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CORD-19:12d35c12218f230c49f4dc892814ba9f1037f251 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-113 Sentence denotes OX40:OX40L axis: emerging targets for improving poxvirus- based CD8 + T-cell vaccines against respiratory viruses
T2 115-123 Sentence denotes Abstract
T3 124-266 Sentence denotes The human respiratory tract is the entry point for over 200 known viruses that collectively contribute to millions of annual deaths worldwide.
T4 267-392 Sentence denotes Consequently the World Health Organization has designated respiratory viral infections as a priority for vaccine development.
T5 393-605 Sentence denotes Despite enormous advances in understanding the attributes of a protective mucosal antiviral immune response, current vaccines continue to fail in effectively generating long-lived protective CD8 + Tcell immunity.
T6 606-762 Sentence denotes To date, the majority of licensed human vaccines afford protection against infectious pathogens through the generation of specific immunoglobulin responses.
T7 763-944 Sentence denotes In recent years, the selective manipulation of specific costimulatory pathways, which are critical in regulating T-cell-mediated immune responses, has generated increasing interest.
T8 945-1237 Sentence denotes Impressive results in animal models have shown that the tumor necrosis factor receptor (TNFR) family member OX40 (CD134) and its binding partner OX40L (CD252) are key costimulatory molecules involved in the generation of protective CD8 + T-cell responses at mucosal surfaces such as the lung.
T9 1238-1407 Sentence denotes In this review, we highlight these new findings with a particular emphasis on their potential as immunological adjuvants to enhance poxvirus-based CD8 + T-cell vaccines.
T10 1409-1614 Sentence denotes The mucosal surfaces of the respiratory tract provide optimal conditions for efficient gaseous exchange and, unfortunately, an ideal portal of entry for innocuous environmental antigen and human pathogens.
T11 1615-1862 Sentence denotes Consequently, the body's respiratory mucosal surfaces contain a complex array of immune regulatory mechanisms that ensures, at least in healthy individuals, a quiescent and non-inflammatory environment that maintains optimal tissue function (1-3).
T12 1863-2188 Sentence denotes However, once a pathogen establishes an infection or the regulatory mechanisms fail, a rapid cascade of events leads to the production of inflammatory cytokines that recruit immune cells in an attempt to eradicate the ensuing pathogen/antigenic stimuli while minimizing the impact on the physiological function of the tissue.
T13 2189-2457 Sentence denotes This ideal conclusion, however, often does not occur, as many human pathogens have evolved virulence and immune modulatory mechanisms that circumvent and disrupt mucosal immune responses resulting in tissue pathology, clinical disease, and possible long-term sequelae.
T14 2458-2906 Sentence denotes To help minimize the detrimental impact of respiratory infections, vaccines were developed against many of the most prevalent respiratory pathogens, including Bordatella pertussis (whooping cough), Corynebacterium diphtheriae (diptheria), Haemophilus influenzae type b (Hib), and Streptococcus pneumoniae (pneumococcal pneumonia and otitis media), successfully reducing infant mortality and the burden of infectious diseases worldwide (4) (5) (6) .
T15 2907-3241 Sentence denotes Yet, despite the continued success and widespread use of respiratory pathogen vaccines, Mycobacterium tuberculosis (TB) and a multitude of respiratory viral infections continue to cause significant morbidity and result in millions of futile deaths each year (7) (8) (9) [World Health Organization (WHO) 2004 Global Burden of Disease].
T16 3242-3344 Sentence denotes Influenza virus alone causes seasonal epidemics that can affect 10-20% of the global population (10) .
T17 3345-3609 Sentence denotes Recent estimates suggest that seasonal influenza viral infections are responsible for between 250,000 to 500,000 deaths annually, which can increase during pandemics caused by the emergence of a novel re-assortment viral strain (WHO 2004 Global Burden of Disease).
T18 3610-3811 Sentence denotes Furthermore, the increasing numbers of deaths attributed to human transmission of highly pathogenic avian influenza strains (H5N1) will elevate influenza virus associated morbidity and mortality (11) .
T19 3812-4129 Sentence denotes In addition to influenza virus, parainfluenza virus, respiratory syncytial virus (RSV), meta-pneumonia virus, severe acute respiratory syndrome coronavirus (SARS-CoV), rhinovirus, measles, and adenovirus are endemic within the human population and can establish acute respiratory tract infection (11) (12) (13) (14) .
T20 4130-4245 Sentence denotes With a few exceptions, existing approaches have failed to develop effective vaccines against these viral pathogens.
T21 4246-4517 Sentence denotes Ominously, the public health impact of respiratory infections is likely to increase in the near future due to aging global populations, increasing antibiotic resistance (in the case of TB and pneumococcus) and altering social attitudes toward vaccination (14) (15) (16) .
T22 4518-4994 Sentence denotes Moreover, the continuing emergence of novel respiratory viruses (through antigenic recombination events and zoonosis), such as the 2009 H1N1 influenza A virus strain, highly pathogenic avian influenza viruses, SARS coronavirus and human cases of monkeypox (11, 17, 18) , taken together with the continued concern of bio-terrorism (anthrax and smallpox) (19, 20) adds to the urgent need to better understand the pathogenesis of respiratory viruses and mechanisms of protection.
T23 4995-5278 Sentence denotes This review discusses the rationale for developing CD8 + T-cell vaccines against existing and emerging human respiratory viruses, and then reviews our current understanding of antigenspecific CD8 + T-cell induction and memory formation in the context of respiratory viral infections.
T24 5279-5394 Sentence denotes The argument will be made that applying this knowledge will be critical in future success of CD8 + T-cell vaccines.
T25 5395-5694 Sentence denotes We then examine how attenuated poxviruses have been developed over the past three decades as candidate vaccines for a variety of mucosal pathogens and discuss how future efforts should focus on understanding in molecular terms why live non-attenuated vaccines result in better CD8 + T-cell immunity.
T26 5695-5961 Sentence denotes In the final section, we discuss how members of tumor necrosis factor receptor (TNFR)/TNF superfamily, specifically, OX40 (CD134) and its binding partner OX40L (CD252), are rapidly emerging as key players in the development of protective CD8 + T-cell memory in lung.
T27 5962-6178 Sentence denotes As such, it will become evident that we have the means today to develop a poxvirus-based vaccine delivery system that establishes far better protective CD8 + T-cell immunity in the lung than anything available today.
T28 6179-6430 Sentence denotes Most if not all of today's licensed vaccines work by promoting a robust and long-lived immunoglobulin response that prevents initial pathogen infection and ensuing replication, resulting in pathogen clearance before the onset of any clinical symptoms.
T29 6431-6675 Sentence denotes This apparently straightforward approach of safely generating neutralizing immunoglobulin via the injection of killed, subunit, or highly attenuated agents has resulted in many successful vaccines that confer life-long protective immunity (6) .
T30 6676-6863 Sentence denotes However, in the context of highly pathogenic or rapidly mutating viruses that target mucosal surfaces such as the respiratory tract, this approach has proven far less successful (6, 21) .
T31 6864-7018 Sentence denotes The limitations of generating solely immunoglobulin-mediated protection are highlighted by the necessity to annually develop a seasonal influenza vaccine.
T32 7019-7269 Sentence denotes The protective mechanism of current subunit or inactivated influenza vaccines is through the generation of neutralizing immunoglobulins against hemagglutinin (HA) and neuraminidase (NA), two integral membrane viral proteins essential for infectivity.
T33 7270-7464 Sentence denotes Although immunoglobulins against these surface proteins are longlived, their functional relevance diminishes rapidly due to antigenic drift in both the HA and NA surface glycoproteins (22, 23) .
T34 7465-7679 Sentence denotes This continual antigen drift or evolution also explains, in part, the difficulty in developing an effective vaccine against other intracellular pathogens such as human immunodeficiency virus (HIV) and malaria (6) .
T35 7680-7899 Sentence denotes Both of these pathogens continually mutate, in the case of HIV, or alter, in the case of the plasmodium parasite, key surface antigens resulting in immunological escape from any induced specific immunoglobulin response.
T36 7900-8154 Sentence denotes This continued dilemma has led to the general acceptance that to develop effective vaccines against these types of intracellular pathogens, a combination of immunoglobulin and a long-lasting memory CD8 + T-cell response must be generated (24) (25) (26) .
T37 8155-8379 Sentence denotes This approach has the added benefit of producing cross-protective immunity mediated by CD8 + T cells that recognize conserved internal components of the specific pathogens, therefore conferring heterosubtypic immunity (27) .
T38 8380-8532 Sentence denotes This would be an ideal strategy in the context of a virus that rapidly mutated its external antigens while maintaining more conserved internal antigens.
T39 8533-8754 Sentence denotes Strictly speaking, CD8 + T cells cannot afford protection against infection per se but can mediate faster viral clearance and provide a substantial degree of protection against challenge with a lethal dose of virus (14) .
