chapter 2: escherisome mediated cytosolic delivery of candida albicans...
TRANSCRIPT
Chapter 2: Escherisome mediated
cytosolic delivery of Candida
albicans cytosolic proteins induces
enhanced cytotoxic T lymphocyte
response and protective immunity
2. Introduction
2.1. Origin and need of the research
Immunologically compromised patients can suffer from mild superficial
lesions to disseminated full blown infections caused by several fungal pathogens
including Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans (Edwards
et. al. 1991, Degregrio et. al. 1982, Sheretz et. al. 1987). The high frequency of life
threatening systemic fungal infections could be attributed to the impaired
immune system of the host because of human immunodeficiency virus infliction
or due to the use of immunosuppressive drugs during organ transplantation or
usage of anticancer agents in treatment of different types of malignancies (Kelin
et. al. 1984). Limitations associated both with diagnosis as well as lack of
effective treatment protocols for most of the fungal diseases by conventional
means argue strongly in favour of pursuing the development of preventive
strategies rather relying on antifungal chemotherapy (Berenguer et. al. 1983,
Reboli et. al. 1993).
During mucosal colonization and systemic fungal infection in mice, Th1
cells arbitrate phagocyte-dependent protection and secrete mediators that help in
evoking protective immunity in the host. In contrast, production of inhibitory
cytokines such as IL-4 and IL-10 by the Th2 cells and high levels of IgE are
associated with disease progression (Romani et. al. 1997, 1999). For example, in
case of C. albicans infection, Th1 type responses correspond to resistance to
establishment of pathogen as seen in healthy humans, whereas Th-2 responses
associated predominantly with pathology and progression of the disease. In
general, antigen presenting cells viz. macrophages and dendritic cells are
uniquely able to initiate immune responses in naïve T-cells. In fact, dendritic
cells, have been reported to actively participate in T-helper cell education
(Steinman et. al. 1994, Bancherean et. al. 1998) and also their interaction with
two different forms of dimorphic C. albicans (Lilic et. al. 1996, Akiyama et. al.
1996, Fidel et. al. 1994, Puccetli et. al. 1995, Steinman et. al. 1994, Bancherean et.
al. 1998, Blasi et. al. 1992). Evidences for the role of T-cell mediated immunity
against disseminated candidiasis has been presented by several groups (Romani et.
al. 1999, Fidel et. al. 1994). Keeping in view, the natural co-operation among the
cells during the elicitation of the immune response, particularly the well described
cytokine-mediated interaction between T-cells and phagocytes such as
neutrophils (Romani et. al. 1997, Romani and Bistoni et. al. 1997), it seems
reasonable to postulate that T-cell mediated immunity may contribute to the
generation of an effective immune response against disseminated candidiasis.
Earlier reports regarding the characterization of T-lymphocytes in controlling
C.albicans infection have categorically described the role of antigen
presenting cells, specially the dendritic cells in processing and presentation
of C.albicans antigens along with MHC Class-I molecules (Fidel et. al. 1994,
Puccetli et. al. 1995, Steinman et. al. 1994, Bancherean et. al. 1998,).
Immunogenic peptides originated from a specific antigen, once expressed along
with MHC Class-I molecules are likely to provoke CTL generation and thereby
possibly help to eliminate the infection from the systemic circulation (Unanue et.
al. 1987).
2.2. Mechanism of Escheriosome entrapped antigen
To be presented to the CD8+ T-lymphocytes, protein antigens must
generally be exposed in the cytoplasm and thereby processed by proteasome
machinery and eventually co-presented along with MHC- I molecules (Braciale et.
al. 1987, Moore et. al. 1988). Soluble antigens are mostly taken up by APC’s
through endocytosis for CD4 T cell generation. However, to generate antigen
specific CTL responses, these antigens have to be delivered to the cytosol of
APC’s. Various approaches used earlier for CTL induction include the use of
fusogenic proteins and virosomes (Polt-Frank et. al. 1997), as well as pH sensitive
liposomes (Reddy et. al. 1991). Though reasonably effective, such vaccines suffer
from several limitations such as toxicity to the host, induction of the
immunological responses against structural integral proteins of virosomes or
fusogenic protein carriers on one hand and cumbersome methods needed for
their preparation on the other (Ames et. al. 1968). Such precincts restrict most
of the presently available tools as vehicle for the delivery of the newer generation
of subunit vaccines against intracellular infections.
2.3. Aim and scope of the present study
E.coli membrane comprises a great majority of anionic phospholipids that
play a pivotal role in membrane-membrane fusion (Balsi et. al. 1992). In the
present study, we report that the liposomes made of E.coli lipid vesicles
(escheriosomes) readily fuse with the plasma membrane, and successfully deliver
the encapsulated antigen to cytosol of the target cells. In vivo administration of
escheriosomes encapsulated antigen (cAg) induced antigen specific strong CTL
responses in the immunized mice. In contrast, the antigen encapsulated in egg
PC-liposomes, in a manner similar to the antigen-IFA emulsion, had limited
access to the cytosolic pathway of MHC-I dependent antigen presentation and
failed to generate antigen specific cell mediated immune response (Syed et. al.
2011, Syed and yan et. al. 2009). Finally, the immunisation with escherisome
encapsulated cytosolic antigen was demonstrated to induce strong protection
against C. albicans infection in Balb/c mice as assessed from survival rate and
fungal burden in vital organs of the infected animals.
2.4. Materials and methods
2.4.1. Chemicals
Egg phophatidylcholine (Egg PC) was isolated and purified using the
standard method as modified in our lab (Puccetli et. al. 1994). Cholesterol (Chol)
was purchased from Centron Research Laboratory, Bombay, India. Nutrient
broth (NB) was from Hi Media Laboratories, Bombay. RPMI, Hank’s balanced
salt solution (HBSS) and Fetal Bovine serum (FBS) was obtained from Life
Technologies (Grand Island, NY). Sodium chromate [51Cr] was bought from
Bhabha Atomic Research Center, Bombay, India. The amount of protein
entrapped in the liposomes was determined by BCA protein assay method (Pierce
Chemical Company).
