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Review

Journal of Pharmacy And Pharmacology

Adenoviral vectors as novel vaccines for influenza Lynda Coughlan, Caitlin Mullarkey* and Sarah Gilbert The Jenner Institute, University of Oxford, Oxford, UK

Keywords clinical evaluation; molecular and clinical pharmacology; whole body pharmacology Correspondence Lynda Coughlan, The Jenner Institute, University of Oxford, ORCRB, Roosevelt Drive, OX3 7DQ Oxford, Oxfordshire, UK. E-mail: [email protected] Received May 9, 2014 Accepted October 5, 2014 doi: 10.1111/jphp.12350 *Present address: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Abstract Objectives Influenza is a viral respiratory disease causing seasonal epidemics, with significant annual illness and mortality. Emerging viruses can pose a major pandemic threat if they acquire the capacity for sustained human-to-human transmission. Vaccination reduces influenza-associated mortality and is critical in minimising the burden on the healthcare system. However, current vaccines are not always effective in at-risk populations and fail to induce long-lasting protective immunity against a range of viruses. Key findings The development of ‘universal’ influenza vaccines, which induce heterosubtypic immunity capable of reducing disease severity, limiting viral shedding or protecting against influenza subtypes with pandemic potential, has gained interest in the research community. To date, approaches have focused on inducing immune responses to conserved epitopes within the stem of haemagglutinin, targeting the ectodomain of influenza M2e or by stimulating cellular immunity to conserved internal antigens, nucleoprotein or matrix protein 1. Summary Adenoviral vectors are potent inducers of T-cell and antibody responses and have demonstrated safety in clinical applications, making them an excellent choice of vector for delivery of vaccine antigens. In order to circumvent pre-existing immunity in humans, serotypes from non-human primates have recently been investigated. We will discuss the pre-clinical development of these novel vectors and their advancement to clinical trials.

Introduction Influenza is a highly contagious respiratory disease responsible for significant annual morbidity and mortality, particularly in the elderly, immunocompromised or the very young.[1] Influenza A viruses can infect humans, birds and other mammals. To date, 18 different haemagglutinin (HA) and 11 different neuraminidases (NAs) have been identified in nature, allowing for the emergence of a large range of subtype combinations (e.g. H3N2 or H5N1). Influenza viruses can accumulate ‘drift’ mutations, largely within HA antigenic sites within the globular head domain,[2] which allow evasion of neutralising antibodies (NAbs) induced by vaccination or prior infection, resulting in seasonal epidemics. Alternatively, influenza viruses can sometimes ‘shift’, exchanging their HA or NA to incorporate a distinct HA or NA subtype. Internal influenza antigens (Ags) can also be exchanged by antigenic shift. Collectively, such major changes can have two outcomes, either leading to the generation of novel subtypes with the potential to cause a pandemic, or these exchanges can negatively impact on virus 382

stability and capacity for replication in a new host. Currently licensed influenza vaccines largely aim to stimulate humoral immunity, inducing NAbs to specific influenza surface glycoproteins, HA or NA. The trivalent inactivated vaccine (TIV) includes one HA/NA from H1N1 strain, one H3N2 and one influenza B virus. However, the efficacy of TIV could be improved as it may fail to adequately protect against drift variants[3] and cannot protect against different influenza subtypes which have the potential to cause pandemics. When the vaccines’ components are well matched to circulating influenza A viruses, the efficacy of TIV ranges from 50% to 75% in healthy adults under 65 years.[4–6] The efficacy of inactivated influenza vaccines in children is ∼59%, though it is largely ineffective in children under the age of two.[7] Furthermore, annual vaccination of children with TIV is thought to impair the development of influenza-specific T-cell responses.[8] Alternatively, the live attenuated influenza vaccine (LAIV), administered as a nasal spray (FluMist in USA or Fluenz in EU, MedImmune,

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MD, USA) is capable of limited replication in human nasal epithelium, initiating an immune response without causing clinical disease. Although LAIV is less effective in adults, it has a high level of efficacy in children between the ages of 2 and 7 years.[9] Therefore, LAIV is preferred for use in children as it permits the development of T-cell responses as well as humoral immune responses. Newly emerging zoonotic influenza viruses such as avian influenza subtypes H5N1, and more recently H7N9, pose a major threat to global public health. Consequently there is a vital need to improve on the performance of seasonal vaccines and develop new vaccines that confer cross-protective responses. Vaccines for potentially pandemic strains can be produced by traditional manufacturing methods. These involve infecting embryonated chicken eggs with influenza vaccine strains, harvesting allantoic fluid and extracting virus. Such vaccines are subsequently inactivated (whole virion) or detergent-treated to make split virion or subunit vaccines.[10] However, work with avian influenza strains with pandemic potential needs to be carried out at high containment levels, increasing the cost of production. Additionally, the speed and magnitude of scale up and the potentially limited availability of eggs during a pandemic scenario would also be problematic if highly pathogenic avian influenza (HPAI) has decimated poultry populations. In clinical studies, inactivated H5N1 vaccines have proven to be poorly immunogenic, requiring high doses of Ag[11] or formulation with potent adjuvants to achieve seroconversion. Furthermore, H5N1 viruses have displayed a high degree of genetic and antigenic variation[12–14] that could complicate vaccine strain selection and stockpiling.[15] Recombinant adenoviruses (Ads) represent an ideal choice of vector for vaccine development as they have been studied extensively both pre-clinically[16,17] and clinically,[18,19] are well tolerated in vivo, possess an excellent safety profile and are capable of stimulating potent immune responses to vaccine Ags.[20,21] Furthermore, vaccines based on viral vectors expressing Ags from influenza represent an attractive alternative to current influenza vaccination approaches and are commercially viable for a number of reasons: (1) they can easily be modified and can incorporate customised transgenes with a capacity of up to 10 kbp, (2) they are rapidly scalable using well-characterised cell lines, with an estimated production time from initial vaccine selection to formulation and filling of 11–13 weeks[22] and (3) several viral vectors are already manufactured to clinical grade and have the potential to be licensed (e.g. Food and Drug Administration China-approved Gendicine (Shenzhen SiBiono GeneTech, China), Ad5-based vector expressing p53 for cancer). In addition to these attributes, it recently has been shown that recombinant Ad vaccine vectors can be desiccated and are thermostable at temperatures of 45°C for up to 6 months following a process of

