1. Introduction

Influenza viruses have been recognized as the main agents responsible for influenza-like and viral lower respiratory tract illnesses, which are significant causes of outpatient visits, hospitalizations and deaths in the community.[1] Their contribution to such illnesses may exhibit wide variations according to the epidemiological picture observed in different seasons and in different countries, age groups, case definitions and detection methods used for surveillance; for example, the proportion of positive samples for influenza viruses in patients with influenza-like illness in the UK ranged between 21% and 33% in children aged <5 years and between 13% and 42% in the elderly during three successive winters.[2]

Although influenza illness affects people of all ages, complications, hospitalizations and deaths are more frequent in the elderly. Mortality rates for influenza illness are higher in older adults than younger adults,[3] reaching 90% of the total number of deaths,[4] although, during the recent pandemic resulting from the circulation of the H1N1 2009 virus, the proportion of influenza deaths in people aged <60 years was markedly higher than during the seasonal epidemics, and a mortality peak among subjects aged <20 years was observed.[5] The elderly are among the groups at highest risk for serious complications, mainly because of a waning immune system, which reduces their overall immune response, and to the high prevalence of risk factors.[6]

Indeed, the impact of influenza is particularly heavy in patients with underlying chronic diseases, such as ischaemic heart disease, stroke, obstructive chronic bronchopneumopathy and diabetes mellitus.[3,79] In the elderly, the risk of hospitalization or death for influenza/pneumonia is 3.6- to 4.8-fold higher in subjects with a high-risk condition compared with healthy, aged adults.[10] The risk is particularly high in older adults with heart and lung diseases and conditions associated with immunosuppression, and these patients exhibited hospitalization or death rates as a result of influenza/pneumonia that were 7- to 10.4-fold and 6.4- to 10.8-fold higher than those observed among healthy adults aged ≥65 years.[10]

The main public health strategy for containing influenza is annual vaccination, which is recommended for the elderly and others in risk-factor categories presenting the highest morbidity and mortality. Several factors affect influenza vaccination in the elderly: immune senescence impairs both the innate and adaptive immunity and reduces the antibody response to vaccine components in many elderly patients.[3,6] There are many changes in an aging immune system that are demonstrated by a reduction in immune response and a reduced capacity for antibody production. The US Centers for Disease Control and Prevention (CDC) estimates 70–90% clinical vaccine efficacy in young adults and this estimate suggests a corresponding clinical efficacy in the elderly of 17–53% depending on circulating viruses. The adjusted odds ratio of responses in elderly versus young adults ranged from 0.24 to 0.59 in terms of seroconversion and seroprotection with respect to all three antigens of influenza vaccine.[11]

The efficacy of vaccines decreases with increasing age: the seroprotection rate against influenza virus strains is only 29–46% in persons aged ≥75 years, compared with 41–58% in persons aged 60–74 years.[1113] The decreasing efficacy of vaccines in this age group is due to several progressive changes, such as a decrease in the number of Langerhans cells, the limited capacity of dendritic cells to produce antigen, defects in the production of Toll-like receptors (TLRs) and reduced production of MHC class I and II molecules. In addition, production of mature naive T cells by the thymus decreases with age, resulting in a reduced number of naive T cells in peripheral blood.[1113]

The thymus begins to regress after birth and a reduction in thymic tissue occurs throughout life, resulting in a decline in thymic output and a reduction in the naive T cell pool.[3,1416] As the T cell total pool does not change with age, the cell pool proliferates to maintain the total number, but this proliferation can lead to replication errors because these cells have a replicative limit. Age-related decline in cellular immunity is the consequence of thymic atrophy, reduced output of new T lymphocytes, accumulation of anergic memory cells, deficiencies in cytokine production and uncertain antigen presentation. Thymic involution leads to a progressive loss of naive T cells, which in turn jeopardizes the success of primary vaccination.[3,1416]

Furthermore, although humoral immunity remains active throughout life, in the aging process, the ability of B cells to produce antibodies against novel antigens diminishes. In addition, aging processes in the bone marrow stroma can endanger the survival of plasma cells and lead to a short duration of immunological protection.[12,17]

