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The Journal of Immunology, 2007, 179, 2187-2194
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Context of Epitope Presentation Can Influence Functional Quality of Recalled Influenza A Virus-Specific Memory CD8+ T Cells1

E. Bridie Day, Weiguang Zeng, Peter C. Doherty, David C. Jackson, Katherine Kedzierska2 and Stephen J. Turner2,3

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lipopeptide constructs offer a novel strategy for eliciting effective cellular and humoral immunity by directly targeting the vaccine Ag to dendritic cells. Importantly, it is not known how closely immunity generated after lipopeptide vaccination mimics that generated after natural infection. We have used a novel lipopeptide vaccine strategy to analyze both the quantity and quality of CD8+ T cell immunity to an influenza A virus epitope derived from the acidic polymerase protein (PA224) in B6 mice. Vaccination with the PA224 lipopeptide resulted in accelerated viral clearance after subsequent influenza virus infection. The lipopeptide was also effective at recalling secondary DbPA224 responses in the lung. Lipopeptide recalled DbPA224-specific CTL produced lower levels of IFN-{gamma} and TNF-{alpha}, but produced similar levels of IL-2 when compared with DbPA224-specific CTL recalled after virus infection. Furthermore, lipopeptide- and virus-recalled CTL demonstrated similar TCR avidity. Interestingly, lipopeptide administration resulted in expansion of DbPA224-specific CTL using a normally subdominant TCRBV gene segment. Overall, these results demonstrate that protective CTL responses elicited by lipopeptide vaccines can be correlated with TCR avidity, IL-2 production, and broad TCR repertoire diversity. Furthermore, factors that impact the quality of immunity are discussed. These factors are important considerations when evaluating the efficacy of novel vaccine strategies that target dendritic cells for eliciting cellular immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Efficient resolution from viral infection often relies on the induction of effective CD8+ cytotoxic T cell (CTL) immunity (1). After infection, viral peptides (p) are presented by the MHC class I (MHCI)4 glycoproteins on the cell surface in an allele-specific manner (2). Upon recognition of pMHCI complexes on activated dendritic cells (DCs) via the clonally expressed TCR (3), naive CTL undergo a program of differentiation resulting in the acquisition of effector functions and rapid expansion (4, 5, 6). After a pathogen has been cleared, CTL numbers contract leaving behind a small population of long-lived memory CTL (4, 6). Memory CTL are characteristically different to naive CTL in that they are now present in higher numbers and display more rapid effector function upon recognition of pMHCI (7). Given the important role for CTL in limiting viral replication and spread, the induction of CTL responses by vaccination is thought to be a means of providing effective protection from the consequences of viral infection (1).

Generally, most current vaccine strategies are based on intact pathogens where the pathogen has been rendered nonpathogenic, either by attenuation or inactivation. The rationale for using a vaccine similar to the "natural" form of the pathogen is the expectation that such vaccines will induce immunity similar to that induced by natural infection. Importantly, while vaccine strategies based on inactivated pathogens are effective at inducing protective Ab responses, they are poor at inducing CTL immunity (8). This is likely due to the inappropriate delivery of Ag to the appropriate Ag-presentation pathway, and the absence of a danger signal required to appropriately activate DCs for priming of CTL responses. Recently, recognition of the importance of DCs in priming effective CTL responses has resulted in vaccine strategies that use either transfer of Ag-loaded DCs (9), or synthetic constructs that target the Ag to DCs in vivo (10).

Synthetic lipopeptide vaccines have demonstrated potential as a novel vaccine strategy for eliciting cellular immunity (11, 12). Their construction consists of minimal peptide determinants (both CD4 and CD8 T cell epitopes) conjugated to a lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys) derived from Mycoplasma fermentans (13). The Pam2Cys component of the vaccine formulation is a potent activator of DCs via the TLR2 (14, 15). In mouse models, lipopeptide vaccination has provided robust cellular immunity against viral, bacterial, and tumor challenge (8, 12, 16). However, it is unclear how the quality of CTL immunity induced after lipopeptide administration mimics that observed after virus infection. To determine this, a comparison of various immune parameters is warranted as it could reveal novel immune correlates that may be useful in evaluating the efficacy of vaccine strategies.

Respiratory infection of C57BL/6 (H2b) mice with influenza A virus causes an acute, localized pneumonia (17) and results in a CTL response that is directed against at least six viral peptides (18). The most prominent, and most well-characterized, responses are directed against peptides derived from the viral nucleoprotein (NP366–374, H2Db binding; Ref. 19) and the acid polymerase (PA224–236, H2Db binding; Ref. 20). Comparison of these influenza-specific CTLs has demonstrated several quantitative and qualitative differences. For example, during primary infection, the magnitude of the CD8+ T cell response to each of these determinants is roughly equivalent (21, 22, 23). However, the DbNP366-specific set dominates the response after secondary challenge (20, 23). Functional analysis shows that relatively more CD8+DbPA224+ compared with CD8+DbNP366+ T cells make TNF-{alpha} and IL-2 after in vitro peptide stimulation (21, 22, 24). This greater functional capacity also correlates with a higher avidity pMHCI interaction (22, 25).

