Enhancing Vaccine Responses in the Elderly: Targeting Inflammation and Immunosenescence

Targeting Inflammation and Immunosenescence to Improve Vaccine Responses in the Elderly Branca Pereira 12 XiaoNing Xu 2 and Arne N Akbar 3 1 HIVGUM Directorate Chelsea and Westminster Hospital NHS Foundation Trust London United Kingdom 2 Faculty of Medicine Imperial College London London United Kingdom 3 Division of Medicine University College London London United Kingdom One of the most appreciated consequences of immunosenescence is an impaired response to vaccines with advanced age While most studies report impaired antibody responses in older adults as a correlate of vaccine efficacy it is now widely appreciated that this may fail to identify important changes occurring in the immune system with age that may affect vaccine efficacy The impact of immunosenescence on vaccination goes beyond the defects on antibody responses as T cellmediated responses are reshaped during aging and certainly affect vaccination Likewise agerelated changes in the innate immune system may have important consequences on antigen presentation and priming of adaptive immune responses Importantly a lowlevel chronic inflammatory status known as inflammaging has been shown to inhibit immune responses to vaccination and pharmacological strategies aiming at blocking baseline inflammation can be potentially used to boost vaccine responses Yet current strategies aiming at improving immunogenicity in the elderly have mainly focused on the use of adjuvants to promote local inflammation More research is needed to understand the role of inflammation in vaccine responses and to reconcile these seemingly paradoxical observations Alternative approaches to improve vaccine responses in the elderly include the use of higher vaccine doses or alternative routes of vaccination showing only limited benefits This review will explore novel targets and potential new strategies for enhancing vaccine responses in older adults including the use of antiinflammatory drugs and immunomodulators Keywords aging immunosenescence and inflammaging vaccine T lymphocytes antiinflammatories INTRODUCTION Human aging is associated with a general decline in physiological functions and increased susceptibility to disease A dysregulation of the immune system known as immunosenescence is characteristic of aging and has been linked with negative clinical outcomes in older adults 1 One of the most appreciated consequences of immunosenescence is an impaired response to new infections and vaccination in older people 2 Four vaccines are currently recommended for individuals over 65 years of age to protect against infections that disproportionately affect older adults including influenza herpes zoster pneumococcal disease and tetanus and diphtheria However Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 1 Edited by Tamas Fulop Universite de Sherbrooke Canada Reviewed by Janet E McElhaney Health Sciences North Research Institute Canada Nadia Maria Terrazzini University of Brighton United Kingdom Birgit Weinberger University of Innsbruck Austria Correspondence Branca Pereira brancapereirachelwestnhsuk Specialty section This article was submitted to Inflammation a section of the journal Frontiers in Immunology Received 14 July 2020 Accepted 23 September 2020 Published 14 October 2020 Citation Pereira B Xu XN and Akbar AN 2020 Targeting Inflammation and Immunosenescence to Improve Vaccine Responses in the Elderly Front Immunol 11583019 doi 103389fimmu2020583019 REVIEW published 14 October 2020 doi 103389fimmu2020583019 responses to these vaccines are often impaired in older individuals placing them at further risk of disease 3 4 This has considerable implications for vaccination against emerging infectious diseases such as COVID19 that have a disproportionately larger effect on older subjects 5 While most studies report antibody responses as a correlate of vaccine efficacy it is now widely appreciated that this may fail to identify important changes occurring in the immune system with age that may affect vaccine efficacy 6 7 The impact of immunosenescence on vaccination goes beyond the defects on T and B cell responses and changes in innate immunity and increased systemic inflammation also referred to as inflammaging may have additional consequences on vaccine efficacy 8 While the mechanisms of immune aging are not yet fully understood it is now apparent that this process is dynamic and multifaceted with a decline in many primordial functions but also gain of new functions as well as changes in the microenvironment Globally agerelated changes in the immune system are better described as a remodeling than a decline in immune functions 9 A better understanding of the full spectrum of changes characterizing immunesenescence is fundamental to the development of novel and improved vaccines for older adults HOW CAN IMMUNOSENESCENCE AND INFLAMMATION AFFECT VACCINE RESPONSES Changes affecting both innate and adaptive immune function with age may lead to impaired vaccine responses in older people Immunosenescence is primarily linked to the involution of primary lymphoid organs bone marrow and thymus resulting in depletion of the peripheral pool of naive B and T cells 10 To maintain peripheral cell numbers there is a clonal expansion of antigenexperienced cells resulting in extreme differentiation and altered functionality 11 Consequently the immune space becomes filled with antigenspecific memory cells leading to a contraction of the immune repertoire and impaired responses to neoantigens 12 In parallel with this the effects of aging on hematopoiesis result in a lineage skewing towards an increase in myeloid versus lymphoid precursor 13 Although the numbers of most circulating innate immune cells may not be significantly reduced with age alterations in their functionality have a particular impact on antigen presentation due to decreased antigen uptake reduced phagocyte functions and altered cytokine production 13 14 In addition to cellintrinsic changes alterations in the microenvironment including a lowgrade chronic inflammatory status and architectural changes occurring in lymph nodes may play previously underappreciated roles in shaping vaccine responses with age 1 15 Excessive baseline inflammation has been recently associated with poor responses to vaccination 16 however more research is needed to reconcile this evidence with the current paradigm that adjuvants enhance immune responses to vaccines by promoting local inflammation It is plausible that stronger local inflammatory signals are needed to overcome background inflammation or that specific inflammatory pathways should be triggered to overcome local inhibitory responses Thus