Effective nasal influenza vaccine delivery using chitosan

Vaccine 23 (2005) 4367–4374

Effective nasal influenza vaccine delivery using chitosan Robert C. Read a,∗ , Simone C. Naylor a , Christopher W. Potter a , Jenny Bond b,1 , Inderjit Jabbal-Gill b,1 , Anthony Fisher b,1 , Lisbeth Illum b,1 , Roy Jennings a a

Academic Unit of Infection and Immunity, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK b West Pharmaceutical Services, Drug Delivery and Clinical Research Centre Ltd., Albert Einstein Centre, Nottingham Science and Technology Park, University Boulevard, Nottingham NG7 2TN, UK Received 24 November 2004; accepted 18 April 2005 Available online 12 May 2005

Abstract Nasal influenza vaccination may prove to be a good alternative to parenteral injection because of the enhancement of the mucosal immune response and the ease of vaccine administration. This study investigated the use of chitosan, a bioadhesive polymer, as a nasal delivery system with inactivated, subunit influenza vaccine. Subjects received nasally 15 or 7.5 ␮g of the standard inactivated trivalent influenza vaccine with chitosan or 15 ␮g of the same vaccine intramuscularly. Serum haemagglutination inhibition (HI) titres for all three vaccine components were measured prior to, and at time points up to 14 weeks after dosing. Serum HI titres following intranasal vaccination with the nasal chitosaninfluenza vaccine met the criteria set by the Committee for Proprietary Medicinal Products in terms of seroprotection rate, seroconversion rate and mean fold increase of HI titre for at least one of the three antigens in the vaccination schedules used. These data show that nasal immunisation with chitosan plus trivalent inactivated influenza is a potentially effective, easily-administered form of vaccination. © 2005 Elsevier Ltd. All rights reserved. Keywords: Nasal influenza vaccination; Chitosan; Serum haemagglutination inhibition

1. Introduction Current influenza vaccines are formulations of inactivated preparations administered parenterally, containing either the surface haemagglutinin (HA) and neuraminidase (NA) proteins or whole virus particles. They have been generally regarded as less than optimal as they induce immunity in only 60–90% of recipients overall [1,2]. This may often be due to a mismatch between the vaccines and antigenically drifting wild virus [3], but also because individuals at the extremes of age respond poorly to the vaccine [3–5]. In addition, current vaccine preparations induce little local IgA antibody responses [6,7], which is unsatisfactory, particularly when it is well known that respiratory mucosal immune ∗ 1

Corresponding author. Tel.: +44 114 271 2027; fax: +44 114 271 3892. E-mail address: [email protected] (R.C. Read). Tel.: +44 115 907 8711.

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.04.021

responses contribute significantly to natural immunity [8,9] and are poor inducers of cellular-immune responses [10,11]. Strategies to enhance the immune responses to influenza vaccines have included the incorporation of adjuvants and use of live attenuated vaccines [12–14]. One alternative strategy to improve influenza vaccination is the administration of inactivated vaccines via the intranasal route. Studies of inactivated vaccines in saline given intranasally to man have generally been disappointing [2,3,15]. However, in both human and animal studies, intranasal influenza virus vaccines, incorporating a variety of adjuvants, have been reported to induce strong local and systemic antibody responses and protection against a subsequent infective challenge [14–19]. Here, we report the results of a study using inactivated, trivalent influenza virus vaccine administered intranasally to subjects together with a chitosan excipient. Chitosan is a cationic bioadhesive polysaccharide derived from crustacean shells, and is widely used as a food additive

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and a slimming aid. Previous studies in mice and guinea-pigs have shown that chitosan was able to significantly enhance both humoral and cellular immune responses of nasally administered influenza, pertussis and diphtheria vaccines [20–25]. In the present study, chitosan-influenza vaccine was given to human subjects as two intranasal doses four weeks apart; and the results compared to or those from subjects given a single dose of standard inactivated trivalent vaccine by the intramuscular route.

