Recombinant Hepatitis B Surface Antigen Research Articles

Isolation and Characterization of Targeting-HBsAg VNAR Single Domain Antibodies from Whitespotted Bamboo Sharks (Chiloscyllium plagiosum).

Immunoglobulin new antigen receptor (IgNAR) is a naturally occurring antibody that consists of only two heavy chains with two independent variable domains. The variable binding domain of IgNAR, called variable new antigen receptor (VNAR), is attractive due to its solubility, thermal stability, and small size. Hepatitis B surface antigen (HBsAg) is a viral capsid protein found on the surface of the Hepatitis B virus (HBV). It appears in the blood of an individual infected with HBV and is widely used as a diagnostic marker for HBV infection. In this study, the whitespotted bamboo sharks (Chiloscyllium plagiosum) were immunized with the recombinant HBsAg protein. Peripheral blood leukocytes (PBLs) of immunized bamboo sharks were further isolated and used to construct a VNAR-targeted HBsAg phage display library. The 20 specific VNARs against HBsAg were then isolated by bio-panning and phage ELISA. The 50% of maximal effect (EC50) of three nanobodies, including HB14, HB17, and HB18, were 4.864 nM, 4.260 nM, and 8.979 nM, respectively. The Sandwich ELISA assay further showed that these three nanobodies interacted with different epitopes of HBsAg protein. When taken together, our results provide a new possibility for the application of VNAR in HBV diagnosis and also demonstrate the feasibility of using VNAR for medical testing.

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.

Study of influence of metal ions in the diagnosis of recombinant hepatitis B surface antigen (HBsAg) using ELISA technique

ObjectivesPreliminary diagnosis of hepatitis B is performed by measuring the levels of serological markers using enzyme-linked immunosorbent assay (ELISA). Errors in diagnosis of the disease using serum markers might occur due to the presence of metallic salts in diagnostic samples. The present study was designed to evaluate the effects of various metal ions on the diagnosis of recombinant hepatitis B surface antigen (HBsAg). MethodsA simple step sandwich ELISA technique was used to evaluate the effects of metal ions on the diagnosis of HBsAg. Further, the zeta potential, electrical conductivity and redox potential of solutions with proteins and metal salts were measured to evaluate the interaction of these metal ions with the protein. ResultsThe results showed that recombinant HBsAg significantly interacted with the tested metal ions, with lithium ion showing the highest interaction, followed by barium, aluminium, and nickel as the percent decrease in the free HBsAg concentration in presence of these metal ions were found to be 95.5, 94.1, 93.5, and 91.6%, respectively. Zeta potential of the protein in reaction mixture also changed considerably and marked decrease in the concentration of free HBsAg was observed. This decrease in the concentration of free HBsAg resulted into error in the ELISA test which was negative in presence of these metal ions due to strong interaction with the HBsAg protein. ConclusionsThe findings of this study clearly demonstrated that the presence of metal ions in pathological samples can result in interaction with the antigen leading to possible diagnostic errors.

CpG-Containing Oligodeoxynucleotides and Freund Adjuvant in Combination with Alum Augment the Production of Monoclonal Antibodies Against Recombinant HBsAg.

Background:Adjuvants are essential to potentiate the immune response to inoculated antigens and play a central role in vaccine development. Alum is generally used as a classic adjuvant, although it does not stimulate proper immunity, and some of the immunized subjects have low or no antibody response. Efforts have been continued to find more efficient adjuvants for better antibody responses. In the present study, the efficacy of three formulations of adjuvants, i.e. Cysteine p Guanine Oligodeoxynucleotide (CpG ODN), alum, and Freund, in the production of monoclonal anti Hepatitis B Surface Antigen (HBsAg) antibodies was investigated.Methods:To immunize mice, regular hepatitis B vaccine containing recombinant HBsAg and alum was used with CpG ODN or Freund adjuvants, and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by Enzyme-Linked Immunosorbent Assay (ELISA) using HBsAg as coating antigen followed by a limited dilution process.Results:The results showed that by using all three formulations of adjuvants, monoclonal antibody (mAb) specific to HBsAg was successfully generated. It was also found that the mice immunized with (HBsAg + Alum) + CpG had the highest concentration of antibody production in serum and hybridoma supernatants as well as positive clones. Based on these findings, the addition of CpG ODN also induced a higher antibody response compared with Complete Freund’s Adjuvant (CFA).Conclusion:Results of this study showed that CpG and Freund adjuvants could be efficient partners for alum in the immunization period of the process of monoclonal antibody production.