In regard to influenza vaccination, the reduced immune response observed in the elderly is particularly evident when immunogenicity is evaluated against drifted circulating strains. Antigen mismatch results in important effects on the immunogenicity of the vaccine: the seroprotection rate against drifted strains in subjects that showed a very good response against the vaccine strains vary widely according to the antigenic distance between vaccine and circulating strains and the patient’s age. For example, the circulation of the drifted variant A/Wyoming/3/2003(H3N2) did not affect the seroprotection rate in subjects vaccinated with A/Panama/2007/1999(H3N2), but the seroprotection rate in subjects vaccinated with A/Wyoming/3/2003(H3N2) dropped to less than 50% against the drifted strains A/California/7/2004(H3N2). The relevance of age emerged from a study by de Jong et al.,[18] in which adults aged >75 years vaccinated with A/Wuhan/359/1995(H3N2) showed a seroprotection rate against the drifted strain A/Sydney/5/1997(H3N2) 60% lower than that observed in adults aged 60–74 years.[1822]

As is examined further in the next section, the effect of an adjuvant addition to a subunit influenza vaccine can lead to higher haemagglutination (HA)-inhibiting seroprotection rates and to higher neutralization antibody titres against drifted strains not included in the vaccine compared with a non-adjuvanted vaccine.[23] The advantage offered by MF59 adjuvant, an oil-in-water emulsion, in terms of higher immunogenicity expressed as higher post-vaccination HA-inhibiting titres, was also observable against viruses showing an antigenic and molecular pattern indistinguishable from the vaccine strain, but became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew.[24]

Novel strategies have been explored to augment the elderly immune response after immunization, including an increased dosage, multiple-dose vaccinations, the use of vaccine adjuvants and more efficient routes of vaccine delivery. This review discusses recent advances in influenza vaccines, with particular attention paid to new adjuvants and new vaccination routes and a focus on the implications for vaccination strategies for the elderly.

2. Licensed Adjuvants for Influenza Vaccination

The use of adjuvants to overcome poor immunogenicity and to spare antigen is not new to vaccinology: the first licensed adjuvant, aluminium-based mineral salts (alum), has been widely used for almost a century[25] and nowadays is a component of diphtheria, tetanus and pertussis (DTP), diphtheria, tetanus and acellular pertussis (DTaP), hepatitis A and B (HB), Lyme disease, rabies and anthrax vaccines, all combinations of DTaP, Haemophilus influenzae type b (HiB) and HB vaccines, and some but not all HiB vaccines. Vaccine adjuvants are molecules, compounds or macromolecular complexes that boost the potency of the specific immune response to antigens, influencing the immune system toward T helper type-1 (Th1)- or Th2-cell immunity.[26]

Adjuvants are effective in several ways, acting both as a delivery system and through immune-potentiating activity: they can modify the cytokine network, favouring antigen conduction to antigen-presenting cells (APCs) and induction of cytotoxic T lymphocyte (CTL) responses, which generally require that the antigen be processed intracellularly. They may prolong antigen release and can also target antigens for presentation by MHC class I or class II molecules, which are critical for directing immunity against intracellular and extracellular pathogens, respectively.[27] The main mechanisms of action of adjuvants and delivery systems used to improve immune response against influenza antigens are shown in table I.

Table I
figure Tab1

Main licensed adjuvants or carrier systems used for influenza vaccine

Although different classes of compounds have shown adjuvant activity in pre-clinical studies, only a few of these have been licensed for human use, because of their often unacceptable safety profile. Despite evaluation of a large number of adjuvants, alum, introduced in the 1920s, remains the standard worldwide adjuvant for human use.[31]

Notably, alum is known to be a poor inducer of T-cell immunity and Th1 response[28,32] and the addition of alum adjuvant to split or subunit influenza vaccines has induced only marginal improvements.