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drying onto a filter-like support membrane in the presence of disaccharide stabilisers.[23] Importantly, storage at 4°C for up to 15 months showed minimal losses in Ad vaccine titer or immunogenicity. There are many advantages to integrating Ad-based influenza vaccines in pandemic preparedness. However, in particular, the potential of Ad-based vectors to stimulate broadly cross-reactive T-cell immunity to conserved influenza Ags is both novel and attractive and may complement existing vaccination regimens, leading to a reduction to the burden on healthcare systems.

Adenoviral Vectors Ads belong to the family Adenoviridae and are nonenveloped viruses with a dsDNA genome (∼36 kbp). The genus Mastadenovirus contains all known human and chimpanzee Ads which are subdivided into species A-G.[24–26] Ads infect a broad range of host species and utilise various cellular receptors for entry including but not limited to, the Coxsackie and Adenovirus Receptor, CD46, sialic acids and heparan sulphate proteoglycans.[24] Wildtype Ads generally cause mild respiratory, gastrointestinal or ocular infections, but more severe disease can be observed in immunocompromised, transplant recipients or paediatric patients.[27] The gene therapy community has exploited Ads as vectors for gene delivery for several decades with varying degrees of success. There has been significant investment in the development of Ads as vectors for disease targets including cancer,[28–31] monogenic disorders[32,33] and more recently as vaccine vectors for infectious disease.[18,20,21,34] First-generation Ad vectors feature deletions in the E1, and in some cases E3 region of the viral genome, rendering these constructs replication incompetent and providing the capacity for insertion of heterologous transgene inserts.[35] However, depending on the disease target, replication-selective as well as replication-competent Ad vectors also are sometimes employed. Although in theory Ad vectors do have the potential to recombine with wildtype Ad viruses following co-infection which could result in a replication-competent virus, this would be a rare event as naturally acquired Ad infections largely affect the upper respiratory or gastrointestinal tract, whereas Ad-based vaccines are delivered intramuscular (i.m.). In addition to their use as vaccine vectors in healthy volunteers, replication-selective, oncolytic adenoviral vectors have been used extensively in immunocompromised patients in clinical trials for cancer and have been shown to have an excellent safety profile following invasive intravenous, intratumoural and intracerebral injection (http:// www.abedia.com/wiley/index.html). By comparison, i.m. injection of replication-deficient Ad vectors as vaccines for infectious disease would be considered to be extremely safe.

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Human adenoviruses as vaccine vectors The most commonly used Ad vector in gene therapy applications is human adenovirus type-5 (Ad5). Although this serotype has been shown to be highly immunogenic,[36,37] its use in clinical vaccination approaches is limited by the high seroprevalence of NAbs in humans.[38,39] Furthermore, an Ad5-based vectored vaccine for Human Immunodeficiency Virus (HIV)-1 failed to demonstrate efficacy in the MERCK STEP trial, despite promising antiviral efficacy in non-human primates.[40] Additionally, several studies have reported that although the T-cell response induced by Ad5 is robust, it displays a dysfunctional exhausted phenotype[41] with decreased anamnestic potential upon subsequent pathogen challenge,[17] although this is largely dependent on the specific Ag used, the vector dose and route of administration. In an effort to overcome these limitations, attention has turned towards the use of rare species human Ads such as species B serotypes Ad35, or species D Ads Ad26, Ad28 or Ad48,[17,42–44] which have low seroprevalence in humans.[16,45] Alternatively, genetically modified chimeric Ad5-based vectors (e.g. Ad5HVR48) which substitute the major antigenic epitopes within the hexon for those derived from rare species, low prevalence serotypes, are also under investigation.[39,46] In pre-clinical studies when compared with Ad5, vaccination with rare species Ad vectors leads to the production of CD8+ T-cells with enhanced recall potential,[17,37] elicitation of more polyfunctional T-cells and improved boosting potential when used to prime heterologous prime-boost regimens.[37] Despite the fact that rare species Ads often display reduced immunogenicity in pre-clinical animal models[17,47] when compared with Ad5, several have been shown to be safe and immunogenic in humans.[19–21,48] Furthermore, the poor immunogenicity observed in pre-clinical models may be complicated by the absence of virus-specific surface receptors in certain animal species. In support of this, a recent doseescalation phase I clinical trial using an Ad26-based vector expressing HIV-1 Env was well tolerated up to doses of 1 × 1011 virus particle (vp) and demonstrated good Envspecific humoral and cellular immunity in healthy HIVseronegative volunteers.[18] Another phase I clinical study coadministering Ad35 expressing HIV gag, reverse transcriptase, integrase and nef or expressing Env was also well tolerated and induced CD4+ and CD8+ T-cell responses that were broad and polyfunctional.[49] These studies demonstrate the potential for future vaccine development using rare species Ad vectors.

