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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.

Please cite this paper as: Svindland et al. (2012) A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses 10.1111/irv.12056000(000), 000–000. 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.

Please cite this paper as: Svindland et al. The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750-2659.2011.00271.x. 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.

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.

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.

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.

Morphine administered nasally to humans as a simple solution is only absorbed to a limited degree, with a bioavailability of the order of 10% compared with intravenous administration. This article describes the development of novel nasal morphine formulations based on chitosan, which, in the sheep model, provide a highly increased absorption with a 5- to 6-fold increase in bioavailability over simple morphine solutions. The chitosan-morphine nasal formulations have been tested in healthy volunteers in comparison with a slow i.v. infusion (over 30 min) of morphine. The results show that the nasal formulation was rapidly absorbed with a T(max) of 15 min or less and a bioavailability of nearly 60%. The shape of the plasma profile for nasal delivery of the chitosan-morphine formulation was similar to the one obtained for the slow i.v. administration of morphine. Furthermore, the metabolite profile obtained after the nasal administration of the chitosan-morphine nasal formulation was essentially identical to the one obtained for morphine administered by the intravenous route. The levels of both morphine-6-glucuronide and morphine-3-glucuronide were only about 25% of that found after oral administration of morphine. It is concluded that a properly designed nasal morphine formulation (such as one with chitosan) can result in a non-injectable opioid product capable of offering patients rapid and efficient pain relief.

Purpose. To investigate whether the widely accepted advantages associated with the use of chitosan as a nasal drug delivery system, might be further improved by application of chitosan formulated as nanoparticles.

A variety of different types of nasal vaccine systems has been described to include cholera toxin, microspheres, nanoparticles, liposomes, attenuated virus and cells and outer membrane proteins (proteosomes). The present review describes our work on the use of the cationic polysaccharide, chitosan as a delivery system for nasally administered vaccines. Several animal studies have been carried out on influenza, pertussis and diphtheria vaccines with good results. After nasal administration of the chitosan-antigen nasal vaccines it was generally found that the nasal formulation induced significant serum IgG responses similar to and secretory IgA levels superior to what was induced by a parenteral administration of the vaccine. Animals vaccinated via the nasal route with the various chitosan-antigen vaccines were also found to be protected against the appropriate challenge. So far the nasal chitosan vaccine delivery system has been tested for vaccination against influenza in human subjects. The results of the study showed that the nasal chitosan influenza vaccine was both effective and protective according to the CPMP requirements. The mechanism of action of the chitosan nasal vaccine delivery system is also discussed.

In recent years microspheres or microparticles produced from biodegradable polymers such as poly( d,l -lactide) (PLA) and poly( d,l -lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been investigated for administration via parenteral, oral, and intranasal routes. These microparticles allow the controlled release of vaccines with an aim to reduce the number of doses for primary immunisation or to develop single dose vaccines. The polymer materials have been widely regarded as being of minimal toxicity. Evaluation of candidate systems in animal studies have shown antibody levels and cell responses similar to or greater than those observed with adjuvants such as alum. However, there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. An alternative approach would be to adsorb antigens to the surface of biodegradable polymer particles. Polymeric lamellar substrate particles (PLSP), produced by a simple precipitation of PLA, are suitable for this purpose. The adsorption of antigens onto these particles is a simple procedure. It avoids pH changes due to bulk polymer degradation and the use of solvents and therefore will be less damaging to the vaccine. Moreover, such systems will be much easier to scale up for a clinical study and eventual manufacture. The aim of this article is to discuss the preparation and physical characteristics of PLSP, antigen adsorption, in vivo efficacy of PLSP antigen systems and to consider the potential of PLSP as controlled release adjuvants for protein, peptide or viral vaccines.

