Mucosal delivery of inactivated influenza virus vaccines in man

Influenza C.W. Potter (editor) © 2002 Elsevier Science B.V. All rights reserved

179

Mucosal delivery of inactivated influenza virus vaccines in man Roy Jennings and Robert Charles Read Section of Infection and Immunity, Division of Genomic Medicine, Universityof Sheffield, UK

Influenza remains an uncontrolled plague of mankind that for centuries has caused severe outbreaks of disease with global morbidity in all age groups. The disease is associated with a significantly increased mortality in most years, often associated with secondary bacterial infections in some high-risk groups. In addition, the threat of a future global pandemic, initiated by a novel strain of influenza and as seen several times in the last century, is an anticipated event. The viruses of influenza A and B are roughly spherical, enveloped particles, approximately 80-120 nm in diameter, containing an RNA genome consisting of eight segments coding for nine structural, and two non-structural proteins. An important property of the type A influenza viruses is their ability to undergo antigenic variation, especially with respect to the haemagglutinin (HA) and neuraminidase (NA) surface proteins. The high mutation rate associated with the genes coding for the HA and NA proteins results in almost annual minor but significant changes in these antigens, a process known as 'antigenic drift'. In addition, the capacity for the reassortment of the genes of influenza viruses causing human disease with those infecting some animal and bird species, termed 'antigenic shift', is a further mechanism by which influenza viruses acquire novel surface proteins [1]. Antigenic shift and drift represent a major problem in the development of influenza virus vaccines. These properties of the type A influenza viruses, together with certain host factors, such as the relatively poor and uneven immune response to immunisation with currently available vaccines, highlight the limitations of these preparations, and has resulted in a search for novel strategies to increase their efficacy. One such strategy is to deliver the vaccine via a mucosal route, and this chapter is concerned with a comparative assessment and evaluation of this approach for the control of influenza virus infection. Acquired resistance to infection by influenza viruses has been associated with the production of circulating antibodies to the HA and NA antigens [2,3]. Both of these surface antigens project through the viral envelope, but are also expressed, together with the internal nucleoprotein antigen of the virus, at the surface of infected cells [4]. Thus, these antigens are available for recognition by the humoral and cellular components of the immune system. Although CD8 ÷ cytotoxic T cells (CTLs), stimulated following influenza virus infection or vaccination, may contribute some degree of protection as a result of cross-reactive killing of infected cells expressing

180 viral nucleoprotein at their surface [5], such protection is probably short-lived and of limited extent. Cellular immune mechanisms probably play a more important role in clearance of virus during the period shortly after natural infection [6-8]. Although this does not preclude some role for immune mechanisms such as antibody-mediated cell cytotoxicity in protection, and as with most virus infections, non-specific defence mechanisms such as natural killer cells and interferon will have a role during the early stages of infection, the weight of evidence suggests that serum and local antibodies directed against virus HA and NA are the major factors in preventing infection by the influenza viruses. The nature and use of current influenza virus vaccines

The first vaccines against influenza were inactivated whole virus preparations developed and used in the late 1950s and in the 1960s (reviewed in [9]). These were relatively crude and impure, and produced both local and systemic reactions in an unacceptable proportion of recipients. Although such saline vaccines, in a more highly purified and markedly less reactive form, remain available commercially today, the most recently developed influenza virus vaccines contain only virus HA and NA [10]; they are well tolerated by recipients following intramuscular (i/m) vaccination [11]. The HA and NA are released from intact virus particles, undergo further purification, and are present in the vaccines as two different forms of micelles; 'stars', formed by the virus haemagglutinin (Fig. la), and 'wheels' formed by the virus neuraminidase (Fig. lb) [12]. The manner by which these micelles can be formed is shown schematically in Fig. 2 [12]. The micellar forms of the influenza virus surface antigens are known to enhance the immune response over that elicited by the proteins as monomers [13], and it has been found that these responses cannot be further improved by the inclusion of an aluminium salt adjuvant, at least in a population primed for the vaccine antigen [14]. A further form of influenza virus vaccine, also commercially available at the present time, is the 'split' vaccine derived from virus particles partially disrupted with a detergent. Besides containing the surface HA and NA proteins of the virus, these vaccines also contain some residual lipid and a fraction of the internal viral proteins. The nature of these vaccines has been reviewed elsewhere [15]. Subunit or surface antigen influenza virus vaccines are the only vaccines recommended for use in children under 12 years of age, as inactivated whole virus vaccine preparations can give rise to systemic reactions in this age-group. However, all unadjuvanted influenza virus vaccine preparations in saline do not demonstrate high efficacy either in terms of the humoral immune responses or protection against subsequent infection [8,16-18]. This is partly due to the intrinsic variability of the virus with respect to its surface HA and NA antigens, but even when these viral antigens present in the vaccine closely match those of strains later circulating in the population, some groups still respond poorly to the vaccine, and remain at risk of infection [19,20]. Thus, protection rates of only about 20% have been reported in the elderly following immunisation with a vaccine that closely matched the circulating