Chimpanzee adenoviruses as vaccine vectors In addition to the use of rare species human Ads, the issue of high seroprevalence of NAbs to Ad5 in humans can be circumvented by using non-human Ads, such as those 384

derived from non-human primates.[50,51] Although there is a high degree of phylogenetic relatedness between Ads derived from different primate hosts,[52] sequence homology within the hypervariable regions (HVRs) of the hexon, the major antigenic target of NAb to Ads[39,53] is more divergent,[54] allowing them to evade pre-existing immunity in humans. The first non-replicating adenoviral vector derived from chimpanzee adenovirus (ChAd) was developed over a decade ago[50] and since then multiple serotypes subsequently have been isolated and engineered, expanding the repertoire of adenoviral vectors available for clinical assessment. In addition to having low seroprevalence in humans,[34] ChAds display robust immunogenicity[21,34,55] and can be manufactured to GMP using standard cell lines for adenoviral production (HEK293[56] or Per.C6®[57] Crucell, Leiden). Several ChAd vectors have been assessed pre-clinically for a range of infectious disease targets including ChAd63,[58,59] AdC6, AdC7 or AdC9 for malaria,[60,61] AdC68,[62] AdC6 or AdC7[63] for HIV-1 and AdC68 for rabies.[64] Moreover, there now have been numerous published reports of clinical trials using ChAd vectors as vaccines for malaria and more recently, influenza. Sheehy and colleagues demonstrated the safety and immunogenicity of ChAd63 expressing malaria Ag merozoite surface protein 1 (MSP1) in a phase Ia clinical trial.[55] Following a boost immunisation with the poxviral vector modified vaccinia virus Ankara (MVA) also encoding MSP1, Ag-specific serum Ab responses as well as robust CD4+ and CD8+ T-cell responses were observed. In a separate clinical study, vaccination of healthy volunteers with a ChAd63 vector expressing an HIV immunogen based on conserved HIV-1 epitopes elicited effector T-cells which could control HIV replication ex vivo in autologous CD4+ T-cells.[65] We have recently tested the safety and immunogenicity of a novel ChAd serotype encoding conserved influenza Ags.[21] This approach will be discussed in more detail in subsequent sections. The ongoing interest in the development of ChAd-based vaccines will see increases in the translation of these new vectors into the clinic in the near future.

Challenges and limitations associated with adenoviral vaccines The use of rare species or non-human Ad vectors clearly provides an opportunity to evade pre-existing NAb responses to Ad5. However, if these vectors become more widely adopted in vaccination programmes it is possible that similar to Ad5, anti-vector immunity to rare or nonhuman species may also become an issue in the future. To counteract this, the increased interest in isolating and identifying novel Ad serotypes would ensure that there is a large pool of suitable vectors to select from. Furthermore, there

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are a number of approaches to circumvent issues related to pre-existing immunity to Ad-based vaccines. Firstly, more detailed characterisation of novel serotypes and subsequent clinical assessment of prime-boost regimens using heterologous Ad serotypes is warranted, in addition to optimisation of prime-boost intervals. Secondly, the generation and clinical assessment of chimeric Ad vectors, such as those with hexon or fiber swaps could help to evade natural or vaccine-induced immunity, which is believed to impact on immunogenicity.[66,67] An alternative approach is the covalent coating of Ad vaccines with polymers such as polyethylene glycol to shield vectors from NAbs.[68] In addition, it is also possible that different routes of vaccine administration could potentially evade pre-existing immunity to Ads. In support of this, recent pre-clinical studies using Ad5-based vaccines have demonstrated that intranasal (i.n.) delivery can overcome existing immunity to Ad5.[69] Additionally, pre-existing NAb responses to Ad5 did not affect the development of anti-H1 HI titers in a recent phase I clinical trial that delivered an Ad5-H1N1 vaccine intranasally.[70] In a separate study, pre-existing immunity to Ad4 did not impact on the induction of cellular or humoral immune responses to H5 delivered via an oral Ad4-H5 vaccine in a recent clinical trial.[71] It will be important to investigate all of these factors in the near future and integrate to these findings into the use of Ad vectors in bi-annual or complementary vaccination programmes for seasonal or pandemic influenza. Furthermore, it would also depend on whether the Ad vaccination approach was to stimulate humoral or cellular immunity, as IgG Abs to influenza HA have been shown persist for the lifetime of the host,[72] thereby potentially negating the necessity to re-administer if the vaccine was effective in stimulating protective levels of NAb titers. Although pre-existing serotype-specific NAb responses can be overcome by using rare species or non-human Ad vectors, cellular immunity to the vector remains a potential issue. CD4+ and CD8+ T-cell responses are directed towards epitopes within conserved regions of the virus, particularly the immunodominant hexon,[73–76] and these can be cross-reactive among different Ad species groups.[75,77] In recent years, a number of studies have demonstrated that pre-existing anti-vector T-cells are boosted in vivo[74,77–79] and ex vivo following vaccination/stimulation with Ad vectors.[80,81] Furthermore, in addition to issues with pre-existing anti-vector NAbs limiting transgene expression, cellular immunity can also eliminate vectortransduced cells, having a negative impact on vaccine immunogenicity. Indeed, high frequencies of pre-existing anti-Ad specific CD4+ T-cells were inversely associated with the magnitude of HIV-specific CD4+ T-cells and decreased the breadth of CD8+ T-cell responses to Ad5HIV vaccine recipients.[74]