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.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.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 effect of L-α-lysophosphatidylcholine (LPC) on the nasal absorption of human growth hormone (hGH) in anaesthetised rats. conscious rabbits and sheep, has been examined. LPC was shown to be an effective enhancer of nasal hGH absorption in three different animal models. In the rat. concentrations of LPC in the range 0–1.0% co-administered with hGH gave absorption values, relative to subcutaneous administration (S/C), from 2.3 to 16.3%. In the rabbit, concentrations of LPC of 0 and 0.2% co-administered with hGH gave absorption values, relative to S/C. of 1.4 and 72.8% respectively. In the sheep, concentrations of LPC in the range 0–0.59% co-administered with hGH gave absorption values, relative to S/C, from 0.2 to 16.0%. The amount of hGH absorbed was directly proportional to the concentration of LPC co-administered, and in sheep this relationship was linear, with a correlation coefficient of 0.99. The shapes of the plasma concentration vs time curves were similar in all three species.

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.

Respiratory syncytial virus (RSV), an important pathogen of the lower respiratory tract, is responsible for severe illness both in new born and young children and in elderly people. Due to complications associated with the use of the early developed vaccines, there is still a need for an effective vaccine against RSV. Most pathogens enter the body via mucosal surfaces and therefore vaccine delivery via routes such as the nasal, may well prove to be superior in inducing protective immune responses against respiratory viruses, since both local and systemic immunity can be induced by nasal immunisation. Previously we have shown that intradermal immunisation of a plasmid DNA encoding the CTL epitope from the M2 protein of RSV induced protective CTL responses. In the present study, the mucosal delivery of plasmid DNA formulated with chitosan has been investigated. Chitosan is a polysachharide consisting of copolymers of N-acetylglucosamine and glucosamine that is derived from chitin, a material found in the shells of crustacea. Intranasal immunisation with plasmid DNA formulated with chitosan induced peptide- and virus-specific CTL responses in BALB/c mice that were comparable to those induced via intradermal immunisation. Following RSV challenge of chitosan/DNA immunised mice, a significant reduction (P

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.

In recent years, several strategies have been under investigation to achieve safe and effective immunisation, in terms of new antigens, adjuvants and routes of vaccination. The latter include mucosal sites such as oral, rectal, vaginal and nasal. Biodegradable microparticles produced from polymers such as poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been extensively studied for immunisation. These microparticles allow controlled release of vaccines with the aim to develop as single dose vaccines. However there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. Polymeric lamellar substrate particles (PLSP) produced by simple precipitation of PLA, form a novel polymeric system for the adsorption of antigens. This procedure avoids pH changes, exposure to organic solvents and hence allows the integrity of the antigen to be retained. The aim of this article is to discuss the factors affecting the characteristics of PLSP and adsorption of antigens onto PLSP and consider their potential as adjuvants for the nasal delivery of protein, peptide or viral vaccines.

This paper investigates the effect of starch microspheres on the absorption enhancing efficiency of various enhancer systems in formulations with insulin after application in the nasal cavity of sheep. The enhancers studied were lysophosphatidylcholine, glycodeoxycholate and sodium taurodihydroxyfusidate, a bile salt derivative. The enhancers were selected on the basis of their perceived or proven mechanism of action and worked predominantly by interacting with the lipid membrane. The bioadhesive starch microspheres were shown to increase synergistically the effect of the absorption enhancers on the transport of the insulin across the nasal membrane. Dependent on the potency of the enhancer system the increment in absorption enhancement was shown to be from 1.4 times to 5 times that obtained for the absorption enhancer in solution.

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 μg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).

Mice were intranasally immunised with a mixture of Bordetella pertussis filamentous haemagglutinin (FHA) and recombinant pertussis toxin, PT-9K/129G (rPT) in combination with chitosan. For both antigens, this formulation induced systemic responses as measured by serum IgG and also mucosal responses as measured by secretory IgA in lung lavage and nasal washes. Immunosorbant assays were used to measure these responses. Both the systemic and mucosal responses were considerably higher than those produced when a mixture of rPT and FHA was administered nasally without chitosan. In comparison, intraperitoneally administered rPT/FHA adsorbed to Alhydrogel elicited only a systemic response, and nasal chitosan solution produced neither systemic nor mucosal response. This study clearly demonstrated that chitosan potentiated the serum and mucosal immune responses to nasally administered FHA and rPT in mice. Hence, this nasal chitosan delivery system has potential as a new non-injectable vaccine for the prophylaxis of whooping cough.