a better understanding of the role of inflammation in vaccination and of the mechanisms of action of adjuvants is needed to be able to fine tune immune responses and selectively stimulate pathways that lead to longlasting immune protection In this review we will describe the most recent data on the effects of aging on immune responses to vaccination and discuss in light of the current knowledge how can immunesenescence and inflammaging be targeted to improve vaccine responses in older adults AgeRelated Changes in Adaptive Immunity Changes in the T Cell Compartment The effects of aging are particularly evident in the T cell compartment and reduced vaccine responses in older people are at least in part due to defective T cell memory responses with age 17 Different mechanisms may be contributing to reduced T cell responsiveness with age 18 but the loss of proliferative capacity 19 and decreased TCR function 2022 and TCR diversity 23 are certainly determining factors Prior antigen exposure in particular latent viral infections such as cytomegalovirus CMV and EpsteinBarr Virus EBV have a significant impact on immunosenescence by shaping the immune repertoire with large proportions of terminally differentiated cells with reduced proliferative capacity and features of replicative senescence 2426 Despite this data on the impact of CMV infection on vaccine responses are controversial with studies showing an association between CMVseropositivity and impaired antibody responses to vaccination in older adults 3 27 while others have found enhanced antibody responses to influenza vaccination in CMVseropositive compared to CMVnegative individuals 28 29 Nevertheless it has been shown that CMV seropositivity is a better predictor of a decline in T cell responses to influenza challenge rather than antibody responses in vaccinated older adults 30 31 When using functional assays of CD8 T cell cytolytic activity upon ex vivo influenza challenge CMV seropositivity was associated with impaired cytolitic responses to influenza measured by granzyme B levels in viruschallenged T cells 30 31 Mechanistically we have described that highly differentiated T cells with features of senescence exhibit decreased TCR responsiveness as a results of loss of key components of the TCR signalossome 20 22 Interestingly these cells concomitantly express NK lineage receptors and acquire TCRindependent functionality 32 Thus nonproliferative senescentlike T cells in particular CD8 T cells are reprogrammed to acquire broad innatelike killing activity regulated by a group of stress sensing molecules known as sestrins 32 Studies in human centenarians have found an expansion of these NKexpressing T cells in old individuals compared to young 33 while others have shown that the expression of NK cell markers on CD8 T cells is particularly evident in individuals with high levels of CD57 indicative of an aged immune system 34 The biological significance of the acquisition of innatelike receptors and functions by T cells is unclear but we believe that this may serve as a beneficial adaptation to ensure broad and rapid effector function with age independently Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 2 of antigenspecificity and this may represent a relatively unexplored opportunity to enhance vaccineelicited immunity 35 36 Despite the loss of proliferative potential aged T cells are metabolically active and exhibit increased production of pro inflammatory cytokines and thus may have detrimental effects on the tissue microenvironment contributing to ageassociated low grade inflammation 3739 Changes in the B Cell Compartment As with T cells there is an agedependent accumulation of late stage memory B cells while the circulating pool of naïve B cells progressively decreases skewing the B cell repertoire and limiting the number of clones available to respond to novel antigens 40 B cells experience significant functional changes with age with reduced proliferative potential and impaired capacity for differentiation into plasma cells after antigen challenge 41 Senescent B cells have also been shown to spontaneously secrete proinflammatory cytokines contributing to agerelated chronic inflammation and further immune dysregulation 42 Overall these changes have been associated with poor health outcomes 43 and diminished responses to vaccination in old age 44 Several studies have shown that older adults have lower antibody responses following vaccination compared to younger adults and have been reviewed elsewhere 45 The quality of these antibody responses is also compromised with reduced diversity in the antibody repertoire 46 47 This is particularly well described for influenza vaccination 48 49 although responses to pneumococcal vaccines are equally compromised 50 Intrinsic defects of B cells such as reduced somatic hypermutation and isotype switch as well as reduced numbers of plasma cells contribute to reduced antibody responses after vaccination and this correlates with decreased vaccine efficacy 41 Changes in Innate Immunity With Age Alterations in the phenotype and function of innate immune cells with age are increasingly well recognized 13 14 and particularly relevant for vaccineinduced immune responses Reduced chemotaxis alterations in signaling pathways following antigen recognition and aberrant cytokine production have been described in neutrophils 51 52 monocytesmacrophages 53 54 and dendritic cells DCs 55 56 derived from older persons further affecting their capacity to process and present antigen to T cells Tolllike receptor TLR signaling has a crucial role in vaccination by linking innate and adaptive immune responses 57 Although the surface expression of TLRs does not show a consistent change with age altered cytokine production and impaired downstream TLR signaling have been described in older adults 58 Interestingly an agedependent decrease in TLR function in human DCs has been linked with poor antibody responses to influenza immunization providing evidence for the impact of an aging innate immune system in vaccine responses 59 Moreover intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals 59 suggesting a dysregulation of cytokine production that may contribute to agerelated inflammation Changes affecting the local microenvironment at the site of injection may have a significant effect on vaccine responses Neutrophils and tissueresident macrophages contribute to a pro inflammatory environment at the site of vaccine injection that is important for recruiting other immune cells and for the priming of adaptive immune responses 60 However as it will be discussed in more detail there is a growing appreciation that excessive local inflammation may be detrimental to vaccine