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Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production and cytolytic activity toward the
immunodominant CTL epitope OVA257–264. In contrast,
administration of OVA with aluminum hydroxide or entrapped in
conventional ester-phospholipid liposomes failed to evoke significant
CTL response. The archaeosome-mediated CD8+ T cell response
was primarily perforin dependent because CTL activity was undetectable
in perforin-deficient mice. Interestingly, a long-term CTL response was
generated with a low Ag dose even in CD4+ T cell deficient
mice, indicating that the archaeosomes could mediate a potent T helper
cell-independent CD8+ T cell response. Macrophages
incubated in vitro with OVA archaeosomes strongly stimulated cytokine
production by OVA-specific CD8+ T cells, indicating that
archaeosomes efficiently delivered entrapped protein for MHC class I
presentation. This processing of Ag was Brefeldin A sensitive,
suggesting that the peptides were transported through the endoplasmic
reticulum and presented by the cytosolic MHC class I pathway. Finally,
archaeosomes induced a potent memory CTL response to OVA even 154 days
after immunization. This correlated to strong Ag-specific up-regulation
of CD44 on splenic CD8+ T cells. Thus, delivery of proteins
in self-adjuvanting archaeosomes represents a novel strategy for
targeting exogenous Ags to the MHC class I pathway for induction of CTL
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Traditional vaccines formulated with protein Ags or attenuated microbes are introduced into the endosomal compartment, and consequently stimulate mainly Ab production and, to varying degrees, Th cell responses. However, these responses cannot eliminate virus-infected or tumor cells that require a strong CTL response. With the identification of protective immunodominant proteins that can be exploited as ideal targets for vaccines, there is an urgent need for efficient strategies for the induction of long-term CTL responses to exogenous Ags. Although coadministering Ags with certain immunostimulating adjuvants may facilitate a CTL response (4, 5), many adjuvants have undesirable side effects such as severe inflammatory responses, precluding their widespread use in human vaccines. Indeed, the only adjuvant currently approved universally for use in humans is aluminum hydroxide (alum),3 which is a relatively weak inducer of cell-mediated immune responses (6).
Liposomes composed of natural or synthetic ester phospholipids (conventional liposomes) have been explored as possible Ag carriers and a liposome-based vaccine against hepatitis A has been licensed for human use (7). However, often the antigenic depot provided by liposomes leads only to MHC class II presentation of the processed Ag. Furthermore, codelivery of immunostimulating agents such as lipid A, cholera toxin, or cytokines is required for effective costimulation and sustained immunity (8, 9, 10).
Archaea consist of organisms distinct from eubacterial and
eukaryotic cells due, in part, to their unique polar lipid structures
(Table I
). The distinct structures of
archaeal lipids confer considerable stability to liposomes
(archaeosomes) formulated from the total polar lipids
(TPL) of the different archaea
(11) or from purified lipid subfractions
(12). In earlier studies, we reported archaeosomes to be
superior adjuvants, capable of facilitating a strong, long-lasting Ab
and CD4+ T cell response to entrapped proteins
(13, 14). Because the limitation of novel adjuvant systems
has often been their inability to evoke a CTL response, we evaluate
herein the possibility that archaeosomes facilitate presentation of
exogenous Ags on MHC class I molecules. Indeed, we show that
archaeosomes can be effectively processed to present soluble entrapped
Ag via the classical cytosolic MHC class I pathway to induce strong
CD8+ T cell memory.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Halobacterium salinarum ("H. cutirubrum") (33170; American Type Culture Collection (ATCC), Manassas, VA), Methanobrevibacter smithii strain ALI (2375; Deutsche Sammlung von Mikroorganismen und Zellkuturen, Gotngen, Germany), Methanosphaera stadtmanae strain MCB-3 (DSM 3091), and Thermoplasma acidophilum strain 122-1B3 (ATCC 27658) were cultivated in 75- to 250-liter fermenters as described earlier (18). Total lipids were extracted from frozen cell pastes, and the TPL were collected as the acetone-insoluble fraction (18).
