The Role of Hepatitis B Surface Antigen in Nucleos(t)ide Analogues Cessation Among Asian Patients With Chronic Hepatitis B: A Systematic Review.

In actual clinical practice, infinite nucleos(t)ide analogues (NAs) treatment for chronic hepatitis B virus (HBV) infection is unrealistic. The most commonly used endpoint is suppression of HBV DNA to undetectable levels with normalization of alanine aminotransferase. However, this criterion for cessation of treatment is associated with various incidences of virological and clinical relapse. Recent studies suggest that decreasing the hepatitis B surface antigen (HBsAg) level at the end of treatment (EOT) to an appropriate cut-off value appears to be a practicable and attainable cessation criterion. We performed a systematic review to explore the optimal cut-off value of HBsAg at EOT for the cessation of NAs treatment. Eleven studies with 1,716 patients were included in this review. When the HBsAg levels at EOT were <100 IU/mL and >100 IU/mL, the respective off-therapy virological relapse rates were 9.1%-19.6% (range) and 31.4%-86.8% (range) at ≥12 months off therapy, regardless of hepatitis B e antigen (HBeAg) status; the respective off-therapy clinical relapse rates were 15.4%-29.4% (range) and 48.1%-63.6% (range) at ≥12 months off therapy, regardless of HBeAg status; and the respective off-therapy HBsAg loss rates were 21.1%-58.8% (range) and 3.3%-7.4% (range) for HBeAg-negative patients at ≥39 months off therapy. Conclusion: Cessation of long-term NAs therapy before HBsAg seroclearance in patients with chronic hepatitis B is a feasible alternative to indefinite treatment. An HBsAg level <100 IU/mL at EOT seems to be a useful marker for deciding when to discontinue NAs therapy. However, regular monitoring is required after the cessation of NAs treatment, and long-term outcomes must be further evaluated.

Mannose-Modified PLGA Nanoparticles for Sustained and Targeted Delivery in Hepatitis B Virus Immunoprophylaxis.

The launched hepatitis B vaccine could induce powerful antibodies, whereas it failed to improve potent cellular immune responses due to that the Th2-type response-induced aluminum adjuvant was adopted. Here, to target antigen-presenting cells under the epidermis and induce potent cellular and humoral immune responses, mannose-modified poly D,L-lactide-co-glycolic acid (PLGA) was synthesized and nanoparticle (MNP)-loaded hepatitis B surface antigen (HBsAg) protein was prepared. HBsAg could be slowly released and highly presented to lymphocytes which facilitated to produce long-lasting immunity based on characters of PLGA. In vitro uptake test results showed that MNPs could enhance internalization in bone marrow-derived dendritic cells (BMDCs) and RAW 264.7 cells. Subcutaneous delivery of MNPs into mice kept humoral immune and strengthened cellular immune responses. Experimental results indicated that MNPs showed significantly modified properties compared with parental PLGA nanoparticles. Thus, the obtained MNPs could be a promising vehicle for hepatitis B vaccine delivery.

The adjuvant system AS01 up-regulates neutrophil CD14 expression and neutrophil-associated antigen transport in the local lymphatic network.

The liposome-based adjuvant system AS01 is under evaluation for use in several vaccines in clinical development. We have shown previously that AS01 injected with hepatitis B surface antigen (HBsAg) induces a distinct cellular signature within the draining lymphatics that enhances local lymphocyte recruitment and antigen-specific humoral immunity. Here, we show that AS01-induced neutrophil recruitment is associated with increased expression of CD14 and enhanced antigen uptake capacity in neutrophils from both afferent and efferent lymphatic compartments during the first 48 h after vaccination. Significant and transient increases in CD14 expression on systemic neutrophils were also observed following primary and boost vaccination with HBsAg-AS01; however, they were not observed following additional encounter with HBsAg-alone or HBsAg-alum. These results show that following immunization with AS01, neutrophils expressing higher levels of CD14 are both more abundant and efficient at antigen uptake, warranting further investigation into the role of neutrophil-associated CD14 in the adjuvanticity of AS01.

Protamine Nanocapsules for the Development of Thermostable Adjuvanted Nanovaccines.