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The STEP trial did not demonstrate vaccine efficacy and identified a trend towards increased HIV-1 acquisition in Ad5 seropositive volunteers vaccinated with an Ad5-based HIV vaccine.[82,83] It is now established that pre-existing NAb responses to Ad vectors do not correlate with T-lymphocyte responses[74,76] and that cellular immunity to Ad vectors is more widespread than serotype-specific NAbs, a reflection of frequent boosting of these cross-reactive T-cells following natural infection with diverse Ad serotypes. Therefore, a number of studies have subsequently implicated pre-existing vector-specific T-cells in the failure of the STEP trial. Although the precise mechanisms have not been elucidated, a number of theories to explain the increase in HIV-1 acquisition following vaccination with Ad5 have been proposed, including (1) the expansion of the CD4+ T-cell pool available for infection with HIV,[80] (2) the upregulation of HIV co-receptors (CCR5 or integrin α4β7),[79,81] (3) the increased homing of HIVsusceptible CD4+ T-cells to mucosal sites[84] or (4) the involvement of Ad5 immune complexes in activating a dendritic cell (DC):T-cell axis which may be permissive to HIV-1 infection.[85] However, these studies are not exhaustive or conclusive and require further investigation in large scale clinical trials in humans. Furthermore, it is worth bearing in mind that any vaccination regimen or natural infection which results in the activation of CD4+ T-cells would theoretically pose the same increased risk of HIV acquisition.

Influenza Vaccine Targets for Humoural Immunity Humoral immunity to TIV is largely characterised by the production of virus-specific Abs, largely directed to influenza surface glycoproteins HA or NA. Based on genetic relatedness, influenza HA subtypes can broadly be classified into two phylogenetic groups, referred to as group 1 and group 2.[86] The vast majority of Abs recognising HA are directed towards the immunodominant, HVRs of the homotrimeric globular head. HA-specific Abs are of particular importance as these inhibit viral attachment and entry and thus are able to confer sterilising immunity.[87] Anti-HA Ab titers of ≥40 in haemagglutination inhibition (HI) assay are considered protective in adults.[88] Furthermore, it has been demonstrated that IgG Abs can persist for up to 90 years post-exposure.[72] Whilst lacking neutralisation capacity, it is thought that Abs formed against NA also contribute to protective immunity. By interfering with NA activity, such Abs can limit viral dissemination and also facilitate antibodydependent cell-mediated cytotoxicity (ADCC), helping to clear virus infected cells.[89,90] Studies in humans have also demonstrated that serum Abs to NA, induced by

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vaccination or natural influenza exposure, can reduce viral replication and disease burden.[91–93]

Adenoviral Vectors Targeting Influenza Haemagglutinin Pre-clinical studies As an alternative to TIV, a number of pre-clinical studies have described the ability of recombinant Ad vectors expressing influenza HA to induce HA-specific humoral immune responses. More than a decade ago it was demonstrated that expression of influenza HA from a recombinant Ad vector could provide partial protection against heterologous influenza challenge.[94] Furthermore, it was found that insertion of HA epitopes into various Ad capsid/ fibre locales could stimulate epitope-specific humoral and cellular immune responses.[95] More recently, Weaver et al. employed a novel approach to inducing broad anti-HA immune responses by constructing a synthetic, centralised consensus HA sequence, derived from phylogenetically related H1 sequences.[96] The rationale behind this strategy was to minimise variations between the vaccine and circulating influenza strains. The authors found that the consensus H1 induced better immune responses and protection against divergent H1 influenza challenge as compared with several wildtype H1 constructs. However, wildtype constructs displayed enhanced protection following homologous challenge. Rao and colleagues compared the protective efficacy of a prime-boost DNA/Ad5-based vaccination using constructs expressing either H5 HA, NP or matrix protein-2 (M2) (from A/Thailand/1(KAN-1)/2004) or combinations thereof in ferrets,[97] the gold-standard animal model for influenza infection. The authors concluded that although immune responses to NP+M2 could provide moderate protection from low-dose HPAI challenge, anti-HA responses were required to protect against a highdose HPAI challenge. In addition to human Ad5-based vectors, efforts to develop rare species Ad vectors as vaccines expressing HA have also yielded promising results in pre-clinical studies. Weaver and colleagues directly compared the efficacy of species C (Ad5, Ad6) and D (Ad26, Ad28 and Ad48) Ads expressing HA following i.m. and mucosal i.n. delivery.[44] Although, species D Ads were less effective than species C Ads following i.m. delivery, they were equally efficient following i.n. administration, with a virus dose of 108 vp providing protection against lethal influenza challenge with A/PR/8/34. Recent work by these authors also has shown that HA expressed by the species D virus Ad48 elicited superior protection following i.n. delivery in transgenic mice expressing CD46, the proposed receptor for species D Ad vectors. 386