responses 16 The effects of age on the phenotype and function of NK cells have been described elsewhere 13 61 and may as well affect the efficacy of vaccination in older people As discussed later NK cells have a previously unrecognized role in vaccination contributing for protection during the early phases postvaccination by mechanisms that involve the generation of innate immune memory 62 Thus the effects of aging on cytotoxicity and cytokine secretion mediated by NK cells may have wider implications for immune responses to vaccination in older adults 63 Agerelated changes in innate T cells are less well described however a decreased frequency and change in phenotype of peripheral gd T cells 64 and mucosalassociated invariant T MAIT cells 65 have been reported in older adults compared to young Recently it has been described that MAIT cells in older adults have an increased baseline inflammatory profile that was associated with reduced Escherichia colispecific responses in aged MAIT cells compared with their young adult counterparts 66 Inflammaging Aging is associated with a chronic and systemic sterile inflammatory state termed inflammaging 67 This is supported by the findings of higher levels of tumor necrosis factor TNF IL6 and other proinflammatory cytokines in the serum of older individuals compared to young 68 69 A variety of stimuli may sustain inflammaging not only chronic antigen stimulation by pathogens but also activation of the inflammasome by endogenous cell debris and misplaced self molecules and microbial translocation due to increased gut permeability 70 Although the innate immune system in particular the monocytemacrophage network are thought to be at the center of inflammaging 70 71 accumulating evidence indicates that senescent cells in general including senescent T and B cells have an important contribution with their senescent associated secretory phenotype SASP 72 Regardless of the origin this lowgrade systemic inflammation is predictive of frailty and earlier mortality 73 and is an established risk factor for many agerelated diseases including heart disease agerelated macular degeneration type II diabetes osteoporosis and cancer 74 75 There is accumulating evidence that increased chronic background levels of inflammation might be detrimental for vaccine responses 7681 Nakaya et al investigated gene signatures predictive of influenza vaccine responses in young and old adults and found that prevaccination signatures associated with T and Bcell function were positively correlated with antibody responses at day 28 after vaccination while Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 3 monocyte and inflammationrelated genes were negatively correlated with antibody responses 76 Similarly studies on HBV vaccination in the elderly revealed that a more pronounced inflammatory gene expression profile at baseline predicted a poorer response to vaccination 77 78 Our group has shown that older individuals exhibit reduced cutaneous immunity to varicella zoster virus recall antigen challenge associated with increased baseline local inflammation 79 Subsequently we demonstrated that infiltrating monocytes play a crucial role in the inhibition of cutaneous immunity by a mechanism driven by increased cyclooxygenase 2 COX2 expression and production of prostaglandin E2 PGE2 ultimately leading to reduced proliferation of skin residentmemory T cells and reduced responses to antigenic stimulation 82 Overall these findings support the concept that elevated baseline inflammation may have a significant role in the agerelated hyporesponsiveness to vaccination and thus reducing background inflammation might be a promising strategy to enhance vaccine responses 83 This may be a particularly important consideration for older subjects who develop severe inflammation after SARSCov2 where reducing inflammation may boost vaccine efficacy 84 CURRENT STRATEGIES TO IMPROVE VACCINE EFFECTIVENESS Current recommendations for vaccination in older adults include vaccines against influenza herpes zoster pneumococcal disease and a booster against tetanus and diphtheria Despite being able to mitigate the severity of the disease to some degree these vaccines often fail to induce protective immunity in the elderly Several approaches are currently in place to improve vaccine effectiveness in this population discussed in detail elsewhere 4 and largely focus on the use of adjuvants higher antigen doses and alternative routes of immunization Influenza Vaccines Adjuvanted influenza vaccines are now the first choice for those over 65 years in countries such as Austria and the United Kingdom UK to overcome the low effectiveness of standard vaccines in the elderly 85 Data from the 201819 influenza season in the UK the first season after the introduction of adjuvanted vaccines for persons above 65 years demonstrated better protection from pneumoniaassociated hospitalizations and laboratoryconfirmed influenza cases with adjuvanted compared to nonadjuvanted vaccines 86 Studies have demonstrated that the addition of MF59 to influenza vaccine enhanced antibody production with increased seroconversion and seroprotection rates 87 improved antibody binding affinity and a more diverse antibody epitope repertoire 88 and induced broader serological protection against drifted strains 89 providing support for the use of adjuvants in influenza vaccination of older populations Despite this a study comparing cellmediated immune responses to vaccination in adults 65 years old randomized to receive one of 4 seasonal influenza vaccinesstandard subunit MF59 adjuvanted subunit and splitvirus vaccines given intramuscularly or intradermally found no benefit of the MF59 adjuvanted formulation over nonadjuvanted formulations delivered by intramuscular and intradermal routes 90 Alternatively the use of highdose influenza vaccines in individuals over 65 years has also been shown to induce higher antibody titers and seroprotection rates compared to standard dose vaccine 91 leading to their approval for clinical use in person aged 65 and older 92 Metaanalysis of randomized controlled trials RCTs showed that highdose vaccines split virus and subunit recombinant hemagglutinin formulations were more effective than standarddose vaccines in preventing influenzalike illness influenza hospitalization and allcause mortality in adults 65 years old 93 When looking at T cell mediated immune responses highdose influenza vaccines had little impact on the development of functional T cell memory in older adults compared to standarddose vaccines 31 Another approach to improve influenza vaccine immunogenicity in older people is the use of alternative routes of vaccination Most vaccines are delivered by intramuscular