Recently, several studies compared immune responses elicited by aluminium-adjuvanted and non-adjuvanted subvirion influenza A (H5N1) vaccines in healthy populations. Bresson et al.,[33] Bernstein et al.[34] and Brady et al.[35] observed no significant differences between adjuvanted and non-adjuvanted vaccines, while Keitel et al.[36] demonstrated that a dosage of 7.5 µg of adjuvanted vaccine elicited a significantly greater immune response than a comparable non-adjuvanted vaccine, and that no adjuvant effects of aluminium hydroxide were observed using other antigen quantities. Recently, a one-dose schedule of a monovalent, whole-virion, inactivated, adjuvanted pandemic H1N1 2009 vaccine with 6 µg HA per 0.5 mL content and aluminium phosphate gel adjuvant showed an encouraging immunogenic profile since it fulfilled all international licensing criteria in both the adult and elderly age groups; however, no comparison with non-adjuvanted vaccine was performed in this trial.[37] The licensing criteria seroprotection rate was reached by a split-virion formulation containing 7.5 µg, 15 µg or 30 µg HA per dose and aluminium hydroxide adjuvant in the elderly, but rates were higher among individuals immunized with non-adjuvanted formulations.[38] The reasons for unsatisfactory augmentation of the immune response with aluminium adjuvant, a finding that has also been reported for other vaccines such as typhoid fever vaccine,[39] have not been completely clarified. Several authors have hypothesized that the aluminium promotes a more efficient antigen degradation, which in this case would not be considered advantageous for small size peptides as it could cause loss of antigenicity.[40] Another possible explanation is partial denaturation of epitopes due to adsorption.[41]

For these reasons, other adjuvants have been developed and, in particular for influenza vaccination, a squalene-based oil-in-water emulsion, MF59, and a virosome-based carrier system were approved for human use more than a decade ago. More recently, AS03, another oil-in-water emulsion, was approved as a component of pre-pandemic H5N1 vaccine and a component of pandemic H1N1 2009 vaccine.[32]

2.1 MF59

MF59 is an oil-in-water adjuvant emulsion composed of 5% v/v (volume of solute/volume of solution %) squalene, 0.5% v/v polysorbate 80 (Tween™ 80) and 0.5% v/v sorbitan trioleate (Span® 85), emulsified under high pressure conditions in a microfluidizer and transformed into small uniform droplets.[42]

The emulsion consists of a drop of oil surrounded by a monolayer of two nonionic surfactants, Tween™ 80 and Span® 85, which are found in many foods, cosmetics and pharmaceutical products, and are used to optimally stabilize the MF59 emulsion droplets. Citrate buffer is used in MF59 to stabilize pH. Finally, MF59 becomes a non-viscous formulation resembling a milky white emulsion. The squalene oil is a natural component of cell membranes, originally obtained from shark liver; it acts as an intermediate in the cholesterol biosynthesis pathway and is essential for the synthesis of cholesterol, steroid hormones and vitamin D.[43] It is noteworthy that administration of MF59 did not elicit a specific humoral response against squalene, removing doubts about potential autoimmunity mechanisms stimulated by this adjuvant.[44] The lack of autoimmunity phenomena has been widely confirmed by post-marketing surveillance.[30]

The precise mechanism of MF59 adjuvanticity has been widely studied. Administration of MF59 induces a significant influx of macrophages and it is capable of triggering the production of chemokines in cells resident at the injection site.[30] This adjuvant combines a strong antigen delivery function with immune-stimulating activity at the injection site, inducing in muscle fibres the production of cytokines, cytokine receptors and adhesion molecules involved in leukocyte migration, and activating local APCs.[29]

MF59 can both improve the antibody responsiveness to influenza and redirect the quality of the antibody response against influenza antigens.[45] This oil-in-water emulsion induced antibody epitope spreading from the HA2 to the HA1 subunit in HA and neuraminidase to a greater degree than non-adjuvanted or aluminium-adjuvanted vaccines and resulted in an increase in the avidity of antibodies binding to HA1.[45] The adjuvant-dependent increase in binding to conformational HA1 epitopes correlated with a broadening of cross-clade neutralization and predicted improved in vivo protection.

In 1997, MF59 was the first new adjuvant to be licensed for human use, after alum, and it is now commercially available in 23 countries worldwide, including 12 member countries of the EU.[30] MF59 has been studied in clinical trials involving more than 26 000 people, including children. It has been licensed since 1997 for use in adults aged >65 years for seasonal influenza vaccine in the EU but is not licensed for sale in the US and Canada.