181

(a)

(b)

Fig. 1. Electron micrographs illustrating the stellar aggregates, 'stars' (a), and circular, 'cartwheel' aggregates (b), of influenza type A virus haemagglutinin and neuraminidase antigens, respectively.

182 HEMAGGLUTININ

"~• ~:~,~ ~ sos ~ sos ~'-~I~L " ~ ~ "~,~.~ t F REMOVED ~ ', 'C" I - - " U - - " IRUS

I I

• (~" HYDROPHOBIC [LIPOPHILIC] SURFACE

~ ~

//

MONOMERS /

h~...~

HYDROPHOBICALLY BONDED POLYMERS

t'/REaOVEOl ~

I

NEURAMINIDASE

Fig. 2. Schematic representation of the removal by detergent (sodium dodecyl sulphate) extraction of the haemagglutinin and neuraminidase antigens from the surface of the influenza virus particle, and their aggregation.

influenza virus strains [21], compared with protection rates of 70% in healthy young adults [22]. In addition many studies, both open and closed, in which volunteers are immunised and subsequently challenged with an attenuated influenza virus strain, show that inactivated influenza virus vaccines at best elicit protective immune responses in only 60-90% of individuals [18]. Elderly recipients respond relatively poorly to influenza virus vaccination because of immune senescence [23-25]. The elderly are particularly at risk from influenza virus infection and secondary bacterial pneumonia, and therefore form a cohort of the population that requires protection. Although current influenza virus vaccines are considered somewhat less than satisfactory with regard to the immune response and protection that follows their administration, it has been reported that these vaccines reduce hospitalisation of elderly individuals with complication of influenza, and also mortality, by 50% [26]. Repeated annual vaccination in the elderly is also suggested, as one study has reported a 75% reduction in mortality in those who had received vaccination previously compared to a 9% reduction amongst patients in this cohort receiving vaccination for the first time [27]. A single dose of current subunit influenza virus vaccine is sufficient to induce an immune response in "primed" individuals, i.e. those who have contacted influenza virus by infection or immunisation at some time in the past [14]. However, in young children or otherwise naive individuals who may not have previously experienced the virus, or any individual infected with a novel strain of influenza virus, two intramuscular doses of these vaccines are required to obtain protective levels of antibody [151. In spite of this, a single i/m dose of the current subunit influenza virus vaccines lacks complete efficacy in any age group of the population. Furthermore, children, who represent important disseminators of the influenza virus [17], are not routinely vaccinated with these vaccines for safety reasons [8].

183 Why should mucosal vaccines be preferable to intramuscular preparations?

There are a number of compelling reasons for development of nasally administered vaccines. People do not like injections, particularly in certain strata of society, but will tolerate nose drops or nasal sprays. It has been argued, that use of mucosal vaccine bypasses the possibility of transmission of blood-borne viruses such as hepatitis B or HIV through inadvertent contamination of needles and syringes by those administering the vaccine [28]. A further problem associated with the use of conventional injected vaccines is compliance. The requirement for annual administration makes this a burden for both patient and physician, with general practitioner policies and immunisation rates varying widely [29]. In the USA, vaccine uptake in the high-risk elderly increased marginally from 33.2% in 1989-90 to 43.9% in 1996-97, while over 75% of those at high risk in all age groups in 1996-97 were not vaccinated against the disease [30]. Amongst the reasons cited for the low vaccine coverage were concerns about adverse reactions and vaccine safety in general, as well as concern about efficacy of the vaccine. In a survey of influenza virus vaccination in primary care in central southern England in 1998 (at a time when national guidelines advised immunisation only for individuals with specified high risk medical conditions or residing in long stay facilities), only 11.5% overall, and 64% of those over 75 years of age, had been immunised [31]. Current influenza virus vaccines are also not popular with physicians, at least in the United Kingdom. In a recent survey [32], only 3% of 477 geriatricians reported offering influenza virus vaccine to all their continuing care patients while 81% never used the vaccine at all. Of the 385 consultants who did not offer the vaccine, it was regarded as unnecessary by 56%, ineffective by a further 33% and too expensive for "blanket" use by 12%. The immunogenicity of the current commercially available intramuscularly administered, inactivated, subunit influenza virus vaccines is assessed primarily by the measurement of circulating serum haemagglutination-inhibiting (HI) antibodies to the viral HA antigens [33]. Serum HI antibody titres of equal to, or greater than, 1 in 40 are generally regarded as equating to a 50% level of protection against homologous challenge virus infection [34], and current Committee for Proprietary Medicinal Products (CPMP) guidelines for Registration of Influenza Vaccines (CPMP Guidelines for Influenza Vaccines, CPMP/BWP/214/96, 1997) recommend that new, experimental influenza virus vaccines should elicit such titres in 75% of recipients. Inactivated influenza vaccines and the immune response