T40 8755-9090 Sentence denotes Towards this end, the scientific community has focused enormous efforts on the development of prophylactic and therapeutic vaccines that can promote CD8 + T-cell responses with the assumption that such a strategy will enhance immunological protection through providing additional support to existing anti-viral immunoglobulin response.
T41 9091-9203 Sentence denotes To date, however, the development of effective T-cell vaccines against many respiratory viruses remains elusive.
T42 9204-9484 Sentence denotes In the past 10 years, immunologists have provided vaccinologists with valuable information on the regulatory mechanisms that govern not only the efficient generation of CD8 + T-cell responses in the lung but also how different memory CD8 + T-cell subsets are maintained over time.
T43 9485-9603 Sentence denotes The current challenge is to apply this knowledge in our endeavors to develop safe and effective CD8 + T-cell vaccines.
T44 9604-9824 Sentence denotes Viruses are obligate intracellular parasites that upon gaining entry into a host cell hijack the host's transcription and protein synthesis machinery to generate progeny virions that initiate the next cycle of infection.
T45 9825-10200 Sentence denotes Consequently, the respiratory epithelium contains an array of virus detection systems such as Toll-like receptors (TLRs), Nod-like receptors (NLRs), and the cytosolic RNA helicases retinoic acid-inducible gene I (RIG I) and melanoma differentiation associated gene 5 (MDA5) family members that act in concert to rapidly detect the presence of an invading virus (14, 28, 29) .
T46 10201-10443 Sentence denotes Upon viral recognition, a complex interplay between these distinct detection systems results in the activation of an array of transcription factors that culminates in the production of antiviral cytokines and inflammatory mediators (14, 28) .
T47 10444-10744 Sentence denotes In addition to establishing a localized inflammatory environment and recruiting innate effector cells, this initial antiviral inflammatory program is important for the activation and differentiation of lung-resident antigen-presenting cells (APCs), namely dendritic cells (DCs) and macrophages (30) .
T48 10745-10896 Sentence denotes Much of our current understanding of CD8 + T-cell immunity stems from studying small rodent influenza and parainfluenza infection models (14, 31, 32) .
T49 10897-11065 Sentence denotes These models have shown the crucial role CD8 + T cells play in controlling viral titers during primary infection and generating protection against subsequent infection.
T50 11066-11249 Sentence denotes Fig. 1 depicts the events occurring in the lung parenchyma, airways, and draining lymph nodes (LNs) during the initial stages of a sublethal intranasal infection with influenza virus.
T51 11250-11379 Sentence denotes Within a couple of days after infection, viral titers in the lung increase rapidly culminating in acute weight loss and cachexia.
T52 11380-11541 Sentence denotes Subsequently, antigen-loaded or infected respiratory DCs migrate to the mediastinal LNs (mLNs) within 6 h, reaching maximal numbers by 18 h post infection (33) .
T53 11542-11708 Sentence denotes Intravital LN microscopy studies suggest that antigen-bearing DCs migrate to and localize in the vicinity of the high endothelial venules (HEVs) (34) (35) (36) (37) .
T54 11709-11852 Sentence denotes This HEV localization event facilitates the DC interaction with naive antigen-specific T cells entering the LN from the peripheral circulation.
T55 11853-12195 Sentence denotes The relatively rare antigen-specific T cells are subsequently selected through Tcell receptor (TCR)/major histocompatibility complex class I (MHC I) engagement and undergo 11 to 15 rounds of clonal expansion, resulting in a large population of antigenspecific effector CD8 + T cells observed during the peak of the primary response (14, 38) .
T56 12196-12359 Sentence denotes During this expansion phase, CD8 + T cells simultaneously differentiate into functionally distinct effector populations based on their cytokine secretion profiles.
T57 12360-12523 Sentence denotes These include T cytotoxic 1 (TC1), TC2, and TC17 cell populations, which correspond to the better described CD4 + T-helper 1 (Th1), Th2, and Th17 cell populations.
T58 12524-12754 Sentence denotes Homeostatic CD8 + Tcell clone frequency, specific antigenic peptide abundance, and presentation kinetics can collectively influence the development and relative frequency of dominant and subdominant CD8 + T-cell populations (39) .
T59 12755-13107 Sentence denotes As early as day four or five post infection, CD8 + T cells are present in the lung tissue and exhibit several effector functions, including the ability to secrete antiviral cytokines [interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα)] and initiate cell killing of virusinfected epithelial cell through Fas, TRAIL or perforin-mediated lysis (14) .
T60 13108-13382 Sentence denotes The migration of CD8 + T cells to the lung tissue and airways does not appear to be dependent on antigen, although infiltrating CD8 + T cells must acquire a sufficient level of activation to downregulate LN homing receptors and acquire lung homing chemokine receptors (40) .
T61 13383-13561 Sentence denotes Following their arrival in the lung, protective virus-specific CD8 + T-cell responses require additional antigen-dependent interactions with respiratory DCs (30, (41) (42) (43) .
T62 13562-13736 Sentence denotes Respiratory DCs critically contribute to the sustained survival of virus-specific effector CD8 + T cells by trans-presentation of interleukin-15 (IL-15) (30, (41) (42) (43) .
T63 13737-13848 Sentence denotes Recent data also suggest that IL-15 mediates the migration of effector CD8 + T cells to the lung airways (44) .
T64 13849-14124 Sentence denotes Together, these data support a two-hit model for promoting effective CD8 + T\[cell responses: a first hit in the lymph node that primes T-cell proliferation and migration to the site of infection, and a second hit that provides a survival signal to the effector T cell (42) .
T65 14125-14321 Sentence denotes The influx of virusspecific CD8 + T cells into the respiratory tract coincides with decreasing viral titers, recovery of body mass and ultimately viral clearance by day eight to 10 post infection.
T66 14322-14530 Sentence denotes After viral clearance the majority of virus-specific effector CD8 + T cells die through apoptosis, while a small population of surviving effector CD8 + T cells persist and seed a long-lived memory pool (14) .
T67 14531-14691 Sentence denotes A cardinal feature of memory T cells is their ability to mediate faster, stronger, and more effective responses to secondary virus challenge than naive T cells.
T68 14692-14895 Sentence denotes In the case of CD8 + T cells, increased numbers, higher activation status, more rapid induction of effector functions, and altered homing patterns contribute to their enhanced recall responses (14, 32) .
T69 14896-15078 Sentence denotes In recent years, accumulating data has highlighted that memory CD8 + T cells are extremely heterogeneous in terms of their phenotype, function, and anatomical distribution (32, 45) .
T70 15079-15387 Sentence denotes Two major subtypes of memory CD8 + T cells have been defined based on the expression of CD62L (also know as L-selectin), a 'homing receptor' for leukocytes to enter secondary lymphoid tissues via HEVs, and CCR7, a chemokine receptor that supports trafficking through secondary lymphoid tissue (46) (Fig. 2) .
T71 15388-15637 Sentence denotes Central memory T cells (T CM ) are CD62L hi /CCR7 hi cells that tend to localize in secondary lymphoid tissues, whereas effector memory T cells (T EM ) are CD62L lo /CCR7 lo cells that localize or traffic through peripheral tissues such as the lung.
T72 15638-15859 Sentence denotes Considerable interest has been focused on elucidating functional differences between these memory T cell subsets and their capacity to confer protection against secondary infection (24, 32, (47) (48) (49) (50) (51) (52) .
T73 15860-16058 Sentence denotes After secondary encounter with antigen, T EM cells can be stimulated by respiratory DCs without the requirement for further division and can demonstrate rapid cytokine production and lytic activity.
T74 16059-16298 Sentence denotes In contrast, resting T CM cells that reside in the lymph nodes require migratory DCs for their reactivation, resulting in de novo proliferation and a 'new' wave of secondary effector T cells that migrate to the lung parenchyma and airways.
T75 16299-16664 Sentence denotes Based on these observations, it has been proposed that T EM cells participate directly in the initiation of protective memory responses by rapidly producing effector molecules (perforin, granzyme and antiviral cytokines) at the site of antigen encounter, whilst T CM cells contribute to the maintenance and/or amplification of the overall secondary T cell response.
T76 16665-16878 Sentence denotes Therefore, the specific subset of memory CD8 T cells generated by vaccination may critically determine the ultimate effectiveness of vaccine induced immune protection against a natural respiratory viral infection.
T77 16879-17067 Sentence denotes There are several key issues that must be taken into account in order to develop effective CD8 T cell vaccines against respiratory viruses reviewed in detail elsewhere (24-26, 32, 53-56) .
T78 17068-17215 Sentence denotes In this section we will focus on the importance of memory subset (T CM vs T EM ) frequency, longevity of memory subsets, and route of immunization.
