Hepatitis B Surface Antigen In Mice Research Articles

Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose : To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods : The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results : In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion : A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses. Keywords : Hepatitis B virus, Plasmid DNA, Vaccine, Spleen cytokines, Humoral and cellular immune responses

Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg.

Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dot-blot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters.

A surfactin lipopeptide adjuvanted hepatitis B vaccines elicit enhanced humoral and cellular immune responses in mice.

WH1fungin (WF), a lipopeptide surfactin, has been verified as an immunoadjuvant previously. In this study, mice were intranasally or parenterally immunized with WF plus Hepatitis B surface antigen (HBsAg), then the immune responses were detected. The results showed 50 µg WF plus 20 μg HBsAg for intranasal and 10 μg WF plus 1 µg HBsAg for parenteral immunization was efficient inducing strong immune response against HBsAg in mice. A high titer and longterm anti-HBsAg IgG was observed for more than 19 weeks in intranasal or parenteral immunizations, much higher than that induced by CpG or Alum adjuvant. The anti-HBsAg IgA was also induced in intestine and lung, indicating that mucosal as well as systemic immune response was elicited for intranasal immunization. The IgG isotype in serum revealed WF induced a Th2-bias immune response with a higher titer of anti-HBsAg IgG1 than IgG2a in mice. Moreover, WF also induced more Th1 cells producing interferon (IFN)-γ and stronger cytotoxic T lymphocytes response than controls for intramuscular administration. These data further confirmed that WF induced Th1- as well as Th2- type immune response toward HBsAg. Taken together, WF-adjuvanted HBsAg elicits more effective immune response than that adjuvanted by Alum or CpG, suggesting its potential for development of more efficient HBV vaccines in the future.

Cationic lipid-formulated DNA vaccine against hepatitis B virus: immunogenicity of MIDGE-Th1 vectors encoding small and large surface antigen in comparison to a licensed protein vaccine.

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.

Screening and appraisal for immunological adjuvant-active fractions from Platycodon grandiflorum total saponins

In this study, the total saponins from the root of Platycodon grandiflorum (PGSt) was subjected to D101 macroreticular resin column chromatography to afford four fractions (PGS30, PGS50, PGS75 and PGS95). PGSt and its four fractions were evaluated and compared for the haemolytic activities and adjuvant potentials on the specific cellular and humoral immune responses of ICR mice against recombinant hepatitis B surface antigen (HBsAg). PGSt, PGS30, PGS50, PGS75, and PGS75 showed a slight haemolytic effect, with their concentration inducing 50% of the maximum haemolysis (HD50) being 16.13 ± 0.81, >200, 17.53 ± 0.24, 20.16 ± 0.76, 76.31 ± 2.20 μg/mL against 0.5% rabbit red blood cell, respectively. PGSt, PGS50, and PGS75 significantly not only enhanced the Con A-, lipopolysaccharide-, and HBsAg-induced splenocyte proliferation, but promoted the killing activities of natural killer (NK) cells from splenocytes in HBsAg-immunized mice (P < 0.01 or P < 0.001). HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody levels in serum were also significantly enhanced by PGSt, PGS50, and PGS75 compared with HBsAg control group (P < 0.05, P < 0.01, or P < 0.001). Moreover, the adjuvant effects of PGS50 and PGS75 on the cellular immune responses and HBsAg-specific IgG2a and IgG2b antibody responses were more significant than those of Alum, PGS30, and PGS95. The results indicated that PGS50 and PGS75 could improve both cellular and humoral immune responses, and elicit a balanced Th1/Th2 response to HBsAg in mice, and that PGS75 may be developed as an ideal candidate adjuvant for hepatitis B vaccine.

