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Keywords:

  • CD8+ T cells;
  • Infectious diseases;
  • Virology;
  • West Nile Virus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

West Nile virus (WNV) is a small, positive-strand RNA virus belonging to the Flaviviridae genus, which causes lethal encephalitis in a subset of infected birds and mammals. In humans, WNV exhibits pronounced age-related morbidity and mortality, but the basis of this effect is unclear, and the molecular and cellular parameters of the host-WNV infection are just beginning to be elucidated. Indeed, numerous mechanisms were implicated in protection in vivo against WNV (IFN-I and IFN-γ, antibody, C’, CD8 and CD4 T cells), but the individual importance of each one of these remains unclear. Here, we show that transfer of highly enriched naïve CD8+ T cells protects the majority of alymphoid mice against lethal WNV infection. To substantiate and expand this finding, we defined the peptide specificity of the CD8 response in H-2b mice and used a panel of identified peptides to map one dominant (NS4b 2248–2256) and several subdominant epitopes. The hierarchy of these epitopes was stably maintained in the memory responses. Most importantly, CTL lines directed against these peptides conferred protection against lethal WNV infection in direct proportion to the epitope immunodominance. These results provide a springboard for future characterization of T cell responses against WNV and demonstrate, for the first time, that CD8 T cells can single-handedly protect from this disease.

Abbreviations:
B6 mice:

C57BL/6 mice

ICCS:

intracellular cytokine staining

WNV:

West Nile virus

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

The West Nile virus (WNV) is a small enveloped virus that contains a single, positive-sense ∼11-kb RNA genome encoding a polyprotein that is post-translationally cleaved into three structural (envelope, E; pre/membrane, pM/M; and capsid, C) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. These ten multifunctional proteins play a role in invasion, entry, viral replication, assembly and modulation of host cell functions, including the immune response (reviewed in 1, 2).

Since its appearance at the Eastern seaboard of the United States (1999), WNV-NY strain 385–99 3, 4 and its immediate descendents have spread through all 48 continental states, infecting more than 10 000 people and killing 264 in 2003 alone 5, 6. The incidence of death is disproportionally frequent in the elderly (median age at death: 75 years) 7. WNV leads to systemic disease in a variable (approximately 20%) percentage of individuals, with the most severe disease being due to neuroinvasion and the consequent meningitis and encephalitis 8, 9. While about 1 in 150 infections results in meningitis or encephalitis, advanced age and impaired immunity are the most significant risk factors for severe neurological disease 10; persons 50–59 years of age have a 10-fold, and those >80 a 43-fold higher incidence of severe disease compared to adults between 20 and 40 years of age 7.

To understand the immunological basis of this age-related susceptibility to WNV, we have developed a model of WNV infection and vulnerability in old C57BL/6 mice (J. B. and J. N.-Ž., unpublished data). Our preliminary results suggested the existence of age-related defects in T cell responses, whereas data from the literature implicated CD8+ T cells in anti-WNV resistance 11, 12. Therefore, we focused on the impact of naïve and memory CD8+ T cells in anti-WNV responses and showed that naïve T cells can protect Rag-KO mice from lethal WNV infection. We then defined H-2b-restricted CD8+ T cell epitopes from multiple WNV protein segments and have demonstrated that these epitopes can protect against lethal WNV infection in direct proportion to their immunodominance, showing that CD8+ T cells can be sufficient for protection against WNV.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

Transfer of naïve CD8+ T cells provides significant protection against WNV in alymphoid mice

CD8+ T cells were implicated in anti-WNV resistance using knockout animals. However, it was not clear whether these cells, by themselves, could protect against the disease. To test whether naïve, unprimed CD8+ T cells can provide protection against lethal WNV, splenic CD8+ T cells, purified to 90–95% purity (<2% CD4+ and <3% B cell contamination), were transferred into RAG-KO animals, and recipients infected with a lethal dose of WNV. While all control recipients, receiving saline, died, transfer of CD8+ T cells provided protection to 75% of recipient animals (Fig. 1). This suggests that naïve CD8+ T cells can provide significant level of protection against WNV, but does not formally rule out other elements of adaptive immunity. We therefore set out to investigate the epitope specificity of the CD8+ T cell response and test whether such epitopes are important determinants of anti-WNV protection.