T79 17216-17501 Sentence denotes There is substantial clinical evidence to suggest that protective T cell immunity does not last for more than a couple of years after resolution of a natural infection or vaccination, unless the response is boosted by re-exposure to the same or cross-reactive antigens (57) (58) (59) .
T80 17502-17707 Sentence denotes Similarly in animal models of influenza and Sendai virus infection the efficacy of protection against secondary challenge wanes rapidly within three to six months after a primary infection (60) (61) (62) .
T81 17708-17931 Sentence denotes This decline in protective T cell immunity occurs despite the fact that the numbers of virus-specific memory CD8 T cells in the lymphoid tissues (T CM cells) remain relatively high for the life span of the animal (61, 62) .
T82 17932-18207 Sentence denotes These studies suggest that in the context of a respiratory viral infection, T CM cells may not respond, expand, or relocate (to the lung) sufficiently quickly to provide immediate protection against disease caused by reinfection (or live pathogen challenge post vaccination).
T83 18208-18416 Sentence denotes More recent studies have indicated that at one-month post infection, the total numbers of virus-specific T EM cells in the lung are substantially higher than the number of T CM cells in the draining LNs (62).
T84 18417-18750 Sentence denotes However, detailed kinetic studies have revealed that despite stable numbers of memory CD8 T cells in the lymphoid organs, the numbers of lung-and airwayresident memory CD8 T cells following a primary Sendai virus or influenza infection gradually decline over the first three to six months before stabilizing at very low levels (62) .
T85 18751-18949 Sentence denotes Interestingly, this decline and stabilization in the number of memory CD8 T cells in the lung directly correlates with a progressive decline in T cell-mediated protection from a secondary challenge.
T86 18950-19307 Sentence denotes Thus, developing strategies that elicit a sufficiently large number of long-lived T EM cells that are not only cytolytic, but also polyfunctional (defined as being able to produce high levels of anti-viral cytokines IFN-γ, TNF, and IL-2) represents a key challenge for next generation vaccines that are able to confer protection against respiratory viruses.
T87 19308-19511 Sentence denotes In this regard, it is becoming increasingly evident that the strength and duration of antigenic stimulation play important roles in determining both the frequency and long-term persistence of T EM cells.
T88 19512-19541 Sentence denotes This will be discussed below.
T89 19542-19799 Sentence denotes Despite continued disagreement on the precise details of how memory T cell generation occurs, most people accept that the amplitude of a memory T cell pool generated after viral clearance is dictated by the magnitude of the primary effector T cell response.
T90 19800-20035 Sentence denotes This phenomenon has been well studied in a number of infectious mouse models including influenza virus where between five and 10 % of the CD8 T cells elicited during the peak of the immune response transition into memory T cells (63) .
T91 20036-20180 Sentence denotes The primary determinant of the effector CD8 T cell size appears to be a function of the initial amount of antigen present during T cell priming.
T92 20181-20396 Sentence denotes It has been shown that brief periods of antigenic stimulation or antigen availability during priming favor the enrichment of T CM cells, whereas prolonged periods support T EM cell generation ( Figure 2 ) (24, 46) .
T93 20397-20722 Sentence denotes This has important implications for vaccine development against respiratory viruses; for example, the level and time of antigen exposure that can be provided by attenuated vaccines, non-replicating vectors, DNA vaccines, or protein subunit vaccines might be significantly less than those provided by livereplicating vaccines.
T94 20723-20904 Sentence denotes This is likely to be linked to the capacity of replicating vaccines to divide multiple times and disseminate faster, thus increasing the amount and the duration of antigen exposure.
T95 20905-21172 Sentence denotes As will be discussed in more detail below, one of the key questions in inducing large effector-cell burst size for new vaccines will be to define the precise mechanisms that regulate the formation of effector T cells in the context of high or persisting antigen load.
T96 21173-21302 Sentence denotes The role of antigen in determining the long-term persistence of memory T cells has been intensely debated over the last 20 years.
T97 21303-21432 Sentence denotes Initial data from mouse models suggested that T cell memory was short lived in the absence of periodic exposure to antigen (64) .
T98 21433-21623 Sentence denotes However, more recent evidence has challenged this idea and suggested that the number of T CM cells can be sustained in the absence of both specific antigen and MHC molecules (65) (66) (67) .
T99 21624-21867 Sentence denotes Further experimental evidence now suggests that a T CM memory pool is maintained, at least in part, through periodic slow cell division and continuous replacement of cells, a process commonly referred to as homeostatic proliferation (68, 69) .
T100 21868-21978 Sentence denotes This turnover of a memory T CM pool is achieved through tonic stimulation by cytokines such as IL-7 and IL-15.
T101 21979-22084 Sentence denotes In contrast, T EM cells are not directly maintained through IL-15-driven homeostatic proliferation (44) .
T102 22085-22239 Sentence denotes Two different (although not mutually exclusive) models have been proposed to explain the longevity of T EM cells in the absence of re-exposure to antigen.
T103 22240-22430 Sentence denotes Initial studies suggested that T EM cell populations in the lung airways are maintained in an antigen-independent manner by continual recruitment of new cells from the circulation (40, 70) .
T104 22431-22655 Sentence denotes The rate of memory cell recruitment was shown to be extremely rapid, resulting in replacement of 90 % of the population every 10 days and that this can be maintained for periods over one year following viral clearance (70) .
T105 22656-22927 Sentence denotes More recent studies extended these observations by demonstrating that antigen can persist in the lung draining LNs for several weeks after influenza virus clearance and that this residual antigen contributes to the maintenance of T EM cells in the lung airways (71, 72) .
T106 22928-23095 Sentence denotes However, a small number of circulating memory CD8 T cells generated after influenza virus infection can migrate to the airways in the absence of cognate antigen (73) .
T107 23096-23265 Sentence denotes Therefore, memory T cell recruitment to the lung airways is controlled by both antigenindependent and antigen-dependent mechanisms immediately following virus clearance.
T108 23266-23482 Sentence denotes As residual antigen is cleared and the number of memory CD8 T cells localized within the lung airways stabilizes at a low level, antigen-independent processes are required to maintain this population over time (73) .
T109 23483-23669 Sentence denotes Thus, it is clear that the role of persisting antigen after vaccination is a crucial point that has to be addressed during an effective T cell vaccine design against respiratory viruses.
T110 23670-23909 Sentence denotes This evidence clearly demonstrates that the use of conventional non-replicating attenuated vaccine strains will fail to induce a robust and long-lived T EM population and therefore have limited application in the design of future vaccines.
T111 23910-24052 Sentence denotes This explains the need to increase the vaccine dose and/or number of booster immunizations when highly attenuated viral vaccines are utilized.
T112 24053-24127 Sentence denotes These approaches however have obvious logistical and economic limitations.
T113 24128-24396 Sentence denotes Thus, in order to fully realize the potential benefits of attenuated vaccines or simple protein/peptide immunizations it will be of great interest to identify specific molecules and/or pathways that can be targeted to safely lower the activation threshold for antigen.
T114 24397-24538 Sentence denotes Currently, intradermal, intramuscular, oral, intranasal, intravaginal, and even rectal routes of immunization are under active investigation.
T115 24539-24754 Sentence denotes Accumulating evidence from many research groups has confirmed that the route of immunization is critical in determining both qualitative and quantitative aspects of a vaccine induced immune response (74) (75) (76) .
T116 24755-25061 Sentence denotes With regards to T cell vaccines against respiratory viruses, recent studies support the concept that long-lived CD8 T cells can be detected in the lung after mucosal, but not systemic immunization, suggesting that the maintenance of memory T cells are also dependent on the particular route of vaccination.
T117 25062-25137 Sentence denotes Two recent studies provide insight into the mechanisms for this phenomenon.
T118 25138-25324 Sentence denotes Verbist et al. (77) demonstrated that generation, maintenance, and function of memory CD8 T cells after respiratory influenza infection is independent of IL-15-mediated survival signals.
T119 25325-25433 Sentence denotes In contrast, systemically (i.v) induced memory T cells were dependent on IL-15 for their long-term survival.
T120 25434-25534 Sentence denotes Thus, CD8 T cells primed at a mucosal site require distinct signals for their long-term maintenance.
T121 25535-25886 Sentence denotes Shiki Takamura et al. (78) extended these results by showing that intranasally primed memory CD8 T cells possess a unique ability to be reactivated by residual antigen in the MLN compared with intraperitoneally primed CD8 T cells, resulting in the preferential recruitment and maintenance of intranasally primed memory CD8 T cells in the lung airways.
T122 25887-26079 Sentence denotes Furthermore, they demonstrated that the inability of intraperitoneally primed memory CD8 T cells to access residual antigen could be corrected by a subsequent intranasal virus infection (78) .
T123 26080-26285 Sentence denotes Thus, initial CD8 T cell priming in the MLN and prolonged presentation of residual antigen in the MLN, are required to maintain large numbers of virus-specific memory CD8 T cells in the lung airways (78) .