Over 50 million doses of seasonal MF59-adjuvanted vaccine (Fluad®, Novartis; Influpozzi® Adiuvato, Novartis) have been delivered in Europe, with more than 60 million doses administered worldwide, demonstrating the good safety profile of this vaccine compared with the non-adjuvanted vaccine, although moderate and mild local reactions showed higher incidence in adjuvanted vaccine recipients compared with the non-adjuvanted vaccine group.[30,42] In terms of immunogenicity, MF59 has been demonstrated to be a potent vaccine adjuvant in humans: data from over 2000 elderly subjects immunized with this adjuvanted vaccine showed a consistently higher immune response with statistically significant increases in post-immunization geometric mean titres (GMTs) and seroconversion and seroprotection rates compared with non-adjuvanted influenza vaccines, particularly for the influenza A (H3N2) and influenza B strains (figure 1). The higher immunogenicity profile of the MF59-adjuvanted vaccine is also maintained after subsequent immunizations.[42,54] This adjuvant vaccine is able to enhance the immune response compared with a subunit influenza vaccine, inducing higher seroprotection rates, especially in frail subjects such as adults with chronic diseases or individuals aged >75 years (figure 1).[5558]

Fig. 1
figure 1

Differences in seroprotection rates against influenza viruses observed in elderly subjects immunized with seasonal virosomal, ID and MF59-adjuvanted vs conventional vaccines (%).[17,21,34,41,43,44,4653] ID = intradermal; IM = intramuscular.

Recent studies have shown that the addition of adjuvants can lead to a broader immune response against variants not included in the vaccine composition (figure 1).[20,23,59] In particular, in the elderly, MF59-adjuvanted vaccine was able to induce a broader immune response against the WHO-recommended egg-grown vaccine viruses and wild-type drifted strains circulating during influenza seasons.[22] During seasons with good or partial matching between vaccine strains and isolates, the addition of MF59 to a subunit influenza vaccine allowed a higher antibody response, which became even higher when the distance between the vaccine and circulating strains increased, compared with that observed in subjects immunized with non-adjuvanted subunit vaccine (figure 1).[24]

The emergence and rapid spread of the new pandemic influenza virus H1N1 2009, identified in April 2009, has focused global efforts on developing a new H1N1 2009 vaccine. The main criticisms of influenza vaccines against pandemic or potentially pandemic strains are (i) the inability to elicit a rapid immune response in naive populations and (ii) the incapacity for rapid and large-scale production.

Studies performed by Stephenson et al.[6062] starting in the first years of this century showed that antibody responses after two doses of MF59-adjuvanted vaccine were higher than those after two doses of non-adjuvanted vaccine against vaccine strain A/duck/Singapore/1997 (H5N3) and viruses belonging to different clades such as A/HongKong/156/1997 and A/HongKong/213/2003. The good priming offered by MF59-adjuvanted vaccine was confirmed after the third vaccine dose, administered after >18 months: it showed a significant booster effect with optimal antibody response against different influenza A (H5N1) clades. This effective priming and boosting effect was also shown in the elderly.[6062]

The higher immunogenicity of MF59-adjuvanted vaccines allows a reduction in the antigen amount required to elicit an effective immune response, i.e. antigen sparing. Focetria® (Novartis), the MF59-adjuvanted monovalent subunit vaccine against the H1N1 2009 pandemic strain, appeared in April 2009 and was authorized for human use by the European Medicines Agency (EMA).[63] It can elicit protective antibody levels with a lower dose, i.e. 7.5 µg of viral antigen versus 15 µg in non-adjuvanted vaccines, potentially resulting in greater vaccine supply.[64]

2.2 AS03

A novel generation of adjuvants named AS was recently developed by GlaxoSmithKline (GSK). In particular, the AS03 adjuvant, a 10% oil-in-water emulsion, was added to split influenza vaccine. The oil phase contained 5% dl-α-tocopherol (11.86 mg) and squalene (10.69 mg) and the aqueous phase contained 2% of the non-ionic detergent Tween® 80 (4.86 mg).[65]

This tocopherol oil-in-water emulsion-based adjuvant system has been tested, during the last few years, in a candidate influenza A (H5N1) pre-pandemic influenza vaccine. GSK Biologicals has used its proprietary adjuvant system AS03 to develop an inactivated split-virus influenza A (H5N1) vaccine containing 3.75 µg HA of the strain A/Vietnam/1194/2004 NIBRG-14, which is a recombinant influenza A (H5N1) from clade 1, engineered by reverse genetics and recommended as a prototype pandemic influenza vaccine strain by the Committee for Medicinal Products for Human Use (CHMP). GSK is currently licensed to market this pre-pandemic influenza vaccine, called Prepandrix™, in all 27 member states of the EU.[66]

