key: cord-0739743-fwxsjiny
authors: Schmidt, Anna; Lapuente, Dennis
title: T Cell Immunity against Influenza: The Long Way from Animal Models Towards a Real-Life Universal Flu Vaccine
date: 2021-01-28
journal: Viruses
DOI: 10.3390/v13020199
sha: c3f138f1b9e1c42201a17e7d3d3733d99cfc21b8
doc_id: 739743
cord_uid: fwxsjiny

Current flu vaccines rely on the induction of strain-specific neutralizing antibodies, which leaves the population vulnerable to drifted seasonal or newly emerged pandemic strains. Therefore, universal flu vaccine approaches that induce broad immunity against conserved parts of influenza have top priority in research. Cross-reactive T cell responses, especially tissue-resident memory T cells in the respiratory tract, provide efficient heterologous immunity, and must therefore be a key component of universal flu vaccines. Here, we review recent findings about T cell-based flu immunity, with an emphasis on tissue-resident memory T cells in the respiratory tract of humans and different animal models. Furthermore, we provide an update on preclinical and clinical studies evaluating T cell-evoking flu vaccines, and discuss the implementation of T cell immunity in real-life vaccine policies.

Influenza viruses are a constant threat to the world community. Globally, 290,000-650,000 seasonal influenza-associated deaths are estimated with the highest mortality rate in sub-Saharan Africa and southeast Asia [1] . The classical risk groups include very young children, the elderly, and individuals with co-morbidities [2] [3] [4] . In addition to seasonal epidemics, influenza A viruses (IAVs) occasionally cause pandemic outbreaks. While the "Spanish flu" from 1918 was the most devastating of these pandemics, with an estimated 50 million deaths [5] , the most recent H1N1 pandemic in 2009 was only moderately pathogenic. Although not caused by a flu strain, the 2020 severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic has revealed the dramatic impact of an emerging respiratory pathogen on healthcare, social life, and the economy in the 21st century.

Influenza viruses belong to the Orthomyxoviridae family, and consist of the four genera A, B, C, and D, with IAV and influenza B virus (IBV) being most relevant for human disease. IBV has a limited host range and strain diversity (Yamagata and Victoria lineages), and does not cause pandemics. In contrast, the genetic instability of IAV constantly creates new virus lineages and subtypes. The error-prone viral polymerase of IAV and IBV lacks a proofreading activity, leading to a continuous accumulation of mutations, especially in the surface proteins hemagglutinin (HA) or neuraminidase (NA) [6, 7] , while the internal virus proteins remain more conserved. This phenomenon called "genetic drift" allows the genetic evolution of seasonal flu strains. "Genetic shift" occurs only in IAV, and describes the exchange of one or more gene segments among different IAV strains upon superinfection, leading to novel virus subtypes. By this mechanism, novel viruses can emerge against which weak or no herd immunity exists in the human population [8] . Thus, the ongoing drift of seasonal flu strains and the occasional emergence of IAV pandemics As early as 1965, Jerome Schulman and Edwin Kilbourne observed that mice recovered from a previous H1N1 infection were partially protected against mortality, virus replication, and lung tissue damage following H2N2 infection [33] . Immunization with inactivated H1N1 was not able to induce Het-I, indicating that humoral responses are not sufficient for the protection. Later animal studies have proven the essential role of cross-reactive T cells for such infection-induced Het-I [34] [35] [36] [37] [38] [39] . Almost forty years ago, McMichael and colleagues generated the first clinical data about Het-I by concluding from a human challenge study that cross-reactive, cytotoxic T cells can suppress nasal virus replication efficiently [40] . More recently, the 2009 H1N1 pandemic provided the unique chance to investigate the impact of pre-existing T cells on infections with a newly emerged pandemic IAV strain in a naturally exposed study population lacking protective antibody responses. One study detected IAV-specific T cell responses at baseline in 43% of the study population, and could prove that the presence of nucleoprotein (NP)-specific T cells correlated with a threefold decreased chance of acquiring a symptomatic, PCR-confirmed IAV infection [41] . Along this line, Sridhar et al. reported that pre-existing, cross-reactive T cells specific for polymerase basic protein 1 (PB1), matrix 1 protein (M1), and NP were inversely associated with illness severity in case of an infection with the novel H1N1 [42] . More specifically, the frequency of IAV-specific, cytotoxic (interferon-γ + , interleukin-2 -), and lung-homing (CD45RA + CCR7 − CD8 + ) T cells showed the strongest inverse correlation to symptom scores. CD4 + T cells were shown to correlate with disease protection as well [43] . In mice and humans, CD8 + T cell responses are predominantly focused on internal virus proteins like NP, polymerase (PA), PB1, polymerase basic 2 (PB2), and M1, while CD4 + T cell responses are more diverse, recognizing the surface proteins HA and NA as well [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] . Although the internal virus proteins are generally considered as more conserved than the surface proteins, viral escape through immune pressure can happen; however, it seems to be connected to a loss of viral fitness, as shown for NP [56, 57] .

When Schulman and Kilbourne conducted their studies in 1965, they could not know that they had observed T cell-mediated Het-I. This specific knowledge was just not available at that time. As immunology has made much progress in the recent decades, we can tell today that they not only did observe T cell immunity, but this immunity was probably also provided by tissue-resident memory T cells (TRMs).

Today we know that T cell responses are diverse in terms of functionality and spatial distribution. For CD8 + T cells, effector and effector-memory T cells (TEFFs and TEMs, respectively), central memory T cells (TCMs), and TRMs exist [58] . While the rather shortlived effector populations have access to peripheral tissues, including the lung, allowing them to directly fight invading pathogens by their cytotoxic functionalities, TCMs are longer-lived and predominantly circulate among lymph nodes to accelerate anamnestic responses in case of reinfection. In contrast to these circulating subsets, TRMs stably reside at the tissue of the primary infection [59, 60] .

Studies in mice have proven the essential role of lung CD8 + and CD4 + TRMs in providing Het-I against secondary IAV infections. Although localized T cell responses cannot prevent an initial infection like nAbs can do, they restrict virus replication, disease severity, and lung pathology [60] [61] [62] [63] . However, CD8 + TRM responses in the lung tissue wane over time. Wu et al. found a substantial loss of TRM immunity within seven months after the primary infection [62] . Work from Takamura and colleagues suggests temporary TRM niches in the lung at the foci of tissue regeneration, because the disappearance of those repair-associated memory depots (the authors call them RAMDs) parallels the waning of TRM responses [60] . Moreover, lung CD8 + TRMs seem to be prone to apoptosis [64] . Interestingly, lung CD4 + TRMs occupy different niches than CD8 + TRMs in the airways and around B cell follicles, and seem to be more stably maintained [60, 65, 66] .

