Hepatitis B Surface Antigen Vaccine Research Articles

LB749. High HBsAb Seroprotection Achieved 4 Weeks after 3 doses of HepB-CpG Vaccine in People Living with HIV (PLWH) without Prior HBV Vaccination (ACTG A5379 Group B Preliminary Results)

Abstract Background Three-dose series of conventional alum-adjuvanted Hepatitis B surface antigen (HBsAg)-based vaccines achieve seroprotection rates (SPRs) of 35-70% in PLWH. HepB-CpG, a HBsAg vaccine adjuvanted with a TLR-9 agonist, achieves high SPR in immunocompetent adults with a 2-dose regimen, but limited data exist in PLWH. Methods A5379 is an ongoing prospective, open-label study to evaluate immunogenicity of the HBV vaccine HepB-CpG in PLWH. The HBV vaccine-naïve group consisted of PLWH without past HBV infection on antiretroviral therapy with CD4 ≥100 cells/mm3 and HIV-1 RNA < 1000 copies/mL. Participants received 0.5 mL of HepB-CpG intramuscularly (20 mcg recombinant HBsAg and 3000 mcg of CpG 1018® adjuvant) at Wks 0, 4, and 24. Primary objectives were to determine the proportion of participants who achieve seroprotection (HBsAb≥10 mIU/mL) at Wk 28 and to assess safety. This study was designed to conclude SPR >55%, with up to 10% loss to follow-up prior to 28 wks. Results Of the 74 eligible participants enrolled at 13 global sites: 46% were male, 66% Asian, 16% Black,15% White and median age was 47 years (range 23-68). 27% were enrolled in the US, 65% in Thailand. Median CD4 was 625 cells/mm3, 96% had HIV-1 RNA < 60 copies/mL, and 9% had diabetes. Primary analysis set per analysis plan consisted of 68 participants (excluded: 3 missed visit, 3 out of visit window). All 68 completed 3 doses and achieved seroprotection (100% [95% CI: 94.7%, 100%]). 88% had HBsAb >1000 mIU/mL (assay upper limit). The SPR was also 100% [CI: 94.2%,100%] in the per protocol analysis set of 62 participants. At 8 wks after the 2nd dose, the SPR was 94.4% followed by 98.5% at Wk 24, prior to the 3rd dose. The proportion of participants with HBsAb >1000 mIU/mL increased from 27.9% at Wk 24 to 83.8% at Wk 28. One or more AEs related to study treatment were experienced by 61% of participants (39% Grade 1, 20% Grade 2, Grade 3 malaise in one participant). Vaccination site pain (40%), malaise (26%), fatigue (23%), myalgia (22%) and headache (22%) were the most frequent AEs. Seroprotection achieved by study week. Percentage of participants achieving seroprotection over the first 28 study weeks. HepB-CpG vaccine was administered at Entry, Week 4, and Week 24. Conclusion In this study of PLWH with no history of HBV vaccination or evidence of prior HBV exposure, 100% seroprotection was achieved at 4 weeks after 3 doses of the HepB-CpG. No unexpected safety issues were observed. Disclosures Kristen Marks, MD, MS, Gilead Sciences: Grant/Research Support Andrea L. Cox, M.D. Ph.D., Janssen: Advisor/Consultant Jennifer C. Price, MD, PhD, AbbVie: Grant/Research Support|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Merck: Grant/Research Support|Theratechnologies: Advisor/Consultant Kelvin McKoy, M.D., M.B.A., Dynavax Technologies: Employee|Dynavax Technologies: Ownership Interest|Dynavax Technologies: Stocks/Bonds Kenneth E. Sherman, MD, PhD, AbbVie: Grant/Research Support|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Helio: Grant/Research Support|Inovio: DSMB|Intercept: Grant/Research Support|MedPace: DSMB|Theratechnologies: Advisor/Consultant|Zydus: Grant/Research Support.

Engineered anti-PDL1 with IFNα targets both immunoinhibitory and activating signals in the liver to break HBV immune tolerance

ObjectiveThe purpose of this study is to develop an anti-PDL1-based interferon (IFN) fusion protein to overcome the chronic hepatitis B virus (HBV)-induced immune tolerance, and combine this immunotherapy with a...

Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses

ObjectiveThis 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. MethodsFirst, 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. ResultsThe 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. ConclusionNLX 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.