One of the main challenges in the development of vaccine has been to improve their stability at room temperature and eliminate the limitations associated with the cold chain storage. In this paper, we describe the development and optimization of thermostable nanocarriers consisting of an oily core with immunostimulating activity, containing squalene or α tocopherol surrounded by a protamine shell. The results showed that these nanocapsules can efficiently associate the recombinant hepatitis B surface antigen (rHBsAg) without compromising its antigenicity. Furthermore, the freeze-dried protamine nanocapsules were able to preserve the integrity and bioactivity of the associated antigen upon storage for at least 12 months at room temperature. In vitro studies evidenced the high internalization of the nanocapsules by immunocompetent cells, followed by cytokine secretion and complement activation. In vivo studies showed the capacity of rHBsAg-loaded nanocapsules to elicit protective levels upon intramuscular or intranasal administration to mice. Overall, our data indicate that protamine nanocapsules are an innovative thermostable nanovaccine platform for improved antigen delivery.

Potential Hepatitis B Vaccine Formulation Prepared by Uniform-Sized Lipid Hybrid PLA Microparticles with Adsorbed Hepatitis B Surface Antigen.

For the purpose of strengthening the immunogenicity of the hepatitis B vaccine, which contains hepatitis B surface antigen (HBsAg), the development of biodegradable poly(lactic acid) (PLA) microparticles (MPs) modified with the cationic surfactant didodecyldimethylammonium bromide (DDAB) was attempted. DDAB-PLA MPs with an uniform size of about 1 µm were prepared in a simple and mild way. DDAB-PLA MPs with increased surface charge enhanced antigen adsorption capacity compared to plain PLA MPs. After immunization, DDAB-PLA MPs induced the gene expression of inflammatory cytokines and chemokines, which facilitated the following immune responses. DDAB-PLA MPs augmented the expression of co-stimulatory molecules along with the activation of bone-marrow-derived dendritic cells (BMDCs). DDAB-PLA MP-based vaccine formulations efficiently induced antibody production more than the aluminum-based vaccine and plain PLA MP-based formulation in vivo. Moreover, DDAB-PLA MPs were more likely to generate the polarization of the Th1 response indicating the cytotoxic ability against infectious pathogens. In conclusion, DDAB-PLA MPs could be a potent vaccine formulation to prime robust cellular and humoral immune responses.

The Lymphatic Immune Response Induced by the Adjuvant AS01: A Comparison of Intramuscular and Subcutaneous Immunization Routes.

The liposome-based adjuvant AS01 incorporates two immune stimulants, 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01 is under investigation for use in several vaccines in clinical development. i.m. injection of AS01 enhances immune cell activation and dendritic cell (DC) Ag presentation in the local muscle-draining lymph node. However, cellular and Ag trafficking in the lymphatic vessels that connect an i.m. injection site with the local lymph node has not been investigated. The objectives of this study were: 1) to quantify the in vivo cellular immune response induced by AS01 in an outbred ovine model, 2) to develop a lymphatic cannulation model that directly collects lymphatic fluid draining the muscle, and 3) to investigate the function of immune cells entering and exiting the lymphatic compartments after s.c. or i.m. vaccination with AS01 administered with hepatitis B surface Ag (HBsAg). We show that HBsAg-AS01 induces a distinct immunogenic cellular signature within the blood and draining lymphatics following both immunization routes. We reveal that MHCII(high) migratory DCs, neutrophils, and monocytes can acquire Ag within muscle and s.c. afferent lymph, and that HBsAg-AS01 uniquely induces the selective migration of Ag-positive neutrophils, monocytes, and an MHCII(high) DC-like cell type out of the lymph node via the efferent lymphatics that may enhance Ag-specific immunity. We report the characterization of the immune response in the lymphatic network after i.m. and s.c. injection of a clinically relevant vaccine, all in real time using a dose and volume comparable with that administered in humans.

Lipid A adjuvanted Chylomicron Mimicking Solid Fat Nanoemulsions for Immunization Against Hepatitis B.