or subcutaneous injection bypassing the mucosal immune compartment Intranasal and intradermal routes for influenza vaccination have been developed with the aim of enhancing immunogenicity particularly cellmediated and mucosal immunity Although studies suggest that intradermal influenza vaccination may enhance immunogenicity compared to standard intramuscular vaccines in persons over 65 years of age 94 pooled analysis of RCT found no significant differences in seroprotection and seroconversion rates in older adults with intradermal vaccine compared to intramuscular 95 and intradermal influenza vaccines are no longer recommended T cell responses were also not different between intramuscular versus intradermal injection in a randomized study comparing influenza vaccines in adults 65 years old 90 It should be noted that when comparing different types of influenza vaccines the formulation may differ Current licensed inactivated influenza vaccines are manufactured using either splitvirus or subunit formulations They are all designed and licensed based on hemagglutinin antibody responses but while they may induce similar antibody responses the differences become more evident when measuring cellular immune responses to vaccination 96 Splitvirus vaccine lack some of the purification steps of subunit vaccines and therefore may contain a larger amount of internal viral proteins such as matrix protein M1 and nucleoprotein 97 that are important to elicit T cell responses 98 Co et al showed that the presence of influenza internal proteins M1 and NP contained in standard dose splitvirus vaccines but not in subunit vaccines were necessary for stimulating CD8 T cell responses measured by IFNgamma production and by cytotoxicity assays in vitro 96 Importantly a study evaluating the clinical effectiveness of split virion versus subunit trivalent influenza vaccines in older adults using a casepositive control testnegative study design demonstrated a vaccine effectiveness of 778 95 confidence interval CI 585903 for the splitvirion compared with 442 95 CI 118 to 709 for the subunit vaccine 99 Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 4 Unfortunately there are not many studies performing headto head comparisons between the different available influenza vaccine options for older adults comparing both humoral and T cell responses A randomized clinical trial comparing immunogenicity of currently available vaccine options for older adultsstandarddose quadrivalent vaccine MF59 adjuvanted trivalent vaccine highdose trivalent vaccine or recombinanthemagglutinin quadrivalent vaccine is currently under way and it will be important for identifying improved vaccination strategies for influenza in older adults 100 Herpes Zoster Vaccines Herpes zoster results from the reactivation of latent varicella zoster virus VZV infection Although the reactivation of VZV can occur throughout life the risk increases substantially with age and in conditions associated with a decline in T cell immunity A liveattenuated VZV vaccine Zostavax is approved for older adults to boost VZVspecific cellmediated immunity CMI Evidence that the vaccine is partially effective in older patients comes from the Shingles Prevention Study that demonstrated a reduction in the incidence of herpes zoster and postherpetic neuralgia by 51 and 67 respectively 101 However the efficacy of the vaccine was agedependent dropping from 64 in the age group 6069 years to 41 in the age group 7079 years In addition to this data on longterm followup indicates that vaccineinduced immune responses decline over time Revaccination can have a booster effect although current evidence is not sufficient to support revaccination of older people 102 A new adjuvanted recombinant zoster vaccine Shingrix has been recently approved to prevent herpes zoster in older adults It consists of recombinant VZV glycoprotein E and a liposome based AS01B adjuvant system This system consists of two adjuvants 3Odesacyl40monophosphoryl lipid A MPL and QS21 formulated in a liposomal delivery system AS01B 103 MPL is a TLR agonist activating the innate immune system at the site of the injection and enhancing antigenpresentation 104 Whist the molecular mechanisms underlying the adjuvant effect of QS21 are not yet fully understood it has been demonstrated that it induces strong and persistent Th2 humoral and Th1 cellmediated immune responses 105 It is thought that the use of liposomal formulations facilitates the escape of the antigen into the cytosol enhancing antigen presentation through MHCI pathway leading to cross presentation to CD8 T cells and an early IFNgamma response that promotes vaccine immunogenicity 106 Interestingly the AS01B adjuvant system seems to require the synergistic action of the three components together for optimal adjuvant effect 107 The efficacy of the adjuvanted recombinant vaccine has been demonstrated in two randomized placebocontrolled Phase III clinical trials where the administration of two doses resulted in 972 protection against HZ in persons over 50 years of age 108 and 898 in adults over 70 years of age 109 While longterm followup is still ongoing robust antibody and CD4 T cell responses were found for at least 3 years after the vaccination although CD8 T cell correlates of protection were not identified 110 A metaanalysis comparing the two vaccines in adults over 50 years of age confirmed the superiority of the adjuvant recombinant subunit vaccine compared to the live attenuated vaccine for the prevention of herpes zoster infection despite a greater risk of adverse events at injection sites 111 An additional advantage of the recombinant zoster vaccine over the liveattenuated vaccine is its suitability to use in immunocompromised patients including HIVinfected patients 112 and in transplant recipients 113 Pneumococcal Vaccines The currently available 23valent polysaccharide vaccine PPV 23 has been used for many years in older adults and is still the first choice in many countries However this vaccine does not generate adequate immunological memory as purified polysaccharides do not induce persistent antigenspecific memory B cells 114 Furthermore responses to PPV23 were impaired in older adults compared to young individuals 115 A 13valent conjugate vaccine PCV13 has been introduced and is now the first line choice for older adults in several countries as it has improved immunogenicity compared to the polysaccharide vaccine 116 Conjugation of polysaccharide antigens enables the uptake and antigen presentation in the context of MHCII to CD4 T helper cells resulting in the generation of memory B cells specific for the polysaccharides 114 A large randomized placebocontrolled trial demonstrated that the conjugate vaccine