Accelerated hepatitis B vaccination: Efficacy in young, regular blood donors

Background and Objectives: Hepatitis B (HB) – a vaccine-preventable, transfusion-transmitted infection – has been estimated to have high residual risk, especially in medium- and high-endemic areas. This potent immunogenic vaccine is routinely injected at 0, 1, and 6 months. Accelerated schedule has been recommended in adults to increase the completion rate. We studied the response rate of accelerated schedule in young, regular blood donors in a high-prevalence area for HB virus (HBV) in Iran. Methods: Young, regular nonimmunized blood donors were vaccinated with recombinant DNA HB surface antigen vaccine (Pasteur Institute of Iran) containing 20 μg of material in 1-ml solution injected intramuscularly to the deltoid muscle at 0, 1, and 2 months. HB surface antibody (HBsAb) was titered 1 month after the completion of doses. Nonresponders took one additional dose and tested a month later. Results: A total of 357 donors with a mean age of 26 ± 3.5 years were enrolled, of whom 287 donors completed three doses of vaccination (80% adherence rate). HBsAb titer was >10 IU/ml in 274 donors (95.5% response rate), whereas after one booster dose injection, the titer in 13 nonresponder persons (4.5%) increased to >10 IU/ml. Conclusion: Accelerated HBV vaccination with near 100% response rate and 80% adherence rate in young, regular blood donors has been successful in the immunization of young blood donors. This strategy improves not only blood safety but also donors' health.

CD4+ T Cell-Released Extracellular Vesicles Potentiate the Efficacy of the HBsAg Vaccine by Enhancing B Cell Responses.

T cells secrete bioactive extracellular vesicles (EVs), but the potential biological effects of CD4+ T cell EVs are not clear. The main purpose of this study is to investigate the effects of CD4+ T cell–derived EVs on B cell responses and examine their role in antigen‐mediated humoral immune responses. In this study, CD4+ T cell EVs are purified from activated CD4+ T cells in vitro. After immunization with the Hepatitis B surface antigen (HBsAg) vaccine, CD4+ T cell EVs‐treated mice show stronger humoral immune responses, which is indicated by a greater Hepatitis B surface antibody (HBsAb) level in serum and a greater proportion of plasma cells in bone marrow. In addition, it is found that EVs released from activated CD4+ T cells play an important role in B cell responses in vitro, which significantly promote B cell activation, proliferation, and antibody production. Interestingly, antigen‐specific CD4+ T cell EVs are found to be more efficient than control EVs in enhancing B cell responses. Furthermore, it is shown that CD40 ligand (CD40L) is involved in CD4+ T cell EVs‐mediated B cell responses. Overall, the results have demonstrated that CD4+ T cell EVs enhance B cell responses and serve as a novel immunomodulator to promote antigen‐specific humoral immune responses.

Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50°C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45°C and only a 10% loss was observed after 6monthsat 50°C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination.

Vaccines targeting preS1 domain overcome immune tolerance in hepatitis B virus carrier mice

These results suggest that preS1 can function as a therapeutic vaccine for the control of CHB. (Hepatology 2017;66:1067-1082).

Activation of TNF-α-AID axis and co-inhibitory signals in coordination with Th1-type immunity in a mouse model recapitulating hepatitis B

Hepatitis B virus (HBV) infection evokes host immune responses that primarily determine the outcome of HBV infection and the clinical features of HBV-associated liver disease. The precise mechanisms by which host factors restrict HBV replication, however, are poorly understood due to the lack of useful animal models that recapitulate immune responses to HBV. Here, we performed comprehensive immunologic gene expression profiling of the liver of a mouse model recapitulating anti-HBV immune response using a high sensitivity direct digital counting system. Anti-HBV cellular immunity with liver inflammation was elicited in mice hydrodynamically injected with a CpG-depleted plasmid encoding hepatitis B surface antigen (HBsAg) gene after preimmunization with HBsAg vaccine. Comprehensive expression analyses revealed the upregulation of Th1-associated genes including tumor necrosis factor (Tnf) and negative regulators of T cell function in the inflamed liver. Interestingly, activation-induced cytidine deaminase (Aicda, termed AID in humans), which reportedly suppresses HBV infection in vitro, was upregulated in hepatocytes in the course of anti-HBV immunity. Hepatocytic expression of Aicda in a Tnf-dependent manner was confirmed by the administration of Tnf antagonist into Aicda-tdTomato mice with anti-HBV immunity. Our findings suggest that activation of Tnf–Aicda axis and co-inhibitory signals to T cells in coordination with Th1-type immunity has critical roles in the immune response against HBV infection.