1066. Immune Escape Mutant Detection Using Commercially Available Methods for Hepatitis B Surface Antigen Serological Testing

BackgroundAlthough overall infection rates of Hepatitis B virus (HBV) in the United States (US) remain stable, as many as 2.2 million persons are still chronically infected with Hepatitis B Virus (HBV)1. Persons who inject drugs (PWID) are at a higher risk of HBV infection and since 2009 three states (KY, TN, WV) have reported up to a 114% increase in cases of acute HBV infection due to higher infection rates among a non-Hispanic white populations (30–39 years), and injection drug users2. Hepatitis B vaccination is recommended as primary prevention for adults who are at increased risk for HBV infection, including PWID. However, data from the National Health Interview Survey indicate that hepatitis B vaccination coverage is low among adults in the general population3, and it is likely to be lower among injection drug users.Hepatitis B Surface Antigen (HBsAg) is the first serological marker to appear after HBV exposure and infection; this marker is included in the recommended panel for acute hepatitis diagnosis and accurate detection is necessary for early and accurate diagnosis. Serological testing challenges exist for HBsAg due to the high degree of genetic variability which can further be exacerbated by endogenous and exogenous pressures. The immuno-dominant region may have one or more mutations described as immune escape mutations which can decrease or abrogate HBsAg binding to antibodies used in immunoassays. Although the prevalence of these mutations is not well documented in the United States, international studies have shown that up to 79% of HBV-reactivated patients (vs 3.1% of control patients; p< 0.001) carry HBsAg mutations localized in immune-active HBsAg regions4.MethodsA study was conducted using a panel of 10 unique recombinant HBsAg immune escape mutants. Panel members were tested by commercially available HBsAg serological immunoassays.ResultsIt was found that although commercially available HBsAg immunoassays are the primary diagnostic tool for HBV diagnosis, not all HBsAg immune escape mutants are detected, with some method detecting as few as 5 out of 10 of these mutant samples.Figure 1 ConclusionImprovement is needed in commercially available methods for the accurate detection of HBsAg.Disclosures Robert Gish, MD, Abbott (Consultant)AbbVie (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Access Biologicals (Consultant)Antios (Consultant)Arrowhead (Consultant)Bayer (Consultant, Speaker’s Bureau)Bristol Myers (Consultant, Speaker’s Bureau)Dova (Consultant, Speaker’s Bureau)Dynavax (Consultant)Eiger (Consultant, Advisor or Review Panel member)Eisai (Consultant, Speaker’s Bureau)Enyo (Consultant)eStudySite (Consultant, Advisor or Review Panel member)Exelixis (Consultant)Fujifilm/Wako (Consultant)Genentech (Consultant)Genlantis (Consultant)Gilead (Consultant, Advisor or Review Panel member, Speaker’s Bureau)GLG (Consultant)HepaTX (Consultant, Advisor or Review Panel member)HepQuant (Consultant, Advisor or Review Panel member)Intercept (Consultant, Speaker’s Bureau)Ionis (Consultant)Janssen (Consultant)Laboratory for Advanced Medicine (Consultant)Lilly (Consultant)Merck (Consultant)Salix (Consultant, Speaker’s Bureau)Shionogi (Consultant, Speaker’s Bureau)Viking (Consultant)

Immunoreactivity pattern of monoclonal antibodies against Hepatitis B vaccine with global Hepatitis B virus genotypes