Traditional parenteral recombinant hepatitis B virus (HBV) vaccines have effectively reduced the disease burden despite being able to induce seroprotective antibody titers in 5-10% vaccinated individuals (non-responders). Moreover, an estimated 340 million chronic HBV cases are in need of treatment. Development of safe, stable, and more effective hepatitis B vaccine formulation would address these challenges. Recombinant hepatitis B surface antigen (rHBsAg) entrapped solid fat nanoemulsions (SFNs) containing monophosphoryl lipid A (MPLA) that was prepared and optimized by quality by design (QbD) using response surface methodology (RSM), i.e., central composite design (CCD). Its immune potential was evaluated with preset immunization protocol in a murine model. Dose escalation study revealed that formulation containing 1 µg of rHBsAg entrapped SFNs with MPLA-induced significant higher humoral, and cellular response compared to the marketed vaccine (Genvac B) administered intramuscularly. SFNs with nanometric morphology and structural similarity with chylomicrons assist in improved uptake and processing to lymphatics. Moreover, the presence of an immunogenic component in its structure further augments delivery of rHBsAg to immune cells with induction of danger signals. This multi-adjuvant based approach explores new prospect for the dose sparing. Improved cellular immune response induced by this vaccine formulation suggests that it could be tested as an immunotherapeutic vaccine as well.

Induction of humoral and cellular immune response to hepatitis B virus (HBV) vaccine can be upregulated by CpG oligonucleotides complexed with Dectin-1 ligand.

A persistent hepatitis B virus (HBV) infection is characterized by a lack of or a weak immune response to HBV, which may be reflective of tolerance to HBV. Efficient induction of HBV-specific immune response leads to the clearance of HBV in patients with a chronic HBV infection. CpG oligodeoxynucleotides (ODN) has a powerful adjuvant effect in HBV vaccination. A recent report demonstrated that the immunization by B/K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand, schizophyllan (SPG), namely K3-SPG, was more effective in the induction of antigen-specific immune response than that by K3. In this study, we examined the efficacy of K3-SPG as a HBV vaccine adjuvant. Wild-type (WT) mice and HBV transgenic (HBV-Tg) mice were subcutaneously immunized with hepatitis B surface antigen (HBsAg) alone, HBsAg and K3, or HBsAg and K3-SPG. The vaccination with HBsAg and K3-SPG significantly enhanced humoral and cellular immune response to HBV antigen compared to the other vaccinations in WT and HBV-Tg mice. K3-SPG induced the accumulation of dendritic cells (DCs) into draining lymph node and the activation of DCs. The expression of cytokines and chemokines related to Th1 and Th2 responses was upregulated after the vaccination including with K3-SPG. In conclusion, these results indicated that the vaccination using K3-SPG may overcome tolerance even in patients with chronic HBV infection.

Vitamin B12 Grafted Layer-by-Layer Liposomes Bearing HBsAg Facilitate Oral Immunization: Effect of Modulated Biomechanical Properties.

Adhesion forces of nanoparticulate materials toward biological membrane are crucial for designing a delivery system for therapeutic molecules and vaccines. The present study aims to investigate the impact of surface roughness of the nanoparticulate system in oral delivery of antigen and its targeting to toward intestinal antigen presenting cells. To evaluate this hypothesis, layer-by-layer coated liposomes (LBL-Lipo) were fabricated using sodium alginate and Vitamin B12 conjugated Chitosan (VitB12-Chi) as anionic and cationic polyelectrolyte, respectively. Change in surface roughness was observed on changes in pH from gastric to intestinal conditions attributed to increase and decrease in charge density on VitB12-Chi. Surface roughness was measured in terms of root-mean-square measured by topographical analysis using atomic force microscopy. LBL-Lipo were further characterized for their size, zeta potential, and release behavior to evaluate the potential for oral vaccine delivery. In vitro cell uptake in macrophage cells (J-744) shows about 2- and 3.1-fold increased uptake of rough LBL-Lipo over smooth LBL-Lipo at 37 °C (endocytosis) and 4 °C (endocytosis inhibition) indicating improved biological interaction. Further in vivo immunization study revealed that prototype formulations were able to produce 4.8- and 3.3-fold higher IgG and IgA levels in serum and feces, respectively, in comparison to smooth LBL-Lipo.

Enhanced immune response to hepatitis B vaccination through immunization with a Pre-S1/Pre-S2/S vaccine.