Development of the CpG Adjuvant 1018: A Case Study.

The development of aluminum salts (alum) as vaccine adjuvants was an empirical process with little understanding of the mechanism of action and, with decades of use, it has become clear that there is a need for alternatives where alum-based adjuvants are suboptimal. Oligonucleotides containing unmethylated CpG sequences represent one alternative as they are potent stimulators of the vertebrate innate immune system through activation of Toll-like receptor-9. This chapter outlines the methods used by Dynavax Technologies to progress a CpG-containing oligonucleotide sequence termed 1018 through preclinical and clinical testing as an adjuvant for immunization against hepatitis B virus (HBV). 1018 is a short (22-mer) oligonucleotide sequence containing CpG motifs active in both rodents and primates. Preclinical testing of hepatitis B surface antigen (HBsAg) + 1018 in comparison to HBsAg + alum demonstrated induction of substantially higher antibody titers and a favorable safety profile for 1018. Most importantly, clinical studies with HBsAg vaccination consistently demonstrate more rapid induction of protective antibody titers with 1018 compared to alum in all populations studied, including groups that are harder to immunize such as the elderly and immunocompromised individuals. These studies represent the basis for use of the CpG-motif-containing oligonucleotide 1018 as an improved adjuvant for HBsAg immunogenicity. HBsAg + 1018 (HEPLISAV-B™) is currently in late-stage clinical testing for prophylactic immunization against HBV.

Novel nanocarrier for oral Hepatitis B vaccine

Oral vaccination is a safe, cost effective and non-invasive method suitable for mass immunization. We fabricated nanoparticle (NP) with 14kd polycaprolactone (PCL) entrapping hepatitis B surface antigen (HBsAg) stabilized with Pluronics® F127 and used it as oral delivery vehicle. We evaluated its efficacy for specific antibody production and compared with parenteral routes of immunization in mice. We found a superior antibody response with a higher titer of anti-HBsAg antibody till 2 months following single oral administration compared to other routes of immunization and conventional alum-based HBsAg vaccine. The NPs with the antigen were found in the macrophages in small intestinal villi, peripheral lymph nodes and other reticulo-endothelial organs 2 months after oral administration. This study suggests the efficacy of the current nanocarrier system for efficient antigen presentation disseminated in peripheral lymphoid tissues following oral administration with a prolonged antibody response, which can minimize the requirement of booster dose.

Oral administration of synthetic selenium nanoparticles induced robust Th1 cytokine pattern after HBs antigen vaccination in mouse model.

The present study proposed that the administration of SeNPs with a conventional HBs antigen vaccine induces a better immune response with a Th1 bias.

Enteric trimethyl chitosan nanoparticles containing hepatitis B surface antigen for oral delivery

Oral vaccination is the preferred route of immunization. However, the degradative condition of the gastrointestinal tract and the higher molecular size of peptides pose major challenges in developing an effective oral vaccination system. One of the most excellent methods used in the development of oral vaccine delivery system relies on the entrapment of the antigen in polymeric nanoparticles. In this work, trimethyl chitosan (TMC) nanoparticles were fabricated using ionic gelation teqnique by interaction hydroxypropyl methylcellulose phthalate (HPMCP), a pH-sensitive polymer, with TMC and the utility of the particles in the oral delivery of hepatitis B surface antigen (HBsAg) was evaluated employing solutions that simulated gastric and intestinal conditions. The particle size, morphology, zeta potential, loading capacity, loading efficiency, in vitro release behavior, structure, and morphology of nanoparticles were evaluated, and the activity of the loaded antigen was assessed. Size of the optimized TMC/HPMCP nanoparticles and that of the antigen-loaded nanoparticles were 85 nm and 158 nm, respectively. Optimum loading capacity (76.75%) and loading efficiency (86.29%) were achieved at 300 µg/mL concentration of the antigen. SEM images revealed a spherical shape as well as a smooth and near-homogenous surface of nanoparticles. Results of the in vitro release studies showed that formulation with HPMCP improved the acid stability of the TMC nanoparticles as well as their capability to preserve the loaded HBsAg from gastric destruction. The antigen showed good activity both before and after loading. The results suggest that TMC/HPMCP nanoparticles could be used in the oral delivery of HBsAg vaccine.