Preparation and characterization of archaeosomes and conventional liposomes
Archaeosomes were prepared from the individual TPL obtained from
the archaea mentioned above.
L-
-dimyristoylphosphatidylcholine (DMPC),
L--dimyristoylphosphatidylglycerol (DMPG), and
cholesterol (CHOL) were purchased from Sigma (St. Louis, MO) for the
preparation of conventional liposomes, defined herein as DMPC:DMPG:CHOL
(1.8:0.2:1.5 molar ratio). Vesicles (archaeosomes and conventional
liposomes) were prepared by pressure extrusion at 23°C
(19). Briefly, 20 mg of dried lipid was hydrated in 1 ml
of PBS (10 mM potassium phosphate, pH 7.14, containing 160 mM NaCl)
containing the protein Ag (10 mg/ml). After an 18-h hydration, the
multilamellar vesicles obtained were pressure extruded several times
using a Liposofast apparatus containing two stacked 400-nm
polycarbonate filters (Avestin, Ottawa, Canada). Ag that was not
associated with the vesicles was removed by ultracentrifugation
(200,000 x gmax for 30 min,
three times) from 7-ml volumes of PBS. Mean vesicle diameters were
determined by number-weighted Gaussian size distributions using a
Nicomp Particle sizer (model 370; Nicomp, Santa Barbara, CA). The
vesicle sizes were in the range of 96–263 nm. The amount of protein
incorporated into the vesicles was estimated by the SDS Lowry method
after lipid removal (20), by comparison with standard
curves constructed for the relevant protein. The ratio of protein to
lipid (microgram per milligram) is based on the salt-free dry weights
of the vesicles.
To ensure that the SDS Lowry method provided a valid comparison between OVA entrapped in M. smithii and conventional liposomes, SDS-PAGE was performed. Electrophoresis of samples heated in SDS reducing buffer was performed under standard conditions using 12.5% polyacrylamide gels in the buffer system of Laemmli at pH 8.8. Gels were stained with Coomassie brilliant blue G-250. Electrophoresis reagents and conditions were obtained from Bio-Rad (Richmond, CA).
Mice and immunizations
Inbred, 6- to 8-wk-old female C57BL/6 mice were obtained from Charles River Breeding Laboratories (St. Constant, Quebec, Canada). C57BL/6 perforin-deficient (C57BL/6-Pfptm1Sdz) and C57BL/6-CD4+ T cell-deficient (C57BL/6J-CD4tm1knw) mice and their controls were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in the animal facility of the Institute for Biological Sciences, National Research Council, in accordance with guidelines from the Canadian Council on Animal Care. Mice were immunized with Ag in PBS (no adjuvant), in alum, or entrapped in either archaeosomes or conventional liposomes. Immunization volume was 100 µl, Ag dose was 15–25 µg/injection, and lipid concentration was 0.6–1.4 mg/injection. For alum immunizations, the Ag was adsorbed onto "Imject Alum" (Pierce, Rockford, IL) according to the manufacturer’s protocol. Immunization routes used included i.p. and s.c. injected at the base of the tail. The immunization schedules were as described in figure legends. OVA Grade V was purchased from Sigma.
Cell lines
EL-4 (T lymphoma) and IC21 (macrophage) cell lines were obtained from ATCC and maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 8% FBS (HyClone, Logan, UT) and 10 µg/ml gentamicin (Life Technologies). EG.7, a subclone of EL-4 stably transfected with the gene-encoding OVA (21), was also obtained from ATCC but maintained in RPMI plus 8% FBS, additionally containing 400 µg/ml G418 (Rose Scientific, Edmonton, Alberta, Canada). A CD8+ T cell hybridoma (CD8OVA1.3) recognizing the CTL epitope of OVA (OVA257–264) was a gift from Dr. C. V. Harding (Case Western Reserve University, Cleveland, OH). For generation of LPS blasts, splenocytes (106/ml) from naive C57BL/6 mice were cultured for 72 h with 2 µg/ml LPS (Sigma) in RPMI 1640 medium supplemented with 8% FBS additionally containing 0.1 ng/ml IL-2. All cells were cultured at 37°C, under 8% CO2 in a humidified atmosphere.
