Towards Future T Cell-Mediated Influenza Vaccines
Thi H. O. Nguyen, Marios Koutsakos, Emma J. Grant, Peter C. Doherty, Katherine Kedzierska
From the Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville 3010, Victoria, Australia (Thi H. O. Nguyen, Marios Koutsakos, Emma J. Grant, Peter C. Doherty, Katherine Kedzierska);Department of Immunology, St Jude Children’s Research Hospital, Memphis, Tennesse 38105, USA (Peter C. Doherty).
Correspondence to:Associate Professor Katherine Kedzierska, Department of Microbiology & Immunology, University of Melbourne, The Peter Doherty Institute for Infection & Immunity, Level 7, 792 Elizabeth Street, Melbourne, 3000, VIC Australia.Email:

Influenza A virus infections (IAVs) impact significantly on global health, being particularly problematic in children, the elderly, pregnant women, indigenous populations and people with co-morbidities. Antibody-based vaccines require annual administration to combat rapidly acquired mutations modifying the surface haemagglutinin (HA) and neuraminidase (NA) glycoproteins. Conversely, influenza-specific CD8+ T cell responses directed at peptides derived from the more conserved internal virus proteins are known to be protective, suggesting that T cell-based vaccines may provide long-lasting cross-protection. This review outlines the importance of CD8+ T cell immunity to seasonal influenza and pandemic IAVs and summarises current vaccination strategies for inducing durable CD8+ T cell memory. Aspectsof future IAV vaccine design and the useof live virus challenge in humans to establish proof of principle are also discussed.

Keyword: Influenza viruses; T cells; Vaccines

Despite annual vaccination, IAV (and influenza B virus, IBV) infectionscause significant morbidity and mortality worldwide, with approximately 250, 000-500, 000 annual deaths in the course of seasonal epidemics and millions more during some historic pandemics (WHO influenza centre website). Seasonal epidemics generally result from point mutations in the surface HA and NAglycoproteins[1], a phenomenon referred to as antigenic drift (Figure 1A, i). In 2014, for instance, the previously circulating A/H3N2/Texas/50/2012 and B/Massachusetts/02/2012 strains were replaced in the southern hemisphere with the “ drifted” A/H3N2/Switzerland/9715293/2013 and B/Phuket/3073/2013, respectively[2], with the consequence that there were two mismatches in the 2014 Fluvax® vaccine. Conversely, “ antigenic shift” , where a novel IAV enters a population that has no pre-existing HA- or NA-specific neutralising antibodies, is the primary cause of influenza pandemics. Such “ shift” is typically a consequence of gene reassortment (or packaging)between one or more IAVs (Fig. 1A, ii) that may have been circulating in humans, domestic livestock or wildlife, typically aquatic birds[3]. The consequence is the emergence of a novel (in the antibody sense) IAVthat can spread rapidly (the 2009 H1N1 IAV) and/or cause high mortality (the 1918 H1N1 IAV).

Figure 1. Variations in influenza A virus.(A) Influenza has two main mechanisms of variation; (i) point mutations occur within the genome which result in slight variations within the virus and thus antigenically drift, and (ii) reassortment, whereby two distinct viruses co-infect the same cell and the segmented genome is reassorted to create a novel virus, and this is typically responsible for antigenic shift. (B) Influenza A viruses circulating during the last century were influenced by pandemics in 1918, 1957, 1968 and 2009 (highlighted by a pink arrow).

The onslaught of the A/H1N1 IAV in 1918 (known as the “ Spanish ‘ flu” ) is thought to have caused the most devastating pandemic on record[1], with > 40 million deaths globally (Fig. 1B). Later pandemics through the 20th and early 21st centuries have been much less severe though, with rapid air travel, they spread across the planet much more rapidly than was the case in 1918/19. The most recent (2009)involved a triple reassortant swine-derived H1N1 virus, A/H1N1/California/7/2009 or pH1N1, which was “ around the world” in less than 6 months and, though generally regarded as mild, caused severe and fatal cases in young, previously healthy individuals, heavily pregnant women, the obese and in indigenous populations[4], raising concerns of further mutations and more deaths. This pandemic strain replaced the previously circulating H1N1 virus and is still co-circulating in the northern and southern hemispheres.