Size-exclusion HPLC provides a simple, rapid, and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens

The assembly of antigen structure is often crucial to the potency of vaccines. Currently adopted methods like animal testing and ultracentrifugation take long time and are difficult to automate for multiple samples. Here we develop a size-exclusion high-performance liquid chromatography (SE-HPLC) method to characterize the assembly of antigen structure during both manufacturing process and storage. Three important vaccine antigens including inactivated foot and mouth disease virus (FMDV), which is a virus vaccine; and two virus-like particles (VLPs) vaccines involving hepatitis B core antigen (HBcAg) VLPs, and hepatitis B surface antigen (HBsAg) VLPs, were successfully analyzed using commercially available TSK gel columns with pore size above 45nm. Combined with other analytical methods including SDS–PAGE, dynamic light scattering, wavelength scan, and multi-angle laser light scattering, the SE-HPLC method was proven to be a simple, rapid, and reliable tool for antigen particles assembly analysis. Specifically, for FMDV whole virus particle, SE-HPLC was used to analyze 146S content in vaccine preparations and the thermal dissociation of the 146S. For HBcAg-VLPs that are expressed in recombinant Escherichia coli, its expression level during cell culture process was quantitatively monitored by SE-HPLC. The SE-HPLC also showed applicability for quality check of HBsAg vaccine preparations by monitoring the product consistency of different lot number and the product stability during storage. Results shown in this work clearly demonstrated that SE-HPLC method has potential as a versatile alternative technology for control of the final product by both manufacturers and the regulatory agencies.

Vaccine Adjuvant Systems containing monophosphoryl lipid A and QS-21 induce strong humoral and cellular immune responses against hepatitis B surface antigen which persist for at least 4 years after vaccination

BackgroundRecombinant hepatitis B surface antigen (HBsAg) was used as a model antigen to evaluate persistence of cellular and humoral immune responses when formulated with three different Adjuvant Systems containing 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and QS-21, in an oil-in-water emulsion (AS02B and AS02V), or with liposomes (AS01B). MethodsThis is an open, 4-year follow-up of a previous randomised, double-blind study. Healthy subjects aged 18–40 years received three vaccine doses on a month 0, 1, 10 schedule and were initially followed for 18 months. A total of 93 subjects (AS02B: n=30; AS02V: n=28; AS01B: n=35) were enrolled in this follow-up and had an additional blood sample taken at Year 4 (NCT02153320). The primary endpoint was the frequency of HBsAg-specific CD4+ and CD8+ T-cells expressing cytokines upon short-term in vitro stimulation of peripheral blood mononuclear cells with HBsAg-derived peptides. Secondary endpoints were anti-HBs antibody titres and frequency of HBsAg-specific memory B-cells. ResultsA strong and persistent specific CD4+ T-cell response was observed at Year 4 in all groups. HBsAg-specific CD4+ T-cells expressed mainly CD40L and IL-2, and to a lesser extent TNF-α and IFN-γ. HBsAg-specific CD8+ T-cells were not detected in any group. A high, persistent HBsAg-specific humoral immune response was observed in all groups, with all subjects seroprotected (antibody titre ≥10mIU/mL) at Year 4. The geometric mean antibody titre at Year 4 was above 100,000mIU/mL in all groups. A strong memory B-cell response was observed post-dose 2, which tended to increase post-dose 3 and persisted at Year 4 in all groups. ConclusionThe MPL/QS-21/HBsAg vaccine formulations induced persistent immune responses up to 4 years after first vaccination. These Adjuvant Systems offer potential for combination with recombinant, synthetic or highly purified subunit vaccines, particularly for vaccination against challenging diseases, or in specific populations, although additional studies are needed.

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response

In this study, for the first time, glycol chitosan (GC) nanoparticles (NPs) were prepared and evaluated to obtain systemic and mucosal immune responses against nasally administered hepatitis B surface antigen (HBsAg). Size, zeta potential and morphology of the NPs were investigated as a function of preparation method. NPs with high loading efficacy ( > 95%) and positively charged surface were obtained with an average particle size of approximately 200 nm. The structural integrity of HBsAg in NPs was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and further confirmed by measuring the in vitro antigenicity using an enzyme immunoassay. During in vivo studies, GC NPs showed the lowest nasal clearance rate and better mucosal uptake when compared with chitosan (CS) NPs. The immunogenicity of NPs-based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum-based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum-based HBsAg induced strong humoral but negligible mucosal immunity. However, GC NPs induced stronger immune response at both of the fronts as compared to generated by CS NPs. This study demonstrates that this newly developed system has potential for mucosal administration of vaccines.