CTL assays
Single cell suspensions from pooled spleens (n =
3–5) of immunized mice were selectively lysed for erythrocytes with
Tris-buffered ammonium chloride, pH 7.2 (Sigma). After washing, 30
x 106 spleen cells were cultured with 5 x
105 irradiated (10,000 rad) EG.7 cells in 10 ml
of RPMI plus 8% FBS containing 0.1 ng/ml IL-2, in
25-cm2 tissue culture flasks (Falcon; Becton
Dickinson, Franklin Lakes, NJ) kept upright. After 5 days (37°C, 8%
CO2), the cells were recovered from the flask and
used as effectors in a standard 51Cr-release CTL
assay. Briefly, for the CTL assay, EL-4 and EG.7 target cells and/or
LPS blasts (5 x 106) were labeled with
51Cr (100 µCi) for 45 min in 50 µl of RPMI
plus 8% FBS. Targets were then washed, and various ratios of effectors
and targets were cocultured for 4 h in 96-well round-bottom tissue
culture plates (Falcon). Supernatants were collected, and radioactivity
was detected by counting. The percent specific lysis was calculated
using the formula: [(cpm experimental - cpm spontaneous)/(cpm
total - cpm spontaneous)] x 100. One lytic unit is defined as
the number of effector cells per 106 spleen cells
that yield 20% specific lysis of a population of 2.5 x
104 target cells.
Depletion of CD8+ T cells
Effectors recovered from 5-day restimulation cultures of spleen cells from immunized mice were depleted of CD8+ T cells before the CTL assay. Briefly, 20 x 106 effectors/ml in RPMI plus 0.3% BSA medium were incubated with anti-CD8 Ab (HO2.2, 10 µg/ml) for 30 min on ice. Cells were washed and rabbit complement (LowTox-M obtained from Cedarlane Laboratories, Hornby, Canada) was added at 10% final concentration in RPMI containing 0. 3% BSA, and incubated for 45 min at 37°C. Cells were then washed, counted, and used as effectors in the CTL assay. The purity of the cells was ascertained by flow cytometric analysis.
Measurement of OVA CTL peptide-specific cytokine production
IC21 macrophage cells (106/ml) were
incubated with 10 µg/ml OVA257–264 peptide
(synthesized at the peptide synthesis facility of our institute) for
2 h in RPMI plus 8% FBS at 37°C under a humidified atmosphere
containing 8% C02. Cells were then washed,
irradiated (10,000 rad), and used as stimulators for spleen cells
obtained from immunized mice. Spleen cells (5 x
105) and stimulator macrophages
(105) were cultured in triplicate in 96-well
microtiter plates for 72 h, and supernatants collected and assayed
for IFN- production by ELISA.
In vitro assays for Ag processing and presentation
Triplicate cultures of IC21 adherent macrophage cells (105/well/100 µl RPMI plus 8% FBS) in 96-well microtiter plates were incubated, in the presence or absence of Brefeldin A (10 µg/ml), with 10 µg OVA entrapped in M. smithii archaeosomes or conventional liposomes. After 4 h at 37°C, in a humidified 8% CO2 incubator, cells were washed with PBS, and fixed with 0.5% paraformaldehyde (15 min at room temperature), washed again, and incubated with 0.2 M lysine (20 min at room temperature). Cells were then washed twice with RPMI 1640 medium, and then CD8OVA1.3 cells (106/well) were added. Cultures were incubated overnight (37°C, 8% CO2, humidified air), and then supernatants were collected and assayed for IL-2 production.