Efficacy and safety of recombinant yeast-derived hepatitis B vaccines for prevention of hepatitis B have been demonstrated unequivocally worldwide as reflected in reduction in HBsAg carrier rates and hepatocellular carcinoma. A new generation of recombinant HBV vaccines expressed in mammalian cells containing Pre-S/S epitopes has been developed in several countries. Such vaccines are useful in special risk groups, i.e., in non-responders to conventional HBV vaccines including older adults, obese people, health care workers, patients with renal failure and on dialysis, transplant patients, patients with HIV as well as travelers on short notice to HBV endemic regions. The future of such vaccines depends on their enhanced immunogenicity and cost profile. Sci-B-Vac™ is a mammalian cell-derived recombinant Pre-S1/Pre-S2/S hepatitis B vaccine which has been shown to be highly immunogenic, inducing faster and higher seroprotection rates against HBV with higher anti-HBs levels at lower HBsAg doses as compared to conventional yeast-derived vaccines. Recently, it has been suggested that such Pre-S/S vaccines against HBV might be efficacious not only for prevention but also for intervention in persistent HBV infection. Data obtained in a recent clinical trial conducted in Vietnam in patients with chronic hepatitis B suggest that repeated monthly i.m. injections of the Sci-B-Vac™ co-administered with daily oral lamivudine treatment can suppress HBV replication and lead to anti-HBs seroconversion in ~50 % of treated patients. Optimization of protocols and efficacy of such an intervention, intended to bypass T cell exhaustion and immune tolerance to HBV remains to be explored.

Aluminum oxyhydroxide-Poly(I:C) combination adjuvant with balanced immunostimulatory potentials for prophylactic vaccines.

The development of high-purity antigens promotes the urgent need of novel adjuvant with the capability to trigger high levels of immune response. Polyinosinic-polycytidylic (Poly(I:C)) is a synthetic double-stranded RNA (dsRNA) that can engage Toll-like receptor 3 (TLR3) to initiate immune responses. However, the Poly(I:C)-induced toxicity and inefficient delivery prevent its applications. In our study, combination adjuvants are formulated by aluminum oxyhydroxide nanorods (AlOOH NRs) and Poly(I:C), named Al-Poly(I:C), and the covalent interaction between the two components is further demonstrated. Al-Poly(I:C) mediates enhanced humoral and cellular immune responses in three antigen models, i.e., HBsAg virus-like particles (VLPs), human papilloma virus (HPV) VLPs and varicella-zoster virus (VZV) glycoprotein E (gE). Further mechanistic studies demonstrate that the dose and molecular weight (MW) of Poly(I:C) determine the physicochemical properties and adjuvanticity of the Al-Poly(I:C) combination adjuvants. Al-Poly(I:C) with higher Poly(I:C) dose promotes antigen-bearing dendritic cells (DCs) recruitment and B cells proliferation in lymph nodes. Al-Poly(I:C) formulated with higher MW Poly(I:C) induces higher activation of helper T cells, B cells, and CTLs. This study demonstrates that Al-Poly(I:C) potentiates the humoral and cellular responses in vaccine formulations. It offers insights for adjuvant design to meet the formulation requirements in both prophylactic and therapeutic vaccines.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings.

BACKGROUND & AIMS: Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. METHODS: Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. RESULTS: In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). CONCLUSIONS: Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.

Evidence for cyclic diguanylate as a vaccine adjuvant with novel immunostimulatory activities.

Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-γ, TNF-α and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization.

Clinical characteristics of hepatitis B virus infection in middle school students born after the universal infant vaccination program in Shanghai, China.

The aim of this study was to investigate the prevalence of hepatitis B surface antigen (HBsAg) and clinical characteristics of middle school students infected with hepatitis B virus (HBV) after initiation of the HBV immunization program in China. A total of 82,156 serum samples were collected from students in 33 junior schools and 25 senior schools. HBsAg was tested by ELISA. Samples from HBsAg-positive students were collected and analyzed for HBV serum markers, alanine aminotransferase (ALT), HBV DNA levels, and HBV genotypes. The overall prevalence of HBsAg was 1.11% in middle school students in Shanghai, China. The prevalence of HBsAg in students born during the immunization program to HBsAg-positive mothers was significantly higher than that in students born during the universal vaccination program (1.47% vs 0.78%, P < 0.01). Only HBV genotypes B and C were found in these infections, and genotype C was the dominant one. Twenty-one (13.0%) of 162 HBsAg-positive students had active hepatitis B, and 18 were hepatitis B e antigen (HBeAg) positive. The universal infant vaccination program has reduced the prevalence of HBsAg significantly. HBeAg-positive hepatitis B, however, needs to be monitored among the students in whom vaccination failed.

Hydrogel-delivered GM-CSF overcomes nonresponsiveness to hepatitis B vaccine through the recruitment and activation of dendritic cells.