Clinical studies VaxArt, Inc recently reported the safety and immunogenicity of a replicating oral Ad5-based vector expressing avian HA along with a dsRNA adjuvant designed to act as a ligand for Toll-like-receptor-3 (TLR3).[98] This was a first-in-human assessment of a recombinant Ad5based vaccine against avian H5 influenza using an oral capsule-based formulation. The study found that the vaccine was well tolerated and 73% of the high dose recipients produced IFN-γ in response to stimulation with H5 peptides. However, the magnitude of the T-cell response was weak. Furthermore, although HA Ab titres were elevated when detected by ELISA, no HI NAbs were detected in any of the participants. A separate vaccination schedule using replicationcompetent Ad4-based vector expressing avian influenza H5 HA delivered orally followed by i.m. boosting with 90 μg of inactivated subvirion H5N1 vaccine, was assessed clinically.[71] Control groups were included who received placebo vaccinations in place of the Ad vaccination, but still received the inactivated H5N1 vaccine. This regimen induced high levels of H5 seroconversion (as measured by a four-fold rise in baseline titre by HA inhibition assay) in groups receiving 1 × 1011 vp Ad4-H5 (100%) whereas only 36% of placebo recipients seroconverted. Furthermore, seroprotection (as measured by HAI titres ≥40) reached 89% in the highest dose cohort, compared with 18% in the placebo control group. In addition to improvements in anti-H5 Ab levels, interferon-γ (IFN-γ) T-cell responses to H5 were also improved, particularly in the high dose group (70%), whereas only 5% of the placebo group had cellular responses to HA. Thus the authors proposed that replication competent vaccines may prove useful in priming immune responses to other H5N1 vaccines with less than desirable immunogenicity. As this was a replicationcompetent vaccine vector, virus shedding was assessed from rectal swabs, and detected in 46% of the vaccinees. Additionally 2/58 household contacts of vaccinees had asymptomatic seroconversions to Ad4, although the vaccine vector was not detected in any swabs taken from these contacts. At doses greater than 109 vp, Ad4 seroconversion and elevation in Ab titres were recorded in both seronegative and seropositive vaccine recipients respectively, however, preexisting Ad4 immunity did not impair the development of anti-H5 cellular or humoural immune responses to the Ad4-based vaccine.

Recent Advances in the Development of Universal Influenza Vaccines The limitations associated with currently licensed influenza vaccines which only stimulate narrow, HA subtype-specific

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immune responses have prompted a dramatic increase in the number of experimental studies to engineer ‘universal’ influenza vaccines. Universal influenza vaccines should have the potential to protect against a broad range of influenza subtypes, including those with pandemic potential. A number of influenza proteins represent a good choice of Ag for stimulating broadly cross-protective heterosubtypic immunity. Such approaches include stimulating humoral responses to (1) conserved regions of the HA stalk, (2) the highly conserved ectodomain of M2 or (3) inducing potent cellular immune responses to T-cell epitopes within highly conserved influenza proteins such as NP or M1. These different approaches will be discussed below.

Haemagglutinin Unlike the highly variable HA globular head region, the stem domain within the HA stalk possesses a greater degree of conservation. Strategies to elicit anti-stem Abs have garnered much interest recently as stalk Abs have demonstrated the ability to neutralise different influenza subtypes.[99–102] Ekiert and colleagues first reported that a human Ab, CR6261, was able to bind a highly conserved region in the HA stem that prevented membrane fusion.[99] This Ab was capable of recognising and neutralising most group 1 HAs. Shortly thereafter the same group described another Ab, CR8020 that neutralised most group 2 HAs.[102] Corti et al. subsequently isolated a mAb from human plasma cells capable of neutralising both group 1 and group 2 HAs.[103] Initially, broadly neutralising anti-stem Abs were thought to be produced at very low levels naturally, although this opinion shifted when Wrammert et al. demonstrated that neutralising cross-reactive anti-stem Abs were stimulated in response to the 2009 pandemic H1N1 influenza strain.[104] Recently, it has been demonstrated that unlike NAbs directed towards the HA head domain, broadly neutralising anti-stem Abs function by mediating FcγR-dependant cytotoxicity of influenza infected cells.[105] Pre-clinical studies investigating the generation of a ‘universal’ influenza vaccine by eliciting heterosubtypic Abs to the HA stem have taken diverse approaches. To date, these include (1) prime-boost immunisation regimes or sequential immunisation, either using different vaccine vehicles (e.g. plasmid DNA prime followed by viral vector expressing a different HA) or identical vaccine vehicles or the (2) construction of and vaccination with chimeric HAs, featuring identical stem regions but different HA head domains. By priming mice with plasmid DNA encoding HA and boosting with TIV, Wei et al. reported the production of broadly neutralising Abs in mice that were capable of recognising diverse H1N1 strains.[106] This regimen also protected mice and ferrets and evoked stem Abs in non-human primates.[106] Both Steel et al. and Bommakanti and colleagues