2. Materials and methods 2.1. Subjects Sixty-eight healthy subjects, aged 18–49 years, 23 of whom were male, participated in the study. The exclusion criteria included pregnancy, hypersensitivity to eggs, poultry or shellfish; allergies to drugs or pharmaceutical agents and a history of influenza virus vaccination in the preceding 6 months. The study was approved by the South Sheffield Research Ethics Committee. 2.2. Study design Prior to immunisation, blood samples were obtained from all subjects, the serum separated and each serum sample tested for haemagglutination-inhibiting (HI) antibodies to influenza A/Sydney/5/97 (A-H3N2) virus by standard procedures [26,27]. Subjects were then coded, ranked by HI titre, and assigned to one of three groups such that the mean geometric titre (GMT) of A-H3N2 serum HI antibody for the three groups was approximately equivalent. Subjects received either conventional, trivalent aqueous subunit vaccine supplied by Solvay Pharmaceuticals B.V. (P.O. Box 900, 1380DA Weesp, The Netherlands) containing surface HA and NA antigens derived from influenza A-H3N2, A/Beijing/262/95 (A-H1N1) and influenza B/Yamanashi/166/98 (B-strain), by intramuscular (i/m) injection (22 subjects), or the same influenza vaccine (either 7.5 ␮g or 15 ␮g), administered intranasally (i/n) as a solution in combination with chitosan glutamate (0.5%, w/w). The antigens used were those recommended for inclusion in the 1999/2000 commercially-available influenza virus vaccine. The two nasal spray formulations were produced according to the principles of GMP, and contained either 15 ␮g total virus subunit protein for each of the three viruses per dose (23 subjects), or 7.5 ␮g total viral subunit protein for each of the three viruses per dose (23 subjects); both were administered in two doses 4 weeks apart. Each dose comprised one spray of 100 ␮l into each nostril. Serum samples were obtained prior to and 4 weeks after the initial intranasal immunisation, prior to and 4 weeks after i/m vaccination, and 14 weeks after the second i/n dose or the single i/m vaccination.

2.3. Haemagglutination inhibition (HI) test Serum antibody responses to the various influenza virus HA in the vaccine were measured by haemagglutinationinhibition (HI) test at Erasmus Medisch Centrum Rotterdam (Aldeling Virologie, Dr. Molewaterplein 40, 40, 3015 GD Rotterdam, The Netherlands). Sera were pre-treated with cholera filtrate (receptor destroying enzyme) at 37 ◦ C for 16 h, then at 56 ◦ C for 1 h to remove non-specific inhibitors. Two-fold serial dilutions of treated serum samples, from 1:20 to 1:1280 in phosphate-buffered saline pH 7.2 (PBS), were incubated with 4 HA units of virus in U-bottom microtitre plates for 30 min at 37 ◦ C. Turkey erythrocytes (1%) were added to each well and incubated for 1 h at 4 ◦ C. The HI titres were taken as the reciprocal of the highest serum dilution that completely inhibited virus haemagglutination. 2.4. Statistical analyses The statistical test (logistic regression analysis) was performed using the SAS programme at the Statistical Services Unit, University of Sheffield. All assumptions of the analysis were checked for each model fitted to the data. Individual significance levels were adjusted to account for the multiple endpoints and comparisons in the analysis. More specifically, individual analyses used a significance level of 0.0014 (Bonferroni correction for 36 analyses) in order to preserve an overall significance level of 5%.

3. Results 3.1. Vaccine tolerance A total of 54 treatment related adverse events, were recorded for subjects given two doses of half-strength vaccine intranasally, and 56 treatment related adverse events for subjects given two doses of full-strength vaccine by the same route. The events were all mild in nature, of short duration after vaccine inoculation, and were mostly the symptoms of rhinorrhea. A total of 12 adverse events were recorded for subjects given vaccine by the i/m route: again, these were all mild in nature, of short duration, occurring immediately following injection, and comprised pain and bruising at the injection site. 3.2. Serum HI antibody response to immunisation In total seven subjects withdrew from the study and one subject, who had received the i/m vaccine, failed to attend for the blood and nasal wash sample collection at 14 weeks post-dose. The numbers that completed the protocol were twenty-two for both the full-strength and half-strength vaccine nasal doses and 16 for the i/m vaccine completed. The serum HI antibody responses are shown in Table 1. The HI responses to the three individual virus components of the

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Table 1 Serum HI antibody responses of subjects given inactivated trivalent influenza vaccine by the intranasal or intramuscular routes Virus type/sub-type

Sample times

Vaccine (15 ␮g) i/n × 2

Vaccine (7.5 ␮g) i/n × 2

HI (GMT)

±S.D.

Pre-dose 4 Weeks post 1st dose 14 Weeks post 1st dose 4 Weeks post 2nd dose 14 Weeks post 2nd dose

48 129

66 132

70 99

60 81

154 146

142 155

129 128

96 102

B/Yamanashi/166/98

Pre-dose

204

212

224

(B-strain)

4 Weeks post 1st dose 14 Weeks post 1st dose 4 Weeks post 2nd dose 14 Weeks post 2nd dose

516

599

507 264

Pre-dose 4 Weeks post 1st dose 14 Weeks post 1st dose 4 Weeks post 2nd dose 14 Weeks post 2nd dose

A/Sydney/5/97 (A-H3N2 strain)

A/Beijing/262/95 (A-H1N1 strain)

HI (GMT)

±S.D.