Cytokine assays
IFN- produced in the supernatants of peptide-stimulated
cultures was measured by a sandwich ELISA (22). Ab pairs
used included RA-6A2 (ATCC HB170) and XMG1.2-biotin (23).
IFN- standards were purchased from ID Laboratories (London, Canada).
Duplicate standard curves were included on each plate. The sensitivity
of the IFN- ELISAs was >100 pg/ml. IL-2 production by the CD8OVA1.3
cells was assayed by a bioassay on HT2 cells as described previously
(22). The sensitivity of the IL-2 bioassay was >2 pg/ml.
Regardless of thresholds, samples from each experiment were tested in
the same assay.
Flow cytometric analysis
To determine whether memory CD8+ T cells generated against OVA would have elevated expression of various cell surface molecules after antigenic restimulation, IC21 macrophage cells (10 x 106) were incubated with the CTL epitope of OVA (OVA257–264) in vitro for 3 h and then injected i.p. into naive and OVA archaeosome-immunized mice. As a control, a group of OVA archaeosome-immunized mice were injected with IC21 cells alone. After 5 days, splenocytes (106) were stained with PE-conjugated anti-mouse CD8 Abs and FITC-conjugated anti-mouse CD44, LFA1, or biotin-conjugated anti-mouse CD28 followed by streptavidin-FITC, for 30 min on ice in 50 µl of RPMI medium containing 8% FBS. Cells were washed and fixed in 1% formaldehyde in PBS, and analyzed by flow cytometry (EPICS XL; Coulter, Hialeah, FL). Anti-mouse CD8, CD44, and CD28 were obtained from PharMingen (San Diego, CA). Anti-mouse LFA (CD11a) was obtained from Cedarlane Laboratories.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were immunized (i.p.) on days 0 and 21 with OVA
entrapped in archaeosomes composed of M. smithii TPL, in
conventional liposomes, or in conjunction with alum. On day 28, the
spleens were removed, and the CTL response evaluated after
restimulation with EG.7 cells for 5 days. In a standard
51Cr release assay, spleen cells from OVA
archaeosome-immunized mice exhibited strong CTL activity toward EG.7
(specific targets expressing OVA) but not EL-4 cells (Fig. 1a). A relatively low amount
of Ag (15 µg OVA entrapped in 
1.0 mg lipid) was used for these
experiments, reiterating the potency of the CTL response. Immunization
with OVA in the absence of an adjuvant, or entrapped in conventional
liposomes, failed to evoke a CTL response. Furthermore, administration
of OVA in conjunction with alum induced only a minimal CTL
response.
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production by the spleen
cell cultures was monitored at 72 h, as a measure of
CD8+ T cell stimulation. Spleen cells of M.
smithii archaeosome-immunized mice responded to
OVA257–264 peptide stimulation by producing
substantial IFN- (Fig. 1
b). There was no nonspecific
activation in the absence of the SIINFEKL peptide. Similarly, spleen
cells from mice immunized with OVA in the absence of adjuvant did not
respond to peptide stimulation.
The dramatically different CTL responses to OVA entrapped in either
conventional liposomes or M. smithii archaeosomes could
conceivably be attributable, in some part, to inaccuracies in
quantitating the amount of Ag entrapped. To address this possibility,
entrapped OVA was estimated as usual by the SDS Lowry method and, based
on this assay, equivalent amounts of protein were separated from lipids
by SDS-PAGE (Fig. 2). Results shown for
0.7 µg protein per lane were representative of several concentrations
tested, and revealed a similar staining intensity of the OVA bands in
unentrapped (lane 1) and conventional liposomes
(lane 3). Although similar, the OVA entrapped in
archaeosomes appears to be slightly overestimated by the Lowry method,
perhaps explained by incomplete removal of Lowry-positive M.
smithii lipids, such as archaetidylserine. In any event, this
would be expected to have either little effect or result in a decrease
in the CTL response observed to OVA entrapped in archaeosomes, rather
than contribute to the dramatically increased CTL activity noted in
Fig. 1.
