OBJECTIVE: This study aimed to investigate the effects of Montanide ISA-720 and Naloxone (NLX) in Hepatitis B surface antigen (HBsAg) vaccine formulation on cytokine and long-lasting antibody responses. METHODS: First, the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10 mg/kg. The experimental mice were immunized three times at a 2-week interval, and then IL-4, IL-2, TNF-α, and IFN-γ cytokines; long-lasting IgG antibody responses 220 days after the last shot; and IgG1/IgG2a isotypes were assessed by ELISA. RESULTS: The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups, whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine responses. In addition, NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ratio. Moreover, HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses, and NLX in Alum- and MON720-based vaccines induced long-lasting antibody responses. CONCLUSION NLX in Alum-based vaccine decreased IL-4 cytokine response, increased IL-2/IL-4 cytokine ratio, and improved long-lasting humoral immune responses in both vaccine formulations. Therefore, the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.
Adjuvants, Immunologic/pharmacology* ; Alum Compounds ; Animals ; Cytokines ; Hepatitis B Surface Antigens ; Hepatitis B Vaccines ; Immunity, Humoral ; Immunoglobulin G ; Interleukin-2 ; Interleukin-4 ; Mice ; Mice, Inbred BALB C ; Mineral Oil ; Naloxone/pharmacology* ; Tumor Necrosis Factor-alpha

<b>Objective:b> To explore the relationship between the status of HBsAg-positive infection of mothers and the non/low-response to hepatitis B vaccine of their infants. <b>Methods:b> A total of 225 pairs of mothers and their infants were recruited in our cohort from June 2011 to July 2013. Infants were given three doses of hepatitis B vaccine at hour 24, first month and month 6(t)h respectively and were followed up for one year after birth. HBV serological markers and HBV DNA in the peripheral blood of both mothers and infants were detected by Electro-chemiluminescence immunoassay and fluorescence quantitative Polymerase Chain Reaction. <b>Results:b> Six HBV infection models were detected in HBsAg-positive mothers, and "HBsAg (+), HBeAg (+), anti-HBc (+)" (model one) and "HBsAg (+), anti-HBe (+), anti-HBc (+)" (model two) accounted for 92.5%(208/225) of all the models. Rate of non/low-response to hepatitis B vaccine in infants born to mothers in model one was lower than those in model two, the differences are statistically significant (χ(2)=4.80, P=0.029). The rate of non/low-response to hepatitis B vaccine in infants showed a downward trend with the rising of HBeAg level in their mothers (χ(2)=4.86, P=0.028). Results from the unconditional logistic regression analysis showed that the HBeAg of the HBsAg-positive mothers was significantly correlated with the low risk of non/low-response to hepatitis B vaccine in infants (OR=0.598, 95%CI: 0.378-0.947). The positive rate of serum HBV DNA in HBsAg-positive mothers was 54.2%, while the rate of non/low-response to hepatitis B vaccine in infants born to HBV DNA positive mothers was similar to those infants born to HBV DNA negative mothers (χ(2)=0.22, P=0.640). <b>Conclusions:b> "HBsAg (+), HBeAg (+), anti-HBc (+)" and "HBsAg (+), anti-HBe(+), anti-HBc (+)" were the common models seen in HBsAg-positive mothers, and the rate of non/low-response to hepatitis B vaccine was different between the two models. HBeAg of HBsAg-positive mothers might have positive effects on the immune response to hepatitis B vaccine in infants but the mechanisms remained not clear. HBV DNA of the HBsAg-positive mothers did not seem to be correlated with the immune response to hepatitis B vaccine in infants.
Adult ; Biomarkers/blood* ; DNA, Viral/blood* ; Diagnostic Tests, Routine ; Female ; Hepatitis B/prevention & control* ; Hepatitis B Antibodies/blood* ; Hepatitis B Surface Antigens/blood* ; Hepatitis B Vaccines/pharmacology* ; Hepatitis B e Antigens/blood* ; Hepatitis B virus/isolation & purification* ; Humans ; Infant ; Infectious Disease Transmission, Vertical/prevention & control* ; Mothers ; Pregnancy ; Pregnancy Complications, Infectious/virology*