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have generated so-called ‘headless’ HA constructs comprised of the conserved HA stem (HA2) and lacking the globular head (HA1).[107,108] Mice immunised with the HA2based ‘headless’ immunogen were protected from a lethal challenge with a homologous virus.[107] Similarly, Steel and colleagues reported that mice immunised with their headless construct generated cross-reactive serum Abs that recognised multiple HA subtypes and were protective in a lethal challenge.[108] Alternatively, using an immunisation schedule that included sequential vaccination with different H3 viruses, Wang and colleagues demonstrated that broadly neutralising Abs to H3 could be induced in vivo.[109] The concept behind this approach of honing the immune response to the conserved regions of HA has been attempted using ‘consensus sequences’ from H1N1 expressed in an Ad vector, as described above.[96] Similarly, chimeric HAs expressing novel head and stalk combinations have recently been described.[110] In these studies mice were repeatedly immunised with constructs with an identical stem but diverse head regions. Repeated exposure of the same stem construct led to the generation of neutralising anti-stem Abs that were shown to be functional in vitro and protective in a heterologous challenge in mice.[110] Efforts to engineer Ad vectors to deliver HAs for the induction of anti-stem Abs remain in their infancy and have not yet been translated to the clinic. However, there is no doubt that the aforementioned approaches will also be exploited by researchers working in the viral vector vaccine field. Prime-boost regimes using Ad vectors expressing different HAs, multivalent Ad vectors expressing multiple HA subtypes as well as chimeric HA constructs are all feasible and may lead to the induction of anti-stem Abs following vaccination. Pre-clinical studies in this area would serve as a proof-of-concept that rational vaccine design may permit the induction of heterosubtypic Abs in humans. However, it remains to be seen if sufficient titres of neutralising Abs can be achieved by these strategies and if these Abs will be broad and long lasting enough to achieve heterosubtypic immunity.[111] Additionally, recent findings by Khurana and colleagues investigating vaccine-associated enhanced respiratory disease (VAERD) indicate that such initiatives should proceed with caution. Using a swine model, animals were vaccinated with an H1N2 strain adjuvanted with an oil-in-water emulsion, challenged with an antigenically mismatched pandemic H1N1 and subsequently developed enhanced pneumonia.[112] The pathological changes in the lungs of these animals were 3-fold higher than those in unvaccinated animals challenged with the same H1N1 virus. In these studies, cross-reactive anti-stem Abs detected in the sera were found to promote virus membrane fusion activity.[112] Given that these experiments have outlined a role for vaccine-induced anti-stem Abs in mediating VAERD, it is essential to gain a clearer understanding of the

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mechanisms of cross-reactive Abs before moving forward with strategies aiming to evoke such responses. However, it is worth considering that the animals used in the aforementioned study were 90% homology.[143] Furthermore, NP and M1 have been shown to be prominent targets of CD4+ and CD8+ T-cells,[144] thus making them an attractive target for stimulating broadly cross reactive T-cell responses. A number of studies have assessed the efficacy of Ad5based vaccines expressing NP. Following i.m. immunisation of chickens with Ad5 encoding NP from human influenza H1N1, potent effector and memory CD8+ T-cell responses to NP were generated which were capable of cross reacting with avian derived NP from H7N2 A/Turkey/Virginia/ 158512/02.[145] Importantly, a prime-boost regimen that consisted of in ovo Ad5-NP+M1 prime followed by an MVA-NP+M1 boost four weeks post-hatch was found to be immunogenic and reduced virus shedding.[146] ChAd vectors expressing NP also have been evaluated pre-

