Adjuvanted influenza vaccines

Why adjuvants

One potential approach to improve influenza vaccines is to include adjuvants. There are a number of reasons adjuvants might be included in a vaccine:

1. Populations with poor immune responses

Adjuvants are used to boost responses in populations with poor immune responses; this includes patients who are immunosuppressed due to either primary immunodeficiencies, transplant treatment or infection – particularly HIV. For example, the inclusion of the adjuvant AS03 improves the anti-influenza antibody quality in HIV positive patients.¹⁷ Likewise, the inclusion of AS03 improved influenza vaccine responses in haemodialysis patients.¹⁸ Vaccination is also less effective in individuals at the extremes of age – the very young and the very old¹⁹ – and adjuvants can help in these situations. Influenza causes the most severe disease in these age groups; infants (under 2 years) and elderly patients (≥65 years) have higher influenza attack rates, more frequent influenza related hospitalisations and greater rates of influenza related mortality.⁴ Globally, influenza infection results in approximately 374,000 hospitalisations in 1 year old children.²⁰ The addition of MF59 to an influenza vaccine induced substantially faster and higher antibody titres in children than a non-adjuvanted vaccine.²¹ There is no global estimate for influenza infection in the elderly, but estimates from the USA put the rate of influenza hospitalisation in elderly patients (≥65 years) at nearly twice that of infants.²² The addition of AS03 improved responses in young and elderly adults,²³ the addition of MF59 improved responses in subjects older than 65²⁴ and virosomes increased the response in geriatric patients.²⁵

2. Boosting the immunogenicity of an antigen

As well as some individuals being poor at making immune responses, some antigens are less immunogenic than others. Many of the longstanding vaccine antigens are pathogen derived, for example diphtheria toxoid, tetanus toxoid and haemagglutinin. Newer vaccines often contain recombinant proteins and these can be less immunogenic than pathogen derived antigens. This is probably because the pathogen derived antigens contain trace levels of inflammatory material from the pathogen and some of the classical vaccine antigens may also have some inherent self-adjuvanting property. It is of note that Flublok – the only licensed recombinant influenza vaccine –does not contain an adjuvant, but it does contain three times as much of each haemagglutinin (45 μg) as Aggripal (15 μg) which is the Seqirus (formerly CSL/Novartis) unadjuvanted egg-derived inactivated virus influenza vaccine.

3. Accelerating responses to a vaccine

Another advantage is that adjuvants can accelerate responses to the vaccine, for example during a pandemic. Most vaccines require more than one administration to reach protective levels in recipients; the addition of an adjuvant can elevate the response to the first dose and push it over the protective threshold. For example HAI titres to an H1N1 vaccine were above the US and European licensure criteria after a single dose only when MF59 was included in both pre-clinical²⁶ and clinical studies.²⁷ For an experimental H9N2 vaccine, antibody titres after the administration of a single dose of MF59 adjuvanted vaccine were similar to those after two doses of nonadjuvanted vaccine.²⁸ Whilst accelerating the response may not lead to enduring immunity, it may be sufficient to protect individuals during the main wave of a pandemic.

4. Dose sparing

The inclusion of adjuvants can enable dose sparing, both for routine and pandemic vaccines. Vaccine antigens are expensive to manufacture and there are limited manufacturing facilities for making vaccines to the required good manufacturing practice (GMP) standards. The addition of AS03 led to a similar response to a lower dose of H5N1 antigen (3.75 μg) compared to the standard 15 μg dose.²⁹ When alum was included as an adjuvant, equivalent responses were seen when doses of influenza antigen were reduced from 15 μg to 6 μg in both young and elderly adults.³⁰ In a phase I study investigating the unlicensed adjuvant Advax (a polysaccharide particulate adjuvant derived from inulin) responses were equivalent between the adjuvant group that used a third of the antigen (15 μg) and the unadjuvanted group that received 45 μg influenza antigen.³¹