2.4.2. Isolation of E. coli lipids
E. coli was cultured in nutrient broth ( 1 % peptone, 0.3 % Beef extract,
0.3 % Yeast extract and 1 % Sodium chloride; pH 7.4 ). The cells were harvested
from mid-log phase (18-20 h). Phospholipids were isolated by the method of
Bligh Dyer, as modified by Ames (Fidel et. al. 1994).
2.4.3. Isolation of cytosolic antigen from Candida albicans
The C. albicans was cultured on YEDP agar plates. The cells were
harvested after 24 hours and homogenized in chilled lysis buffer (2% Triton X-
100 (w/v), 1% SDS, 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA
(pH 8.0), 1mM PMSF). The homogenate was sonicated for 45 min at 4 C using a
bath sonicater. After sonication the homogenate was vortexed for 1 hr by
intermittent cooling to 4 C. The preparation was pelleted at 2000 g for 15 min
and the supernatant collected. The concentration of the protein was determined
by BCA method as described earlier (Smith et. al. 1985).
2.4.4. Preparation of liposomes
The cAg bearing liposomes were prepared using E. coli lipid or egg PC
essentially by following the published procedure as standardized in our lab
(Unanue et. al. 1987). Briefly, egg PC/cholesterol (2:1 molar ratio, total lipid 20
mg) or E. coli lipids (total lipid 20 mg) were reduced to thin dry film under N2
atmosphere. The film was hydrated, followed by sonication in a bath-type
sonicator for 1 h at 4 oC under N2 atmosphere. The liposomes thus formed were
mixed at this stage with an equal volume of cAg (30 mg/ml). The mixture was
flash frozen and thawed (3 cycles), and then lyophilized. The free-flowing, dried
powder thus obtained was rehydrated with distilled water (120 l) and finally re-
constituted with PBS. The preparation was centrifuged at 14,000g and the pellet
was further washed at least 3 times with PBS to remove the traces of the
unentrapped solute. The protein entrapped in the liposomes was estimated as
described elsewhere (Romani et. al. 1997). Briefly, the liposomes (given volume)
were lysed with 10% Triton X-100 solution (the final concentration of Triton X-
100 was maintained 1%). Protein concentration was determined using the BCA
reagent and a calibration curve prepared in presence of triton X-100.
2.4.5. Animals
Inbreed female Balb/c mice (8-10 weeks old), of 20 2 g of weight, were
obtained from the Institute’s Animals House Facility.
2.4.6. Immunization
The animals were divided in various groups. Each group was consisted of
10 animals. The animals were immunized with cAg-IFA or cAg entrapped in
various types of liposomes. The immunization schedules varied from experiment
to experiment depending upon nature of the study, and described accordingly.
2.4.7. Preparation of CD4+ T cells
Animals were immunized either with single dose of free cAg, cAg
encapsulated in egg PC/chol or E. coli-lipid liposomes (100 g cAg /animal)
through sub-cutaneous route (near the base of tail vein). On day 7, five animals
from each group were sacrificed and their spleens removed aseptically. The
splenic CD4+ T cells were isolated as described elsewhere (Cesborn et. al. 1993).
For lymphokine assay experiments, the animals were immunised intravenously
using two different immunising schedules. In one set, ten animals in each group
were administrated with a single dose of cAg (100 g cAg/animal), while in the
other, the animal were immunized with a total of three doses of cAg (100 g cAg
per animal, on day 0, 7, and 14). On day 21, the animals were sacrificed and CD4+
T cells were isolated as described earlier. The enriched population of CD4+ T
cells were stained with anti-CD4 and anti-CD3 Abs and the purity was found to
be >98%, as revealed by FACScan.
2.4.8. CD4+
T cell proliferation
The CD4+ T cells (2x104/well) obtained from pool of the splenic cells of
five mice from different groups were cultured in triplicate wells. The cells were
incubated with macrophages (6x104/well) pulsed with different doses (0.001-100
g/ml) of free cAg or that encapsulated in egg PC/chol or escherisomes. The
cultures were incubated for 72 h at 37 C/7% CO2. The cells were pulsed with
1.0 Ci [3H]-thymidine for 16 h before harvesting with an automatic cell harvester
(Skatron, Tranby, Norway). The [3H]-thymidine incorporation was measured by a
standard liquid scintillation counting method. The results are expressed as mean
cpm of triplicate cultures.
2.4.9. Lymphokine assays
The cultures were set up as described for Th cell proliferation. The
supernatants were collected after 48 h for estimation of IL-2, IL-4 and IFN-
levels. The IL-2, IL-4 and IFN- were assayed by ELISA method as standardized
in our lab.
2.4.10. Preparation of CD8+ T cells
Various groups of Balb/c mice were injected via sub-cutaneous route,
with a total three doses (day 0, 7, and 14) of IFA- cAg, cAg encapsulated in egg
PC/chol or escheriosomes and also with free cAg mixed with empty
escheriosomes [100 g cAg (which correspond to 500 nmoles of
lipid)/animal/week] for 3 weeks. On day 21, the animals (five animals each
group) were sacrificed and spleens were taken out aseptically. The CD8+ cells
were prepared as described elsewhere (Cesborn et. al. 1993). Cells obtained from
different animals in a given group were pooled, purified and used for the cyto-
toxicity assay. The enriched population stained with anti-CD8 Ab, was >98%
pure, as evaluated by FACScan.
2.4.11. CD8+ T lymphocyte response
P815 (H-2d macrophage cell line) cells were used as target cells for
performing cytotoxic T lymphocyte assay following published protocol as
standardized in our lab (Syed et. al. 2003) . Beside P815 cells, we used thio-
glycollate elicited peritoneal macrophages as target cells as well. Briefly, Balb/c
mice were injected with 3 ml of thioglycollate broth inter peritoneally. On day 4,
the macrophages were isolated from the peritoneal cavity elicited cells (PEC) by
adherence on Petri plates. The harvested cells (2x107cells/ml) were washed 3
times with HBSS and incubated at 37 C for 3-4 hr with either free cAg, cAg
entrapped in egg PC/chol or encapsulated in escheriosomes. The cells were again
washed thrice to remove free antigen. This was followed by incubation with 51Cr
(100 Ci/2x107 cells) for 45-60 min at 37 C. The cells were finally washed with
RPMI solution and used as target cells.