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demonstrate that all archaeosome types
tested can induce a CTL response, and that a single injection (15-µg
Ag dose) is sufficient for induction of activity. However, M.
stadtmanae vesicles were comparatively less efficient at inducing
a CTL response.
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To specifically identify the cell population inducing the CTL
activity after archaeosome OVA immunization, CD8+
T cells were depleted from restimulated spleen cell effectors before
the CTL assay. Flow cytometric analysis of the depleted effectors
indicated the population to be >99% CD8+ T cell
depleted, and there was no nonspecific depletion of
CD4+ T cells (data not shown). Depletion of
CD8+ T cells completely abrogated the lytic
activity of OVA-M. smithii effectors toward EG.7 cells (Fig. 4), clearly demonstrating that the CTL
response is CD8+ T cell mediated.
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CD8+ T cells kill infected targets by the
perforin/granule exocytosis mechanism and to a limited extent by the
Fas pathway (2). To determine the contribution of these
mechanisms, control and perforin-deficient mice were immunized with OVA
archaeosomes, and the CTL activity was tested. In the absence of
perforin, no detectable CTL activity was noted on EG.7 target cells by
effectors obtained from OVA-M. smithii archaeosome-immunized
mice (Fig. 5a). The absence of
CTL activity suggests that the archaeosome-induced OVA-specific
CD8+ T cells primarily kill by the
perforin/granule exocytosis pathway. In accordance with this,
perforin-deficient effectors also failed to kill LPS blasts (pulsed
with the OVA CTL peptide) known to express high levels of Fas
(Fig. 5b).
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CD4+ T cell help is often a requirement for
strong and sustained CTL responses (25). Our previous
studies had indicated that archaeosomes induce strong
CD4+ Th1 and Th2 responses to entrapped protein
Ags (13), suggesting that CD4+ T
cell help may be available for the CTL response. To assess whether CTL
response induced by archaeosomes can occur in the absence of
CD4+ T cells, control and
CD4+ T cell-deficient mice were immunized once
s.c. with OVA-M. smithii archaeosomes, and then CTL
responses were evaluated 15 and 30 days later. Data in Fig. 6 demonstrate that after immunization
with OVA archaeosomes, CD4+ T cell-deficient mice
evoked Ag-specific CTL activity comparable to controls. The CTL
activity in the absence of CD4+ T cell help was
evident even at 30 days postimmunization. Furthermore, these results
were obtained after a single s.c. immunization with a relatively low Ag
dose (15 µg), reiterating the potency of archaeosomes as
adjuvants.
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Induction of CTL responses by OVA archaeosomes, and IFN-
production by spleen cells obtained from immunized mice to the
immunodominant CTL epitope of OVA (OVA257–264),
suggested efficient processing of soluble Ag in vivo. We have
previously shown that macrophages can efficiently phagocytose
archaeosomes in vitro, with a maximum phagocytosis achieved by 4–6 h
(26). To determine the mechanism of Ag processing, IC21
macrophage cells were incubated in vitro for 5 h with OVA
archaeosomes in the absence or presence of brefeldin A, washed, fixed,
and then tested for their ability to stimulate CD8OVA1.3 cells. IL-2
production by CD8OVA1.3 cells was used as an indicator of stimulation.
As a control we also compared the ability of OVA entrapped in
conventional liposomes to stimulate CD8OVA 1.3 cells. As shown in
Fig. 7, OVA archaeosomes were efficiently
processed by IC21 macrophages, resulting in potent stimulation of
CD8OVA1.3 cells for IL-2 production. However, when brefeldin A was used
in preculture, IL-2 production was undetectable. In contrast,
OVA-conventional liposomes did not induce activation of CD8OVA 1.3
cells even in the absence of brefeldin A, indicating lack of MHC class
I presentation ability. Brefeldin A is an inhibitor of the conventional
MHC class I presentation pathway and blocks the anterograde movement of
peptide-loaded MHC class I molecules from endoplasmic reticulum (ER) to
the cell surface (27). Therefore, our results are
consistent with a mechanism where archaeosomes use the classical MHC
class I presentation pathway for processing and presentation of
entrapped Ags.