Given the potential of lipopeptide vaccination to elicit enhanced CTL responses and the need for identifying novel immune correlates, we were interested in how lipopeptide vaccination impacted various measures of CTL quality. We investigated the capacity of lipopeptide vaccination to elicit DbPA224-specific responses, and protect from subsequent influenza A virus challenge. We also compared the quantity and functional quality of the DbPA224-specific CTL generated after both lipopeptide vaccination and infection to establish how closely this vaccination strategy mimics the immune quality established after infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Synthesis and purification of lipidated and nonlipidated epitope-based vaccines

Synthetic peptide and lipopeptide constructs were synthesized as previously described (13). The constructs incorporated an immunodominant H2Db-restricted CD8+ T cell determinant from the acid polymerase of influenza A virus (SSLENFRAYV; PA224) and a CD4+ T cell epitope derived from the fusion protein of the morbillivirus, canine distemper virus (KLIPNASLIENCTKAEL) (26). Peptides were assembled linearly on F-moc-Wang resin (Auspep) using conventional solid-phase F-moc chemistry. The nonlipidated construct Th-PA, contained the CD4+ and CD8+ T cell epitopes, separated in sequence by a lysine residue. The lipidated construct, Th-P2C-PA, incorporated the lipid moiety Pam2Cys, derived from the macrophage-activating lipopeptide 2 isolated from M. fermentans, attached to the central lysine through two serine residues (13). Constructs were purified by HPLC and authenticated by analytical reverse-phase chromatography and mass spectrometry.

Inoculation/infection of mice

C57BL/6J (B6, H2Db) mice were bred at the University of Melbourne (Parkville, Australia). To study various aspects of the immune response to epitope-based vaccines, a variety of inoculation/infection schedules were used, with mice receiving either a 9 or 45 nmol dosage. For analysis of primary responses, naive B6 mice were anesthetized with methoxyflurane and inoculated intranasally (i.n.) with the peptide immunogens delivered in 30 µl of PBS or infected i.n. with 104 PFU influenza virus A/HK-x31 (HKx31, H3N2). To study recall responses, mice were primed by i.p. injection with 1.5 x 107 PFU A/PR8/34 (PR8, H1N1) virus at least 6 wk before inoculation with peptide constructs or secondary challenge with 104 PFU HKx31 virus. For viral challenge experiments, mice were either primed with 1.5 x 107 PFU PR8 virus or inoculated i.n. with peptide constructs. Six weeks postpriming, mice were infected i.n. with 104 PFU of a mutant HKx31 virus (HKx31 NP N5Q), which contained a glutamine substitution at position 5 of the immunodominant viral epitope derived from the influenza A nucleoprotein (NP366) (27). This mutation completely abolishes presentation of the NP366 epitope by H2Db and was used to assay immune protection in the absence of a DbNP366-specific CTL response. Ethics approval for animal experiments was obtained from the University of Melbourne Animal Experimental Ethics Committee.

Virus stocks were grown in the allantoic cavity of day 10 embryonated chicken eggs, and titers were determined by plaque assay as PFU on monolayers of Madin Darby canine kidney cells (8).

Tissue sampling and cell preparation

Spleen and lung lymphocyte populations were recovered from mice at the indicated time points and processed as previously described (22). Briefly, single-cell suspensions were prepared from perfused lungs following enzymatic digestion with collagenase A (Roche Applied Science) and passed through 70-µm cell strainers (BD Biosciences). RBC were lysed by treatment with Tris-buffered ammonium chloride. Spleens were disrupted by grinding between frosted slides and enriched for CD8+ cells by incubation on tissue culture dishes coated with goat anti-mouse IgG and goat anti-mouse IgM (Jackson ImmunoResearch Laboratories).

IFN-{gamma} ELISPOT assay

Wells of 96-well Millipore Multiscreen-HA filter plates were coated overnight with 10 µg/ml rat anti-mouse IFN-{gamma} capture Ab (R4-6A2; BD Biosciences/BD Pharmingen). Wells where then blocked with complete RPMI 1640 medium (JRH Biosciences) containing 10% v/v heat-inactivated FCS (JRH Biosciences), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 5 mM HEPES, 55 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). Spleen and lung suspensions were prepared and added to wells in 2-fold dilutions starting at 5 x 105 cells/ml. Cells were incubated at 37°C, 5% CO2 for 18 h in the presence of 10 U/ml recombinant human IL-2 (Roche Diagnostics), together with 5 x 105 irradiated (2200 rad) naive splenocytes pulsed with or without 2 µg/ml PA224 peptide (Auspep). IFN-{gamma} production was detected by staining with rat anti-mouse biotinylated IFN-{gamma} detecting Ab (XMG1.2; BD Biosciences/BD Pharmingen) at 5 µg/ml in PBS with 0.05% Tween 20 (APS Chemicals) and 1% heat-inactivated FCS, followed by incubation for 1 h with streptavidin-alkaline-phosphatase diluted 1/500 in PBS with 0.05% Tween 20. ELISPOT substrate containing 1 mg of 5-bromo-4-chloro-3-indlylphosphate (Roche Diagnostics) per milliliter of 2-amino-2-methyl-1-propynol buffer (Sigma-Aldrich) was added to the wells and spots were developed at 37°C, 5% CO2 for 30 min. Spots were counted on an AID EliSpot Reader System version 3.1.1 (Autoimmun Diagnostika).

