Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles

Alginate coated chitosan nanoparticles were previously developed with the aim of protecting the antigen, adsorbed on the surface of those chitosan nanoparticles, from enzymatic degradation at mucosal surfaces. In this work, this new delivery system was loaded with the recombinant hepatitis B surface antigen (HBsAg) and applied to mice by the intranasal route. Adjuvant effect of the delivery system was studied by measuring anti-HBsAg IgG in serum, anti-HBsAg sIgA in faeces extracts or nasal and vaginal secretions and interferon-gamma production in supernatants of the spleen cells. The mice were primed with 10 microg of the vaccine associated or not with nanoparticles and associated or not with 10 microg CpG oligodeoxynucleotide (ODN) followed by two sequential boosts at three week intervals. The association of HBsAg with the alginate coated chitosan nanoparticles, administered intranasally to the mice, gave rise to the humoral mucosal immune response. Humoral systemic immune response was not induced by the HBsAg loaded nanoparticles alone. The generation of Th1-biased antigen-specific systemic antibodies, however, was observed when HBsAg loaded nanoparticles were applied together with a second adjuvant, the immunopotentiator, CpG ODN. Moreover, all intranasally vaccinated groups showed higher interferon-gamma production when compared to na?ve mice.     

Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.     

Alginate coated chitosan core shell nanoparticles for oral delivery of enoxaparin: In vitro and in vivo assessment

The objective of present research work was to develop alginate coated chitosan core shell nanoparticles (Alg-CS-NPs) for oral delivery of low molecular weight heparin, enoxaparin. Chitosan nanoparticles (CS-NPs) were synthesized by ionic gelation of chitosan using sodium tripolyphosphate. Core shell nanoparticles were prepared by coating CS-NPs with alginate solution under mild agitation. The Alg-CS-NPs were characterized for surface morphology, surface coating, particle size, polydispersity index, zeta potential, drug loading and entrapment efficiency using SEM, Zeta-sizer, FTIR and DSC techniques. Alginate coating increased the size of optimized chitosan nanoparticles from around 213 nm to about 335 nm as measured by dynamic light scattering in zeta sizer and further confirmed by SEM analysis. The performance of optimized enoxaparin loaded Alg-CS-NPs was evaluated by in vitro drug release studies, in vitro permeation study across intestinal epithelium, in vivo venous thrombosis model, particulate uptake by intestinal epithelium using fluorescence microscopy and pharmacokinetic studies in rats. Coating of alginate over the CS-NPs improved the release profile of enoxaparin from the nanoparticles for successful oral delivery. In vitro permeation studies elucidated that more than 75% enoxaparin permeated across the intestinal epithelium with Alg-CS-NPs. The Alg-CS-NPs significantly increased (p < 0.05) the oral bioavailability of enoxaparin in comparison to plain enoxaparin solution as revealed by threefold increase in AUC of plasma drug concentration time curve and around 60% reduction in thrombus formation in rat venous thrombosis model. The core shell Alg-CS-NPs showed promising potential for oral delivery and significantly enhanced the in vivo oral absorption of enoxaparin.     

Maturation of recombinant hepatitis B virus surface antigen particles

The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immuno-genicity of this widely-used vaccine component.     

Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.     

RhinoVax is an efficient adjuvant in oral immunisation of young chickens and cholera toxin B is an effective oral primer in subcutaneous immunisation with Freund's incomplete adjuvant