TRM populations can differ phenotypically due to tissue-specific adaptions, but two commonly used markers are the C-type lectin CD69 and the integrin CD103. Both markers are expressed on murine CD8 + TRM cells in the respiratory tract [60, 62, 67] . Apart from this constitutive expression of adhesion molecules, the limited ability of TRM to recirculate is further caused by a lack of molecules that enable tissue egress and promote migration towards lymphatic tissues, such as S1PR1, CCR7, or CD62L. Accessory TRM markers like CD11a, CD49a, or PD-1 have been described as well [62, 65, 66, 68] . However, the exact phenotype and the mechanisms that maintain specific TRM populations might differ depending on the inductive conditions. Even different immunodominant T cell clones against the same pathogen might show divergent transcriptomic profiles [67] . Lung CD4 + TRM populations and their phenotypes are substantially less well studied. Nevertheless, CD69 is a stringent marker for resident CD4 + T cells, while CD103 might indicate a regulatory phenotype rather than being a good marker for effector TRMs [61, 69, 70] . Apart from phenotypic occurrences, intravascular staining can be used to delineate TRMs from circulatory T cells [71] .

Mouse models provide several advantages for immunological research, like the availability of tools and transgenic strains or economic husbandry. However, mouse models do not optimally represent human IAV disease, immunology, or lung anatomy. Thus, in a translational regard, other animal models might be more suitable to study TRM-mediated flu immunity. Particularly important for TRM research, methods to define and quantify localized T cell populations like parabiosis [59] [60] [61] , intravascular staining [71] , or specific phenotypic profiles were first established in mice, but can be principally transferred to less common laboratory animals (although parabiosis is technically difficult and ethically controversial). In the following, we will discuss the current knowledge about respiratory TRMs in other animal models and in humans (Table 1) . As early as 1933, ferrets were shown to be permissive to human influenza strains [91] . Nowadays, ferrets are a valuable model organism to study flu disease and novel vaccine candidates, due to their similarities in IAV pathogenesis and transmission compared to humans [92, 93] . Immunologic studies in ferrets have been hampered so far by the lack of relevant reagents. However, efforts have been made to identify commercially available antibodies with cross-reactivity in the ferret model, leading to a basic selection of reagents [76, 94] . A further expansion of this toolbox to analyze adaptive immune responses in ferrets is part of the strategic plan for a universal flu vaccine, communicated by the National Institute of Allergy and Infectious Diseases [95] . Several studies have shown that Het-I in ferrets induced by experimental infections, leading to reduced pulmonary virus replication and virus transmission to naïve ferrets [81, [96] [97] [98] [99] [100] [101] [102] [103] . TRMs were unfortunately not investigated in these studies. Also, a recent study demonstrated Het-I after H1N1 infection against a secondary H2N2 challenge [78] . Animals with pre-existing immunity experienced reduced virus replication, weight loss, and fever (a symptom not evident in mice, since they get hypothermic upon infection). The analysis of systemic T cells exposed cross-reactive responses biased towards the recognition of NP, non-structural protein 1 (NS1), and PA. Mucosal T cells were not analyzed after the priming infection. However, the authors established lung perfusion in ferrets to remove blood contaminations from the respiratory organ (although perfusion is not an optimal method to identify lung TRMs [71] ) and isolated flu-specific T cells from the nasal turbinate and lung tissue after the secondary infection, which at least suggests the establishment of local T cells after IAV infections in ferrets. Intravascular staining or a rough phenotypic analysis, both feasible with available antibody clones against CD3, CD4, CD8, CD103, CD11a, and Ly6C [76] , were not conducted.

H1N1, H1N2, and H3N2 strains are endemic in pig populations all over the world [104, 105] , and domestic pigs are a source of pandemic IAV strains, while also being economically of great importance. Therefore, this model organism is increasingly used in flu research, as outlined in a separate review [106] . Importantly, pigs show several anatomical and immunological similarities to humans [107] . Similar to the ferret model, the immunological toolbox is limited, but has grown in recent years for example by the identification of immunodominant T cell peptides and the development of respective MHC-multimers for NP, HA, NA, M1, and PB2specific CD8 + T cells [80, 108, 109] . LAIVs are licensed for vaccination in pigs [110] and induce protection against matching and divergent IAVs [111] [112] [113] . T cell responses were not assessed in these studies, but others report an induction of T cells by LAIV vaccination [114] [115] [116] and by experimental infection with H1N2 [117] . Tungatt and colleagues used newly developed swine multimers to stain NP-specific CD8 + T cells in blood, lymph node, and bronchoalveolar lavage (BAL) samples of IAV-experienced pigs [80] . The latter ones showed the greatest T cell responses, with up to 13% multimer-positive cells. A separate study demonstrated that 90% of BAL T cells are protected from intravascular staining, suggesting that this population is mainly composed of TRM cells [81] . In contrast, T cells isolated from lung tissue contained only about 40% TRMs, indicating significant contamination by vascular T cells, as commonly described in mouse models [71] . Moreover, a rough phenotypic analysis of BAL TRM in pigs showed a predominant CD27 -CCR7phenotype [82] .

Non-human primates (NHPs) are the animal model with the highest degree of similarity to humans [118, 119] . In particular, their adaptive immune systems are largely comparable and therefore NHPs present a valuable model for viral infections [120] . Likewise, human immunological reagents often show cross-reactivity to NHPs, resulting in a diverse toolbox. On the other hand, NHP models are expensive, and ethical aspects must be considered. This might be the reason why, despite great advantages, they are not used extensively to study (mucosal) flu immunity. Nevertheless, T cell-mediated Het-I has been reported in macaques [121, 122] . The work of Pichyangkul and colleagues demonstrated a significant induction of local humoral and cellular responses following pulmonary exposure to the 2009 pandemic H1N1 virus [83] . NP-specific CD69 + CD103 + lung TRMs (both CD4 + and CD8 + ) were highly prevalent in the lung, while only marginally found in blood. Another study described TRM phenotypes induced by Mycobacterium tuberculosis in macaques via intravascular staining and reported the expression of CD103 and CD69 on BAL und lung TRMs [84] .