5. Immunomodulation

Adjuvants can also change the quality of the immune response to antigen. The use of different adjuvants can change the pattern of cytokines and chemokines released, leading to the recruitment of different cells.³² This is particularly noticeable with T cell responses where the combination of different adjuvants with the same antigen can lead to very different outcomes. In a recent study the combination of influenza antigen with MF59 or Alum gave a strong IgG1 antibody responses associated with IL-5 producing T cells indicating Th2 skewing, whilst combination of haemagglutinin with the cationic liposomal adjuvant CAF01 led to a more Th1 and Th17 biased cellular response.³³ Since current influenza vaccines primarily confer immunity through antibody, shaping the CD4 T helper response may not be necessary to improve protective efficacy. However, the addition of adjuvant can also potentially improve the quality of the B cell response and CD8 T cell responses both of which may be important for the development of a universal flu vaccine. Additionally, there may be subtle effects if the adjuvant leads to a switch in antibody subtype, when non-neutralising antibody functions, such as antibody dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), are important.

6. Mucosal vaccine delivery

A final use is to enable mucosal delivery of vaccines. Through the induction of local immunity at sites of infection, mucosal vaccination may be more appropriate than systemic vaccination. However, mucosal surfaces are much harder to vaccinate for a number of reasons – they are broadly tolerogenic and they also have mechanical (cilia, gap junctions), chemical (mucus) and biochemical (proteolytic enzymes) barriers to antigen. Specific adjuvants may be required to protect the antigen in this environment and to induce a local immune response. One adjuvant that was licensed for this purpose was the heat labile enterotoxin (LT) of Escherichia coli, which was included in Nasalflu.³⁴

How adjuvants work

Before describing what is known of the mechanism of specific adjuvants included in licensed influenza vaccines, it is necessary to give a brief overview of their general mechanism of action (reviewed in depth elsewhere³⁵). Fundamentally, adjuvants improve the ability of the host immune system to recognise the administered antigen as foreign and respond to it; beyond this simple description they are extremely diverse in their molecular and cellular mechanisms of action.

The first requirement is for the vaccine antigen to be seen by the immune system. One problem is that soluble antigen is quickly cleared by the lymphatics and therefore is never seen by the immune system. Adsorbing (sticking) the antigen onto an insoluble complex (Alum) or in an oil-in-water emulsion (MF59/ AS03/ AF03) leads to the retention of antigen at the injection site. In theory, the antigen/adjuvant depot can then be sampled by antigen presenting cells, which then take antigen to the lymph nodes. However, recent studies in mice have shown that removing the site of the depot even as early as two hours after immunisation had no negative effect on the immune response, suggesting that formation of a depot at the injection site is not essential.³⁶

Adjuvants can also increase antigen visibility by increasing the recruitment of cells to the site of injection.³⁷ The adjuvant can either recruit cells directly³⁸ or indirectly by inducing local sentinel cells to release cytokines and chemokines.³⁹ Another method of increasing antigen visibility to the immune system is to increase uptake by antigen presenting cells. This can also occur directly by acting on antigen presenting cells⁴⁰ or indirectly by inducing antigen shuttling to lymph nodes by other cell types.⁴¹

However, seeing the antigen is not sufficient to induce an adaptive immune response, cells also need to be licensed to respond. Some adjuvants promote dendritic cell (DC) maturation, via increased expression of MHCII and the co-activation markers CD80 and CD86.⁴² This effect is not limited only to DCs, as Alum and MF59 have both been shown to upregulate MHCII and CD86 expression on other antigen presenting cells including monocytes and macrophages.⁴³

Underpinning the recruitment and activation of antigen presenting cells is the ability of adjuvants to stimulate the innate response, with a particular focus on triggering pattern recognition receptors (PRR). PRRs are expressed by innate cells and enable them to recognise infections. It covers a broad range of families including the Toll like receptors (TLR), Rig-like receptors (RLR) and the inflammasomes. Some adjuvants act directly by engaging these receptors, for example the TLR5 ligand flagellin.⁴⁴ However other adjuvants, particularly particulate adjuvants, act more indirectly by inducing local damage which is in turn detected by inflammasome complexes,⁴⁵ though the exact pathway by which this occurs is not fully characterised.

