Removing N-terminal sequences in pre-s1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine

Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.     

Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: a mouse potency study

A standard mouse potency test was performed to evaluate the immunogenicity of recombinant hepatitis B surface antigen (HBsAg) produced in the baculovirus/insect cell expression system. Groups of NIH Swiss mice were immunized with serial four-fold amounts of either baculovirus-derived HBsAg adsorbed to aluminum sulfate or a commercially available yeast-derived recombinant HBsAg vaccine preparation. Results from these experiments showed that the effective dose of baculovirus- and yeast-derived HBsAg vaccine preparations necessary to seroconvert 50% of the animals were similar. The duration of the antibody response to HBsAg was studied in mice immunized with the highest doses of the two recombinant vaccine preparations 3 and 6 months after injection. No decrease in the anti-HBs response was observed 6 months after injection. No decrease in the anti-HBs response was observed 6 months after immunization with either of the two vaccine preparations. These results indicate that the baculovirus-derived recombinant HBsAg could serve as an alternative vaccine candidate for hepatitis B virus.     

One Step Membrane Incorporation of Viral Antigens as a Vaccine Candidate Against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures.

Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines.

The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.     

Properties of recombinant hepatitis B vaccine

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.     

Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration.

BACKGROUND: Attempts to optimize DNA vaccines in mice include using different routes of administration and different formulations. It may be more relevant to human use to carry such studies out in nonhuman primates. Here we compare different approaches to delivery of a DNA vaccine against the hepatitis B virus (HBV) in Aotus monkeys. MATERIALS AND METHODS: Thirty-two adult Aotus l. lemurinus monkeys divided into 8 groups of four were immunized with 400 microg of a DNA vaccine which encoded hepatitis B surface antigen (HBsAg). DNA in saline was administered by intradermal (ID) or intramuscular (IM) injection with needle and syringe, IM injection with the Biojector needleless injection system or combined ID (needle) and IM (Biojector). DNA formulated with cationic liposomes (CellFECTIN) was injected IM with needle or Biojector. DNA with added E. coli DNA (100 microg) was injected IM with the Biojector or ID. A ninth group of 4 monkeys was injected IM (needle) with Engerix-B, a commercial vaccine containing recombinant HBsAg (10 microg) adsorbed onto alum. Monkeys were boosted in an identical fashion to their prime at 8 weeks, but all received the protein vaccine (Engerix-B) at 16 weeks. Sera was assessed for antibodies against HBsAg (anti-HBs) by enzyme-linked imunosorbent assay (ELISA). RESULTS: The primary humoral response induced by IM delivery of the DNA vaccine was very poor. In most cases there was no detectable anti-HBs even after 2 DNA doses but the kinetics of the response to subsequent protein indicated that a memory B cell response had been induced. In contrast, following IM-administration of DNA using the Biojector, detectable anti-HBs were observed in 3 of 8 animals and evidence for immunological priming was apparent in an additional 4 of the 8 monkeys. ID injection of DNA vaccine in saline induced a potent antibody response which was augmented 6-fold by the addition of E. coli DNA. Combining ID and IM administration did not improve humoral immunity over ID injection alone. CONCLUSIONS: For immunization of primates with DNA vaccines, ID may be a preferable route to IM, although it is not clear whether the Aotus monkey is a relevant model for humans in this respect. Nevertheless, the use of the Biojector needleless injection system may improve responses with IM delivery of DNA vaccines. As well, the immunostimulatory action of E. coli DNA may be used to augment the humoral response induced by a DNA vaccine.     

One step membrane incorporation of viral antigens as a vaccine candidate against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures. Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines. The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

CPG ODN allows lower dose of antigen against hepatitis B surface antigen in BALB/c mice

We have evaluated alum, immunostimulatory cytosine guanine dinucleotide-containing oligodeoxy-nucleotides (CPG ODN), and an alum/CPG ODN combination as adjuvants with hepatitis B surface antigen, to compare their potential to allow lower doses of antigen to be used for induction of humoral responses. BALB/c mice were immunized by intramuscular injection with 0.01, 0.1 or 1.0 micro g recombinant hepatitis B surface antigen without adjuvant or with alum and/or CPG ODN added. When given without adjuvant or with alum, each 10-fold decrease in amount of antigen resulted in a similarly reduced titre of antibody against hepatitis B surface antigen. In contrast, CPG ODN, on its own or combined with alum, allowed high anti-hepatitis B surface antigen titres even with a 1000-fold reduction in amount of antigen. These findings may have important immunological and economical consequences for vaccine development.     

