Final Report Summary - NASPANVAC (Nasal pandemic influenza vaccine)
Executive summary:
The NASANVAC project was undertaken in response to the need to develop an effective user friendly, heat stable vaccine to control the highly pathogenic form of avian influenza virus (H5N1) which has the potential to cause another global influenza pandemic. A significant number of deliverables have been achieved, despite the challenges that arose during the project. Crucially, the guanosine monophosphate (GMP) surface subunit antigen of H5N1 strain NIBRG-14 raised in eggs was promptly sourced from a new supplier (Novartis) and a material transfer agreement (MTA) signed permitting its use in preclinical studies after the first supplier (Solvay) could not supply the antigen subsequent to changes in corporate structure and management. A bridging study in the mouse confirmed that the Novartis antigen was sufficiently similar to the egg-based antigen originally sourced from Solvay and thus, would be suitable for use in preclinical and clinical studies.
Formulation development work has confirmed that liquid and powder formulations of H5N1 adjuvanted with the biopolymer chitosan are stable and suitable for clinical use. Serological assays (surrogate correlates of protection) in mouse and ferret sera were initially established to enable the evaluation of vaccine efficacy. Preclinical studies in mice and ferrets have confirmed that both chitosan glutamate (CSN) and trimethyl-chitosan (TM-CSN) as solutions are efficacious in augmenting serology responses. Mouse studies further indicated that it is possible to enhance the humoral and cellular responses obtained after administering two doses of an intranasal CSN-adjuvanted vaccine by first priming with a subcutaneous injection of antigen. CSN was also shown to be a safe mucosal adjuvant and significantly no pro-inflammatory response was observed in the hypothalamus of the brain (allaying some concerns over Bell's palsy-like adverse effects which have manifested in clinical practice after administration of some intranasal influenza vaccines). Based on collective findings, the consortium initially selected a CSN liquid formulation to proceed to a phase I proof-of-principle clinical study, which could potentially be supported without the need for further preclinical toxicology. Following the decision by Novartis not to supply antigen for clinical use a definitive challenge study to establish efficacy was conducted in the ferret model using liquid vaccine formulations based on both CSN and TM-CSN. A significant aspect of this study was as a comparison of intratracheal and intranasal challenge, the first time such studies have been attempted.
In this project we planned to develop a prototype nasal H5N1 vaccine, which could be delivered through a commercial nasal delivery applicator and which showed a positive immunogenicity response in humans; thereby forming the basis for further exploitation of the project results in terms of phase II / III clinical trials. The specific objective of conducting a phase I clinical trial was not met due to non-availability of suitable GMP antigen. Nevertheless, the excellent challenge study data obtained in the ferret should allow strategic decisions to be made over further development of an intranasal vaccine. The data clearly demonstrated protective immunity against lethal H5N1 A/Vietnam/1194/2004 virus infection following intranasal vaccination adjuvanted with both CSN and TM-CSN, though the latter appeared to be more efficacious in the recognised model of human influenza, the ferret. In view of the excellent safety profile of chitosan, these data are highly encouraging given that traditionally vaccine adjuvants tend to be relatively more toxic moieties. Thus, the risk: benefit ratio for a chitosan-based vaccine may be favourable when compared with that of more potent adjuvants. A H5N1 pandemic could have a major impact on public services, making self-reliant treatment and management of the disease mandatory. The NASPANVAC project has clearly demonstrated the potential to develop a nasal vaccine which could be self-administered and also enable rapid mass vaccination with minimum aid from specialist healthcare providers.
Project context and objectives:
Annual outbreaks of influenza A and B, and pandemics of influenza A, are responsible for substantial mortality and morbidity, particularly in high-risk groups including the elderly and those with chronic underlying medical conditions. Vaccines are the most effective means of defence against influenza to prevent infection and control its spread. Despite the availability of effective parenteral vaccines, influenza still incurs considerable medical and socio-economic costs. Barriers limiting vaccine uptake include the intramuscular (IM) route of injection, and the perception of vaccine ineffectiveness. Furthermore the capacity to manufacture to meet a global demand is limited.
Delivery of an influenza vaccine by the nasal route, targeted to the mucosal site of virus entry and principal location of its replication, offers potential advantages over parenteral delivery of the vaccine. While conventional IM influenza vaccines are effective at inducing serum immunoglobulin G (IgG) neutralising antibodies, they are poor at stimulating mucosal IgA. Mucosal IgA exhibits both heterosubtypic cross-reactivity to influenza virus strains and potent immunological memory, properties that offer potential wider protection against variants of influenza that have drifted antigenically from the vaccine strain.
Immunity to influenza infection in man is multifactorial, and the precise contribution of innate immunity, serum IgG to haemagglutinin (HA) and neuraminidase (NA), local secretory IgA and T-helper (Th1)- and Th2-type immune responses has been difficult to ascertain. Live virus vaccines and inactivated vaccine induce different arms of the immune response, with sufficient antibody in either serum or nasal secretions being capable of conferring resistance.
Stimulation of both local and systemic immune responses following influenza vaccination may enhance vaccine efficacy, particularly among the elderly, who exhibit age-related reductions in immunity to vaccination. In addition, induction of mucosal responses could be important if stockpiled vaccine, prepared in advance of a pandemic, differs significantly antigenically from the emergent pandemic strain or subtype. The simpler intranasal route of administration also offers the possibility for self-administration and could reduce the healthcare costs of delivery and increase vaccine uptake.
This project is being undertaken in response to the need to develop an effective user friendly, heat stable vaccine to control the highly pathogenic form of avian influenza virus (H5N1) which has the potential to cause another global influenza pandemic.
The principal challenges to pandemic vaccine deployment:
a) avian HA (H5 and H7) is poorly immunogenic compared to H1 and H3 HA;
b) there is limited manufacturing capacity for subunit vaccines;
c) H5 and other avian influenza strains are continuously evolving limiting the usefulness of pandemic vaccine stockpiling; and
d) dose-sparing strategies for the antigen component of the vaccine are not optimised.
The overall objective of this programme was to develop a nasal avian influenza vaccine using ChiSys (chitosan), which is a chitosan-based vaccine delivery technology of Archimedes Development Ltd. The most effective way of controlling a pandemic flu would be via the nasal route as this route has the advantage of allowing the generation of both systemic and mucosal immune responses. Mucosal antibodies may facilitate control of the virus at its point of entry. In comparison, the currently used injectable vaccine cannot induce mucosal immune responses. The nose is an excellent route for vaccination as the nasal cavity has a large surface area available for absorption, with a highly vascularised underlying epithelium as well as its own lymphoid tissue. Intranasal vaccination has several advantages over parenteral delivery:
- combats the pathogen both at its point of entry and also systemically;
- avoids the need for injection and for purchase and safe disposal of syringes;
- eliminate the risk of HIV transmission through re-use of syringes or accidental contact with body fluids for example through needle-stick injuries, especially in the developing world;
- have greater public compliance;
- well-suited to rapid mass global vaccination programmes.
The scientific objectives of the project were to:
- develop an intranasal influenza vaccine that induces both systemic and mucosal immunity;
- develop an influenza vaccine that induces a substantial level of cross-immunity against a drifted (diverse) strain of H5;
- develop an influenza vaccine that is effective at low dose, to meet the global demands from limited vaccine stockpiles;
- develop an influenza vaccine that has thermal stability, so as to avoid refrigeration for storage and transportation;
- employ a user friendly cost effective vaccine applicator;
- evaluate, adjuvant combinations, dose, schedule, efficacy and toxicity in pre-clinical studies;
- evaluate efficacy and safety profile in humans (proof of principle);
- disseminate the findings to scientific community and industry.
The project was structured into the following interconnectd parts:
1. supply of antigen, formulation development and selection of a device;
2. carry out pre-clinical studies in mouse and ferret models to establish the optimum antigen dose, schedule and formulation; vaccine efficacy was to be evaluated using antibody and T-cell assays and challenge tests;
3. carry out toxicity and proof-of-principle phase I clinical studies using GMP antigen;
4. coordination of the project, data management of all the results from the work-packages and dissemination of results.
Project results:
Supply of antigen and formulation development
Supply of antigen
Bulk antigen (inactivated purified surface subunit antigen of H5N1 strain NIBRG-14, propagated in chicken eggs), batch 1 as supplied by Solvay Pharmaceuticals in July 2008 was concentrated to a level necessary for use in the programme (primarily to facilitate the conduct of immunology studies in the mouse) using membrane-based tangential flow filtration (TFF) equipment and was found to retain its potency. The concentrated antigen (SU) was used for developing solution and powder formulations utilising chitosan as potential adjuvant. A further supply of the bulk antigen (inactivated purified surface subunit antigen of H5N1 strain NIBRG-14 (SU-c) propagated in cell culture), batch 2 was received in January 2010.
Due to changes in corporate structure and management, Solvay declined to supply subunit antigen of GMP H5N1 (A/Vietnam/1194/2004 (NIBRG-14)) propagated in eggs or cell culture, for continuing the work. Hence the consortium looked for another supplier who could supply a similar antigen. Participant 3 opened the communications with Novartis Vaccines and Diagnostics Srl and participant 1 proceeded with subsequent communications and negotiations. Under an MTA agreement, Novartis initially supplied relevant GMP subunit antigen (NIBRG-14) for preclinical work with indications for supply of antigen for a clinical study at a later stage, as it would require more extensive legal paper work. However, on 23 October 2011, Novartis declined to supply the GMP subunit antigen for clinical work. As a result the clinical study could not be carried out; hence the attention was focused on a challenge study in ferrets to obtain robust protection efficacy data.