The analysis of localized T cell responses directly in human tissues is most meaningful regarding clinical applications. However, the investigation of human TRMs is difficult, since most immunological methods used in preclinical research are not applicable to humans. Thus, so far no direct investigation of TRM-mediated protection against flu infections has been conducted, but some insights could be generated in BAL samples, lung tissue biopsies [85, 86, 88, 90] , and human cadavers [74, 87] , as well as in the context of lung transplantation [123] . These studies have shown that CD4 + and CD8 + TRMs in the airways and lungs express CD69 and less stringently CD103. Other studies could further define accessory markers like chemokine receptors (CXCR3, CCR5, CCR6, CXCR6), adhesion molecules (CD49a, CD97), and checkpoint molecules (CTLA-4, PD-1) [86, 88] . In a seminal study, Snyder et al. followed lung transplant recipients longitudinally for the maintenance of existing TRM phenotypes in the donor organ and the de novo generation of new TRM populations [89] . Donor TRM populations persisted for more than 15 months after lung transplantation, and expressed canonical TRM markers like CD69, CD103, CD49a, and PD-1. Two studies from Christopher Chiu's lab investigated CD8 + and CD4 + TRM responses in experimental human respiratory syncytial virus (RSV) infection [85, 90] . Immune responses were assessed longitudinally in BAL samples showing an accumulation of CD69 + CD103 + TRM cells in the airways after convalescence. Moreover, the CD8 + TRM responses before the challenge correlated with reduced symptoms and viral replication. BAL CD4 + T cells were mainly CD69 + , and about 20% showed additional expression of CD103. Thus, these studies reported protective effects of airway TRMs against human respiratory viruses for the first time. Similar experimental human challenge studies are essential to investigate TRM-mediated immunity against influenza.

In contrast to current IAV immunization strategies, which primarily induce humoral responses, numerous preclinical vaccine candidates exploit T cell immunity to induce protection against a broad spectrum of IAV strains. Considering the important contribution of localized T cell responses to Het-I, several strategies aim at the induction of TRM responses in the respiratory tract. A main prerequisite for respiratory TRMs is a local delivery of antigens [60, 62, 63] or specific adjuvants that bypass the need for local antigens [124] . Nevertheless, one of these vaccine components must be administered into the airways. Moreover, an induction of lung CD8 + TRMs relies on antigen cross-presentation by dendritic cells (DCs), mainly by migratory CD103 + type I conventional DCs [125] . This antigen cross-presentation is much more efficient after genetic vaccination, leading to endogenous antigen production in the host, compared to protein-or peptide-based strategies that need further enhancement by adjuvants or other mechanisms.

Several genetic vaccine platforms have been established for intranasal delivery, such as DNA formulated with polyethylenimine [126, 127] , adenoviral vectors [75, [128] [129] [130] [131] [132] , recombinant Sendai virus [133, 134] , modified vaccinia Ankara virus (MVA) [135] , or murine cytomegalovirus vectors [136] . By the vector-driven expression of conserved internal flu proteins, all these approaches (and many more not mentioned here) are able to induce Het-I. However, the extent of lung TRM establishment might differ among these platforms. Even vectors based on different subtypes of the same virus family evoke divergent immune profiles [137] . Thus, an induction of lung TRMs per se might not be problematic with genetic vaccines, but the amplitude and the long-term maintenance are parameters to be improved by refined strategies. First, an increased number of initial TRMs could lead to longer maintenance of the protective levels of T cell immunity. The co-delivery of inflammatory factors in genetic vaccinations has been used extensively to modulate systemic immune responses [138] [139] [140] [141] [142] [143] [144] [145] . We could show that the mucosal co-delivery of interleukin (IL)-1β can increase the induction of lung CD4 + and CD8 + TRMs significantly [73] , thus illustrating that genetic adjuvants might be an important tool to boost TRM responses. Second, repetitive intranasal immunizations over time can maintain and refresh the existing TRM pool [146] . Third, optimized heterologous prime-boost regimens or even simultaneous immunizations at different body sites might lead to synergistic effects between local and systemic immune responses to ensure long-term immunity [147] .

When it comes to protein-and peptide-based vaccines, additional tricks must be exploited to enhance the induction of T cell immunity in the respiratory tract, especially regarding CD8 + TRMs. Some of these tricks rely on the use of specific adjuvants or antigen-targeting to cell types of interest. Wakim and colleagues used an antibody-targeted intranasal vaccination to specifically address a model antigen to CD103 + DCs. As a result, antigen-targeting to Clec12a or Dec205 could increase induction of CD103 + CD8 + TRM cells in the lung, leading to improved protection against a subsequent IAV challenge [148] . Another study from the same lab describes the use of zymosan as a mucosal adjuvant, allowing an establishment of CD103 + CD69 + CD8 + TRMs independent of local antigen application to the lungs [124] . However, both studies used a model where in vitro activated, T cell receptor-transgenic T cells were administrated before immunization. The physiological priming process of naïve CD8 + T cells was not assessed in these models. Therefore, it would be interesting to see whether these strategies work in a less artificial vaccination setting as well. Another study used the conserved matrix 2 protein ectodomain fused to a protein-adjuvant called CTA1-DD (cholera toxin A1 derivative and S. aureus fragment D dimer) as an intranasal vaccine [69] . The vaccine induced CD103 low CD69 + CD4 + TRM responses that were crucial for flu immunity, and the protective effects partially relied on IL-17A production. These data illustrate the important role that CD4 + TRMs can play in Het-I and the feasibility to induce protective levels of CD4 + TRMs by protein vaccines.

While numerous vaccine strategies that evoke cross-reactive T cell responses have been developed in animal models, a fairly low number also progressed towards human clinical trials. Even fewer of these studies have exploited local or mucosal administration strategies. A comprehensive review about clinical trials with universal flu vaccine candidates is given elsewhere [149] . Here, we will predominantly focus on vaccines intended or expected to induce T cell responses and for which clinical data have already been published.

A few trials evaluated epitope-based peptide vaccines, but so far none of them have induced efficient protective immunity. Flu-v consists of conserved peptides derived from M1, M2, and NP formulated with Montanide ISA-51 as an adjuvant. The vaccine induces cellular immunity in humans, and could reduce symptoms and viral shedding in a small human challenge study [150, 151] . A recently published phase IIb study showed only a limited capacity of the vaccine to protect against mild to moderate influenza disease in experimental infections. One but not two vaccine doses reduced disease burden, but an impact on virus shedding could not be found in any of the vaccinated groups [152] . BiondVax's Multimeric-001 peptide vaccine contains nine conserved B cell, CD4 + , and CD8 + T cell epitopes from HA, NP, and M1 of IAV and IBV [153] . While it has been proven safe and immunogenic, recent data from a phase III trial (NCT03450915) with more than 12,000 participants did not demonstrate significant VE against flu infections [154] . Another peptide vaccine is FP-01.1, which combines peptides from NP, M1, PB1, and PB2. Two doses of the vaccine, given four weeks apart, resulted in a responder rate of 83% in the high-dose group. CD4 + and CD8 + T cell responses against the conserved vaccine immunogens were induced and peaked at day 7 post-immunization. By now, no data about longevity of the immune responses or VE were published, but an experimental challenge study was conducted (NCT02071329).