Natural infection by influenza virus is acquired via the respiratory tract, with the virus initiating its replication in the lining mucosal epithelial cells, subsequently being shed to neighbouring cells throughout the respiratory tract and to the exterior, as replication proceeds. It is therefore apparent that specific and/or non-specific defence mechanisms operating at this site would have an important role in preventing or controlling infection by influenza viruses.

184 The current inactivated influenza virus vaccines are administered by the i/m route, usually into the deltoid muscle of the upper arm. Whilst these vaccines promote a systemic immune response in the form of circulating IgG antibody, their ability to induce antibody, either IgA or IgG, at local sites in the nasal passages is questionable at best [33,35,36]. Current opinion suggests that administration of inactivated influenza virus vaccines via a mucosal route is a reasonable future strategy for improving the efficacy of these preparations for the prevention of influenza infection [18,33,37]. Whilst there is a wealth of evidence to indicate that inactivated whole or subunit influenza virus vaccines can elicit serum IgG immune responses in most groups of the population, and that this antibody correlates with protection [2,14,34], it is unlikely that this antibody would be present at the mucosal surface when wild virus enters the nasopharynx. It has been proposed that the observed protective efficacy of circulating anti-influenza IgG antibody induced following vaccination [18], is due to the presence of high levels of transudated, plasma-derived neutralising IgG antibody, relative to IgA antibody, in the secretions bathing the lower respiratory tract [37-39]. This is pertinent because it is likely that most cases of influenza are a result of successful viral replication in the lower respiratory tract [40]. While this hypothesis can account for the partial success of parenterally administered inactivated influenza virus vaccines, it might equally well serve to highlight the shortcomings of these vaccines when given by intramuscular injection. Thus, the failure of such vaccines to induce significant amounts of IgA antibody in the upper passages of the respiratory tract, where the IgA isotype constitutes more than 90% of detectable antibody [41], and where a significant proportion of influenza virus infections are probably initiated [40], may permit the establishment of such infections in individuals immunised by the parenteral route. One strategy currently under consideration for improving the efficacy of influenza virus vaccination specifically addresses this problem, and involves the simultaneous administration of an inactivated influenza virus vaccine parenterally, and a live, attenuated preparation by a mucosal route [36]. Thus, by simultaneous administration to volunteers aged 50 to 75 years, of a live attenuated, recombinant, cold-adapted vaccine by the intranasal route, and an inactivated, baculovirus-derived, recombinant HA vaccine intramuscularly, these workers were able to increase the percentage of both serum HI and HA-specific nasal wash antibody responses over those seen in subjects receiving either the live attenuated or inactivated vaccine by the same route alone (Table 1). The same workers also found significantly less laboratory-documented influenza A infection, influenza-like illness and outbreak-associated respiratory illness, in the group receiving combined live and inactivated vaccination, compared to the group given inactivated vaccine alone, on case monitoring through the following winter season [36]. These results indicate the additional protective efficacy afforded by supplementation of a parenterally administered vaccine with vaccination via an intranasal route using a live vaccine preparation. The use of such a combined vaccination strategy on a commercial scale is, however, subject to the difficulties surrounding the use of live attenuated influenza virus vaccines in general, including the theoretical

185 Table 1 Serum and nasal wash antibody titres in studies involving live or inactivated influenza virus vaccines or both (modified from [36]) Vaccine

No.