The continuing threat from novel, and potentially pandemic IAVs was emphasised in 2013 following a substantial incidence (in China) of fatal disease following bird to human transmission of avian A/H7N9 virus[5]. Live poultry markets were rapidly shut down to prevent further avian to human spread and to limit the possibility of subsequent transmission between people. Exacerbated by the fact that this H7N9 IAV causes asymptomatic infections in birds, which make control more difficult, further human H7N9 outbreaks have occurred, with the mortality rate being 38% (principally in the elderly) in over 600 cases, > 99% of whom were hospitalised with severe pneumonia (97.3%)[5, 6]. So far, like the earlier concern about the extremely virulent (for both birds and humans) avian H5N1 viruses, though, this H7N9 virus has not changed to spread readily between people.

The inactivated and attenuated influenza vaccines that are currently available operate principally by inducing sterilising immunity in the form of neutralising serum IgG antibodies directed at the IAV and IBV HA and NA surface glycoproteins. Most commonly used[7]are trivalent inactivated influenza vaccines (TIV), with current products consisting of purified, inactivated, “ split” virions from the three circulating strains A/H1N1, A/H3N2 and IBVvariants predicted by the WHO to have “ epidemic potential” . Similarly, the quadrivalent inactivated vaccine (QIV), is formulated with inactivated split virus and comprises an additional IBV component. The approach is cumbersome, partly because these seasonal vaccines need to be updated annually to match the current circulating strains, due to continuing antigenic changes in the HA and NA proteins. Another disadvantage is thatfailures occur when the circulating strains do not match the vaccine components[8]that, for production reasons, must be predicted months ahead. And, of course, there is no protection against any novel, pandemic IAV. Broadly cross-reactive (for HA) neutralising antibodies are rarely detected in humans following seasonal infection or vaccination, but have been demonstrated after non-exposed individuals were vaccinated with the monovalent pandemic H1N1 vaccine[9]. Additionally, patients hospitalised with H7N9-infections had minimal H7N9-specific neutralising antibody titreson admission[10], further highlighting issues with the antigenicity of some (particularly avian) HAs. Such findings also raise the question whether molecular manipulation of these proteins (or added adjuvants) might enhance responsiveness to vaccine formulations. Nevertheless, neutralizing antibody-based vaccines have shown protection against laboratory-confirmed infection with 50% influenza vaccine efficacy for general practice patients and 39% for patients admitted to hospital[11]. Multiple groups have sincecharacterised broadly neutralising antibodies for “ next generation” antibody-based vaccines, with such approaches being reviewed elsewhere[12, 13]. However there is, as yet, no obvious way of making an antibody-based vaccine that routinely mediates cross-protection against mutant “ seasonal” or novel pandemic strains.

It has been known for some time that established CD8+ T cell-mediated immunity offers a measure of broad, if not necessarily immediate, protection against influenza infection across different virus strains, offering an alternative approach (to annual vaccination) for achieving longer-lasting protection against severe disease caused by novel pandemic IAVs and mutant (in the HA and NA) IAVs and IBVs. Influenza-specific CD8+ T cells recognise peptides derived from the relatively conserved internal viral proteins (matrix protein, M1; nucleoprotein, NP; and polymerase basic protein 1, PB1) presented on the surface of variant MHCclass I molecules. In this review, we probe the possibilityof developing vaccine strategies that promote effective, long-lasting CD8+ T cell memory that might be recalled following infection with novel (in the neutralising antibody sense) IAVs and IBVs.The advantage of such T cell based vaccines offer broader protection without the need for annual vaccination. However, current T-cell based vaccines using live attenuated influenza viruses have produced only modest results and are challenged by the need to optimally design vaccines to ensure effective priming, boosting and maintenance of the T cell response against influenza viruses, which will also be discussed.