2.4.12. Cytotoxicity assay
The 51Cr-labelled macrophages/P815 cells (5x103/well) were used as
target cells in the cytotoxicity assay. The antigen primed target cells were
incubated with CD8+ T cells (effector cells isolated from the spleen of the five
mice from various groups pooled, and used for assay) at an effector to target
(E/T) ratios of 2.5:1-20:1. The cells were incubated at 37 C for 6 hr, after
stipulated time period, the cells were pelleted at 3000g (15 min, 5 C) and the
amount of released 51Cr was determined by measuring the radioactivity in the
supernatant. The experiments were performed three times and the error bars
represent standard deviation of the means from three different experiments. Total
51Cr release was calculated by treating an aliquot of the target cells with Triton X-
100 (10% final concentration). The spontaneous release of 51Cr in the supernatant
was determined by incubating the labeled macrophages for 6 h. Amount of auto-
release was subtracted from the total release to determine the extent of
macrophage lysis. In most of the experiments, the auto-release was less than 25%.
The percent specific release was calculated as the (mean sample cpmmean
spontaneous cpm/mean maximum cpm-mean spontaneous cpm) x 100%.
2.4.13. Phenotype of T lymphocytes isolated from spleen of immunized animals.
To determine phenotype of various T lymphocytes developed in cAg
immunized animals, the splenic cells were stained with fluorescent probe
conjugated antibodies as per manufacturer’s instruction 1×106 spleen cells of
mouse (n = 10 from each group) were stained with fluorescein isothiocyanate
(FITC)-conjugated monoclonal antibodies specific for mouse CD8 T-cell
receptor and phycoerythrin (PE)-conjugated monoclonal antibodies specific for
mouse CD4 T-cell receptors. The cells were incubated for 30 min at 4 0C, washed
three times with dilution buffer (0.01M phosphate buffer saline, pH 7.4
containing 1% bovine serum albumin, 0.1% sodium azide) and resuspended in
500 µl of 2% paraformaldehyde. The percentage of positive cells was measured
with a fluorescence-activated cell sorter (Guava) and data was analyzed
accordingly.
2.4.14. Determination of nitric oxide production in peritoneal macrophages
Nitric oxide production in peritoneal macrophages of the immunized
animals was assessed as described earlier (Syed and Yan et. al. 2009). Briefly,
peritoneal macrophages (1 x 106 cells/animal) from the immunized mice were
cultured in complete RPMI. The cells were incubated with various forms of cAg
(final concn. 10 g/well). After 24 h of incubation, 100 l of culture supernatant
was collected from each well and subsequently mixed with an equal volume of
Griess reagent [1 % sulphanilamide and 0.1 % n-(1-napthyl) ehtylenediamine
dihydrochloride in 2.5 % H3PO4] and further incubated for 10 min at room
temperature. The absorbance of colored complex was determined at 550 nm in
an ELISA reader. The amount of nitrite in culture supernatant was determined by
extrapolation from the standard curve that was plotted with analytical grade
sodium nitrite (NaNO2) diluted in culture medium.
2.4.15. Determination of cAg-specific IgG isotypes by ELISA
The production of cAg specific antibodies was measured in the sera of
the immunized mice. The animals were injected, via sub-cutaneous route, with
two doses of free cAg, cAg entrapped in the egg PC/chol or E.coli lipid
liposomes (100 g cAg/animal) on day 0 and 7, and bled on day 14 to monitor
the presence of antibodies as described earlier (Flynn et. al. 1993). Briefly, a
ninety-six well microtitre plate was incubated overnight with 50 l of cAg (25
g/ml) in carbonate-bicarbonate buffer (0.05 M, pH 9.6) at 4 oC. After usual
washing and blocking steps the plate was finally incubated with 1:500 dilutions of
test and control sera at 37 oC for 2h. After excessive washing of the plate, it was
further incubated with 50 l of biotinylated goat anti mouse IgG1 and IgG2a
antibodies. The plate was incubated at 37 oC for 1h. After the usual washing
steps, 50 l of streptavidin-HRP were added to each well and the plate was
incubated at 37 oC for 1h. The plate was washed again before adding 50 l ABTS
and was finally incubated at 37 oC for 20 min. The reaction was terminated by the
addition of 50 l of 70% H2SO4. The absorbance was read at 492 nm with a
microtitre plate reader (Eurogenetics, Torino, Italy).
2.4.16. Challenge of animals with Candida albicans Infection
Candida albicans (ATCC 18804, preserved in 10 % glycerol at –20 C) was
cultured in 5% dextrose. The cells were harvested after 24 hours and
pelleted at 2000g for 20 minutes at 4 C. The cells were counted by
hemocytometer and were diluted with normal saline in such a way that each 200
l contains 6.5105 cells. On the day 25th (fourth day after the final booster)
each animal was challenged with 6.5105 cells (in 200l) of C. albicans
intravenously (I.V route). The establishment of infection was assessed on the
basis of survival rate and CFU count in various vital organs of mice challenged
with C. albicans infection.
2.4.17. Determination of fungal load in various vital organs
The establishment of infection in immunized mice was assessed by determining
fungal load in different vital organs. The animals belonging to various immunized
groups were sacrificed on day 6th post infection. Vital organs viz. liver, kidney,
lung, spleen, were taken out aseptically. The organs were minced separately in
normal saline (5ml) and an aliquot (200l) of this suspension was plated on
YPD agar plates after appropriate dilution. The plates were incubated for
48-72 hours at 37 C .The development of colonies in a given vital organ
suspension belonging to a specific group was noted and the fungal load was
calculated by multiplying with the respective dilution factor. The animals
survived on day 60 post-infection were also screened for fungal load in various
vital organs using same method.