The standard hepatitis B surface Ag (HBsAg) vaccine fails to induce anti-hepatitis B surface Abs in 5-10% of healthy subjects, a phenomenon known as HBsAg nonresponsiveness, which is closely related to HLA class II alleles and impaired Th cell responses to HBsAg in these subjects. We hypothesized that GM-CSF, a potent adjuvant in enhancing the Ag-presentation activity of APCs, might help to generate Th cell responses in nonresponders, subsequently providing help for B cells to produce anti-hepatitis B surface Abs. We used a thermosensitive biodegradable copolymer (hydrogel) system to codeliver HBsAg and GM-CSF to achieve maximal local cytokine activity at the injection site. In responder mouse strains, hydrogel-formulated HBsAg plus GM-CSF (Gel/HBs+GM) vaccine elicited much greater anti-hepatitis B surface Ab titers and Th cell proliferative responses than a commercial aluminum-formulated HBsAg vaccine or free HBsAg. The adjuvant effect of the Gel/HBs+GM vaccine was dependent upon the local release of GM-CSF. More importantly, the Gel/HBs+GM vaccine elicited high HBsAg-specific Ab titers and Th cell responses in B10.M mice, a mouse strain that does not respond to the current HBsAg vaccine because of its H-2 haplotype. Analysis of the draining lymph nodes of Gel/HBs+GM vaccine-treated mice revealed an elevated number of CD11c(+) dendritic cells showing enhanced expression of MHC class II and a variety of costimulatory molecules. These results demonstrate that hydrogel-formulated GM-CSF might represent a simple and effective method to generate next-generation hepatitis B virus vaccines for inducing anti-hepatitis B surface Abs in nonresponders.

Formulation, characterization and optimization of hepatitis B surface antigen (HBsAg)-loaded chitosan microspheres for oral delivery.

CONTEXT: Approximately 400 million persons worldwide have chronic hepatitis B. This is due to problems associated with vaccine delivery, stability and cost. Hence the present challenge in vaccinology is to develop safer, cheaper and easy-to-deliver forms of vaccines. A novel needle-free oral vaccine will be an ideal tool to fight this silent killer disease. OBJECTIVE: The aim of this work was to prepare and evaluate chitosan-loaded HBsAg microspheres for oral delivery. MATERIALS AND METHODS: Chitosan microspheres were prepared by emulsion solvent evaporation technique. To overcome the enzymatic and permeation barrier, protease inhibitors and permeation enhancers were also added. Studies were conducted to find the effect of stabilizer concentration, stirring speed, cross-linking agent and polymer concentration on microsphere size and entrapment efficiency. Formulations were characterized for their particle size, entrapment efficiency. They were also evaluated for the in vitro drug release, in vivo performances and the effect of different storage conditions. RESULTS: HBsAg-loaded chitosan microspheres with bacitracin as protease inhibitor showed better protective levels of immunity after oral administration comparing with aprotinin as protease inhibitor. Stability at room temperature up to a period of four months reduces incomplete vaccine coverage and logistic requirements. CONCLUSION: The study signifies the potential of the formulated chitosan microspheres for effective oral administration of HBsAg.

"Prevention of structural perturbation and aggregation of hepatitis B surface antigen: screening of various additives".

The instability of protein and antigen(s) during encapsulation in biodegradable polymers by water-in-oil-in-water (w/o/w) encapsulation is well established. The aim of present study is to screen various additives to prevent the inactivation and loss of immunogenicity of HBsAg upon its exposure to the water/CH(2)Cl(2) (methylene chloride) interface by simulating the formulation steps involved in the preparation of microspheres. The secondary structure of HBsAg, recovered under different conditions after primary emulsification, was investigated by FTIR spectroscopy and Circular Dichorism. Subsequently, PLGA microspheres were formulated and characterized for their size, shape, incorporation efficiency, antigen integrity, and immunogenicity. The immunogenicity and the HBsAg recovery under different conditions were tested in BALB/c mice. Inulin and trehalose were found to be better stabilizing agents to prevent the aggregation, the structural perturbations and immunogenicity of HBsAg. This study substantiated that inulin could overcome the aggregation and denaturing effects of the water/CH(2)Cl(2) interface upon HBsAg during emulsification step and upon encapsulation.

Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B.

