solids from the supernatant. Supernatants were frozen at -70°C until the IgA assay was performed.
Splenocytes (1 x 108/mouse), Peyers’ patch lymphocytes (1 x 107/mouse), and lamina propria lymphocytes (1.5 x 106/mouse) were recovered by routine methods 12 wk after the initiation of immunization (20). Briefly, spleens were harvested and teased to make single-cell suspensions. Intestines were isolated and stripped of mesenteric fat, and the Peyer’s patches were excised. The tissue was washed and incubated in digestion medium (collagenase VIII, 300 U/ml; Sigma, St. Louis, MO; DNase, 1–1.5 µg/ml; Sigma) for 1 h. Single-cell suspensions were obtained by pouring the digestion mixture over a fine nylon sieve. Cells were subsequently washed, and Peyers’ patch lymphocytes were separated on a 75%/40% Percoll gradient. Lamina propria lymphocytes were isolated by opening residual intestinal tissue longitudinally, washing extensively, cutting intestines into short segments, and incubating in 1 mM EDTA to remove the epithelial layer. After EDTA treatment, the tissue was washed in RPMI (Irvine Scientific, Irvine, CA) supplemented with 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 2 mM L-glutamine (Cellgro, Natham, VA), 100 U/ml penicillin-100 µg/ml streptomycin (Pen/Strep; Cellgro), and Fungizone (Life Technologies). The tissue was poured over a coarse sieve, and residual tissue was incubated with digestion medium. The lamina propria lymphocyte digestion mixture was poured over a fine nylon sieve to obtain a single-cell suspension, and then lymphocytes were purified on a 75%/40% Percoll gradient. These procedures resulted in >90% viability of all lymphocyte preparations.
Immunological assays
Antibody levels were determined by routine ELISA techniques (21). Briefly, microtiter plates were coated overnight with 5 µg/ml gp120 in borate-buffered saline (pH 9.2). Plates were then washed and blocked with 1% BSA. Plates were again washed, and serially diluted serum samples were added. After overnight incubation at 4°C, plates were washed, and alkaline phosphatase-labeled goat anti-mouse IgG, IgG2a, or IgA (Southern Biotechnology Associates, Birmingham, AL) was added for 2 h. After plates were washed, a solution of p-nitrophenyl phosphate (1 mg/ml; Boehringer Mannheim, Indianapolis, IN) was added and OD405 was read 1–4 h after addition of the substrate. Ab titers were determined by end point titration of the samples and reflect the amount of dilution required to achieve twice-background OD readings. ELISA plates were analyzed using the DeltaSOFT II v. 3.66 program (Biometallics, Princeton, NJ).
For CTL assays, 7 x 106 splenocytes, Peyer’s patch lymphocytes, or lamina propria lymphocytes were cultured in supplemented RPMI with 6 x 106 mitomycin C-treated naive splenocytes in the presence of recombinant human IL-2 (50 IU/ml; BD PharMingen, San Diego, CA) and an HIV-1 class I (H2d)-restricted gp120 peptide, which has been described in previous publications (p18-I10; RGPGRAFVTI; 4 µg/ml) (22, 23). After 5 days, restimulated cells were harvested, and specific lysis of target cells was measured using the Cytotox 96 assay kit according to the manufacturer’s instructions (Promega, Madison, WI).
IFN-, macrophage-inflammatory protein (MIP) 1
, and MIP1
responses were evaluated by incubation of splenocytes, Peyer’s patch
lymphocytes, or lamina propria lymphocytes at 5 x
105 cells/ml in 96-well plates in a final volume
of 200 µl supplemented RPMI with gp120 (10 µg/ml) or p18-I10 (4
µg/ml). Culture supernatants were harvested at 72 h and analyzed
by ELISA for IFN- (BD PharMingen), MIP1, MIP1, or RANTES (R&D
Systems, Minneapolis, MN) content, according to the manufacturers’
recommendations. Each culture supernatant was compared with the
standard curve on the plate using the DeltaSOFT II v. 3.66 program.