During infection, CD8+cytotoxic T lymphocytes (CTLs) promote pathogen clearance by killing infected cells and reducing viral spread by releasing cytotoxic granules (containing perforin and granzymes) and secreting pro-inflammatory cytokines[14, 15, 16], such as IFN-, TNF〈 and IL-2. Conversely, “ helper” CD4+ T cells[17]facilitate dendritic cells (DC) maturation, drive Ig class-switching in B cells, and promote stronger, higher affinity antibody responses[18]. The CD4+set also secretes antiviral and pro-inflammatory cytokines to recruit elements of innate immunity to sites of infection. Many experiments have shown over the decades[19, 20, 21, 22] that naï ve C57Bl6 (B6) and BALB/c mice primed with one IAV strain (e.g. H1N1) clear virus more quickly, and suffer less severe disease following intranasal (i.n.) challenge with a heterologous IAV (e.g. H3N2), though this protection can be over-ridden when extremely virulent IAVs are used. Similarly, isogenic guinea pigs primed with pH1N1-2009 intratracheally showed partial protection against H7N9 challenge[23]. Building on earlier findings in animal studies, the importance of CD8+ T cell-mediated immunity in human influenza was first established when Andrew McMichael and colleagues showed that the magnitude of CTL responsesprior to infection in healthy volunteers correlated positively with the capacity to clear an A/H1N1/Munich/1/79 virus following respiratory challenge[24]. Furthermore, the findings of a retrospective surveillance study suggested that individuals previously infected with H1N1 in the early 1950s were less susceptible to the 1957 H2N2 pandemic IAV[25], again supporting the idea of cross-protection, or heterosubtypic immunity, although the techniques (and understanding of CTL responses) were not available at that time.

The conservation of internal IAV proteins (M1, NP and PB1) and, as a consequence, the immunogenic peptides that bind to widely expressed HLA types in Caucasians (e.g. HLA-A2[26] and Han Chinese (e.g. HLA-A2, HLA-A11, HLA-A24[27, 28]) allows IAV-specific CD8+CTLs to provide a measure of cross-reactive, or heterosubtypic, immunity[29, 30, 31, 32, 33]. The protective capacity of heterosubtypic T cell immunity against the 2009 pH1N1 was evident in a household cohort study, where the presence of pre-existing CD8+ T cell responses in people who were seronegative at baseline prior to pH1N1 infectionsuffered less severe illness[34]. Conversely, establishedIAV-specific CD4+, and to a lesser extend CD8+, T cell memory correlated with lower virus shedding and fewer clinical symptoms following human challenge of seronegative donors with mild H3N2 strain[35]. Additionally, CD8+ T cells specific for highly conserved epitopes can persist in the blood of healthy individuals over a 13-year time-course[33], providing evidence on longevity of established cross-protective CD8+ T cell immunity towards influenza viruses.

One of the most abundant, and well-characterised, IAV-specific CD8+ T cell responses[36] is that to HLA-A* 02:01/M158-66.This prominence is reflected in the fact that theGILGFVFTL M158-66peptide sequence is present in the 1918 pandemic IAV[32], with there being a history of only sporadic mutations over the ensuing 97 years (Valkenburg SA and KK, PNAS, in press). Other immunodominant “ cross-protective” influenza-specific CD8+ (and CD4+) T cell epitopes, restricted by various HLA alleles, have been identified by screening T cells with overlapping peptides from a range of IAV proteins[31, 35, 37, 38]. For instance, CD8+ and CD4+ T cell responses were observed (mainly towards M1 and NP) in H5N1-seronegative but H1N1-seropositive individuals following stimulation with overlapping peptides spanning the H5N1 proteome[31]. Similar studies have shown that NP and M1 are major targets for IAV-specific T cell immunity[34, 35, 37, 38].

With the emergence of the novel H7N9 virus, Quinones-Parra and colleagues identified 6 universal CD8+ T cell epitopes that were 100% conserved in known human IAVs (from the 1918 pandemic virus to this H7N9 strain), suggesting that such pre-existing CTL-mediated immunity may, if at significant levels, provide a measure of protection against novel IAVs[32]. These include HLA-A* 02:01/M158-66, HLA-A* 03:01/NP265-273, HLA-B* 08:01/NP225-233, HLA-B* 18:01/NP219-226, HLA-B* 57:01/NP199-207 and HLA-B* 27:05/NP383-391 (although mutants emerged with time in H3N2) epitopes, reflecting that these HLA types are common in Caucasian populations[39]. Interestingly, only limited CTL responses were detected for H7N9-derived peptides that bind HLA-A* 01:01, A* 68:01, B* 15:01 and A* 24:02, suggesting that ethnicities favouring these HLA types, in particular the Alaskan and Australian Indigenous populations[32], should be more vulnerable to H7N9 infection, as they have been in the past to novel H1N1 pandemic strains. Thus, notwithstanding the high frequency of CD8+ T cell responses directed at HLA-A* 02:01/M158-66and other well-known, highly conserved epitopes, designing peptide-based T cell vaccines that only cover the major HLA types clearly disadvantages such Indigenous populations[40].