If the induction of strong CD8 + T cell responses is desired, genetic immunizations with DNA, RNA, or viral vectors that lead to endogenous antigen production in the vaccinees have an intrinsic advantage over protein-and peptide-based vaccines. One of the most characterized vector-based universal flu vaccine approaches is MVA-M1/NP. In total, nine clinical trials were initiated with this vaccine candidate, which encodes full-length NP and M1 derived from IAV. MVA-M1/NP was shown to induce CD4 + and CD8 + T cell responses specific for the conserved vaccine epitopes. Analyses of the vaccine immunogenicity in different age groups presented a decreasing induction of T cell responses in older individuals. While vaccine-induced T cell responses remained statistically significant over the baseline for 52 weeks in 50-59-year-old participants, the responses in >70-year-old participants were only significant over the baseline for three weeks after vaccination [155] . In a human challenge study, the vaccine showed a slightly decreased infection rate in the vaccine group (2 out of 11) compared to the placebo group (5 out of 11), but an interpretation of the vaccine effect was hampered by the unexpectedly low number of infections in the placebo group [156] . Of note, the flu infections in the placebo group induced stronger T cell responses than vaccination with MVA-M1/NP, suggesting that vaccine-induced immunity is weaker compared to naturally acquired immunity. VE is currently being evaluated in larger trials, both as a standalone vaccine (NCT03883113) and as adjunct to a licensed QIV (NCT03880474). ChAdOx1-NP + M1 is based on a replication-deficient chimpanzee adenovirus. Similar to the MVA-M1/NP, it encodes full-length NP and M1 proteins. In a phase I study, the vaccine was shown to induce T cells against the vaccine antigens, peaking 14 days after immunization. A boost with the aforementioned MVA-M1/NP 7 or 14 weeks after ChAdOx1-NP + M1 could further increase T cell responses [157] .

While all the above mentioned vaccine trials exploited systemic immunization routes, mucosal vaccine administration is key to engage local immune responses, especially TRMs in the respiratory tract. Altimmune's NasoVax is an adenovirus serotype 5-based vaccine (Ad5) encoding full-length HA from an H1N1 strain. No trial data have been published yet, but results presented at a conference suggest superior immunogenicity with a high dose of NasoVAX compared to a licensed QIV [158] . Besides a 100% seroprotection rate, T cell responses were six-fold higher compared to the QIV. It must be investigated to which extent those responses provide Het-I, but cross-reactive T cell responses against conserved epitopes of HA are described [159, 160] .

In addition, two orally given vaccines have been in human trials. One trial was assessing replication-competent adenovirus serotype 4 (Ad4) encoding HA from an H5N1 strain [161] . The "vaccine take", defined as the percentage of vaccinees being PCR-positive and/or seropositive for Ad4 after vaccination, was strongly dose-dependent but reached 96% after three high-dose immunizations. A total of 70% of the participants in these highdose groups mounted antigen-specific T cell responses, but these consisted predominantly of CD4 + T cells. While HA seroconversion was achieved in only a minority after Ad4-HA immunization (4-19%), a parenteral boost vaccination with inactivated H5N1 led to increased seroconversion rates in a dose-dependent manner like the previous Ad4-HA immunizations. Similarly, an Ad5-vectored vaccine encoding HA from H5N1 from VaxArt induced T cell responses in 75% of the participants, but failed to induce nAbs [162] . Unfortunately, the specific contribution of CD4 + and CD8 + T cells were not deciphered in these analyses. It is important to note here that the low seroconversion rates reported in these studies are not due to a low immunogenicity of the vaccine platforms; instead, this is rather attributed to a generally low immunogenicity of some avian HA variants [163] .

Another vaccine from VaxArt, VXA-A1.1, relies on a non-replicating Ad5 encoding HA and an immunostimulatory dsRNA as a TLR3 agonist. VXA-A1.1 is given as a tablet and targets epithelial cells in the small bowel [164] . Due to the tablet formulation and its stability at room temperature, this vaccine enables distribution independent of healthcare professionals, for example by mail delivery. Moreover, it seems that the oral administration evades pre-existing anti-vector immunity to some extent. The respond rate after one dose of the vaccine was 92% with regard of humoral responses (four-fold increase in HAI) [164] . In a recent phase II study, the VE against a vaccine homologous strain was evaluated by experimental infections. Compared to a study group that received a licensed QIV, VXA-A1.1 generated a similar protective immunity against infection [165] . Since serum HAI titers were about nine-fold higher in the QIV group compared to VXA-A1.1, additional immune parameters must be responsible for the observed VE. Immunoglobulin A (IgA)-and immunoglobulin G-secreting cells, as well as polyfunctional T cells, have been correlated with protective immunity. Mucosal immunity in the respiratory tract was not directly assessed in this study, although preclinical studies report an induction of mucosal IgA in the respiratory tract [166] . Whether orally administered vaccine platforms are able to establish respiratory TRMs has not been reported so far, but the "gut-lung axis" allows some tissue-resident immune populations to traffic between both organs [167] .

In conclusion, vaccine strategies aiming at the induction of cross-reactive T cell immunity have been evaluated in clinical trials, but no breakthroughs with clear and effective Het-I were reported so far. Some vaccines induce T cells but lack efficacy; others show a relatively rapid decline of T cell immunity. While a few mucosal vaccines have entered clinical trials, none of the studies really assessed local immune responses directly. However, the analysis of vaccine-induced TRMs is essential to define new mucosal correlates of protection that are not accessible in peripheral blood mononuclear cells (PBMCs).

Preclinical studies have illustrated the indispensable role of localized T cell responses in the protection against IAV. Concomitantly, many different vaccination strategies have been developed in these animal models to establish TRMs and eventually led to broad and effective Het-I. However, it is still a long way to establish T cell immunity as a protective correlate in humans. In the following, we will discuss essential steps to define novel COPs and give an outlook about a potential implementation of T cell immunity in current vaccination guidelines.

Early-phase clinical trials are not only important to show the safety of novel vaccines, but are also key to estimate their efficacy and to prioritize on the most promising candidates for later phases. To this end, either a direct correlate of protection or a surrogate marker must be assessed to interpolate protective efficacy from vaccine immunogenicity. Classically, the HAI is such a correlate with a clearly defined protective threshold, although there are debates about the general validity of a hemagglutination inhibition titer of 1:40 for protective immunity [12, 168] . Such specific thresholds of protective immunity parameters (directly assessed COPs or surrogate parameters) are urgently needed for T cell-mediated flu immunity.

