Chitosan:ß-glucan particles as a new adjuvant for the hepatitis B antigen.

The development of new vaccine adjuvants is urgently needed not only to enable new routes of vaccine administration but mostly to go beyond protective humoral immunity, often insufficient to fight infectious diseases. The association of two or more immunopotentiators or mimicking pathogen physicochemical properties are strategies that can favor powerful and more balanced Th1/Th2 immune responses. Therefore, the present work aimed to combine both chitosan and ß-glucan biopolymers in the same particle, preferably with surface ß-glucan localization to simulate the cell wall of some pathogens and to stimulate the immune cells expressing the Dectin-1 receptor. Chitosan:ß-glucan particles (ChiGluPs) were developed through a chitosan precipitation method. The chitosan was precipitated into a ß-glucan alkaline solution followed by genipin crosslink. The optimized method produced particles with a mean diameter of 837 nm for ChiPs and 1274 nm for ChiGluPs. ß-glucan surface location was confirmed by zeta potential measurements (+24 mV for ChiGluPs and +36 mV for ChiPs) and zeta potential titration. These new particles showed high antigen loading efficacy and low cytotoxicity. Mice vaccination studies revealed that both ChiPs and ChiGluPs had an adjuvant effect for the hepatitis B surface antigen (HBsAg), with ChiGluPs resulting in serum anti-HBsAg total IgG 16-fold higher than ChiPs, when administered with 1.5 µg HBsAg per dose. Specifically, IgG1 subclass was 5-fold higher and IgG3 subclass was 4-fold higher for ChiGluPs comparing to ChiPs. Overall, the preparation method developed allowed the advantageous combination of ß-glucan with chitosan, without chemical functionalization, which represents an additional step toward tailor-made adjuvants production using simple precipitation techniques.

A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine.

Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding protein that was discovered through screening a human liver cDNA expression library, was introduced into hepatitis B vaccine. A good laboratory practice, non-clinical safety evaluation was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vaccine. The results indicate that SBP could enhance the HBsAg-specific immune response, thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained here warrant further investigation of SBP as a vaccine adjuvant.

Oral delivery of microparticles containing plasmid DNA encoding hepatitis-B surface antigen.

The role of albumin-based chitosan microparticles on enhancing immune response of plasmid DNA (pDNA) to hepatitis-B surface antigen (HBsAg) vaccine after oral administration was investigated in mice. The pDNA encoding HBsAg was entrapped in albumin microparticles using a one-step spray drying technique optimized in our laboratory. The encapsulated particles were also characterized in vitro for their shape, size, encapsulation efficiency, content, and stability. Albumin microparticles could protect the DNA from nuclease degradation as confirmed in our agarose gel study. Further immune modulating effect was studied in our formulation by measuring IgG antibodies in serum as well as IgA antibodies in fecal extracts. The mice were immunized with a prime dose of 100 µg of pDNA in microparticle formulations with and without interleukins biweekly until week 7 followed by a booster dose of equivalent strength on week 33 to compare the response with the subcutaneous group. The oral immunization with the pDNA to HBsAg microparticles gave significantly higher titer level of both sIgA and IgG at week 9 and 34, respectively, in oral vaccine with interleukins group when compared with the subcutaneous group. Thus, we observed an augmentation of both humoral and cellular immune responses for prolonged periods after immunization.

CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study.

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) act as potent Th1-like immune enhancers with many antigens in animal models. We have extended these observations to the first clinical evaluation of the safety, tolerability and immunogenicity of CPG 7909 when added to a commercial HBV vaccine. In a randomized, double-blind phase I dose escalation study, healthy volunteers aged 18-35 years were vaccinated at 0, 4 and 24 weeks by intramuscular injection with Engerix-B (GlaxoSmithKline). The regular adult dose of 20 microg recombinant hepatitis B surface antigen (HBsAg) adsorbed to alum was administered mixed with saline (control) or with CPG 7909 at one of three doses (0.125, 0.5 or 1.0 mg). HBsAg-specific antibody responses (anti-HBs) appeared significantly sooner and were significantly higher at all timepoints up to and including 24 weeks in CPG 7909 recipients compared to control subjects (p< or = 0.001). Strikingly, most CpG 7909-vaccinated subjects developed protective levels of anti-HBs IgG within just two weeks of the priming vaccine dose. A trend towards higher rates of positive cytotoxic T cell lymphocyte responses was noted in the two higher dose groups of CPG 7909 compared to controls. The most frequently reported adverse events were injection site reactions, flu-like symptoms and headache. While these were more frequent in CPG 7909 groups than in the control group (p<0.0001), most were reported to be of mild to moderate intensity regardless of group. In summary, CPG 7909 as an adjuvant to Engerix-B was well-tolerated and enhanced vaccine immunogenicity. CPG 7909 may allow the development of a two-dose prophylactic HBV vaccine.

The assessment of local tolerance, acute toxicity, and DNA biodistribution following particle-mediated delivery of a DNA vaccine to minipigs.

Particle-mediated DNA delivery was used to administer a DNA vaccine against Hepatitis B to minipigs. The study represented one arm of the safety evaluation program for this product and was designed to assess local tolerance, acute toxicity, and biodistribution of the DNA plasmid. The vaccine was given to 4 groups of minipigs that were sacrificed at 2, 28, 56, or 141 days after treatment. The procedure was well tolerated with mild local skin reactions at 2 days postdosing and no evidence of systemic toxicity. By 28 days the skin lesions had regressed apart from a low grade perivascular mononuclear cell infiltrate in the upper dermis, together with a small number of phagocytosed gold particles. This infiltrate persisted up to 141 days. The expressed HBsAg was detected by immunohistochemistry in keratinocytes (usually in association with an intranuclear gold particle) at 2 days but not at later time points. Polymerase chain reaction (PCR) was used to assay treatment sites and selected internal organs to evaluate biodistribution and persistence of the DNA plasmid. At 2 days the plasmid was detected in the treatment sites and also in the inguinal lymph nodes. At day 57 it was present in the treatment sites only and by day 141 appeared to have cleared. The results from this study demonstrate that particle-mediated gene delivery was well tolerated in the minipig. The biodistribution and persistence of the plasmid was within acceptable limits for this type of vaccine. As the minipig is regarded as a good model for humans these data support the concept that particle-mediated DNA delivery will be safe in human clinical applications.