Furthermore, an epitope that is prominent in the overall CTL response generated within one individual may be much less emphasized, or even absent, in another who also expresses the relevant HLA allele. Such poorly understood (in the mechanistic sense) immunodominance hierarchies have been well definedin murine models, with variations in CTL population size being variously related (reviewed in [41]) to the relative magnitudes of the naï ve CD8+CTL precursor (CTLp) sets[42, 43], the route of infection[44], the type of antigen-presenting cell (APC), protein abundance in such APCs[45], the efficiency of processing and presentation of a particular peptide, the affinity of the peptide for HLA and the affinity of the TCR for the pHLA complex[46, 47, 48]. These immunodominance hierarchies are less well defined for outbred human populations than for inbred mouse strains, but they could impacton CTL response magnitudes to particular vaccine preparations.

Interestingly, the degree of epitope sequence homologybetween different viruses does not necessarily correlate with immunodominance. Protection against H7N9 challenge in mice primed with an H1N1 or H9N2 virus correlated with the size of the primary influenza-specific memory CD8+ T cell pool, not the extent of epitope conservation between the different viruses[19]. Furthermore, differentimmunodominance hierarchies were observed in the primary and secondary (recall) responses and could not be entirely explained by the degree of epitope conservation, thus further highlighting the complexity of the mechanisms that underpin heterosubtypic immunity and cross-protection.

In Wang et al.’ s unique human natural infection study, some hospitalised H7N9-infected patients discharged within 2-3 weeks displayed an early, robust H7N9-specific CD8+ CTL response (6-8 days after disease onset), which was then followed by the neutralising antibody response (9-17 days after disease onset). In contrast, the IAV-specific CTL response in patients that later succumbed to infection was delayed, or minimal[10, 49]. These rapid CD8+ T cell responses identified in surviving individuals likely originated from pre-existing, heterosubtypic memory, though lack of knowledge of previous IAV infection history makes this a speculative conclusion. Nevertheless, global H7N9-specific CD8+ and CD4+ T cell responses were successfully measured in patients ex vivo following PBMC stimulation with live whole H7N9 virus, without the need for prior knowledge of immunodominant epitopes or the HLA types of the subjects[10].

There is, therefore, mounting evidence that human heterosubtypic CD8+ T cell immunity resulting from natural IAV infection provides an important degree of cross-protection against new and emerging viruses in the absence of neutralising antibodies. Whether T cell-based vaccines can be generated to maximally enhance cross-protection, i.e. mimic natural infection, remains to be elucidated.


Although the TIV and QIV vaccines induce strong humoral immune responses, they typically elicit little, if any, CD8+ T cell memory. Similarly, although aiming to induce both humoral and CTL responses, the live attenuated influenza vaccine (LAIV) FluMist® (licenced in selected countries)generally elicits poor T cell responses. Interestingly, the immunogenicity of these vaccines was dependent on the type of vaccine and age group. Adults vaccinated with TIV or LAIV experienced limited increases from baseline in IFN-γ production by influenza-specific CD8+ T cell populations following restimulation (< 0.1% increase on d28 over d0, n=20)[50]. Vaccination with a H5N1 LAIV[51] did increase the number of HLA-A* 02:01/M158-66-specific CD8+ T cells in HLA-A2+ individuals, but only marginally (< 0.1% over pre-exposure). In children aged 5-9 years old, giving a TIV did not induce any change in IAV-reactive T cell prevalence, whilst a LAIV only slightly increased the frequency of IFN-+CD8+ T cells (< 0.5% on day 28 over day 0). Conversely, children aged 0.5-4 years old without previous influenza vaccination experience showed a limited (< 0.3%) rise in CD8+ T cell numbers on d10 following the first TIV dose but these increases reverted to baseline on d10 after the second TIV dose[50].