Stimulation and intracellular cytokine staining

Enriched T cell populations (0.5–2 x 106 cells) from the spleen and lung were stimulated for 5 h in 200 µl of complete RPMI 1640 medium, 10 U/ml recombinant human IL-2, and 1 µg/ml Golgi-Plug (BD Biosciences/BD Pharmingen) in the presence or absence of 1 µM PA224 peptide. Cells were washed in FACS Buffer (0.02% sodium azide (Sigma-Aldrich) and 1% BSA (Invitrogen Life Technologies) in PBS) before staining with anti-CD8{alpha}-PerCP-Cy5.5 Ab (BD Biosciences/BD Pharmingen) for 30 min on ice. After washing, cells were permeabilized by paraformaldehyde fixation, using reagents supplied in a Cytofix/Cytoperm kit (BD Biosciences/BD Pharmingen) according to the manufacturer’s instructions. Cells were stained with anti-IFN-{gamma}-FITC (XMG1.2; BD Biosciences/BD Pharmingen), anti-IL-2-PE (JES6-5H4; BD Biosciences/BD Pharmingen), and anti-TNF-{alpha}-allophycocyanin (MP6-XT22; BD Biosciences/BD Pharmingen) for 30 min on ice. After further washing, cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Immunocytometry Systems) and data were analyzed using CellQuest Pro software (BD Immunocytometry Systems).

Tetramer staining and tetramer dissociation analysis

Lymphocytes (0.5–2 x 106 cells) from the spleen and lung were stained with DbPA224 tetramer conjugated to either streptavidin-PE or streptavidin-allophycocyanin (Molecular Probes) for 60 min at room temperature. Cells were washed in FACS Buffer before staining with anti-CD8{alpha}-FITC or anti-CD8{alpha}-allophycocyanin for 30 min on ice. Where TCR Vbeta analysis was performed, a panel of FITC-conjugated Abs specific for various TCR Vbeta families was included at this staining step. Cells were washed and resuspended for flow cytometry analysis.

As a measure of TCR avidity, enriched lymphocyte populations from the spleen and lung were used in a tetramer dissociation assay according to La Gruta et al. (25). Briefly, after staining with DbPA224-PE tetramer as described above, cells were washed and incubated at 37°C in the presence of anti-H2Db Ab at 5 µg/ml (28-14-8; BD Biosciences/BD Pharmingen) to prevent tetramer rebinding. At designated time points, cells were removed into cold FACS Buffer, washed, and stained with anti-CD8{alpha}-FITC Ab (BD Biosciences/BD Pharmingen) on ice for 30 min. After further washes, tetramer staining was analyzed by flow cytometry.

Single-cell analysis of TCR usage

For comparison of CDR3beta diversity, PR8-primed mice were either infected i.n. with 104 PFU HKx31 or inoculated with 45 nmol Th-P2C-PA lipopeptide. At the acute time point (day 8), enriched T cell populations from the spleen were stained with DbPA224 tetramer, anti-CD8{alpha}-allophycocyanin Ab and anti-Vbeta7-FITC Ab (recognizes the TCR encoded by the TRBV29 gene segment). Single CD8+Vbeta7+DbPA224+ cells were isolated with a MoFlo sorter (Cytomation) fitted with a Cyclone single-cell deposition unit. Cells were sorted directly into wells of 96-well PCR plates (Eppendorf) containing 5 µl/well of cDNA reaction mix. The cDNA reaction mix contained 0.25 µl of Sensiscript reverse transcriptase, 1x cDNA buffer, 0.5 mM dNTP (all Qiagen), 0.125 µg of oligo dT(15) (Promega), 100 µg/ml gelatin (Roche Diagnostics), 0.5 nM spermidine (Sigma-Aldrich), 100 µg/ml tRNA (Roche Diagnostics), 10 U of RNase Out (Invitrogen Life Technologies), and 0.1% Triton X-100 (Sigma-Aldrich). Plates were incubated at 37°C for 90 min to synthesize cDNA. Reverse-transcriptase activity was stopped by incubation of plates at 95°C for 5 min. The Vbeta7+ transcripts were amplified by nested PCR and sequenced as described elsewhere (28, 29). TCR gene segment nomenclature is according to the IMGT database (30).