The immune modulator capacity of antigen-pulsed dendritic cells (DC) has been documented in patients with cancers and in animal models of chronic viral infections. Cancer antigen-pulsed DC are now used for treating patients with cancer. But viral antigen-pulsed DC are not used in chronic viral-infected patients because safety of antigen-pulsed DC has not been evaluated in these patients. DC were isolated from human peripheral blood mononuclear cells by culturing with human-grade granulocyte-macrophage colony stimulating factor and interleukin-4. Human blood DC were cultured with hepatitis B surface antigen (HBsAg) for 8h to prepare HBsAg-pulsed DC. After immunogenicity assessment of HBsAg-pulsed DC in vitro, five million HBsAg-pulsed DC were administered intradermally to five patients with chronic hepatitis B (CHB) 1-3 times. HBsAg-pulsed DC were immunogenic in nature because they produced significantly higher levels of interleukin-12 and interferon-γ compared to unpulsed DC (P<0.05). Also, HBsAg-pulsed DC induced proliferation of HBsAg-specific T lymphocytes in vitro. CHB patients injected with HBsAg-pulsed DC did not exhibit generalized inflammation, exacerbation of liver damage, abnormal kidney function, or features of autoimmunity. Administration of HBsAg-pulsed DC induced anti-HBs in two patients and HBsAg-specific cellular immunity in 1 patient. This is the first study about preparation of antigen-pulsed DC using human consumable materials for treating patients with CHB. Because HBsAg-pulsed DC were safe for all patients with CHB and had immune modulation capacity in some patients, phase I and phase II clinical trials with antigen-pulsed DC in CHB and other chronic infections are warranted.

Enhanced specific immune responses by CpG DNA in mice immunized with recombinant hepatitis B surface antigen and HB vaccine.

BACKGROUND: Hepatitis B vaccine adjuvant, alum, is generally used for vaccination although it does not stimulate Th1 immunity and 10% of the population has low or no antibody response. Efforts have been continued to find more efficient vaccine adjuvants for better antibody response as well as stimulation of Th1 immunity. METHODS: CpG DNA was used as an adjuvant for recombinant HBsAg to immunize 6- to 8-week-old female BALB/c mice with or without alum for different dosages. The production of HBsAb, CD80 and CD86 from dendritic cells, and cytokines IL-10, IL12, etc., were analyzed and compared for the performance of immunization. RESULTS: 5-20 µg CpG DNA had the best co-stimulation effect of HBsAb serum conversion for mice vaccinated with recombinant expressed HBsAg. The mice vaccinated with recombinant 20 µg CpG DNA and regular vaccine (containing alum adjuvant) had the highest concentration of antibody production. IL-12b, IL-12a and IL10 mRNA reached to the peak level between 3 and 6 hours after the CpG DNA induction in splenocytes. The expression levels of CD80 and CD86 leucocyte surface molecules were increased with 20 µg CpG DNA alone or with 20 µg CpG DNA and 4 µg HBsAg. CONCLUSIONS: Our results confirmed the adjuvant effect of CpG DNA for HBsAg in the mouse model. The increase of IL10 and IL12 production suggested the involvement of Th1 cell activation. The activation of CD80 and CD86 molecules by CpG-ODN might be part of the mechanism of T/B cells coordination and the enhancement of recombinant HBsAg induced immune response.

Aerosolized PLA and PLGA nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis B vaccine.

Porous poly(L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) nanoparticles were tested for pulmonary delivery of hepatitis B vaccine. In particular, the effects of particle size and hydrophobicity on mucosal and cell-mediated immune responses were investigated. Three formulations of PLA and PLGA nanoparticles containing a fixed amount of hepatitis B surface antigen (HBsAg) were prepared by a double-emulsion-solvent-evaporation method and characterized for surface morphology, charge, size, density and in vitro release. The immune responses were studied by measuring secretory IgA levels in mucosal fluids and quantitating cytokine levels in rat spleen homogenates. Particle uptake was studied in rat alveolar macrophages. Scanning electron microscopy revealed particles with smooth surfaces. Zeta potential measurements indicated that the particles carried negative surface charges. The antigen was continuously released for 42 days in phosphate buffer. Hydrophobic particles >500 nm elicited a more robust increase in secretary IgA, interleukin-2 and interferon-γ levels compared to hydrophilic particles <500 nm. Large hydrophobic particles were more efficiently internalized by rat alveolar macrophages compared to smaller hydrophilic particles. Calu-3 cell viability studies indicate that the viability of cells is not affected by nanoparticulate formulations. This study demonstrates that inhalable nanoparticles of HBsAg produce an enhancement of immune responses.