Formulation development and selection of device
a) Solutions
Various grades of chitosan, CSN which is the proprietary nasal delivery system of participant 1 (Archimedes), carboxy-methyl chitosan (CM-CSN), TM-CSN and (for the first time in any investigative study) diquaternary piperazine chitosan (DP-CSN) were assessed to determine the most efficacious adjuvant. Of all the chitosans CM-CSN was found to be most compatible with the antigen and the solution formulation was stable for at least 6 months when stored at 2-8 degrees of Celsius in terms of potency measure by participant 4 using single radial immunodiffusion (SRID) assay. Of the chitosan stock solutions (without antigen), CM-CSN, TM-CSN and DP-CSN were stable at all storage conditions (2-8 or 25 degrees of Celsius) up to 12 months. With increasing temperature and with time the viscosity of CSN solution decreased while that of CM-CSN at higher concentration (20 mg / ml) increased and solution ultimately formed a gel.
A commercially available Pfeiffer metered dose nasal spray pump with paediatric nozzle attached to a 5 ml amber bottle was found to be most suitable as a delivery device for solution formulations after assessment for delivery in terms of fill volume, the number of priming sprays required and delivered dose weight.
A clinical study was planned to evaluate two nasal formulations containing H5N1 antigen either with or without CSN. In both cases, it was proposed to supply each formulation to the study site in two separate containers to be mixed just prior to use; one container to hold antigen solution and the other to hold either CSN solution or saline diluent (for use in preparing chitosan-free formulation). A study was carried out to evaluate stability of the antigen solution when stored at 2-8 degrees of Celsius in glass vials and in plastic syringes over a period of 12 weeks in order to select a suitable package for clinical use. Results of this study clearly indicated that the antigen was stable in terms of appearance, potency and integrity using SRID assay in both glass vial and BD Plastipak syringe; therefore either package would be suitable for the clinical supplies. Further investigations were carried out to study the effect of pH on antigen potency and integrity which were evaluated by SRID assay. The results showed that the antigen lost its potency significantly when pH = 6; however when pH > 6 (e.g. 6.2 and 7) the antigen was stable for at least 4 hours, ample time to enable administration.
b) Powders
In order to facilitate the development of a thermally stable vaccine, powder formulations were developed. The effect of various excipients (mannitol, sucrose and trehalose) and freezing methods on the potency of freeze dried antigen was studied. Based on the handling properties of the freeze dried solids and final powder formulations, mannitol was identified as a necessary bulking agent for use in the freeze drying process. Prototype powder formulations were prepared satisfactorily. The antigen content of the final powder formulations could not be measured using the SRID assay by participant 4 due to interference with the assay when the powder was solubilised Investigations were carried out to enable potency measurement of the antigen in powder formulations containing chitosan using the SRID. The antigen was found to be soluble in both glycerine and dimethyl sulfoxide (DMSO) but failed to be detected by the SRID method in powder extracts. Solid phase extraction of the antigen from chitosan formulations followed by high-pressure liquid chromatography (HPLC) resulted in only 70 % recovery possibly due to interaction between the antigen and chitosan. The effect of fast and slow freezing, excipients and buffer on antigen potency was also examined. The results confirmed that fast freezing in liquid nitrogen was a suitable process, while mannitol and phosphate buffered saline (PBS) were appropriate excipient and buffer respectively, for preparing the powder formulations (prior to blending with chitosan).
The delivery characteristics of the CSN powders were assessed using the commercially available Pfeiffer unit dose nasal powder devices. When loaded with 10 - 10.5 mg of powder approximately 98% of the dose was delivered when the device was fired. The mean particle size was approximately 37 µm, indicating suitability of this device for intranasal dosing to humans.
Manufacture of vaccine formulations
Solution vaccine formulations were manufactured for three mouse studies performed by participant 7 and four mouse studies by participant 5. Both solution and powder formulations were manufactured for two immunogenicity studies in ferret performed by participant 4. Solution formulations were also manufactured for the challenge study in ferret subcontracted to Viroclinics by participant 4.
Due to unavailability of the GMP subunit antigen, toxicology and clinical studies could not be carried out. Hence the formulations were not manufactured for these studies for the 12 and 9 month stability studies to support the toxicology and the clinical studies respectively.
In-Vitro studies
To induce an effective mucosal immune response, a successful vaccine should be directed toward effectively triggering the innate immune response. Innate immunity is critical for orchestrating the adaptive immune response through the activation of antigen-presenting cells (APCs) such as the dendritic cell (DC). Initial systemic responses are governed by the innate immune system, which produces pro-inflammatory responses (cytokines and chemokines) to the initial insult of an infectious agent. Furthermore, this initial rapid pro-inflammatory response is considered to be the critical trigger provided by traditional immunological adjuvants. Thus, many vaccine formulations strategies include innate immune triggers to provide this adjuvant signal.
In vitro studies were performed to examine the ability of various chitosan solutions to activate / potentiate an innate immune response. The results showed that both CSN and TM-CSN solutions gave positive results; with TM-CSN being more efficient in its ability to enhance toll-like receptor (TLR)-induced cytokine production skewing the T cell response towards a mixed Th1 / Th17 phenotype. Further studies to evaluate the effects of chitosan on TLR-induced cytokine production by plasmacytoid DCs (pDCs) showed that TM-CSN enhanced CpG induced IP-10 which is a Th1 cell chemokine, whereas CSN induced LPS induced IL-12p40 and IL-1beta which are Th17 polarising cytokines. CSN had significant suppressive effect on both LPS and CpG induced LP27 which is known to stimulate the production of interferon (IFN)-alpha and has been shown to exhibit both pro- and anti-inflammatory properties. Further studies would be required to investigate this effect.
The chitosan formulations that exhibited the best ability to potentiate the innate immune response in vitro supported the identification of formulations with the best chance of succeeding in vivo.
In vivo mouse studies
Several in vivo studies were carried out in mice to evaluate various chitosans, antigen dose response, efficacy of the adjuvanted antigen, effect of previous immunity, establish vaccination regime and examine the effect of nasally administered chitosan on local brain cytokine production.
Evaluation of different chitosans and dose response
Of the three chitosans (CSN, carboxymethyl chitosan and diquaternary piperazine chhitosan) tested, CSN produced the highest overall immune response (IgG, nasal wash IgA and serum radial haemolysis (SRH)). Furthermore CSN enhanced SRH titres after one dose as compared to the antigen alone. A dose response study in mice showed that CSN augmented the highest serum IgG and IgA, nasal wash IgA, seroprotection (SRH titres) shown in Figures 1 and 2 respectively below and the quality of the T cell responses. The SU (7.5 ug HA) formulated with CSN induced good nasal wash IgA and serum IgG responses after the second immunisation and low frequencies of double cytokine producing CD4+ cells (not shown).
Evaluation of vaccine regime
To discover the most efficacious vaccination regime, parenteral priming with either alum or cytosine-phosphate-guanine dinucleotide (CpG) followed by nasal vaccination with the antigen formulated with CSN and TM-CSN was explored. CSN and TM-CSN enhanced HA-specific IgG1 in sera of alum and CpG primed mice, whereas TM-CSN strongly enhanced HA-specific IgG2a in sera of CpG primed mice. Priming with antigen in the presence of alum polarised the antigen specific T cell response towards a Th2 phenotype with high concentrations of antigen-specific IL-4 and IL-5 detected in both the spleen and lymph nodes of immunised mice. In contrast, priming with antigen in the presence of CpG followed by intranasal boosting with antigen and TM-CSN induced a more mixed Th1 / Th2 profile in the spleen of immunised mice.
Effect of previous H3 or H5 immunity
As the general human population is already exposed to H3 influenza viruses the effect of previous H3 immunity on nasal immunisation with H5N1was investigated. The results showed that two nasal doses of H5 / CSN administered intranasally following H3 or H5 priming in the presence of CpG considerably enhanced the antibody responses. This indicated that subcutaneous prime / nasal boost strategy could provide protection against H5 viral infection.
Further prime / boost studies confirmed that two doses of nasal chitosan adjuvanted H5 vaccine could produce both humoral and cellular immune responses. These responses could be further enhanced by priming the mice subcutaneously with CpG adjuvanted H5 vaccine followed by two doses of chitosan adjuvanted H5 vaccine. A comparative study examining mucosal versus parenteral priming showed that CSN elicits a strong IFN-gamma response when administered intranasally and also induces a cytokine IL-22 which is known to play an important role in the regulation of immunity and inflammation at barrier surfaces.
Effect of nasally administered CSN on local brain cytokine production
When vaccinating via the nasal route in the presence of an adjuvant, it is important to investigate its effect on the local lymphatic tissues as well as on the brain (due to its close proximity to the nasal cavities). The nasal associated lymphoid tissue (NALT) is a mucosal lymphoid organ which is thought to play a pivotal role in directing the development of B and T cells that can protect the respiratory tract. This study aimed to investigate the effectiveness of CSN used in the vaccine formulations at activating immunity in the NALT. CSN activated the production of IL-1beta, IL-6 (important for Th17 responses) and IL-12p35, a critical polarising cytokine for Th1 development. In the NALT CSN also induced the production of IL-22 which is known to play an important role in the regulation of immunity and inflammation at barrier surfaces. Early studies have indicated that IL-22 does not appear to play a substantial direct role in immunity to viral pathogens. However, the importance of IL-22 in vaccine induced protective immunity to influenza would require further investigation.
On evaluating the cytokine production in the hypothalamus region of the brain the results showed that CSN did not augment the production of pro-inflammatory cytokines, IL-1beta and IL-6 after intranasal immunisation; thereby supporting the claim that chitosan is a safe adjuvant to use intranasally.