2.4.18. Statistical Analysis
The data were analysed by one-way analysis of variance (ANOVA)
following Dunnet’s t test method. P < 0.05 was considered statistically significant.
2.5. Results
Immunization with escheriosome encapsulated cAg antigen evokes
effective cell mediated as well as humoral immune responses in the immunized
animals.
2.5.1. Cell mediated immune response
2.5.1.1. CD8+ T Lymphocyte response
Strong fusogenic properties of escheriosomes facilitate the delivery of the
entrapped antigen into the cytosol of the APCs for processing and presentation
via MHC class I pathway. We evaluated the potential of escheriosomes entrapped
cAg to undergo MHC-I processing and presentation to generate a CD8+ T cell
response. The pilot experiments suggest that immunization with a total amount
of 300 g of administrated cAg (100 g cAg per dose/per animal, three doses
each at week interval) generated substantial target lysis at an effector to target
ratio of 10:1 (data not shown). This dose was selected for subsequent studies to
perform 51Cr release assay. Interestingly, immunisation with cAg entrapped in the
escheriosomes (EL-cAg), but not it other forms viz. cAg -IFA or cAg entrapped
in egg PC/chol liposomes and free cAg mixed with empty escheriosomes (sham
liposomes), generated cytotoxic T cells. A considerably high degree (30-40%) of
target cell lysis occurred when the cAg was encapsulated in the escheriosomes, as
compared to less than 1% specific lysis in cAg -IFA or cAg incorporated into the
egg PC/chol liposomes or sham escheriosomes (P<0.001) (figure 1). The result
of the present study clearly suggested that incubation of target cells with
escheriosomes encapsulated cAg led to the co-presentation of processed peptide
with MHC I molecules that eventually recognized by specific effector cells, while
other forms of cAg (free or egg PC encapsulated) were ineffective. Moreover, the
recognition of target cells was confined to the MHC compatible cells only, thus
effector cells were able to induce lysis of P 815 cells, while allogenic EL-4 cells
(H-2b) were not recognized by some effector cells (data not shown). In the next
set of experiments we demonstrated that beside P815 cells, escherisome mediated
delivery of cAg to peritoneal macrophages causes its processing and presentation
along with MHC I pathway. The results of the present study demonstrate that
peritoneal macrophages can also behave as efficient target cells to evoke antigen
specific CTL response. As evident from figure 1b the lysis of target cells increased
with increasing E: T ratio (figure 1).
2.5.1.2. DTH response
Delayed type hypersensitivity, which is an in vivo manifestation of cell-mediated
immune response, has direct correlation with protection of mice against various
intracellular infections. The immunization of mice with escheriosome
encapsulated cAg induced antigen specific cell mediated immune responses.
Measuring the increase in footpad thickness of immunised animals assessed the
cell-mediated immune response. As evident from figure 2, among the various
prepration of vaccine studied, only escheriosome-encapsulated antigen was able
to induce remarkable CMI response (0.1320.015 x 10-3 mm), and no other form
of cAg was able to induce cell-mediated immune responses in immunized animals
(P values; Control PBS Vs EL-cAg < 0.001; IFA-cAg Vs EL-cAg and PC-Ag Vs
EL-cAg < 0.01).
Induction of cell-mediated immune response upon immunization with various
cAg based vaccines was further confirmed by assessment of cytokine level in
plasma of immunized animals. The animals immunised with single dose of
antigen yielded very marginal expression of IL-2 and IFN- in both
escheriosomes (20 pg/ml IL-2, 28.4 pg/ml IFN-) and egg PC/chol liposomes
(12.0 pg/ml IL-2, 16.0 pg/ml IFN-) treated groups (personal observation).
However, boosting thrice with escheriosomes resulted in substantial increase in
IL-2 and IFN- levels (11510 pg/ml IL-2, 12018 pg/ml IFN-), while not so
significant increase in level of type I cytokines was observed upon multiple
immunizations with IFA-cAg formulations. Interestingly, multiple immunization
schedules were successful in evoking type I cytokine response in egg PC/chol
liposomes group as well (P values; Control PBS Vs EL-cAg < 0.001; IFA-cAg Vs
EL-cAg and PC-Ag Vs EL-cAg < 0.05) (Figure 3). No detectable amount of
lymphokines was observed in the case of control animals inoculated either with
PBS, sham egg PC/chol or sham escheriosomes (no antigen).
2.5:1 5:1 10:1 20:1
Effector : Target Ratio
Perc
en
t S
pe
cific
Lysis
0
10
20
30
40
EL-cAg
PC-cAg
sham EL
IFA-cAg
free-cAg
sham EL+cAg
Figure 1A Escheriosome provide an efficient means of sensitizing target cells to class 1-
restricted CTL recognition. Balb/c mice were immunized with cAg encapsulated in
escheriosomes. Cytotoxic T cells isolated from the spleen of 5 mice were pooled and
used as effector cells for cytotoxic assay. The MHC restriction as well as antigen
specificity of the generated CTLs was demonstrated by 51Cr release assay. The target cells,
P815 (H-2d) were pulsed with various form of cAg and co-cultured with effector cells,
isolated from EL-cAg treated animals in varying effector-to-target cell ratio (2.5:1-20:1).
In another set, target cells, EL-4 (H-2b) were pulsed with escheriosome-encapsulated cAg
and incubated with same set of effector cells. The lysis of the target cells was measured
by 51Cr release assay. As evident from data the lysis of target cells increased with
increasing E: T ratio. Moreover, the recognition of target cell is confined to the MHC
compatible cells only, thus effector cells were able to induce lysis of P815 cells, while
allogenic EL-4 cells were not recognized at all by the same effector cells. (P Value: El-
cAg Vs PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P; <0.001).