T124 26286-26409 Sentence denotes Clearly, these findings have to be considered for the rational design of CD8 T cell vaccines against respiratory pathogens.
T125 26410-26500 Sentence denotes Several approaches have been used to elicit protective CD8 T cell responses (25, 79, 80) .
T126 26501-26825 Sentence denotes These include the delivery of proteins in replication-competent attenuated recombinant viruses (live attenuated vaccines), in replication-defective attenuated recombinant viruses, in DNA vaccines, protein/peptide subunit vaccines, and through combining one or more of these modalities in a heterologous prime-boost strategy.
T127 26826-26998 Sentence denotes Currently the two most extensively utilized recombinant viral vectors in preclinical and clinical studies are derived from poxviruses and adenoviruses (25, (79) (80) (81) .
T128 26999-27202 Sentence denotes Detailed discussion on adenovirus vectors, DNA vaccines, and protein subunit vaccines will not be provided here, and for further information the reader is referred to the following reviews (25, 79, 80) .
T129 27203-27467 Sentence denotes The focus of the remaining sections of this review center around the poxvirus-based vectors listed in Table 1 , and more specifically how the pursuit of safer vaccines has paradoxically resulted in largely ineffective CD8 T cell vaccines against mucosal pathogens.
T130 27468-27665 Sentence denotes Poxviruses are a family of large enveloped viruses that contain a linear double-stranded DNA genome, ranging in size between 130 to 300 kb pairs, that is capped by a hairpin loop at each end (82) .
T131 27666-27821 Sentence denotes They are also unique in that they encode their own transcription machinery enabling poxviruses to replicate in the cytoplasm of an infected cell (82, 83) .
T132 27822-27997 Sentence denotes The concept of utilizing live-attenuated poxviruses as an immunizing agent against respiratory viruses can be traced back to the global smallpox eradication program (82, 83) .
T133 27998-28155 Sentence denotes The strategy for this program involved mass vaccination with VACV, an antigenically similar virus to the etiological agent of smallpox disease Variola major.
T134 28156-28265 Sentence denotes Many live-attenuated VACV strains (first generation vaccines) were used in different parts of the world (83):
T135 28266-28563 Sentence denotes Lister or Lister/ Elstree strain was the most widely used vaccine globally; New York City Board of Health (NYCBOH; later produced by Wyeth Laboratories as Dryvax) strain was used in the Americas and in West Africa; Copenhagen strain (Denmark), and the Ankara strain (Turkey) were also widely used.
T136 28564-28719 Sentence denotes These VACV variants are known to differ in expression of several virulence factors that determine their replicative capacity and safety profiles (83, 84) .
T137 28720-29008 Sentence denotes It was the ability of these VACV strains to induce robust humoral and cell mediated immunity (82, 83, 85 ) that led to the World Health Organization declaring the eradication of smallpox in 1980 and shortly thereafter routine vaccination with VACV could be discontinued in most countries.
T138 29009-29218 Sentence denotes Serendipitously, at the same time as the global VACV vaccination programme was terminated, novel molecular biological tools had identified VACV as an extremely versatile eukaryotic expression vector (86, 87) .
T139 29219-29417 Sentence denotes One of the main properties of VACV, and poxviruses in general, that provides an advantage over other viral vectors is the relative ease by which large inserts of foreign DNA can be cloned into them.
T140 29418-29507 Sentence denotes This allows for the insertion of multiple genes and the creation of multivalent vaccines.
T141 29508-29730 Sentence denotes Furthermore, poxviruses have the potential to be administered by different routes, such as intradermal, intramuscular, oral, intranasal, intravaginal, and intrarectal routes to generate immunoglobulin and T cell responses.
T142 29731-30014 Sentence denotes Over the last 30 years, a growing number of reports have described the utility of recombinant vaccinia viruses (rVACVs) expressing relevant antigens from multiple viral, bacterial, and parasitic pathogens to confer protection to immunized animals (81, (88) (89) (90) (91) (92) (93) .
T143 30015-30202 Sentence denotes The VACV strain most extensively utilized in animal models has been the highly virulent Western Reserve (WR) strain, which was derived from the NYCBOH strain by multiple passages in mice.
T144 30203-30318 Sentence denotes For obvious safety reasons, the WR strain does not represent a viable strain for use in human vaccine applications.
T145 30319-30606 Sentence denotes Moreover, the well-documented severe, albeit rare, complications following vaccination with first generation VACV strains, and the increasing number of immune compromised individuals have raised legitimate concerns about their widespread use as a vaccine-delivery system in humans (84) .
T146 30607-30888 Sentence denotes Thus, during the past three decades, a major focus of research has been on developing novel, highly attenuated, recombinant VACV strains (rVACV) that demonstrate significantly improved safety profiles and can be used in humans as potential vaccines candidates (83, 84) (Table 1) .
T147 30889-31105 Sentence denotes As briefly mentioned above, the safety concerns surrounding the WR and first-generation VACV-based vaccine candidates have been addressed through the development of highly attenuated VACV strains (83, 84) (Table 1 ).
T148 31106-31222 Sentence denotes These include, attenuated modified vaccinia virus Ankara (MVA; (94) ) and the Copenhagen derived NYVAC strain (95) .
T149 31223-31438 Sentence denotes MVA is replication deficient in mammalian cells, but importantly retains the capacity to synthesize viral proteins, whilst NYVAC is a highly attenuated virus that is unable to produce infectious particles in humans.
T150 31439-31556 Sentence denotes Both virus vectors are extremely safe and can be used in young children and immuno-compromised individuals (84, 96) .
T151 31557-31686 Sentence denotes In addition, an attenuated Avipoxvirus vaccine vector, the canary pox like ALVAC, was also developed for use in humans (97, 98) .
T152 31687-31800 Sentence denotes Due to its restricted tissue tropism to avian cells, ALVAC can be safely used in immune compromised humans (84) .
T153 31801-32020 Sentence denotes In recent years, MVA, NYVAC and ALVAC have all been utilized, with varying degrees of success, as promising vaccine vectors for many infectious diseases including influenza, SARS, HIV, HSV, TB and malaria (81, 84, 99) .
T154 32021-32223 Sentence denotes Furthermore, these novel vaccine vectors have been increasingly used, again with varying degrees of success, in the development of therapeutic vaccines against several common cancer antigens (81, 100) .
T155 32224-32380 Sentence denotes The large number of clinical trials has produced a complicated picture with regards to using these vaccine vectors to generate a robust CD8 T cell response.
T156 32381-32652 Sentence denotes Despite a small number of clinical trials providing promising preliminary evidence for CD8 T cell induction (101, 102) , unfortunately, many trials demonstrate a limited induction of antigen specific CD8 T cell against the infectious disease or cancer target of interest.
T157 32653-32863 Sentence denotes For example, the immunogenicity of an attenuated ALVAC HIV-1 vaccine in humans was shown to be low, eliciting HIV-1-specific CD8 T cell responses in less than 25 % of normal volunteers (103) (104) (105) (106) .
T158 32864-33063 Sentence denotes Similarly, the immunogenicity of MVA expressing the GAG protein (consensus of HIV-1 clade A) and several immunodominant CD8 T cell epitopes was also shown to be unsatisfactory (17 % response) (107) .
T159 33064-33244 Sentence denotes A smaller study using the same vaccines at a higher dose showed enhanced immunogenicity (40 % response), but the T cell responses induced were exclusively CD4 T cell driven (108) .
T160 33245-33615 Sentence denotes With regards to respiratory viruses, a small phase I clinical trial in healthy adults investigating a novel MVA-based heterosubtypic influenza A vaccine (MVA NP +M1) demonstrated that all volunteers had a significant increase in the number of IFN-γ producing CD8 T cells in their blood at weeks one, three and eight after intradermal or intramuscular vaccination (109) .
T161 33616-33735 Sentence denotes This response, however, rapidly declined over time, reaching pre-vaccination levels by week 52 post-vaccination (109) .
T162 33736-34023 Sentence denotes This is consistent with an earlier preclinical study that demonstrated MVA induced T cell responses are followed several weeks later by a dramatic (20-40 fold) contraction, a phenomenon that also occurred with DNA-poxvirus regimens in which NYVAC and ALVAC were used for boosting (110) .
T163 34024-34104 Sentence denotes Another interesting characteristic of MVA vaccines was revealed by Pillai et al.
T164 34105-34250 Sentence denotes 2011 , in which MVA-primed HIV-1 Gag-specific memory cells were found to be predominantly of the T CM phenotype (CD62L and IL-2 positive) (111) .
T165 34251-34499 Sentence denotes Similarly, after a single immunization with MVA vector expressing pre-erythrocytic malaria antigens, Reyes-Sandoval et al. (112) found that MVA induced accelerated T CM rather than T EM generation and consequently failed to protect against malaria.