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clinically and have demonstrated promise. A single shot of chimpanzee vector AdC7 expressing NP elicited potent anti-NP T-cell responses and partially protected mice from challenge with H5N1 avian influenza.[147] Furthermore, a novel approach using ChAd vector AdC68 expressing NP fused to the immunomodulatory C-terminal domain of Herpes Simplex Virus glycoprotein D significantly augmented NP-specific CD8+ T cell responses in aged mice compared with the corresponding AdC68-NP lacking this domain.[148] Clinical To date, the majority of clinical trials using viral vectored vaccines for influenza have employed the poxviral vector MVA. In a phase I study with healthy adult volunteers, MVA-NP+M1 was found to be both safe and immunogenic, eliciting potent CD8+ T-cell responses with a CD27(+)CD45RO(+)CD57(–)CCR7(–) phenotype to the vaccine Ag.[141] Importantly, this candidate vaccine was also found to be safe and immunogenic in individuals over 50 years[149] and when co-administered with the seasonal influenza vaccine TIV, allowed for the simultaneous induction of both T-cell responses to NP and M1 and increased haemagglutination-inhibition (HI) titres to HA.[150] Furthermore, immunogenicity was found to be similar in younger and older volunteers, demonstrating the potential for viral vectored vaccines to at least partially overcome immunosenescence in groups considered highrisk for severe influenza infection.[149] The efficacy of MVANP+M1 was subsequently tested in a human influenza challenge study using influenza A/Wisconsin/67/2005.[142] In this study, fewer vaccinees than controls went on to develop laboratory confirmed influenza, and those that did experienced less severe illness and shed virus for significantly fewer days than control subjects who also developed confirmed infections. More recently, our group has recently reported the firstin-human clinical assessment of a novel chimpanzee Ad vector ChAdOx1 which is based on species E virus isolate Y25.[34] This non-replicating vector has been engineered to express influenza NP and M1 as a fusion protein.[21] The vaccine was well tolerated up to doses of 2.5 × 1010 vp and demonstrated potent Ag-specific T-cell responses to the vaccine insert, as measured by IFN-γ ELISPOT.[21] Due to the small number of volunteers in the aforementioned study, the phenotype or functional capacity of the T-cell response was not characterised. However, it will be important to assess this in future studies in order to help identify novel correlates of protection which could act as a reliable predictor of vaccine efficacy. A current clinical trial (Clinical Trials.gov Identifier; NCT01818362) is now investigating the potential for using ChAdOx1-NP+M1 in a prime-boost

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vaccination regimen with MVA-NP+M1.[21] This study will be a head-to-head comparison of the immunogenicity of a heterologous prime vaccination with either ChAdOx1NP+M1 or MVA-NP+M1 followed by a boost vaccination at 8 weeks or 52 weeks with the alternate vector. The larger number of volunteers (n = 48) recruited in this study will allow us to comparatively assess the phenotype of the immune responses following ChAd/MVA prime as well post-boost. Similar to a previous clinical study where we demonstrated the safety and immunogenicity of MVANP+M1 in adults aged over 50 years,[149] we are also currently testing ChAdOx1-NPM1 alone or in a ChAdOx1NP+M1 prime MVA-NP+M1 boost in this age group.

mice have indicated that i.n. delivery of Ad vectors may induce superior immune responses and better protection than i.m. vaccination,[127] these differences are often not as pronounced in ferrets.[152] It will be important to establish whether or not different routes of administration in humans result in comparable immunogenicity and are costeffective. Furthermore, a better understanding of the basic mechanisms underlying the inherent immunogenicity of Ad vectors, and indeed rare species or simian Ad vectors, following different routes of delivery is warranted. This will greatly aid in the engineering of optimised Ad vectors for vaccine trials in humans.

Importance of route of delivery

Understanding immune responses to adenoviral vectored vaccines

In addition to the immunogenicity of the vaccine vector, inserted Ag and choice of vaccination regimen (primeboost, co-administration), the route of delivery can greatly influence the success of vaccination strategies. Influenza vaccines delivered orally (e.g. Ad4-H5), intramuscularly (e.g. TIV), intranasally (e.g. LAIV) and intradermally (i.d.) delivered (e.g. TIV) have been shown to be immunogenic,[151] albeit with varying degrees of efficacy. However, extensive clinical studies to investigate the optimal route of administration for Ad-based vaccines have not yet been carried out. It is thought that the induction of immune responses at mucosal sites, particularly at the site of pathogen entry, can lead to protective immune responses.[152] In contrast to TIV which is inactivated and delivered i.m., this is one advantage of the cold-adapted vaccine LAIV (FluMist) which is administered as a nasal spray and is capable of replication in the upper respiratory tract.[153] Many Ad vectors have a natural tropism for the respiratory tract and therefore possess the potential for i.n. delivery or via aerosolised immunisation. As a result, several pre-clinical studies have comparatively assessed the efficacy of Ad-based vaccines following different routes of administration for influenza and other pathogens with respiratory tropism. A comparison of a prime-boost regimen in mice using Ad5-NP+M1/MVA-NP+M1 showed that i.n. boosting following i.m. prime led to elevations in CD8+ T-cell IFN-γ responses in the bronchoalveolar lavage. Mice vaccinated via i.m./i.m. or i.m./i.n. were partially protected against heterologous challenge whereas mice vaccinated i.d./i.d. were not protected.[154] Therefore, these findings suggested that there was no greater level of protection when vaccinating i.n. vs i.m. A separate study showed that i.n. immunisation with an Ad5-based vector expressing NP induced potent CD8+ T-cell responses, strong mucosal IgA and protected mice against both homologous and heterologous influenza challenge.[155] Although pre-clinical studies in