Determination of pulmonary viral titers

Mice were sacrificed by cervical dislocation and perfused lungs aseptically transferred into RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Life Technologies) and 24 µg/ml gentamicin (DBL). Lungs were disrupted by passage through a fine mesh sieve and cells pelleted by centrifugation. The supernatant containing infectious virus was divided into aliquots and stored at –70°C until use. Lung viral titers were determined by plaque assay on monolayers of Madin Darby canine kidney cells as previously described (8).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Administration of the Th-P2C-PA lipopeptide vaccine elicits primary DbPA224-specific CTL responses

Lipopeptide vaccination has been demonstrated to be effective at providing CTL immunity against viral and bacterial challenge (8, 12, 16). To demonstrate that a lipopeptide vaccine construct could elicit DbPA224-specific responses, B6 mice were inoculated with 45 nmol Th-P2C-PA, the unlipidated construct, or infected i.n. with HKx31. DbPA224-specific responses could be measured in both the spleen and lung on day 9 after Th-P2C-PA administration, although the numbers were significantly lower in mice that had received the lipopeptide vaccine compared with those observed after virus infection (Fig. 1A, p < 0.001, and B, p < 0.0004, respectively). Although the magnitude of DbPA224-specific memory CTL found in the spleen was greater in mice that had resolved virus infection compared with lipopeptide-vaccinated mice (Fig. 1C, p < 0.015), there were equivalent numbers in the lung (Fig. 1D). Importantly, the difference in splenic memory CTL numbers between these groups (2-fold) at day 31 (Fig. 1C) after infection did not reflect the difference in effector CTL numbers at day 9 (10-fold; Fig. 1A). At each time point, the number of DbPA224-specific CTL was always greater for the lipidated peptide vaccine construct compared with the unlipidated construct (Fig. 1).


Figure 1
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  		FIGURE 1. Quantitation of primary and memory responses induced by lipopeptide vaccine constructs. Naive B6 mice were inoculated with 104 PFU x31 virus (i.n.), 45 nmol Th-P2C-PA (i.n.), 45 nmol Th-PA (i.n.), or 30 µl of PBS (i.n.). On day 9 (A and B) or 31 (C and D), spleens (A and C) and lungs (B and D) were harvested and single-cell suspensions prepared. DbPA224-specific CD8+ cells were identified by staining with DbPA224-PE tetramer and anti-CD8{alpha}-FITC Ab. Data show mean numbers of DbPA224-specific CD8+ cells ± SD of four mice (except PBS lung day 9, n = 3 mice). The statistical analysis compares samples to PBS groups on each separate panel (*, p < 0.05; **, p < 0.01).

 
Administration of Th-P2C-PA lipopeptide results in decreased viral lung titers after challenge

To examine whether Th-P2C-PA vaccination would provide protection from influenza A virus, naive mice and mice previously primed i.n. with 45 nmol Th-P2C-PA or PR8 virus were infected with A/HKx31 and viral lung titers were determined on days 2, 6, and 8 after infection (Table I). The HKx31 virus used had a mutation introduced into the NP366 peptide (position 5 Asn-Gln mutation) abrogating peptide binding to H2-Db. As a consequence, the major DbNP366-specific CTL response is not present (27). Viral growth peaked to similar levels 2 days postinfection in both primed and naive mice. By day 6, both the PR8- and Th-P2C-PA-primed mice demonstrated ~15- to 20-fold lower lung viral titers compared with unprimed mice (Table I). This result contrasts previous reports describing poor protection after vaccination with PA224 peptide-pulsed DCs (31, 32). Despite the absence of a large DbNP366-response, the PR8 primed mice had completely cleared virus by day 8 postinfection and two of three of the lipopeptide mice had also completely cleared virus. In contrast, two of three of the naive mice had significantly higher levels of virus still present in the lungs. These results demonstrate that cellular immunity generated after DbPA224-specific lipopeptide vaccination is capable of limiting viral replication after challenge.


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  		Table I. Pulmonary viral titers after viral challenge of lipopeptide-inoculated mice

 
The magnitude of IFN-{gamma}+DbPA224-specific CTL responses was equivalent in both the spleen (Fig. 2A) and lung (Fig. 2B) of mice previously primed with either the lipopeptide vaccine or PR8 virus. There was no difference in the proportion of IFN-{gamma}+DbPA224-specific CTL producing either TNF-{alpha} (Fig. 2, C and D) or IL-2 (Fig. 2, E and F). Importantly, vaccination with the unlipidated PA224 construct did not result in establishment of robust memory as the recall response was significantly diminished in both the spleen (Fig. 2A, p < 0.001) and lung (Fig. 2B, p < 0.05). Therefore, DbPA224-specific CTL primed with lipopeptide exhibited protective responses that were functionally equivalent to CTL primed with virus infection.