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Figure 1. Protective effect of naïve CD8 T cells Rag2–/–. Splenic CD8+ T cells (2 × 106–5 × 106) from naïve B6 mice were isolated by positive selection (90–95% purity) and transferred to B6 Rag2–/– mice. At 24 h after transfer, mice were challenged with 300 PFU WNV s.c. Significant difference according to the log-rank test: ** p>0.005.

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WNV protein overload provides means to identify protein targets for CD8 cells

Previously, it was shown that in vitro incubation of antigen-presenting cells (APC) with massive amounts of proteins (mg range) could result in processing and presentation of the MHC class I epitopes of the protein 13, 14. This “protein overload” could theoretically either lead to uptake of some of the protein and its leaking into MHC class I biosynthetic/antigen-processing pathway; or to the extracellular cleavage of just enough of the correct peptide from imperfectly synthesized or even fully synthesized proteins to produce MHC targeting (where only a few peptides, and even a single one 15, 16 would suffice to detect biological activity); or by other, unknown mechanisms. Regardless of the exact mechanism(s), we reasoned that this strategy could allow us to screen for those WNV segments that contain the H-2b-restricted CD8+ epitopes. We expressed each of the WNV protein segments as GST fusion proteins in E. coli, as described in the Materials and methods, and illustrated in Supporting Information Fig. S1 for representative proteins, (arrows pointing to the molecular weight of indicated GST proteins before and after induction with IPTG). Each of the proteins was incubated at 0.1 mg/well with H-2b splenocytes from infected mice for 12 h and production of IFN-γ by splenic CD8+ T cells measured by the ICS assay as described in the Materials and methods. A dominant response was detected against NS4b, a prominent response against E and a borderline response to M and NS5 segments (Table 1). The combined magnitude of the response against all proteins amounted to 50–120% of the IFN-γ response obtained with polyclonal stimulation with anti-CD3 mAb (not shown), and was typically higher than the response to virally infected cells (not shown, but see below for values shown for “total”). Importantly, reactivity against each of the segments was confirmed using pools of overlapping peptides (Table 1), consistent with the idea that epitopes indeed existed within these proteins.

Table 1. CD8 T cell response to WNV proteins and peptide poolsa)
GST-proteinPercent responsePeptide pool (15-mers)Exp. 1 (% response)Exp. 2 (% response)Exp. 3 (% response)
  1. a) Immune spleen cells were treated by the protein-overload approach (second column) or with overlapping peptide pools (last three columns) as described in the Materials and methods and were then scored for production of IFN-γ by CD8 T cells. Percent response was calculated by adding the response to all protein segments or all peptide pools, respectively, and using that value as 100%; responses to individual proteins/peptide pools were calculated from those values.

Capsid1.8Capsid0.471.450.87
Pre-membrane+ membrane7.8Pre-membrane+ membrane7.455.061.09
Envelope31.9Envelope26.3613.7410.67
NS10.6NS10.710.000.52
NS20.5    
NS2aNDNS2a0.590.540.25
NS2bNDNS2b1.060.180.93
NS30.4NS30.350.360.02
NS4a0.8NS4a0.350.360.59
NS4b55.8NS4b60.4072.5183.15
NS5NDNS52.255.792.79

Definition of dominant and subdominant WNV epitopes that elicit CD8+ T cell responses in vivo

To identify optimal epitopes within the above WNV proteins, we screened smaller peptide pools and finally individual 15-mer peptides. From the identified 15-mers, we aligned the amino acid sequence based on known H-2Kb and H-2Db peptide binding motifs 17, and synthesized the optimal predicted peptides, as well as their potential N- and C-terminal extensions, to identify optimal peptides that represent major targets for CD8+ T cells. These peptides were then used in T cell function assays to narrow down and confirm the optimal epitopes. Representative examples of T cell reactivity by IFN-γ secretion against 15-mers and optimal peptides are shown in Supporting Information Fig. S2, and the summary of the results for all epitopes are shown in Table 2, which lists all identified peptides and their restriction elements. All peptides described are cited with their inclusive amino acid numbers the first time in the text, as well as in Table 2. Subsequently, abbreviated nomenclature is used, based upon designation of the protein component from which the peptide is derived, e.g. E (envelope), etc., followed by the initial amino acid at which the peptide begins, counting from the beginning of the polyprotein; therefore, the envelope peptide 347–354 is designated E347, etc..