Forty-five approximately 50% in-bred 14-day-old White Leghorn female chickens (Gallus domesticus) originating from 11 hens were distributed into 5 treatment groups containing one sister in each treatment group. Phase I involved oral administration of an antigen, Bovine Serum Albumin (BSA), in combination with various adjuvant preparations, either Cholera Toxin B-subunit (CTB) and/or RhinoVax (RV). A positive control group received BSA emulsified in Freund's Incomplete Adjuvant (FIA) by subcutaneous injection. All chickens responded with immunospecific IgA, IgM and IgG antibodies in their circulation. Classical parenteral immunisation with FIA was generally the most potent mode of antigen administration. The highest immunospecific IgG concentrations recorded in the orally-immunised chickens were in the group immunised with 20% RV as the adjuvant. The concentration in this group was approximately 5 times lower than that recorded in the FIA group. For practical egg yolk polyclonal antibody production purposes, the oral regime using 20% RV as adjuvant seems an attractive alternative to the more invasive technique of injecting the antigen in FIA emulsions. In Phase 2 all chickens were subjected to traditional subcutaneous immunisation with a new antigen, human IgG emulsified in FIA. The two groups of chickens that had received CTB orally during Phase I responded with significantly higher immunospecific antibody concentrations than did the other chickens, indicating that oral administration of CTB prior to traditional parenteral immunisation may have a priming effect on the humoral immune system. The immunospecific antibody response varied between the 11 families of chickens. There was no correlation between familial responsiveness to oral and subcutaneous immunisations. Families that were high responders to oral immunisation were not high responders to parenteral immunisation and vice versa.     

Serum hepatitis B surface antigen levels in the natural history of chronic hepatitis B infection

BACKGROUND The production of hepatitis B surface antigen (HBsAg) may evolve during long-lasting virus-host interactions in chronic hepatitis B (CHB). The impact of age on HBsAg production remains unclear. AIM To determine the age-specific distribution patterns of HBsAg and related factors during the natural course of CHB infection. METHODS Seven hundred and sixty-eight untreated HBsAg carriers were enrolled in the study. The parameters and distribution patterns of HBsAg were evaluated in relation to age and immune phases. RESULTS The HBsAg levels were significantly lower in the HBeAg-negative stage, with the lowest levels in inactive carriers. The HBsAg tended to decrease from hepatitis to cirrhosis and to hepatocellular carcinoma, and from Child-Pugh class A to B and to C. Age and HBV DNA were independently associated with HBsAg levels. In HBeAg-positive patients, the HBsAg levels were distributed in a triphasic-like decline pattern by 2 logs across age strata. For HBeAg-negative patients, the titres in inactive carriers exhibited a 2-log reduction, but remained unchanged over age strata in patients with HBeAg-negative hepatitis. The ratios of HBsAg/HBV-DNA were highest, but steadily decreased with age in inactive carriers, whereas the levels remained largely unchanged over the entire age strata in patients with HBeAg-negative hepatitis. CONCLUSIONS Age and HBV DNA levels are independent parameters of HBsAg levels. During the natural course of CHB infection, HBsAg levels decrease with age and disease progression, but the patterns are significantly different between the immune phases of CHB. This information may contribute to our understanding of the immunopathogenesis of chronic hepatitis B and management involving HBsAg quantification.     

新型乙型肝炎病毒表面抗原中蛋白核酸疫苗的免疫原性研究

目的 观察新型乙型肝炎病毒（HBV）表面抗原中蛋白（MHBs)核酸疫苗的免疫原性。方法 以新型人体应用载体质粒pSW3891构建MHBs核酸疫苗（pSW3891/MHBs/adr)，对照组和实验组Balb/c小鼠分别基因枪法免疫对照载体质粒（pSW3891)和MHBs核酸疫苗，采用酶联免疫吸附试验检测抗HBs,LDH释放测定法检测小鼠特异性细胞毒T淋巴细胞（CTL）杀伤活性。结果 MHBs核酸疫苗可在体外高效表达，免疫小鼠后可产生高滴度抗HBs，血清阳性终点滴度达1：97200,小鼠脾细胞HBs特异性细胞毒性T淋巴细胞（CTL）杀伤活性达78.81%。结论 新型MHBs核酸疫苗在Balb/c小鼠实验中表现出良好的体液和细胞免疫原性。     

Viral protein complexed liposomes for intranasal delivery of hepatitis B surface antigen