ELISPOT assays were performed using nitrocellulose-backed 96-well
plates (Millipore, Bedford, MA). Plates were coated with 50 µl PBS
containing rat anti-mouse IFN- Ab (BD PharMingen) at 10 µg/ml
or goat anti-mouse MIP1 Ab (R&D) at 5 µg/ml and incubated
overnight at 4°C. Wells were washed with borate-buffered
saline-0.05% Tween 20 and then blocked with 200 µl of supplemented
RPMI for 1 h at 37°C. Serial dilutions of splenocytes
from each mouse starting at 2 x 106
cells/well were then plated and incubated in triplicate wells in medium
alone or with gp120 (10 µg/ml) or P18-I10 (4 µg/ml). After 24
h, wells were washed, and biotinylated anti-IFN- (PharMingen) or
biotinylated anti-MIP1 (R&D Systems) was added to the
appropriate wells for 2 h at room temperature. Wells were then
washed and HRP-streptavidin conjugate (Zymed, South San Francisco, CA)
was added for 1 h at room temperature. Plates were then developed
by adding TMB membrane substrate (Kierkegaard & Perry Laboratories,
Gaithersburg, MD) per the manufacturer’s instructions. Plates were
dried, and spots were counted using a dissecting microscope. The number
of peptide-specific cytokine-secreting cells was determined as a
frequency of total CD8 T cells by using a correction factor based on
the fraction of CD8 T cells present in spleens of untreated and
CD4-depleted mice as determined by flow cytometry (data not shown).
Statistical analyses
Statistical analyses were performed using the GraphPad Prism program (GraphPad Software, San Diego, CA). The significance of differences in means between multiple groups was determined using one-way ANOVA with Bonferroni’s posttest analysis. When only two groups were compared, the significance of differences in means between the two groups was determined by unpaired t test. Significant differences were defined as p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the optimal antigenic targets for HIV vaccine development
have not yet been established, in the present studies gp120 should be
considered as a model Ag for the application of ISS-based immunization
strategies to generate improved immunity to better HIV target Ags as
they are identified. Recent studies in the literature suggest that ISS
coadministered with HIV particles can elicit some potentially
beneficial immune parameters (24, 25, 26). Conjugation of ISS
to protein Ags, including gp120, has previously been shown to improve
some aspects of their immunogenicity (12, 19, 27, 28).
Therefore, we generated the gp120:ISS conjugate to determine whether
ISS conjugation might generate an improved immune response to this
relatively poorly immunogenic HIV Ag (29). ISS-containing
ODN (7.5 kDa) or ODN containing a mutated, nonstimulatory motif (mODN,
7.5 kDa) were conjugated to gp120 protein (120 kDa) as described in
Materials and Methods. Coomassie Blue staining after
SDS-PAGE of the gp120:ODN conjugates revealed a 140-kDa band,
reflecting a protein-ODN ratio of 
1:3 (Fig. 1
a). Western blot analysis
with anti-gp120 Ab (Fig. 1b), UV shadowing of the
oligonucleotides (Fig. 1c), and Southwestern blot analysis
with radioactively labeled complementary oligonucleotides (data not
shown) confirmed successful conjugation.
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To determine whether ISS could improve humoral and cytokine responses to gp120, BALB/c mice were immunized i.d. with coadministered gp120 plus ISS or with gp120:ISS conjugate. For comparison, control mice were immunized with gp120 alone, gp120 plus mODN, or gp120:mODN conjugate. In pilot experiments, ISS coadministered with gp120 at a dose similar to that present in the conjugate (1.3 µg ISS per mouse) led to immune responses similar to those seen after immunization with gp120 alone (data not shown). Therefore, in subsequent studies in which unconjugated ISS was codelivered with gp120, a 40-fold higher dose of ISS (50 µg) was used.