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To determine the longevity of the CTL response induced, mice were
immunized with OVA-M. smithii archaeosomes (on days 0 and
21), and then the CTL response was monitored periodically in
representative mice (n = 3 per time point). For a more
imperative and stringent comparison of CTL activity, the data were
converted to lytic U/106 spleen cells, wherein 1
lytic unit represents the number of effectors yielding 20% specific
lysis of 2.5 x 104 EG.7 targets. In Fig. 8, the data demonstrate that OVA
archaeosomes induced a strong recall CTL response with dramatic
increases initially, stabilizing by 100 days postimmunization. The
potent recall response was evident even at 154 days postimmunization.
Similar trends were obtained when data were expressed as lytic units
per whole spleen (data not shown). Thus, delivery of soluble Ags
entrapped in archaeosomes not only evokes a CTL response in the short
term, but also induces potent CD8+ T cell
memory.
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Memory T cells are characterized by the high expression of cell
surface molecules such as CD44, and are usually more responsive to Ag
challenge. To ascertain the development and activation of memory cells,
mice immunized with OVA archaeosomes on days 0 and 21 were rechallenged
in vivo with OVA257–264 peptide-pulsed IC21
macrophages on day 140, and the expression of cell surface markers on
splenic CD8+ T cells was analyzed 5 days later. A
dramatic up-regulation (2.5-fold) of CD44 in comparison to naive
controls was observed in OVA archaeosome-immunized mice (Fig. 9a). This induction of CD44
expression in OVA archaeosome-immunized mice was Ag specific, as
boosting with IC21 cells without peptide did not enhance the expression
(Fig. 9a, middle). As
CD44high cells are classically associated with
the memory phenotype (28), the strong up-regulation of
CD44 observed even at 140 days postimmunization using a relatively low
Ag dose (25 µg/injection) indicates the establishment of a potent
memory response. The modulation of other cell surface molecules
such as LFA1 and CD28 also suggests efficient activation and
responsiveness of the memory CD8+ T cells to Ag
challenge (Fig. 9b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) all showed CTL activity. We
have earlier related the structural properties of the TPL from the
human archaea, M. smithii and M. stadtmanae, to
their contrasting humoral adjuvant activity (15). High
amounts of archaetidyl inositol and ß-Glcp-(1, 6)-ß-Glcp-(1, 1)-archaeol (present in M. stadtmanae)
correlated with low adjuvant activity, whereas major lipids having
phosphoserine head groups were found in M. smithii TPL.
Interestingly, in this study, we found M. stadtmanae
archaeosomes to be comparatively less efficient inducers of CTL
responses as well. However, the divergent lipid compositions of other
archaeosomes that facilitate strong CTL response suggest that a feature
in common to all archaeal lipid structures may confer adjuvant
activity, at least in part. Entrapment of Ags in conventional liposomes composed of synthetic esters failed to induce CTL responses. Additionally, conventional liposomes lacked the ability to process OVA for MHC class I presentation even in vitro. Indeed, incubation of APCs with OVA-conventional liposomes for an extended period (24 h) also did not facilitate MHC class I presentation of the Ag (data not shown). This may partly be attributable to the decreased phagocytosis by APCs of conventional liposomes compared with archaeosomes (26). In earlier studies, we have noted conventional liposomes of varying ester lipid compositions to be poor adjuvants for induction of Ab and CD4+ T cell responses as well (13, 14). Some studies have reported that entrapment of Ags in positively charged or pH-sensitive liposomes facilitates CTL activity (31, 32). However, large Ag doses and/or coincorporation of other immune modulators such as monophosphoryl lipid A, Quil A, and/or cholera toxin are often required (8, 9, 10, 32, 33). In one study, activation of APCs by surface incorporation of anti-CD40 ligand on liposomes facilitated induction of CTLs (34). In comparison, archaeosomes constitute a simple and potent delivery system.