Intranasal studies evaluating cyclic diguanylate monophosphate (c-di-GMP) adjuvanted antigen with or without CSN
A novel experimental adjuvant c-di-GMP was found to enhance systemic and local antibody responses in terms of serum IgG1, IgG2a, SRH, HA inhibition (HI) and virus neutralisation (VN) antibody titres. VN titres against the homologous strains in all the adjuvanted groups were significantly higher than in the non-adjuvanted group. The evaluation of cross clade reactive antibodies against the clade 2.1 virus A/Indonesia/5/05 (IDON) showed no neutralisation of this virus in the antigen alone groups, whilst some animals in the adjuvanted groups were seropositive. However no reactive antibodies against A/Indonesia/05/2005 were observed.
A clear dose response was observed for all vaccine groups, with adjuvants augmenting the highest local nasal wash IgA response at an antigen dose of 7.5 ug after the second administration; whereas no local IgA was found in the control animals that had been dosed with PBS. At two and three weeks after the second dose no influenza specific serum IgG or IgA were detected in the control group, whereas the c-di-GMP or combination c-di-GMP+CSN groups had the highest humoral responses closely followed by the CSN group. Both CSN and c-di-GMP effectively enhanced splenocyte proliferation as compared to antigen alone. The evaluation of Th-cell profile showed that c-di-GMP adjuvanted vaccine produced a Th1 / Th17 biased response, whilst the CSN adjuvanted vaccine group had a cytokine profile indicating a more Th2 skewed response.
Overall this study showed that both CSN and c-di-GMP boosted functional antibody responses. The c-di-GMP adjuvant provided significant dose-sparing in terms of seroprotection, whereas the adjuvant combination did not. However, the adjuvant combination skewed the immune responses towards a more balanced Th profile than either of the adjuvants alone. This study highlights the importance of assessing the humoral and cellular immune responses following immunisation to allow direct evaluation novel mucosal influenza vaccines for finding the optimal vaccine formulation.
Comparison of CSN and TM-CSN adjuvanted influenza (H5N1) vaccine
Since conclusive immunological results were not obtained from two ferret studies, a statistically powered study was performed to compare the efficacy of CSN and TM-CSN as mucosal adjuvants. Both CSN and TM-CSN augmented the antibody response, with SRH titres above the protective threshold of 25 mm2 and no significant difference was observed in HI titres to the two chitosans but CSN resulted in significantly higher SRH titres than TM-CSN. The homologous neutralising antibody response was significantly higher in the CSN group than all the other groups. In terms of the heterologous antibody response cross-reactive neutralising antibody response to A/Indonesia/1/2005 strain was observed in majority of the CSN immunised animals (7 of 10) and none in the other groups.
Low concentrations of local nasal wash IgA were detected after the first dose with the highest response detected in the two chitosan groups. The second dose significantly augmented the local IgA particularly in the chitosan groups, with the CSN group at two weeks and the TM-CSN group at two and three weeks post vaccination (Figures 9 B and C).
The splenocyte proliferation assay showed a significant adjuvant effect of CSN, and TM-CSN suggesting an enhancement of the cellular immune response. Of the two chitosan adjuvants CSN appeared to be more effective than TM-CSN. In terms of the cytokine response the antigen alone and chitosan adjuvants elicited Th1 (IL2, IFN-gamma), Th2 (IL4, IL5 and IL10) and Th17 response (Figure 10). A significantly high IL-17 response was observed in two chitosan groups.
Overall this study confirmed that both CSN and TM-CSN boosted functional antibody as well as local IgA responses with CSN appearing to be marginally more efficacious than TM-CSN. Similar observations were made in terms of T cell proliferation. However TM-CSN showed a significantly higher IL-23 response than CSN.
Comparison of Solvay and Novartis antigens
Since the source of the antigen was changed during the project, an immunogenicity bridging study was carried out to compare the Solvay and Novartis antigens. Both the antigens elicited similar IgG1titres pre-boost and post-2nd booster vaccination. Slight enhancement in IgG2a titres was observed in pre-boost sera in response to Solvay antigen but comparable IgG2a titres were observed in response to both antigens in the final samples. Both the antigens showed some enhancement of SRH titres compared to the PBS control. Solvay antigen performed slightly better than Novartis antigen in that 2 / 6 animals reached the protective level. The Solvay antigen also performed slightly better in the homologous VN assay. Moreover, the cytokine data appeared to indicate Solvay antigen to elicit a rather more robust mixed T cell response with respect to Th1 and Th17 and similar Th2-type response.
Overall the results indicated that the antigens from the two sources were sufficiently similar for use in further preclinical and clinical studies.
In vivo ferret studies
The immunogenicity ferret study 1
The immunogenicity of novel intranasal formulations, both as liquid and powders containing H5N1 antigen, was evaluated in the ferret model. Antibody titres obtained in ferrets were were relatively low and inconsistent, for example the positive control (i.m.) in this study performed poorly as no SRH seroprotection was observed despite some HI seroconversion. Although the results of this study were inconclusivethere were indications that chitosan enhanced immune response and that the liquid formulation possibly was more efficacious than powders. The consortium concluded that the non-responsiveness of powders was probably due to technical issues, i.e. not delivering the powders appropriately in the nasal cavity of the ferrets rather than the antigen immunogenicity. It also appears that.
The immunogenicity ferret study 2
A further immunogenicity study was carried out to evaluate liquid and powder formulations containing H5N1 antigen, in combination with two different chitosans (CSN or TM-CSN), via the nasal route in the ferret model. The study included a chitosan-free nasal liquid formulation and a control IM injection of the antigen.
The overall results of this study were compromised for a number of reasons. The background SRH values (day 0) were high in all the groups. Furthermore the serum samples for days 0 and 21 were observed to be coloured indicating contamination with plasma proteins, thereby possibly contributing to non-specific cross-reaction. The outcome of the results was further complicated by the administration of antibiotics to animals in two groups as well as a reduced number of animals completing the study in another two groups due to poor health.
The immune responses indicated that chitosan liquid formulations were efficacious in enhancing both the seroconversion and seroprotection rates following two nasal vaccinations, with TM-CSN possibly performing similarly but possibly slightly better than CSN. The positive control (i.m.) group also enhanced immune responses following both one and two vaccinations. However the nasal and the i.m. groups could not be directly compared as the latter group had a limited number of animals.
It was not possible to select the most appropriate formulations for further evaluation from the two ferret studies. Hence the consortium decided to carry out a statistically powered study in mice to compare the efficacy of CSN and TM-CSN as mucosal adjuvants in solution formulations (section 1.3.5.3).
The challenge study
Following the decision by Novartis not to supply antigen for clinical use, a definitive challenge study to establish efficacy of intranasally administered influenza H5N1 A/Vietnam/1194/2004 vaccine (supplied by Novartis) was conducted in the ferret model using liquid vaccine formulations based on both CSN and TM-CSN. The study was subcontracted to a contract research organisation, Viroclinics. The study protocol was specifically designed by several members of the consortium in collaboration with Viroclinics.
The objective of this study was to investigate the immunogenicity and protective efficacy of intranasally administered adjuvanted vaccines in the ferret H5N1 wild type model. The intranasal candidate vaccines were aqueous solutions containing subunit of inactivated NIBRG-14 influenza vaccine prepared in egg, GMP grade, obtained from Novartis. The potency of the antigen was defined by its HA content quantified by SRID method. Candidate vaccines were adjuvanted with CSN or TM-CSN prior to testing. For control purposes, a chitosan-free vaccine formulation was tested along with appropriate placebo PBS controls. The design of this study is summarised.
The vaccination phase:
This study consisted of six groups each comprising 6 ferrets:
- groups 1 and 2 were intranasally immunised with CSN adjuvanted vaccine;
- group 3 was intranasally immunised with non-adjuvanted vaccine;
- group 4 was intranasally immunised with TM-CSN adjuvanted vaccine;
- groups 5 and 6 were treated with PBS.
Intranasal vaccine were administered twice, once on study day 0 and then again on study day 21. During the immunisation phase and just prior to challenge blood samples were taken to evaluate the immune response to vaccination.
The challenge phase:
Animals were challenged individually with a dose of 105 TCID50 H5N1.
A/Vietnam/1194/2004 HPAI virus on study day 49. Animals from Groups 1 and 5 were challenged via the intranasal route and animals of groups 2, 3, 4 and 6 were challenged via the intratracheal route. Ferrets were monitored for infection related clinical and virological responses and for vaccine induced immunological responses. Animals were sacrificed on study day 54 (i.e. 5 days post challenge) to evaluate pathologic and additional virological parameters.
Summary of results
i. Antibody response induced by vaccine candidates
Antibody responses were tested using three different assay methodologies, namely SRH, VN and HI. The serum HI antibody response against H5N1 A/Vietnam/1194/2004 was greater in animals immunised with the TM-CSN adjuvanted vaccine compared to CSN and was absent in animals immunised with non-adjuvanted vaccine. The results were similar for SRH and VN assays. CSN and TM-CSN adjuvanted vaccines also induced cross protective antibodies in-vitro against H5N1 viruses from representatives from clade 2.1 and 2.2.
ii. Results of challenge phase of the study
The inoculation route of the challenge with H5N1 A/Vietnam/1194/2004 HPAI virus had significant influence on the clinical, virological and pathological outcome of the virus infection. Intratracheal challenge with H5N1 A/Vietnam/1194/2004 influenza virus was shown to be lethal and to result in high level virus replication in lung and trachea. Intranasal challenge with H5N1 A/Vietnam/1194/2004 influenza virus was not lethal and resulted in high level virus replication in the upper respiratory tract and the majority of animals also exhibited virus replication in the olfactory bulb. In accordance, histopathological findings in the lung were more severe after intratracheal challenge and encephalitis was more common after intranasal challenge. The serum antibody response correlated well with the protection from intratracheal infection with influenza H5N1 A/Vietnam/1194/2004.