Figure 2
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  		FIGURE 2. Qualitative analysis of DbPA224-specific CD8+ T cell responses after viral challenge of previously primed mice. Naive B6 mice were primed with either 1.5 x 107 PFU PR8 virus (i.p.), 9 nmol Th-P2C-PA (i.n.), 9 nmol Th-PA (i.n.), or PBS alone. Fifty days after vaccination, mice were infected with 104 PFU HKx31 (i.n.). Lungs and spleens were sampled on day 8 after infection and splenocytes enriched for CD8+ cells. Cells were stimulated with 1 µM PA224 peptide for 5 h in the presence of brefeldin A. Cells were analyzed for expression of CD8, IFN-{gamma}, TNF-{alpha}, and IL-2 as previously described. Results for DbPA224-specific CTL derived from spleen (A, C, and E) and lung (B, D, and F) are shown. The number of CD8+IFN-{gamma}+ DbPA224-specific CTL are shown in A and B. The proportion of IFN-{gamma}+ CTL coexpressing TNF (C and D) and IL-2 (E and F) are shown. Shown is the mean ± SD (n = 4), representative of three experiments. Significance was determined using a Student t test (*, p < 0.05; **, p < 0.01 with respect to samples from PR8/x31).

 
Comparison of memory DbPA244-specific CTL recalled after either lipopeptide or influenza A virus infection

Given the magnitude of immunity was equivalent for lipopeptide- and virus-primed memory CTL recalled with virus infection, it was of interest to determine whether CTL primed with lipopeptide were functionally equivalent to those generated after virus infection. However, the numbers of DbPA224-specific CTL elicited after primary lipopeptide administration were too low for reasonable assessment of the response using conventional assays (16). Therefore, the qualitative characteristics of virus-primed memory cells, recalled with either Th-P2C-PA lipopeptide or heterologous virus challenge, were compared. Lymphocytes were isolated from either the spleen or lung and intracellular cytokine staining performed to assay both the magnitude and functional quality of recalled DbPA224-specific CTL. As previously described (22), cytokine production by both lipopeptide- and virus-recalled DbPA224-specfiic CTL followed a hierarchy with TNF-{alpha} producers a subset of the IFN-{gamma}+ producers (Fig. 3). Although administration of the Th-P2C-PA lipopeptide vaccine resulted in a smaller recall response in the spleen when compared with virus challenge (Fig. 4A, p < 0.001), the recall response was equivalent in the lung (Fig. 4B). Importantly, the Pam2Cys moiety was required for efficient expansion of memory CTL as the number of DbPA224-specific CTL recalled with the unlipidated construct was significantly lower than either A/HKx31 or lipidated Th-P2C-PA challenge (Fig. 4, A and B).


Figure 3
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  		FIGURE 3. Comparison of cytokine profiles for secondary DbPA224-specific CTL recalled with either lipopeptide or virus inoculation. PR8-primed B6 mice were given either 104 PFU HKx31 (A and B), or 45 nmol Th-P2C-PA (C and D) i.n. Tissues were harvested 8 days later and lymphocytes were stimulated with 1 µM PA224 peptide for 5 h in the presence of brefeldin A, stained with anti-CD8{alpha}-PerCPCy5.5, fixed, permeabilized, and stained for intracellular IFN-{gamma} and TNF-{alpha}. The numbers shown on the FACS profiles are the percentage of TNF-{alpha}+ of IFN-{gamma}+ CD8+ T cells. Shown are representative cytokine profiles for the spleen (A and C) and lung (B and D).

 

Figure 4
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  		FIGURE 4. Mode of challenge impacts DbPA224-specific CTL cytokine profiles. PR8-primed B6 mice were given 104 PFU x31, 45 nmol Th-L-PA, 45 nmol Th-PA, or 30 µl of PBS i.n. Lymphocytes were isolated from the spleen and lung and stimulated with PA224 peptide as described in Fig. 2. Samples were then stained simultaneously for IFN-{gamma}, TNF-{alpha}, and IL-2. Results for DbPA224-specific CTL derived from spleen (A, C, and E) and lung (B, D, and F) are shown. The number of CD8+ DbPA224-specific CTL are shown in A and B. The proportion of IFN-{gamma}+ CTL coexpressing TNF (C and D) and IL-2 (E and F) are shown. Data show mean ± SD of four mice and are representative of three experiments. Significance was determined using a Student t test (*, p < 0.05; **, p < 0.01 with respect to samples from PR8/HKx31).