Hepatitis B surface antigen (HBsAg) specific monoclonal antibodies (mAbs) are potentially valuable therapeutic and diagnostic tool. We have previously established and characterized a panel of mAbs derived from immunized BALB/c mice with a yeast-derived recombinant HB vaccine subgentoype A2 and HBsAg subtype adw2. This study was conducted to evaluate the reactivity pattern of this anti-HBs mAbs panel with various genotypes and subgenotypes of HBV using the first WHO HBV genotype reference panel containing 15 serum samples representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H.Ten out of 21 anti-HBs mAbs were able to strongly recognize all gentopye/subtypes of HBsAg provided in the WHO reference panel. However, 10 out of 21 anti-HBs mAbs showed a moderate to profound loss of reactivity with HBV genotypes/HBsAg subtypes D2/ayw3, E/ayw4, F2/adw4, and H/adw4. Two mAbs from the second group displayed a profoundly reduced reactivity with only 1 out of 3 C2/adr genotype/subtype samples. The amino acid alignment of these 3 samples showed that this particular sample contains amino acid substitution at residue 127, which is located inside “a” determinant. This amino acid substitution, which profoundly affected the reactivity of anti-HBs antibodies, has been previously reported only in D/ayw3, E/ayw4, F/adw4, and H. Interestingly, the amino acid alignment of the samples in this WHO panel showed that P127T substitution can also be found in C2/adr. Comparing amino acids sequences inside the antigenic loop (AGL) showed that D2/ayw3 contains a T118A/P127T double substitution, E/ayw4 contains P127L/T140S, F2/adw4 contains P127L/T140S/ F158L, and H/adw4 contains P127L substitution. Therefore, amino acid variability at positions 118, 127, 140, and 158 was found to cause significant loss of reactivity with anti-HBs mAbs.Since HBsAg variability in different genotypes of HBV can profoundly affect the reactivity of anti-HBs mAbs, analytical sensitivity for HBsAg assays should be considered based on the circulating and common HBV variants in the relevant countries.

Screening and identification of hepatitis B surface antigen binding to lactoferrin in human milk

Objective Human milk of mothers with positive hepatitis B surface antigen (HBsAg) contains hepatitis B virus (HBV). However, breastfeeding does not increase the risk of mother-to-infant transmission of HBV. Previous investigations demonstrated that breast milk has a property of binding with HBsAg. This study aimed to identify the component in human milk that can bind to HBsAg. Methods This study was performed in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, from June 2015 to February 2017. Human milk samples from two postpartum women with negative HBV markers and two control samples of cow milk and goat milk were analyzed by Far-Western blot, in which highly purified recombinant yeast HBsAg was used to bind with whey proteins. Based on the results of mass-spectrum analysis, competition inhibition test was used to confirm the functioning component. Results Far-Western blot showed remarkable protein bands at the relative molecular weight of about 80 000 in both lanes of human milk, but none in the lane of cow or goat milk. Mass-spectrum analysis of the protein band indicated there were proteins sharing 28.4%-93.4% homology in amino acid sequences with five proteins with the highest homology to lactoferrin (93.4%). Further Far-Western blot with purified recombinant lactoferrin showed that lactoferrin could bind to the recombinant HBsAg. Competition inhibition test suggested that the purified recombinant lactoferrin inhibited the binding of HBsAg to its antibody in a dose-dependent manner. Conclusions This study confirms the capability of lactoferrin in human milk to combine with HBsAg, suggesting that lactoferrin can bind to HBV. Further study on whether lactoferrin can inhibit the infectivity of HBV would be valuable to clarify the reason for not increasing the risk of mother-to-infant transmission of HBV by breastfeeding. Key words: Milk, human; Lactoferrin; Hepatitis B surface antigens

Construction of bicistronic cassette for co-expressing hepatitis B surface antigen and mouse granulocyte-macrophage colony stimulating factor as adjuvant in tobacco plant

Context: The co-delivery of adjuvant and antigen has shown to be more effective for targeting the immune response than antigen alone. Therefore, designing an efficient bicistronic system is more assuring for production of both elements in the same tobacco cells as a plant model system.Objective: Comparing the efficient transient co-expression of hepatitis B surface antigen (HBsAg) and mouse granulocyte macrophage colony stimulating factor (mGM-CSF) in tobacco leaves by designing either mono or bicistronic cassettes.Materials and methods: Four expression cassettes containing tobacco etch virus (TEV) leader sequence were constructed with and without above genes in different orders. The cassettes were transferred into tobacco, Nicotiana tabacum L. (Solanaceae), leaves by agroinfiltration technique. The expression levels were compared using ELISA and western blotting and bioactivity of cytokine was assessed by in vitro proliferation of mouse GM-CSF-responsive progenitor cells.Results: Agroinfiltrated leaves contained recombinant HBsAg protein at 20–50 ng/mg and mGM-CSF at 0.2–4 ng/mg in both nonglycosylated and glycosylated forms. The highest expression obtained in HBsAg and mGM-CSF monocistronic co-agroinfiltrated leaves. The expression of mGM-CSF was 1.1 and 0.2 ng/mg in two different orders of bicistronic cassettes. The growth frequency of GM progenitors was approximately 1/187 cells for standard rGM-CSF and 3.2 times less activity for the plant produced.Discussion and conclusions: The recombinant mGM-CSF was produced less in bicistronic cassette than other forms; however, co-presenting of both vaccine candidate and adjuvant is confirmed and could be promising for amelioration of plant expression system as a means for vaccine production.