To assess the ability of the vaccination reagents under study to
augment humoral immune responses, Ag-specific total IgG and IgG2a
(which is Th1 dependent) levels from serum collected 12 wk after
initiation of immunization were measured (Fig. 2a). Compared with controls,
mice immunized i.d. with gp120 plus ISS or gp120:ISS conjugate showed
significantly higher levels of total IgG and IgG2a
(p < 0.001). In addition, both gp120 plus ISS
coadministration and gp120:ISS immunization improved IgG1 responses
relative to control immunizations (data not shown). Previous studies
have shown that gp120 is a poor target for the generation of
HIV-neutralizing Abs (29, 30); therefore, neutralization
studies were not pursued. Future studies with HIV-related Ags effective
at eliciting neutralizing Abs will need to verify the ability of ISS to
augment these neutralizing Ab responses.
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production is a hallmark feature of Th1-biased immunity and
contributes to protection against many viral infections (31, 32). Therefore, the CD4 T cell IFN- response of immunized
mice was determined by culture of splenocytes with gp120 and analysis
of supernatants by ELISA (Fig. 2
b). IFN- production was
significantly higher for mice immunized with gp120 plus ISS
(p < 0.05) or gp120:ISS conjugate
(p < 0.001) when compared with control
immunized mice. Furthermore, gp120:ISS conjugate was more effective at
inducing an IFN- response than gp120 plus ISS
(p < 0.001).
CCR5 acts as a coreceptor for HIV entry into cells
(33, 34, 35, 36, 37), and competitive inhibition of this
virus-coreceptor interaction by CCR5-specific -chemokines (MIP1,
MIP1, and RANTES) may inhibit the intercellular spread of HIV and
the natural progression of the infection to AIDS (38). The
ability of ISS to induce -chemokine production from macrophages in
an Ag-independent manner (A. A. Horner, S. K. Datta, and E.
Raz, unpublished observations) led us to investigate whether ISS-based
vaccines could elicit their Ag-specific production. Ag-specific
secretion of MIP1, MIP1, and RANTES was assessed by ELISA of
supernatants from splenocytes cultured with gp120 (Fig. 2, c
and d). Mice immunized with gp120:ISS conjugate demonstrated
significantly stronger MIP1 and MIP1 responses than controls
(p < 0.001) or gp120 plus ISS-immunized mice
(p < 0.001 for MIP1 and p
< 0.05 for MIP1). However, although less effective than gp120:ISS
conjugate, gp120 plus ISS coadministration also elicited significant
MIP1 but not MIP1 production. Interestingly, whereas significant
levels of RANTES have been observed upon stimulation of APCs by ISS in
vitro, gp120-specific RANTES production was not appreciably induced
above background levels in this series of experiments (data not
shown).
Immunization i.n. with ISS-based gp120 vaccines elicits systemic and mucosal immune responses
Protection against HIV infection is likely to require immunity at
mucosal sites because 1) its spread is principally by sexual
transmission, 2) the intestinal mucosa represents an important site for
the initial replication of the virus, and 3) mucosal immunity is
important for protection against mucosal challenge in published models
of HIV viral infection (20, 39). As mucosal immunity is
best elicited by vaccine delivery to mucosal sites (20, 40), the immunization reagents described in the previous
sections were administered i.n. to mice at the same doses used for i.d.
immunization, and both systemic and mucosal immune parameters were
measured. Similar to i.d. immunization, i.n. immunization with gp120
plus ISS or gp120:ISS conjugate elicited significantly higher levels of
serum IgG and IgG2a than controls (p < 0.001;
Fig. 3a). Furthermore, i.n.
immunization with gp120 plus ISS or gp120:ISS conjugate also induced a
vigorous secretory IgA response detected in vaginal washes and fecal
samples (p < 0.001 vs controls) (Fig. 3b). In contrast, i.d. immunization with these reagents
failed to elicit a significant mucosal IgA response. Finally, i.n.
immunization with either gp120 plus ISS or gp120:ISS conjugate elicited
significantly more gp120-specific IFN-, MIP1, and MIP1
production than control immunizations (p <
0.001 for IFN-, p < 0.05 for MIP1 and MIP1;
Fig. 3, c–e).