Animals immunised with the TM-CSN adjuvanted vaccine were protected against intratracheal infection-related findings in terms of lethality, body weight loss, fever, virus replication in respiratory and brain tissues and lung pathology (data not shown). Animals immunised with CSN- and in particular TM-CSN-adjuvanted vaccine were protected against intratracheal infection. Performance of the control group immunised with the non-adjuvanted vaccine and intratracheally challenged was, with respect to the evaluated clinical, virological and pathological parameters, low and not substantially different from that of PBS treated animals following intraracheal challenge.
In conclusion the severity of the H5N1 A/Vietnam/1194/2004 HPAI virus infection was, with respect to clinical, virological and pathological outcome, defined by the route of challenge. The outcome of infection was dependent on route of inoculation. Intranasal immunisation with the CSN or TM-CSN adjuvanted vaccines induced serum antibody responses that were protective against inoculation with H5N1 A/Vietnam/1194/2004 HPAI virus, whereas intranasal immunisation with the non-adjuvanted vaccine did not. Antibody responses were cross protective amongst different clades of H5N1 viruses. The highest level of protection from an intratracheal challenge with H5N1 A/Vietnam/1194/2004 HPAI virus was induced by the TM-CSN adjuvanted vaccine and this corresponded with the highest serum HI antibody responses.
Overall, immunisation with CSN adjuvanted vaccine was less effective than immunisation with TM-CSN adjuvanted vaccine. Immunisation with the CSN adjuvanted vaccine induced comparable levels of protection against an intranasal and intratracheal challenge with H5N1 A/Vietnam/1194/2004 HPAI virus. Moreover the comparison of intratracheal and intranasal challenge with a lethal dose of live virus in ferret is groundbreaking research as it has highlighted the advantages and disadvantages of each route of challenge; the normal practice has been to challenge only via the intratracheal route.
Clinical study
The relevant consortium members made preparations to perform a clinical proof-of-principle efficacy study. Whilst awaiting the supply of GMP antigen by Novartis, participant 2 wrote an outline of the study protocol and participant 1 started to prepare the CTA in preparation for submission of the application for the planned clinical trial to the regulatory authorities.Considerations were also given to the type of containers that might be used for the delivery of vaccine to the humans and hence a stability study of the antigen in either glass vials or plastic syringes was performed and the suitability of spray devices was confirmed (section 1.3.1.2). Furthermore to optimise B cell assays, participant 7 used test samples from a Phase I clinical trial of an H5N1 (RG14) vaccine using Matrix M as an adjuvant performed at University of Bergen for a project other than NASPANVAC. B cells called antibody secreting cells (ASC) in the ELISPOT assay were successfully optimised and enumerated. Good levels of long term memory B cell response at 12 months post vaccination was also observed with the highest responses being detected in the adjuvanted groups.
When Novartis declined the supply of GMP antigen for a clinical study on 23 October 2011, it became apparent that it was not possible to do a clinical study; no alternative supplier could be identified at that point. Hence the consortium unanimously agreed to focus the efforts and resources to perform a challenge study in an established in-vivo efficacy (ferret). This study showed highly promising results which are discussed above (section 1.3.5.3).
Conclusion
Although, for reasons not under the control of the consortium the objective of performing a proof-of-principle clinical study was not accomplished, the robust efficacy data in the ferret model along with the immense immunogenicity (both systemic and local antigen specific Igs, protective / functional antibodies, several cytokines) mouse data including the evaluation of the most efficacious chitosan type, dose response, effect of previous immunity, vaccine regime and the effect of nasally administered chitosan on local brain cytokine production obtained in this project, forms an excellent comprehensive preclinical package for submission to the regulatory authorities to develop a nasal H5N1 vaccine using chitosan as the mucosal delivery platform. Stability data and administration techniques/devices are confirmed to facilitate such a study.
Potential impact:
Socio-economic impact
To date, 606 cases of human H5N1 infections have been reported in 15 different countries of which almost 60 % have been fatal. However, any initiative that formulated transmission to or between humans could produce catastrophic levels of infection. A number of strategies and approaches are being brought into play to confront an influenza pandemic. Certain antiviral drugs (Tamiflu, Relenza) are commercially available and are the first line of treatment for health care providers. However, for these drugs to be effective the treatment has to commence within 48 hours of the onset of the symptoms. These drugs belong to a class of medicines called NA inhibitors which prevent the influenza virus from spreading inside the body and are designed to be active against all clinically relevant influenza virus strains. Due to the rapid production of drifted antigens of the H5N1 virus, there are already indications of development of resistance to these drugs. Hence, the use of antiviral drugs may not provide the treatment that would be required for an avian influenza pandemic.
Vaccination is the cornerstone of influenza prophylaxis. In the event of a pandemic, rapid production and deployment of a pandemic vaccine will limit viral spread, hospitalisations, serious complications of disease and ultimately death. The pandemic virus strain could significantly differ from the virus vaccine strain being stockpiled by various governments as a mean of strategic planning. In recent years there has been an increase in manufacturing of seasonal trivalent influenza doses. There are only a few countries with influenza vaccine manufacturing capacity and these will probably reserve the scarce supply of a pandemic vaccine for their own citizens. The World Health Organisation (WHO) aims for equitable and timely access to vaccines for all peoples of the world. However, the reality is that only a few countries manufacture influenza vaccine; therefore populations in other countries most affected by the pandemic may not have access to a pandemic vaccine. During the swine origin influenza pandemic in 2009, the virus spread globally within two months, highlighting the importance of rapid deployment of vaccine. Developing countries had limited access to pandemic vaccines and a needle free mucosal influenza vaccine would have allowed vaccination with limited numbers of public health providers. The NASPANVAC programme investigated a number of adjuvants, alone and as co-adjuvants, that could enhance the immunogenicity of current vaccines to allow dose-sparing formulations and allow the limited vaccine supply to be used as effectively as possible which could have a real positive effect on controlling a pandemic.
The history of pandemic vaccine development since 1997 provides worrying lessons in the difficulties of preparing influenza vaccines containing novel influenza subtypes. The hope is that these lessons will allow the vaccine industry to be ready to immediately start production of the most efficacious vaccine once a pandemic has been declared. Each manufacturer has its own licensed facility and process for manufacturing and these may not be the best vaccine formulation to provide the best protection in a pandemic. There is an urgent need for research and comparative clinical trials comparing different vaccine formulations with or without adjuvant, vaccine strength, number of doses and routes of administration to provide information on what is the appropriately formulated vaccine and optimal use, to save valuable time in preparation of a pandemic vaccine. The preclinical studies carried out under the NASPANVAC project clearly showed that nasally delivered chitosan adjuvanted vaccine enhanced both local and systemic immune responses thereby combating the virus at its point of entry and also systemically, induced specific cytokines giving insight into Th1 / Th2 balance as well as their role in regulation of immunity and inflammation, demonstrated heterologous cross-reactive neutralising antibody response against a couple of H5N1 strains and most of all provided protection against the challenge with the live homologous virus. Although the NASPANVAC programme did not manage to demonstrate the efficacy of a nasally delivered pandemic vaccine in humans, the various issues were extensively addressed and investigated in preclinical studies which now form the basis for further exploitation of the project results in terms of phase II / III clinical trials, applications for regulatory approval in major jurisdictions, implementation of commercial scale manufacture and global commercialisation of a nasal vaccine.
Wider societal implications of the project
In view of the excellent safety profile of chitosan and the robust preclinical data produced in the NASPANVAC programme are highly encouraging given that traditionally vaccine adjuvants tend to be relatively more toxic moieties. CSN and TM-CSN were shown to elicit protective immunity against lethal H5N1 challenge in a recognised model of human influenza, the ferret. Thus, the risk: benefit ratio for a chitosan-based vaccine may be favourable when compared with that of more potent adjuvants. A H5N1 pandemic could have a major impact on public services, making self-reliant treatment and management of the disease mandatory. The NASPANVAC project has clearly demonstrated the potential to develop a nasal vaccine which could be self-administered and also enable rapid mass vaccination with minimum aid from specialist healthcare providers.
Main dissemination activities and exploitation of results
a) The dissemination activities to date have been as follows:
1. Jabbal-Gill I. Nasal vaccine innovation. J Drug Target. 2010. Dec; 18(10):771-86
2. Jabbal-Gill I, Watts P, Smith A. Chitosan-based delivery systems for mucosal vaccines. Expert Opinion on Drug Delivery, Posted online on 19 June 2012 (doi:10.1517/17425247.2012.697455)
3. Signe C. Svindland, Åsne Jul-Larsen, Solveig Andersen, Rishi Pathirana, Abdullah Madhun, Emanuelle Montomoli, Inderjit Jabbal-Gill, Rebecca J. Cox. 'The mucosal and systemic immune responses elicited by a chitosan adjuvanted intranasal influenza H5N1 vaccine' Poster presented at The Oxford influenza conference, Influenza 2010.
4. Svindland SC, Jul-Larsen Å, Pathirana R, Andersen S, Madhun A, Montomoli E, Jabbal-Gill I, Cox RJ. The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine. Influenza Other Respi Viruses. 2012 Mar; 6(2):90-100. doi: 10.1111/j.1750-2659.2011.00271.x. Epub 2011 Jul 12.
5. Signe C. Svindland1, Gabriel Kristian Pedersen, Rishi Pathirana*, Geir Bredholt*, Jane Kristin Nøstbakken*, Åsne Jul-Larsen*, Carlos Guzman, Emanuele Montomoli, Giulia Lapini Inderjit Jabbal-Gill*, Michael Hinchcliffe*, Rebecca J. Cox1*. A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine. Submitted to Influenza and other Respiratory Viruses (2012)
b) Publish the mouse data, especially the cytokines and the ferret data demonstrating the efficacy of a nasally delivered chitosan adjuvanted vaccine in prestigious peer-reviewed scientific journals by the end of 2012.
c) Present the efficacy data in ferrets initially at an international meeting in Q4 2012 and others thereafter.