Vaccination with a DNA vaccine encoding CD317-targeting HBs antigen elicits enhanced immunity in mice

BackgroundConventional hepatitis B virus (HBV) vaccines fail to induce protective antibody titers in 5–10% of immune-competent vaccines. Therefore, safe and effective HBV vaccines are still clinically needed. MethodsIn this study, we developed a plasmid DNA vaccine encoding CD317 single-chain fragment variable (α317scFv) linked with the hepatitis B surface antigen (HBsAg) and detected the humoral and cellular immune responses elicited by this vaccine in BALB/c mice. ResultsVaccination with this fusion DNA vaccine in BALB/c mice induced more robust antiviral T cell and antibody immunity against HBsAg. Compared with mice vaccinated with control vaccine encoding HBsAg, the level of serum-circulating anti-HBsAg antibody (HBsAb) was nearly double in fusion DNA-vaccinated mice. More interesting, splenic lymphocytes isolated from fusion DNA-vaccinated mice showed more potent proliferation and IFN-γ production after being re-stimulated with recombinant HBsAg in vitro. And not only that, the cytotoxicity of fusion DNA vaccine-sensitized splenocytes was ∼3-fold higher than that of controls. ConclusionTaken together, our results reveal that the fusion DNA vaccine can induce more effective immunological protection against HBV, and is a promising candidate for preventing HBV infection.

Host cell protein testing strategy for hepatitis B antigen in Hexavalent vaccine – Towards a general testing strategy for recombinant vaccines

BackgroundRecombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine. MethodsPolyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein. ResultsUsing the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans. ConclusionsAnalysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required.

Wide dynamic range of surface‐plasmon‐resonance‐based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA

Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098-0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA.

Hepatitis B Surface Antigen Promotes the Invasion of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells by Upregulation of Toll-Like Receptor 2.

Hepatitis B virus (HBV) infection is one of the major risk factors leading to the development of hepatocellular carcinoma (HCC). Hepatitis B surface antigen (HBsAg) plays a pivotal role in HBV-related HCC pathogenesis, and Toll-like receptor (TLR) 2 is also considered to mediate tumor progression. However, the interaction between HBsAg and TLR2 in HCC progression remains unclear. Thus, the aim of the study was to explore the effect of HBsAg-TLR2 pathway on growth and invasion of HBV-related HCC cells and examine the potential mechanisms been involved. The expression of TLR2 was measured in two different HCC cell lines (HepG2 and HepG2.2.15) with or without recombinant HBsAg by real-time reverse polymerase chain reaction and Western blot. Cellular proliferation, invasion, cytokine productions, and downstream signaling pathways were also measured in TLR2-silencing HepG2.2.15 cells in response to HBsAg stimulation. The mRNA and protein levels of TLR2 were significantly elevated in HepG2.2.15 cells than those in HepG2 cells. HBsAg simulation increased proinflammatory cytokine production and invasion of HepG2.2.15 cells, while this process was inhibited by TLR2 silence. However, TLR2 siRNA transfection alone did not affect the bioactivities of tumor cells. Moreover, HBsAg increased expression of MyD88 and phosphorylation of NF-κB p50 and p38MAPK. Downregulation of TLR2 inhibited HBsAg-induced MyD88 and p-NF-κB, but not p-p38MAPK in HepG2.2.15 cells. In conclusion, HBsAg stimulation promotes the invasion of HBV-related HCC cells. TLR2/MyD88/NF-κB signaling pathway may be involved in this procession by upregulation of cytokine production. The interaction between TLR2 and HBsAg may contribute to the poor prognosis of HBV-related HCC.