Recently, the AS03 adjuvant has been adopted in the licensed formulation of a current H1N1 2009 pandemic vaccine (Pandemrix®, GSK). Good immunogenicity, together with an optimal safety and acceptable tolerability profile, have been demonstrated with these new adjuvanted vaccines in healthy adults, using a 3.75 µg antigen dosage.[67,68] The AS03-adjuvanted vaccine with 4-fold less antigen elicited an immune response comparable with that of the non-adjuvanted vaccine.[68] Dose reduction is an important benefit if there is an insufficient antigen supply as in the case of a pandemic threat. Several studies have evaluated the immunogenicity and the safety of the AS03-adjuvanted pre-pandemic influenza vaccine in adults,[6769] but a complete evaluation of this formulation is required in young children, the elderly and individuals with chronic disease.

The AS03-adjuvanted vaccine has been demonstrated to elicit a cross-reactive antibody response against heterologous viral challenge in adults aged 19–61 years.[69] Preliminary results have also been reported with an AS03-adjuvanted seasonal influenza vaccine administered to older adults (aged ≥65 years); the new adjuvant system was able to enhance the immune response compared with a non-adjuvanted vaccine, although higher reactogenicity was recorded in recipients in the adjuvanted group.[70,71] A higher incidence of adverse events, such as pain, swelling, irritability and fever, when compared with whole vaccine, was recently confirmed in healthy children aged <5 years.[72,73]

3. Virosomes as Antigen Carriers

Virosomes are liposomes consisting of a biodegradable, non-toxic and non-immunogenic phospholipid membrane[74] that can be used to reconstitute a virus-like particle, i.e. a virus envelope, complete with surface antigens (e.g. HA and neuraminidase proteins), but without the internal genetic material of the virus. Virosomes have been demonstrated to be a versatile and efficient carrier system for a variety of antigens, e.g. proteins, peptides, nucleic acids and carbohydrates.[75] Virosomes represent a novel vaccine presentation form that closely mimics the native virus.

An influenza virosome can be regarded as an antigen delivery system for immunogenic antigens without the pathological consequences of cell infection. Because of the repetitive arrangement of the HA molecules on its surface, and like the actual influenza virus itself, the virosome is taken up avidly by APCs via receptor-mediated endocytosis and enters the host cell in an endosome. The slightly acidic pH of the endosome allows fusion of the virosomal membrane with the endosomal membrane, such that the interior of the virosome becomes continuous with the cytosol. In this way, any antigens inside the virosome have access to the cytosolic MHC class I presentation pathway, activating CTLs. Antigens on the surface of the virosome (as well as antigens derived from degraded virosomes) enter the MHC class II presentation pathway and activate Th cells.[46] Thus, influenza virosomes have the potential to stimulate both the humoral as well as the cellular immune pathway.[47,76]

Virosomes are one of five potentiating systems approved by regulatory authorities for human use; the others are alum, MF59, monophosphoryl lipid A (MPL) and alum, and AS03. Of these, only virosomes have carrier capabilities. Immunopotentiating reconstituted influenza virosomes (IRIVs) have an excellent tolerability, as shown by the two products already on the market for immune prophylaxis against hepatitis A and influenza (Inflexal® V, Crucell; Isiflu® V, Crucell).[48,7779] In particular, recent studies have confirmed the good safety and tolerability profile of a virosomal influenza vaccine in the elderly and in healthy, chronically ill, and immunocompromised adults, with an incidence of adverse events similar to that observed with split or subunit vaccine recipients.[49,80,81] The effect of IRIVs on immunogenicity in the elderly has been widely studied during the last decade and a number of studies have compared the immune responses elicited by virosomal and standard influenza vaccines; however, results have been inconsistent. Several clinical investigations reported a greater immune response elicited by virosomal vaccines compared with a non-adjuvanted formulation in children and older adults (figure 1).[50,8284] Virosomal vaccines also showed similar immunogenicity to conventional vaccines in studies performed by de Bruijn et al.[51,81] However, other authors have shown that the immune response conferred by virosomal vaccines is inferior to that elicited by other adjuvanted influenza vaccines.[49,8587]