To compare different adjuvant formulation and explore the impact of Calcineurin B subunit(CnB) as adjuvant with a novel HBV protein particle (HBSS1) vaccine in mice, female C57BL/6 mice were immunized HBSS1 with Al(OH)3 only, or a normal dose (5 μg) CnB only, or (CnB+ Al(OH)3) mixture as the adjuvant. All immunized groups were primed twice at 4-week intervals; followed by boosting with recombinant adenoviral based HBV vaccine(rAdSS1) at 10-week intervals. We detected the antigen specific humoral response in mice, including total IgG antibody and IgG subtyping. Then, we characterized the specific cell-mediated immune (CMI) response by detection of γ-interferon secreting splenocytes after stimulaton with S or PreS1 peptide pools. No enhancement of immunity was found among the mice with 5 μg of CnB alone or combined with Al(OH), adjuvanted vaccine,which could not induce higher level of anti-PreS1 and anti-S antibodies and CMI than that of HBSS1 alone or Al(OH)3 adjuvanted vaccines. We concluded that CnB is not an effective adjuvant for a novel HBV subunit vaccine.
Adjuvants, Immunologic ; pharmacology ; Animals ; Calcineurin ; pharmacology ; Female ; Hepatitis B Vaccines ; immunology ; Mice ; Mice, Inbred C57BL ; Protein Subunits

While currently therapeutic vaccines for chronic hepatitis B virus (HBV) infection are actively being developed to complement standard antiviral treatments, their immune activity, especially T cell activity, remains to be further improved. Here, we investigated the role of heat shock proteins HSP70 and gp96 on cellular and humoral immunity, using the main structure antigens of hepatitis core (HBcAg) and surface (HBsAg) as the DNA vaccine. By ELISPOT (enzyme linked immunospot assay), IFN-gamma intracellular staining, [3H]-thymidine incorporation and ELISA (enzyme linked immunosorbent assay) analyses, we showed that immunization with HBsAg/HBcAg DNA formulation along with HSP70 or gp96 induced significant increase of T-cell (about 1-6-fold) and antibody (about 20%-60%) immunity against HBsAg and HBcAg. These results may provide bases for designing HSP70- and gp96-based vaccines aimed at eliciting T-cell responses for therapeutic applications.
Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; HSP70 Heat-Shock Proteins ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; immunology ; therapy ; Humans ; Immunoglobulin G ; immunology ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; immunology ; Vaccines, DNA ; immunology

Hepatitis B virus core (HBc) proteins have been used as carrier for foreign epitopes since the 1980s. They could self-assemble into icosahedral particles. Foreign epitopes could be inserted into HBc protein in various protein regions, including the N- or C-terminal and the major immunodominant region (MIR). The factors relevant in the design of HBc particles for vaccine purpose are summarized in this review.
Adjuvants, Immunologic ; pharmacology ; Drug Carriers ; Epitopes ; genetics ; immunology ; Genetic Vectors ; Hepatitis B Core Antigens ; genetics ; immunology ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, Synthetic ; biosynthesis ; immunology

We previous reported the development of novel hepatitis B virus(HBV) vaccine containing the surface antigen(S) plus PreS1 fusion derived from Chinese hamster ovary (CHO) cells system. In this study, we analyzed the impact of different adjuvants on immunogenicity of the HBV particle vaccine in Balb/C mice, including alum alone, CpG oligodeoxynucleotides (CpG-ODN) alone and CpG-ODN in combination with alum adjuvant. We first detected the antigen specific humoral response in mice, including total IgG antibody and IgG subtyping. Then, we characterized the specific cell-mediated immune (CMI) response by detection of gamma-interferon secreting splenocytes after stimulating with S or PreS1 peptide pool. Our results showed that: CpG-ODN adjuvanted vaccine could rapidly induce higher level of anti-PreS 1 and anti-S antibodies, and a higher ratio of IgG2a/IgG1 antibody than that of alum adjuvanted vaccine. At the same time, CpG-ODN adjuvanted vaccine induced robust antigen-specific cellular immune responses in mice, which was superior to that of alum adjuvanted vaccine and CpG-ODN in combination with alum adjuvanted vaccine; however, the vaccine candidate with CpG-ODN in combination with alum adjuvant induced highest anti-S antibody and mixed IgG subclasses in mice after twice immunization. There exists dominant HBV CMI epitopes in the N-terminal of S antigen. These results provided important evidence that CpG-ODN adjuvanted HBSS1 particles vaccine may serve as a novel candidate in the development of new preventive and therapeutic agents against hepatitis B infection.
Adjuvants, Immunologic ; pharmacology ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; biosynthesis ; immunology ; Immunization ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides ; immunology ; Protein Precursors ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; biosynthesis ; immunology