2.5: 1 5:1 10:1 20:1
Effector : Target Ratio
Perc
en
t sp
ecif
ic L
ysis
0
10
20
30
40
50
Figure 1B Escheriosome encapsulated cAg can induce T lymphocytes with CD8+
phenotype. Effector cells obtained from the cAg (encapsulated in escheriosome) primed
animals were pre-treated with anti-CD4+ or anti-CD8+ monoclonal antibodies followed
by incubation with baby rabbit complement. The effector cells were incubated with cAg
pulsed, 51Cr-loaded target cells (thioglycolate elicited macrophages). The lysis of the target
cells was measured by 51Cr release assay. Interestingly, immunization with cAg entrapped
in the escheriosomes, but not other forms of cAg viz. cAg -IFA or cAg entrapped in egg
PC/chol liposomes and free cAg mixed with empty escheriosomes (sham liposomes),
generated cytotoxic T cells. A considerably high degree (30-40%) of target cell lysis
occurred when the cAg was encapsulated in the escheriosomes, as compared to less than
1% specific lysis in cAg -IFA or cAg incorporated into the egg PC/chol liposomes or
sham escheriosomes. (P Value: El-cAg Vs PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P;
<0.001).
Figure 2 DTH response in animals immunized with various preparation of cAg vaccine.
The Balb/c mice were immunized in their foot pad route with various forms cAg vaccines
subcutaneously. The left hind leg was inoculated with 50 µl saline while right hind leg was
administered with equal volume of different antigen preparations. The thickness of the foot
pad was measured by Vernier’s caliper. (P Value: El-cAg Vs PC-cAg ; P<0.001, EL-cAg vs
IFA-cAg P; <0.001).
0
20
40
60
80
100
120
140
160
Saline IFAcAg Sham EL + cAg
PC-cAg EC-cAg
Foo
t P
ad T
hic
knes
s (x
10
-3 m
m)
0
20
40
60
80
100
120
140
EC-cAg PC-cAg Sham EL Sham PC Saline
IL-2
Co
nce
ntr
atio
n (
pg/
ml)
0
50
100
150
200
250
300
350
400
450
EC-cAg PC-cAg Sham EL Sham PC Saline
IL-4
Co
nce
ntr
atio
n (
pg/
ml)
Figure 3 Cytokine profile of animals immunized with escheriosome encapsulated
cAg. (A) IL-2, (B) IL-4, and (C) IFN-γ. The culture was set as mentioned in material
and method section, In-vitro T-cells proliferation induced by EC-cAg, PC-cAg, Sham-
EL, and saline. T-cells were obtained from spleen of mice immunized with cAg
entrapped Escheriosome. The spleen T cells were stimulated with a mixture of cAg
entrapped Escheriosome, PC-cAg, Sham-EL, and saline with different Conc. (0.001-
100μg/ml). Induction of cell mediated immune response was further confirmed by the
assessment of cytokine level in plasma of immunized animals. No detectable amount of
lymphokines was observed in the case of control animals inoculated with either saline,
sham egg PC/chol or sham escheriosomes. (P Value: El-cAg Vs PC-cAg ; P<0.001, EL-
cAg vs IFA-cAg P; <0.001).
2.5.1.3. CD4+ T cell response
0
20
40
60
80
100
120
140
160
EC-cAg PC-cAg Sham EL Sham PC Saline
IFNγ
Co
nce
ntr
atio
n (
pg/
ml)
Cell mediated immune responses can also be manifested by assessment of
transformation of lymphocytes in presence of specific antigen. In the present
study, we analyzed the stimulatory potential of various forms of cAg on the
proliferation of antigen specific T cells. T lymphocytes obtained from mice
immunized with antigen encapsulated in the escheriosomes induced significantly
higher proliferation when compared to the T lymphocytes obtained from these
immunized with egg PC/chol liposomes (Figure 4). T-cell responsiveness to
antigen was observed in a dose dependent manner. Control cultures containing
cells obtained from either cAg immunized animals or the groups immunized
with PBS or fusogenic lipids only (sham liposomes with no cAg), gave
background levels of <2000 cpm of 3H-thymidine incorporation.
2.5.1.4. Escheriosomes enhance the expression of CD4+ and CD8+ T-cells.
Flow cytometric analysis was conducted to evaluate the expression of CD4+ and
CD8+ T-cells in spleenocytes isolated from animals administrated with normal
Saline, IFA-cAg, Sham Liposomes + Free cAg, PC-cAg and EC-cAg. The data of
present study clearly reveal that T-cells isolated from animals immunized with
EL-cAg showed significantly higher level of expression of both CD4+ (35.16%)
and CD8+ (41.56%) on their surface when compared to control animal (normal
saline, 5.34%, 7.22% for CD4+ and CD8+ T-cells respectively), animal immunized
with IFA-cAg expressed 12.68% of CD4+ and 12.96% of CD8+ T-cells
respectively. In contrast, T-cells isolated from animal vaccinated with PC-cAg
express relatively less expression of CD4+ (26.57%) and CD8+ (14.52%) (Figure
5). P values: Control Vs EL-cAg < 0.001; IFA-cAg Vs EL-cAg < 0.001; EL-cAg
Vs PC-cAg < 0.01.
2.6. Humoral Immune response
2.6.1. Upregulation of both IgG2a as well as IgG1-isotype by immunizing the
animals with escheriosomes encapsulated antigen.
Immunization of female Balb/C mice with various forms of cAg resulted in
induction of cAg specific antibodies. Among various experimental groups, EL-
cAg combination demonstrated greater adjuvant potential, while relatively less
Antigen Conc (g/ml)
0.03 0.16 0.8 4 20 100
CP
M
0
5000
10000
15000
20000
25000
30000
saline
Sham EL
PC-cAg
EC-cAg
Figure 4 T-cells proliferation induced by cAg entrapped Escheriosome, T-cells were
obtained from spleen of mice immunized with cAg Escheriosome entrapped. The spleen
T cells were stimulated with a mixture of cAg entrapped Escheriosome, PC-cAg, Sham-
EL, and saline with different Conc. (0.001-100μg/ml). After 72 hr cultivation, the
proliferation of T cells was determined by [3H]-thymidine incorporation. (P Value: El-cAg
Vs PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P; <0.001).