242 892 424

640 1267 494

226

386

479

391

316

998 351

10 373

436 198

558 206

411 209

18 82

45 140

30 77

93 98

9 427 179

10 549 218

109 75

167 121

114 105

117 107

HI (GMT)

±S.D.

Vaccine (15 ␮g) i/m × 1

GMT: geometric mean titre.

vaccine are shown for pre-dose, 4 weeks after the first, 4 and 14 weeks after the second i/n vaccination, and for pre-dose, 4 and 14 weeks following the single i/m injection. The results are expressed as geometric mean titres (GMT ± S.D.). The Committee for Proprietary Medicinal Products (CPMP) has indicated that for registration of current injectable inactivated influenza vaccines, in the 18–60 year age group, at least one of the following criteria must be fulfilled for the vaccine strains contained in the vaccine [28]: (i) achieve a serum HI antibody titre of ≥40 in 70% of subjects (seroprotection rate, SP), (ii) induce ≥four-fold increase in HI antibody titres in 40% of subjects (seroconversion rate, SC), (iii) attain ≥2.5-fold increase over the pre-immunisation HI antibody titre level (mean fold increase, MFI). For each of the three separate components of the inactivated vaccine given i/n or i/m the seroprotection and seroconversion rates as well as the mean fold increase were calculated. For subjects given the full-strength vaccine i/n, each of the strains met at least two of the above criteria either after one dose or two doses 28 days apart (Fig. 1). The A-H3N2 and B-strains achieved seroprotection rate ≥70% and MFI of >2.5% while the A-H1N1 strain achieved the seroconversion rate ≥40% and MFI of 2.5%. For subjects given the half-strength vaccine i/n, each of the strains met either two or one of the above criteria after one or two doses (Fig. 2). The A-H1N1 achieved a seroconversion rate ≥ 40% and MFI of 2.5%, the B-strain achieved seroprotection rate ≥70% and MFI of >2.5%, whereas A-H3N2 strain met only the protection rate criterion. Furthermore, each of the strains after 14 weeks following the second vaccination with full or half i/n doses met either one or two of the above criteria (Figs. 1 and 2). For subjects vaccinated with full-strength, the A-H3N2 strain met all the three criteria, the A-H1N1 strain

induced seroprotection rate ≥40% and MFI of 2.5%, while the B-strain achieved the seroprotection rate and the MFI criteria. For subjects vaccinated with half-strength the A-H1N1 strain met the seroprotection and MFI criteria while the A-H3N2 and B-strains achieved only the seroprotection rate ≥70%. Better responses were seen following one i/m immunization and the three strains met the above criteria (Fig. 3). The MFI values were comparatively much higher and a larger percentage of subjects showed seroconversion of ≥4 but again, for the B-strain virus component, the seroconversion rate had fallen to <40% at 14 weeks after vaccination. Overall the results indicate satisfactory serum HI antibody responses to one and two doses of full-strength or half-strength i/n vaccine. The results were not statistically different from those obtained following administration of the non-adjuvanted, commercial influenza vaccine by the i/m route.

4. Discussion Although inactivated influenza viruses in saline induce significant protection against influenza virus infection, these vaccines remain less than optimal: thus they do not induce protective levels of serum HI antibody in all subjects [27], induce little local IgA antibody [3], and immunity in only 60–90% of vaccinees [2]. Attempts to improve and broaden the immune response by alternative methods of vaccine preparation or incorporation of carrier/adjuvant systems have been mixed to date [3]. Alternative methods of immunisation are therefore under consideration. The use of high doses of purified influenza haemagglutinin antigen as a vaccine [29] may prove unacceptable in certain target groups of the population, such as the elderly, even though such preparations in healthy young adults can enhance both local and systemic antibody responses. However, the recognised importance of local

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Fig. 1. Response to 15 ␮g intranasal vaccine with chitosan. Seroprotection rate is given as percentage achieving a serum HI antibody titre ≥40. Seroconversion rate is percentage subjects exhibiting a ≥ four-fold increase in HI antibody titre.