List of websites: http://www.naspanvac.com
The NASANVAC project was undertaken in response to the need to develop an effective user friendly, heat stable vaccine to control the highly pathogenic form of avian influenza virus (H5N1) which has the potential to cause another global influenza pandemic. A significant number of deliverables have been achieved, despite the challenges that arose during the project. Crucially, the guanosine monophosphate (GMP) surface subunit antigen of H5N1 strain NIBRG-14 raised in eggs was promptly sourced from a new supplier (Novartis) and a material transfer agreement (MTA) signed permitting its use in preclinical studies after the first supplier (Solvay) could not supply the antigen subsequent to changes in corporate structure and management. A bridging study in the mouse confirmed that the Novartis antigen was sufficiently similar to the egg-based antigen originally sourced from Solvay and thus, would be suitable for use in preclinical and clinical studies.
Formulation development work has confirmed that liquid and powder formulations of H5N1 adjuvanted with the biopolymer chitosan are stable and suitable for clinical use. Serological assays (surrogate correlates of protection) in mouse and ferret sera were initially established to enable the evaluation of vaccine efficacy. Preclinical studies in mice and ferrets have confirmed that both chitosan glutamate (CSN) and trimethyl-chitosan (TM-CSN) as solutions are efficacious in augmenting serology responses. Mouse studies further indicated that it is possible to enhance the humoral and cellular responses obtained after administering two doses of an intranasal CSN-adjuvanted vaccine by first priming with a subcutaneous injection of antigen. CSN was also shown to be a safe mucosal adjuvant and significantly no pro-inflammatory response was observed in the hypothalamus of the brain (allaying some concerns over Bell's palsy-like adverse effects which have manifested in clinical practice after administration of some intranasal influenza vaccines). Based on collective findings, the consortium initially selected a CSN liquid formulation to proceed to a phase I proof-of-principle clinical study, which could potentially be supported without the need for further preclinical toxicology. Following the decision by Novartis not to supply antigen for clinical use a definitive challenge study to establish efficacy was conducted in the ferret model using liquid vaccine formulations based on both CSN and TM-CSN. A significant aspect of this study was as a comparison of intratracheal and intranasal challenge, the first time such studies have been attempted.
In this project we planned to develop a prototype nasal H5N1 vaccine, which could be delivered through a commercial nasal delivery applicator and which showed a positive immunogenicity response in humans; thereby forming the basis for further exploitation of the project results in terms of phase II / III clinical trials. The specific objective of conducting a phase I clinical trial was not met due to non-availability of suitable GMP antigen. Nevertheless, the excellent challenge study data obtained in the ferret should allow strategic decisions to be made over further development of an intranasal vaccine. The data clearly demonstrated protective immunity against lethal H5N1 A/Vietnam/1194/2004 virus infection following intranasal vaccination adjuvanted with both CSN and TM-CSN, though the latter appeared to be more efficacious in the recognised model of human influenza, the ferret. In view of the excellent safety profile of chitosan, these data are highly encouraging given that traditionally vaccine adjuvants tend to be relatively more toxic moieties. Thus, the risk: benefit ratio for a chitosan-based vaccine may be favourable when compared with that of more potent adjuvants. A H5N1 pandemic could have a major impact on public services, making self-reliant treatment and management of the disease mandatory. The NASPANVAC project has clearly demonstrated the potential to develop a nasal vaccine which could be self-administered and also enable rapid mass vaccination with minimum aid from specialist healthcare providers.
Project context and objectives:
Annual outbreaks of influenza A and B, and pandemics of influenza A, are responsible for substantial mortality and morbidity, particularly in high-risk groups including the elderly and those with chronic underlying medical conditions. Vaccines are the most effective means of defence against influenza to prevent infection and control its spread. Despite the availability of effective parenteral vaccines, influenza still incurs considerable medical and socio-economic costs. Barriers limiting vaccine uptake include the intramuscular (IM) route of injection, and the perception of vaccine ineffectiveness. Furthermore the capacity to manufacture to meet a global demand is limited.
Delivery of an influenza vaccine by the nasal route, targeted to the mucosal site of virus entry and principal location of its replication, offers potential advantages over parenteral delivery of the vaccine. While conventional IM influenza vaccines are effective at inducing serum immunoglobulin G (IgG) neutralising antibodies, they are poor at stimulating mucosal IgA. Mucosal IgA exhibits both heterosubtypic cross-reactivity to influenza virus strains and potent immunological memory, properties that offer potential wider protection against variants of influenza that have drifted antigenically from the vaccine strain.
Immunity to influenza infection in man is multifactorial, and the precise contribution of innate immunity, serum IgG to haemagglutinin (HA) and neuraminidase (NA), local secretory IgA and T-helper (Th1)- and Th2-type immune responses has been difficult to ascertain. Live virus vaccines and inactivated vaccine induce different arms of the immune response, with sufficient antibody in either serum or nasal secretions being capable of conferring resistance.
Stimulation of both local and systemic immune responses following influenza vaccination may enhance vaccine efficacy, particularly among the elderly, who exhibit age-related reductions in immunity to vaccination. In addition, induction of mucosal responses could be important if stockpiled vaccine, prepared in advance of a pandemic, differs significantly antigenically from the emergent pandemic strain or subtype. The simpler intranasal route of administration also offers the possibility for self-administration and could reduce the healthcare costs of delivery and increase vaccine uptake.
This project is being undertaken in response to the need to develop an effective user friendly, heat stable vaccine to control the highly pathogenic form of avian influenza virus (H5N1) which has the potential to cause another global influenza pandemic.
The principal challenges to pandemic vaccine deployment:
a) avian HA (H5 and H7) is poorly immunogenic compared to H1 and H3 HA;
b) there is limited manufacturing capacity for subunit vaccines;
c) H5 and other avian influenza strains are continuously evolving limiting the usefulness of pandemic vaccine stockpiling; and
d) dose-sparing strategies for the antigen component of the vaccine are not optimised.
The overall objective of this programme was to develop a nasal avian influenza vaccine using ChiSys (chitosan), which is a chitosan-based vaccine delivery technology of Archimedes Development Ltd. The most effective way of controlling a pandemic flu would be via the nasal route as this route has the advantage of allowing the generation of both systemic and mucosal immune responses. Mucosal antibodies may facilitate control of the virus at its point of entry. In comparison, the currently used injectable vaccine cannot induce mucosal immune responses. The nose is an excellent route for vaccination as the nasal cavity has a large surface area available for absorption, with a highly vascularised underlying epithelium as well as its own lymphoid tissue. Intranasal vaccination has several advantages over parenteral delivery:
- combats the pathogen both at its point of entry and also systemically;
- avoids the need for injection and for purchase and safe disposal of syringes;
- eliminate the risk of HIV transmission through re-use of syringes or accidental contact with body fluids for example through needle-stick injuries, especially in the developing world;
- have greater public compliance;
- well-suited to rapid mass global vaccination programmes.
The scientific objectives of the project were to:
- develop an intranasal influenza vaccine that induces both systemic and mucosal immunity;
- develop an influenza vaccine that induces a substantial level of cross-immunity against a drifted (diverse) strain of H5;
- develop an influenza vaccine that is effective at low dose, to meet the global demands from limited vaccine stockpiles;
- develop an influenza vaccine that has thermal stability, so as to avoid refrigeration for storage and transportation;
- employ a user friendly cost effective vaccine applicator;
- evaluate, adjuvant combinations, dose, schedule, efficacy and toxicity in pre-clinical studies;
- evaluate efficacy and safety profile in humans (proof of principle);
- disseminate the findings to scientific community and industry.
The project was structured into the following interconnectd parts:
1. supply of antigen, formulation development and selection of a device;
2. carry out pre-clinical studies in mouse and ferret models to establish the optimum antigen dose, schedule and formulation; vaccine efficacy was to be evaluated using antibody and T-cell assays and challenge tests;
3. carry out toxicity and proof-of-principle phase I clinical studies using GMP antigen;
4. coordination of the project, data management of all the results from the work-packages and dissemination of results.
Project results:
Supply of antigen and formulation development
Supply of antigen
Bulk antigen (inactivated purified surface subunit antigen of H5N1 strain NIBRG-14, propagated in chicken eggs), batch 1 as supplied by Solvay Pharmaceuticals in July 2008 was concentrated to a level necessary for use in the programme (primarily to facilitate the conduct of immunology studies in the mouse) using membrane-based tangential flow filtration (TFF) equipment and was found to retain its potency. The concentrated antigen (SU) was used for developing solution and powder formulations utilising chitosan as potential adjuvant. A further supply of the bulk antigen (inactivated purified surface subunit antigen of H5N1 strain NIBRG-14 (SU-c) propagated in cell culture), batch 2 was received in January 2010.
Due to changes in corporate structure and management, Solvay declined to supply subunit antigen of GMP H5N1 (A/Vietnam/1194/2004 (NIBRG-14)) propagated in eggs or cell culture, for continuing the work. Hence the consortium looked for another supplier who could supply a similar antigen. Participant 3 opened the communications with Novartis Vaccines and Diagnostics Srl and participant 1 proceeded with subsequent communications and negotiations. Under an MTA agreement, Novartis initially supplied relevant GMP subunit antigen (NIBRG-14) for preclinical work with indications for supply of antigen for a clinical study at a later stage, as it would require more extensive legal paper work. However, on 23 October 2011, Novartis declined to supply the GMP subunit antigen for clinical work. As a result the clinical study could not be carried out; hence the attention was focused on a challenge study in ferrets to obtain robust protection efficacy data.