is effective in persons over 65 years of age reducing the number of hospitalizations due to communityacquired pneumonia caused by vaccinetype strains by 456 and the number of cases of invasive pneumococcal disease by 75 117 It is still debatable which pneumonoccal vaccine is more suitable to the elderly and this is largely reflected in the heterogeneity of the recommendations for pneumococcal vaccines from country to country PCV13 induces stronger and longlasting memory responses compared to PPV23 however PPV 23 covers more serotypes This is particularly relevant in the context of the serotype replacement that is seen as a consequence of routine childhood vaccination with PCV13 leading to the reduction in the incidence of pneumococcal disease caused by vaccine serotypes while other serotypes become more prevalent 118 Tetanus and Diphtheria Vaccines Antibody responses to tetanus and diphtheria vaccines are also suboptimal in old age In addition to reduced antibody concentrations in the elderly protection is shortlasting and a second booster after 5 years did not lead to additional long lasting immunity in older people 119 Overall immune responses to currently recommended vaccines are suboptimal in older people Despite the important successes achieved with strategies currently in place to improve vaccine responses in the elderly most available vaccines still fail to elicit longlasting immune responses and insufficiently trigger cellmediated and mucosal immunity Therefore novel approaches should be explored to enhance immunogenicity and efficacy of vaccines in this population Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 5 NOVEL STRATEGIES FOR ENHANCING VACCINE RESPONSES Implementing New Correlates of Vaccine Efficacy Although real estimates of vaccine efficacy can only be established in randomized placebocontrolled trials against laboratoryconfirmed cases the standard of practice is to use surrogate markers of vaccineinduced protection against disease 120122 Hemaglutinin inhibition HI assays detecting antibody responses to vaccine strains are the most widely used correlates of protection induced by vaccines Nevertheless studies in older adults have found a poor correlation between antibody responses to influenza vaccine and protection against laboratoryconfirmed cases of influenza 7 123 The limitations associated with overreliance on HI assays to ascertain vaccine responses have been reviewed elsewhere 124 however there is growing appreciation that the use of HI antibody titers as a sole measure of vaccine efficacy may fail to detect important changes in cellular immunity that occur with age 6 7 It has been shown that older adults exhibit lower T cell responses to influenza compared to young controls 125 and that preexisting CD4 T cells against conserved internal influenza proteins are important for limiting virus replication and disease severity 126 Additionally Sridhar et al showed that in the absence of crossreactive neutralizing antibodies CD8 T cells specific to conserved viral epitopes correlated with crossprotection against symptomatic influenza 127 However T cell correlates of protection based on the frequency of IFNgammaproducing CD4T 126 and CD8 T cells 128 have only been established in young adults and have not yet been validated in older adults On the other hand other studies have demonstrated that ex vivo T cell parameters eg interferon IFNgamma and IL10 ratio granzyme B levels measuring cellular immune responses to influenza challenge performed better than antibody titers as correlates of vaccine efficacy in older adults 7 129 Correlates of protection based on functional assays of CD8 T cell cytolytic activity are important to better predict vaccine efficacy and should ideally be incorporated into the evaluation of protective immunity in the elderly 7 Nevertheless there is still limited data on functional T cell responses to vaccines particularly in older adults such as CD8 T cellmediated ex vivo virus inhibition assays as described in HIV vaccine development 130 Although recent data indicates that innate immune cells may be important contributors for developing effective cytolytic mediated immunity to infection this requires a functional readout of the response to vaccination Novel correlates of vaccine effectiveness are needed and an evolving area of interest is the contribution of neutralizing and crossreactive antibodies induced by vaccination to enhanced protection against disease 131 The use of functional assays such as antibodydependent cell mediated cytotoxicity ADCC and serum neutralization assays to detect crossreactive antibodies that may not necessarily be detected in HI assays has been suggested as alternative correlates of protection however they are difficult to standardize across laboratories Likewise the incorporation of methods to assess antibody binding affinity specificity and epitope diversity of polyclonal antibodies would be important for a more comprehensive assessment of the quality of immunizationinduced antibody responses and for developing more effective vaccines 132 Sequencing B and T cell receptors to analyze repertoire clonality and diversity could represent a valuable tool to predict vaccine efficacy by identifying vaccineinduced clones that will respond better and for longer to a given immunogen 133 134 Although difficult to implement as routine measure of vaccine efficacy assessment of repertoire clonality and diversity would be important to direct the development of nextgeneration vaccines that provide longlasting immunity against infection Searching for Novel Adjuvants to Stimulate the Immune System Adjuvants act as enhancers of vaccineinduced immunogenicity at multiple dimensions inducing local proinflammatory cytokine production recruiting and activating innate immune cells stimulating antigen presentation and ultimately boosting humoral and cellular immune responses 135 For many years aluminium salts have been the only adjuvant in use in human vaccines In recent years highthroughput screening approaches have led to the discovery of many novel adjuvants However to date only two adjuvants MF59 and AS01B are currently licensed for persons older than 65 years while the majority failed to translate to effective therapeutics mostly due to their sideeffects 136 As our understanding of the mechanisms that boost immunogenicity rapidly increases new adjuvants are being developed with focus on generating multifaceted immune responses Recent research efforts have also focused on developing new ways to deliver old adjuvants in order to improve their function while reducing sideeffects 137 The requirements for effective novel adjuvants are to boost innate and adaptive immune responses to vaccines