<b>OBJECTIVEb>To investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice.

<b>METHODSb>Eukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3 cells, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively. The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine (pcDS) by intramuscular injection. HBsAg-specific activity of splenic cytotoxic T lymphocyte (CTL) in the immunized mice was measured by LDH release assay. The splenic memory CD8+ T cells, and intracellular IFN-gamma and IL-4 of splenic lymphocytes and CD8+ T cells after immunization were detected by flow cytometry.

<b>RESULTSb>The NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein. HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization (P<0.05). Compared to mice immunized with pcDS alone, CD44high and CD127(IL-7R) were all significantly up-regulated in memory CD8+ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization (P<0.05 and P<0.01). The pcDS plus CD137L group also elicited higher levels of IFN-gamma secreted by CD8+ T cells and splenic lymphocytes than pcDS alone at a week, 12 and 13 weeks after immunization, respectively (all P<0.01).

<b>CONCLUSIONb>DNA, viral/immunol; Co-stimulatory molecule CD137L can enhance the Tc1 (type I) cell-mediated immunity, HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine, and may be an efficient adjuvant in priming HBV-specific T cell response.

4-1BB Ligand ; immunology ; pharmacology ; Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccination ; methods ; Vaccines, DNA ; immunology

<b>OBJECTIVEb>To explore the effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation (LT).

<b>METHODSb>Brown-Norway liver allografts were transplanted into Lewis recipients. The transplanted Lewis rats were injected with EK506 (2 mg/kg) and randomly divided into two groups: rats in HBsAg-DCs group (n = 15) were intraperitoneally injected with HBsAg pulsed DCs at 14 d and 28 d after LT, and rats in the HBsAg group (n = 15) were injected with HBsAg (200 mul) once a week for 12 weeks. Rats without any immunosuppressive treatment after LT served as controls (n = 5). IL-2 and IFN-gamma mRNA expression in spleen were analyzed by RT-PCR, serum IL-2, IFN-gamma and anti-HBs were detected by ELISA.

<b>RESULTSb>High dose of FK506 resulted in the immunosuppressed in LT rats, as evident by low production of IL-2 and IFN-gamma, and without liver rejection compared to rats in the control group. HBsAg-DCs induced high titer of anti-HBs antibody, however, titer of anti-HBs were seldom detectable in the HBsAg group at 1, 2 and 3 mouth after vaccination.

<b>CONCLUSIONb>The capacity of HBsAg-DCs to induce anti-HBs in immunosuppressed rats suggested that DC vaccine may prevent HBV recurrence in liver transplanted patients.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cytokines ; blood ; genetics ; metabolism ; Dendritic Cells ; immunology ; Disease Models, Animal ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Antibodies ; blood ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; Immunosuppression ; Immunosuppressive Agents ; administration & dosage ; Liver Transplantation ; immunology ; Male ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred BN ; Rats, Inbred Lew ; Secondary Prevention ; Spleen ; immunology ; metabolism

Antiviral Agents ; pharmacology ; therapeutic use ; Drug Administration Schedule ; Hepatitis B Vaccines ; therapeutic use ; Hepatitis B, Chronic ; drug therapy ; therapy ; Humans ; Interferons ; therapeutic use ; Nucleotides ; pharmacology ; therapeutic use

Adjuvants, Immunologic ; pharmacology ; Animals ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Interleukin-15 ; pharmacology ; Killer Cells, Natural ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Vaccines, DNA ; immunology