Besides CD8+ T cells, cytolytic activity is exhibited by other cell types, including components of innate immunity such as NK cells (35) and some CD4+ T cells (2). However, NK cell activation is often transient, and CD4+ T cell-mediated killing is less potent than that mediated by effector CD8+ T cells. Immunostimulatory agents such as CpG motifs can promote innate immunity (36). However, we observed that CTL activity induced by archaeosomes was peptide specific, suggesting that killing was not mediated by NK cells. Furthermore, elimination of CD8+ T cells completely abrogated CTL activity, indicating that Ag-specific CD8+ T cells were induced. The perforin/granule exocytosis pathway is often the dominant mechanism of killing by CD8+ T cells, whereas both CD4+ and CD8+ T cells can kill by the Fas pathway that principally regulates immune responses (2). The lack of killing after archaeosome-Ag immunization by perforin-deficient CD8+ T cells highlights the importance of perforin. Furthermore, CD8+ effectors induced in perforin-deficient mice failed to kill LPS blasts (known to express high levels of Fas), suggesting lack of Fas-mediated killing. However, it is possible that the Fas ligand expression on effectors was not optimal or the duration of assay was not sufficient to reveal Fas-dependent killing.
Several studies indicate that CD8+ T cell responses require CD4+ T cell help for effective stimulation and long-standing immunity. CD4+ T cells prevented the rapid depletion of CD8+ T cells after a transient response to Ag (37). Protective CD8+ T cell responses to chronic lymphocytic choriomeningitis virus and hepatitis virus infection required CD4+ T cell help (38, 39). Adjuvants composed of particulate viral Ags (virus-like particles) require CD4+ T cell help for evoking CD8+ T cell response to exogenous Ag (40). CTL responses that result from cross-priming, as after DNA vaccination, are also dependent on CD4+ T cell help (41). The dependency of CD8+ T cells on CD4+ T cells may be attributed to their low levels of cytokine production, insufficient to sustain their proliferation (42). Interestingly, archaeosomes effectively bypassed CD4+ T cell help for induction of CTL responses to entrapped Ag. Thus, archaeosomes may be effective even in immunocompromised individuals. High Ag doses can surpass the need for CD4+ T cell help as it may lead to higher density of MHC-peptide complexes being presented to CD8+ T cells (43). However, our results indicate that single immunization with a relatively low dose of Ag (15 µg) entrapped in archaeosomes can induce a potent T helper cell-independent CTL response. Effective costimulation and activation of APCs such as dendritic cells help bypass CD4+ T cell help (44, 45, 46). Archaeosomes are phagocytosed by APCs to a greater extent than conventional liposomes (26). Whether this interaction leads to efficient activation and consequent costimulation by APCs needs further study.