Nanoparticles encapsulating hepatitis B virus cytosine-phosphate-guanosine induce therapeutic immunity against HBV infection

Infection with hepatitis B virus (HBV) is the most common cause of liver disease worldwide. However, because the current interferon (IFN)-based treatments have toxic side effects and marginal efficacy, improved antivirals are essential. Here we report that unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) from the HBV genome (HBV-CpG) induced robust expression of IFN-α by plasmacytoid dendritic cells (pDCs) in a Toll-like receptor 9 (TLR9)-dependent manner. We also identified inhibitory guanosine-rich ODNs in the HBV genome (HBV-ODN) that are capable of inhibiting HBV-CpG-induced IFN-α production. Furthermore, nanoparticles containing HBV-CpG, termed NP(HBV-CpG), reversed the HBV-ODN-mediated suppression of IFN-α production and also exerted a strong immunostimulatory effect on lymphocytes. Our results suggest that NP(HBV-CpG) can enhance the immune response to hepatitis B surface antigen (HBsAg) and skew this response toward the Th1 pathway in mice immunized with rHBsAg and NP(HBV-CpG). Moreover, NP(HBV-CpG)-based therapy led to the efficient clearance of HBV and induced an anti-HBsAg response in HBV carrier mice. Endogenous HBV-CpG ODNs from the HBV genome induce IFN-α production so that nanoparticle-encapsulated HBV-CpG may act as an HBsAg vaccine adjuvant and may also represent a potent therapeutic agent for the treatment of chronic HBV infection.

Detection of lamivudine-resistant variants and mutations related to reduced antigenicity of HBsAg in individuals from the cities of Santos and São Paulo, Brazil

BackgroundContinuous long-term treatment is recommended to reduce the hepatitis B virus (HBV) viral load. However, as a consequence, resistance mutations can emerge and be transmitted to other individuals. The polymerase (POL) gene overlaps the surface (S) gene. Thus, during treatment, mutations in the POL gene may lead to changes in hepatitis B surface antigen (HBsAg). The purpose of this study was to evaluate the frequency of lamivudine and vaccine escape mutations in HBsAg-positive blood donors from the city of Santos and in untreated HBV mono-infected patients from the city of São Paulo, Brazil.MethodsHBV DNA was extracted from 80 serum samples, of which 61 were from volunteer blood donors and 19 were from untreated HBV patients. A fragment of the POL/S genes containing 593 base pairs was amplified using nested PCR. Thirty four were PCR-positive and sequencing was performed using an ABI Prism 3130 Genetic Analyzer. Alignments and mutation mapping were performed using BioEdit software.ResultsHBV DNA from 21 blood donors and 13 untreated patient samples were characterized using nucleotide sequencing PCR products from the POL/S genes. We were able to detect one sample with the resistance mutation to lamivudine rtM204V + rtL180M (2.94%), which was found in a volunteer blood donor that has never used antiviral drugs. The other samples showed only compensatory mutations, such as rtL80F (5.88%), rtL80V (2.94%), rtL82V + rtV207L (2.94%), rtT128P (5.88%), rtT128N/S (2.94%) and rtS219A (5.88%). We found modifications in the S gene in 14 of the 34 samples (41.16%). The mutations detected were as follows: sM133L + sI195T (2.94%), sI195M (2.94%), sP120T (2.94%), sY100S/F (2.94%), sY100C (17.64%), sI/T126P + sQ129P (2.94%), sM198I + sF183C (2.94%) and sS210R (5.88%).ConclusionsOur results suggest the transmission of lamivudine-resistant forms. Thus, the evaluation of HBV-infected subjects for lamivudine resistance would improve treatment regime. Moreover, the mutations in the S gene may impair HBsAg antigenicity and contribute to HBsAg failure detection and vaccine escape.

TH1 and TH2 responses are influenced by HLA antigens in healthy neonates vaccinated with recombinant hepatitis B vaccine.