Within the same cohort study, phenotypic evidence of antigen-specific responses (CD27 and perforin expression)was found to be dependent on the age group and type of vaccine for IAV-specific CD8+T cells. In adults (n=10), modest changes in frequency forCD27+ (~10% decrease on d10 vsd0) and perforin+(~2% increase on d10 vs d0) were detected in IFN-+ IAV-specific CD8+ T cells after LAIV (but not TIV) priming. Moreover, the d10 “ activation” phenotypes were transient and revertedto baseline levels by d28, consistent with the observed lack of change inIFN-+ IAV-specific CD8+T cell frequencies. Children (5-9 years) showed similar patterns for both LAIV and TIV vaccines. In 9 IAV-naï ve children (0.5-4 years), the phenotype of IAV-specific CD8+ T cells changed significantly after two doses of TIV, with a ~20% decrease in CD27 (p< 0.01) and a ~30% increase for perforin (p< 0.01) on d10 compared to d0. Whether the phenotypic changes observed in the 0.5-4 years old group following two doses of TIV were stable or transient was not assessed[52].

Other studies have looked at the consequences of vaccinating children (12-35 months old) with combinations of TIV or LAIV using prime-boost protocols (TIV/TIV, TIV/LAIV, LAIV/TIV, LAIV/LAIV). Children (n=10-13 per group) who received at least one dose of LAIV displayed only a small increase (< 2-fold) in the numbers of proliferating IFN-+CD8+ T cells following virus challenge in vitroon d60 after vaccination, although this was statistically significant (p< 0.05)[53]. In a subsequent study, 0.5-3 year olds were vaccinated with an adjuvanted (n=14) or non-adjuvanted (n=15) inactivated pH1N1 vaccine. Those given the adjuvanted vaccine (vs the unadjuvanted counterpart) displayed higher levels of IFN-+ production for T cells restimulated in vitro with peptides spanning the M1, NP and NS1 IAV proteins.Boosting with a seasonal TIV vaccine a year later indicated that the number of IFN--secreting cells was increased in both groups (~3-fold), but most of the responders were CD4+ T cells (88% of IFN- producers) rather than the CD8+CTLs[54]. Overall, none of the currently licenced influenza vaccines have been able to induce strong and robust CD8+ T cell responses.

Novel vaccine formulations have been assessed for their ability to induce CD8+ T cell responses and provide protection. One such formulation is the modified vaccinia virus Ankara (MVA)-NP+M1 recombinant. This engineered poxvirus encodes two internal proteins, NP and M1 of a H3N2 IAV (A/Panama/2007/99) under the control of the viral promoter. In various cohorts (including the elderly), the extremely attenuated (compared with the historic vaccinia used to eradicate smallpox) MVA-NP+M1 has been deemed safe and well tolerated[55, 56, 57]. Vaccination of healthy volunteers with MVA-NP+M1 increased the number of IFN-γ -secreting CD8+ T cells in adults on d56(< 0.5% over d0, n=8)[56] and the elderly on d7 and d21 (< 0.2% over d0, n=30)[55]. Similarly, vaccination of HLA-A2-expressing individuals (n=6) increased the number of tetramer+ HLA-A* 02:01/M158-66CD8+T cells (< 0.5% over controls)[57]. Additionally, these HLA-A* 02:01/M158-66-specific CD8+T cellsshowed higher levels of effector molecules such as granzyme A and perforin[58]. Encouragingly, MVA-NP+M1-vaccinated human volunteers challenged i.n. with A/Wisconsin/67/2005 displayed increased protection against influenza infection compared to unvaccinated controls[58]. Furthermore, of those that were infected, vaccinated individuals had lower overall symptom scores andthese observations translated into 60% vaccine efficacy[57].Although the cohort was small (n=11 per group) and results need to be replicated in larger field studies.

Flu-v (comprising 4 peptides, 3 from IAV and 1 from IBV) offers a novel approach for influenza vaccination[59, 60, 61] and is deemed safe and well tolerated in Phase I clinical trials. PBMCs from Flu-v primed individuals (vs unvaccinated controls)showed < 4x increase in IFN- production (probably by CD8+ T cells) following in vitrostimulation with peptides[59, 62]. However, following challenge with A/H3N2/Wisconsin/67/2005, no statistically significant differences in mean viral shedding or symptom score were detected for vaccinated versus unvaccinated volunteers. There was a significant negative correlation between IFN-γ levels and total virus shedding, but this correlation did not hold true for IFN-γ production and total symptom score[62].