and induce longterm protective memory as well as to counterbalance the lowgrade inflammatory state that might hamper vaccine responses 136 138 The incorporation of pathogens associated molecular patterns PAMPs in vaccine formulations that act as ligands for pattern recognition receptors PRRs on innate immune cells is a strategy already in place for enhancing vaccinespecific responses PRR activation leads to inflammatory cytokine and type I IFNs production facilitating antigen crosspresentation and activation of cytotoxic T cells 135 Due to their ability to induce strong cellmediated responses TLR ligands are attractive sources for developing new adjuvants 57 139 140 Some TLR agonists are already in clinical stage as vaccine adjuvants Monophosphoryl lipid A is among the first of a new generation of TLR agonists to be already approved and in clinical use worldwide as an adjuvant in several vaccine formulations including a vaccine against hepatitis B virus FENDrix and human papilloma virus Cervarix 141 Another TLR4 agonist glucopyranosyl lipid adjuvant GLA formulated in a squaleneinwater emulsion SE has been Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 6 shown in a firstinhuman trial to improve magnitude and quality of humoral and Thelper 1 type cellular responses elicited by the ID93 tuberculosis vaccine 142 The stimulatory effect of GLASE is well preserved in older adults 143 and in vitro studies in the context of vaccination with a splitvirus influenza vaccine in older adults confirmed the activation of DCs to induce a Th1 response increasing the interferong to IL10 ratio and the cytolytic granzyme B response to influenza virus challenge both of which have been shown to correlate with protection against influenza in older adults 144 However the response to TLR agonists was impaired in aged compared to young mice 145 and the age related defects in TLR function and cytokine production might limit the utility of TLR ligands in older adults 58 59 Although more research is needed the use of combinations of TLR agonists has been proven effective in experimental models and might be a possible strategy for more effective vaccination in the older population 140 Triggering Innate Immune Memory Effective vaccination strategies should aim at inducing protective adaptive immunity but also incorporate novel means of triggering innate immune memory to induce lifelong protection against infection 146 Recent findings suggest that NK cells may play important roles in vaccination through the modulation of adaptive immune responses and generation of innate immune memory 62 63 NK cells can be activated following immunization through cytokines produced in response to adjuvants 147 or by direct stimulation of receptors including TLRs 148 Thus vaccine adjuvants can be optimized to promote activation and recruitment of NK cells to target tissues where they can positively or negatively regulate antigen presenting cells and downstream T cell responses 149 Additionally NK cells may contribute to enhanced vaccine responses through the generation of longlived memory NK cells capable of mediating rapid effector functions following reexposure to antigen reminiscent of T cell memory responses 62 150 151 The concept of innate immune memory is relatively new and a better understanding of how memory NK cells are generated and can mediate specific recognition of antigen is important to define strategies promoting the development of these cells during vaccination Targeting T Cells to Induce Broad Protective Immunity An ongoing challenge in vaccination is the development of vaccines that are able to induce broad protective immunity This is particularly relevant for influenza where next generation vaccines inducing T cell immunity may potentially overcome the limitations of current available vaccines that rely on antibodies to provide narrow subtypespecific protection and are prone to antigenic mismatch with circulating strains The concept of universal vaccines is based on the possibility of inducing heterosubtypic immunity whereby T cells can target diverse influenza strains by recognizing highly conserved peptides 127 152 Studies conducted during the 2009 H1N1 pandemic provided key insights into the role of crossreactive T cells in mediating heterosubtypic protection in humans We conducted influenza studies to map T cell responses before and during infection in adults with no detectable antibodies to pandemic H1N1 and found that preexisting CD4 T cells targeting highly conserved protein epitopes exhibited cytotoxic activity across strains and were important to limit viral replication and disease severity 126 By mapping the type of epitopes that were able to generate heterotypic responses across strains the results of this work and others 153 can aid the development of broadly protective T cell vaccines 154 This may be particularly important in the context of pandemics where there is no preexisting immunity Interestingly a recent study done in COVID19 convalescent patients detected circulating SARSCoV2reactive CD4 T cells in 4060 of unexposed individuals supporting the importance of crossreactive heterotypic T cell responses for clinical protection and limiting disease severity 155 Exploring New Pathways for the Development of Broadly Protective Vaccines Innate Tcells MAIT cells gd cells and NKT cells are attractive vaccine targets as they can link both innate and adaptive immunity by mediating TCRdependent and independent innatelike functions 156 A common feature of innate T cells is their capacity to respond rapidly to danger signals and proinflammatory cytokines such as IL12 15 18 and Type I IFNs in a TCRindependent mechanism and participate in the early stages of defense against certain infections MAIT cells are abundant in human lungs where they have been shown to contribute to protection against influenza infections 157 and mucosal tissues such as the intestinal mucosa making them attractive targets for mucosal vaccine design Recent studies have shown that MAIT cell frequencies can be rapidly boosted through mucosal administration of synthetic MAIT cell ligands with TLR agonists 157 158 and this could be particularly beneficial for the elderly who have impaired MAIT cell immunity Bystander activation by cytokines is a feature shared by a subset of conventional T cells particularly CD8 T cells We have recently shown that as T cells differentiate toward senescence they become less responsive to TCR conventional signaling while acquiring innatelike functions 32 The reprogramming of highly differentiated CD8 T cells from TCR to NKR functional activity provides them broad protective functions that can be beneficial in the context of aging 35 and might be also relevant