Formulation development and selection of device
a) Solutions
Various grades of chitosan, CSN which is the proprietary nasal delivery system of participant 1 (Archimedes), carboxy-methyl chitosan (CM-CSN), TM-CSN and (for the first time in any investigative study) diquaternary piperazine chitosan (DP-CSN) were assessed to determine the most efficacious adjuvant. Of all the chitosans CM-CSN was found to be most compatible with the antigen and the solution formulation was stable for at least 6 months when stored at 2-8 degrees of Celsius in terms of potency measure by participant 4 using single radial immunodiffusion (SRID) assay. Of the chitosan stock solutions (without antigen), CM-CSN, TM-CSN and DP-CSN were stable at all storage conditions (2-8 or 25 degrees of Celsius) up to 12 months. With increasing temperature and with time the viscosity of CSN solution decreased while that of CM-CSN at higher concentration (20 mg / ml) increased and solution ultimately formed a gel.
A commercially available Pfeiffer metered dose nasal spray pump with paediatric nozzle attached to a 5 ml amber bottle was found to be most suitable as a delivery device for solution formulations after assessment for delivery in terms of fill volume, the number of priming sprays required and delivered dose weight.
A clinical study was planned to evaluate two nasal formulations containing H5N1 antigen either with or without CSN. In both cases, it was proposed to supply each formulation to the study site in two separate containers to be mixed just prior to use; one container to hold antigen solution and the other to hold either CSN solution or saline diluent (for use in preparing chitosan-free formulation). A study was carried out to evaluate stability of the antigen solution when stored at 2-8 degrees of Celsius in glass vials and in plastic syringes over a period of 12 weeks in order to select a suitable package for clinical use. Results of this study clearly indicated that the antigen was stable in terms of appearance, potency and integrity using SRID assay in both glass vial and BD Plastipak syringe; therefore either package would be suitable for the clinical supplies. Further investigations were carried out to study the effect of pH on antigen potency and integrity which were evaluated by SRID assay. The results showed that the antigen lost its potency significantly when pH = 6; however when pH > 6 (e.g. 6.2 and 7) the antigen was stable for at least 4 hours, ample time to enable administration.
b) Powders
In order to facilitate the development of a thermally stable vaccine, powder formulations were developed. The effect of various excipients (mannitol, sucrose and trehalose) and freezing methods on the potency of freeze dried antigen was studied. Based on the handling properties of the freeze dried solids and final powder formulations, mannitol was identified as a necessary bulking agent for use in the freeze drying process. Prototype powder formulations were prepared satisfactorily. The antigen content of the final powder formulations could not be measured using the SRID assay by participant 4 due to interference with the assay when the powder was solubilised Investigations were carried out to enable potency measurement of the antigen in powder formulations containing chitosan using the SRID. The antigen was found to be soluble in both glycerine and dimethyl sulfoxide (DMSO) but failed to be detected by the SRID method in powder extracts. Solid phase extraction of the antigen from chitosan formulations followed by high-pressure liquid chromatography (HPLC) resulted in only 70 % recovery possibly due to interaction between the antigen and chitosan. The effect of fast and slow freezing, excipients and buffer on antigen potency was also examined. The results confirmed that fast freezing in liquid nitrogen was a suitable process, while mannitol and phosphate buffered saline (PBS) were appropriate excipient and buffer respectively, for preparing the powder formulations (prior to blending with chitosan).
The delivery characteristics of the CSN powders were assessed using the commercially available Pfeiffer unit dose nasal powder devices. When loaded with 10 - 10.5 mg of powder approximately 98% of the dose was delivered when the device was fired. The mean particle size was approximately 37 µm, indicating suitability of this device for intranasal dosing to humans.
Manufacture of vaccine formulations
Solution vaccine formulations were manufactured for three mouse studies performed by participant 7 and four mouse studies by participant 5. Both solution and powder formulations were manufactured for two immunogenicity studies in ferret performed by participant 4. Solution formulations were also manufactured for the challenge study in ferret subcontracted to Viroclinics by participant 4.
Due to unavailability of the GMP subunit antigen, toxicology and clinical studies could not be carried out. Hence the formulations were not manufactured for these studies for the 12 and 9 month stability studies to support the toxicology and the clinical studies respectively.
In-Vitro studies
To induce an effective mucosal immune response, a successful vaccine should be directed toward effectively triggering the innate immune response. Innate immunity is critical for orchestrating the adaptive immune response through the activation of antigen-presenting cells (APCs) such as the dendritic cell (DC). Initial systemic responses are governed by the innate immune system, which produces pro-inflammatory responses (cytokines and chemokines) to the initial insult of an infectious agent. Furthermore, this initial rapid pro-inflammatory response is considered to be the critical trigger provided by traditional immunological adjuvants. Thus, many vaccine formulations strategies include innate immune triggers to provide this adjuvant signal.
In vitro studies were performed to examine the ability of various chitosan solutions to activate / potentiate an innate immune response. The results showed that both CSN and TM-CSN solutions gave positive results; with TM-CSN being more efficient in its ability to enhance toll-like receptor (TLR)-induced cytokine production skewing the T cell response towards a mixed Th1 / Th17 phenotype. Further studies to evaluate the effects of chitosan on TLR-induced cytokine production by plasmacytoid DCs (pDCs) showed that TM-CSN enhanced CpG induced IP-10 which is a Th1 cell chemokine, whereas CSN induced LPS induced IL-12p40 and IL-1beta which are Th17 polarising cytokines. CSN had significant suppressive effect on both LPS and CpG induced LP27 which is known to stimulate the production of interferon (IFN)-alpha and has been shown to exhibit both pro- and anti-inflammatory properties. Further studies would be required to investigate this effect.
The chitosan formulations that exhibited the best ability to potentiate the innate immune response in vitro supported the identification of formulations with the best chance of succeeding in vivo.
In vivo mouse studies
Several in vivo studies were carried out in mice to evaluate various chitosans, antigen dose response, efficacy of the adjuvanted antigen, effect of previous immunity, establish vaccination regime and examine the effect of nasally administered chitosan on local brain cytokine production.
Evaluation of different chitosans and dose response
Of the three chitosans (CSN, carboxymethyl chitosan and diquaternary piperazine chhitosan) tested, CSN produced the highest overall immune response (IgG, nasal wash IgA and serum radial haemolysis (SRH)). Furthermore CSN enhanced SRH titres after one dose as compared to the antigen alone. A dose response study in mice showed that CSN augmented the highest serum IgG and IgA, nasal wash IgA, seroprotection (SRH titres) shown in Figures 1 and 2 respectively below and the quality of the T cell responses. The SU (7.5 ug HA) formulated with CSN induced good nasal wash IgA and serum IgG responses after the second immunisation and low frequencies of double cytokine producing CD4+ cells (not shown).
Evaluation of vaccine regime
To discover the most efficacious vaccination regime, parenteral priming with either alum or cytosine-phosphate-guanine dinucleotide (CpG) followed by nasal vaccination with the antigen formulated with CSN and TM-CSN was explored. CSN and TM-CSN enhanced HA-specific IgG1 in sera of alum and CpG primed mice, whereas TM-CSN strongly enhanced HA-specific IgG2a in sera of CpG primed mice. Priming with antigen in the presence of alum polarised the antigen specific T cell response towards a Th2 phenotype with high concentrations of antigen-specific IL-4 and IL-5 detected in both the spleen and lymph nodes of immunised mice. In contrast, priming with antigen in the presence of CpG followed by intranasal boosting with antigen and TM-CSN induced a more mixed Th1 / Th2 profile in the spleen of immunised mice.
Effect of previous H3 or H5 immunity
As the general human population is already exposed to H3 influenza viruses the effect of previous H3 immunity on nasal immunisation with H5N1was investigated. The results showed that two nasal doses of H5 / CSN administered intranasally following H3 or H5 priming in the presence of CpG considerably enhanced the antibody responses. This indicated that subcutaneous prime / nasal boost strategy could provide protection against H5 viral infection.
Further prime / boost studies confirmed that two doses of nasal chitosan adjuvanted H5 vaccine could produce both humoral and cellular immune responses. These responses could be further enhanced by priming the mice subcutaneously with CpG adjuvanted H5 vaccine followed by two doses of chitosan adjuvanted H5 vaccine. A comparative study examining mucosal versus parenteral priming showed that CSN elicits a strong IFN-gamma response when administered intranasally and also induces a cytokine IL-22 which is known to play an important role in the regulation of immunity and inflammation at barrier surfaces.
Effect of nasally administered CSN on local brain cytokine production
When vaccinating via the nasal route in the presence of an adjuvant, it is important to investigate its effect on the local lymphatic tissues as well as on the brain (due to its close proximity to the nasal cavities). The nasal associated lymphoid tissue (NALT) is a mucosal lymphoid organ which is thought to play a pivotal role in directing the development of B and T cells that can protect the respiratory tract. This study aimed to investigate the effectiveness of CSN used in the vaccine formulations at activating immunity in the NALT. CSN activated the production of IL-1beta, IL-6 (important for Th17 responses) and IL-12p35, a critical polarising cytokine for Th1 development. In the NALT CSN also induced the production of IL-22 which is known to play an important role in the regulation of immunity and inflammation at barrier surfaces. Early studies have indicated that IL-22 does not appear to play a substantial direct role in immunity to viral pathogens. However, the importance of IL-22 in vaccine induced protective immunity to influenza would require further investigation.
On evaluating the cytokine production in the hypothalamus region of the brain the results showed that CSN did not augment the production of pro-inflammatory cytokines, IL-1beta and IL-6 after intranasal immunisation; thereby supporting the claim that chitosan is a safe adjuvant to use intranasally.