Unimpaired immunogenicity of yeast-expressed hepatitis B surface antigen stored at elevated temperatures.

Global adoption of hepatitis B virus (HBV) vaccines has greatly reduced new HBV infections. Current HBV vaccines are liquid suspensions containing recombinant hepatitis B surface antigen (HBsAg) particles mixed with aluminum phosphate or aluminum hydroxide. Refrigeration (2-8°C) as recommended for vaccine transport and storage may be unachievable in certain HBV-prevalent developing countries or regions. In this study, we stored yeast-expressed HBsAg and aluminum hydroxide separately at the standard (4°C) and elevated temperature (25, 37, or 45°C) for 14, 23, and 30 days, then mixed them and used the mixture to vaccinate mice with a prime-boost program. The antisera from all the vaccinated mice successfully inhibited HBV infection of HepG2 cells stably expressing HBV receptor sodium taurocholate cotransporting polypeptide. Furthermore, no serum HBsAg was detected in vaccinated mice on Day 1 post-hydrodynamic injection (HDI) of an HBV replicon plasmid and onwards, accompanied with an increasing anti-HBsAg antibody (HBsAb) level. In contrast, serum HBsAg in phosphate-buffered saline (PBS)-injected mice peaked on Day 4 post-HDI and was cleared on Day 14 post-HDI, which coincided with appearance of HBsAb on Day 7 post-HDI, suggesting a typical HBV acute infection process. HBV DNA peaked on Day 4 post-HDI in vaccinated mice, its level significantly lower than that in PBS-injected mice on Day 7 post-HDI, and showed a higher clearance rate. Taken together, we conclude that storing recombinant HBsAg at 25, 37, or 45°C for 14-30 days does not impair its immunogenicity in mice.

Immune memory response induced in vitro by recombinant hepatitis B surface antigen challenge 13-18 years after primary vaccination.

The question of whether booster doses are required to maintain long-term protection against hepatitis B virus (HBV) after primary vaccination remains to be determined. Thus, the aim of this study was to evaluate the immune memory responses to hepatitis B surface antigen (HBsAg) challenge in vaccinated individuals through an in vitro-specific stimulation assay. Peripheral blood mononuclear cells (4 × 10(6) cells/ml) were stimulated with 50 ng/ml of recombinant HBsAg. In vitro anamnestic antibody responses, as shown by detection of high avidity antibody in culture supernatants, were found 13-18 years after primary vaccination and were not correlated with serum antibodies (r = -0.177; P = 0.377). In addition, the findings from this study indicate that immune memory against hepatitis B was well preserved in 40.0% and 60.0% of vaccinees with anti-HBs levels less than 10 IU/L or lacking serum antibodies altogether, respectively. In conclusion, the data suggest the presence of immunological memory in vaccinated individuals, including those who showed anti-HBs <10 IU/L or undetectable antibody.

Immunization and chemical conjugation of Bm95 obtained from Pichia pastoris enhances the immune response against vaccinal protein and Neisseria meningitidis capsular polysaccharide

The ectoparasite Rhipicephalus (Boophilus) microplus causes severe economic losses to the cattle industry in tropical and subtropical regions, and transmits endoparasites, such as Babesia bovis. The glycoprotein Bm95 is homologous to Bm86, a surface membrane protein of gut epithelial cells in R. microplus, and has been shown to efficiently control this ectoparasite in regions of the Americas. The immunostimulant properties of Bm86 have already been demonstrated after its coinjection with hepatitis B surface antigen (HBsAg) and the infectious bovine rhinotracheitis virus. This study evaluated the carrier and immunostimulant properties of Bm95 using low immunogenic Neisseria meningitidis capsular C polysaccharide (Men CpS) and HBsAg. We produced two polysaccharide-Bm95 conjugates by carbodiimide (MenCpSBm-c) and reductive amination (MenCpSBm-ra) methods. These conjugates were characterized and evaluated in mice. Antibody titers against Men CpS were significantly higher in mice immunized with MenCpSBm-ra (2,350±250, P<0.01) than in those immunized with MenCpSBm-c (250±75) or Men CpS (570±104). The study data indicate effective immunological memory after booster inoculation in mice immunized with MenCpSBm-ra. Additionally, significant humoral immunity against HBsAg was documented in mice coimmunized via the intranasal route with recombinant Bm95 (11,400±345) and HBsAg (128,000±250) compared with mice immunized only with HBsAg (400±40) or Bm95 (5,461±150, P<0.01). In conclusion, the immunostimulatory properties of recombinant Bm95 make it a useful element for developing safer conjugated vaccines against bacterial pathogens and for evaluation against ticks and tick-borne diseases in the context of a polyvalent veterinary vaccine.