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Because an effective CD8 CTL response is important in preventing
and controlling HIV infection (41, 42, 43, 44), the ability of
ISS-based gp120 immunization to elicit Ag-specific CTL activity was
determined. Both i.d. (Fig. 4a) and i.n. (Fig. 4b) administration of gp120:ISS conjugate elicited similar
levels of high specific lysis in splenic CTL assays. However, although
i.d. administration of gp120 plus ISS elicited CTL activity that was
similar to the conjugate, i.n. administration of gp120 plus ISS
elicited a significantly lower CTL response (p
< 0.05). In addition to systemic CTL activity, i.n. gp120:ISS
conjugate delivery, and to a much lesser extent gp120 plus ISS
coadministration, induced mucosal CTL responses as measured with lamina
propria (Fig. 4c) and Peyer’s patch lymphocytes (Fig. 4d). However, consistent with the poor secretory IgA
response seen after systemic vaccination, both i.d. gp120 plus ISS and
gp120:ISS conjugate immunizations induced only weak CTL responses at
these mucosal sites.
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The cytokine and chemokine data described in the previous sections
include MHC class II-dependent responses, as intact gp120 protein was
used to stimulate cells. Cytokine and chemokine secretion by CD8 T
cells, in addition to CTL responses, are important for controlling HIV
infection, whereas CD4 T cell deficiency is a characteristic feature of
AIDS (38, 45). Therefore, the ability of ISS-based
vaccines to induce cytokine and chemokine responses from CD8 T cells
was investigated. Splenocytes from immunized mice were restimulated in
vitro with MHC class I (H2d)-restricted gp120
peptide, and cytokine and chemokine production in culture supernatants
was subsequently determined by ELISA. Mice immunized i.d. with either
gp120 plus ISS or gp120:ISS conjugate demonstrated significant CD8 T
cell production of IFN-, MIP1, and MIP1 compared with control
immunized mice (Fig. 5, a–c).
Similar results were seen with i.n. gp120 plus ISS and gp120:conjugate
vaccination (data not shown).
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During the course of HIV infection, CD4 T cells are depleted.
Therefore, it would be important for a therapeutic AIDS vaccine to
elicit robust immunity in the absence of CD4 T cells. The ability of
ISS-based vaccines to elicit cytokine, chemokine, and CTL responses
from CD8 T cells led us to investigate whether these responses required
CD4 T cell help. Previous investigations have demonstrated that OVA:ISS
conjugate vaccination induces equivalent CTL responses in CD4 knockout
and wild-type mice, whereas the CTL response in CD4 knockout mice
immunized with OVA plus ISS is compromised (12).
Therefore, gp120:ISS conjugate was used to i.d. immunize wild-type and
CD4 T cell-depleted mice to compare their CD8 T cell responses.
Splenocytes from gp120:ISS conjugate-immunized, CD4 T cell-depleted
mice that were restimulated with a class I-restricted gp120 peptide
demonstrated a retained ability to secrete Ag-specific IFN- (Fig. 6a), MIP1 (Fig. 6b), and MIP1 (Fig. 6c) relative to
splenocytes from immunized mice that were not CD4 T cell-depleted.
Furthermore, by ELISPOT analysis, CD4 T cell-depleted and nondepleted
mice immunized with gp120:ISS conjugate had equivalent frequencies of
CD8 T cells producing IFN- and MIP1 in response to incubation
with a class I-restricted gp120 peptide (Fig. 6d).
Consistent with these results, Ag-specific CTL activity was also
retained in CD4 T cell-depleted mice (Fig. 6e). As expected,
restimulation of splenocytes from immunized CD4 T cell-depleted mice
with gp120 protein failed to elicit cytokine or chemokine responses
(data not shown). Furthermore, CD4 T cell-depleted mice were unable to
generate a detectable Ab response after gp120:ISS conjugate
immunization despite their development of CD8 T cell immunity (data not
shown). Similar to i.d. immunized mice, CD4 T cell-depleted mice
immunized i.n. with gp120:ISS conjugate also showed retained CD8 T cell
responses, including CTL responses at both systemic and mucosal sites
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines such as MIP1 and MIP1, which have been shown to
protect against cellular invasion by HIV (33, 34, 35, 36, 37, 38).