Table 2. Identification of optimal CD8 T cell epitopes using overlapping WNV peptidesa)
15-mer peptideOriginSequenceOptimal epitope/stimulusPeptide sequence/restrictionExp. 1%CD8+IFN-γ+cellsExp. 2%CD8+IFN-γ+cellsExp. 3%CD8+IFN-γ+ cellsExp. 4%CD8+IFN-γ+ cells
  1. a) Experiments were performed as in Table 1, except that individual peptides were used, and that results are not represented relative to total reactivity, but rather as % of total splenic CD8+ cells secreting IFN-γ. Boldfaced are the sequences and the peptide designations for those peptides that represent optimal epitopes that produced unambiguous T cell reactivity. ND: not determined.

   HSV gB-8pSSIEFARL (Kb)0.140.290.150.15
   2c11 17.664.8015.125.13
Peptide 59 (294–303)ENVFNCLGMSNRDFLEGVENV294-302LGMSNRDFL(Db)NDND0.770.52
Peptide 69 (347–354)ENVANLAEVRSYCYLATVENV345-353EVRSYCYLA0.230.480.25ND
Peptide 70ENVVRSYCYLATVSDLSTENV347-354RSYCYLAT (Kb)2.170.813.221.84
   ENV347-355RSYCYLATV0.350.070.18ND
Peptide 104 (521–529)ENVSSAGSTVWRNRETLMENV518-528AGSTVWRNRET0.190.540.26ND
Peptide105ENVTVWRNRETLMEFEEPENV521-529TVWRNRETL(Db)0.590.620.460.26
Peptide 155 (771–778)ENVIALTFLAVGGVLLFLENV771-778IALTFLAV(Kb)NDND2.280.64
Peptide 498 (2488–2496)NS4bGASSVWNATTAIGLCNS4b2488-2496SSVWNATTA(Db)5.492.906.003.14
 NS4bGASSVWNATTAIGLCNS4b2488-2497SSVWNATTAI5.02ND5.173.0
Peptide 573 (2863–2872)NS5PWDTITNVTTMAMTDNS52863-2872DTITNVTTM(Db)NDND0.270.21
Peptide 635 (3171–3181)NS5GKGPKVRTWLFENGENS53171-3181GKGPKVRTWLNDND0.16ND
   NS53172-3181KGPKVRTWLNDND0.17ND
   NS53177-3184RTWLFENG (Kb)NDND0.150.21
Peptide 643 (3172–3181)NS5LHFLNAMSKVRKDIQNS53216-3224AMSKVRKDINDND0.160.18

In agreement with the reactivity to whole proteins, the strongest reactivity was observed against one of the NS4b 15-mers, amounting to 50–70% of the reactivity to the whole virus. From that 15-mer, we identified the NS4b nonamer peptide 2488–2496, SSVWNATTA (Table 2), which was shown to bind in a quantitative manner to H-2Db (Fig. 2), to cause robust IFN-γ secretion (Fig. 3A) and to readily sensitize target cells for CTL lysis (Fig. 3B). In the same manner, we have identified other WNV epitopes: the H-2Kb binder E347–354 RSYCYLAT, which is responsible for 17–30% of the whole response, the H-2Db binder E521–529 TVWRNRETL, the H-2Db binder E293–301 LGMSNRDFL, the H-2Kb-restricted NS4b2286–2295 ISSLFGQRI, and the H-2Db-restricted NS52863–2871, DTITNVTTM peptide (Figs. 2 and 3, Table 2, and data not shown), with smaller and more variable contributions to the response. Of the peptides tested, the NS4b2488 peptide bound strongly to Db, whereas the two next peptides on the immunodominance scale, E347 and, in particular, E521, exhibited weaker binding (Fig. 2), possibly accounting for the weaker responses. Of interest, direct ex vivo CTL activity directly correlated to the observed immunodominance by intracellular cytokine staining (ICCS) (Fig. 3B). We also identified several other candidate peptides from NS5, with less prominent or borderline contribution to the overall response (sum of these peptides accounts for >3% of total reactivity, not shown), which were not tested extensively (Table 2 and not shown).