Other products intended to promote CD8+ T cell-mediated immunity are the FP-0.01 and Multimeric-001 vaccines, both of which are currently in Phase I clinical trials. FP-0.01 consists of fluorocarbon-conjugated peptides from IAV NP, M1, PB1 and PB2 proteins. Although the frequency of IFN-+CD8+ T cells was marginally increased (< 0.1% over day 0), PBMCs derived from FP-0.01-vaccinated individuals produced significantly more (compared to unvaccinated controls) IFN- following stimulation with peptides or with IAV-infected, HLA-matched cells[63]. Similarly, the Multimeric-001candidate vaccine consists of a large protein comprising 9 highly conservedpeptides from IAV HA, NP and M1 proteins. Compared to PBMCs from unvaccinated controls, PBMCs from Multimeric-001-vaccinated individuals displayed an increase in proliferation and cytokine production following stimulation with peptides, the Multimeric-001 vaccine or live virus[64].


Many different factors can potentially influence the immune response to virus infections (summarised in Fig. 2), and should be taken into consideration for future influenza vaccine design, as discussed below.

Figure 2. Future influenza vaccinations require multiple considerations. Multiple factors influence the efficacy of vaccines and thus for effective future influenza vaccine design, many facets need to be considered including: (i) the route of vaccination to induce protective immunity at the site of infection, (ii) the vaccination regimen that maximises the generation of long-lasting protective immunity, (iii) which parts of the innate and/or adaptive immune system to target, and (iv) the DC subset required to activate these immune correlates.

4.1 Targeting distinct DC subsets may enhance CD8+CTL activation

Efficacy presumably reflects the efficient delivery of vaccine antigens to the professional APCs that activate CD8+ T cells. Cross-presenting DCs may perhaps be the most important APC, as depleting CD11c+ DCs using diphtheria toxin receptor abrogates CD8+CTL primingin murine models[65].

Though DC biology is complex, they have (at least for mice) been categorised into crude (albeit distinct) lineages with defined functions[66]. Intriguingly, both CD8+ and CD103+ DCs are vital for the activation of naï ve CD8+ T cells (reviewed in [66]). Studies utilising wild type (C56BL/6) or Batf-/- mice (deficient in CD8+and CD103+CD11b-DC subsets) demonstrated that influenza-specific CD8+CTLs from naï ve Batf-/- mice displayed diminished expansionon d10after infection with the PR8 strain of IAV[67], indicating that it is important to target the relevant DC subsets for maximal IAV-specific CTL stimulation[67], both in the natural course of infection and for future IAV vaccine design. Expectedly, DC subsets are more complicated in humans than murine models. Even so, there is evidence that comparable, distinct DC subsets with defined functions actually exist in humans (reviewed in [68]).

Limited studies in mouse models suggest that the activation of naï ve and memory CD8+ T cells may depend on the involvement of different DC populations (reviewed in [69]). This is evidenced by one study where CD8a-CD11b- lung-derived DCs (isolated from mice infected with IAV presenting the HSV gB-1 peptide) were capable of activating naï ve but not memory gBT-I-specific CD8+ T cells derived from gBT-I transgenic mice[70]. Human vaccination studies by He et al.[50] showed that priming with a LAIV induces ~0.3% increases in CD8+ T cell immunity forchildren, however, though this was not detected in adults. It would not be surprising if the rules for vaccinating naï ve individuals (children) versus boosting pre-existing immunity in individuals who have memory IAV-specific immunitywere indeed different. However, the available evidence suggests thatmost children have been exposed to influenza by the age of 7[71], suggesting that a single vaccine formulation designed to boost pre-existing, heterosubtypic immunity would be suitable for the majority of the population.

4.2 Routes of vaccination may also influence CD8+ T cell responses

At least in the mouse, distinct DC subsets (reviewed in [66]) are found in different anatomical locations and exhibit varied functions (reviewed in [72]). This may explain why the route of vaccination impacts the quality and magnitude of the resulting immune response[22, 73, 74]. Giving different vectors via different routes will likely lead to variation in the location of DC priming and the prominence of one or other DC subset. For example, vaccination with therecombinant MVA.HIVA-NP vector vaccine in miceintravenously (i.v.) led to extensive, antigen-specific division of CD8+ T cells in the spleen[74]. Conversely, intradermal (i.d.) exposure led to responses localised to the lymph nodes nearest to the site of infection, whilst intramuscular (i.m.) administration resulted in limited CD8+ T cell proliferation in the non-draining lymph nodesand spleen[74]. Others have shown that i.d.priming is more effective than i.m. or subcutaneous vaccination against multiple viral infections including rabies, measles, polio, HBV and influenza (reviewed in [73]). More relevantly, DC priming and immune activation has been assessed in mice given infectious IAVsvia non-respiratory routes that do not permit virus replication[22]. DC priming by i.m. IAVinfection is localised to the draining lymph nodes, whilst i.p. exposure results in more systemic priming of DCs[22]. Together, these studies suggest that the route may be important for generating an optimal immune response(reviewed in [75]) and, though there may be little relevant information for memory CTL generation in humans, this variable should be considered in future influenza vaccine design.