Hepatitis B virus surface antigen as delivery vector can enhance Chlamydia trachomatis MOMP multi-epitope immune response in mice

Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. There is currently no commercially available vaccine against C. trachomatis. Major outer membrane protein (MOMP) of C. trachomatis is considered to be an ideal candidate for prophylactic vaccine. We designed a MOMP multi-epitope containing T- and B-cell epitope-rich peptides and developed hepatitis B surface antigen (HBsAg) as antigen delivery vehicle. In order to study the immunogenicity and efficacy of the candidate vaccine in a murine model of chlamydial genital infection, we engineered a recombinant plasmid expressing HBsAg and MOMP multi-epitope genes. Results of reverse transcription polymerase chain reaction and immunofluorescence assay revealed successful expression of the recombinant HBsAg/MOMP multi-epitope gene at both the transcription and translation levels. Intramuscular administration in mice was able to elicit not only antibodies against Chlamydia and HBsAg but also cytotoxic T lymphocyte activity against Chlamydia. In addition, mice inoculated with the rHBsAg were highly resistant to C. trachomatis genital infection. The rHBsAg DNA with MOMP multi-epitope appended at the C terminus of the HBsAg stimulated a stronger immune response and protective response than that appended at the N terminus. Together, our results suggested that use of a recombinant HBsAg encoding the MOMP multi-epitope could be a powerful approach to developing a safe and immunogenic C. trachomatis vaccine.

Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties

Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.

In vitro lymphocyte stimulation by recombinant hepatitis B surface antigen: A tool to detect the persistence of cellular immunity after vaccination

The study of the human immune response to hepatitis B virus (HBV) has been hampered by the lack of an adequate model to evaluate the hepatitis B surface antigen (HBsAg) specific cell response. Thus, this study was conducted to perform an in vitro analysis of the antigenic properties of recombinant HBsAg and demonstrate the influence of variables such as culture time, antigen concentration and cell density on lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) were isolated from the venous blood of vaccinated individuals, and in vitro cellular immune responses were evaluated using an HBsAg-specific proliferation assay. Lymphoproliferative responses were detected in culture systems, despite the lack of serum antibodies. Optimal results were obtained when lymphocytes were stimulated at a seeding density of 4×106cells/mL, with 50ng/mL of recombinant HBsAg protein vaccine for 3 days. Data from the present study may contribute to the development of an adequate system to evaluate the cellular immune responses to HBsAg in vaccine recipients.

Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen

To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication. Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids). The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.

Monoclonal Antibodies Against 24 kDa Surface Antigen of Hepatitis B Viruses

Hepatitis B virus (HBV) infection is one of the major public health problems worldwide. Effective control of HBV transmission in areas of high and intermediate endemicity would not be possible without vaccination of the vulnerable group. The diagnosis of acute and chronic hepatitis B infection is based on the detection of hepatitis B surface antigen (HBsAg). In this study, we prepared nine hybridomas that produced monoclonal antibodies specific for HBsAg. BALB/c mice were immunized with 24 kDa HBsAg and the immune spleen cells were fused with SP2/0 myeloma cell line. We obtained seven IgG1, one IgM, and one IgG2b positive clones. The stable hybrids were sub-cloned and ascitic fluid was prepared in the BALB/c mice. After antibody purification by protein G, the affinity column was prepared and the pure 24 kDa HBsAg from cell extract was eluted from the column. Western blot analysis showed that all monoclonal antibodies are specific for 24 kDa antigen. Since 24 kDa HBsAg is used for vaccination against hepatitis, these monoclonal antibodies are the best candidate for the isolation and purification of recombinant HBsAg from yeast expression vector by affinity column to be used for vaccination.