 
Analysis of the proportion of IFN-{gamma}+ CTL producing either TNF-{alpha}+ or TNF-{alpha}+/IL-2 has previously been used to determine qualitative differences in CTL populations after influenza A virus infection (22, 25). To determine whether lipopeptide administration altered the capacity of DbPA224-specific CTL to produce cytokine, the proportion of IFN-{gamma}+DbPA224-specific CTL producing multiple cytokines was determined. A significantly lower proportion of TNF-{alpha}+ DbPA224-specific CTL from both spleen (Fig. 4C) and lung (Fig. 4D) was observed in lipopeptide-challenged mice compared with those infected with heterologous virus. This apparently lower quality cytokine response was also reflected in a significantly lower proportion of IL-2+ CTL (Fig. 4, E and F). Although CTL recalled with either the Th-P2C-PA or Th-PA constructs resulted in a lower proportion of TNF-{alpha} producers compared with virus challenge (Fig. 4C, D), it was only Th-P2C-PA administration that resulted in a lower proportion of IL-2 producers (Fig. 4, E and F). The high proportion of IL-2+ CTL found in the Th-PA and PR8-PBS group reflects the lack of expansion of pre-existing memory CTL that typically demonstrate maturation to multiple cytokine production (22).

Analysis of the amount of cytokine produced, determined by measuring the mean fluorescence intensity (MFI) of staining, demonstrated that splenic and lung DbPA224-specific CTL recalled after Th-P2C-PA administration expressed lower levels of IFN-{gamma} (Fig. 5, A and B, p < 0.01) and TNF-{alpha} (Fig. 5, C and D, p < 0.01) compared with CTL recalled after virus infection. Interestingly, there was no difference in the MFI for IL-2 staining between CTL recalled after either lipopeptide or virus administration (Fig. 5, E and F) supporting the notion that despite the observed hierarchy of cytokine production, they have been observed independently regulated of other cytokine profiles (22).


Figure 5
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  		FIGURE 5. DbPA224-specific CTL recalled with lipopeptide exhibit lower levels of IFN-{gamma} and TNF-{alpha}, but not IL-2. Using the samples described in Fig. 4, the relative abundance of each cytokine was measured by the MFI of staining. Results show the average MFI of IFN-{gamma} (A and B), TNF-{alpha} (C and D), and IL-2 (E and F) staining for the DbPA224-specific CTL isolated from either spleen (A, C, and E) or lung (B, D, and F). Shown is the mean ± SD (n = 4), representative of three experiments. Significance was determined using a Student t test (*, p < 0.05; **, p < 0.01 with respect to samples from PR8/HKx31).

 
Comparison of tetramer dissociation and TCR repertoire diversity of DbPA224-specific CTL recalled by either virus or lipopeptide challenge

Previous analysis of influenza A virus-specific CTL populations demonstrated that an increased capacity to produce multiple cytokines correlated with a higher TCR-binding avidity (23, 25). Given the decreased capacity of lipopeptide-induced DbPA224-specific CTL to produce IFN-{gamma} and TNF-{alpha}, the TCR-binding avidity was analyzed (Fig. 6). Despite the observed differences in cytokine production (Figs. 4 and 5), there was no difference in the tetramer disassociation kinetics of DbPA224-specific CTL after virus or lipopeptide challenge. This was the case for both splenic (Fig. 6A) and lung (Fig. 6B) CTL populations.


Figure 6
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  		FIGURE 6. Comparison of TCR avidity by tetramer dissociation. A tetramer dissociation assay was used to compare the TCR avidity of DbPA224-specific CD8+ cells recovered from the spleens and lungs of PR8-primed mice inoculated with 104 PFU HKx31 ({diamondsuit}), 45 nmol Th-P2C-PA({square}), 45 nmol Th-PA ({blacktriangleup}) or PBS (•). Cells were stained with DbPA224 tetramer and then incubated at 37°C in the presence of anti-H2Db Ab to prevent rebinding of dissociated tetramer. Tetramer staining of CD8+ cells was evaluated at the indicated time points. Results are plotted as the natural log of the normalized fluorescence vs time for the spleen (A) and lung (B). Shown is the mean ± SD (n = 3), representative of three experiments.

 
After influenza A virus infection, the vast majority of DbPA224-specific CTL express TCRs that are comprised of the TRBV29 (otherwise described as Vbeta7.1) and TRBJ2-7 (otherwise described as Jbeta2S7) gene segments (28). Furthermore, there is also a preferred CDR3beta-region length of 6–7 aa. The TRBV, TRBJ, and CDR3beta length was analyzed for DbPA224-specific CTL isolated from naive mice either infected with A/HKx31 or vaccinated i.n. with the lipopeptide (Table II and data not shown). DbPA224-specific CTL isolated from mice that received either virus infection or lipopeptide demonstrated preferred selection of the TRBV29 gene-segment (Table II and data not shown). Lipopeptide-induced TRBV29+DbPA224-specific CTL maintained the preferred usage of the TRBJ2-7 gene segment and a CDR3beta length of 6 aa (Table II and data not shown). Furthermore, there was no difference in the TRBV29 TCR repertoire diversity of DbPA224-specific CTL recalled with either lipopeptide or virus challenge (Table II). Interestingly, there was an increase in the use of the TRBV13-2/3 (formerly Vbeta8.1/2) gene segment by lipopeptide induced DbPA224-specific CTL (data not shown). In summary, lipopeptide administration results in a broader repertoire with expanded CTL using a normally subdominant TRBV13-2/3 gene segment without impacting on repertoire diversity of preferred TRBV29 TCR gene-segment usage.