Figure 5 cAg-escheriosome evoke antigen specific T cells in immunized mice. The
animals were immunized with (a) normal Saline (b) IFA-cAg (c) Sham Liposomes + Free
antigen. (d) PC-cAg and (e) EC-cAg as described in material and methods. The freshly
isolated spleen cells from the BALB/c mice of each experimental and control groups (n
= 5 each group) were pooled and stained with FITC conjugated anti-CD4 (A) as well as
anti-CD8 (B) monoclonal Abs (Sigma) before analyzing them by flow cytometry (BD
Biosciences). The cells with log fluorescence intensities > 101 were gated as CD4+ T-cells
and compared in the histogram. X-axis parameter: Log fluorescence of FITC-
CD8+/CD4+ T-cells; Y-axis parameter: counts of CD8+/CD4+ T-Cells which are positive
for the marker used. (P Value: El-cAg Vs PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P;
<0.001).
Figure 6A Humoral immune response in mice immunized with various cAg
formulations. The immunized animals were analyzed for the presence of antigen specific
antibodies using ELISA method. The antibody titer was expressed as dilution which produced >
0.2 optical density. (P Value: El-cAg Vs PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P; <0.001).
0
10000
20000
30000
40000
50000
60000
70000
80000
Control Sham EL+ cAg
IFA cAg PC-cAg EC-cAg
Ab
sorb
ance
at
49
2 n
m
Figure 6B Generation of antigen specific IgG isotypes in sera of animals
immunized with various forms of cAg. BALB/c mice immunized with various
preparation of cAg were bled on day 7th of last booster to determine antigen specific
antibody isotypes (IgG1 and IgG2a) in their sera. Sera (1:500 dilution) obtained from the
control and experimental animals were analyzed for the presence of cAg-specific IgG-
isotype by ELISA method as described earlier. The levels of two major isotypes of IgGs
were expressed as absorbance (A492) of the colored complex developed in the
immunosorbent assay.The values obtained here are the mean ± S.D. of the sera of five
individual animals. No detectable concentration of antibody was observed in sera of
animals immunized with sham EL or PC liposomes. (P Value: El-cAg Vs PC-cAg ;
P<0.001, EL-cAg vs IFA-cAg P; <0.001).
0
0.2
0.4
0.6
0.8
1
1.2
EL-cAg PC-cAg
Ab
sorb
ance
at
49
2 n
m
IgG1
IgG2a
interim of antibody response was observed in groups of animals, which were
immunized with IFA-cAg or PC-cAg. The animals belonging to the groups which
were immunized with antigen entrapped in escheriosomes demonstrated
significantly enhanced antibody titre (A492 0.640.2) when compared to both PC-
cAg (A492 0.320.2) and IFA-cAg (A492 0.140.2). P values: Control Vs EL-cAg <
0.001; IFA-cAg Vs EL-cAg < 0.001; EL-cAg Vs PC-cAg < 0.01. As shown in
figure 6, there was significant enhancement (P values PBS only Vs EL-cAg <
0.001; IFA-cAg Vs EL-cAg < 0.005; and PC-cAg Vs EL-cAg < 0.01) in total IgG
antibody titre in animals immunized with EL-cAg (A492 1.40.2) and PC-cAg (A492
0.80.1) as compared to other control groups after second booster (P values:
Control Vs EL-cAg < 0.001; IFA-cAg Vs EL-cAg < 0.001; EL-cAg Vs PC-cAg
< 0.01). The isotyping results demonstrated that there was substantial amount of
IgG1 (A492 0.98 0.08) and IgG2a (A492 0.74 0.06) titre in animals immunized
with EL-cAg group (figure 6b). The animals immunized with PC-cAg have
demonstrated relatively low level of IgG2a (A492 0.21 0.04), while moderate
amount of IgG1 isotype (A492 0.59 0.01) of antibodies (P values; EL-cAg vs PC-
cAg < 0.01). No IgG1 and IgG2a-isotypes were detected in the control group of
mice injected with either sham EL, egg PC/chol liposomes (no antigen) or PBS.
2.6.2. Nitric oxide production
A number of cytotoxic substances produced by activated macrophages as
a measure to counter the pathogen attack. When, peritoneal macrophages (1 X
106 cells/animals) from the immunized mice were cultured in complete RPMI and
incubated with various form of cAg (final conc. 10µg/well). After incubation,
100µl of supernatant was collected from each well and NO production was
estimated as described earlier in material and methods section. Immunization
protocol involving EC-cAg resulted in expression of high level of NOS (nitric
oxide synthetase), an enzyme that oxidizes L-arginine to yield L-citruline and
nitric oxide (NO) as compared to PC-cAg. On Day 8,
EC-cAg PC-cAg
NO
Co
ncen
tart
ion
(
mo
l)
0
5
10
15
20
25
30
35
Day 8
Day 45
Figure 7 cAg escheriosome evoke nitric oxide production in macrophages. Nitric
oxide production profile of animals immunized with various antigen prepration of
liposomes at day 8 and 45. The culture was set as described in material and method.
peritoneal macrophages (1 X 106 cells/animals) from the immunized mice were cultured
in complete RPMI and were incubated with various form of cAg (final conc. 10µg/well).
After incubation, 100µl of supernatant was collected from each well to determine NO
production. No significant amount of nitric oxide was detected in the control animals
inoculated with either saline, sham egg PC/chol or sham escheriosomes. The data is
mean ± S.D. of three mice per group and representative of three different experiments.
(P Value: El-cAg Vs PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P; <0.001).
supernatant from EC-cAg produced 15±2 µmol NO, while PC-cAg yielded only
9±2 µmol of NO. Similar pattern was followed on day 45, EC-cAg generated
much higher amount of NO (30±2 µmol) than PC-cAg (12±1.5 µmol)
formulation.