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Figure 2. Epitope binding kinetics of selected peptides to H-2Kb and H-2Db as estimated by the RMA-S MHC stabilization assay. Optimal peptides, used at indicated concentration, were tested for their ability to stabilize empty MHC class I molecules on RMA-S cells. RMA-S stabilization assay was performed exactly as described in the Materials and methods, and stable expression of H-2Kb (left panel) and H-2Db (right panel) detected using FCM and mAb AF6 and B22–249, respectively. Results are plotted as % maximal class I expression, calculated as mean relative fluorescence intensity compared to that of class I molecules expressed by RMA-S cells at 29°C, which was taken as 100%. Given that optimal peptides often induce higher expression of class I molecules than that achieved with empty molecules at 29°C, results of this assay can (and do) exceed 100%. Peptides known to bind to Kb and Db (HSV gB498–505 and SV40 large T404–412, respectively) were used as positive controls. Representative results for one out of three experiments are shown.

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Figure 3. CD8 T cell response to the WNV epitope. B6 mice were infected with WNV; their splenic cells were isolated 7 days later and tested by three functional assays. (A) Peptide-stimulated cytokine secretion of IFN-γ was determined following 6-h stimulation by ICCS. (B) Cytotoxicity was tested by a direct ex vivo51Cr-release assay. (C) NS4b2488:Db pMHC tetramer staining of immune CD8+ T cells on days 7 and 50 post infection as gated on CD8+ T cells. Numbers under the panels denote percentage of CD8+ cells secreting IFN-γ in samples corresponding to the above tetramer staining. All experiments were performed exactly as in the Materials and methods. Results representating a minimum of three experiments are shown in each figure.

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We next confirmed the above results using pMHC tetramers against the dominant NS4b2488 peptide. This analysis independently confirmed that the response to identified epitopes occurs in vivo, and is a direct consequence of viral infection (Fig. 3C). Phenotypic analysis of epitope-responding CD8+ T cells (Supporting Information Fig. S3) further confirmed their correct identification as WNV-specific cells. Not surprisingly, on day 7 post infection, the cells responding to the epitopes in vivo exhibited the expected phenotypic characteristics of effector cells during the acute phase of the response: CD11ahiCD62LloCD44hi, secreting IFN-γ, with a minor population secreting IL-2 as well (Supporting Information Fig. S3). However, these cells did not down-regulate CD127 at this time point (Supporting Information Fig. S3), and also remained CD27hi. A thorough kinetic analysis of these and other activation markers will be needed to resolve whether CD127 down-regulation indeed fails to occur in WNV infection, or whether the kinetics of T cell marker expression in response to WNV may differ from that published for other viral infections 18.

At this point in the course of our study, we became aware that the group at Washington University was performing a search for WNV epitopes using computer-assisted epitope prediction. To compare methods and validate each other's results, we swapped the identified sequences, and have realized that we identified largely overlapping, but not identical, epitope sets. Thus, our approach had failed to identify one important epitope, the H-2Kb binder E771–778 IALTFLAV, which makes up about 12–20% of the response. This was probably due to the problems surrounding synthesis of the 15-mer peptide, which was subsequently showed not to be stable in functional assays. Nevertheless, we synthesized this epitope and used it in subsequent studies to examine epitope hierarchy.