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  		Table II. Comparing TCR diversity of DbPA224+CD8+ cells recalled with either HKx31 virus or Th-P2C-PA lipopeptide

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study compared the quality of cellular immunity generated after lipopeptide vaccination to that observed after virus infection. The number of resident memory CTL in the lungs was equivalent to that observed after virus infection despite greater numbers of DbPA224-specific CTL in infected mice at the acute time point. This may reflect the fact that the lipopeptide construct contains a Pam2Cys moiety and has the capacity to activate DCs, presumably via TLR2 activation, with minimal inflammation (12, 14, 15). Vaccination with activated, peptide-loaded DCs resulted in rapid establishment of Listeria monocytogenes-specific cellular immune memory and the resulting secondary responses were equivalent to those observed after infection (9). Importantly, the generation of memory CTL was accelerated in DC-vaccinated animals due to the lack of inflammation (9, 33). It is therefore tempting to speculate that lipopeptide vaccines may, in fact, result in accelerated memory CTL formation without the need for large expansion of effector CTL. This notion warrants further investigation because an important implication is that vaccine regimens could be administered more rapidly, helping improve vaccine coverage.

Of particular interest was comparison of qualitative measurements of DbPA224-specific CTL when recalled with either the Th-P2C-PA lipopeptide or virus infection. Importantly, there were distinct functional differences in the capacity of Th-P2C-PA recalled CTL to produce IFN-{gamma} and TNF-{alpha}, but not IL-2, compared with virus-recalled CTL. Interestingly, there was no functional difference in memory CTL that were established after either primary infection or lipopeptide vaccination, then recalled after heterologous viral challenge. Therefore, this suggests that it is the mode of challenge that alters functional outcome of recalled CTL, rather than the mode of priming used to establish CTL memory. Why would the mode of Ag challenge alter the functionality of recalled CTL? Given the repertoires and magnitudes of recalled responses in the lung were similar for virus- and lipopeptide-recalled CTL, the observed functional differences most likely reflect differential modulation of cytokine production rather than differential recruitment of memory CTL. This modulation may be influenced by regulatory mechanisms controlling the biochemical modification of gene-specific promoters (34, 35, 36). Such modifications can be influenced by the inflammatory milieu induced after immune challenge (34). Given lipopeptide administration may not induce as much inflammation as viral infection, it is tempting to speculate that perhaps lipopeptides may not induce the appropriate biochemical modifications that would otherwise result in full effector function. If this was indeed the mechanism, it might also explain why there was no observed difference in cytokine production when lipopeptide- and virus-primed memory CTL are recalled with heterologous virus challenge. However, this is notion is speculative and warrants further investigation.

A more simple explanation may relate to differences in the kinetics of CTL responses after recall with either lipopeptide vs virus infection. We have previously demonstrated that the capacity of influenza A virus-specific CTL to produce multiple cytokines varies over the course of both primary and recall responses to infection (22). Therefore, it is possible that earlier analysis of CTL recalled with lipopeptide may have revealed CTL with similar cytokine profiles to that observed for CTL recalled after virus infection. In summary, while the data suggest that presentation of epitopes via lipopeptides has the capacity to alter effector T cell function, it does not impact on the establishment of robust cellular immunity.

Interestingly, while the proportion of IL-2 producers was lower in lipopeptide-recalled CTL populations, the level of IL-2 production (measured by MFI) was not altered compared with both IFN-{gamma} and TNF-{alpha}. Previous reports have associated the level of cytokine production with CTL TCR avidity for a given pMHC (22, 25). There was no difference in the TCR avidity of DbPA224-specific CTL recalled with either lipopeptide or virus. Therefore, measurement of immune correlates such as TCR avidity and the level of IL-2 production may provide novel measures for evaluating cellular immunity induced after vaccination.

Ag-specific TCR repertoire diversity has recently emerged as an important facet of cellular responses to pathogens (37). There is growing evidence that increased TCR diversity is associated with protection from virus infection (38), minimizing CTL escape during persistent infections (39), and maximizing the potential for cross-reactive CTL responses to different pathogens (40). Given pMHC structures can impact on TCR diversity (41), selection of vaccine peptide Ags that elicit the appropriate TCR repertoire diversity is likely to be critical for vaccine efficacy. Interestingly, the DbPA224-specific CTL recalled with lipopeptide demonstrated a broader TCR repertoire compared with those recalled with heterologous virus challenge. Normally after influenza A virus infection, TRBV13-2/3 usage by DbPA224-specific CTL is subdominant to the more prevalent TRBV29 gene segment (42). Given the low CTL numbers induced after primary administration of lipopeptide, it is difficult to ascertain whether a similar increase in TRBV13-2/3 usage is observed (data not shown). It will be of interest to determine whether similar expansions of subdominant TCR biases are observed for other lipopeptide constructs (38, 39).