2.7. Protection Studies
2.7.1. Escheriosome encapsulated cAg imparts protection against C. albicans
infection in Balb/c mice
Vaccine potential of C. albicans antigens bearing escheriosome based vaccine was
evaluated against experimental candidiasis by immunizing mice with following
antigen preparations viz. normal saline; sham escheriosomes; sham
escheriosomes + cAg (physical mixture); IFA-cAg; PC-cAg and EL-cAg as
described in materials and methods. The animals belonging to various groups
were injected with total three boosters of cAg subcutaneously (100g/animal on
day 0, 7 and 14) for three weeks. The immunized mice were subsequently
challenged with C.albicans spores (6105 spores per animal) by intravenous route
to induce the experimental disseminated candidiasis. The survival data on day 15
post infection showed almost 100 % survival of the animals which were
immunized with escheriosome encapsulated cAg (P value: Saline Vs EL-cAg
0.001) were alive while around 75 % of the animals survived in the group which
was immunized with PC-cAg (P value: saline Vs PC-cAg 0.01; EL-cAg Vs PC-
cAg < 0.05). No animal survived in the saline control group beyond day 15 post
infection (Figure 8). On day 60 post challenge with infection, there was around 80
% survival of the animals which were treated with escheriosome encapsulated
cAg antigen, while 40 % of the animals survived in the group which was
immunized with egg PC liposomes. (P value: Control Vs EL-cAg 0.001; EL-cAg
Vs PC-cAg < 0.01).
The prophylactic potential of escheriosome against systemic candidiasis
was further established by determining residual fungal load in various vital organs.
The animals were sacrificed on day sixth post infection to determine fungal load
in systemic circulation as well as in different vital organs. The fungal load data
demonstrated that the animals treated with escheriosome encapsulated antigen
had several fold less fungal load in various vital organs when compared to the
Time (in days)
0 10 20 30 40 50 60 70
Perc
en
t S
urv
ival
0
20
40
60
80
100
120Saline
IFA + cAg
Sham EL + cAg
PC-cAg
EC-cAg
Figure 8 Prophylactic potential of escheriosomes in terms of survival rate of animals that
were challeged with C. albicans infection. After immunisation with various preparation of
cAg antigen as described in material and methods, the animals were challenged with
6.5105 cells (in 200l) of C. albicans intravenously. Each group consisted of 10 animals.
The animals were monitored for survival till 60th day post challenge. Data is
representative of five different experiments. (P Value: El-cAg Vs PC-cAg ; P<0.001, EL-
cAg vs IFA-cAg P; <0.001).
Saline
IFAcAg
Sham E
L + c
Ag
PC-cAg
EC-cAg
CF
U/m
l (x1
03 )
0
20
40
60
80
100
Kidney
Liver
Spleen
Lungs
Sham E
L + c
Ag
IFAcA
g
PC-c
Ag
EC-c
Ag
CF
U/m
l (x
10
5)
0
50
100
150
200
250
300
Figure 9 Effect of immunisation on establishment of infection in animals
immunised with various form of cAg. (A) Fungal burden in vital organs (liver, spleen,
kideney and lungs) and in (B) blood of immunized mice was determined on 6th day post
challenge with 6.5105 cells (in 200l) of C. albicans. The data is mean ± S.D. of three
mice per group and representative of three different experiments. (P Value: El-cAg Vs
PC-cAg ; P<0.001, EL-cAg vs IFA-cAg P; <0.001).
group treated with egg PC liposomes (Figure 9A). The fungal load in the systemic
circulation was in compliance with the severity of infection (fungal load) in
various vital organs, and there was almost 10 fold fewer fungal burden (CFU)
present in animals that were immunized with EL-cAg group, when compared to
that immunized with PC-cAg group (Figure 9B). The animals, which survived on
day 60, were also screened for residual fungal load in various vital organs. While
none of the animals survived in other groups, except the animals that were
immunized with EL-cAg. Interestingly, the animals were found to be virtually free
of any fungal load except one animal, which showed the presence of 140 CFU in
the lung.
2.8. Discussion
In general pathogen specific antibodies play major role in containing the
establishment of various pathogens in the host. In fact, antibodies protect the
host against invading pathogens from the entry point itself. However, the
protective immunity against intracellular pathogens is a bit complicated as such
organisms adapt intracellular parasitism to avoid antibody recognition. To
suppress intracellular pathogens, host immune system relies on activation of
Interferon producing cytolytic T lymphocytes (Tarleton et. al. 1992, Cesborn et.
al. 1993, Syed et. al. 2003, Flynn et. al. 1992, Muller et. al. 1992, Doherty et. al,
1992). The role of immunlogical responses against yeast like fungi, such as
C.albicans, has remained a contentious issue. Several research groups contend that
C. albicans specific antibodies may be protective aganist experimental
desseminated candidiasis (Ding et. al, 1988). On the contrary, various lines of
evidences militate against protective function of pathogen specific antibodies to
play any role in constraining the infection (Kirby et. al. 1984). In fact, the
controversies can be attributed to the ability of the fungus to reversibly switch
between unicellular yeast to filamentous forms in the infected host (Han et. al.
1995). It is imperative that successful elimination of infection like C. albicans could
be made possible by activation of humoral as well as cell mediated responses
directed against both forms of the C. albicans (Wajner et. al. 1996).
While immunization of animals using exogenous antigen generally leads to
the generation of antigen specific antibodies, activation of cytotoxic T
lymphocytes has always been problematic. Earlier we have demonstrated
fusogenic potential of escheriosomes with various living cells including antigen
presenting cells. Keeping into consideration the strong membrane membrane
fusion potential of escheriosomes, one can speculate that subsequent to the
fusion, the encapsulated antigen can access to cytosol of the antigen
presenting cells. In cytosol, the exogenous antigen is likely to follow MHC-I
processing and presentation that ultimately lead to the elicitation of cytotoxic
CD8+ T lymphocyte response (Odds et. al. 1998). Besides, the fraction of
escheriosomes which escape the membrane fusion would be avidly taken up
by the antigen presenting cells through endocytosis and finally undergo MHC-
II processing and presentation and, contribute in activation of humoral
immune responses (Braciale et. al. 1987). In the present study, we have tried
to develop a liposome based vaccine which can induce appropriate
immunological responses against C. albicans infection.