group

subjects Serum HI; Mean log 2 titre

Live Inact~ated Both

7 8 11

Mean log 2 titre ± SEM and % response by: Nasal HA-IgA ELISA; Mean log: titre

Pre

Post

%

Pre

Post

%

3.0±0.4 1.9±0.4 2.5±0.5

3.5±0.2 4.0±0.6 5.6±0.4

14 75 82

3.6±0.7 3.9±0.5 2.6±0.5

4.1±0.9 3.2±0.5 3.4±0.4

43 12 54

P < 0.05 compared with inactivated vaccine alone.

possibility of co-infection with human or, worse non-human wild-type virus and consecutive hazardous reassortment, and transmission of vaccine virus to other species [42]. The special problem of the use of live-attenuated vaccines in immunocompromised patients is also an important consideration, as some degree of immunosuppression is fairly common in the elderly [25]. These potential concerns are further compounded by problems of vaccine cost-effectiveness and supply inherent in the use of such a dual approach, in addition to problems of uptake and compliance that beset the use of parenterally administered, inactivated influenza vaccines. The studies described above suggest an important role for antibodies present at the mucosal surface in protection against influenza virus infection, and also indicate that such antibodies can be stimulated by intranasal immunisation. However, other factors such as cellular immune mechanisms may also be involved in protection against influenza virus infection at the mucosal surface. Following antigen activation, proliferation and partial differentiation in the nasally associated lymphoid tissue (NALT), effector B- and T-cells travel via the lymph nodes to the circulation [43]. Their subsequent trafficking to mucosal tissue appears to be dependent, at least with respect to gut-associated lymphoid tissue (GALT), on adhesion molecules or homing receptors expressed on their surface [44]. Although T-lymphocytes are known to participate within the common mucosal immune system [45], the role of CTLs in the nasal passages seems to be limited to clearance of and recovery from, an already established influenza virus infection [8,46]. However, a CD4 ÷, T-helper lymphocyte response is necessary to provide help to B-cells and permit antibody production, and influenza virus vaccines should clearly elicit activation of these cells. Whether the induction of some form of effector cellular immune response would add a further significant contribution towards protection against influenza virus infection locally in the respiratory tract following parenteral immunisation with an inactivated influenza vaccine is doubtful. It is probable that influenza-specific CTLs operating in the subepithelial layers of the respiratory tract reduce the local spread of virus thereby limiting the duration of the infection through assisting in virus clearance [6,8]. Cytotoxic lymphocyte responses have been demonstrated in man

186 following parenterally administered inactivated virus vaccine, but the response is short-lived [7]. Recent studies in mice having a targeted disruption in either the chain of the CD8 + molecule or the immunoglobulin ~ heavy chain, i.e. CD8 ÷ T cell or B cell deficient animals, revealed that heterosubtypic immunity, the cross protection against infection with different influenza A virus subtypes, to influenza A virus infection requires B cells, but not CD8 ÷, cytotoxic T lymphocytes [47], emphasising the importance of promoting a strong B cell response for protection against this infection. Mucosal immunisation and influenza virus vaccines

The work described above implies that the presence of both local, mucosal IgA, and systemic IgG antibodies, acting together with non-specific defence mechanisms, may be sufficient to elicit protection against the establishment of cognate influenza virus infection, providing there is reasonably close concordance between the vaccine and infecting strains of virus. Limiting and clearing an already established infection almost certainly involves cellular immune defence mechanisms. Studies in both animals [48,49] and man [50] have established that complete protection against natural influenza virus infection is due to locally secreted IgA antibodies in the respiratory tract, together with virus-neutralising IgG antibodies present both in the circulation and in respiratory tract transudate [51]. The NALT, the principal mucosal-associated lymphoid tissue of the respiratory tract, is highly vascularised with a large absorption area due to the presence of microvilli on non-ciliated ceils [52]. Microorganisms and antigens impacting at the surface of the nasal mucosa will induce initial responses through defined micro-compartments, the inductive lymphoid sites [53] which contain M (membrane) cells facilitating the sampling and transport of antigens to the underlying lymphoid tissues [54]. In these tissues antigen-presenting cells such as dendritic cells and activated B-cells, interacting together with T-cells, will take up and process the antigens thereby eventually generating antigen-specific effector cells. In the NALT these are primarily IgAl-secreting plasma cells [55,56]. Successful studies with live intranasal vaccines

Studies in humans with varying titres of influenza-specific nasal IgA antibodies but similar levels of influenza-specific serum HI antibodies have shown that individuals with nasal IgA are relatively more immune to infection compared to those who are relatively deficient of this antibody [57]. Other workers have reported a correlation between resistance to influenza virus infection and secretory IgA (slgA) in the nasal passages [50,58], and other, earlier studies in humans have reported an important protective role for slgA antibody [59]. In one study, an intranasaUy administered, live attenuated influenza virus vaccine induced greater local IgA antibody levels in nasal washings than were seen following parenteral immunisation with inactivated vaccine [60]. The cold-adapted, intranasally administered live attenuated influenza virus