The first obvious question is this: where should T cell responses be sampled? While PBMCs might be an adequate biological sample to estimate systemic T cell immunity, mucosal vaccines hold the greatest promise to evoke local immune responses. Thus, sampling at the mucosal site of interest is key to evaluate the actual purpose of these vaccines. However, what is the actual mucosal site we would like to address? In animal models, lung TRMs are the most studied mucosal T cell COP [62, 63] . However, it is unlikely that human vaccines will exploit administration routes directly aiming at the lower respiratory tract, due to the invasive nature of such procedures and potential side effects. Instead, it is more likely that mucosal next-generation vaccines will target tissues of the upper respiratory tract, like the nasal mucosa. In mice, CD8 + TRMs in the nasal epithelia were shown to protect against severe disease by blocking pulmonary spread of the IAV infection [169] . Sampling of nasal tissue in clinical trials would be much less elaborate compared to sampling of the lung tissue. However, human challenge studies with RSV as well as studies from the Farber lab illustrate the feasibility of assessing CD4 + and CD8 + TRM responses in the lower respiratory tract by bronchial biopsies and BALs in humans [85, 89, 90 ]. An interesting alternative induction and sampling site for mucosal immunity are nasopharynx-associated lymphoid tissues, which include the adenoids and tonsils. In particular, studies from Rebecca Cox showed that immunization with LAIV in children rapidly evokes B cells, CD4 + T cells, and CD8 + T cells in the tonsils [170, 171] . While it seems clear that tonsillar T cells are induced upon intranasal LAIV immunization, and that these responses correlate with serum HAI, their contribution to or significance in mucosal immunity is not clear yet. Neither their direct protective effect (used as direct COP), nor any correlation with tissue TRM responses (used as surrogate marker) have been described so far. Interestingly, tonsillar immunization with genetic vaccines in NHPs induce T cell responses in BAL samples, which are considered as a stringent TRM population [172] . Thus, there are several mucosal sites where immune responses can be induced and assessed.

Future studies must investigate the relevance of each of these compartmentalized responses in respect to protection.

Preclinical animal models are essential to investigate the basic immunological principles of anti-flu immunity and have helped us in the past to develop concepts like T cell cross-reactivity, TRM responses, and universal flu vaccine approaches. However, the critical step is to translate these concepts into real-life human vaccines. A combination of controlled human challenge studies that employ in-depth immunological analyses and large efficacy trials seem to be required to establish novel COPs. This was also outlined at the "Immunological Assays and Correlates of Protection for Next-Generation Influenza Vaccines" meeting in 2019 [173] . Once specific COPs are identified, it is important to define protective thresholds of the respective responses (absolute COPs). For systemic T cell responses, Forrest et al. found that >100 spot-forming cells per 10 6 PBMCs in interferon-γ ELISPOT analyses correlated with protection against symptomatic flu infections in young children vaccinated with LAIV [174] . Although this value is discussed within the field, this type of protective threshold is needed to estimate VE in clinical trials from immunogenicity data. Likewise, the sample collection at different (mucosal) body sites and the actual immunological assays must be standardized to allow direct comparisons of clinical trials among study sites and labs [173] . This also helps to prioritize the most promising vaccine candidates already after early clinical phases.

Once effective flu vaccines that rely on T cell responses are market-ready, it must be discussed how those are integrated in vaccination practices and recommendations. Elderly people aged over 65 years, people with underlying diseases (diabetes, chronic obstructive pulmonary disease, asthma, heart and kidney diseases), and pregnant women are the most vulnerable groups. Since sterilizing immunity is the most efficient way to protect them against flu complications, the induction of nAbs must remain the most important COP in these risk groups. Moreover, the induction of nAbs in pregnant women is important for a maternal antibody transfer to the offspring-an important risk group that is unlikely to receive genetic or mucosal flu vaccines within the first months of life.

All parts of the community would benefit from the presence of cross-reactive T cell immunity against flu. In risk groups, T cell immunity would present a safeguard in case of vaccine mismatches and emerging pandemic strains. Young and healthy individuals do not necessarily require sterile protection against influenza, since potential infections in these groups are less likely to result in severe disease. Nevertheless, cross-reactive T cell responses can further decrease the rate of flu-related complications and might also to some extent provide population-wide protection against emerging flu strains with higher pathogenicity. At the same time, the non-sterile nature of T cell immunity would still allow mild IAV infections, which can naturally boost systemic and mucosal T cell responses [175] . Thus, regular boosting of T cell immunity by T cell-inducing vaccines and natural infections might be a way to maintain long-term mucosal Het-I in humans. Table 2 summarizes our recommended vaccination practices for different target groups. So far, the effect of such broad and pronounced T cell immunity in large parts of the community on virus evolution is unknown. As pointed out by others, one result could be a general decrease of flu infections, while decreasing virus replication per person is also slowing down virus evolution. On the other hand, immune pressure on conserved virus proteins exceeding natural immunity could select for escape mutants [176] . However, viral escape might be limited due to functional constraints and loss of viral fitness [57] .

Regarding the vaccine platforms, a full switch to genetic and favorably mucosal vaccines in all target groups should take place if the safety profile of the respective vaccine allows it. In those vaccines, the antigen components can be combined and updated as needed. All vaccine formulations should include conserved flu proteins like NP, M1, or polymerase proteins in order to induce cross-reactive T cell responses. In the mentioned risk groups, HA-encoding components can be easily added (and adapted annually) in order to evoke nAbs, preferentially in the respiratory tract. This strategy combines the advantages of genetic vaccines regarding immunogenicity, manufacturing, and adaptability, while it considers the vulnerability of specific target groups and the benefits of infection-permissive immunity in healthy individuals at the same time.

Recent influenza vaccines are not appropriate to protect the community against seasonal and pandemic flu strains. The time has come to implement modern immunology and vaccine technology in human flu vaccines. In the context of the latest Ebola and SARS-CoV-2 outbreaks, several efficient genetic vaccines have been approved. Thus, the aim should be to employ these technologies for routine flu shots also in order to enable new T cell-based COPs. Many years of preclinical research prove the protective potential of cross-reactive T cell immunity and more recently of respiratory TRMs. Substantial knowledge could be gathered to understand and induce TRM responses in animal models. It is now critical to illuminate remaining knowledge gaps and their translation into clinical approaches. Current preclinical data indicate that local inflammation in the respective mucosa is a minimal prerequisite for the establishment of local T cells. Local expression of antigens seems to be additionally required for the induction of lung TRMs. Therefore, local vaccination techniques currently seem inevitable to evoke TRM responses. However, the respiratory tract is an immunologically fragile environment, and the unintentional induction of autoimmunity or allergies must be avoided. This becomes even more important if mucosal vaccines are used in individuals with pre-existing respiratory diseases. For the long-term maintenance of immunity, it is essential to develop vaccine strategies that either induce long-lived TRM populations or refresh short-lived TRMs regularly. Eventually, the consideration of novel vaccine technologies and cross-reactive T cell responses holds the promise of decreasing flu mortality in seasonal and pandemic outbreaks significantly. 