Furthermore, these studies establish that ISS-based immunization
elicits gp120-specific cytokine, chemokine, and CTL responses from CD8
T cells in CD4 T cell-deficient mice, suggesting that this vaccination
strategy could have clinical utility even in CD4 T cell-depleted AIDS
patients for whom CD8 T cells are likely to play a crucial role in
controlling infection (38, 41, 42, 43, 44, 45). The ability of i.n. ISS-based immunization to induce mucosal immunity, consistent with previously published data using other Ags (46, 47, 48), represents another attractive feature of this vaccination strategy. Mucosal immunity is important because HIV is transmitted primarily by mucosal routes and the presence of mucosal IgA in human vaginal secretions has been correlated with resistance to infection in commercial sex workers (49), although mucosal IgA did not provide mucosal protection in models of other viral infections (50, 51). Immunization i.n., especially with the gp120:ISS conjugate, also effectively elicits systemic immune responses, although certain immune parameters are not as robust as with i.d. vaccination. It remains to be seen whether delivery of an ISS-based HIV vaccine by mucosal routes alone is sufficient or whether a combination of systemic and mucosal immunizations is needed to optimize systemic and mucosal immunity.
Similar to previously described results with other Ags (12, 19, 27, 28), the present series of experiments demonstrates that i.d. gp120:ISS conjugate vaccination induces a stronger immune response than coadministration of an equivalent dose of gp120 with 40-fold higher amounts of unconjugated ISS and further establish the improved immunogenicity of gp120:ISS conjugate for mucosal vaccination. The immunological potency of the gp120:ISS conjugate relative to gp120 mixed with ISS can best be explained by reviewing the known mechanisms of action of ISS. As previously discussed, ISS, like other PAMPs, exert their effects mainly on APCs, such as macrophages and dendritic cells, stimulating them to secrete cytokines and up-regulate Ag processing and costimulatory molecules. ISS-activated APCs are thus primed to instruct lymphocytes and efficiently promote adaptive immune responses (4, 8, 52, 53). The gp120:ISS conjugate physically colocalizes the Ag to these ISS-activated APCs, whereas without ISS conjugation only a fraction of ISS-activated APCs will encounter, process, and present the target Ag for the orchestration of the subsequent adaptive immune response. The present results, as well as previous work (12), suggest that for difficult-to-elicit immune responses, such as the induction of systemic immunity with mucosal immunization or the induction of immunity in the absence of CD4 T cells, gp120:ISS conjugate vaccination offers a distinct advantage over coadministration of gp120 and ISS.
Our previous investigations have shown that OVA:ISS conjugate elicits
equivalent systemic CTL responses in CD4 knockout and wild-type mice
(12). The present investigations expand on this
observation, demonstrating that gp120:ISS conjugate vaccination elicits
equivalent systemic and mucosal CD8 T cell effector responses in mice
that have been depleted of CD4 T cells compared with nondepleted mice.
These CD8 T cell effector responses include cytokine and chemokine
secretion as well as CTL responses. The level of cytokines and
chemokines secreted by CD8 T cells (Fig. 5) is less than that seen with
CD4 T cell responses (Fig. 2), but this CD8 T cell response may be
important, especially in states of CD4 depletion such as AIDS. ELISPOT
investigations further establish that CD4 T cell depletion does not
reduce the frequency of CD8 T cells that are generated in response to
gp120:ISS vaccination. These results raise intriguing mechanistic
questions about how ISS exert their profound effects on CD8 T cell
immunity. The current model for CD8 T cell activation invokes the
concept of licensing of APCs (54, 55, 56). In this model,
CD40-CD40 ligand interaction between APCs and CD4 T cells results in
up-regulation of costimulatory molecules and other factors on APCs,
which licenses them to subsequently interact with and activate CD8 T
cells. ISS appear to replace the licensing function of CD4 T cells
(12), presumably by initiating similar downstream
signaling events in APCs (57, 58).