4.3 Tissue resident memory cells confer protection against IAV

Tissue-resident memory (TRM) CD8+ T cells play an important role in protecting mice against localized HSV challenge[76]. Primed at the site of infection, the TRMs characteristically express CD69 and CD103(reviewed in [77]) and remain localized following antigen clearance to provide a first line of regional defense[76]. Recently, studies in murine models have suggested that lung-resident CD8+ T cells provide some protection against IAV challenge. Wu et al.[78] showed that the presence of TRM CD8+ T cellsdeveloped by i.n. IAV priming correlated with early virus clearance in the lung-draining mediastinal lymph node following challenge with a heterosubtypic IAV strain[78]. Similarly, Wakim et al.[79] induced IAV-specific lung-resident CD8+ T cells using antibody-targeted vaccination, and showed that these TRM CTL cells protect mice from lethal challenge with PR8-OVA[79]. Perhaps more physiologically relevant, IAV-specific TRMs have been identified in the lungs of rhesus macaques following i.n. prime and intratracheal boost with pH1N1 virus, however their role in protection against subsequent IAV challenge is yet to be elucidated[80].

Unfortunately, little is known about the protective capacity of TRMs in humans. To date, the most informative studies have utilised transgenic mice (NOD-SCID B2m-/-) lacking a functional DC compartment. These mice had their DC subsets reconstituted using human CD34+ hematopoietic progenitor cells (HPCs), then vaccinated with LAIV (or their DCs were harvested and incubated with LAIV in vitro) and the functional capacity of the DC subsets was assessed. They showed that CD1c+ DCs were important for the generation of TRM (CD103+CD69+) CD8+ T cells following LAIV vaccination, with an important role being assigned to TGFb[81]. Thus, as previously suggested (reviewed in [82]), it is also important to consider the TRMs when designing future IAV vaccines that depend (at least in part) on heterosubtypic CTL immunity.


Looking at the “ experiment of nature” provided by IAV “ seasonal” epidemics and pandemics, Epstein et al.[25] and Sridhar et al.[34] have provided important insights into T cell-mediated cross-protection against novel IAVs. With increased global awareness, better surveillance networks and advances in immunological/virological analysis, well designed prospective studies are critical for understanding the spectrum of factors influencing IAV severity, from mild seasonal strains[83] to the more serious avian-derived H5N1[84] and H7N9 viruses[10] that have caused significant morbidity and mortality in South-East Asia. Such studies are, however, rare and time-consuming because they rely on the recruitment of large cohorts of seronegative participants prior to natural heterosubtypic infection. Additionally, as pandemic events are unpredictable and often far apart, relying on such intermittent research strategy is not ideal.

Human live virus challenge studies offer a promising opportunity for establishing a “ proof-of-principle” into the possible utility and durability of protective T cell-based vaccines. Seronegative humans can be vaccinated or infected with a mild dose of influenza and subsequently challenged with a heterosubtypic virus in a clinically controlled environment, allowing the collection of valuable clinical, virological and immunological data at multiple time points. Given that good products are available for testing, such studies would allow the rapid development, evaluation and implementation of novel T cell-based vaccines and anti-viral therapies. In addition, the power of modern technologies (which require relatively few PBMCs for analysis) means that we would gain key mechanistic insights into the important determinants underpinning IAV protection and disease severity under controlled conditions that are not readily replicated in field studies.Though we cannot assess highly pathogenic or lethal strains using this approach, it may be possible to use avirulent, engineered variants for some studies. Interestingly, human infection models are now being utilised to advance vaccine design and development for dengue viruses[85, 86] and malaria[87].