Vaccine against human hepatitis B virus prepared from antigen derived from human hepatoma cells in culture

Vaccine against human hepatitis B was prepared using antigen derived from hepatitis B carrier hepatoma cells grown in the interstices of a Diaflo hollow filter unit. Hepatitis B surface antigen (HBsAg) produced by these cells was purified by immune affinity chromatography, digestion with DNase and pepsin, and Sephadex G-150 separation. The Formalin-treated antigen was formulated in 20-micrograms dose on alum adjuvant with thimerosal added as a preservative. This cell culture vaccine was as potent as human plasma-derived vaccine as measured in a mouse potency assay. The vaccine proved safe in tests in chimpanzees and in human subjects who were in late stages of cancer of the central nervous system and who were receiving therapy for their condition. None of five subjects who received the vaccine developed untoward clinical reactions. Two of the subjects who received all three doses of vaccine developed antibody against HBsAg. Three persons, two given only the primary doses and one who was given all three doses but was lost to follow-up, demonstrated no response. The slow and relatively low antibody responses to the vaccine were similar to those in other immunosuppressed persons who were given vaccine of human plasma origin.     

A 49,000-dalton polypeptide bearing all antigenic determinants and full immunogenicity of 22-nm hepatitis B surface antigen particles

Spherical 22-nm hepatitis B surface antigen (HBsAg) particles with a subtype adr were purified from plasma of asymptomatic carriers of hepatitis B virus. When purified HBsAg preparation was treated with sodium dodecylsulfate in the absence of reducing agents, it yielded spherical particles with a diameter smaller than 22 nm, and in addition, a polypeptide with a molecular size of 49,000 daltons, which seemed to constitute the outer coat of HBsAg particles. The recovery of the polypeptide on the basis of optical density at 280 nm was 2%, starting from 22-nm HBsAg particles. The 49,000-dalton polypeptide apparently represented a structural unit of the surface of HBsAg particles, since it bore all common (a, Re) and subtypic (d, r) determinants with essentially the same antigenic titers as intact HBsAg particles. Furthermore, this polypeptide was equally immunogenic as 22-nm HBsAg particles in raising corresponding antibodies in mice. When the 49,000-dalton polypeptide was reduced in the presence of 2-mercaptoethanol, it cleaved into 22,000- and 27,000-dalton polypeptides with a drastic decrease in both antigenicity and immunogenicity. These results indicate the different molecular arrangements between outer coat and inner portion of HBsAg particles, and a potential application of the 49,000-dalton polypeptide as a component vaccine, owing to its strong antigenicity both in vitro and in vivo.     

Enhancing the immunogenicity of exogenous hepatitis B surface antigen-based vaccines for MHC-I-restricted T cells

Vaccination with either exogenous hepatitis B surface antigen (HBsAg) lipoprotein particles without adjuvants, or plasmid DNA encoding secreted small HBsAg stimulate long-lasting, potent antibody responses in H-2d (BALB/c) and C57Bl/6 (H-2b) mice. Vaccination with exogenous HBsAg primes MHC-I restricted cytotoxic T lymphocyte (CTL) responses to HBsAg in H-2d but not H-2b mice, while DNA vaccination primes HBsAg-specific CTL responses in both mouse strains. We defined vaccination strategies that could elicit CTL responses to exogenous HBsAg in 'low responder' C57Bl/6 mice. We found that the bacterial plasmid DNA itself, synthetic oligodeoxynucleotides containing immunostimulating sequences, or recombinant Th1 cytokines (IL12, IFNgamma) efficiently support priming of CTL responses to exogenous HBsAg in 'low responder' H-2b mice, but have only minor effects on CTL priming in 'high responder' H-2d mice in the high dose range tested. These molecularly well defined adjuvants can thus efficiently support priming of anti-viral T cell responses under 'low responder' conditions.     

不同佐剂对含S+PreS1融合抗原的乙型肝炎病毒颗粒疫苗免疫原性的影响

本研究在前期工作基础上,用CHO细胞表达的含PreS1+S融合抗原的新型基因工程HBV颗粒疫苗(HBSS1)与Al(OH)3、CpG及CpG+Al(OH)3等佐剂配伍,在Balb/C小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFNELISpot检测)。结果表明:CpG佐剂结合HBSS1颗粒疫苗可快速诱导(单针免疫)高水平的抗PreS1及S抗体,IgG2a/IgG1比率1,同时可诱导较高抗原特异的细胞免疫应答;Al(OH)3+CpG双佐剂组一次免疫后可诱导产生最高的抗S抗体滴度(1:105),其产生的抗体亚类包括IgG1、IgG2a与IgG2b;在S抗原N端(13~49aa)存在优势CTL表位。结论:CpG佐剂结合HBSS1颗粒疫苗应是发展新型治疗性乙肝疫苗的较佳选项。     

Immunoglobulin immobilized liposomal constructs for transmucosal vaccination through nasal route

