Comparative evaluation of hepatitis B surface antigen

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Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 110 – 118 www.nanomedjournal.com

Original Article

Comparative evaluation of hepatitis B surface antigen–loaded elastic liposomes and ethosomes for human dendritic cell uptake and immune response Dinesh Mishra, PhD a,⁎, Pradyumna Kumar Mishra, PhD b , Sunil Dabadghao, DM b , Vaibhav Dubey, MPharm a , Manoj Nahar, MPharm a , Narendra K. Jain, PhD a a

Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar, India b Bhopal Memorial Hospital and Research Center, Bhopal, India Received 16 July 2008; accepted 17 April 2009

Abstract The aim of the present study was to evaluate two vesicular carrier systems, ethosomes and elastic liposomes loaded with hepatitis B surface antigen, for in vitro qualitative and quantitative uptake by human dendritic cells (DCs) and ability to stimulate T lymphocytes. Quantitative uptake of antigen-loaded carriers was documented by flow cytometry, and internalization of the systems by the DCs was studied using spectral bioimaging. Ability of antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes and levels of TH1/TH2 cytokines were also examined using flow cytometry. Both vesicular carrier systems as antigen delivery modules and DCs as antigen-presenting cells were able to generate a protective immune response. However, ethosomes were found to have higher internalizing ability and immunogenicity in comparison with elastic liposomes. These properties of ethosomes coupled with their skinnavigating potential, make it an attractive vehicle for development of a transcutaneous vaccine against hepatitis B in preference to elastic liposomes. From the Clinical Editor: Two carrier systems for more potent vaccine administration - ethosomes and elastic liposomes loaded with hepatitis B surface antigen – are compared. Ethosomes demonstrated higher internalizing ability and immunogenicity. Due to their known skin-navigating potential, ethosomes may represent an attractive vehicle for development of a transcutaneous vaccine against hepatitis B. © 2010 Elsevier Inc. All rights reserved. Key words: Dendritic cells; Hepatitis B vaccine; Elastic liposomes; Ethosomes; Transcutaneous immunization

Hepatitis B is a serious global public health problem, with about two billion people infected by a small enveloped DNA virus in the liver. Approximately 360 million people have chronic infection, and 600,000 die worldwide each year from complications related to the disease including hepatocellular carcinoma.1 The prevalence of chronic hepatitis B virus (HBV) infection varies markedly in different geographic areas of the world, from 8% to 10% in high-endemic areas to 0.1% in low-prevalence areas.2 Infection with the virus leads to a Financial support was provided by University Grant Commission, New Delhi, India. ⁎ Corresponding author: Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470003, India. E-mail address: [email protected] (D. Mishra).

wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis leading to cirrhosis and hepatocellular carcinoma.3 Hepatitis B vaccination is the most effective measure to prevent HBV infection. Universal hepatitis B immunization has also been shown to be highly cost-effective in low-income countries with intermediate endemicity.4 Although the available vaccines produce a good and sustained immune response, their effectiveness drops with age; they are also less efficient in special groups like patients with chronic renal failure, liver transplant recipients, and individuals infected with the human immunodeficiency virus.5 There is a need to develop more potent prophylactic vaccines against HBV and better regimens of therapeutic vaccines for chronic carrier states. Animal models have shown therapeutic

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.04.003 Please cite this article as: D. Mishra, et al, Comparative evaluation of hepatitis B surface antigen–loaded elastic liposomes and ethosomes for human dendritic cell uptake and immune response. Nanomedicine: NBM 2010;6:110-118, doi:10.1016/j.nano.2009.04.003