T166 34500-34710 Sentence denotes These studies suggests that MVA in its current form may not be the most effective poxvirus vector for induction of strong and long-lasting protective CD8 T-cell responses in peripheral tissues such as the lung.
T167 34711-34945 Sentence denotes If one looks back in the literature it becomes evident that these somewhat disappointing clinical results with non-replicating highly attenuated poxviruses could have been predicted based on studies carried out more than 20 years ago.
T168 34946-35107 Sentence denotes A seminal study by Morgan et al. (113) demonstrated that the virulence of a VACV vector critically influences the protective efficacy of the recombinant vaccine.
T169 35108-35372 Sentence denotes Cottontop tamarins vaccinated with a VACV-WRbased recombinant expressing the gp340 envelope antigen of Epstein-Barr virus (EBV) were protected against EBV-induced lymphoma, but animals inoculated with the attenuated Wyeth (VACV-NYCBOH)-based recombinant, were not.
T170 35373-35489 Sentence denotes The precise mechanism for this was not investigated in detail but it appeared to be independent of humoral immunity.
T171 35490-35839 Sentence denotes In summary, 30 years of increasing safety and regulatory concerns surrounding replication competent recombinant poxviruses has unfortunately resulted in the widespread use of vaccine delivery vectors that are ineffective at inducing robust and long-lived memory CD8 T cells necessary to confer protection against highly virulent respiratory viruses.
T172 35840-36080 Sentence denotes At the dawn of recombinant poxvirus-vaccine research, relatively little was known about the precise molecular mechanisms that govern the quantity, phenotype, and quality of memory T cells following infection with a live (replicating) virus.
T173 36081-36236 Sentence denotes In the interim we have gained a number of important insights, which must be considered in our pursuit of safe and effective poxvirus-based T cell vaccines.
T174 36237-36449 Sentence denotes First, replication-competent or highly virulent poxviruses, in general, promote a more potent primary and memory CD8 T cell response compared to that of replication-defective or highly attenuated vaccine strains.
T175 36450-36555 Sentence denotes Second, replication-defective poxviruses predominantly drive the induction of T CM cells over T EM cells.
T176 36556-36813 Sentence denotes Third, in many instances, multiple dosing, or higher dosing (10 7 -10 9 ), with replication-defective or highly attenuated poxvirus vaccine strains are necessary to achieve comparable levels of CD8 T cells to those produced by live poxvirus vaccine strains.
T177 36814-36972 Sentence denotes Lastly, while attenuated poxvirus strains provide good short-term T cell protection, they are not effective at conferring longterm T cell mediated protection.
T178 36973-37137 Sentence denotes As discussed previously, these limitations are logically related to antigen load, antigen persistence and the ability to effectively stimulate CD8 T cell responses.
T179 37138-37356 Sentence denotes Thus, a convincing argument can be made that a key challenge in utilizing attenuated poxvirus vectors to develop efficacious T cell vaccines is to define the molecular mechanisms that link virulence and immunogenicity.
T180 37357-37725 Sentence denotes Towards this end, several recent studies have indicated that key events during initial T cell priming, including inflammatory stimuli (24, 54) , co-stimulation (114), CD4 T cell help (115) , and the cytokine mileu (54), have long lasting programming effects on the quantity, phenotype, and quality of the memory T cells generated in response to a live viral infection.
T181 37726-38043 Sentence denotes In the following sections we will discuss the importance of T cell costimulatory receptors, specifically, we will focus on the importance of TNF/TNFR family members, OX40 and OX40L, in eliciting protective T cell memory and their potential use as immunological adjuvants to enhance poxvirus-based CD8 T cell vaccines.
T182 38044-38249 Sentence denotes It has been hypothesized that the co-evolution of pathogens and their hosts has been a major driving force behind the development of a highly sophisticated and extremely adaptable vertebrate immune system.
T183 38250-38564 Sentence denotes This co-evolution is clearly demonstrated by the fact that many large DNA viruses, such as poxviruses and herpesviruses, possess multiple homologues of cytokines, chemokines, or the associated receptors of these molecules, that can neutralize or modulate host immunity to favour the virus (116) (117) (118) (119) .
T184 38565-38835 Sentence denotes It is assumed that the main goal of these immune modulatory strategies is to enhance viral replication and survival, however it is highly plausible that there exists unknown consequences of viral immune evasion strategies and how these impact the host's immune response.
T185 38836-39103 Sentence denotes Could it be possible that certain molecular pathways that regulate alternative immune responses, such as those involved in autoimmunity, allergic or even anti-tumor responses, might have evolved or be differentially utilized as a consequence of viral evasion tactics.
T186 39104-39313 Sentence denotes An obvious example is the large number of co-stimulatory receptors, especially in the immunoglobulin (Ig) and TNF/ TNFR superfamilies that are expressed on T cells, and known to be critical for their activity.
T187 39314-39590 Sentence denotes CD28-B7, ICOS-ICOSL, and CD2-LFA-3 typify co-stimulatory molecules of the Ig superfamily (120, 121), whereas TNFR/TNF family members are typified by, but are not exclusive to, OX40-OX40L; 4-1BB-4-1BBL; CD27-CD70; GITR-GITRL; CD30-CD30L and HVEM-LIGHT (114, (122) (123) (124) .
T188 39591-39794 Sentence denotes Members of the TNF/TNFR superfamily are recognized for their ability to stimulate T cells and provide signals to promote sustained T cell clonal expansion and long-term survival (114, (122) (123) (124) .
T189 39795-39979 Sentence denotes This is achieved through the selective targeting of intracellular signalling molecules that promote effector cytokine production, cell division and suppress apoptosis (114, 122, 123) .
T190 39980-40164 Sentence denotes Interestingly, there are a number of receptor-ligand pairs within the TNF/TNFR superfamily that are not constitutively expressed but are induced during specific inflammatory scenarios.
T191 40165-40309 Sentence denotes They are thought to provide 'late' signals that subtly regulate T cell responses in both a quantitative and qualitative manner (114, 120, 122) .
T192 40310-40448 Sentence denotes Why so many co-stimulatory molecules exist, with often apparently similar and overlapping functional attributes, remains to be determined.
T193 40449-40676 Sentence denotes Several pox and herpes viruses encode TNFR superfamily homologues (TNFR itself, CD30, and HVEM) that act as decoy receptors to modulate host immune responses to favour viral replication and persistence (125) (126) (127) (128) .
T194 40677-40908 Sentence denotes It is of interest, that recent analysis of the teleost genome, an organism in which the first fully functional adaptive immune system was thought to have developed, has revealed the presence of many human TNF superfamily orthologs.
T195 40909-41048 Sentence denotes This provides evidence to suggest that many of the TNF superfamily members co-emerged around the same time as did antigen receptors (129) .
T196 41049-41203 Sentence denotes Interestingly, however, four ligands were not found to exist within the teleost genome, namely OX40L, CD70 (the ligand for CD27), GITRL, and CD30L (129) .
T197 41204-41498 Sentence denotes As briefly discussed above, these are important regulators of the later phases of T cell expansion and survival, leading to the hypothesis that these ligands may have arisen more recently to allow immune adaptation, plasticity and effective development of protective immunity against pathogens.
T198 41499-41715 Sentence denotes To test this hypothesis, we recently utilized several poxvirus infection models and investigated the use and role of select TNF/TNFR and Ig family members in initiating and sustaining protective CD8 T cell responses.
T199 41716-41980 Sentence denotes Utilizing a panel of VACV strains with varying degrees of virulence in mice, our laboratory recently demonstrated that the relative virulence of a virus/vaccine dictates the quantity and protective capacity of the resulting memory CD8 T cells generated (76, 130) .
T200 41981-42129 Sentence denotes The three VACV strains used in our study included the highly virulent WR strain and two clinically relevant VACV vaccine strains, Lister and NYCBOH.
T201 42130-42266 Sentence denotes All VACV strains are known to differ in their expression of several virulence factors that impact on their replicative capacity in vivo.
T202 42267-42531 Sentence denotes Consequently, we found that after intranasal or intraperitoneal infection only the WR VACV strain replicated rapidly, reaching high titers across multiple tissues (76 and Salek-Ardakani unpublished observations), and resulting in severe and sustained inflammation.
T203 42532-42657 Sentence denotes In contrast, the Lister and NYCBOH strains were cleared more rapidly and were unable to disseminate to the same extent as WR.
T204 42658-42939 Sentence denotes Mice infected with an attenuated VACV vaccine strain (Lister or NYCBOH) via the intranasal or intraperitoneal route generated five to ten-fold fewer lung-resident virusspecific memory CD8 T cells compared to mice infected with the virulent, replication sufficient, WR strain (76) .