Primary and memory response of CD8 T cells to WNV epitopes

We next used the identified epitopes to examine and compare effector and memory stages of the CD8+ response to WNV. ICCS was performed using both peptide pools (not shown) or optimal peptides (Fig. 4 and Supporting Information Table S1) on day 7 (effector phase) and day 50 (memory phase) post infection. Frequencies of the responding cells dropped from the peak of 5–8% of responding cells/total CD8+ T cells in the primary to ∼1–1.5% of the cells in the memory phase, consistent with the expected expansion and contraction of the primary response (Supporting Information Table S1; Fig. 4). In this experiment, E347 yielded a very strong response; however, it still ranked behind NS4b2488 in dominance. Otherwise, the hierarchy was similar to that noted in other experiments at the peak of acute infection (day 7, Fig. 4), and showed little change at 50 days after infection, in the memory phase of the response (Fig. 4, Supporting Information Table S1), regardless of whether we used peptide pools (not shown) or individual peptides (Fig. 4). If anything, the immunodominance was more pronounced in favor of the NS4b and the two major E epitopes in the memory phase. Thus, whereas seven of the peptides tested gave signals above the background in the primary response, accounting for >90% of the response to the whole virus (Supporting Information Table S1 and Fig. 4), the response to NS4b2866 and E521 epitopes in the memory phase was no longer reliably detectable, consistent with further focusing of the immune response (Fig. 4). Moreover, the response to individual peptides was always at least equal, and often significantly larger than the response to WNV-infected cells (Fig. 4 and not shown), probably due to peptide competition, unequal temporal expression of all epitopes or a combination of these and other, unknown, factors. Regardless of the exact reason, this suggested that we have indeed identified the vast majority of WNV epitopes in this MHC haplotype.

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Figure 4. Primary and memory response to dominant and subdominant epitopes. ICCS was performed at indicated times post infection using peptide pulse or WNV infection of the H-2b macrophage cell line, IC-21. Results represent four animals/group (x ± SEM) from one out of three experiments with comparable results.

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In vivo relevance of the anti-WNV epitope CD8 T cell responses

The above epitopes were all identified using CD8+ T cells from infected animals, and therefore would be expected to represent physiological targets of the cellular immune system. As the choice of the epitopes is dictated by both host and pathogen factors, and because factors such as precursor frequencies, p:MHC affinities, pMHC:TCR affinities, T cell avidities and viral immune evasion all influence the relative protective value of a response to a given epitope, we sought to investigate whether responses to individual epitopes can influence resistance to WNV infection. We therefore sought to test whether peptide-specific CTL lines can confer protection against the lethal WNV infection to alymphoid RAG-2–/– mice. Splenocytes from infected animals were explanted on day 7 p.i. and were restimulated in vitro with the indicated peptides. After 7 days, we obtained cell lines that were further purified by immunomagnetic sorting to obtain lines that contained <0.5% CD4 T cells and B cells. These CTL lines were transferred into RAG-2–/– recipients, engraftment was confirmed and animals were infected 24 h later. In the absence of transferred cells, recipients exhibited high mortality typical of animals lacking adaptive immune system (J.B. and J.N.-Ž., unpublished results; 19), and transfer of an irrelevant CTL line (specific for the HSV-1 gB-8p epitope) did not improve their survival (Fig. 5). By contrast, cell lines directed against the two major WNV epitopes (NS4b2488 and E347) conferred a high degree of protection (>75%); the CTL line directed against a minor epitope E521 provided clear, but less strong, protection, which did not reach statistical significance. Therefore, we conclude that CD8+ CTL lines, directed against the major WNV epitopes identified in this study, can provide significant protection against lethal WNV in vivo, in the absence of other components of the adaptive immune system. This protection appeared to correlate to immunodominance for the peptides tested.

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Figure 5. Antigen-specific CD8+ T cells are sufficient for protection against lethal WNV encephalitis. CTL cell lines specific for indicated WNV or control epitopes were obtained as described in the Materials and methods, and were transferred into RAG-2–/– recipient, 1 day prior to challenge with a lethal dose (300 PFU) of WNV. Both NS4b2488 (open circles) and E347 (open triangles) significantly increased the resistance of RAG-2–/– mice to WNV challenge in comparison to RAG-2–/– mice that received gB-specific CD8 T cells (log-rank test p>0.01), whereas CTL lines directed against E521 (closed triangles) provided some protection, which did not reach statistical significance. This is the compilation of two experiments with each group containing a total of 10–12 mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

In this study, we investigated the ability of CD8+ T cells to protect alymphoid mice from lethal infection, and identified the molecular targets that are the focus of attack by these CD8+ T cells.