Nanoparticle-mediated drug delivery represents the future in terms of treating inner ear diseases. Lipid core nanocapsules (LNCs), 50 nm in size, were shown to pass though the round window membrane (RWM) and reached the spiral ganglion cells and nerve fibers, among other cell types in the inner ear. The present study aimed to evaluate the toxicity of the LNCs in vitro and in vivo, utilizing intact round window membrane delivery in rats. The primary cochlear cells and mouse fibroblast cells treated with LNCs displayed dosage dependant toxicity. In vivo study showed that administration of LNCs did not cause hearing loss, nanoparticle application-related cell death, or morphological changes in the inner ear, at up to 28 days of observation. The cochlear neural elements, such as synaptophysin, ribbon synapses, and S-100, were not affected by the administration of LNCs. However, expression of neurofilament-200 decreased in SGCs and in cochlear nerve in osseous spiral lamina canal after LNC delivery, a phenomenon that requires further investigation. LNCs are potential vectors for the delivery of drugs to the inner ear.     

Solution conformation of an immunodominant epitope in the hepatitis B virus preS2 surface antigen

We have determined the solution conformation of the major B cell epitope (residues 123-145, adrl23 hereafter) in the preS2 region of hepatitis B virus known to be associated with infection neutralization. The adrl23 shows an "L" shaped helix-turn-helix topology with two beta-turns formed by residues Ala(130)-Asp(133) and Asp(133)-Val(136) intervening the N- and C-terminal helices. The N-terminal alpha-helix consists of residues Ser(124)-Gln(129) whereas the C-terminal 3(10) helix is formed by residues Val(136)-Tyr(140). The beta-turns overlap partially with the putative "conformational" epitope. The overall topology of adrl23 is primarily maintained by hydrophobic interactions involving Phe(127), Leu(131), Leu(132), Val(136), and Tyr(140) that are clustered on one side of the molecule. An additional hydrophobic stabilization comes from Phe(141) that is buried inside the concave side of the molecule. A network of hydrogen bonds formed among Thr(125), His(128), and Arg(137) further contribute to the "boomerang-shaped" architecture of adrl23. The N-terminus of adrl23 is immobile due to a hydrogen bond between the N-terminal amide proton of Asn(123) and the hydroxyl oxygen of Thr(126). The side chains of Asp(133), Arg(135), Val(136), Leu(139), and Tyr(140) that were shown to be important for binding to a monoclonal antibody H8 mAb are surface exposed.     

Oral hepatitis B vaccine: chitosan or glucan based delivery systems for efficient HBsAg immunization following subcutaneous priming

The World Health Organization encourages “the development of oral formulations to simplify their transport, storage and administration in poor countries”, and to “facilitate an effective immunization program to prevent sexually transmitted hepatitis B”. Thus, two distinct and promising delivery systems were developed: recombinant hepatitis B surface antigen (HBsAg) encapsulated into alginate-coated chitosan particles (AlgChiPs) and into glucan particles (GPs) mainly composed of β-1,3-d-glucan. In vitro preliminary studies showed that both could be internalized by peripheral blood mononuclear cells and murine Peyer’s patches, an imperative aspect regarding oral immunization. Chitosan particles (ChiPs) have shown interesting immunostimulating properties as mast cells activators. Vaccination studies reveal that three oral immunizations induced serum anti-HBsAg Immunoglobulin G (IgG) in 60 % of the animals and anti-HBsAg secretory IgA in faeces for both formulations. When subcutaneous (SC) priming was done, followed by two oral boosts, all mice were responder and much higher serum anti-HBsAg IgG titers were observed, besides mucosal protective immunity.     

抗变异乙型肝炎表面抗原单克隆抗体与8种乙型肝炎检测试剂盒检测效果的比较

目的评价本实验室制备的抗变异乙型肝炎(乙肝)表面抗原单克隆抗体检测野生及变异表面抗原的能力。方法用酶联免疫吸附试验(ELISA)评价本实验室制备的抗变异乙肝表面抗原的单克隆抗体,以及国内常用的8种乙肝检测试剂盒,对3种野生乙肝表面抗原和15种变异乙肝表面抗原的检测效果。结果本实验室制备的抗乙肝变异表面抗原单克隆抗体对变异表面抗原的检测能力明显优于国内常用检测试剂盒。结论该单克隆抗体为进一步研制高敏感性乙肝检测试剂盒提供了前提。     

Hepatitis B surface antigen in notified cases of viral hepatitis.