T205 42940-43193 Sentence denotes Similar differences were observed at the peak (days seven and eight) of the initial effector CD8 T cell response, implying that altered molecular regulation at this phase of the response could explain the difference in size of the memory pool generated.
T206 43194-43535 Sentence denotes To highlight the importance of CD8 T cells in protecting against subsequent viral encounter, MHC class-II-deficient mice (MHC II −/− ), that lack CD4 T cells and therefore can not generate VACV-specific humoral immunity, were vaccinated with WR, Lister, and NYCBOH and challenged 70 days later with a lethal intranasal dose of VACV-WR (76) .
T207 43536-43667 Sentence denotes All mice vaccinated with the WR strain, the most virulent virus, survived the lethal infection and presented mild disease symptoms.
T208 43668-43877 Sentence denotes In contrast, no protection was evident in mice vaccinated with either attenuated strain (Lister or NYCBOH), resulting in comparable mortality and disease severity to that observed in naïve (unvaccinated) mice.
T209 43878-44153 Sentence denotes This demonstrated that a VACV strain capable of replicating to greater titres, in multiple tissues and over a longer period of time promotes greater numbers of persisting antigen-reactive CD8 T cells that afford protection against a highly lethal respiratory virus challenge.
T210 44154-44315 Sentence denotes In humans, immunization with live-attenuated VACV (Lister or NYCBOH) elicits what was previously thought to be a good memory CD8 T cell response (85, 131, 132) .
T211 44316-44604 Sentence denotes The VACVspecific memory CD8 T cell pool has been shown to persist for many years in the majority of vaccine recipients, however recent observations have highlighted a rapid decline in memory CD8 T cell numbers does occur in a significant group of individuals over time (133) (134) (135) .
T212 44605-44740 Sentence denotes This raised concerns as to whether optimal long-lived CD8 T cell immunity can in fact be generated using first generation VACV strains.
T213 44741-44910 Sentence denotes Our data provides considerable evidence to warrant these concerns as vaccination using the virulent VACV-WR promoted far superior numbers of protective memory CD8 cells.
T214 44911-45112 Sentence denotes This result raised the question as to whether a specific molecular mechanism exists that is engaged during an infection with the virulent WR strain but not the attenuated Lister or NYCBOH VACV strains.
T215 45113-45278 Sentence denotes If alternate molecular control mechanisms are evident, they could potentially be harnessed to enhance CD8 T cell responses generated by attenuated poxvirus vaccines.
T216 45279-45435 Sentence denotes OX40 is a member of the TNFR family and is not constitutively expressed on naïve T cells but is induced hours to days after TCR engagement (114, 122, 124) .
T217 45436-45646 Sentence denotes Maximal expression is typically observed between two and five days following T cell activation after which OX40 is down-regulated, implying a delayed mode of action in primary immune responses (114, 122, 124) .
T218 45647-45833 Sentence denotes Consistent with this, we recently reported that OX40 was seen on a proportion of VACV specific CD8 T cells as early as day four and peaked on day five post-infection with VACV-WR (136) .
T219 45834-46058 Sentence denotes In our initial studies we found that the development of high numbers of effector CD8 T cells capable of producing both IFNγ and TNFα was strongly impaired when OX40 was lacking on a CD8 T cell responding to VACV (124, 136) .
T220 46059-46354 Sentence denotes Analysis of the main immunodominant CD8 T cell populations (B8R, A3L, A8R, A23R, B2R), which account for close to 70 % of the whole VACV-specific response, demonstrated a global impairment in CD8 T cell priming in the absence of OX40 such that the response was reduced by between 70-80 % (136) .
T221 46355-46577 Sentence denotes We further demonstrated that the engagement and activity of OX40 during a virulent viral infection ensured the generation of a high frequency of persisting and functional memory CD8 T cells that located to the lung (136) .
T222 46578-46768 Sentence denotes Most interestingly, OX40 driven induction of lung-resident memory CD8 T cells correlated directly with a robust protection against a highly lethal intranasal infection with VV-WR (76, 136) .
T223 46769-46882 Sentence denotes Subsequently, we assessed the requirement for OX40 after infection with the Lister and NYCBOH VACV strains (76) .
T224 46883-47047 Sentence denotes In striking contrast to VACV-WR, little difference in the frequency of virus-specific effector and memory CD8 T cells was observed in the absence of OX40 signaling.
T225 47048-47197 Sentence denotes Thus, virulence and host evasion mechanisms of VACV-WR result in persistent viral replication and the engagement of the co-stimulatory receptor OX40.
T226 47198-47335 Sentence denotes This then results in enhanced induction of memory CD8 T cells that afford protection against a highly lethal respiratory virus challenge.
T227 47336-47473 Sentence denotes In light of these studies, we postulated whether the differential engagement of OX40 applies to other T cell stimulatory receptors (76) .
T228 47474-47795 Sentence denotes In this regard, recent data from our group and others have shown that in addition to OX40, CD28 (a co-stimulatory receptor in the Ig superfamily) (137, 138) and CD27 (another TNFR family member) (139) , can be utilized during a VACV-WR infection and each can contribute to the generation of effector CD8 T cell responses.
T229 47796-48067 Sentence denotes CD28 and CD27 are distinguishable from OX40 in that they are constitutively present on resting CD8 T cells, but their respective ligands, B7.2 and CD70, are similar to OX40L in that they are induced once an APC receives certain inflammatory stimuli (120, 137, 140, 141) .
T230 48068-48250 Sentence denotes In contrast to our OX40 studies, we found that CD28 was indispensible for optimal effector CD8 T cell generation regardless of whether mice were infected with Lister or NYCBOH (76) .
T231 48251-48394 Sentence denotes However, similar to OX40, deficiency in CD27 resulted in impaired CD8 T cell responses to WR but little or no defect to Lister or NYCBOH (76) .
T232 48395-48584 Sentence denotes Another interesting distinction between infection with the virulent WR and the attenuated viruses was only evident when VACV-specific T cell responses were assessed during the memory phase.
T233 48585-48799 Sentence denotes CD28-deficient mice exhibited defective memory CD8 T cell generation in response to Lister and NYCBOH but interestingly not to WR, even though CD28 was required for an optimal effector response to this virus (76) .
T234 48800-48949 Sentence denotes This indicates that in response to VACV-WR infection, there exists a CD28-dependent phase of CD8 T cell priming followed by a CD28-independent phase.
T235 48950-49187 Sentence denotes CD27 signaling has previously been shown to prolong CD8 T cell survival beyond the effector phase of the immune response and thus serves to increase the number of memory cells by inhibiting effector T cell death (124, (142) (143) (144) .
T236 49188-49414 Sentence denotes Consistent with these results, blocking the CD27 and CD70 interactions in CD28-deficient mice late in the primary response (between days 5 and 8) completely abrogated the CD28-independent CD8 T cell responses to VACV-WR (76) .
T237 49415-49680 Sentence denotes These results support a model in which a temporal sequence of events is necessary for optimal virus-specific CD8 T cell proliferation, survival and memory generation, brought about by a specific sequential engagement of costimulatory molecules CD28, OX40, and CD27.
T1 49681-49985 Sentence denotes In summary, we show that viral virulence and evasion strategies that impact viral replication, dissemination and invariably the inflammatory milieu, can lead to differential use of co-stimulatory receptors for T cells which dictate the magnitude and effectiveness of the CD8 T cell response ( Figure 3 ).
T2 49986-50323 Sentence denotes Furthermore, these data are of high potential significance to vaccination, and promote the notion that the use of attenuated viruses may not elicit the best long-term T cell memory because they do not allow molecules such as OX40 and CD27 or their ligands, that may have evolved for the purpose of promoting T cell memory, to be engaged.
T3 50324-50606 Sentence denotes This provides a hypothetical model that might in part explain the abundance of such stimulatory receptors for T cells, and demonstrate that molecular plasticity can occur during anti-viral responses where certain immune mechanisms thought non-essential could become highly relevant.
T4 50607-50828 Sentence denotes Contrary to our VACV-WR studies, but similar to the data with VACV-Lister and VACV-NYCBOH discussed above, the regulation of CD8 T cell responses in other viral settings is variably dependent on OX40 and OX40L signalling.
T5 50829-51182 Sentence denotes Initial studies in OX40 −/− and OX40L −/− mice independently infected with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), Theiler's murine encephalomyelitis (TMEV) and influenza virus demonstrated no defect in control of viral replication or the generation of effector CD8 T cell populations (124, (145) (146) (147) (148) .
T6 51183-51455 Sentence denotes However, recent studies investigating the impact of OX40/OX40L signalling on memory and recall CD8 T cell responses demonstrated a reduction in the number and secondary expansion of memory CD8 T cells in the lung, despite not influencing initial CD8 T cell priming (148) .
T7 51456-51624 Sentence denotes Similarly, OX40 signalling regulates the accumulation of CD8 T cells reactive with the persistent-phase epitopes during a mouse cytomegalovirus (MCMV) infection (149) .