The factors which contribute to the potent immunogenicity of Ad vectors in pre-clinical animal models and the differences between Ad5 and various rare species Ads remain unclear. Therefore it follows that their mechanism of action in humans is as yet undefined. Intramuscular immunisation of mice with adenoviral vectors leads to efficient transduction of APCs and often sustained, localised Ag expression or viral genome persistence in muscle tissue.[156] These conditions provide the ideal scenario for direct and cross-presentation of Ag to CD8+ T-cells (Figure 3). In addition, the migration of professional APCs to draining lymph nodes allows processing and presentation of vaccine Ag to T-cells. The direct uptake of virus by various cell types in the lymph node such as APCs and parenchymal cells, as well as cells at the injection site are believed to contribute to the ensuing immune response to adenoviral vaccines.[157] However, depending on the viral dose used, vector leakage and systemic dissemination to other organs could impact on the kinetics of Ag presentation independently of interactions taking place at the injection site and within local lymphatics. In a review by Holst and colleagues, the authors propose various mechanisms, highlighting the relative contributions that multiple cell types may have in Ag-presentation and in driving Ag-specific T-cell responses following vaccination with Ad vectors.[158] These potential virus:host cell interactions are summarised schematically in Figure 3. Although a great deal is known about Ad virus:cell interactions following intravenous delivery of Ad5-based vectors,[16,159–161] little is known about the spatial and temporal dynamics of virus:cell interactions at the injection site or within draining lymphatics following vaccination[156,162] and almost nothing is known for rare species Ads or ChAd vectors. However, in the future, advances in intravital imaging methods such as 2-photon microscopy, along with the combined use of genetically modified mouse strains, molecular virology and basic immunological techniques

392

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(a)

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DIRECT PRESENTATION

(b)

AFFERENT (INCOMING) LYMPHATIC VESSEL

CROSS DRESSING

CD8+ T-cell

CD8+ T-cell DRAINING LYMPH NODE

(c)

CROSS PRESENTATION

CD8+ T-cell

Ag presentation to CD8+ T-cells

Ag presentation to CD8+ T-cells

Ag presentation to CD8+ T-cells

APC transduction & endogenous Ag processing

Muscle cell transduction & uptake of antigen by APCs

Muscle cell transduction & uptake of peptide: MHC complexes by APCs

APC

MUSCLE

(d)

LOCAL PRESENTATION

(e)

LOCAL PRESENTATION WITHOUT APCs

Ag presentation to local CD8+ Tcells

Ag presentation to CD8+ T-cells Muscle cell transduction, cell death & uptake of peptide: MHC complexes or antigen sampling by APCs

MUSCLE

Muscle cell transduction and presentation of peptide: MHC complexes on cell surface

Adenoviral vector

CD8+ T-cells

Tissue resident cell

Adenovirus receptor

APC

Antigen

MHC I CD8

Figure 3 Mechanisms of antigen presentation following vaccination with adenoviral vectors. (a) Direct presentation. APCs are transduced by an Ad vector following i.m. vaccination, migrate to the draining lymph node and present Ag to CD8+ T cells. (b) Cross-presentation. Tissue resident cells (e.g. muscle) are transduced and present Ag to APCs which process it, migrate to the draining lymph node and present it to the CD8+ T cells via MHC I. (c) Cross-dressing. Tissue resident cells process and present Ag. MHC/peptide complexes are released and taken up by APCs, which migrate to the draining lymph node and present the Ag to the CD8+ T cells on MHC molecules derived from originally infected tissue resident cells. (d) Local presentation by APCs. Tissue resident cells are transduced, but Ag remains at the injection site. Resident and inflammatory APCs present Ag to local CD8+ T cells, either from processing of cell debris or from presentation of MHC molecules already loaded with antigen in the parenchymal cells. (e) Local presentation without APC’s. Tissue resident cells are transduced, process Ag and present it to local CD8+ T cells via MHC class I molecules. Schematic adapted from Bassett et al. and Holst et al.[157,158]

will allow for detailed characterisation of the mechanisms responsible for the immunogenicity of Ad vectors. This knowledge could subsequently be applied to the design of optimised platform vectors for Ad-based influenza vaccines.

Final Summary It is clear that optimal influenza vaccines should simultaneously induce both T-cell and B-cell NAb-mediated immunity, and evoke long lasting immune responses that

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are broadly cross-protective. With the exception of LAIV in young children with limited prior exposure to influenza, seasonal influenza vaccines largely fail to induce cellular immunity. However, LAIV is not efficacious in all at-risk target populations. Mismatches between influenza strains contained in vaccines and those circulating in humans, in addition to the protracted production of inactivated vaccines grown in eggs, highlight the importance of investigating alternative options for influenza vaccine production. Viral vectors represent a promising option, particularly for stimulating cellular immunity to influenza and their potential for combination with the currently licensed vaccines has already been demonstrated,[150] although this has not yet been formally investigated for Ad vectors. Another advantage is the relative speed of scale up in comparison to traditional influenza vaccines which may allow these vectors to be deployed at early stages in pandemic scenarios until a strain matched TIV vaccine is manufactured.[22] The evolving interest in developing improved influenza vaccines in recent years has brought renewed excitement to the field, though this is not without its challenges. Apart from the HI, the field lacks standardised and widely accepted immune correlates of protection against influenza infection.[163] There remains a large degree of variation in assay techniques, interpretation and identification of endpoints that correlate with protection from lab to lab, par-

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Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.

Acknowledgement S.C.G is a named inventor on a patent application describing the ChAdOx1 vector (GB Patent Application No. 1108879.6) and on patents for other vaccination approaches for influenza.

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Adenoviral vectors as novel vaccines for influenza.

Influenza is a viral respiratory disease causing seasonal epidemics, with significant annual illness and mortality. Emerging viruses can pose a major ...
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