The Age Distribution of Mortality Due to Influenza: Pandemic and Peri-Pandemic

Populations at Risk for Severe or Complicated Influenza Illness: Systematic Review and Meta-Analysis

Risk Factors for Serious Outcomes Associated with Influenza Illness in High-versus Low-and Middle-Income Countries: Systematic Literature Review and Meta-Analysis

Updating the Accounts: Global Mortality of the 1918-1920

Mapping the Antigenic and Genetic Evolution of Influenza Virus

Comparison of the Mutation Rates of Human Influenza A and B Viruses

Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans

The Role of Serum Haemagglutination-Inhibiting Antibody in Protection against Challenge Infection with Influenza A2 and B Viruses

Haemagglutination-Inhibiting Antibody to Influenza Virus

Relationship between Haemagglutination-Inhibiting Antibody Titres and Clinical Protection against Influenza: Development and Application of a Bayesian Random-Effects Model

Correlates of Protection against Influenza in the Elderly: Results from an Influenza Vaccine Efficacy Trial

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

Recent H3N2 Influenza Virus Clinical Isolates Rapidly Acquire Hemagglutinin or Neuraminidase Mutations When Propagated for Antigenic Analyses

Optimisation of a Micro-Neutralisation Assay and Its Application in Antigenic Characterisation of Influenza Viruses

Influenza Vaccine Effectiveness in the Community and the Household

Re-Arranged: Support for Policies to Vaccinate Elderly People against Influenza

Interim Estimates of 2014/15 Vaccine Effectiveness against Influenza A(H3N2) from Canada's Sentinel Physician Surveillance Network

Protective Effect of Vaccination Against Induced Influenza A 1

Effectiveness of MF59-Adjuvanted Seasonal Influenza Vaccine in the Elderly: A Systematic Review and Meta-Analysis

Comparative Effectiveness of High-Dose Versus Standard-Dose Influenza Vaccines Among US Medicare Beneficiaries in Preventing Postinfluenza Deaths During

Efficacy and Safety of High-Dose Influenza Vaccine in Elderly Adults: A Systematic Review and Meta-Analysis

Efficacy, and Immunogenicity of Flublok in the Prevention of Seasonal Influenza in Adults

Epidemic Season: A Step towards Improved Influenza Vaccine Effectiveness

Genetic Bases of the Temperature-Sensitive Phenotype of a Master Donor Virus Used in Live Attenuated Influenza Vaccines: A/Leningrad/134/17/57 (H2N2)

Restricted Replication of the Live Attenuated Influenza A Virus Vaccine during Infection of Primary Differentiated Human Nasal Epithelial Cells

Efficacy of Vaccination with Live Attenuated, Cold-Adapted, Trivalent, Intranasal Influenza Virus Vaccine against a Variant (A/Sydney) Not Contained in the Vaccine

Live and Inactivated Influenza Vaccines Induce Similar Humoral Responses, but Only Live Vaccines Induce Diverse T-Cell Responses in Young Children

Influenza Vaccine Effectiveness Against 2009 Pandemic Influenza A(H1N1) Virus Differed by Vaccine Type During 2013-2014 in the United States

Influenza Vaccine Effectiveness in the United States during the 2015-2016 Season

Vaccine Failure and Serologic Response to Live Attenuated and Inactivated Influenza Vaccines in Children during the

Next-Generation Influenza Vaccines: Opportunities and Challenges

Induction of Partial Specific Heterotypic Immunity in Mice by a Single Infection with Influenza A Virus

Transfer of Specific Cytotoxic T Lymphocytes Protects Mice Inoculated with Influenza Virus

Biological Properties of an Influenza A Virus-Specific Killer T Cell Clone. Inhibition of Virus Replication in Vivo and Induction of Delayed-Type Hypersensitivity Reactions

In Vivo Effector Function of Influenza Virus-Specific Cytotoxic T Lymphocyte Clones Is Highly Specific

Kinetics and Specificity at the Clonal Level of the Cytotoxic T Lymphocyte Response to Influenza Pneumonia

Heterosubtypic Immunity to Influenza Type A Virus in Mice. Effector Mechanisms and Their Longevity

Profound Protection against Respiratory Challenge with a Lethal H7N7 Influenza A Virus by Increasing the Magnitude of CD8+ T-Cell Memory

Cytotoxic T-Cell Immunity to Influenza

Natural T Cell-Mediated Protection against Seasonal and Pandemic Influenza. Results of the Flu Watch Cohort Study

Cellular Immune Correlates of Protection against Symptomatic Pandemic Influenza

Preexisting Influenza-Specific CD4+ T Cells Correlate with Disease Protection against Influenza Challenge in Humans

The Epitopes of Influenza Nucleoprotein Recognized by Cytotoxic T Lymphocytes Can Be Defined with Short Synthetic Peptides

Identification of Viral Molecules Recognized by Influenza-Specific Human Cytotoxic T Lymphocytes

Anti-Influenza Virus Cytotoxic T Lymphocytes Recognize the Three Viral Polymerases and a Nonstructural Protein: Responsiveness to Individual Viral Antigens Is Major Histocompatibility Complex Controlled

Murine Cytotoxic T Lymphocyte Recognition of Individual Influenza Virus Proteins. High Frequency of Nonresponder MHC Class I Alleles

Recognition of the PB1, Neuraminidase, and Matrix Proteins of Influenza Virus A/NT/60/68 by Cytotoxic T Lymphocytes

A Previously Unrecognized H-2Db-Restricted Peptide Prominent in the Primary Influenza A Virus-Specific CD8+T-Cell Response Is Much Less Apparent Following Secondary Challenge

Diversity of Epitope and Cytokine Profiles for Primary and Secondary Influenza A Virus-Specific CD8 + T Cell Responses

Uneven Distribution of MHC Class II Epitopes within the Influenza Virus

Immunomic Analysis of the Repertoire of T-Cell Specificities for Influenza A Virus in Humans

Genome-Wide Screening of Human T-Cell Epitopes in Influenza A Virus Reveals a Broad Spectrum of CD4+ T-Cell Responses to Internal Proteins, Hemagglutinins, and Neuraminidases

Analyses of the Specificity of CD4 T Cells During the Primary Immune Response to Influenza Virus Reveals Dramatic MHC-Linked Asymmetries in Reactivity to Individual Viral Proteins

Immunodominant CD4+ T-Cell Responses to Influenza A Virus in Healthy Individuals Focus on Matrix 1 and Nucleoprotein

Sequence Variation in the Influenza A Virus Nucleoprotein Associated with Escape from Cytotoxic T Lymphocytes

Functional Constraints of Influenza A Virus Epitopes Limit Escape from Cytotoxic T Lymphocytes

Transcriptional Control of Effector and Memory CD8+ T Cell Differentiation

Dynamics of Blood-Borne CD8 Memory T Cell Migration In Vivo

Specific Niches for Lung-Resident Memory CD8 + T Cells at the Site of Tissue Regeneration Enable CD69-Independent Maintenance