Highly enriched naïve CD8 T cells transferred into RAG-KO mice protected 75% of the mice from WNV encephalitis, suggesting that, in a large number of cases, CD8 T cells can be sufficient for protection against this flavivirus. This finding is consistent with previous observations of Shresta et al.11 and Wang et al.12, which implicated CD8+ T cells as necessary in the recovery from primary WNV infection. Further experiments will be needed to determine whether naïve CD8 T cells can confer complete protection, and what other elements of the immune system, previously implicated in WNV resistance, may be necessary and sufficient to confer full protection.

We next investigated the molecular complexity of the anti-WNV CD8 T cell response at the target epitope level. Six H-2b WNV epitopes recognized by the CD8+ T cells were identified, using a combination of protein overload (suitable for rapid identification of proteins that contain T cell epitopes 13) and overlapping peptide library approaches. Of these six, the strongly immunodominant peptide NS4b2488, was responsible for 50–70% of the total reactivity to peptide pools. Next in the hierarchy was E347, which consistently carried 17–25% of the reactivity, followed, in order, by E521, E293, NS4b2286 and NS52863, which gave smaller and more variable responses.

As mentioned above, concurrently with our studies, a group from the Washington University had used a computer-based epitope prediction algorithm to identify epitopes from this same pathogen, also in the H-2b haplotype (Purtha et al., published in this issue of EJI). Our results were largely overlapping but not identical. Of importance, both approaches identified the same immunodominant NS4b2488 epitope as the main CD8 target, albeit the computer algorithm approach identified the longer version, SSVWNATTAI, which, in our hands, tended to stimulate slightly smaller functional responses compared to the nonamer SSVWNATTA (Table 2). Of more interest were the differences between the two approaches. Thus, while some of the peptides listed in Table 1 and 2 were not identified by the computer algorithm approach, we also realized that our approach had failed to identify one epitope, E771, (making up about 12–20% of the response). This was probably due to the problems surrounding synthesis of the 15-mer peptide, which was subsequently shown not to be stable in functional assays. This peptide was subsequently used in functional assays.

Using all of the above peptides, we have assigned the immunodominance hierarchy to the identified epitopes in the order NS4b2488 > E347 > E771 > all other epitopes. The three most dominant epitopes could readily account for >80% of the total response. Therefore, they provide means to easily track the CD8+ T cell response to WNV in C57BL/6 (B6) mice in a single ICCS sample. An interesting finding was that in the primary response, there was no down-regulation of CD127 in response to any of the epitopes tested (Supporting Information Fig. S2). It is unclear whether this is due to the nature of the viral infection or due to a possible difference in kinetics of activation marker expression. Nevertheless, immunological memory was clearly formed against the key epitopes. In fact, in the course of the memory responses, the CD8+ T cell response to WNV focused further: while the response to these three epitopes dominated even more, the responses to other epitopes diminished and were often not detectable.

Most importantly, when a few identified peptides were tested for their ability to elicit CTL lines that could protect animals against lethal WNV, those cell lines showed protective activity in vivo, and peptide immunodominance appeared to correlate with protective activity. We conclude that the identified epitopes present not only targets of CD8 attack, but also have the capacity to elicit in vivo protective CD8+ T cells. These epitopes should therefore help unravel the kinetics of CD8+ T cell response to WNV, and help us provide insight into the presence or the absence of specific defects in WNV adaptive immunity in old mice.