D. Mishra et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 110–118

efficacy of vaccination with antigen-pulsed dendritic cells (DCs) in various chronic and human diseases.6 DCs represent a complex network of antigen-presenting cells (APCs) that function in the initiation of primary immunity as well as maintaining the balance between tolerance and immune recognition. DCs are very efficient in capturing, processing, and presentation of antigens with stimulation of naive T lymphocytes and are even capable of priming naive CD8+ T lymphocytes.7 The ability of DCs to invoke a primary response is due to expression of cell surface markers along with costimulatory signals that are essential for T-cell activation, and this potential has been exploited in recent years to develop nextgeneration vaccines for chronic and malignant human diseases using antigen-pulsed DCs.8,9 Nanotechnology, building on its ability to control structures at the atomic level, promises to develop effective drug delivery systems. The convergence of nanoparticles and technology offers major advantages like improved efficacy with reduced toxicity, thereby improving convenience and patient compliance.10,11 Although engineered nanocarrier systems in vaccine formulation have been studied for many years, the recent emergence of targeting antigen specifically to DCs using these systems has invited significant interest.12 Two such nanoengineered vesicular carrier systems, elastic liposomes and ethosomes, have been shown as versatile carrier agents in their ability to incorporate a diverse range of antigens to DCs with potential for maximizing immune responses.13 Previously, we investigated the uptake and generation of immune response by murine DCs pulsed with hepatitis B surface antigen (HBsAg)–loaded elastic liposomes. We have shown protective immune response with HBsAg-loaded elastic vesicles as antigen delivery modules and DCs as APCs.14,15 Ethosomes are interesting and innovative vesicular carriers that have appeared in the fields of pharmaceutical technology and drug delivery in recent years.16 These are soft, malleable structures tailored for enhanced delivery of active agents. It has been shown that the physicochemical characteristics of ethosomes allow this vesicular carrier to transport active substances more efficaciously through the stratum corneum into the deeper layers of the skin than conventional liposomes.17-19 In the current investigation we compared the uptake and immune response generation by human DCs pulsed with HBsAg-loaded elastic liposomes and ethosomes.

Methods Materials The HBsAg (source, genetically modified yeast cells) was obtained as a gift sample from the National Institute of Immunology (New Delhi, India); bovine serum albumin (BSA; fraction V), fluorescein isothiocyanate (FITC), Rhodamine, and soya phosphatidyl choline (SPC) were purchased from SigmaAldrich (St Louis, Missouri). Span 80 (an ester of plain sorbitan with fatty acids) was purchased from Himedia (Mumbai, India). RPMI-1640, Alamar blue, fetal calf serum (FCS), and sodium azide were procured from Hyclone (Logan,

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Utah). Bromodeoxyuridine (BrdU) was obtained from Roche (Mannheim, Germany). Annexin V–FITC/propidium iodide (PI) apoptosis assay kit, buffer for fluorescence-activated cell sorting (FACS), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) were purchased from BD Biosciences (San Jose, California). Dulbecco's modified Eagle medium was purchased from Gibco BRL Life Technologies (Carlsbad, California).

Preparation of vesicular system Elastic liposomes were prepared by the method as reported elsewhere with slight modifications.15 In brief, an ethanolic solution of SPC was mixed with Span 80 (ratio 86%:14% wt/wt) in phosphate buffer (pH 6.5) containing HBsAg solution (10 μg/mL), and the suspension was pushed through a series of 0.45, 0.22-μm polycarbonate filters (Millipore, Billerica, Massachusetts). The ethosomes were prepared by classical mechanical dispersion method as reported elsewhere.20,21 Ethosomes investigated here were composed of 2.0% (wt/vol) SPC, 25% (vol/vol) ethanol, antigen (HBsAg, 10 μg/mL). Briefly, SPC was dissolved in chloroform-methanol (3:1) in a clean, dry, roundbottom flask. Organic solvents were then removed by rotary vacuum evaporator above the lipid main phase transition temperature of 60-C (rotary evaporator; Superfit, Ambala, India). Finally, the traces of solvent were removed from the deposited lipid film under vacuum overnight. The lipid film was then hydrated with phosphate-buffered saline (PBS; pH 6.5) containing 25% ethanol and HBsAg (10 μg/mL) by rotation (60 rpm, 1 hour) at the corresponding temperature (60°C). Similarly, elastic liposomes and ethosomes labeled with FITC-BSA and Rhodamine-BSA were also prepared for FACS and spectral bioimaging studies, respectively.