Eighty-five patients notified to the local medical officer of health as infectious or serum hepatitis during the period 1 September 1974-1 September 1975 were investigated by questionnaire. Serum from 73 cases was tested for the presence of HBsAg. Sixteen cases were notified as serum hepatitis but only 13 were shown to have HBsAg in their serum. Sixty-eight cases were notified as infectious hepatitis and 21 HBsAg positive cases were found amongst the 57 sera tested. Two-thirds of the HBsAg positive cases were not suspected clinically. The epidemiology of hepatitis in Christchurch is discussed and suggestion is made that all notified cases of hepatitis should have blood tested for HBsAg.     

T-cell responses to hepatitis B surface antigen in lung transplant recipients

STUDY OBJECTIVE: To determine whether lung transplant recipients would have a less vigorous T-cell response to hepatitis B surface antigen (HBsAg) than that of patients awaiting lung transplantation and healthy subjects, we sought to measure and compare T-cell responses among these three groups. DESIGN: Prospective study. SETTING: Lung transplant clinic at a university hospital. SUBJECTS: Twelve lung transplant recipients, 12 patients awaiting lung transplantation, and 15 healthy subjects. All participants had received the hepatitis B vaccine series and had a documented antibody response to it. INTERVENTION: Blood samples were obtained from each participant. MEASUREMENTS AND MAIN RESULTS: Participants' sex, age, time since lung transplantation (if applicable), and time since hepatitis B immunization were recorded. Peripheral blood mononuclear cells were isolated from the participants' blood samples for the trans vivo delayed-type hypersensitivity (DTH) assay. These cells were mixed with saline, tetanus toxoid, or HBsAg and injected into the footpads of immunodeficient mice. Resultant swelling of the footpad was used as an index of human T-cell response. The healthy subjects were younger than the patients in both transplant groups. However, we found no significant difference in DTH response elicited by HBsAg among the healthy subjects, patients awaiting lung transplantation, and lung transplant recipients (mean +/- standard error [SE] 34.7 +/- 4.3, 32.1 +/- 3.1, and 33.5 +/- 4.0 x 10(-4) in., respectively, p>0.8) or when tetanus toxoid was used as a positive control (15.7 +/- 2.8, 22.8 +/- 6.5, and 21.7 +/- 3.9 x 10(-4) in., respectively, p>0.3). No correlation between age or time since immunization and DTH response was noted. CONCLUSION: Lung transplant recipients maintained a T-cell response to HBsAg that was similar in vigor to that of both patients awaiting transplantation and healthy subjects even though their antibody concentrations waned rapidly after transplantation. The role of these T cells as a correlate of protection from infection remains to be investigated.     

Metallothionein overexpression suppresses hepatic hyperplasia induced by hepatitis B surface antigen

Transgenic mice that express the viral coat proteins of hepatitis B virus (HBV) in the liver display hepatocellular damage, inflammation, regeneration, hyperplasia, and, eventually, neoplasia that is similar to that of people with chronic, active hepatitis caused by HBV infection. Hepatocellular regeneration, in the context of chronic injury and inflammation, is thought to expose dividing cells to excessive oxygen radicals, which are believed to lead to DNA damage and, ultimately, neoplasia. Because metallothioneins scavenge free radicals in vitro, we generated mice that express excess (>10-fold) metallothionein I (MT-I* mice) and the HBV surface antigens (HBsAg) to ascertain whether MT-I* would ameliorate aspects of the pathology induced by HBsAg. Markers of hepatocyte injury and tumorigenesis in HBsAg mice were compared to those in double transgenic (HBsAg and MT-I*) mice. Hepatic hyperplasia, histology, aneuploidy, and accumulation of an oxidative DNA adduct, 8-oxo-2'-deoxyguanosine, were examined. Although hepatitis and neoplasia were not prevented by MT-I* expression in the HBsAg mice, there was less hyperplasia and less aneuploidy. We conclude that MT-I produces a beneficial effect in this in vivo model of HBV-induced hepatitis.