IgA antibody [2,11,30] has stimulated interest in intranasal immunisation. In previous studies, a single unadjuvanted intranasal inoculation with inactivated influenza vaccine in saline has been reported to elicit poor serum antibody responses, but to be a good inducer of local antibody [2,8]. However, repeated doses of such vaccines have been reported to induce both local and systemic responses [31,32]. Improved local and systemic immune responses have also been reported when vaccines are administered with an adjuvant/carrier vehicle, and out of the large number of potential vehicles available [33,34], many have been tested in animal models [17–19,35–39]. From these results a limited number have

been tested in subjects. Problems of toxicity which prohibit the use of many adjuvants for parenteral immunisation [40] may be less evident when used in intranasal vaccines. Of the potent adjuvants identified from animal studies [3], and tested in subjects, the adjuvant MF59 has been shown to enhance both humoral and local antibody responses to influenza antigens following two doses [14]. Similar results have been obtained using Escherichia coli toxin B (B subunit; holotoxin) in animal and volunteer studies [41] and again using E. coli B toxin plus virosomes [16,42]. With a single exception these reports are incomplete since the duration of the local and systemic antibody responses, and the stimulation of cell-mediated immune responses which

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Fig. 2. Response to 7.5 ␮g intranasal vaccine with chitosan. Seroprotection rate is given as percentage achieving a serum HI antibody titre ≥40. Seroconversion rate is percentage subjects exhibiting a ≥ four-fold increase in HI antibody titre.

may have a role to play in immunity against influenza [6], as well as protection against challenge infection, remain to be determined. However, Durrer et al., in a controlled, randomised, double-blind phase 2 clinical trial [42], showed that a virosome-based influenza virus vaccine, adjuvanted with enterotoxigenic E. coli heat-labile toxin (HLT), given intranasally, could elicit a spectrum of humoral and cellmediated immune responses, and neutralising IgA antibody at the mucosal surface, in healthy adult subjects aged 18–60 years. These findings lend support to the strategy of i/n immunisation with an adjuvanted, inactivated influenza vaccine proposed in the present studies. A recent study using Proteosome (nanospheres of purified N. meningococcal outer membrane proteins) as an adjuvant showed that subjects

produced positive results in terms of seroprotection, nasal IgA and some protection after a challenge following the administration of two doses. These observations were apparent only in subjects who had baseline serum HI titres [43]. The most recent large scale study carried out by the same group showed that their nasal vaccine was effective in preventing influenza in the field but neither the single nor the two-dose groups were statistically different from the placebo [44]. The potential use of chitosan as a delivery system for inactivated influenza vaccines given intranasally has been clearly demonstrated in mice [9,23,24]. Bacon and colleagues showed that both serum and local antibody responses were greater for chitosan mixed with subunit vaccine than for saline vaccine alone or together with another carrier, gellan [9]. In

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Fig. 3. Response to 15 ␮g intramuscular Influvac® . Seroprotection rate is given as percentage achieving a serum HI antibody titre ≥40. Seroconversion rate is percentage subjects exhibiting a ≥ four-fold increase in HI antibody titre.

the current study, the commercially available trivalent, inactivated influenza vaccine was administered intranasally with chitosan, in human subjects. Although the design of the study was not optimal (a non-adjuvanted control group was not included and we did not measure local IgA production) the results suggest that the tested vaccine has potential to be safe and efficacious. A single dose of this vaccine preparation induced serum HI antibody titres that met CPMP requirements. The vaccine was safe and well-tolerated by the subjects. Overall, the geometric mean serum HI antibody titres induced by the vaccine met CPMP requirements, and the seroconversion rate and protective antibody levels achieved, were not statistically different from those for saline vaccines given intramuscularly. This accords with similar results obtained with other adjuvant/carrier systems [16,41,42]. There was no

clear evidence that antibody titres were further enhanced by a second dose of the current intranasal influenza virus vaccine. Although the induction of mucosal antibodies following vaccination with the chitosan influenza vaccine has not been investigated in the present study, evidence from animal studies using vaccines formulated with chitosan, strongly support the capacity of such vaccines to promote mucosal antibody [9,20–22]. Moreover, the intranasal delivery of a detoxified diphtheria toxin vaccine formulated with chitosan to humans, has resulted in the induction of systemic T cell responses, and in particular, significantly augmented Th2-type responses [46] while studies in humans have indicated that the administration of inactivated influenza vaccines via the intranasal route, with some form of carrier or delivery system, can induce mucosal antibody [3,14,16,42–45]. In addition, a recent

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study has demonstrated that the nasal delivery of a plasmid expressing certain respiratory syncytial virus epitopes, accompanied by chitosan as an adjuvant, can induce cytotoxic CTL responses in Balb/c mice that protected these animals against this virus [47]. The present study suggests that chitosan is a potentially successful nasal delivery system for vaccination against influenza. Chitosan also has potential utility for other bacterial and viral vaccines. The observations reported here are highly encouraging and further studies are now needed to determine optimal dosage schedule and dosage size.

Acknowledgements We acknowledge the assistance of sisters and staff of Ward E3, Royal Hallamshire Hospital, Sheffield.

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