Cutting Edge: Tissue-Retentive Lung Memory CD4 T Cells Mediate Optimal Protection to Respiratory Virus Infection

Lung-Resident Memory CD8 T Cells (TRM) Are Indispensable for Optimal Cross-Protection against Pulmonary Virus Infection

Vaccine-Generated Lung Tissue-Resident Memory T Cells Provide Heterosubtypic Protection to Influenza Infection

Dynamics of Influenza-Induced Lung-Resident Memory T Cells Underlie Waning Heterosubtypic Immunity

Lung Niches for the Generation and Maintenance of Tissue-Resident Memory T Cells

Biased Generation and In Situ Activation of Lung Tissue-Resident Memory CD4 T Cells in the Pathogenesis of Allergic Asthma

TCR-PMHC Encounter Differentially Regulates Transcriptomes of Tissue-Resident CD8 T Cells

Cutting Edge: CD69 Interference with Sphingosine-1-Phosphate Receptor Function Regulates Peripheral T Cell Retention

M2e-Tetramer-Specific Memory CD4 T Cells Are Broadly Protective against Influenza Infection

CD103hi Treg Cells Constrain Lung Fibrosis Induced by CD103lo Tissue-Resident Pathogenic CD4 T Cells

Cutting Edge: Intravascular Staining Redefines Lung CD8 T Cell Responses

Quantifying Memory CD8 T Cells Reveals Regionalization of Immunosurveillance

IL-1β as Mucosal Vaccine Adjuvant: The Specific Induction of Tissue-Resident Memory T Cells Improves the Heterosubtypic Immunity against Influenza A Viruses

CXCR6 Regulates Localization of Tissue-Resident Memory CD8 T Cells to the Airways

Long-Term Maintenance of Lung Resident Memory T Cells Is Mediated by Persistent Antigen

Improving Immunological Insights into the Ferret Model of Human Viral Infectious Disease

Pulmonary Surfactant-Biomimetic Nanoparticles Potentiate Heterosubtypic Influenza Immunity

Systemic and Respiratory T-Cells Induced by Seasonal H1N1 Influenza Protect against Pandemic H2N2 in Ferrets

Aerosol Delivery of a Candidate Universal Influenza Vaccine Reduces Viral Load in Pigs Challenged with Pandemic H1N1 Virus

Induction of Influenza-Specific Local CD8 T-Cells in the Respiratory Tract after Aerosol Delivery of Vaccine Antigen or Virus in the Babraham Inbred Pig

Comparison of Heterosubtypic Protection in Ferrets and Pigs Induced by a Single-Cycle Influenza Vaccine

Distribution of Droplets and Immune Responses After Aerosol and Intra-Nasal Delivery of Influenza Virus to the Respiratory Tract of Pigs. Front

Tissue Distribution of Memory T and B Cells in Rhesus Monkeys Following Influenza A Infection

Prevention of Tuberculosis in Macaques after Intravenous BCG Immunization

RSV-Specific Airway Resident Memory CD8+ T Cells and Differential Disease Severity after Experimental Human Infection

Programs for the Persistence, Vigilance and Control of Human CD8+ Lung-Resident Memory T Cells

Human Tissue-Resident Memory T Cells Are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites

Trigger-Happy Resident Memory CD4+ T Cells Inhabit the Human Lungs

Generation and Persistence of Human Tissue-Resident Memory T Cells in Lung Transplantation

Epitope-Specific Airway-Resident CD4+ T Cell Dynamics during Experimental Human RSV Infection

A Virus Obtained from Influenza Patients

Animal Models for Influenza Viruses: Implications for Universal Vaccine Development

Animal Models in Influenza Research

Moving Forward: Recent Developments for the

A Universal Influenza Vaccine: The Strategic Plan for the National Institute of Allergy and Infectious Diseases

Vaccination against Seasonal Influenza A/H3N2 Virus Reduces the Induction of Heterosubtypic Immunity against Influenza A/H5N1 Virus Infection in Ferrets

Infection of the Upper Respiratory Tract with Seasonal Influenza A(H3N2) Virus Induces Protective Immunity in Ferrets against Infection with A(H1N1)Pdm09 Virus after Intranasal, but Not Intratracheal, Inoculation

Evaluation of the Humoral and Cellular Immune Responses Elicited by the Live Attenuated and Inactivated Influenza Vaccines and Their Roles in Heterologous Protection in Ferrets

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Immunogenicity and Protection of A(H3N2) Live Attenuated Influenza Vaccines Containing Wild-Type Nucleoprotein in a Ferret Model

Extensive T Cell Cross-Reactivity between Diverse Seasonal Influenza Strains in the

Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge

Heterosubtypic Cross-Protection Correlates with Cross-Reactive Interferon-Gamma-Secreting Lymphocytes in the

Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between

A Veterinary Diagnostic Laboratory Web-Based Platform to Monitor the Temporal Genetic Patterns of Influenza A Virus in Swine

Cell Immune Responses to Influenza Viruses in Pigs

Comparative Distribution of Human and Avian Type Sialic Acid Influenza Receptors in the Pig

Identification of Swine Influenza Virus Epitopes and Analysis of Multiple Specificities Expressed by Cytotoxic T Cell Subsets

Identification of Cross-Reacting T-Cell Epitopes in Structural and Non-Structural Proteins of Swine and Pandemic H1N1 Influenza A Virus Strains in Pigs

Live Attenuated Influenza Virus Vaccine Reduces Virus Shedding of Newborn Piglets in the Presence of Maternal Antibody

Efficacy in Pigs of Inactivated and Live Attenuated Influenza Virus Vaccines against Infection and Transmission of an Emerging H3N2 Similar to the 2011-2012 H3N2v

Swine Influenza Virus Vaccine Serologic Cross-Reactivity to Contemporary US Swine H3N2 and Efficacy in Pigs Infected with an H3N2 Similar to

Comparison of Adjuvanted-Whole Inactivated Virus and Live-Attenuated Virus Vaccines against Challenge with Contemporary, Antigenically Distinct H3N2 Influenza A Viruses

Immunogenicity and Protective Efficacy of an Elastase-Dependent Live Attenuated Swine Influenza Virus Vaccine Administered Intranasally in Pigs

Vaccination with NS1-Truncated H3N2 Swine Influenza Virus Primes T Cells and Confers Cross-Protection against an H1N1 Heterosubtypic Challenge in Pigs

Heightened Adaptive Immune Responses Following Vaccination with a Temperature-Sensitive, Live-Attenuated Influenza Virus Compared to Adjuvanted, Whole-Inactivated Virus in Pigs

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung

Development and Homeostasis of T Cell Memory in Rhesus Macaque

Comparative Phylogenetic Studies of Rhesus Monkey (Macaca Mulatta) and Human (Homo Sapiens) Using G-Banding Pattern