The results of the study show that:
a) Escheriosomes mediate cellular immune response against entrapped C. albicans
antigen.
b) Adminstration of escheriosome encapsulated C. albicans antigen induce
strong CD4+/CD8+ T lymphocyte immune responses.
c) Besides CD8+ T lymphocyte induction, escherisome encapsulated C. albicans
antigen activate B lymphocytes for IgG2a/IgG1 antibodies production.
d) Immunization with escheriosome encapsulated antigen imparts protection
against C. albicans infection.
e) Escheriosomes based vaccines can suppress virulence of C. albicans.
The escheriosome based cAg vaccine was evaluated for its potential to
evoke desirable immune response in Balb/c mice. Interestingly, cell mediated
response was observed in animals, which were immunized with escheriosomes
encapsulated antigen (EL-cAg), but not in control groups including that
immunized with egg PC liposome encapsulated antigen (figure 2). Beside DTH,
immunization with EL-cAg formulation elicits strong T cell proliferation in dose
dependent manner (figure 4). The escheriosome mediated antigen delivery
ensues in up-regulation of Type I cytokines that facilitate induction of CD8+ T
lymphocyte response. Thus immunizations with EL-cAg formulation induced Il-
2 as well as IFNγ cytokine, while immunization with PC-cAg and IFA-cAg led to
the production of IL-4 mainly. Beside Th1/Th2 polarization in favor of cell
mediated immune response, delivery of cAg to the cytosol of the antigen
presenting cell ensues its processing and presentation along with MHC class I
pathway and thereby facilitates induction of antigen specific CTL response in
immunized animals.
Our results also show that immunization with escheriosomes
encapsulated antigen generate high antibody response when compared to egg PC
liposome encapsulated or free form of the antigen (Figure 6). Surprisingly, both
escheriosome as well as egg PC liposome encapsulated cAg formulations were
found to have biased induction for IgG1 type of specific antibodies when
compared to IgG2a isotype. Recently, other research groups have also shown that
antigen encapsulated in leptosome (liposome prepared from Letospira biflexa
membrane lipids) and smegmosome (liposome prepared from Mycobacterium
smegmatis membrane lipids) preferentially evoked IgG1 type of antibodies over
IgG2a isotype in-spite of elevated level of IFN-g in the immunized animals (Syed
et. al. 2011, Syed and Yan et. al. 2009). In general, IFN-γ produced in response to
LPS promotes the secretion of IgG2a isotype of antibodies in the immunized
mice. It seems escheriosome in a manner similar to other liposome based vaccine
delivery systems such as smegmosome or leptosome do not follow same pathway
of IFN-γ driven IgG2a induction as reported for LPS (Markine-Goriaynoff et. al.
2000). Alternatively, there are several reports that argue that IgG2a is not solely
induced by type 1 cytokine IFN-γ rather than other factors might also play
important role in class switching. As escheriosome encapsulated cAg facilitate
induction of both Th1 and Th2 type cytokines the later might be inducing more
of IgG1 isotype than IgG2a isotype of antibodies (Szomolanvi-Tsuda et. al.
2001). Lastly, being mixture of more than one type of proteins cAg might
comprise of both B cells as well as T cells antigen, that was eventually facilitating
activation of B cells for production IgG1 type of antibody mainly (Blum et. al.
1993).
The protection studies demonstrate remarkable survival rate of
animals which were immunised with escheriosome encapsulated antigen
only, in comparison to those immunised with the PC-cAg based liposome
(figure 8). The observed results advocate the hypothesis that successful
elimination of C. albicans from systemic circulation require active involvement of
both humoral as well as cell mediated immune response concomitantly. The data
of the present study substantiates this assumption as escheriosome based
vaccination successfully elicited desired CTL (cell mediated) and antibody
(humoral) response. In contrast, egg PC liposomes entrapped antigen generally
activate of humoral immune response. It seems the PC-cAg based liposoemes
eliminate mainly the yeast form of the invading C. albicans, while the filamentous
form escapes antibody mediated killing by adapting intracellular parasitism in the
cytosol of antigen presenting cells (Christiana et. al. 2000), which ultimately
leading to recurrence of infection and subsequent death.
Immunization with EC-cAg led to activation of cell mediated immunity
that obliterate infection completely by killing both forms of C. albicans (figure 8).
This can be explained on the premise that escheriosome mediated activation of
antibodies as well as cytotoxic T lymphocytes can specifically recognize both
unicellular yeast as well as filamentous intra-cellular form of C. albicans.
The escheriosome mediated protection against C.albicans infection
was further confirmed by the results showing fungal burden in systemic
circulation determined on day 8th post-infection (figure 9A). We hypothesised
that immunisation with escheriosome leads to the generation of antibodies which
subdue virulence of infection and delay establishment of infection. The
remarkable reduction in fungal load in various vital organs observed the
initial phase of exposure with infection in escheriosome treated animals
support our hypothesis (figure 9B).
Since protective immunity against C.albicans infection need to address
both the yeast as well as hyphal form of the pathogen, the presence of
hyphae form in the cytosol of antigen presenting cells need an
immunization protocol which can eradicate intracellular pathogens, we used
escheriosome based carrier system to induce pathogen specific humoral as well as
cell mediated immune responses. As both forms of C.albicans differ in
external morphology (Wajner et. al. 1996), and contain several common
cytosolic antigens (data not shown), we opted to use cytosolic proteins
while performing this study, with the idea that use of the multiple antigens
can provoke both humoral as well as cell mediated immunity effective
against both form of the C. albicans. At present, we are trying to establish role of
individual cytosolic antigen in imparting protection against C.albicans
infection. Our preliminary studies show that among different proteins
escheriosomes encapsulated Hsp 60 is highly immunogenic for both humoral as
well as cell mediated immune response (Khan et. al. 2009).
We infer from the present study that exogenous antigens can be tailored
to the simultaneous presentation with MHC- I as well as MHC-II processing
and presentation pathways and thereby induce both humoral as well as cell
mediated immunity.
Finally, it can be concluded that nano-particles based antigen delivery
system facilitates slow release of antigen over extended time period. The delivery
system can also help in simultaneously activation of both type I and type II
cytokines in the immunized subjects thereby successfully evoke both humoral as
well as cell mediated response against C. albicans.