Platycodin D2 Improves Specific Cellular and Humoral Responses to Hepatitis B Surface Antigen in Mice

Platycodin D2 (1), a less hemolytic saponin from the root of Platycodon grandiflorum than platycodin D (2), was evaluated for the potential to enhance specific cellular and humoral immune responses to hepatitis B surface antigen (HBsAg) in mice. It significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and HBsAg-induced splenocyte proliferation in HBsAg-immunized mice (P<0.05, P<0.01, and P<0.001, resp.). HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the serum were also markedly enhanced by 1 compared to the HBsAg control group (P<0.01 or P<0.001). Moreover, 1 significantly promoted the production of Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-10) cytokines from splenocytes in the HBsAg-immunized mice (P<0.001). The adjuvant potential of 1 on splenocyte proliferation, serum HBsAg-specific IgG2a and IgG2b antibody response, as well as Th1-cytokine secretion from splenocytes in the HBsAg-immunized mice was higher than that of Alum. The results suggest that 1 could improve both cellular and humoral immune responses to HBsAg in mice. Hence, 1 might be a promising adjuvant for hepatitis B vaccine with dual Th1- and Th2-potentiating activity.

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant

Background & Aims Present therapy for chronic hepatitis B attains control only in limited proportions. Small interfering RNA (siRNA) offers a new tool with potential therapeutic applications for hepatitis B virus (HBV). Given the importance of sequence identity in the effectiveness of siRNA and the heterogeneity of HBV sequences among different isolates, a short hairpin RNA (shRNA)-expressing plasmid, pSuper/HBVS1, was developed to target a region conserved among major HBV genotypes and assess its effectiveness control of HBV. Methods HBV replication-competent plasmid was cotransfected with pSuper/HBVS1 to HuH-7 cells or to mice. The levels of viral proteins, RNA, and DNA were examined in transfected cells and animals. The effects of pSuper/HBVS1 on clinical isolates with genotypes B and C were also determined. Results pSuper/HBVS1 significantly decreased levels of viral proteins, RNA, and DNA for HBV genotype A in cell culture and in mice. Comparable suppressive effects were observed on clinical isolates of genotypes B and C. A clone with a silent mutation in the target region was identified from a patient with genotype C. This mutant revealed diminished sensitivity to pSuper/HBVS1 and could be selected out in the presence of pSuper/HBVS1 in cell culture. Conclusions These findings indicated that shRNA could suppress HBV expression and replication for genotypes A, B, and C, promising an advance in treatment of HBV. However, the emergence of resistant mutants in HBV quasispecies should be considered.

Oligodeoxynucleotides containing synthetic immunostimulatory motifs augment potent Th1 immune responses to HBsAg in mice

Toll-like receptor 9 (TLR9) modulators have potent Th1-adjuvant activity. We recently reported the development of immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) or R'pG (R'=1-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs. IMOs activate TLR9 pathways, resulting in cytokine secretion profiles different from those induced by CpG DNA. In the present study we evaluated the adjuvant activity of IMOs containing CpG, CpR, or R'pG motifs in combination with hepatitis B surface antigen (HBsAg) in a mouse model. Mice immunized with HBsAg plus IMO produced higher levels of IgG2a and lower levels of IgG1 than did mice immunized with HBsAg alone or with alum. High IgG2a responses were found at week 4 and remained high until 14 weeks after immunization. Adoptive transfer of splenocytes from HBsAg/IMO-immunized mice to naïve mice resulted in strong IgG2a production in response to antigen boost. Splenocytes of mice immunized with HBsAg/IMO produced high levels of IFN-γ, but not Th2 cytokines IL-4 and IL-5, in antigen-recall experiments in vitro. The use of IMOs as adjuvants to HBsAg resulted in the production of strong anti-HBsAg antibodies at antigen doses as low as 0.2 μg. These data demonstrate that IMOs enhance the immunogenicity of HBsAg through potent Th1 immune responses, which may allow lower doses of antigen in vaccination.

Induction of immune tolerance toward tumor-associated-antigens enables growth of human hepatoma in mice.