There was no difference in the TCR repertoire diversity within the prominent TRBV29 TCR gene segment of DbPA224-specific CTL recalled with either lipopeptide or virus. The same finding was made when comparing the TCR repertoire diversity of DbPA224-specific CTL when the PA224 epitope was ectopically expressed in the neuraminidase of influenza A virus (23). Together, this supports the notion that structural characteristics of a given pMHC antigenic complex, rather than epitope context, determines TCR repertoire diversity (37, 41). Overall, given recent positive associations between increased breadth of CTL responses and control of virus infection, lipopeptides may provide robust cellular immunity due to increased breadth of TCR repertoire diversity not typically observed after infection.

Administration of the Th-P2C-PA lipopeptide resulted in accelerated viral clearance correlating with increased DbPA224-specific CTL responses. This is in contrast to studies demonstrating that priming of DbPA224-specific CTL using DC vaccination resulted in delayed viral clearance compared with that observed after infection of naive mice (31, 32). It was suggested that delayed viral clearance was a consequence of poor presentation of the PA224 peptide presentation on lung epithelial cells at the site of infection (32). However, the protection observed after lipopeptide vaccination supports the notion that DbPA224-specific CTL are important in protection from influenza A virus infection (27). Importantly, recent evidence suggests that under appropriate inflammatory conditions, PA224 presentation can be demonstrated for nonprofessional APCs (43).

One possible explanation for the observed discrepancy is that perhaps the DbPA224-specific CTL response, induced after the DC priming used by Crowe et al. (31, 32), was in some way of a lower quality to that observed after infection. Use of DCs previously activated with activating agents such as CpG or LPS have been demonstrated to induce similar levels of CTL responses to that observed after L. monocytogenes infection (9). Treatment of DCs with synthetic lipopeptide constructs, like that used in this study, result in high levels of activation, similar to those observed after CpG oligonucleotides or LPS treatment (12, 14, 15). The priming strategy used by Crowe et al. (31), however, used DCs that were not activated by pretreatment with these agents. Therefore, it is conceivable that differences in DC activation during priming resulted in different outcomes to infection. This highlights the fact that measurement of cell surface markers, such as increased expression of MHC class II (31), as a surrogate for DC activation may not suffice as measure of for priming of CTL responses.

Another possible explanation for the discrepant results is the different routes of vaccination used between the studies. The lipopeptide was administered intranasally where it resulted in the establishment of long-lived lung-resident memory CTL (31). In the studies by Crowe and colleagues, the DCs were given i.v. and it is not clear whether lung-resident, memory DbPA224-specific CTL were generated. The presence of resident, long-lived CTL in peripheral tissues after resolution of infection has been described for a number of pathogens (44, 45, 46, 47) and are thought to play an important role in initial control of secondary infection (45, 48). Respiratory lipopeptide administration resulted in similar levels of resident DbPA224-specific memory CTL in the lung tissue to that seen after virus infection. Therefore, it is possible that the protective effect offered by lipopeptide vaccination was due, in part, to establishment of memory DbPA224-specific CTL in the appropriate anatomical location before secondary challenge.

This study has, for the first time, evaluated qualitative aspects of the CTL response induced after lipopeptide administration. Importantly, our results have highlighted how appropriate activation of DCs and route of vaccination are important considerations when designing and evaluating novel vaccine strategies for inducing cellular immunity. Furthermore, lipopeptides also provide an important tool for dissecting the factors that contribute to robust cellular immunity such at TCR avidity, TCR repertoire diversity, and function. Such tools are essential to better identify the measurements of CTL function that correlate with protective immunity.


   Acknowledgments
 
We thank Drs. Nicole La Gruta and Lorena Brown for critical review and discussion, and Dina Stockwell for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an Australian National Health and Medical Research Council (NHMRC) Dora Lush Postgraduate Scholarship awarded to E.B.D., an NHMRC Burnet Fellowship awarded to P.C.D., an NHMRC R. D. Wright Fellowship awarded to S.J.T. and K.K., and Science Technology Innovation funds from the Government of Victoria, Australia. Back

2 K.K. and S.J.T. contributed equally. Back

3 Address correspondence and reprint requests to Dr. Stephen J. Turner, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail address: sjturn{at}unimelb.edu.au Back

4 Abbreviations used in this paper: MHCI, MHC class I; DC, dendritic cell; Pam2Cys, S-[2,3-bis(palmitoyloxy)propyl]cysteine; i.n., intranasal; MFI, mean fluorescence intensity. Back

Received for publication March 7, 2007. Accepted for publication June 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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