Protection against WNV in mice has received considerable attention over recent years, and it is clear that both innate and adaptive immune systems play a role 2022. This includes natural and acquired early antibodies 23, complement 24, type I IFN and IFN-γ 20, 25, 26 and T cells 27, including CD8+ T cells 11, 12, 28, and perforin 29. What is less clear is which of these mechanisms is/are absolutely required and is/are both necessary and sufficient for protection against WNV. With regard to this last point, we show (Fig. 5) that virus-specific CD8+ T cells are not only necessary, but can be sufficient to mediate protection against WNV, in that they can protect the majority of adoptive hosts in the absence of other components of adaptive immunity. Therefore, a vaccine that would include CD8+ epitopes may be a viable strategy to prevent severe WNV disease.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

Cloning and production of GST-WNV proteins

WNV RNA was isolated using Tri-reagent (MRC), and was converted into cDNA using SuperScript III (Invitrogen, Carlsbad, CA) and random hexamers. cDNA was used as template and PCR products were generated using Accuprime PFX enzyme (Invitrogen). PCR products were enzyme digested and ligated into the pGEX 4T-1 vector (GE Healthcare Life Sciences, Piscataway, NJ) to produce 10 individual plasmids encoding a GST-fusion protein of each of the 10 WNV protein subunits. Individual GST-WNV proteins were produced in BL-21 (DE3) cells (Stratagene, La Jolla, CA) as inclusion bodies after induction with IPTG. Inclusion bodies were solubilized with urea, and the GST proteins refolded during dialysis in the presence of protease inhibitors. Following refolding, proteins were purified using glutathione-Sepharose 4B beads (Pharmacia, Piscataway, NJ). Purified proteins were dialyzed overnight in 1× PBS, concentrated using a Centricon membrane (Millipore, Freehold, NJ) and protein concentration determined using a BCA kit (Bio-Rad, Hercules, CA). A small aliquot was run on a SDS-PAGE gel, transferred to a PVDF membrane (Bio-Rad), and probed with anti-WNV serum from immune mice. Remaining portions of protein were frozen at –80°C until further use.

Mice

Adult (2–6 months old) male B6 mice were purchased from the National Cancer Institute Breeding Program (Frederick, MD). B6.Rag2–/– mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and bred at the VGTI vivarium (Oregon Health & Science University); they were used at 2–4 months of age. All animals were housed and bred under specific pathogen-free conditions at the Oregon Health & Science University. All WNV experiments were completed within a United States Department of Agriculture (USDA, Frederick, MD) approved Biosafety Level (BSL) 3 facility, and were approved by the Institutional Animal Care and Use Committee, and the Institutional Biosafety Committee in accordance with the applicable federal, state, and local regulations.

Virus, peptides, and cell lines

WNV strains NY99-crow, 31A and 385–99 were used and all virus strains yielded similar results. WNV strains NY99 and 385–99 were kind gifts of Drs. W. Ian Lipkin (Columbia University, New York, NY) and Robert Tesh (University of Texas Medical Branch, Galveston, TX), respectively; strain 31A was provided by the USDA reagent program (Ames, IA). An overlapping peptide library covering the entire length of the viral polyprotein (15-mers overlapping by 10 amino acids) was obtained from Sigma Aldrich (St. Louis, MO). Additional synthetic peptides were purchased at >95% purity from Sigma Aldrich and 21st Century Biochemicals (Marlboro, MA), diluted in 10% H2O/90% DMSO, stored at –80°C and subsequently used at indicated concentrations. Virus was grown in mycoplasma-negative Vero cells, cultured under aseptic conditions as described previously; mycoplasma-negative EL-4, IC-21 and MC57g cells (all H-2b) were used in target-cell and stimulation assays. Cells were infected using variable MOI as indicated; however, for ICCS and for 51Cr assays or for any experiments using MC57g cells, the cells were infected at the MOI of 10 for 30 h prior to use.