The aim of the present investigation was to evaluate the prospective of surface-engineered vesicular carriers for mucosal immunization via the nasal route. IgG antibody was immobilized on the surface of hepatitis B surface antigen (HBsAg) antigen–loaded liposomes. The developed formulations were characterized on the basis of physicochemical parameters, such as morphology, particle size, polydispersity index, entrapment efficiency, and zeta potential. Liposomal formulations were then evaluated for in-process antigen stability and storage stability. In vivo studies were conducted to visualize targeting potential, localization pattern, and immunogenicity. In addition, immune response was compared with alum-HBsAg vaccine injected intramuscularly. The serum anti-HBsAg titer, obtained from the postnasal administration of IgG-coupled liposomes, was significantly higher than plain liposomes. Moreover, IgG-coupled liposomes generated both humoral (i.e., systemic and mucosal) and cellular immune responses upon nasal administration, while the alum-adsorbed antigen displayed neither cellular (cytokine level) nor mucosal (IgA) response. The formulation also displayed enhanced transmucosal transport, improved in vitro stability, and effective immunoadjuvant property. To conclude, IgG antibody-coupled liposomes may serve as novel carriers to augment the secretory immune response of antigen encapsulated in the liposomes, apparently by escalating liposome uptake via M cells, thereby rationalizing their use as a carrier adjuvant for nasal subunit vaccines.     

Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund’s adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8⁺ T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D^b-restricted and three HLA-A^*0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.     

Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies

Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system. Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1-7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg-expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN and DNA vaccines remained weak in the presence of maternally transferred anti-HBs antibodies.     

Accelerated immune response to DNA vaccines

DNA vaccines offer considerable promise for improvement over conventional vaccines. For the crucial step of delivering DNA vaccines intracellularly, electroporation (EP) has proven to be highly effective. This method has yielded powerful humoral and cellular responses in various species, including nonhuman primates. In an attempt to further improve DNA vaccination we used micron-size gold particles (which do not bind or adsorb DNA) as a particulate adjuvant which was coinjected with DNA intramuscularly into mice, followed by EP of the target site. The presence of gold particles accelerated the antibody response significantly. Maximum titers against hepatitis B surface antigen (HBsAg) were reached after one boost in 6 weeks, whereas 8 weeks were required without particles. These immunizations were effective in protecting mice against tumor challenge with cancer cells expressing HBsAg as a surrogate cancer antigen. Computer modeling of electric fields and gene expression studies indicate that gold particles do not stimulate EP and subsequent antigen expression. The particles may act as an attractant for immune cells, especially antigen presenting cells. We conclude that particulate adjuvants combined with DNA vaccine delivery by EP reduces the immune response time and may increase vaccine efficacy. This method may become valuable for developing prophylactic as well as therapeutic vaccines. The rapid response may be of particular interest in countering bio-terrorism.     

Liposomes as a Mucosal Adjuvant System: An Intranasal Liposomal Influenza Subunit Vaccine and the Role of IgA in Nasal Anti-Influenza Immunity

Abstract

Liposomes exhibit potent immunoadjuvant activity in a variety of experimental vaccine formulations. We have investigated the mucosal adjuvant activity of liposomes in an influenza subunit vaccine. Mice were immunized intranasally (I.N.) with the major surface antigen of influenza virus, hemagglutinin (HA), mixed with negatively charged liposomes. Inclusion of the liposomes in the vaccine resulted in a marked stimulation of the serum IgG response against the antigen. In addition, the liposomal preparation, but not the antigen alone, induced a significant secretory IgA (s-IgA) response, not only in the lungs and nasal cavity, but also at the mucosa of the urogenital tract. The adjuvant activity of the liposomes appeared to be independent of a physical association of the antigen with the liposomes: Stimulation of antibody responses was observed even when liposomes and antigen were administered separately in time. Serum IgG and local s-IgA responses to I.N. immunization with the liposomal vaccine were comparable to the corresponding responses induced by an influenza infection. Mice immunized with the liposomal vaccine or mice recovered from an influenza infection were completely protected from (re)infection. Protection from nasal infection was abrogated by treatment of the mice with an anti-IgA antiserum, while anti-IgG had no effect, indicating that s-IgA plays an essential role in nasal anti-influenza immunity.     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].     

Immunological and biophysical alteration of hepatitis B virus antigens by sodium hypochlorite disinfection.

Sodium hypochlorite (NaOCl) was examined as an effective disinfectant in hepatitis laboratories. Concentrations of NaOCl containing 5,600 ppm (5,600 microgram/ml) of available chlorine were found to be effective in destroying the antigenicity of hepatitis B surface antigen (HBsAg) in virion-rich plasma after an exposure time of 1 min or more. In the treatment of protein-deficient solutions containing HBsAg, smaller concentrations of available chlorine (less than 500 pm) are equally effective. Neither 17-to 25-nm HBsAg particles nor 45-nm virion particles could be detected by electron microscopy after treatment. chemical interaction of protein and NaOCl was confirmed by isoelectrofocusing of 125I-labeled HBsAg. More than 90% of the labeled material was found at pH 3.0 or lower, indicating complete antigen oxidation. Labeled HBsAg was reduced in density from 1.21 g/cm3 in CsCl to approximately 1.07 g/cm3 after treatment with NaOCl. Both hepatitis B core antigen and deoxyribonucleic acid polymerase activity were significantly reduced after interaction with hypochlorite solutions. These results show that NaOCl destroys hepatitis B antigenicity and virus structures and therefore may be utilized as a disinfectant for the virus.