T8 51625-51733 Sentence denotes Collectively these data suggest that no two viruses initiate the same combination of costimulatory pathways.
T9 51734-51929 Sentence denotes This raises many questions surrounding the specific signals that initiate the 'program' of co-stimulatory pathways required for induction, maintenance and recall of memory CD8 T cell populations.
T10 51930-52215 Sentence denotes As discussed above, the level of virus replication and persistence, brought about by virulence and immune evasion strategies, is one parameter that dictates the differential use of co-stimulatory receptors and subsequently the magnitude and efficacy of the ensuing CD8 T cell response.
T11 52216-52403 Sentence denotes Furthermore, the inflammatory signature initiated by a specific virus strain or the presence of virus specific virulence factors may also authorize particular co-stimulatory interactions.
T12 52404-52571 Sentence denotes APC derived cytokines such as IL-1, IL-2, IL-12, and TNFα have been shown to influence the extent and length of co-stimulatory receptors/ligand expression (114, 124) .
T13 52572-52774 Sentence denotes Whilst a recent LCMV study revealed a differential requirement for type I IFNs in controlling the initial expansion and generation of CD8 T cells that is not used in response to a VACV infection (150) .
T14 52775-52909 Sentence denotes Furthermore the CD40 engagement, TLR ligation and IL-18 derived signals can also regulate the expression of OX40L on APCs (114, 140) .
T15 52910-53151 Sentence denotes In summary, the extent of viral replication, in terms of peak titre and tissue tropism, and the unique inflammatory signature of a specific viral infection is likely to influence the use of a specific combination of co-stimulatory molecules.
T16 53152-53432 Sentence denotes Further studies to investigate the spatial and temporal expression profiles of both co-stimulatory ligands and receptors in different viral settings will almost certainly add to our understanding and help in the development of safe yet effective vaccines against viral infections.
T17 53433-54077 Sentence denotes Accumulating evidence from our laboratory and others support the notion that a critical component of any future CD8 T cell vaccine against highly virulent respiratory viruses must be the capacity to (1) promote robust expansion of naïve precursor T cells reactive with viral antigenic peptides; (2) allow a high frequency of these virus-specific effector T cells to survive over time as memory T cells; (3) allow high numbers of these memory cells to persist in the lung parenchyma and airways in the absence of persisting antigen; and (4) to retain high effector function in order to provide optimal surveillance against subsequent infections.
T18 54078-54249 Sentence denotes As alluded to earlier, replication-defective highly attenuated poxvirus vectors, although extremely safe, are unlikely to satisfy a number of these important requirements.
T19 54250-54608 Sentence denotes Consequently, a number of innovative strategies have been developed to facilitate and enhance the immunogenicity of attenuated poxvirus vectors, including the use of heterologous prime-boost regiments (151) , co-administration of TLR agonists (152) , cytokines (153) (154) (155) (156) (157) , and targeting T cell co-stimulatory receptors (158) (159) (160) .
T20 54609-54806 Sentence denotes Our studies, highlighted above, suggest that attenuated VACV-vaccine strains will not elicit the most effective CD8 T cell memory responses in the lungs due to the lack of OX40 and CD27 engagement.
T21 54807-54964 Sentence denotes Therefore, we investigated whether engagement of OX40 would boost the response to attenuated VACV vaccine strains by treating with an agonist antibody (76) .
T22 54965-55137 Sentence denotes Agonistic OX40 treatment during the initial priming of CD8 T cells markedly enhanced the numbers of effector and memory cells not only in the spleen, but also in the lungs.
T23 55138-55248 Sentence denotes This was observed regardless of whether vaccination was via the intraperitoneal or dermal scarification route.
T24 55249-55462 Sentence denotes Most significantly, agonistic OX40 provided strong protection against a lethal respiratory virus challenge in MHC II −/− mice vaccinated with Lister or NYCBOH, which alone were ineffective at preventing lethality.
T25 55463-55643 Sentence denotes The extent of protection when mice were vaccinated with the combination of attenuated vaccine strains and agonistic OX40 was similar to mice vaccinated with the virulent WR strain.
T26 55644-55810 Sentence denotes To extend these results, we focused on priming CD8 T cells by subcutaneous immunization with the immunodominant VACV peptide epitope, B8R 20-27 , given in IFA (161) .
T27 55811-56001 Sentence denotes This strategy allowed us to assess the role of memory CD8 T cells in the absence of any preexisting VACV-specific immunoglobulin and in the presence of an intact naïve CD4 T cell population.
T28 56002-56120 Sentence denotes Several weeks later, once memory cells had developed, mice were challenged intranasally with a lethal dose of VACV-WR.
T29 56121-56391 Sentence denotes Correlating with our prior data, agonistic OX40 provided strong protection in a CD8-dependent CD4-independent manner in mice vaccinated with a high dose of B8R [20] [21] [22] [23] [24] [25] [26] [27] peptide, in that no disease, as measured by weight loss, was observed.
T30 56392-56726 Sentence denotes It should be stressed that prevention of weight loss during the first seven days of infection is extremely difficult to achieve, especially with such high challenge doses (x 300 LD 50 ) that were used in our experiments, and therefore an important demonstration of the utility of agonistically targeting OX40 in anti-viral protection.
T31 56727-56895 Sentence denotes More interestingly, targeting OX40 induced almost complete protection in mice immunized with a low dose of B8R 20-27 that alone was ineffective at preventing lethality.
T32 56896-57000 Sentence denotes This was reflected in both minimal loss of weight and mortality, as well as lung immunopathology (161) .
T33 57001-57235 Sentence denotes As before, the extent of protection directly correlated with the number of IFN-γ producing B8Rspecific memory CD8 cells that were generated in the lung after OX40 engagement, and the number that accumulated after intranasal infection.
T34 57236-57463 Sentence denotes These findings are reminiscent of those obtained with secondary influenza or Sendai virus infection, in which the presence of significant numbers of CD8 T cells in the lung before challenge correlated with immunity (162, 163) .
T35 57464-57642 Sentence denotes The development of polyfunctional CD8 T cells, which are considered the most potent memory cells for anti-viral immunity, is a powerful metric for potency of adjuvanticity (55) .
T36 57643-57923 Sentence denotes Agonist OX40 treatment at the time of vaccination elicits highly polyfunctional B8Rspecific CD8 T cells that persisted, specifically, in the lung tissue in high numbers for at least 18-months after secondary challenge with VACV and importantly, was independent of CD4 T cell help.
T37 57924-58101 Sentence denotes As previously discussed, to effectively promote lung tropic memory CD8 T cells both the route of vaccination and the virulence/immunogenicity of the vaccine vector is paramount.
T38 58102-58370 Sentence denotes Collectively, our studies demonstrate that intraperitoneal infection with attenuated VACV vaccine strains or subcutaneous vaccination of a viral peptide in the presence of an agonistic OX40 antibody can generate long-lasting memory CD8 T cell that persist in the lung.
T39 58371-58598 Sentence denotes Therefore, targeting OX40 during priming of VACV-specific CD8 T cells elicits fully protective and long-lived anti-viral CD8 T cell responses in the lung irrespective of the site of vaccination and the context of viral antigen.
T40 58599-58750 Sentence denotes Several other groups have raised the possibility that triggering OX40 could enhance vaccination efficacy against other viral (and non viral) pathogens.
T41 58751-59033 Sentence denotes The use of pox-viral vectors that encode an antigen of interest in combination with OX40L has demonstrated enhanced splenic CD4 and CD8 T cell responses to a hepatitis B surface antigen (HBsAg) (164) and improved protective efficacy of a foot-and-mouth disease (FMD) vaccine (165) .
T42 59034-59260 Sentence denotes Furthermore, a recombinant poxvirus vector encoding OX40L in combination with B7-1, ICAM-1 and LFA-3 enhanced both CD4 and CD8 T cell proliferative capacity and cytokine production after antigen re-stimulation in vitro (166) .
T43 59261-59511 Sentence denotes A similar approach, in which a HIV-1 canary pox vaccine vector was co-administered intramuscularly with an OX40L-expressing canarypox vector resulted in the expansion of HIV-specific CD8 T cells detected in the spleen six weeks post infection (167) .
T44 59512-59611 Sentence denotes The use of an agonistic OX40 antibody and fusion proteins has also produced very promising results.
T45 59612-59779 Sentence denotes Co-administration of an agonistic OX40 antibody with SIV-gp130 in rhesus monkeys resulted in enhanced virus-specific T cell and immunoglobulin recall responses (168) .
T46 59780-59947 Sentence denotes Whilst the targeting of OX40 using an agonistic mAb elicited strong antiviral CD8 T cells during a MCMV infection, resulting in enhanced clearance of the virus (149) .