To the best of our knowledge, human experimental studies involving sequential exposures with different live IAVshave not been conducted. Rather, a small handful of human single infection models have been reported. The early work of McMichael and colleagues demonstrated the safety and efficacy of infecting human volunteers i.n.with live unattenuated A/H1N1/Munich/1/79 virus grown in hen’ s eggs, which showed a correlation between CD8+ T cell responses and viral clearance[24]. Similarly, Wilkinson and colleagues[35]challenged healthy adult volunteers with either cell-grown A/H3N2/Wisconsin/67/05 or egg-grown A/H1N1/Brisbane/59/07, in a study that suggested CD4+ T cells responses correlated with less illness severity. In an earlier A/H1N1/Brisbane/59/07-infected cohort, subsequent analysis of the B cell response showed that virus-specific antibody-secreting cells were present in the blood at 7dafter infection, most likely originating from the B cell memory compartment[88].

Recently the pH1N1-2009 A/California/2009 strain was utilised in a clinical trial. Individuals from three separate cohorts were inoculated i.n.with 1/1000 (n=5, ~3.5× 106 TCID50), 1/100 (n=12) or 1/10 dilution (n=12) of neat virus, respectively. Symptom scores, virus shedding, antibody titres, nasal discharge weight and haematology cell countswere assessed, though there was no concurrent description of the CTL response[89]. It is anticipated that this influenza strain will be used in forthcoming clinical trials to assess the potency of novel vaccines and anti-viral therapies.


It is important to note that immunological analysis of PBMCs does not necessarily correlate with what’ s happening in the lymphoid tissue or at the site of infection in the lung. This was addressed in a prospective study of 84 naturally infected individuals (including infants), where cellular responses, viral loads and cytokines were assessed in the nasal lavage and blood to determine correlates of disease severity[83]. Although there were no clear associations between clinical outcome, age or viral load, differential immune profiles were observed for the airways and the blood, while increased levels of nasal MCP-3, nasal IFN-a2 and plasma IL-10 at the time of confirmed influenza by PCR predicted progression to severe disease[83].

Although we have numerous assays to assess CD8+CTLs in whole blood and PBMCs, continuing improvements in cellular immunological techniques (i.e. multi-parameter flow cytometry, peptide/MHC tetramers and TCR profiling) in combination with high-throughput systems biology techniques down to the single-cell level (i.e. Fluidigm, RNA-seq) will also facilitate novel in-depth analysis of specimens, thus further advancing our efforts to develop an effective broadly cross-protective T cell based vaccine. Furthermore, additional work is needed to develop easy and robust methods to measure cellular immunity in the airways, particularly with very limited material. Additionally, recent findings suggest that both arms of the adaptive immune response[10], together with the innate response[10, 83] and associated inflammatory mediators[49], play an important role in fine-tuning the host’ s immune response to limit IAV severity and disease in humans, and that all these factors can (and should) be assessed when probing the possible utility of future influenza vaccines.


It is long been known (from experiments with laboratory animals and now from correlative studies in humans) that cross-reactive CD8+ CTL-mediated immunity plays an important part in controlling IAV infections. With the continuing threat of novel pandemics, evidence for heterosubtypic CTL protection in the absence of neutralising antibodies, suggests that there is a casefor developing effective and long-lasting vaccines that depend, at least in part, on priming CD8+T cell memory. Substantial research is required to develop effective vaccines, and to establish optimal regimes for priming, boosting and maintaining protective IAVCTLs including (perhaps) those localised in the respiratory tract. Knowledge gained from these studies should provide a platform for new T cell-based vaccine strategies that target both influenza A and B viruses. Very little is known about influenza B-specific T cell-mediated immunity (reviewed in [90, 91]), yet the IBVscan (every 3-4 years) be more important than the IAVs and cause severe clinical impairment. Overall, future clinical infection/human challenge studies are needed to establishthe proof-of-principal that a T cell-based vaccine will be useful for (in particular) IAV pandemic preparedness.


This work is supported by a University of Melbourne International Fee Remission Scholarship (awarded to M.K.); a University of Melbourne International Research Scholarship (awarded to M.K.); Australian National Health and Medical Research Council (NHMRC) CJ Martin Biomedical Early Career Fellowship (awarded to E.J.G.); NHMRC Senior Research Fellowship Level B (awarded to K.K.); and NHMRC Program Grant 1071916 (awarded to P.C.D. and K.K.).

The authors have declared that no competing interests exist.

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