187 vaccines developed in the 1980s have been reported to stimulate broader immune responses, particularly in respiratory secretions, compared to inactivated, parenterally delivered preparations [61-64]. Numerous other reports have described the ability of live attenuated influenza vaccine preparations to elicit good intranasal IgA responses in humans (reviewed by [33,37]), although there is also evidence that such vaccines are less effective at inducing good responses in the elderly [65,66]. Secretory IgA is known to effectively neutralise influenza virus infectivity, primarily through the inhibition of viral attachment and penetration into susceptible cells. It is also more efficacious than IgG antibody in this respect, at least in vitro [67,68]. The importance and role of IgG present in nasal secretions is probably supplementary to that of slgA, although it has been claimed that it correlates with protection in man [69]. Influenza virus vaccines and mucosal immunisation in man

Immunisation via the intranasal route with unadjuvanted vaccines

Over the past few years there has been considerable interest in intranasal or oral administration of inactivated, saline influenza virus vaccines, or, alternatively, the same vaccines associated with an adjuvant or carrier (reviewed by [33]). The problems associated with the use of live attenuated influenza vaccines, particularly with attenuation and the failure of preparations such as the live, cold-adapted influenza strains to come to commercial fruition, have fuelled research and developmental work in this area. In a recent study, intranasal immunisation of a group of community-residing elderly individuals, average age 66.9 years, with two doses of an inactivated, unadjuvanted whole influenza virus vaccine, showed that 50% responded with a local IgA antibody response as defined by a 1.4-fold increase in nasal antibody levels above those detectable prior to vaccination [70]. In contrast, only 20% of volunteers receiving intramuscular immunisation with a commercially available inactivated, split influenza virus vaccine achieved similar responses. The figure of a 1.4-fold or greater increase in IgA antibody in post-, as compared to pre-immunisation nasal secretions is currently taken as indicative of a significant local antibody response [71,72]. Other studies using inactivated saline-based influenza vaccines for intranasal immunisation, with administration via aerosol or drops have been undertaken and are reviewed elsewhere [33]. In brief, these studies indicate that one, two or three doses of saline-based, inactivated influenza virus vaccines given intranasally can promote local antibody, which correlates in some cases with protection against subsequent challenge virus infection [73,74]. However, this local antibody response is short-lived, reaching a peak at 2-4 weeks postimmunisation, but undetectable three months later. In general, single doses of these inactivated preparations induced less than satisfactory serum antibody responses, although further booster doses could improve this. In spite of these shortcomings and the relatively few studies carried out to date, the overall findings are considered encouraging and warrant further investigation [33]. A more recent publication describes the use of a novel, inactivated, intranasally delivered influenza virus

188 vaccine in a volunteer general practice population through a prospective, doubleblind, placebo-controlled trial. Although full details of the nature of the vaccine are not reported, a single dose was found to increase significantly the number of volunteers possessing protective levels of serum HI antibody against both type A influenza strains, and the B influenza virus strain present in the preparation. Over the subsequent winter period, follow-up monitoring of the volunteers showed that both 'respiratory illness events' and 'respiratory illness days', were significantly reduced in the vaccinated, as compared to the control group [75]. Immunisation via the intranasal route with adjuvanted vaccines

The results of studies using intranasally administered, inactivated influenza virus vaccines containing carriers or adjuvants are summarized in Table 2. In 1996, Hashigucci and co-workers reported that the intranasal administration of a trivalent inactivated influenza virus vaccine together with the heat-labile enterotoxin B subunit of Escherichia coli containing a trace amount of the holotoxin, in two doses four weeks apart, elicited both local, salivary IgA antibody and also serum antibody to at least one of the three vaccine components. These responses were

Table 2 R e s p o n s e s to adjuvanted influenza vaccines given intranasally to volunteers Vaccine

Adjuvant/

No.

