A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine.

BACKGROUND: Highly pathogenic avian influenza A/H5N1 virus remains a potential pandemic threat, and it is essential to continue vaccine development against this subtype. A local mucosal immune response in the upper respiratory tract may stop influenza transmission. It is therefore important to develop effective intranasal pandemic influenza vaccines that induce mucosal immunity at the site of viral entry. OBJECTIVES: We evaluated the humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c-di-GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co-adjuvanting an experimental adjuvant (c-di-GMP) with chitosan. METHODS: BALB/c mice were intranasally immunised with two doses of subunit NIBRG-14 (H5N1) vaccine (7·5, 1·5 or 0·3 µg haemagglutinin (HA) adjuvanted with chitosan (CSN), c-di-GMP or both adjuvants. RESULTS: All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 7·5 µg HA CSN and c-di-GMP-adjuvanted groups. The c-di-GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 0·3 µg HA group. CSN elicited a Th2 response, whereas c-di-GMP induced higher frequencies of virus-specific CD4+T cells producing one or more Th1 cytokines (IFN-γ+, IL-2+, TNF-α+). A combination of the two adjuvants demonstrated effectiveness at 7·5 µg HA and triggered a more balanced Th cytokine profile. CONCLUSION: These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines.

Chitosan-based delivery systems for mucosal vaccines.

INTRODUCTION: Mucosal vaccine development faces several challenges and opportunities. Critical issues for effective mucosal vaccination include the antigen-retention period that enables interaction with the lymphatic system, choice of adjuvant that is nontoxic and induces the required immune response and possibly an ability to mimic mucosal pathogens. Chitosan-based delivery systems are reviewed here as they address these issues and hence represent the most promising candidates for the delivery of mucosal vaccines. AREAS COVERED: A comprehensive literature search was conducted, to locate relevant studies published within the last 5 years. Mucosal delivery via nasal and oral routes is evaluated with respect to chitosan type, dosage forms, co-adjuvanting with novel adjuvants and modulation of the immune system. EXPERT OPINION: It is concluded that chitosan derivatives offer advantageous opportunities such as nanoparticle and surface charge manipulation that facilitate vaccine targeting. Nevertheless, these technologies represent a longer-term goal. By contrast, chitosan (unmodified form) with or without a co-adjuvant has significant toxicology and human data to support safe mucosal administration, and thus has the potential for earlier product introduction into the market.

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine.

BACKGROUND: Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose-sparing strategies and an efficient mass-vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. OBJECTIVES: This study has investigated the immune response and the dose-sparing potential of a chitosan-adjuvanted intranasal H5N1 (RG-14) subunit (SU) vaccine in a mouse model. METHODS: Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)-adjuvanted SU vaccine [7·5, 15 or 30 µg haemagglutinin (HA)] or with a non-adjuvanted SU vaccine (30 µg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG-14) virus (WV) vaccine (15 µg HA), and the control group consisted of unimmunised mice. RESULTS: The chitosan-adjuvanted SU vaccine induced an immune response superior to that of the non-adjuvanted SU vaccine. Compared with the non-adjuvanted SU group, the chitosan-adjuvanted SU vaccine elicited higher numbers of influenza-specific antibody-secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T-helper 1/T-helper 2 cytokine response in the chitosan-adjuvanted SU groups, and these groups had an increased percentage of virus-specific CD4(+) T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non-adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan-adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T-helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4(+) Th1 cells simultaneously secreting three different cytokines (INFγ(+) , IL2(+) and INFα(+) ). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. CONCLUSION: The cross-clade serum reactivity, improved B- and T-cell responses and dose-sparing potential of chitosan show that a chitosan-adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.

Nasal vaccine innovation.

The current vaccine market is gaining momentum in the development of alternative administration routes namely intranasal, oral, topical, pulmonary, vaginal, and rectal; the nasal route offers the most promising opportunity for vaccine administration. It can enhance convenience, safety, elicit both local and systemic immune responses; thus potentially provide protection from pathogens at the site of entry. Nasal vaccine innovation comes with both opportunities and challenges. The innovative strategies used by industry and researchers to overcome the hurdles are discussed in this article: these include live-attenuated vaccines, adjuvants, mucoadhesives, particulate delivery systems, virus-like particles, vaccine manufacture, challenges of regulatory authorities, and the nasal vaccine impact on market potential. Critical issues for effective nasal vaccination are the antigen-retention period that enables its interaction with the lymphatic system and choice of an adjuvant that is nontoxic and induces the required immune response. Co-adjuvanting by means of a mucoadhesive technology addresses some of these issues. ChiSys(®), a natural bioadhesive with proven intranasal safety profile, has already demonstrated efficacy for several nasally delivered vaccines including norovirus. With the looming threat of a pandemic, alternatives such as intranasal vaccination will ultimately facilitate greater public compliance and rapid mass global vaccination.

Intranasal delivery of recombinant human growth hormone (somatropin) in sheep using chitosan-based powder formulations.

The effectiveness of chitosan in promoting the intranasal bioavailability of recombinant human growth hormone (hGH) has been evaluated. hGH was formulated with chitosan to produce a powder blend (Formulation A) and granules (Formulation B) for intranasal administration. The in vivo pharmacokinetic performance of the formulations was evaluated in a group of six sheep in a randomised crossover study. A subcutaneous injection of hGH solution was administered as a control. The intranasal and subcutaneous doses of hGH were 0.3 and 0.03 mg/kg, respectively. The intranasal formulations appeared to be well tolerated. Mean bioavailabilities of hGH from Formulations A and B were 14 and 15%, respectively relative to subcutaneous injection. It is concluded that chitosan-based intranasal powder formulations may provide a practical means for non-injectable delivery of hGH and, potentially, other therapeutic protein molecules.