The intracellular events involved in presentation of Ags by MHC class I molecules classically involves the cytosolic pathway wherein Ags are processed by the proteasome and transferred to the ER, where they are loaded onto MHC class I molecules and then transported via the Golgi to the cell surface (1). However, alternative noncytosolic mechanism(s) that do not require the Ag trafficking through the ER have been proposed to explain the presentation of exogenous Ags by MHC class I (47). In the presence of Brefeldin A, an inhibitor of the classical pathway (27), APCs were unable to present archaeosome-delivered Ag on MHC class I molecules, indicating ER trafficking of Ag and processing by the cytosolic pathway. Exogenous Ag bound to micron-sized particles derived from bacteria, latex beads, iron, or silica can be presented by MHC class I (43, 48). This effect appears to be due to internalization of the large particles (1–10 µm) by phagocytes into phagosomes, which then transfer Ag into the cytosol. Thus, microparticles smaller than 0.5 µm often fail to evoke an efficient CTL response (48). In contrast, archaeosomes used by us are unilamellar or oligolamellar, and consistently <300 nm in size. Interestingly, phosphatidylserine on apoptotic cells is recognized by APCs and facilitates uptake by phagosomes (49). Similarly, it is possible that archaetidylserine and caldarchaetidylserine (phosphatidylserine analogs) present in the TPL of M. smithii facilitate phagosomal uptake of these archaeosomes. However, other archaeosomes that lack phosphatidylserine (T. acidophilum and H. salinarum) also stimulate a CTL response. Thus, mechanism(s) such as fusion with cell membrane and macropinocytosis, which have been proposed to explain the cytoplasmic delivery of drugs entrapped in conventional liposomes (50), may also be operational for archaeosomes.
An important aspect of vaccination is the ability to induce long-term memory. Thus, the ability of archaeosomes to induce potent memory CTL responses and up-regulate the expression of memory markers on splenic CD8+ T cells emphasizes its usefulness as a vaccine adjuvant. Although live viral infections often result in long-lasting immunity, the factors that determine establishment and maintenance of long-term memory appear complex. A strong primary response may correlate to establishment of a larger pool of circulating memory T cells, and stimulation of potent APCs like dendritic cells may be beneficial (28). As archaeosomes are phagocytosed effectively, they may efficiently target Ag to APCs, facilitating a strong primary response and consequent long-term memory. Ag persistence may be another factor for effective memory, though recent studies have suggested that maintenance of memory T cells does not require continued MHC-Ag interaction (51).
Several strategies are being explored for the induction of CD8+ T cell response to exogenous Ags (47, 52). One approach is the integration of Ag genes into live viral or bacterial vectors that will invade the cytoplasm of the APC and introduce Ags to the MHC class I pathway. However, such organisms bear the potential to cause disease and hence safety remains a key deterrent. An alternate approach is the use of plasmid expression vectors, which can be transcribed and translated by host cells. In this case the integration of the plasmid with host chromosomes could potentially disrupt or mutate important host genes. Immunostimulatory agents such as CpG-DNA motifs have also been shown to facilitate induction of CTL responses to coadministered Ag by activation of dendritic cells (36). The use of MHC class I binding peptides conjugated with lipids or in conjunction with adjuvants, and peptide-pulsed dendritic cells, which can bypass the Ag processing pathway, are effective for CTL induction (53, 54, 55). However, MHC haplotype restriction of peptides often limits the use of this approach. Particulate systems such as virus-like particles, heat shock protein complexes, ISCOMS, and biodegradable microspheres are also being explored as potential CTL-evoking adjuvants (4, 47, 52). In comparison, the advantage of archaeosomes may be in their unique ability to independently adjuvant different facets of the immune response to the entrapped Ag, including CTLs, T helper cells (13), and Ab (14), even in the long term. Furthermore, like other carrier systems, archaeosomes allow for efficient entrapment of hydrophilic or hydrophobic, protein, peptide, and/or small molecular Ags with relative ease. Moreover, M. smithii is a natural nonpathogenic habitant of the human colon (56), suggesting that lipids from such archaea may be more readily acceptable, from a regulatory perspective, in vaccine formulations. Overall, archaeosomes represent versatile, potentially universal, vaccine delivery adjuvants.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lakshmi Krishnan, Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Room 3016, Ottawa, Ontario, Canada K1A 0R6.
3 Abbreviations used in this paper: alum, aluminum hydroxide; TPL, total polar lipids; ER, endoplasmic reticulum.
Received for publication March 23, 2000. Accepted for publication August 2, 2000.
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References |
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vß3). J. Immunol. 161:6250.This article has been cited by other articles:
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