Nonhuman Primate Models of Human Viral Infections

Cross-Reactive T Cells Are Involved in Rapid Clearance of 2009 Pandemic H1N1 Influenza Virus in Nonhuman Primates

Multigenic DNA Vaccine Induces Protective Cross-Reactive T Cell Responses against Heterologous Influenza Virus in Nonhuman Primates

Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma

Zymosan By-Passes the Requirement for Pulmonary Antigen Encounter in Lung Tissue-Resident Memory CD8+ T Cell Development

Optimal Generation of Tissue-Resident but Not Circulating Memory T Cells during Viral Infection Requires Crosspriming by DNGR-1+ Dendritic Cells

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-Protective Immunity against Influenza Viruses

Intranasal DNA Vaccine for Protection against Respiratory Infectious Diseases: The Delivery Perspectives

Protection against Multiple Influenza A Subtypes by Vaccination with Highly Conserved Nucleoprotein

Single-Dose Mucosal Immunization with a Candidate Universal Influenza Vaccine Provides Rapid Protection from Virulent H5N1, H3N2 and H1N1 Viruses

A Universal Influenza A Vaccine Based on Adenovirus Expressing Matrix-2 Ectodomain and Nucleoprotein Protects Mice From Lethal Challenge

Vaccination to Conserved Influenza Antigens in Mice Using a Novel Simian Adenovirus Vector, PanAd3, Derived from the Bonobo Pan Paniscus

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2

Recombinant Sendai Virus as a Novel Vaccine Candidate for Respiratory Syncytial Virus

Induction of Influenza-Specific Mucosal Immunity by an Attenuated Recombinant Sendai Virus

Mucosal CD8+ T Cell Responses Induced by an MCMV Based Vaccine Vector Confer Protection against Influenza Challenge

Evaluation of Adenovirus 19a as a Novel Vector for Mucosal Vaccination against Influenza A Viruses

Genetic Adjuvants for DNA Vaccines

Regulation of DNA-Raised Immune Responses by Cotransfected Interferon Regulatory Factors

Super-Activated Interferon-Regulatory Factors Can Enhance Plasmid Immunization

Activation of Innate Immunity, Inflammation, and Potentiation of DNA Vaccination through Mammalian Expression of the TLR5 Agonist Flagellin

Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity

Interference of Retroviral Envelope with Vaccine-Induced CD8+ T Cell Responses Is Relieved by Co-Administration of Cytokine-Encoding Vectors

Genetic Co-Administration of Soluble PD-1 Ectodomains Modifies Immune Responses against Influenza a Virus Induced by DNA Vaccination

Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA

Repeated Antigen Exposure Extends the Durability of Influenza-Specific Lung-Resident Memory CD8+ T Cells and Heterosubtypic Immunity

Combined Local and Systemic Immunization Is Essential for Durable T-Cell Mediated Heterosubtypic Immunity against Influenza A Virus

Antibody-Targeted Vaccination to Lung Dendritic Cells Generates Tissue-Resident Memory CD8 T Cells That Are Highly Protective against Influenza Virus Infection

A Decade in Review: A Systematic Review of Universal Influenza Vaccines in Clinical Trials during the 2010 Decade

A Synthetic Influenza Virus Vaccine Induces a Cellular Immune Response That Correlates with Reduction in Symptomatology and Virus Shedding in a Randomized Phase Ib Live-Virus Challenge in Humans

Safety, and Efficacy of a Standalone Universal Influenza Vaccine, FLU-v, in Healthy Adults

Efficacy of FLU-v, a Broad-Spectrum Influenza Vaccine, in a Randomized Phase IIb Human Influenza Challenge Study

Safety and Immunogenicity of Multimeric-001-A Novel Universal Influenza Vaccine

BiondVax BiondVax Announces Topline Results from Phase 3 Clinical Trial of the M-001 Universal Influenza Vaccine Candidate

A T Cell-Inducing Influenza Vaccine for the Elderly: Safety and Immunogenicity of MVA-NP+M1 in Adults Aged over 50 Years

Preliminary Assessment of the Efficacy of a T-Cell-Based Influenza Vaccine, MVA-NP+M1, in Humans

Clinical Assessment of a Novel Recombinant Simian Adenovirus ChAdOx1 as a Vectored Vaccine Expressing Conserved Influenza A Antigens

Safety and Immunogenicity of NasoVAX, a Novel Intranasal Influenza Vaccine

Murine Helper T Lymphocyte Response to Influenza Virus: Recognition of Haemagglutinin by Subtype-Specific and Cross-Reactive T Cell Clones

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

Safety and Immunogenicity of an Oral, Replicating Adenovirus Serotype 4 Vector Vaccine for H5N1 Influenza: A Randomised, Double-Blind, Placebo-Controlled, Phase 1 Study

Oral Administration of an Adenovirus Vector Encoding Both an Avian Influenza A Hemagglutinin and a TLR3 Ligand Induces Antigen Specific Granzyme B and IFN-γ T Cell Responses in Humans

Vaccines for Pandemic Influenza: Summary of Recent Clinical Trials

High Titre Neutralising Antibodies to Influenza after Oral Tablet Immunisation: A Phase 1, Randomised, Placebo-Controlled Trial

Efficacy, Immunogenicity, and Safety of an Oral Influenza Vaccine: A Placebo-Controlled and Active-Controlled Phase 2 Human Challenge Study

Orally Administered Adenoviral-Based Vaccine Induces Respiratory Mucosal Memory and Protection against RSV Infection in Cotton Rats

S1P-Dependent Interorgan Trafficking of Group 2 Innate Lymphoid Cells Supports Host Defense

Granzyme B: Correlates with Protection and Enhanced CTL Response to Influenza Vaccination in Older Adults

Resident Memory CD8+ T Cells in the Upper Respiratory Tract Prevent Pulmonary Influenza Virus Infection

Live-Attenuated Influenza Vaccine Induces Tonsillar Follicular T Helper Cell Responses That Correlate With Antibody Induction

Early Induction of Cross-Reactive CD8+ T-Cell Responses in Tonsils After Live-Attenuated Influenza Vaccination in Children

Novel Vaccine Regimen Elicits Strong Airway Immune Responses and Control of Respiratory Syncytial Virus in Nonhuman Primates

Meeting Report and Review: Immunological Assays and Correlates of Protection for Next-generation Influenza Vaccines

Correlation of Cellular Immune Responses with Protection against Culture-Confirmed Influenza Virus in Young Children

Yearly Influenza Vaccinations: A Double-Edged Sword?

Beyond Clinical Trials: Evolutionary and Epidemiological Considerations for Development of a Universal Influenza Vaccine

We would like to thank Matthias Tenbusch for critically reading the manuscript and for stimulating discussions during the preparation of this review.

The authors declare no conflict of interest.