T47 59948-60118 Sentence denotes Furthermore, stimulation of OX40, in cooperation with 4-1BB, during vaccination with an OVA-expressing poxvirus vector also enhanced OVA-specific memory responses (169) .
T48 60119-60265 Sentence denotes This approach also promoted fungal clearance and enhanced T cell responses against the respiratory fungal pathogen Cryptococcus neoformans (170) .
T49 60266-60514 Sentence denotes Collectively, these studies suggest that OX40 is a promising target for enhancing T cell responses against a variety of antigens, and that agonist reagents, to OX40, or OX40L encoded within a vaccine vector, might be useful in vaccination regimens.
T50 60515-60734 Sentence denotes This strategy could enable the highly desirable ability to retain the use of attenuated vaccines or simple peptide or protein immunization whilst promote both immunoglobulin and T cell components of the immune response.
T51 60735-60884 Sentence denotes For a more comprehensive insight into the molecular mechanisms of agonistic OX40 therapies refer to (171) (172) (173) (174) (175) (176) (177) (178) .
T52 60885-61065 Sentence denotes Without doubt the greatest concern with enhancing CD8 T cell responses through the use of agonistic OX40 antibodies or vector encoded OX40L is a hyper-inflammatory immune response.
T53 61066-61290 Sentence denotes A clinical trial investigating the propensity of inducing Treg development through the use of an anti-CD28 super-agonist antibody (TGN1412) resulted in six human volunteers experiencing life-threatening complications (179) .
T54 61291-61450 Sentence denotes Consequently, future clinical trials targeting T cell stimulatory receptors will undoubtedly face more stringent safety requirements by regulatory authorities.
T55 61451-61674 Sentence denotes To this end, toxicity trials in non-human primates have already demonstrated that agonistic OX40 antibody therapy is well tolerated and safe, therefore providing a strong rationale to pursue clinical tests in humans (168) .
T56 61675-61816 Sentence denotes Direct interaction between viral pathogens and OX40 itself also raise concerns surrounding the targeting of OX40 to enhance T cell responses.
T57 61817-61949 Sentence denotes Recently, OX40 was identified as cell entry receptor for both feline (FIV) (180, 181) and human immunodeficiency virus (HIV) (182) .
T58 61950-62141 Sentence denotes By replicating in and subsequently killing OX40 expressing cells, both FIV and HIV would preferentially deplete virus-specific T cells that would otherwise mediate antiviral immune responses.
T59 62142-62228 Sentence denotes T cell tropic viruses may also exploit the OX40-OX40L axis to aid their dissemination.
T60 62229-62458 Sentence denotes For example, infection with Human T cell leukemia virus type 1 (HTLV-1), the causative agent of adult T cell leukemia (ATL), enhances OX40 expression in a cooperative process involving NF-κB and the viral oncoprotein, Tax (183) .
T61 62459-62696 Sentence denotes Intriguingly, a small number of ATL patients displayed OX40 dependent adhesion of leukemic cells to endothelial cells (184) , suggesting that virus-induced OX40 expression could enhance leukemic cell infiltration and viral dissemination.
T62 62697-62812 Sentence denotes Viruses can hijack transcriptional activation events triggered by OX40 to induce the expression of their own genes.
T63 62813-63037 Sentence denotes A number of viruses including cytomegalovirus (185, 186) and HIV (187) have NF-κB response elements incorporated into their genomes which can enhance viral reactivation, in the case of CMV, and viral replication (182, 188) .
T64 63038-63193 Sentence denotes The possible influence of OX40 on the replication, survival and/or reactivation from latency of viruses represents an interesting area for future research.
T65 63194-63318 Sentence denotes Finally, research into the potential pathogenicity of enhanced T cell responses upon natural virus re-exposure is necessary.
T66 63319-63509 Sentence denotes An inappropriate memory T cell response upon re-exposure to RSV after vaccination with a formalin-inactivated vaccine resulted in several infant deaths and countless hospitalizations (189) .
T67 63510-63737 Sentence denotes Consequently it would be necessary to determine the safe upper limit for the number of memory CD8 T cells desired and also whether maintaining large numbers of tissue resident (lung) memory CD8 T cells is detrimental over time.
T68 63738-64080 Sentence denotes In summary, although OX40 is a promising target for enhancing protection against infection and reducing viral-induced immunopathology, the positive effect OX40 may have on virus replication and survival in infected host cells raises new considerations for its manipulation, making the infectious status of the patient of paramount importance.
T69 64081-64320 Sentence denotes Accumulating evidence now indicates that targeting human OX40 and OX40L holds great promise for future vaccine strategies against highly virulent respiratory viruses and other unmet clinical infectious diseases such as HIV, Malaria and TB.
T70 64321-64634 Sentence denotes The literature discussed above provide compelling evidence to suggests the targeting of co-stimulatory pathways such as OX40/OX40L, or other similar molecules in this family, can promote robust expansion of antigen-specific effector T cells in response to safe attenuated VACV vaccine strains and peptide antigen.
T71 64635-64887 Sentence denotes Furthermore, OX40 stimulation generates long-lived memory effector T cells that are capable of surviving at mucosal sites in the absence of persisting antigen whilst maintaining their effector function to provide protection against natural re-exposure.
T72 64888-65180 Sentence denotes There is no doubt that increasing our understanding of basic CD8 T cell biology and the dynamic regulatory influences the many co-stimulatory pathways have on the generation, phenotype, maintenance and re-activation of CD8 T cells will facilitate the development of novel CD8 T cell vaccines.
T73 65181-65459 Sentence denotes A key area of research that will provide important insight into the plasticity and adaptation of co-stimulatory regulation of T cell responses is identifying the differences in inflammatory signature between viruses that elicit potent CD8 T cell responses and those that do not.
T74 65460-65625 Sentence denotes This will enable us to recapitulate the signature of a virulent virus whilst using a safe attenuated vaccine virus to promote life long CD8 T cell mediated immunity.
T75 65626-65965 Sentence denotes (1) Upon viral infection, lung resident immature dendritic cells (DC) take up viral antigen, differentiate, and migrate to the regional draining lymph nodes. (2) Antigen-bearing DCs migrate to and localize in the vicinity of high endothelial venules (HEV) and interact with naïve T cells that enter the lymph node (LN) via the circulation.
T76 65966-66744 Sentence denotes Viral specific T cells are subsequently selected through TCR/MHC I engagement and undergo rapid clonal expansion and differentiation. (3) Effector T cells migrate to the site of infection via the circulation where they receive survival signals, in the form of IL-15, from respiratory DCs (4) and through the release of anti-viral cytokines and effector molecules kill virus infected epithelial cells. (5) After viral clearance the majority of virus-specific effector CD8 T cells die through apoptosis, whilst a hand full of surviving effector CD8 T cells persist and seed a long-lived memory pool (6) comprised of central memory T cells (CD62L hi , CCR7 hi ), that reside in lymphoid tissue, and effector memory T cells (CD62L lo , CCR7 lo ) that reside at mucosal surfaces (7).
T77 66745-66866 Sentence denotes Upon activation, naive CD8 T cells develop into either central memory T cells (T CM ) or effector memory T cells (T EM ).
T78 66867-66980 Sentence denotes As indicated T CM and T EM cells are distinguishable based on their phenotype, function, and anatomical location.
T79 66981-67083 Sentence denotes The T CM and T EM fate decision occurs early during priming, determined by antigen access and/or dose.
T80 67084-67253 Sentence denotes Brief periods of antigenic stimulation or antigen availability during priming favor the enrichment of T CM cells, whereas prolonged periods support T EM cell generation.
T81 67254-67358 Sentence denotes T EM and T CM have the ability to interchange phenotype, dictated by the presence or absence of antigen.
T82 67359-67379 Sentence denotes Modified from (46) .
T83 67380-67597 Sentence denotes The model explains why the strongly replicating (live) vaccinia virus (VACV) Western Resrve strain (VACV-WR) results in better CD8 T cell immunity as compared with attenuated VACV strains (VACV-Lister or VACV-NYCBOH).
T84 67598-67849 Sentence denotes The level of virus replication, brought about by virulence and immune evasion tactics, can lead to differential use by a CD8 T cell of stimulatory receptors in the TNFR and Ig superfamilies, and that this dictates the magnitude of the T cell response.
T85 67850-68036 Sentence denotes Two TNFR family members, OX40 and CD27, drive the generation of memory CD8 T cells with virus that replicates strongly, or when higher doses of attenuated virus are used for inoculation.
T86 68037-68261 Sentence denotes In contrast, CD28/B7 interactions, but not OX40/OX40L or CD27/CD70 interactions, are used to generate memory responses to attenuated viruses that are rapidly cleared, and this corresponds with strongly reduced T cell memory.
T87 68262-68411 Sentence denotes This differential molecular use and altered CD8 T cell memory determines the ability of the host to protect against subsequent respiratory infection.