For the effective induction of an antibody response, there needs to be an interaction between T and B cells. B cells do not directly interact with antigen presenting cells, but they do respond to some of the same signals,⁴⁶ so it may be that adjuvants activate them in this way. Alternatively, improving the T cell quality with adjuvant, for example increasing the number of T follicular helper cells,⁴⁷ may lead to improvement in the antibody response.

Whilst many of the adjuvants that are used have been developed empirically, greater insight about the induction of the innate immune response and how that shapes the adaptive immune response has led to immunologically designed adjuvants, for example MPLA targeting TLR4 which is incorporated into AS04. However, for many of the adjuvants in wide use, mechanistic knowledge is incomplete, but this doesn't prevent vaccine licensure; provided a vaccine works and is safe, the mechanism of action is a secondary consideration.

Adjuvants in licensed influenza vaccines: Characterisation and mechanism

Alum is the most commonly included adjuvant in influenza vaccines, but even then is only included in five vaccines. The other adjuvants used are virosomes (Inflexal V), MF59 (FluAd), AS03 (Pandemrix). AF03 was licensed for as part of Humenza, but this product was never marketed. Heat labile enterotoxin (LT) was licensed as part of Nasalflu, but this has been withdrawn.

Impact of adjuvants on the immune response to flu

The inclusion of an adjuvant increases anti-influenza antibody responses. When compared against unadjuvanted vaccines, virosome adjuvanted vaccines were more immunogenic in both children⁷⁵ and the elderly.²⁵ In children, the addition of MF59 induced greater antibody⁷⁶ and cellular⁷⁷ responses than vaccine without adjuvant. MF59 also induced a better response in immune naïve individuals⁷⁸ to a potential pandemic antigen. Likewise the inclusion of the AF03 adjuvant boosted responses compared to unadjuvanted vaccine in 6–35 month old children.⁷⁹ H5N1 influenza vaccine formulated with AS03 induces stronger B and T-cell responses than vaccine alone.⁸⁰ When MF59 was compared directly against virosomes, it led to a significantly greater number of elderly patients seroconverting (fourfold increase in antibody titre),²⁴ but both have been shown to have efficacy against influenza infection in the elderly.⁸¹ Comparisons have also been performed between AS03, MF59 and unadjuvanted H7N9 antigen; both the adjuvants induced seroconversion in significantly more patients than no adjuvant,⁸² in this study AS03 inclusion led to a higher antibody titre. A couple of meta-analyses indicated that inclusion of MF59 increased Haemagglutination inhibition (HI) titres by 1.14–1.4 fold,^5,83 but it was argued that this would not have a big impact on efficacy, as based on human challenge studies,¹⁰ this difference in HI titre is not large enough to have an effect.

Safety/Tolerability of adjuvanted flu vaccines

In general adjuvants are well tolerated, though they may increase some local site symptoms, particularly injection site pain. Two Phase III studies of AS03 adjuvanted H5N1 have been performed covering 10,000 adults.^84,85 In these studies, the adjuvanted vaccine solicited local and general symptoms more frequently, including pain, fatigue, headache and myalgia. Immunisation of children with an AS03 adjuvanted vaccine was also associated with transient injection site pain.⁸⁶ Similar results were seen with an AS03 adjuvanted H1N1 vaccine, the most frequently reported symptom was injection site pain,⁸⁷ and local and general symptoms were reported more frequently for AS03-adjuvanted H1N1 vaccine recipients than for controls.^88,89 In children, the incidence of some reactions, especially fever (axillary temperature ≥37.5°C), increased after the second dose.⁹⁰ A meta-analysis of MF59 usage in clinical trials in elderly adults suggested that local reactions were slightly more common for vaccine with adjuvant, but fever was very uncommon in either group.⁹¹ A retrospective review over the lifespan of the virosome adjuvanted vaccine,⁹² Inflexal V,⁹³ suggest that virosomes are well tolerated.