ICCS and surface flow cytofluorometric staining

Cytokine-producing T cells were detected using the Cytofix-Cytoperm Kit (BD-PharMingen, San Diego, CA), as described. Single-cell splenocyte suspension was depleted of red blood cells and was incubated with 1 μM peptide or infected with WNV in the presence of 5 μg/mL Brefeldin A (Sigma Aldrich) for 6 h at 37°C. After 6 h, the cells were washed and blocked with Fc block (anti-mouse FcγRI/III; BD-PharMingen) and incubated overnight in the presence of a saturating dose of surface antibodies against CD8, CD3, CD4, CD11a, CD43 (Clone 1B11), CD44 and CD62L (BD-PharMingen). After washing, the cells were fixed, permeabilized and intracellular antibodies (anti-IFN-γ, or anti- IL-2; BD-PharMingen) added for 30 min. The samples were then washed and analyzed using either a FACSCalibur or LSR II cytometer (Becton Dickinson Immunocytometry Systems, Sunnyvale, CA) instrument. Where indicated, pMHC tetramers, conjugated to allophycocyanin (NIH Tetramer Facility, Atlanta, GA) were added to unstimulated cells in conjunction with other markers for surface staining. Flow cytometry analysis (FCM) was performed by collecting a minimum of 5 × 104 events and gates set on lymphocyte population based on forward and orthogonal light scatter, followed by marker positioning to denote fluorescence greater than that of control stained or unstained cells.

pMHC stabilization assay

This assay was performed using the RMA-S cell line, exactly as previously described 30, 31, using mAb Y3 and B22/249, followed by secondary, allotype-specific antibodies conjugated to PE (Southern Biotech, Huntsville, AL).

Infection, immunization, CTL line generation and CTL assays

Mice were immunized subcutaneously (s.c.) between the shoulder blades with 20–600 PFU WNV, or with 1 μg of the indicated WNV peptides emulsified in the adjuvant TiterMax (CytRx, Norcross, GA), exactly as previously described 32. After 7 days, splenocytes were isolated and subjected to FCM, ICCS or CTL assay analysis as described above, or were restimulated in vitro to generate CTL lines for adoptive transfer experiments. Briefly, splenocytes were co-cultured with irradiated (30 Gy), peptide-coated (0.1 μg/mL) syngeneic spleen cells for 7 days. Cells were then harvested, purified using anti-CD8-coated magnetic beads (Miltenyi Biotech, Santa Cruz, CA) and transferred as described below. Infection was performed s.c. using pre-titrated virus dose lethal for B6.Rag-2–/– (300 PFU).

Direct ex vivo CTL activity was determined using peptide-coated and control EL-4 thymoma cells as targets. Radioactivity was measured using TopCount Packard δ/γ radioactivity reader (Packard Co., Detroit, MI), using a standard 51Cr-release assay, exactly as described previously 32.

Adoptive transfer and virus challenge experiments

For naïve CD8+ T cell transfer, spleens from 4-week-old B6 mice, containing less than 5% CD44hi (memory) splenic T cells, were coated with anti-CD8-coated beads, and CD8+ cells isolated at 90–95% purity. These cells were transferred i.v. (at 5 × 106–10 × 106 cells/recipient), transfers were monitored and challenge performed as below.

Peptide-specific CTL lines generated by in vitro restimulation of WNV-primed spleen cells, as above, were purified to deplete CD4+ and B220+ cells to <1%, and were injected (2 × 106–5 × 106 cells/recipient) i.v. into RAG-2–/– recipients and engraftment success was evaluated by FCM 24 h later. Animals were challenged 24 h after cell transfer as described above. Survival was scored on a daily basis. Death occurred between days 10 and 18, and all animals surviving this period remained disease free for 60–90 days at which point the experiment was terminated. Data are shown as percent survival at the termination of the experiment, with the statistical significance determined using Fisher's exact test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

We thank Dr. Ilhem Messaoudi and Ms. Anna Lang for help and assistance, and Dr. Michael Diamond and his colleagues for communicating results prior to publication. Supported by the USPHS awards N01 50027 (J.N-Z.), T32 AI007472 (J.B.) and RR0163 (to the ONPRC) from the National Institute of Allergy and Infectious Diseases and the National Institute for Research Resources, National Institutes of Health. The authors have no conflict of interest.

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Note added in proof

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

The article cited in this text by Purtha et al., as “published in this issue of EJI” concluding that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines, has the following [ ]reference:

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Note added in proof
  9. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2007/37196_s.pdf or from the author.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.