Adoptive transfer of immunity against hepatitis B surface antigen (HBsAg) was previously shown to facilitate suppression of experimental human hepatocellular carcinoma (HCC) expressing HBsAg in athymic mice. We have shown that oral tolerance induces antigen-specific immune suppression of HBsAg by feeding hepatitis B virus (HBV) antigens. In the present study we evaluated the effect of oral tolerance induction toward HBV or HCC antigens on the growth of experimental HCC-expressing HBsAg in mice. Tolerance induction was induced in mice by 5 oral feedings of 1 microg HBV antigens or HCC-extracted proteins (50 microg protein) before vaccination with recombinant HBsAg. Splenocytes (2 x 10(6)) from these mice were transferred to sublethally irradiated athymic BALB/c mice previously transplanted subcutaneously with 10(7) human hepatoma Hep3B cells. Adoptive transfer of splenocytes immunized toward HBsAg prevented tumor growth. At 4 weeks after splenocyte transplantation, tumor volume and serum alpha-fetoprotein (AFP) levels in athymic mice transplanted with splenocytes immunized to HBsAg were undetectable as compared with 1,048 +/- 738 mm(3) and 2,500 +/- 1,431 ng/ml in recipients of naïve splenocytes (p < 0.0001). Mice receiving splenocytes tolerized toward Hep3B cells, as manifested by reduced serum HBs antibody levels, reduced HBV-specific stimulation index and reduced HBV-specific-IFN gamma spot-forming cells, had early tumor growth evident by elevated AFP serum levels, weight loss and mortality, which were suppressed at 6 weeks. Mice transplanted with splenocytes tolerized toward HBV antigens did not have direct evidence of tumor growth. Induction of oral tolerance toward HCC-extracted proteins enabled transient tumor growth in this model. This effect was mediated through downregulation of the anti-HBV immune response.

Molecular analysis of the modulatory factors of the response to HBsAg in mice as an approach to HBV vaccine enhancement

Mice were injected with immune complexes containing the recombinant hepatitis B surface antigen (HBsAg) vaccine (S + preS2) bound to different monoclonal antibodies (mAbs), in order to determine whether an enhancement of the response to a human vaccine could be obtained and observed. Enhancement and indifference were observed, as well as a decrease in immunogenicity. N relationship could be established between any effect and affinity or isotypy of the bound mAbs. The preS2 region was rendered more immunogenic when an IgG2a mAb was bound to the S region of the HBsAg. The response to the S region was not modulated, whereas immunogenicity of the preS2 collinear region was decreased by antibody shielding. The mAb which was the most efficient as an enhancer of the antibody response also increased binding of the complexed immunogen to antigen presenting cells. The binding of a human mAb to the sole S region, but not to the preS2 region, should be tested as a potentiating agent of the anti-preS2 human immune response.

Molecular analysis of the modulatory factors of the response to HBsAg in mice as an approach to HBV vaccine enhancement.

Mice were injected with immune complexes containing the recombinant hepatitis B surface antigen (HBsAg) vaccine (S + preS2) bound to different monoclonal antibodies (mAbs), in order to determine whether an enhancement of the response to a human vaccine could be obtained and observed. Enhancement and indifference were observed, as well as a decrease in immunogenicity. No relationship could be established between any effect and affinity or isotype of the bound mAbs. The preS2 region was rendered more immunogenic when an IgG2a mAb was bound to the S region of the HBsAg. The response to the S region was not modulated, whereas immunogenicity of the preS2 colinear region was decreased by antibody shielding. The mAb which was the most efficient as an enhancer of the antibody response also increased binding of the complexed immunogen to antigen presenting cells. The binding of a human mAb to the sole S region, but not to the preS2 region, should be tested as a potentiating agent of the anti-preS2 human immune response.

T-cell dependence of immune response to hepatitis B antigen in mice.

THE presence of hepatitis B surface antigen (HBsAg) in blood is a marker of infection with hepatitis B virus (HBV). Persistence of the antigen in the blood of ‘carriers’ is a manifestation of proliferation of HBV in liver cells and this carrier state can be associated with various types of liver disease1. In guinea pigs and chimpanzees, immunisation with HBsAg in Freund's complete adjuvant (FCA) elicits a cutaneous delayed type hypersensitivity (DTH) response2,3. It is believed that integrity of the thymus-dependent cell-mediated immune system in man is necessary for resistance to HBV, and that deficiency of cell-mediated immunity could predispose to development of the carrier state4,5. We therefore investigated the contribution of thymus-derived cells to the immune response to HBsAg in mice by immunisation of BALB/c mice, mice heterozygous for the nude mutant gene (nu/+), and congenitally athymic mice of the nu/nu strain, using purified HBsAg.