Intranasal studies evaluating cyclic diguanylate monophosphate (c-di-GMP) adjuvanted antigen with or without CSN
A novel experimental adjuvant c-di-GMP was found to enhance systemic and local antibody responses in terms of serum IgG1, IgG2a, SRH, HA inhibition (HI) and virus neutralisation (VN) antibody titres. VN titres against the homologous strains in all the adjuvanted groups were significantly higher than in the non-adjuvanted group. The evaluation of cross clade reactive antibodies against the clade 2.1 virus A/Indonesia/5/05 (IDON) showed no neutralisation of this virus in the antigen alone groups, whilst some animals in the adjuvanted groups were seropositive. However no reactive antibodies against A/Indonesia/05/2005 were observed.
A clear dose response was observed for all vaccine groups, with adjuvants augmenting the highest local nasal wash IgA response at an antigen dose of 7.5 ug after the second administration; whereas no local IgA was found in the control animals that had been dosed with PBS. At two and three weeks after the second dose no influenza specific serum IgG or IgA were detected in the control group, whereas the c-di-GMP or combination c-di-GMP+CSN groups had the highest humoral responses closely followed by the CSN group. Both CSN and c-di-GMP effectively enhanced splenocyte proliferation as compared to antigen alone. The evaluation of Th-cell profile showed that c-di-GMP adjuvanted vaccine produced a Th1 / Th17 biased response, whilst the CSN adjuvanted vaccine group had a cytokine profile indicating a more Th2 skewed response.
Overall this study showed that both CSN and c-di-GMP boosted functional antibody responses. The c-di-GMP adjuvant provided significant dose-sparing in terms of seroprotection, whereas the adjuvant combination did not. However, the adjuvant combination skewed the immune responses towards a more balanced Th profile than either of the adjuvants alone. This study highlights the importance of assessing the humoral and cellular immune responses following immunisation to allow direct evaluation novel mucosal influenza vaccines for finding the optimal vaccine formulation.
Comparison of CSN and TM-CSN adjuvanted influenza (H5N1) vaccine
Since conclusive immunological results were not obtained from two ferret studies, a statistically powered study was performed to compare the efficacy of CSN and TM-CSN as mucosal adjuvants. Both CSN and TM-CSN augmented the antibody response, with SRH titres above the protective threshold of 25 mm2 and no significant difference was observed in HI titres to the two chitosans but CSN resulted in significantly higher SRH titres than TM-CSN. The homologous neutralising antibody response was significantly higher in the CSN group than all the other groups. In terms of the heterologous antibody response cross-reactive neutralising antibody response to A/Indonesia/1/2005 strain was observed in majority of the CSN immunised animals (7 of 10) and none in the other groups.
Low concentrations of local nasal wash IgA were detected after the first dose with the highest response detected in the two chitosan groups. The second dose significantly augmented the local IgA particularly in the chitosan groups, with the CSN group at two weeks and the TM-CSN group at two and three weeks post vaccination (Figures 9 B and C).
The splenocyte proliferation assay showed a significant adjuvant effect of CSN, and TM-CSN suggesting an enhancement of the cellular immune response. Of the two chitosan adjuvants CSN appeared to be more effective than TM-CSN. In terms of the cytokine response the antigen alone and chitosan adjuvants elicited Th1 (IL2, IFN-gamma), Th2 (IL4, IL5 and IL10) and Th17 response (Figure 10). A significantly high IL-17 response was observed in two chitosan groups.
Overall this study confirmed that both CSN and TM-CSN boosted functional antibody as well as local IgA responses with CSN appearing to be marginally more efficacious than TM-CSN. Similar observations were made in terms of T cell proliferation. However TM-CSN showed a significantly higher IL-23 response than CSN.
Comparison of Solvay and Novartis antigens
Since the source of the antigen was changed during the project, an immunogenicity bridging study was carried out to compare the Solvay and Novartis antigens. Both the antigens elicited similar IgG1titres pre-boost and post-2nd booster vaccination. Slight enhancement in IgG2a titres was observed in pre-boost sera in response to Solvay antigen but comparable IgG2a titres were observed in response to both antigens in the final samples. Both the antigens showed some enhancement of SRH titres compared to the PBS control. Solvay antigen performed slightly better than Novartis antigen in that 2 / 6 animals reached the protective level. The Solvay antigen also performed slightly better in the homologous VN assay. Moreover, the cytokine data appeared to indicate Solvay antigen to elicit a rather more robust mixed T cell response with respect to Th1 and Th17 and similar Th2-type response.
Overall the results indicated that the antigens from the two sources were sufficiently similar for use in further preclinical and clinical studies.
In vivo ferret studies
The immunogenicity ferret study 1
The immunogenicity of novel intranasal formulations, both as liquid and powders containing H5N1 antigen, was evaluated in the ferret model. Antibody titres obtained in ferrets were were relatively low and inconsistent, for example the positive control (i.m.) in this study performed poorly as no SRH seroprotection was observed despite some HI seroconversion. Although the results of this study were inconclusivethere were indications that chitosan enhanced immune response and that the liquid formulation possibly was more efficacious than powders. The consortium concluded that the non-responsiveness of powders was probably due to technical issues, i.e. not delivering the powders appropriately in the nasal cavity of the ferrets rather than the antigen immunogenicity. It also appears that.
The immunogenicity ferret study 2
A further immunogenicity study was carried out to evaluate liquid and powder formulations containing H5N1 antigen, in combination with two different chitosans (CSN or TM-CSN), via the nasal route in the ferret model. The study included a chitosan-free nasal liquid formulation and a control IM injection of the antigen.
The overall results of this study were compromised for a number of reasons. The background SRH values (day 0) were high in all the groups. Furthermore the serum samples for days 0 and 21 were observed to be coloured indicating contamination with plasma proteins, thereby possibly contributing to non-specific cross-reaction. The outcome of the results was further complicated by the administration of antibiotics to animals in two groups as well as a reduced number of animals completing the study in another two groups due to poor health.
The immune responses indicated that chitosan liquid formulations were efficacious in enhancing both the seroconversion and seroprotection rates following two nasal vaccinations, with TM-CSN possibly performing similarly but possibly slightly better than CSN. The positive control (i.m.) group also enhanced immune responses following both one and two vaccinations. However the nasal and the i.m. groups could not be directly compared as the latter group had a limited number of animals.
It was not possible to select the most appropriate formulations for further evaluation from the two ferret studies. Hence the consortium decided to carry out a statistically powered study in mice to compare the efficacy of CSN and TM-CSN as mucosal adjuvants in solution formulations (section 1.3.5.3).
The challenge study
Following the decision by Novartis not to supply antigen for clinical use, a definitive challenge study to establish efficacy of intranasally administered influenza H5N1 A/Vietnam/1194/2004 vaccine (supplied by Novartis) was conducted in the ferret model using liquid vaccine formulations based on both CSN and TM-CSN. The study was subcontracted to a contract research organisation, Viroclinics. The study protocol was specifically designed by several members of the consortium in collaboration with Viroclinics.
The objective of this study was to investigate the immunogenicity and protective efficacy of intranasally administered adjuvanted vaccines in the ferret H5N1 wild type model. The intranasal candidate vaccines were aqueous solutions containing subunit of inactivated NIBRG-14 influenza vaccine prepared in egg, GMP grade, obtained from Novartis. The potency of the antigen was defined by its HA content quantified by SRID method. Candidate vaccines were adjuvanted with CSN or TM-CSN prior to testing. For control purposes, a chitosan-free vaccine formulation was tested along with appropriate placebo PBS controls. The design of this study is summarised.
The vaccination phase:
This study consisted of six groups each comprising 6 ferrets:
- groups 1 and 2 were intranasally immunised with CSN adjuvanted vaccine;
- group 3 was intranasally immunised with non-adjuvanted vaccine;
- group 4 was intranasally immunised with TM-CSN adjuvanted vaccine;
- groups 5 and 6 were treated with PBS.
Intranasal vaccine were administered twice, once on study day 0 and then again on study day 21. During the immunisation phase and just prior to challenge blood samples were taken to evaluate the immune response to vaccination.
The challenge phase:
Animals were challenged individually with a dose of 105 TCID50 H5N1.
A/Vietnam/1194/2004 HPAI virus on study day 49. Animals from Groups 1 and 5 were challenged via the intranasal route and animals of groups 2, 3, 4 and 6 were challenged via the intratracheal route. Ferrets were monitored for infection related clinical and virological responses and for vaccine induced immunological responses. Animals were sacrificed on study day 54 (i.e. 5 days post challenge) to evaluate pathologic and additional virological parameters.
Summary of results
i. Antibody response induced by vaccine candidates
Antibody responses were tested using three different assay methodologies, namely SRH, VN and HI. The serum HI antibody response against H5N1 A/Vietnam/1194/2004 was greater in animals immunised with the TM-CSN adjuvanted vaccine compared to CSN and was absent in animals immunised with non-adjuvanted vaccine. The results were similar for SRH and VN assays. CSN and TM-CSN adjuvanted vaccines also induced cross protective antibodies in-vitro against H5N1 viruses from representatives from clade 2.1 and 2.2.
ii. Results of challenge phase of the study
The inoculation route of the challenge with H5N1 A/Vietnam/1194/2004 HPAI virus had significant influence on the clinical, virological and pathological outcome of the virus infection. Intratracheal challenge with H5N1 A/Vietnam/1194/2004 influenza virus was shown to be lethal and to result in high level virus replication in lung and trachea. Intranasal challenge with H5N1 A/Vietnam/1194/2004 influenza virus was not lethal and resulted in high level virus replication in the upper respiratory tract and the majority of animals also exhibited virus replication in the olfactory bulb. In accordance, histopathological findings in the lung were more severe after intratracheal challenge and encephalitis was more common after intranasal challenge. The serum antibody response correlated well with the protection from intratracheal infection with influenza H5N1 A/Vietnam/1194/2004.