More recently, Baldo et al.[88] compared the immunogenicity of non-adjuvanted, MF59-adjuvanted and virosomal inactivated influenza vaccines against homologous and heterologous strains in elderly subjects with chronic diseases. Vaccination with MF59-adjuvanted vaccine resulted in slightly but consistently higher post-vaccination GMTs than the other two vaccination groups. In particular, virosomal vaccine induced statistically significantly lower corrected GMTs versus all three vaccine strains and type A heterologous strains compared with MF59, while no difference was seen with a split vaccine. The differences in seroprotection rates against drifted strains observed in elderly subjects immunized with virosomal and conventional vaccines are shown in figure 1.[88]

4. Intradermal Vaccines

Non-adjuvant strategies have been assessed for their ability to enhance antibody response in the elderly and in other populations that respond poorly to vaccines. The route of immunization plays a very important role in determining the level and breadth of induced responses. Most vaccines are administered intramuscularly but other routes are used to administer vaccine: transcutaneous and epidermal (measles, mumps, rubella and yellow fever), intradermal (Bacille Calmette-Guérin and rabies), intranasal (live attenuated influenza) and oral (poliomyelitis, cholera, rotavirus and typhoid fever) routes have been tested and widely used in both the distant and recent past.[89]

In particular, the potential of intradermal vaccination is well known, but the standard Mantoux intradermal technique is difficult to perform correctly and requires highly skilled personnel.[90]

The availability of an appropriate injection system and a technique (BD Soluvia™, Becton, Dickinson and Co.) combining simplicity, safety and ease of use has allowed large-scale evaluation of the tolerability, safety and immunogenicity of intradermal influenza vaccine. Vaccination via the intradermal route involves the administration of the antigen into the dermal layer of the skin. This vaccine uses the skin immune system by delivering antigen directly to dermal dendritic cells (APCs) that naturally amplify the immune response by efficiently migrating and presenting antigen to T cells in the draining lymph nodes.[91]

The targets of intradermal immunization are Langerhans cells and dermal dendritic cells that are resident in and recruited from the blood. Because of the high concentration of specialized immune cells in this skin layer and their ability to effectively stimulate an immune response, intradermal vaccination provides direct and efficient access to the immune system. After antigen capture and maturation, these cells migrate through lymphatic vessels to the draining nodes where they trigger T- and B-cell activation. Free antigen can also migrate directly to the draining node, where it is captured and presented to lymphocytes by blood-derived resident dendritic cells.[92]

The safety profile of intradermal immunization has been documented in many inactivated vaccine studies, particularly for vaccines against rabies, HB and influenza. As far as the safety profile of influenza intradermal vaccine in the elderly is concerned, a large randomized clinical trial performed in four European countries, which included more than 3700 participants, showed that the incidence of CHMP solicited and unsolicited systemic and injection site reactions is similar in subjects immunized by intradermal and intramuscular routes.[93] Solicited injection site reactions in the week after vaccination, such as pain, pruritus, erythema, swelling and induration, were more frequent among subjects immunized with intradermal vaccine. These reactions occurred mainly within 3 days of vaccination and were transient, lasting 3 days or less in the large majority of cases. An acceptable safety and tolerability profile was recently confirmed by studies conducted by other authors, both in adults and in the elderly.[52,53,94,95]

Intradermal influenza vaccine administered using an intradermal microinjection system elicits a significantly higher antibody response compared with intramuscular vaccination with an identical dose of antigen in adults aged ≥60 years (figure 1).[93] A 60% dose intradermal vaccine yielded seroprotection rates and GMTs comparable to those of conventional full-dose influenza vaccination in elderly subjects.[52] An intradermal injection of trivalent inactivated influenza vaccine, containing 6 µg of HA for each antigen (40% of the usual dose), resulted in similarly vigorous antibody responses among persons aged 18–60 years but was slightly less effective among those aged >60 years compared with the response elicited by the standard dose of 15 µg of HA for each antigen (figure 1). In any case, intradermal vaccination with a lower antigen dose than that used for a recently available vaccine met the CHMP criteria in both adult and elderly age groups.[53]

Recently, Van Damme et al.[94] compared the immunogenicity elicited by the standard-dose vaccine administered intradermally and MF59-adjuvanted vaccine administered intramuscularly and showed a comparable response in elderly individuals.