Vacci-

Antibody response

Reactions

Carrier

doses

nation route

Serum

Mucosal secretions

Local Systemic

2 (NS)

i/n

++

++

++

++

2 (NS)

i/n

+

+

-+

-+

1 (15/zg)

i/n

+

+

+_

___

2 (7.5/~g) 2 (7.5/.tg) 1 (15 ~g)

i/n i/n i/m

++ -+ +

++ -+ +

---+ +--

-+ + +-

Trivalent (H3; H I ; B) Native E. coli (Virosomal) HLT

2 (15/zg)

i/n

++

ND

++

___

78

Trivalent (H3; H1; B) MF59 (WVV) Nil

2 (15/xg) 2 (15 ~g)

i/n i/n

+ +

++ ++

___ +-

+__ +-

84

Trivalent (H3; H1; B) Chitosan

2 (7.5/~g) 2(15~g)

i/n i/n i/m

+ + +++

+ + -+

___ -+ -+

___ -+ -+

83

Trivalent (H3; H1; B) E. coli B subunit + holotoxin "(WVV) Nil Trivalent (H3; H1; B) Native E. coli HLT " " " Nil "(Virosomal) Nil

1 (15~g)

i/m = intramuscular; i/n = intranasal; NS = not stated; W V V = whole virus vaccine.

Ref.

76

77

189 detected four weeks following the second vaccination, in approximately half the volunteers [76], and compared favourably with both local and systemic antibody responses elicited by the inactivated vaccine administered intranasally without adjuvant. However, the E. coli B subunit/holotoxin vaccine formulation did induce some side effects, albeit short-lived, in a relatively high proportion of volunteers. These consisted of local effects such as discomfort, sneezing, runny/stuffy nose and cough, in 59%, and systemic symptoms of malaise, headache, abdominal pain and fever in 23% of volunteers, following the initial dose of the vaccine; the incidence of these side effects was considerably lower following second administration of the vaccine preparation [76]. Other workers have utilized the E. coli heat-labile enterotoxin (HLT), but in its native form, and not as the B subunit alone, as an adjuvant for the intranasal administration of inactivated influenza virus vaccine to volunteers [77,78]. The results of a phase I clinical study (summarized in Table 2), indicate that two nasal spray vaccinations containing 7.5 ~g of the HA and NA subunits of three influenza virus strains in a virosomal (liposomal) formulation [79,80], were able to induce a humoral immune response comparable to that of a single intramuscular injection of the same vaccine formulation containing 15 ~g of each HA antigen. In addition, a significantly greater incidence of salivary, influenza-specific IgA was observed following two doses of the trivatent influenza HA/HLT vaccine containing 7.5/zg HA of each influenza virus strain than was seen following a single intranasal dose of the same vaccine preparation containing 15/xg HA of each influenza virus strain [77]. A similar HLT mucosal vaccine administered to a group of healthy 18-60 year-olds induced similar levels and incidence of serum HI antibody. Both local and systemic reactions were mild and transient (Table 2). The production of local, mucosal antibody was not determined in this study [78]. This vaccine preparation has been available commercially in Switzerland. Other substances have been used in an adjuvant or carrier capacity for influenza virus vaccines delivered intranasally. MF59 is an oil-in-water adjuvant consisting of droplets of squalene oil stabilized by Tween 80 and sorbitan trioleate surfactants [81]. Chitosan is a cationic polysaccharide consisting of repeating units of N-acetylD-glucosamine and D-glucosamine, produced by partial deacetylation of chitin obtained from the shells of crustaceans [82,83]. The MF59 adjuvant has recently been shown to be both safe and immunogenic in humans [84], eliciting mucosal IgA and serum HI antibody responses in healthy adult volunteers aged 18-40 years when administered in conjunction with a formalin-inactivated whole virus vaccine preparation (Table 2). However, both local and humoral antibody responses were not significantly different from those induced following intranasal immunisation with the equivalent, unadjuvanted vaccine [84]. Chitosan, a deacetylated form of chitin, currently used as a food additive and an over-the counter slimming aid, has mucoadhesive properties, enhancing the bioavailability of peptide and protein drugs following intranasal administration. It is safe when administered to humans [82,85,86]. In mice, intranasal immunisation with influenza B virus surface antigens elicits strong local and systemic antibody responses