The immune response to hepatitis B surface antigen (HBsAg) is influenced by several factors, of which HLA antigens and balanced secretion of Th1/Th2 cytokines play important roles. The aim of this study was to evaluate the influence of HLA antigens on cytokine secretion by HBsAg-stimulated peripheral blood mononuclear cells (PBMC) from healthy neonates vaccinated with recombinant HBsAg. PBMCs were isolated from 48 Iranian neonates vaccinated with a recombinant HBV vaccine. The cells were stimulated in vitro with rHBsAg and the concentration of IL-4, IL-10, IL-12 and IFN-γ were quantitated in culture supernatant by sandwich ELISA. HLA typing was performed by microlymphocytotoxicity method. Significant diminished secretion of both Th1 (IFN-γ) and Th2 (IL-4, IL-10) cytokines was observed in HBsAg-stimulated PBMC from vaccinees expressing the HLA-DR7 compared to DR7 negative vaccinees. Similarly, lower production of these cytokines was also observed in vaccinees with DR7-DR53-DQ2, B7-DR7-DR53-DQ2 and A2-DR7-DR53-DQ2 haplotypes (p<0.05, p <0.005). While HBsAg-stimulated PBMC of DR13+ subjects produced lower levels of Th2-type cytokines (IL-4 and IL-10), those of HLA-B8+ or HLA-A9+ subjects produced higher levels of Th2-type cytokines. Cytokine secretion in response to PHA mitogen was not associated with a given HLA antigen or haplotype and was similarly represented in all groups of subjects irrespective of their HLA complex. These results indicate that HLA antigens may differentially influence cytokine secretion by HBsAg-specific T-cells of healthy neonates vaccinated with recombinant HB vaccine. This phenomenon may have an important implication for control of the immune response to HBsAg vaccine.

Novel membrane extraction procedure for the purification of hepatitis B surface antigen from Pichia pastoris

The recombinant hepatitis B surface antigen (HBsAg) vaccine provides excellent protection against hepatitis B virus (HBV). However, high costs of its production prevents many underdeveloped and developing nations from implementing HBsAg vaccination. This in turn increases the risk of contracting HBV related diseases. Majority of the commercial HBV vaccines are derived from purified HBsAg expressed in recombinant yeasts. Most of the cost in production of the vaccine is incurred during the downstream processing. The costs associated with HBsAg purification can be decreased by optimizing the pre-chromatography steps and by reducing the impurity burden on chromatography operations. Here in this work we present a novel strategy for the enriched extraction of recombinant HBsAg from Pichia pastoris membranes. We have also developed a simple, easy to operate process for the purification of HBsAg VLPs from the membranes of P. pastoris. This novel strategy, while utilizing a single column chromatographic step in the purification scheme results in the highest recovery of HBsAg VLPs reported in the literature. The yield of HBsAg at the end of purification was nearly 5% (85μg/g of induced wet cell biomass). The HBsAg purified from this process has shown the presence of VLPs. The immunization of these VLPs in BALB/c mice with alhydrogel adjuvant has shown good titers of neutralizing antibodies.

Posintro™-HBsAg, a modified ISCOM including HBsAg, induces strong cellular and humoral responses

To improve the hepatitis B vaccines on the market new adjuvant systems have to substitute aluminium. In this study the hepatitis B surface antigen (HBsAg) was incorporated into a novel adjuvant system, the Posintro™, a modification of the traditional immune stimulatory complexes (ISCOMs). This new HBsAg vaccine formulation, Posintro™-HBsAg, was compared to two commercial hepatitis B vaccines including aluminium or monophosphoryl lipid A (MPL) and the two adjuvant systems MF59 and QS21 in their efficiency to prime both cellular and humoral immune responses. The Posintro™-HBsAg induced the strongest humoral response with high titers of HBsAg specific antibody, high number of antigen specific B-cells and a strong T helper 1 (Th1) antibody profile when compared to the other adjuvant formulations. The Posintro™-HBsAg was also a strong inducer of cellular immune responses with induction of delayed type hypersensitivity (DTH) reaction and CD4 + T-cell proliferation. In addition, Posintro™-HBsAg was the only vaccine tested that also induced a strong cytotoxic T lymphocyte (CTL) response, with high levels of antigen specific CD8 T-cells secreting IFN-gamma mediating cytolytic activity. The results demonstrate that this novel experimental vaccine formulation, the Posintro™-HBsAg, is strongly immunogenic and can induce both class I and class II responses in experimental animals. This shows promise both for the protection against hepatitis B virus infection and as a potential therapeutic vaccine.