The EMA recently approved use of the intradermal influenza vaccine in humans, with indications for use in subjects aged 18–59 years (Intanza® 9 µg, Sanofi Pasteur MSD) or ≥60 years (Intanza® 15 µg/IDflu® 15 µg, Sanofi Pasteur, MSD).

5. High-Dose Vaccines

An antigen dose-dependent increase in immune response has been observed by several authors following vaccination with an increased amount of influenza virus HA antigen.[96100] Thus, one approach to improving immune response among elderly persons is to increase the antigen dose in the vaccine formulation. Standard-dose inactivated trivalent influenza vaccine contains a total of 45 µg (15 µg of each of the three recommended strains) of influenza virus HA antigen per 0.5 mL dose. This approach has been experimented with for both pandemic and seasonal vaccines. Early trials with H5N1 vaccine and other avian influenza vaccines suggested that at least two doses of vaccine containing a high antigen dose were needed to induce acceptable antibody responses.[101,102] Treanor et al.[102] administered two doses of a subvirion H5N1 vaccine at 7.5, 15, 45 or 90 µg per dose to healthy adults. All doses were well tolerated but doses of <90 µg induced serum HA-inhibiting titres or neutralization titres of ≥40 in <50% of subjects.

The immunogenicity of conventional vaccine with a higher-than-standard antigen dose has also been evaluated against the new pandemic H1N1 2009 virus. One dose of vaccine containing 30 µg of antigen was highly immunogenic in adults, suggesting that this dose afforded sufficient protection against this pandemic influenza A H1N1 virus. However, seroprotection rates observed in the elderly are very similar using 7.5, 15 and 30 µg doses, i.e. ranging between 93% and 95%.[103]

In December 2009, the US FDA licensed an injectable inactivated trivalent influenza vaccine (Fluzone® High-Dose, Sanofi Pasteur) for people aged >65 years.[104] The vaccine contains a total of 180 µg (60 µg of each strain) of influenza virus HA antigen per 0.5 mL dose.

Immunogenicity data in older adults indicated that this high-dose formulation elicited significantly higher HA-inhibiting titres against all three influenza virus strains included in seasonal influenza vaccines recommended during the study periods, compared with the standard-dose split vaccine.[97,100,105] Superior immune responses to high-dose vaccines were demonstrated by both the higher rates of seroconversion and the GMT ratios for the two influenza A virus strains included in the vaccine and by higher seroprotection rates for all strains for those who received high-dose vaccine than for those who received the standard vaccine. Improved immunogenicity was demonstrated in subjects aged ≥75 years and those with cardiopulmonary disease.

A safety and tolerability evaluation in adults aged ≥65 years showed that solicited injection site reactions and systemic adverse events were more frequent after vaccination with the high-dose formulation compared with the standard-dose split vaccine, but typically were mild and transient.[97,100,105] In the largest study,[105] significantly more high-dose vaccine recipients reported greater injection site pain ≤7 days after vaccine administration and moderate-to-severe fever, compared with standard-dose vaccine recipients.

Concerns about increased reactogenicity and reliability of supply have also been raised in relation to use of the high-dose vaccine.[3]

6. Conclusions: New Perspectives and Real Problems

The availability of new first-generation adjuvants (i.e. TLR agonists, cytokines, CD1d agonists, cell wall components, etc.), the almost endless combinations of adjuvants already tested and used with success (i.e. MPL and AS04 in human papillomavirus, and tocopherol and squalene in influenza vaccines) and the increasingly deeper understanding of their mechanisms of action offer attractive opportunities for the future in terms of eliciting an immune response, especially in high-risk groups and low-responder individuals, such as the elderly, patients with chronic diseases and immunocompromised patients.

However, the weak point in the use of currently available and tested adjuvanted, intradermal or higher-dose vaccines is that, although greater immunogenicity has been observed in a number of studies, it is not yet clear how this will translate into protection against influenza and influenza-related complications and deaths. Observational studies or comparisons of the adjuvanted, intradermal or higher-dose vaccines with standard vaccines may provide an answer to some doubts about the advantages offered by such ‘enhanced’ vaccines in terms of clinical effects.

Another challenge for the future involves the health systems, which face difficulties in collecting new evidence. Defined goals, a clear vaccination plan, vaccine implementation policies and programme evaluation should be introduced to support new vaccination tools, but the use of new vaccines must be supported by strong evidence, not only in terms of higher immunogenicity but also greater effectiveness.