When adjuvants didn't work

However, there have been notable cases where adjuvanted influenza vaccines have had to be withdrawn (Nasalflu) or the recommended usage altered (Pandemrix). Separating the specific contribution of adjuvant to the adverse effect is complicated as they are always administered in combination with the antigen. However, it is likely that the adjuvant played a role in the adverse effects seen. There are a range of possible mechanisms by which the inclusion of an adjuvant might have increased the incidence of severe adverse effects including increased inflammation caused by the adjuvant, as seen with LT adjuvanted Nasalflu, or altered responses to the antigen including increased immunogenicity of sub-dominant epitopes, as possibly seen with AS03 and narcolepsy.

Future of flu vaccines and adjuvants

Clinical trials of adjuvanted flu vaccine studies in humans have a long history, with one of the earliest studies being performed by one of the founders of modern vaccinology, Maurice Hilleman, who used a stabilised water-in-oil formulation in 1967.¹⁰² The appetite for new adjuvants has ebbed and flowed, at times they are heralded as the next big thing that will change vaccinology, but at other times they are seen as a red herring. The number of experimental adjuvants that have been used pre-clinically is too large for the scope of this review. Whilst there is a huge range of pre-clinical vaccine adjuvants in development, a smaller number have made it into clinical trials (Table 2). There are a number of reasons why the pre-clinical adjuvants have not moved forwards: some of them simply do not work, some have limited efficacy in animal models that fails to translate into human responses, some would be too expensive to manufacture for a mass market and some are just too weird and wonderful to have a pathway to commercial and clinical development. There have been cycles of development of the substances used, from empirical approaches to immunological design based on better understanding of immune sensing. The adjuvants that have been tested clinically fall into four broad categories: toll like receptors (TLR) ligands, formulation, cytokines and immunostimulators with unknown mechanism. Where results are reported, experimental adjuvants have mostly increased the antibody response to influenza, though in some cases the increases have been marginal.

Adjuvants in licensed vaccines other than influenza

In addition to those that have been tested in early phase clinical trials, there are adjuvants that have been included in licensed vaccines that may be included in influenza vaccines in the future. GSK has two other adjuvant formulations that are used in licensed vaccines. AS01 is a liposomal adjuvant containing the TLR4 ligand monophosphoryl lipid A (MPLA) and the saponin QS-21 and is part of the anti-malaria vaccine Mosquirix. AS01 was designed specifically to boost cell-mediated immunity, with a particular focus on CD8 T cells.¹¹⁷ A vaccine containing this adjuvant has now completed phase III clinical trials,^118,119 conferring medium-term moderate protection to malarial disease. AS04, which contains Alum and MPLA, is used in Cervarix (human papilloma virus) and Fendrix (Hepatitis B). AS04 was first used in Fendrix in 2005 and is currently licensed in Europe.¹²⁰

Future of adjuvanted flu vaccines

The biggest question is whether any of the adjuvants in development will be included in a licensed commercial product. There are two hurdles to overcome – the cost of manufacturing the adjuvant to GMP standard at a scale required for influenza vaccine and the risk of an unforeseeable adverse effect occurring when the vaccine is deployed at a population level. Realistically for the current generation of influenza vaccines, particularly in the face of existing adjuvants from the major vaccine manufacturers, in our opinion it is unlikely that a new adjuvant will be included in a seasonal influenza vaccine. However, there is still scope for research into adjuvants to support the next generation of influenza vaccines. Speculatively this could focus on the following areas:

• 1.

  Stabilising the haemagglutinin stem region. The holy grail of influenza vaccine research is the ‘universal flu vaccine’. This would be one vaccine that covers all current and future strain variations. One speculative approach to achieve this extremely difficult goal has been to target the stem region of the haemagglutinin antigen.¹²¹ Very approximately speaking, haemagglutinin is shaped like a mushroom, with a head and a stem. The head is immunologically dominant, but is also the region that changes the most, the stem is more conserved across different flu stains.¹²² Potentially antibodies raised against this region may be able to cross neutralise different strains of virus. Whilst natural infection does raise some anti-stem antibodies,¹²³ raising them with a vaccine has proved tricky. One application of adjuvants could be to stabilise structures that expose the stem region without the head domain.

• 2.

