[Effect of polyethylene glycol as adjuvant on hepatitis B virus DNA vaccine in vitro].

OBJECTIVE: To explore whether polyethylene glycol (PEG) as adjuvant could enhance the humoral and cellular immune responses on hepatitis B virus DNA vaccine. METHODS: C57BL/6J mice were immunized with PEG and pVAX-S2 or alone pVAX-S2. After these mice were finally immunized for 14 days, the anti-HBs IgG, T cell proliferation, the expression of cytokines and CTL in vivo were detected. RESULTS: Compared to mice immunized with alone pVAX-S2, PEG as adjuvant could increase the production of anti-HBs IgG and HBsAg specific T cell proliferation. In addition, the expression of IL-4, IFN-gamma in CD4+ T cells and IFN-gamma in CD8+ T cells was higher than control groups. The PEG/ pVAX-S2 groups could elicit significantly in vivo HBsAg specific CTL responses. CONCLUSIONS: PEG as adjuvant could enhance humoral and cellular immune responses, as well as in vivo CTL activity.

Mannosylated niosomes as adjuvant-carrier system for oral genetic immunization against hepatitis B.

Aim of the present study was to develop mannosylated niosomes as oral DNA vaccine carriers for the induction of humoral, cellular and mucosal immunity. Niosomes composed of span 60, cholesterol and stearylamine as constitutive lipids were prepared by reverse phase evaporation method and were coated with a modified polysaccharide o-palmitoyl mannan (OPM) in order to protect them from bile salt caused dissolution and enzymatic degradation in the gastrointestinal tract and to enhance their affinity towards the antigen presenting cells of Peyer's patches. Prepared niosomes were characterized in vitro for their size, shape, entrapment efficiency, ligand binding specificity and stability in simulated gastric fluid and simulated intestinal fluid. OPM coated niosomes were found to better stable in simulated GIT conditions. The immune stimulating activity was studied by measuring serum anti-HBsAg titer, secretory IgA level in intestinal and salivary secretions and cyokines level (IL-2 and IFN-gamma) in spleen homogenates following oral administration of niosomal formulations in Balb/c mice and compared with naked DNA as well as pure recombinant HBsAg injected intramuscularly. The serum anti-HBsAg titer obtained after oral administration of OPM coated niosomal formulations was although less as compared to that elicited by naked DNA and pure HBsAg administered intramuscularly, but the mice were seroprotective within 2 weeks and antibody level far above the clinically protective limit for humans was achieved. Intramuscular naked DNA and recombinant HBsAg did not elicited sIgA titer in mucosal secretions that was induced by oral administration of OPM coated niosomes. Similarly, cellular response (cytokines level) was absent in pure HBsAg treated animals. OPM coated niosomes produced humoral (both systemic and mucosal) and cellular immune response upon oral administration. The study signifies the potential of OPM coated niosomes as DNA vaccine carrier and adjuvant for effective oral immunization.

Augmented humoral and cellular immune responses to hepatitis B DNA vaccine adsorbed onto cationic microparticles.

Plasmid expressing HBV small envelope antigen was formulated with poly(lactide-co-glycolide-acid) (PLGA) and cetyltrimethylammonium bromide (CTAB) to generate highly uniform microparticles. Controlled release of DNA from these microparticles was demonstrated in vitro and in vivo using flow cytometry and confocal laser scanning microscopy with the focus on localization and quantitatively evaluation of antigen-presenting cells (APCs) involved in the expression of target antigen. Compared to mice vaccinated with naked DNA, mice immunized with PLGA-CTAB-DNA microparticles displayed a much higher percentage of CD11c+, HBsAg-expressing APCs in the draining lymph nodes at 24 h and day 14 postinoculation. In addition, a prolonged transcription of plasmid DNA was detected by RT-PCR in mice immunized with the microparticles. A significantly enhanced immunogenicity of PLGA-CTAB-DNA over naked DNA was observed in immunized mice, including higher levels of antibody production, interferon gamma (IFN-gamma) secretion and cytotoxic T lymphocyte activity. Mice immunized with PLGA-CTAB-DNA microparticles also showed greater efficacy of immunoprotection against challenge of transplanted HBsAg-expressing tumor cells. Our data suggest that controlled release of the PLGA-CTAB-DNA microparticles might involve in the mechanisms of its augmented immunogenicity and enhanced immunoprotection.

Non-ionic surfactant based vesicles (niosomes) for non-invasive topical genetic immunization against hepatitis B.

DNA vaccines are capable of eliciting both humoral as well as cellular immune responses. Liposomes have been widely employed for DNA delivery through topical route; however, they suffer from certain drawbacks like higher cost and instability. In present study, non-ionic surfactant based vesicles (niosomes) for topical DNA delivery have been developed. DNA encoding hepatitis B surface antigen (HBsAg) was encapsulated in niosomes. Niosomes composed of span 85 and cholesterol as constitutive lipids were prepared by reverse phase evaporation method. Prepared niosomes were characterized for their size, shape and entrapment efficiency. The immune stimulating activity was studied by measuring serum anti-HBsAg titer and cyokines level (IL-2 and IFN-gamma) following topical application of niosomes in Balb/c mice and results were compared with naked DNA and liposomes encapsulated DNA applied topically as well as naked DNA and pure recombinant HBsAg administered intramuscularly. It was observed that topical niosomes elicited a comparable serum antibody titer and endogenous cytokines levels as compared to intramuscular recombinant HBsAg and topical liposomes. The study signifies the potential of niosomes as DNA vaccine carriers for effective topical immunization. The proposed system is simple, stable and cost effective compared to liposomes.

