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.