for vaccination Another area of potential interest is the use of monoclonal antibodies that selectively block inhibitory receptors to boost T cell function In light of the unprecedented results obtained with the use of checkpoint inhibitors eg PD1 CTLA4 in cancer new avenues of research are open for the use of these immunomodulators in other settings including vaccination 159 160 Interestingly improved vaccine responsiveness has been linked to reduced frequencies of CD4 and CD8 T Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 7 lymphocytes expressing PD1 For instance immunological responses to the liveattenuated zoster vaccine in individuals over 50 years of age were correlated with prevaccination levels of regulatory T cells and PD1expressing T cells regardless of the age of the vaccine 161 Ex vivo blocking experiments corroborated a role of PD1 and CTLA4 as modulators of decreased VZV responses 161 A study on the responses to a trivalent inactivated influenza vaccine in lung cancer patients receiving PD1 blockade therapy compared to agematched healthy controls showed comparable serological protection but an increased rates of immunerelated adverse events IRAEs 162 although a subsequent study found no increase in incidence or severity of IRAEs in patients on immune checkpoint inhibitors who received the flu vaccine 163 While more research is needed on the safety and efficacy of such combinations of immune checkpoint inhibitors with vaccines this combinatorial approach has been tested and proved efficient in preclinical and clinical trials using therapeutic cancer vaccines with antiPD1 164 165 or anti CTL4 166 monoclonal antibodies As the expression of inhibitory receptors on T cells has been shown to increase with age and differentiation 37 167 the selective blockade of inhibitory receptors known to regulate T cell activity could be explored as means of boosting cellular responses in the elderly prior to or during vaccination Blocking Baseline Inflammation to Boost Vaccine Responses Responses to vaccination vary widely across individuals and are generally poorer in particular groups including not only the elderly but also individuals with autoimmune diseases HIV infection 168 and cancer 169 A common feature among these groups is the presence of a chronic inflammatory background that has been associated with adverse health outcomes 170 Furthermore there is a growing appreciation that preexisting inflammation may be a determinant of vaccine responsiveness and thus modulating baseline inflammation prior to vaccination has become an attractive area of research to boost vaccine responses 16 83 171 Using highthroughput technology researchers have identified baseline transcriptional signatures that predict protective immune responses to vaccines 76 7881 Most of the signatures identified so far are indicative of broad immune activation and excessive inflammation For example a study comparing responses to the yellow fever vaccine in an African cohort compared with a Swiss cohort found that an activated immune profile of NK cells monocytes and differentiated T and B cell subsets was associated with reduced responses to vaccination 81 Our group has previously shown that older individuals have decrease ability to mount recall responses to VZV antigen challenge in the skin 172 and this was subsequently associated with increased baseline local inflammation 79 Ingenuity pathway analysis indicated that this inflammation was driven by the activation of p38 MAP kinase pathway in the skin of old individuals compared with young Shortterm systemic treatment with an oral p38 MAPK inhibitor Losmapimod significantly increased the cutaneous VZV response in older subjects 79 supporting the concept that antiinflammatory interventions may be promising strategies for boosting immunity during aging Furthermore oral administration of an mTOR inhibitor Rapamycin prior to influenza vaccination of older adults resulted in increased antibody titers against all three strains of a trivalent influenza vaccine by more than 20 in individuals aged above 65 years 173 Other immunomodulator agents such as metformin imiquimod 174 and antiinflammatory drugs inhibiting COX2 expression 175 eg aspirin and NSAIDS that are currently approved for clinical use in other settings may represent attractive approaches to promote more effective vaccine responses by transiently alleviating chronic inflammation prior to vaccination Finally it is likely that targeting other sources of inflammaging by changing the composition of the microbiome 176 or selectively removing senescent cells using senolytic drugs 177 may represent further opportunities for enhancing vaccine immunity in the setting of chronic inflammation REFLECTIONS ON COVID19 VACCINATION STRATEGIES FOR THE ELDERLY The discussion about the impact of aging on immunity and vaccination is particularly relevant at the moment as the COVID19 pandemic placed again the spotlight on the vulnerability of older adults to emerging infectious diseases Epidemiological data reveals that individuals over 60 years of age are disproportionately affected by SARSCoV2 infection experiencing the most severe forms of disease and the highest hospitalization rates 178180 Age is a strong predictor of death among patients hospitalized with COVID19 181 182 and a review of epidemiological data from different countries revealed an exponential increase in case fatality rates with age regardless of the geographic region 183 Despite being the most affected risk group older adults are the least likely to respond to a new vaccine This represents a major challenge for vaccine development and thus it is critical to understand how immunosenescence and inflammaging impact on vaccine responses to ensure that vaccination remains effective in this age group 184 To meet this need leading vaccine developers Oxford UniversityAstraZeneca ClinicalTrailsgov number NCT04516746 NIAIDModerna Therapeutics NCT04405076 and BioNTechPfizer NCT04368728 are currently recruiting adults over 55 years of age to evaluate efficacy safety and immunogenicity of their vaccine candidates in older individuals However due to intricacies of clinical trial design with strict inclusionexclusion criteria most COVID19 vaccine studies may fail to include a sufficient number of older individuals in particular those in their 70s and 80s As of 3 of September 2020 the COVID19 vaccine development landscape includes 33 vaccine candidates in clinical trials of which 6 candidates are currently in phase III clinical trials 185 Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 8 Despite the promising preliminary reports of their phase III trials 186 187 current vaccine frontrunners have not yet published results on