  Universal T cell vaccines. Whilst most influenza vaccine research has focussed on vaccines that can induce antibody, an alternative might be to induce T cells. Adjuvants that stimulate T cell responses e.g. CAF09¹²⁴ or IC31¹²⁵ may potentiate stronger CD8 responses which may be beneficial. Because they often recognise conserved regions of influenza, T cells can possibly offer better cross neutralisation.^126,127 There is a history of pre-clinical studies indicating that cross protection can be achieved with CD8 T cells¹²⁸ and in human challenge studies T cells correlated with viral shedding.¹²⁹ More recently, studies have shown that individuals with elevated T cell responses experienced less severe disease on exposure to pandemic influenza.¹³⁰ Interestingly CD4 T cells have also been shown to correlate with protection against challenge.¹³¹

• 3.

  Mucosal protection. It is becoming clear that local, mucosal immune responses may be more protective than systemic responses. For example, we have recently shown that local IgA is a correlate of protection against influenza challenge.¹³² Likewise lung resident T cells (Trm) correlate with protection against challenge in both mouse¹³³ and human infection studies.¹³⁴ However, mucosal vaccination has to date been sub-optimal, the addition of an adjuvant may improve mucosal responses. The addition of an adjuvant to a mucosal influenza vaccine is challenging as the only licensed mucosal vaccine containing an adjuvant (LT/ NasalFlu) had to be withdrawn due to the association with Bell's Palsy.⁹⁸

• 4.

  Protection in diverse age groups. One of the major priorities for an adjuvanted influenza vaccine is the ability to induce a strong response in individuals who are most susceptible to infection – the elderly and the very young.¹⁹ It is likely that since the reasons vaccines are less effective in these age groups are different, different approaches will be required. The use of GLA with an RSV antigen improved anti-RSV responses in adults over the age of 60, suggesting that it may also be effective with influenza.^135,136 Studies in children are more complex to perform, with a greater risk of unforeseen complications, but this doesn't stop there being a need for infant specific adjuvants. AS02 has been tested in infants (under 1 year of age) in the context of a malaria vaccine, increasing T cell responses.¹³⁷

• 5.

  Pandemic protection. Of all the viruses, influenza remains one of the most likely to cause a pandemic. This has occurred multiple times, most recently with the emergence of the 2009 H1N1 strain. Under these circumstances, the addition of an adjuvant would enable faster responses and dose sparing to achieve greater coverage. Any of the adjuvants described in this review could potentially perform this function and a number of clinical trials have been performed to pre-test adjuvanted vaccines in this capacity. There are also a number of pandemic vaccines that are pre-licensed to cover the emergence of new strains.

• 6.

  Boosting recombinant and neoantigens. The majority of the current influenza vaccines are egg derived and therefore contain some degree of other viral material which may boost the immune response to the antigens. However, recombinant antigens, especially those that have been specifically designed using structural vaccinology approaches, may need boosting by adjuvant.¹³⁸ This is particularly important for neoantigens from newly emerged pandemic influenza strains for which there is no pre-existing adaptive immunity, notably avian derived antigens have lower immunogenicity in humans.

• 7.

  Resetting original antigenic sin. An individual's lifetime influenza exposure history is complex with a mixture of vaccination and natural infection. This repeated exposure shapes the antibody and T cell responses, often focussing the response on immunodominant regions.¹³⁹ The concern is that original antigenic sin may reduce the ability to generate responses to novel antigens. It is possible that an adjuvant could reset the B cell response or present new antigens in such a way that B cell memory is altered.

• 8.

  Altering isotype for maternal vaccination. One usage of influenza vaccines is maternal immunisation, this protects both the mother and the offspring in early life by passive antibody transfer. During pregnancy, maternally derived antibodies are actively transported through placenta from the mother to the foetus, which can provide passive immunity for infants up to 6 months against infection.¹⁴⁰ There are four subclasses of human IgG (IgG1-4). Placental transfer of IgG depends on the subclass, IgG1 is best followed by IgG4, IgG3 and IgG2.¹⁴¹ Since adjuvants can alter the IgG subclass,³³ potentially the inclusion of a minimally inflammatory adjuvant that preferentially boosts the IgG1 response could boost the amount of antibody transferred to the foetus.