190 [82]. In a recently-conducted clinical trial using a trivalent, inactivated, surface antigen, commercially-available influenza virus vaccine admixed with chitosan and administered intranasally to volunteers (Table 2), both local IgA and circulating HI antibodies to the A/Sydney (H3N2), A/Beijing (H1N1) and B/Yamanashi HA antigens could be stimulated at levels that were not significantly different from those induced by the unadjuvanted saline vaccine delivered intramuscularly at similar dosage levels [83]. This study also further demonstrated the safety and tolerability of this preparation following intranasal administration to humans. The mechanism of action of chitosan in enhancing bioavailability remains poorly understood. It has been suggested that it may facilitate increased uptake of antigens across the nasal mucosa by slowing down mucociliary clearance thereby prolonging contact of antigen with the mucosal surface [87,88]. The use of chitosan as a mucosal adjuvant in mice for intranasal delivery of a vaccine against diphtheria, consisting of the crossreacting material (CRM197) of diphtheria toxin has also recently been reported [89]. It was found that the CRM1:7 preparation, poorly immunogenic when delivered intranasally in solution, induced high levels of antigen-specific IgG, secretory IgA, toxin-neutralising antibodies and T cell responses, predominantly of the Th2 subtype, when given to mice intranasally in conjunction with chitosan [89]. Immunisation via the oral route

Although most recent studies on mucosally administered influenza virus vaccines have utilised the intranasal route, there has also been considerable interest in immunising against influenza by the oral route, and several studies, both in animals and in humans, have been conducted (reviewed in [37]). The rationale of oral immunisation is two-fold. Firstly, the gastrointestinal tract is rich in lymphoid tissue with approximately 10% of lymphoid cells in humans associated with organised structures, such as Peyer's patches, in the gut [90]. Secondly, the concept of a common mucosal immune system [91] allows for stimulation of other mucosal surfaces such as the nasal passages and the respiratory tract, sites of initial infection by influenza viruses. In addition, immunisation by the oral route would be more acceptable than a parenteral injection. However there are a number of problems associated with delivery of antigen into the gastrointestinal tract that are not encountered with intranasal delivery. These include the induction of specific systemic tolerance which occurs following the administration of large doses of antigen by the oral route [92], and also the degradation of antigens by gastric acid and proteolytic enzymes [93]. Nevertheless the possibility of immunisation by this route has attracted attention. Influenza virus vaccine incorporated in gelatine capsules coated with cellulose acetate phthalate and delivered orally to healthy volunteers aged between 20 and 45 years in a dosage 10 times that of the conventional, systemic influenza virus vaccine on five consecutive mornings, induced relatively high levels of IgA in both saliva and nasal washings compared to those elicited following parenteral or intranasal immunisation, but virtually no serum antibody [94]. Furthermore, the IgA antibody

191 response in saliva, but not in nasal washings was still rising 35 days following oral immunisation, and this was not observed following intranasal immunisation, while systemic immunisation, although eliciting high levels of circulating IgG, induced only low levels of secretory IgA in saliva and nasal washings [94]. An earlier study [95] found that oral immunisation of five volunteers with an 'enteric-coated' inactivated influenza virus vaccine elicited a significant rise in influenza-specific IgA antibodies in tears, nasal secretions and saliva which was maximal 5-7 weeks after completion of the vaccination schedule. As in the later study, Bergmann and co-workers delivered their vaccine in multiple doses, on various days over a two week period. The apparent requirement for large doses of antigen coupled with the necessity to protect against degradation indicates that oral immunisation could be a relatively costly procedure, as well as being scientifically untenable due to the possibility of inducing tolerance to the antigen(s) used in the vaccine.

Other strategies for immunisation against influenza Over recent years there has been considerable research and developmental activity directed into DNAvaccine technology (reviewed by [96]). Although most of the work with influenza virus vaccines in this respect has been concerned with immunisation via parenteral routes, there is no theoretical reason why such vaccines should not be administered at mucosal surfaces. DNA vaccines provide a number of advantages over more conventional preparations, not least over live-attenuated vaccines, in that they are non-replicating, and therefore safe [97]. As DNA stability is not affected by high temperature, they may be easier to use in tropical areas [58]. In addition, DNA vaccines are reported to stimulate both humoral and cellular immune responses [98], and to be particularly effective at priming immune responses. These properties are often sufficient to provide protection, at least in laboratory animal models [99,100]. Nevertheless, the immunogenicity of DNA vaccines remains relatively low in large animals and non-human primates compared to mice [99,101]. This necessitates the inclusion of targeting and/or delivery systems to enhance efficacy [102], protect against nuclease degradation in the lungs and augment immunological responses [97]. In early studies, the intranasal inoculation of a plasmid expression system for the influenza virus HA antigen into mice was found to promote some resistance to a lethal influenza virus challenge [103]. More recently intranasal administration to mice of a plasmid expressing influenza virus HA, in combination with cholera toxin has been shown to induce influenza-specific B cell responses in both the lung and spleen tissue [58]. However, specific antibodies in both serum and mucosal fluids were undetectable in these studies, although there was some, limited, evidence of protection [58]. More recently, the complete protection of mice against a lethal, homologous influenza challenge virus infection was achieved following intranasal immunisation of a liposome-encapsulated plasmid encoding the influenza virus HA at a vaccine dose that was 2.5-fold lower than that needed to achieve the same degree of protection following intramuscular injection of the same vaccine preparation [97].