Animals immunised with the TM-CSN adjuvanted vaccine were protected against intratracheal infection-related findings in terms of lethality, body weight loss, fever, virus replication in respiratory and brain tissues and lung pathology (data not shown). Animals immunised with CSN- and in particular TM-CSN-adjuvanted vaccine were protected against intratracheal infection. Performance of the control group immunised with the non-adjuvanted vaccine and intratracheally challenged was, with respect to the evaluated clinical, virological and pathological parameters, low and not substantially different from that of PBS treated animals following intraracheal challenge.
In conclusion the severity of the H5N1 A/Vietnam/1194/2004 HPAI virus infection was, with respect to clinical, virological and pathological outcome, defined by the route of challenge. The outcome of infection was dependent on route of inoculation. Intranasal immunisation with the CSN or TM-CSN adjuvanted vaccines induced serum antibody responses that were protective against inoculation with H5N1 A/Vietnam/1194/2004 HPAI virus, whereas intranasal immunisation with the non-adjuvanted vaccine did not. Antibody responses were cross protective amongst different clades of H5N1 viruses. The highest level of protection from an intratracheal challenge with H5N1 A/Vietnam/1194/2004 HPAI virus was induced by the TM-CSN adjuvanted vaccine and this corresponded with the highest serum HI antibody responses.
Overall, immunisation with CSN adjuvanted vaccine was less effective than immunisation with TM-CSN adjuvanted vaccine. Immunisation with the CSN adjuvanted vaccine induced comparable levels of protection against an intranasal and intratracheal challenge with H5N1 A/Vietnam/1194/2004 HPAI virus. Moreover the comparison of intratracheal and intranasal challenge with a lethal dose of live virus in ferret is groundbreaking research as it has highlighted the advantages and disadvantages of each route of challenge; the normal practice has been to challenge only via the intratracheal route.
Clinical study
The relevant consortium members made preparations to perform a clinical proof-of-principle efficacy study. Whilst awaiting the supply of GMP antigen by Novartis, participant 2 wrote an outline of the study protocol and participant 1 started to prepare the CTA in preparation for submission of the application for the planned clinical trial to the regulatory authorities.Considerations were also given to the type of containers that might be used for the delivery of vaccine to the humans and hence a stability study of the antigen in either glass vials or plastic syringes was performed and the suitability of spray devices was confirmed (section 1.3.1.2). Furthermore to optimise B cell assays, participant 7 used test samples from a Phase I clinical trial of an H5N1 (RG14) vaccine using Matrix M as an adjuvant performed at University of Bergen for a project other than NASPANVAC. B cells called antibody secreting cells (ASC) in the ELISPOT assay were successfully optimised and enumerated. Good levels of long term memory B cell response at 12 months post vaccination was also observed with the highest responses being detected in the adjuvanted groups.
When Novartis declined the supply of GMP antigen for a clinical study on 23 October 2011, it became apparent that it was not possible to do a clinical study; no alternative supplier could be identified at that point. Hence the consortium unanimously agreed to focus the efforts and resources to perform a challenge study in an established in-vivo efficacy (ferret). This study showed highly promising results which are discussed above (section 1.3.5.3).
Conclusion
Although, for reasons not under the control of the consortium the objective of performing a proof-of-principle clinical study was not accomplished, the robust efficacy data in the ferret model along with the immense immunogenicity (both systemic and local antigen specific Igs, protective / functional antibodies, several cytokines) mouse data including the evaluation of the most efficacious chitosan type, dose response, effect of previous immunity, vaccine regime and the effect of nasally administered chitosan on local brain cytokine production obtained in this project, forms an excellent comprehensive preclinical package for submission to the regulatory authorities to develop a nasal H5N1 vaccine using chitosan as the mucosal delivery platform. Stability data and administration techniques/devices are confirmed to facilitate such a study.
Potential impact:
Socio-economic impact
To date, 606 cases of human H5N1 infections have been reported in 15 different countries of which almost 60 % have been fatal. However, any initiative that formulated transmission to or between humans could produce catastrophic levels of infection. A number of strategies and approaches are being brought into play to confront an influenza pandemic. Certain antiviral drugs (Tamiflu, Relenza) are commercially available and are the first line of treatment for health care providers. However, for these drugs to be effective the treatment has to commence within 48 hours of the onset of the symptoms. These drugs belong to a class of medicines called NA inhibitors which prevent the influenza virus from spreading inside the body and are designed to be active against all clinically relevant influenza virus strains. Due to the rapid production of drifted antigens of the H5N1 virus, there are already indications of development of resistance to these drugs. Hence, the use of antiviral drugs may not provide the treatment that would be required for an avian influenza pandemic.
Vaccination is the cornerstone of influenza prophylaxis. In the event of a pandemic, rapid production and deployment of a pandemic vaccine will limit viral spread, hospitalisations, serious complications of disease and ultimately death. The pandemic virus strain could significantly differ from the virus vaccine strain being stockpiled by various governments as a mean of strategic planning. In recent years there has been an increase in manufacturing of seasonal trivalent influenza doses. There are only a few countries with influenza vaccine manufacturing capacity and these will probably reserve the scarce supply of a pandemic vaccine for their own citizens. The World Health Organisation (WHO) aims for equitable and timely access to vaccines for all peoples of the world. However, the reality is that only a few countries manufacture influenza vaccine; therefore populations in other countries most affected by the pandemic may not have access to a pandemic vaccine. During the swine origin influenza pandemic in 2009, the virus spread globally within two months, highlighting the importance of rapid deployment of vaccine. Developing countries had limited access to pandemic vaccines and a needle free mucosal influenza vaccine would have allowed vaccination with limited numbers of public health providers. The NASPANVAC programme investigated a number of adjuvants, alone and as co-adjuvants, that could enhance the immunogenicity of current vaccines to allow dose-sparing formulations and allow the limited vaccine supply to be used as effectively as possible which could have a real positive effect on controlling a pandemic.
The history of pandemic vaccine development since 1997 provides worrying lessons in the difficulties of preparing influenza vaccines containing novel influenza subtypes. The hope is that these lessons will allow the vaccine industry to be ready to immediately start production of the most efficacious vaccine once a pandemic has been declared. Each manufacturer has its own licensed facility and process for manufacturing and these may not be the best vaccine formulation to provide the best protection in a pandemic. There is an urgent need for research and comparative clinical trials comparing different vaccine formulations with or without adjuvant, vaccine strength, number of doses and routes of administration to provide information on what is the appropriately formulated vaccine and optimal use, to save valuable time in preparation of a pandemic vaccine. The preclinical studies carried out under the NASPANVAC project clearly showed that nasally delivered chitosan adjuvanted vaccine enhanced both local and systemic immune responses thereby combating the virus at its point of entry and also systemically, induced specific cytokines giving insight into Th1 / Th2 balance as well as their role in regulation of immunity and inflammation, demonstrated heterologous cross-reactive neutralising antibody response against a couple of H5N1 strains and most of all provided protection against the challenge with the live homologous virus. Although the NASPANVAC programme did not manage to demonstrate the efficacy of a nasally delivered pandemic vaccine in humans, the various issues were extensively addressed and investigated in preclinical studies which now form the basis for further exploitation of the project results in terms of phase II / III clinical trials, applications for regulatory approval in major jurisdictions, implementation of commercial scale manufacture and global commercialisation of a nasal vaccine.
Wider societal implications of the project
In view of the excellent safety profile of chitosan and the robust preclinical data produced in the NASPANVAC programme are highly encouraging given that traditionally vaccine adjuvants tend to be relatively more toxic moieties. CSN and TM-CSN were shown to elicit protective immunity against lethal H5N1 challenge in a recognised model of human influenza, the ferret. Thus, the risk: benefit ratio for a chitosan-based vaccine may be favourable when compared with that of more potent adjuvants. A H5N1 pandemic could have a major impact on public services, making self-reliant treatment and management of the disease mandatory. The NASPANVAC project has clearly demonstrated the potential to develop a nasal vaccine which could be self-administered and also enable rapid mass vaccination with minimum aid from specialist healthcare providers.
Main dissemination activities and exploitation of results
a) The dissemination activities to date have been as follows:
1. Jabbal-Gill I. Nasal vaccine innovation. J Drug Target. 2010. Dec; 18(10):771-86
2. Jabbal-Gill I, Watts P, Smith A. Chitosan-based delivery systems for mucosal vaccines. Expert Opinion on Drug Delivery, Posted online on 19 June 2012 (doi:10.1517/17425247.2012.697455)
3. Signe C. Svindland, Åsne Jul-Larsen, Solveig Andersen, Rishi Pathirana, Abdullah Madhun, Emanuelle Montomoli, Inderjit Jabbal-Gill, Rebecca J. Cox. 'The mucosal and systemic immune responses elicited by a chitosan adjuvanted intranasal influenza H5N1 vaccine' Poster presented at The Oxford influenza conference, Influenza 2010.
4. Svindland SC, Jul-Larsen Å, Pathirana R, Andersen S, Madhun A, Montomoli E, Jabbal-Gill I, Cox RJ. The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine. Influenza Other Respi Viruses. 2012 Mar; 6(2):90-100. doi: 10.1111/j.1750-2659.2011.00271.x. Epub 2011 Jul 12.
5. Signe C. Svindland1, Gabriel Kristian Pedersen, Rishi Pathirana*, Geir Bredholt*, Jane Kristin Nøstbakken*, Åsne Jul-Larsen*, Carlos Guzman, Emanuele Montomoli, Giulia Lapini Inderjit Jabbal-Gill*, Michael Hinchcliffe*, Rebecca J. Cox1*. A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine. Submitted to Influenza and other Respiratory Viruses (2012)
b) Publish the mouse data, especially the cytokines and the ferret data demonstrating the efficacy of a nasally delivered chitosan adjuvanted vaccine in prestigious peer-reviewed scientific journals by the end of 2012.
c) Present the efficacy data in ferrets initially at an international meeting in Q4 2012 and others thereafter.
List of websites: http://www.naspanvac.com