Isolation and culture of human DCs and fibroblasts culture The study was approved by the Institutional Review Board of H.S. Gour University (HSGU-BMHRC-IRB-17/0407-1A). An informed consent was obtained from all human subjects included in the study. DCs were generated from human peripheral blood monocytes (PBMCs) as reported elsewhere.22,23 PBMCs were isolated by density gradient centrifugation using Ficoll-Plaque plus (GE Healthcare, Amersham, Buckinghamshire, United Kingdom). Cells were plated in six-well tissue culture plates (BD Discovery Labware, Franklin Lakes, New Jersey) at 2 × 106 cells/well in RPMI 1640–10% FCS. After incubation for 1 hour at 37°C in 5% CO2, nonadherent cells were removed by gentle washing and adherent cells were cultured with RPMI 1640–10% FCS enriched with 500 U/ mL GM-CSF and 8 ng/mL IL-4 to generate DCs. The culture medium was replaced on days 3 and 6. NIH3T3 cells (embryonic fibroblast cells) were obtained from American Type Culture Collection (ATCC, Manassas, Virginia) and were grown in Dulbecco's modified Eagle medium with 4 mM l-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 90%; FCS, 10%; 95% air; 5% CO2.

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Figure 1. Kinetics of uptake of ethosomes and elastic liposomes conjugated to FITC-BSA loaded with HBsAg measured by flow cytometry. Cell-associated fluorescent FITC on the FL1-H axis was evaluated to assess intracellular trafficking of the carriers. Represented plots were taken from five experiments, and values are expressed as mean ± SE for M2 zone (uptake). (A) FSC/SSC dot plot showing cluster of DC population. (B) DCs pulsed with soluble HBsAg (2.58% ± 0.79%). (C) Uptake of elastic liposomes by human DCs following 4 hours' incubation (51.20% ± 2.35%). (D) Uptake of ethosomes by human DCs following 4 hours' incubation (84.88% ± 0.68%).

Flow-cytometric analysis for evaluation of kinetics of uptake

Spectral bioimaging analysis for internalization studies

On day 7 of culture, 100 μL FITC-BSA–conjugated ethosomes or elastic liposomes loaded with HBsAg were added to wells. Kinetics of uptake of the system by the cells was studied at intervals of 0, 1, 2, 4, and 8 hours. At the end of each interval the cells were harvested; excess of formulations was removed by washing with ice-cold PBS containing 0.01% sodium azide and 5% FCS, and the cells were resuspended in FACS buffer. Phagocytosis was measured by BD FACSCalibur (BD Biosciences) equipped with an argon ion laser at an excitation wavelength of 488 nm. For each sample, 10,000 events were collected. Cell-associated FITC (FL1-H) was measured by Cell Quest software (BD Biosciences). To further confirm the process of uptake, we also followed a dual-labeling strategy with cells labeled with anti-CD11c monoclonal antibody (mAb)–conjugated FITC to confirm the gating for DCs with Rhodaminelabeled ethosomes or elastic liposomes. Cell-associated FITC (FL1-H) and Rhodamine (FL2-H) was measured simultaneously using Cell Quest software (BD Biosciences). In both cases DCs pulsed with soluble HBsAg and ethosomes or elastic liposomes not loaded with antigen were used for control.14

For spectral bioimaging, following day 7 of culture 2 × 105 cells/300 μL were transferred to LabTek chambered slides (Nalgene Nunc Corp., Rochester, New York). The cells were treated with 100 μL of Rhodamine-BSA conjugated ethosomes and elastic liposomes formulations loaded with HBsAg. After 6 hours the cells were washed with ice-cold PBS containing 0.01% sodium azide and 5% FCS. The cells were then fixed with 4% paraformaldehyde for 30 minutes and finally washed with PBS. Cells without treatment were considered as control and referred to for comparisons. The investigations were carried out by using an upright fluorescence microscope Axioscope 2 plus (Carl Zeiss AG, Oberkochen, Germany) equipped with highpressure mercury lamp (HBO 100) for excitation and triplebandpass filter set. All images were captured using a 100× oil immersion objective lens. In the spectral range from 400 to 700 nm the objective lens has minimal fluctuations ranging from 85% to 90%. The optical head attached to the microscope is composed of a Sagnac common-path interferometer and imaging optics including a cooled charge-coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan). Image acquisition


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Figure 2. Measurement of uptake of ethosomes and elastic liposomes conjugated with Rhodamine-BSA loaded with HBsAg and DCs labeled with CD11c mAbs conjugated with FITC. Cell-associated fluorescence dual-labeled (i.e., positive for both FITC [FL1-H] and Rhodamine [FL2-H]) was considered. Represented plots were taken from five experiments. (A) FSC/SSC dot plot showing cluster of DC population. (B) DCs pulsed with soluble HBsAg expressing CD 11c– FITC. (C) Uptake of elastic liposomes by human DCs following 4 hours' incubation. (D) Uptake of ethosomes by human DCs following 4 hours' incubation.