The present studies demonstrate that ISS-based immunization induces CTL activity and other class I-restricted T cell responses against exogenous, soluble Ags. In addition to licensing APCs to become more potent activators of CD8 T cells, ISS appear to allow cross-presentation of Ag, which involves rerouting of phagocytosed, exogenous protein from the MHC class II pathway to the MHC class I pathway. The means by which ISS-mediated cross-presentation occurs have only begun to be elucidated. ISS have been shown to up-regulate expression of MHC class I molecules, as well as other components of the class I Ag processing pathway, such as TAP. Furthermore, studies in TAP knockout mice have demonstrated that induction of CTL activity by ISS-based vaccines is dependent on TAP (H. J. Cho and E. Raz, manuscript in preparation). However, the exact mechanisms by which ISS shunt exogenous proteins into the class I Ag processing pathway after phagocytosis require further investigation.
Cytokines induced by ISS, some of which are known to be involved in the Th1-biased immune response seen with ISS-based immunization, probably also play a role in the initiation and maintenance of the CD8 T cell response. For example, IL-15, which is induced by ISS in a type I IFN dependent manner (K. Takabayashi and E. Raz, unpublished observations) has been recently implicated in the proliferation of activated CD8 memory T cells (59, 60). Therefore, in addition to licensing APCs and allowing cross-presentation of Ag, ISS may create an appropriate cytokine milieu for the induction of vigorous CD8 T cell immunity.
The potency of ISS-based immunization demonstrated in this report validates the concept of using PAMPs (i.e., activators of innate immunity) in conjunction with Ags (i.e., activators of adaptive immunity) to elicit robust immune responses. Reports of enhanced immunity to certain microbial Ags delivered with PAMPs derived from other pathogens (61, 62) confirm the viability of this approach for future vaccine development. These studies further demonstrate the potential for harnessing the innate immunostimulatory properties of PAMPs from one pathogen to improve immune responses to Ags from unrelated pathogens.
With respect to the design of vaccines against HIV, our unpublished
studies have demonstrated that the HIV genome has only 15% of the
ISS predicted by chance, with a genome frequency approaching that of
its human host (J. Van Uden and E. Raz, manuscript in preparation).
Furthermore, HIV, like other retroviruses, does not generate large
amounts of genomic DNA during infection and replication. These
considerations suggest that ISS are unlikely to be an important PAMP
for the activation of a host immune response during HIV infection and
that the reduced frequency of ISS in the HIV genome may have been
selected for in the evolution of this virus. Conjugating PAMPs such as
ISS, which are especially potent activators of innate immunity, to
relevant Ags derived from HIV, which does not naturally express these
PAMPs, results in novel immunogens that colocalize Ag to PAMP-activated
APCs. Our results demonstrate the viability of this strategy for the
generation of improved immune responses to this immunologically evasive
pathogen.
The present series of experiments demonstrates that ISS-based vaccines,
such as the gp120:ISS conjugate, elicit a multifaceted immune response
with characteristics thought to be important for protection against
HIV. These include 1) an Ag-specific Ab response, 2) a Th1 cytokine
response, 3) the secretion of CCR5-specific -chemokines, 4) a CTL
response, 5) mucosal immunity, and 6) a CD8 T cell response that is
independent of CD4 T cell help. These results add to a growing body of
evidence suggesting that PAMPs such as ISS, and their conjugation to
relevant Ags, provide a means for recruiting and activating the innate
immune system for the purposes of enhancing and diversifying the
adaptive immune response to Ags from infectious agents such as
HIV.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eyal Raz, Department of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663. E-mail address: eraz{at}ucsd.edu
3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; PRR, pattern recognition receptor; ISS, immunostimulatory DNA sequences; ODN, oligodeoxynucleotide; mODN, mutated oligodeoxynucleotide (not containing CpG motif); i.d., intradermal; i.n., intranasal; PKR, dsRNA-activated protein kinase; MIP, macrophage-inflammatory protein.
Received for publication March 12, 2001. Accepted for publication June 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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