the vaccine safety and immunogenicity in elderly Relaxing the eligibility criteria and ensuring an adequate representation of the groups most affected by COVID19 disease such as elderly people those with comorbidities and people from black Asian and minority ethnic groups is of key importance for successful vaccination strategies for COVID19 Trials in older adults are also important to understand why immune responses to COVID19 infection and vaccination may vary from person to person A recent study performed deep immune profiling of 125 COVID19 patients and identified immune profiles associated with poor clinical outcomes 97 Severe COVID19 disease was associated with an immunotype characterized by the paucity of circulating follicular helper cells and the presence of highly activated CD4 and CD8 T cells with increased frequencies of highly differentiated CD8 T cell EMRAs and exhausted PD1 CD8 T cells providing evidence for the association between an immunosenescent phenotype and disease severity Other studies have shown that severe COVID19 disease correlated with elevated serum concentrations of inflammatory cytokines including interleukin 6 IL6 granulocyte colonystimulating factor GCSF IP10 MCP1 macrophage inflammatory protein 1a MIP1a and tumor necrosis factor TNF 188191 Among these IL6 has received particular attention 189 providing support for several clinical trials on IL6 receptor antagonists as potential treatments for severe COVID19 disease 192 Accumulating evidence suggests that the pathophysiological hallmark of COVID19 disease is severe inflammation with descriptions of a cytokine storm syndrome 193 194 induced by a dysregulated monocyte macrophage response 195 196 As previously discussed the presence of lowgrade sterile inflammation characterized by high baseline serum concentrations of proinflammatory cytokines including IL6 is a hallmark of aging 70 and is predictive of early mortality 73 Thus it can be speculated that inflammaging is one of the mechanisms underlying increased morbidity and mortality due to SARSCoV2 infection in older adults 196 As preexisting inflammation may also be detrimental to vaccine responses it has been proposed that reducing inflammation with shortterm course of mTOR or p38 MAPK inhibitors and possibly other antiinflammatory agents eg steroidal drugs such as dexamethasone may be used as a strategy for improving COVID19 vaccine responses in older people 84 CONCLUDING REMARKS AND UNSOLVED QUESTIONS Despite the important successes achieved with current vaccines most available vaccines still fail to elicit longlasting immunity in older adults Current vaccine strategies must evolve to be able to enhance cellmediated and mucosal immunity in addition to inducing longlasting antibody responses However to date most clinical trials leading to vaccine approval in older adults rely entirely on antibody responses as correlates of protection and thus novel correlates of vaccine effectiveness are needed that fully reflect the changes occurring with age in the immune system The use of system vaccinology approaches can aid researchers in identifying signatures that predict protective immune responses and this information can be used for optimization of current vaccination strategies Responses to vaccination vary widely across individuals and baseline immune profiles matter to determine the outcome of vaccination Recent data suggests that excessive baseline inflammation is deleterious and may hamper immune responses and thus novel approaches aimed at reducing inflammation may offer novel opportunities to improve vaccine responses in older individuals Yet the prevailing view is that adjuvants improve vaccine responses by promoting local inflammation Thus more research is needed to understand the role of inflammation in vaccine responses and to reconcile these seemingly paradoxical observations It could be speculated that the effects of systemic versus local inflammation are distinct and that the beneficial effects of antiinflammatory drugs on vaccine response result from the systemic reduction of the lowlevel chronic inflammation Additionally chronic immune activation may be associated with desensitization or tolerance to new antigenic stimulation resulting in poor immune responses Thus stronger adjuvants may be needed to overcome this tolerogenic state and alleviate the consequences of chronic inflammation There is a need to develop newer and more specific adjuvants able to fine tune immune responses and selectively stimulate pathways that lead to longlasting immune protection As our understating of immunosenescence and inflammaging increases new individualized approaches could point towards the development of more effective vaccines for older individuals AUTHOR CONTRIBUTIONS BP has done the literature search and writing AA and XNX contributed for the writing and revision of the manuscript All authors contributed to the article and approved the submitted version FUNDING BP is funded by the Joint Research Committee of Chelsea and Westminster Hospital NHS Foundation Trust and Imperial College London UK AA was supported by the Medical Research Council MRC Grand Challenge in Experimental Medicine MICA Grant MRM0038331 XNX was supported by the Department of Health and Social Care using UK Aid funding and is managed by the Engineering and Physical Sciences Research Council EPSRC grant number EPR013764 1 Note the views expressed in this publication are those of the authors and not necessarily those of the Department of Health and Social Care Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology wwwfrontiersinorg October 2020 Volume 11 Article 583019 9 REFERENCES 1 NikolichZugich J The twilight of immunity emerging concepts in aging of the immunesystem Nat Immunol 2018191109 doi 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inflammation in patients with COVID19 a key role for monocytes and macrophages Nat Rev Immunol 2020 20 635562 doi 101038s4157702003314 196 Pence BD Severe COVID19 and aging are monocytes the key Geroscience 2020 424105161 doi 101007s11357020002130 Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Copyright 2020 Pereira Xu and Akbar This is an openaccess article distributed under the terms of the Creative Commons Attribution License CC BY The use distribution or reproduction in other forums is permitted provided the original authors and the copyright owners are credited and that the original publication in this journal is cited in accordance with accepted academic practice No use distribution or reproduction is permitted which does not comply with these terms Pereira et al Immunosenescence Inflammation and Vaccination Frontiers in Immunology 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