was performed using Case Data Manager software (Edingen Neckarhausen, Germany), and the spectral analysis was done with Spectral Imaging 4.0 software (Applied Spectral Imaging, Edingen Neckarhausen, Germany). Internalization studies were also conducted in NIH3T3 cells using a protocol similar to that mentioned above except using FITC-BSA–conjugated elastic liposomes and ethosomes loaded with HBsAg. Data presentation was performed with Adobe Photoshop 6.0 (Adobe Systems, San Jose, California).14,24

Evaluation of cell growth, apoptosis, necrosis, and cytotoxicity Three days after culture with the ethosomes and elastic liposomes loaded with HBsAg, cells were seeded into 96-well plates at a high density of 10,000 cells/well. After 24 and 48 hours, cell growth was assessed with Alamar blue assay (Hyclone), which incorporates a colorimetric growth indicator, based on detection of metabolic activity. At the same time points, cells were harvested and an apoptosis/necrosis assay was

performed using Annexin V–FITC/PI apoptosis assay kit (BD Biosciences) according to the manufacturer's recommendations. From each cell, forward light scatter (FSC), orthogonal light scatter (SSC), the FITC (FL1-H), and PI (FL2-H) fluorescence were measured using Cell Quest software (BD Biosciences). The gate was applied in the FSC/SSC dot plot to restrict the analysis to DCs only. For the gated cells the percentage Annexin V–FITC positive or negative or PI positive or negative cells were evaluated. In each case a total of 10,000 events was recorded in HI mode with 10/10 log quadrant gate. Release of lactate dehydrogenase into the culture supernatant, a marker of cytotoxicity, was measured spectrophotometrically.14,25 DCs pulsed with soluble HBsAg and ethosomes or elastic liposomes not loaded with antigen were used for control.

T-cell proliferative response assay Mononuclear cells were isolated from the human peripheral blood by Ficoll density gradient centrifugation. PBMCs

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thus isolated were cultured in RPMI 1640 medium containing autologous serum for 1 hour. The nonadherent cells were removed, and the T cells were purified by nylon wool separation. They were co-cultured in the medium with human DCs pulsed with HBsAg-loaded ethosomes and elastic liposomes in a 10:1 ratio for 5 days. T-cell proliferation was assessed by BrdU incorporation method.14,21 DCs pulsed with soluble HBsAg and DCs pulsed with elastic and/or ethosomes not loaded with antigen were used for control. Following culture the cells were harvested, washed in PBS, and adjusted to a final concentration of 2.0 × 106/mL. Detection of BrdU incorporation was studied following standard labeling protocol with anti-BrdU mAb–FITC conjugated (Santa Cruz Biotechnology, Santa Cruz, California) and dual labeling with anti-CD3 mAb–phycoerythrin conjugated (Chemicon, Temecula, California). Assay was performed on a flow cytometer platform using FSC/SSC gating strategy for identification of the cluster of T cells and FL1/ FL2 for measuring resultant fluorescence emitted from FITC and phycoerythrin channels. A total of 10,000 events were recorded in HI mode with 10/10 log quadrant gate.14

Figure 3. Time course of uptake of ethosomes and elastic liposomes loaded with HBsAg conjugated to FITC by DCs measured via flow cytometry (n = 5).

Multiplex cytometric bead array analysis for secreted TH1/TH2 cytokine profile Supernatants collected from cultures were subjected to measuring TH1/TH2-mediated response by determining levels of TH1 (IL-2, interferon-γ, tumor necrosis factor-α) and TH2 (IL4, IL-6, IL-10) cytokines. BD Cytometric Bead Array (CBA) Human TH1/TH2 Cytokine Kit (BD Biosciences) was used, and the assay was performed as per the manufacturer's instructions. Data acquisition and analysis were carried out on a flowcytometric platform using BD CBA software (BD Biosciences).14