Effect of chitosan on the intranasal absorption of salmon calcitonin in sheep.

The effects of a chitosan-based delivery system on the pharmacokinetics of intranasally administered salmon calcitonin (sCT) were investigated in a sheep model. In particular, the feasibility of producing a formulation with a comparable or improved bioavailability and/or less variability than the currently marketed nasal product (Miacalcin nasal spray, Novartis Pharmaceuticals) was assessed. A comparator (control) formulation comprising sCT solution was also tested. Sheep (n=6) were dosed intranasally according to a randomized crossover design. The intranasal sCT dose was 1100 IU (equivalent to approximately 17 IU kg-1). After completion of the nasal dosing legs, five of the sheep received 300 IU sCT (equivalent to approximately 5 IU kg-1) by subcutaneous injection to estimate relative bioavailability. After intranasal or subcutaneous dosing, serial blood samples were taken and plasma separated by centrifugation before measuring sCT concentrations by ELISA. Pharmacokinetic (non-compartmental) and statistical (analysis of variance or non-parametric alternative) analyses were performed. No systemic or local adverse effects were observed following intranasal or subcutaneous administration of sCT. The mean relative bioavailability of sCT from the chitosan solution was improved twofold compared with Miacalcin nasal spray and threefold compared with sCT control solution. Inter-animal variability in sCT absorption appeared to be lower with use of the chitosan-based solution compared with the control solution or commercial product. Based on the reported sheep data, a chitosan delivery system could offer the potential to significantly improve the intranasal absorption of sCT and reduce the variability in absorption. In the clinical setting, this may allow relatively lower doses of the drug to be given intranasally and/or lead to improvements in the efficacy or quality of intranasal therapy.

Effective nasal influenza vaccine delivery using chitosan.

Nasal influenza vaccination may prove to be a good alternative to parenteral injection because of the enhancement of the mucosal immune response and the ease of vaccine administration. This study investigated the use of chitosan, a bioadhesive polymer, as a nasal delivery system with inactivated, subunit influenza vaccine. Subjects received nasally 15 or 7.5 microg of the standard inactivated trivalent influenza vaccine with chitosan or 15 microg of the same vaccine intramuscularly. Serum haemagglutination inhibition (HI) titres for all three vaccine components were measured prior to, and at time points up to 14 weeks after dosing. Serum HI titres following intranasal vaccination with the nasal chitosan-influenza vaccine met the criteria set by the Committee for Proprietary Medicinal Products in terms of seroprotection rate, seroconversion rate and mean fold increase of HI titre for at least one of the three antigens in the vaccination schedules used. These data show that nasal immunisation with chitosan plus trivalent inactivated influenza is a potentially effective, easily-administered form of vaccination.

Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan.

We previously reported that intranasal immunization with a non-toxic mutant cross-reacting material (CRM)197 of diphtheria toxin, formulated with chitosan, generated protective neutralizing antibodies in mice and guinea pigs. Furthermore, we demonstrated that intranasal delivery of a powder formulation of the CRM197-based vaccine was well tolerated and significantly boosted antibody responses in adult volunteers. Here we report that intranasal booster immunization with CRM197 alone or with chitosan induced systemic T cell responses. We addressed for the first time the induction of T cell subtypes following intranasal vaccination in humans. Intranasal vaccination with CRM197, like parenteral immunization with a conventional diphtheria toxoid vaccine, enhanced antigen-specific IFN-gamma production. However, formulation of the nasal diphtheria vaccine with chitosan significantly augmented Th2-type responses, which correlated with protective levels of toxin-neutralizing antibodies in intranasally boosted individuals. The results suggest that vaccines capable of inducing strong Th2-type responses, such as CRM197 formulated with chitosan, have potential for the development of a protective mucosal vaccine against diphtheria in humans. Furthermore, our findings demonstrate that mucosal subunit vaccines with appropriate delivery systems have considerable potential for booster immunization of adults.

Protective levels of diphtheria-neutralizing antibody induced in healthy volunteers by unilateral priming-boosting intranasal immunization associated with restricted ipsilateral mucosal secretory immunoglobulin a.

Subunit intranasal vaccines offer the prospect of inducing combined systemic-mucosal immunity against mucosally transmitted infections such as human immunodeficiency virus. However, although human studies have demonstrated the induction of active immunity, secretory immunoglobulin A (sIgA) responses are variable, and no study has demonstrated protection by accepted vaccine-licensing criteria as measured by direct toxin-neutralizing activity. Using the genetically inactivated mutant diphtheria toxoid CRM(197) in a bioadhesive polycationic polysaccharide chitosan delivery system, we found that a single nasal immunization was well tolerated and boosted antitoxin neutralizing activity in healthy volunteers, which could be further boosted by a second immunization. The neutralizing activity far exceeded accepted protective levels and was equivalent to that induced by standard intramuscular vaccine and significantly greater than intranasal immunization with CRM(197) in the absence of chitosan. A striking but unexpected observation was that although unilateral intranasal immunization induced circulating antitoxin antibody-secreting cells, a nasal antitoxin sIgA response was seen only after the second immunization and only in the vaccinated nostril. If these data are reproduced in larger studies, an intranasal diphtheria vaccine based on CRM(197)-chitosan could be rapidly licensed for human use. However, a restricted sIgA response suggests that care must be taken in the priming-boosting strategy and clinical sampling techniques when evaluating such vaccines for the induction of local mucosal immunity.