192 The excellent priming capability of DNA vaccines [99,100] together with the necessity of delivering such vaccines in some form of carrier, has led to the concept of a heterologous or homologous 'prime-boost' strategy for the use of DNA vaccines, in conjunction with conventional, inactivated vaccine preparations administered as boosters [104]. Such regimens have not yet included a mueosal route for administration of either type of vaccine preparation. Other recent studies focusing on enhancement of DNA immunisation, have been concerned with the trapping of DNA vaccines in the lung with macroaggregates, in order to enhance mucosal immunity [105]. Although the use of cytokines, chemokines and other costimulatory molecules, such as ILl2 or GM-CSF [106] and CpG DNA [107], are also under consideration for promotion of immunity to nucleic acid vaccines, considerably more work is needed before such preparations can be seriously considered for use in mucosal immunisation against influenza virus infection. Conclusion

The current optimism surrounding vaccination against infection by the influenza viruses has arisen from technological advances that have permitted the development and use in humans of novel, adjuvanted vaccine preparations with enhanced immunogenicity [76,77,79,84]. Indeed, one of these vaccine preparations has been available commercially in Switzerland. In addition, there have been important leaps forward in the understanding of factors most relevant for immunity against influenza virus infection [18,33,37,46,50,55]. It has been known for many years that the administration of live attenuated influenza virus vaccines to man via the intranasal route induces immunity that is almost equivalent to that elicited by natural virus infection [33]. However, the use of live attenuated vaccines has been dogged by problems such as the degree of attenuation attainable, concerns about co-infection and reassortment with wild-type virus, and their use in the most important target populations such as the elderly and chronically ill, who may have compromised immunity. Recent advances in technology have resulted in the development of highly immunogenic, adjuvanted, killed influenza virus vaccines. It is probable that non-living, adjuvanted vaccines may not stimulate long-term, broad, cross-reactive immunity or induce all major facets of the immune response, including the effector arm of the cell mediated response. However, the safety, uniformity and relative ease of manufacture associated with inactivated influenza virus vaccines, suggests that such preparations, administered by the intranasal route, are potentially effective future vaccines. It is possible that the relatively short duration of the immune response, both locally and systemically, to adjuvanted intranasal vaccines may be overcome by a regimen of multiple doses at intervals throughout the months of prevalence of influenza virus infection. Such a regimen may be very acceptable if the vaccine under development for intranasal delivery, can be simply and effectively self-administered by the patient in the form, for instance, of a nasal spray. However, studies that incorporate these multiple dosage regimens have yet to be conducted.

193 Although the potential of inactivated, adjuvanted intranasal vaccines is very great in primed populations, their efficacy in unprimed or immunocompromised populations remains to be confirmed. One possibility for protecting such groups may be the use of a combined immunisation strategy involving a parenterally administered inactivated vaccine in conjunction with an intranasally delivered, appropriately attenuated live influenza virus vaccine [36]. Such an approach may represent the most effective strategy for cohorts of the population which are at risk from influenza virus infection and subsequent secondary bacterial pneumonias. A dual approach to vaccination with some form of inactivated influenza virus vaccine alone being used for control of the infection in healthy immunocompetent individuals, and a combined live attenuated/inactivated vaccination strategy being reserved for the elderly and other at risk groups, may be appropriate. Such an overall influenza vaccination strategy would be less costly and more easily implemented than blanket use of either a live attenuated vaccine alone or a combined live attenuated/inactivated vaccination strategy. Nevertheless it is encouraging that a study carried out by Gluck and co-workers using the virosomal influenza vaccine in a volunteer group 63-102 years of age, showed that, in addition to being well tolerated, the vaccine induced protective levels of serum HI antibody in 67% (influenza B), 79% (H3N2) and 83% (H1N1) of the 63 volunteers receiving the trivalent vaccine preparation [79]. New vaccine technology has been fully exploited over the past few years to build on improvements in current influenza virus vaccines. It seems probable that within the coming five years, a new generation of efficacious, inactivated, intranasally delivered, adjuvanted influenza virus vaccines will become commercially available and make considerable clinical and economic impact against this disease. References

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