Results Using FACS analysis, cell-associated fluorescence was evaluated to assess intracellular trafficking of the carriers. In the present study, cells were incubated with FITC-BSA– conjugated elastic liposomes or ethosomes loaded with HBsAg and analyzed at different time intervals (0, 1, 2, 4, and 8 hours). A steady increase in the uptake percentage was recorded as well as maximum cell-associated fluorescence for both the systems tested. However, at 4 hours for DCs pulsed with ethosomes loaded with HBsAg, the percentage uptake was 84.88% ± 0.68% in comparison to 51.20% ± 2.35% observed in the case of elastic liposomes. The kinetics of uptake is presented with mean fluorescence intensity recorded in FL1 versus counts by FACS analysis (Figure 1). Confirmation of the findings with anti-CD11c mAb–conjugated FITC to confirm the gating for DC and Rhodamine-BSA–labeled ethosomes or elastic liposomes using dual-labeling strategy also showed similar results (Figure 2). Percentage uptake for both the systems has been graphically represented (Figure 3). The observations on a comparative basis clearly suggest that ethosomal systems possess a better internalizing ability than elastic liposomes.

Besides quantitative determination by FACS analysis, the qualitative uptake study of Rhodamine-BSA–labeled elastic liposomes and ethosomes loaded with HBsAg by human DCs after 4 hours' incubation was carried out using a spectral bioimaging system. Figures 4 and 5 clearly delineate the intracellular delivery efficacy of ethosomes and elastic liposomes loaded with HBsAg to human DCs (Figure 4) and fibroblasts (Figure 5). The results clearly suggest better internalization efficacy of ethosomes than elastic liposomes. Detection of apoptosis and necrosis evaluated by dual staining with Annexin V–FITC and PI in DCs pulsed with elastic liposomes after 48 hours recorded very few changes (4.23% ± 0.35%), whereas in the case of DCs pulsed with ethosomes 20.65% ± 0.72% apoptotic index was observed (Figures 6 and 7). According to the growth curve studied with Alamar blue staining and evaluation of cytotoxicity by measuring lactate dehydrogenase activity in the culture supernatant after 48 hours, recorded values could be well corroborated with cell death studies (data not shown), suggesting that ethosomes are more toxic than elastic liposomes. The ability of DCs pulsed with ethosomes and elastic liposomes loaded with HBsAg to stimulate human T-cell proliferation was assessed by the BrdU incorporation method, and secreted amounts of cytokines produced by T cells were measured by multiplex CBA assay. Co-culture of T cells with pulsed DCs resulted in a higher T-cell proliferation rate comparable to antigen alone (HBsAg) (Figure 8). HBsAgloaded ethosomes and elastic liposomes induced approximately a two- to threefold increase in IL-2 concentrations, four- to fivefold increase in interferon-γ concentrations, and twofold increase in tumor necrosis factor-α levels as compared with control (P b .001) (Figure 9). Concentrations of TH2 cytokines, however, recorded an increase, but the data did not delineate a clear skewing effect. Thus, the significant effect of HBsAg-


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Figure 4. Spectral bioimaging micrographs showing intracellular fluorescence (Rhodamine) and nucleus counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Higher internalizing ability of ethosomes conjugated to Rhodamine-BSA loaded with HBsAg is evident. (A) DCs pulsed with soluble HBsAg. (B) DCs following 4 hours of incubation with elastic liposomes conjugated to Rhodamine-BSA loaded with HBsAg. (C) DCs following 4 hours of incubation with ethosomes conjugated to Rhodamine-BSA loaded with HBsAg.

Figure 5. Spectral bioimaging analysis of NIH3T3 cells showing intracellular fluorescence (FITC) and nucleus counterstained with DAPI. Higher intracellular fluorescence clearly suggests ethosomes as better carrier systems than elastic liposomes. (A) NIH3T3 cells pulsed with HBsAg. (B) NIH3T3 cells following 4 hours of incubation with elastic liposomes conjugated to FITC-BSA loaded with HBsAg. (C) NIH3T3 cells following 4 hours of incubation with ethosomes conjugated to FITC-BSA loaded with HBsAg.

loaded ethosomes and elastic liposomes in inducing a TH1 response could be observed (Figure 9).