A novel microencapsulated peptide vaccine against hepatitis B.

A 48 amino acid synthetic peptide (S121/48) representing residues 121-167 of the major envelope protein of hepatitis B virus (HBsAg) was successfully encapsulated into polylactide co-glycolide microspheres. A single immunization of the microspheres in BALB/c (H-2d) mice resulted in the production of high-titre anti-HBs antibodies (IgG1-type). The response was long lasting and was superior to that obtained using the same peptide adjuvanted with Freund's complete adjuvant. A T-cell memory response was detected 10 weeks after a booster immunization (approximately 35 weeks after initial immunization) as measured by in-vitro re-stimulation of splenocytes. This study illustrates the feasibility of a single dose vaccine for hepatitis B and is, to our knowledge, the first demonstration of a synthetic peptide immunogen inducing anti-native protein antibodies of comparable titre to those obtained with conventional vaccines for hepatitis B. The suitability of a synthetic peptide vaccine for hepatitis B is discussed.

Liposome-mediated DNA vaccination: the effect of vesicle composition.

Liposome-entrapped DNA has been shown to enhance the potency of DNA vaccines, possibly by facilitating uptake of the plasmid by antigen-presenting cells (APC). In this paper, we have investigated the influence of the liposomal composition and surface charge on such potency. Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was entrapped within cationic liposomes of various compositions and surface charges with high efficiency (88-97% of the amount used) by the dehydration-rehydration method that generates dehydration-rehydration vesicles (DRV). Cryo-electron microscopy revealed that DNA-containing DRV (DRV(DNA)) were multilamellar. In immunisation studies, female Balb/c mice were given two to four intramuscular injections of 10 microg naked or liposome-entrapped pRc/CMV HBS and bled at time intervals. Results indicate that the lipid composition of the DRV(DNA) influences the strength of the humoural response (immunoglobulin (Ig)G subclasses) with inclusion of dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylethanolamine (PE) in the liposomal structure contributing to greater responses. DRV(DNA) in which the DOPE or PE were omitted or substituted with cholesterol led to significant reduction of humoural responses against the encoded antigen. Replacing phosphatidylcholine (PC) in the DRV(DNA) with the high-melting distearoyl phosphatidylcholine also contributed to lower responses. In other experiments, IgG responses were monitored in mice immunised with pRc/CMV HBS entrapped in DRV composed of PC and DOPE as before but incorporating increasing amounts of DOTAP (1-16 micromol). Maximal IgG responses were observed at 10 weeks after the first of four injections and suggested a trend of higher responses when 4 or 8 micromol DOTAP was present in the DRV(DNA) formulation. Cell-mediated immunity (measured in terms of endogenous antigen-specific splenic interferon-gamma) in mice immunised with pRc/CMV HBS entrapped in liposomes composed of PC, DOPE and DOTAP (16:8:4 molar ratio) was much greater than in animals treated with naked plasmid. These results indicate that liposome-mediated DNA immunisation is more effective than the use of naked DNA, and also suggest that the presence of fusogenic phosphatidylethanolamine in DRV in conjunction with a low-melting phosphatidylcholine and an appropriate content of cationic lipid might contribute to more effective liposomal DNA vaccines. The notion that liposomes improve immune responses to the plasmid-encoded vaccine by facilitating the latter's uptake by APC was supported by the observation that in Balb/c mice injected intramuscularly with liposome-entrapped pCMV. Enhanced green fluorescent protein, expression of the gene in terms of fluorescence intensity in the draining lymph nodes, was much greater than in animals treated with the naked plasmid.

Cationic polymers that enhance the performance of HbsAg DNA in vivo.

In this paper, different cationic polymers were investigated as a DNA delivery system both in vitro in dendritic and muscle cells and in vivo, in a murine model. Expression of the reporter gene beta-galactosidase was used in order to determine the in vitro transfection efficiency of these polymer-DNA complexes (polyplexes) and both specific mRNA and protein expression were monitored in parallel with polyplex toxicity on the cells. Interestingly, the enhancing expression activities of the different polyplexes were tissue-dependent, implying that they may gain entrance to the cells through specific receptors. Subsequently, complexes of polymers and DNA plasmid (pCMV-S) encoding the human hepatitis B virus (HBV) surface antigen (HBsAg) were injected into the skeletal muscles of BALB/c mice. Higher levels of both HBsAg local expression in the tibial anterior muscles and systemic humoral immune responses were detected when the selected polymers complexed with pCMV-S were compared to those complexed with pCMV-S alone. Induction of immunoglobulin G2a (IgG2a) against HbsAg in the serum of pCMV-S-polyplex vaccinated mice varied with the polymer used, suggesting that polyplex-mediated DNA vaccination can potentially modulate the type of helper T cell immunity (Th). The effect of some polyplexes to switch the host immune response more towards a Th1 response may be associated with their differential efficiency to transfect dendritic cells and/or other antigen-presenting cells (APC) as was observed in vitro. These results suggest that the investigated cationic polymers can be effective as delivery/adjuvant compounds for DNA.