Discussion In the present study we have examined and compared the immune response generated by recombinant HBsAg using two nanoparticle-based delivery systems, namely elastic liposomes and ethosomes, and DC as APCs, in vitro. HBsAg-loaded liposomes are clearly superior to soluble HBsAg in terms of their ability to stimulate the T lymphocytes and generation of TH1type immune response. Because the intracellular fate of engineered nanoscale drug delivery systems for determining the initial mode of inter-

nalization and intracellular trafficking by DCs are prerequisites to a vigorous T-cell and cytotoxic T lymphocyte response, we used a combinatorial approach of flow cytometry and spectral bioimaging that addresses all concerns in this regard.14 We observed that HBsAg-loaded ethosomes were rapidly internalized by human DCs, reaching a maximum percentage uptake at 4 hours, which is significantly higher than elastic liposomes. Cytoplasmic localization studies performed using spectral bioimaging in both HBsAg-loaded ethosomes and elastic liposomes–pulsed DCs also show that ethosomes are internalized faster than elastic liposomes by the DCs and fibroblasts as well. These antigen-loaded liposomes were able to stimulate T cells in vitro and induced a TH1-type immune response that was clearly superior to soluble HBsAg. A comparison of elastic liposomes and ethosomes for delivery of

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Figure 6. Flow-cytometric analysis for apoptosis and necrosis in DCs. Represented plots were taken from five experiments. (A) FSC/SSC plot showing the population of DCs; (B) DCs pulsed with soluble HBsAg; (C) following 24 hours of incubation with elastic liposomes loaded with HBsAg; (D) after 48 hours; (E) following 24 hours of incubation with ethosomes loaded with HBsAg; (F) after 48 hours.

HBsAg antigen to the DCs shows that the latter are more potent carriers in terms of internalization by DC and generation of TH1-type responses. However, despite faster internalization into the DC and a stronger TH1-type immune response, ethosomes produced greater toxicity to the DCs as compared with elastic liposomes, as suggested by increased apoptosis. This could be due to the presence of ethanol as a constituent of ethosomes. Whether reducing the concentration of ethanol in the ethosomes can

reduce the amount of apoptosis without affecting their stability or their property of transcutaneous migration must be evaluated. Antibodies to HBsAg are neutralizing and provide lasting protective immunity after immunization and recovery of acute infections. However, patients who spontaneously recover from HBV infection typically mount vigorous multiepitope-specific CD4+ and CD8+ T-cell responses that are readily detectable in blood samples. By contrast, patients with chronic hepatitis B tend to have late, transient, or narrowly focused T-cell responses.


Figure 7. Histogram showing apoptotic index of DCs following flowcytometric analysis using Annexin V–FITC and PI assay. Apoptotic index (%) is the sum of cells positive for Annexin V–FITC (early apoptotic) and positive for both Annexin V–FITC with PI (late apoptotic).

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Figure 9. Histogram showing concentrations of TH1/TH2 cytokines in culture supernatant following multiplex CBA analysis. The results suggest DCs pulsed with ethosomes loaded with HBsAg induce a strong TH1 response in comparison to elastic liposomes pulsed with DCs.

to hold the key for the generation of therapeutic vaccines against hepatitis B for chronic carrier state and in immunesuppressed conditions.

References

Figure 8. T-cell proliferation analysis by BrdU assay. Represented plots were taken from five experiments, and values are expressed as mean ± SE. (A) DCs alone; (B) DCs pulsed with soluble HBsAg; (C) DCs pulsed with elastic liposomes alone (not loaded with antigen); (D) DCs pulsed with ethosomes (not loaded with antigen); (E) DCs pulsed with ethosomes loaded with antigen; (F) DCs pulsed with elastic liposomes loaded with antigen.

A role for T-cell responses in hepatitis B has been proven in the chimpanzee model by the finding that in vivo depletion of either CD4+ or CD8+ T cells prevents viral clearance and clinical recovery.26 Studies on T-cell responses done on patients who successfully recovered from acute hepatitis B infections show a TH1 cytokine profile.27 Our study demonstrates the feasibility and immunogenicity of a DC vaccine pulsed with ethosomes or elastic liposomes loaded with HBsAg, and outlines the potential usefulness of this approach in the generation of DC-based vaccine against HBV. DC-based vaccines using ethosomes as the carrier system have the further potential of being delivered transcutaneously. Vaccines capable of generating potent T-helper responses and in particular those that can augment a TH1 response are thought

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