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Journal of Virology, December 2008, p. 11734-11741, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.00435-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Lymphocytic Choriomeningitis Virus Infection Yields Overlapping CD4⁺ and CD8⁺ T-Cell Responses

Courtney Dow,^1,2 Carla Oseroff,¹ Bjoern Peters,¹ Courtney Nance-Sotelo,² John Sidney,¹ Michael Buchmeier,³ Alessandro Sette,¹ and Bianca R. Mothé^2*

The La Jolla Institute for Allergy and Immunology, La Jolla, California 92037,¹ Department of Biological Sciences, California State University, San Marcos, California 92096,² The Scripps Research Institute, La Jolla, California 92037³

Received 27 February 2008/ Accepted 2 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activation of CD4⁺ T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4⁺ T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2^d. This is quite disparate to the H-2^b setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2^d or whether additional CD4⁺ T-cell epitopes could be identified in the setting of the H-2^b background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4⁺ T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4⁺ epitopes, four of them also stimulate CD8⁺ T cells in a statistically significant manner. Furthermore, we assessed these CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4⁺ and CD8⁺ T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CD4⁺ T-cell immune responses have been known to perform several important functions in the setting of host defense. CD4⁺ T cells provide cognate help to B cells, a required step for immunoglobulin switching and affinity maturation of B cells, specifically those that produce neutralizing antibodies. CD4⁺ T-cell immune responses are also needed for the proper development and activation of cytotoxic CD8⁺ T cells and are critical for their expansion and persistence as memory cells. Finally, CD4⁺ T cells may participate directly in pathogen clearance via cell-mediated cytotoxicity and/or through production of cytokines (41).

Immune responses resulting in viral clearance have been associated with the activation and expansion of virus-specific CD4⁺ T cells. This effect is mediated by direct effector mechanisms and through the priming and maintenance of cytotoxic T-lymphocyte (CTL) responses, which act to clear viral infection (3, 7, 17, 22, 23, 28, 51). However, certain viruses, such as human immunodeficiency virus, lymphocytic choriomeningitis virus (LCMV), and hepatitis C virus, have developed mechanisms to establish persistent infection. Despite potent responses early in the course of infection, CTL responses are often ineffective at clearing these viral infections (1, 5, 13, 19, 46). Several different mechanisms have been shown to contribute to the failure of T-cell responses in the course of persistent viral infections, including clonal exhaustion, overexpression of programmed death 1, and the rapid appearance of viral mutations resulting in escape variants (2, 11-14, 18, 29, 52, 55). Lack of effective CD4⁺ T-cell help early in infection, during the priming stage of the CD8⁺ T-cell response, may also contribute to the ultimate failure of the CTL response to clear infection (28, 55). Thus, an accurate measurement of CD4⁺ T-cell responses early during infection is critical in determining how these responses promote development of effective CD8⁺ T-cell responses and more in general to understand the profile of successful viral clearance during acute infection.

We analyzed the role of CD4⁺ T cells in viral murine infection with LCMV (32, 33, 49). The LCMV genome consists of two single-stranded RNA segments, the 3.4-kb small (S) segment and 7.2-kb large (L) segment. The L segment encodes the viral polymerase (L) and zinc-binding protein (Z), while the S segment encodes the nucleoprotein (NP) and glycoprotein precursor (GP), which is posttranslationally cleaved to yield a signal peptide (SP) and the two mature envelope glycoproteins, GP1 and GP2 (26, 47).

CD8⁺ T-cell responses to LCMV have been characterized primarily by measuring responses directed against numerous well-established major histocompatibility complex (MHC) class I restricted CTL epitopes in enzyme-linked immunospot (ELISPOT), tetramer and intracellular cytokine staining (ICCS) assays (16, 55). In contrast, CD4⁺ T-cell responses against LCMV are not as well characterized. Recently, we have shown that nine epitopes were detectable after LCMV Armstrong in the H-2^d setting. These responses were directed against the NP, GP, and Z proteins. Furthermore, one of the responses contained a nested CD8⁺ T-cell epitope. These responses determined, for the first time, the breadth of CD4⁺ T-cell responses against LCMV infection.

In contrast, only two LCMV-specific IA^b-restricted CD4⁺ T-cell epitopes, GP 61-80 and NP 309-328, have been described (34). The transgenic SMARTA mouse, solely expressing T cells specific for LCMV GP 61-80, has aided in understanding the role of the CD4⁺ T-cell response in LCMV infection (6, 35, 36, 53). However, a more thorough understanding of the complexity of the CD4⁺ T-cell responses is important to study viral clearance and/or chronicity and may provide insights into how to develop interventions to prevent or resolve persistent infections. We were interested in determining whether other CD4⁺ T-cell responses could be detected in the setting of IA^b or whether a broad set of helper responses is unique to LCMV infection of H-2^d mice.

To address this question, we infected C57BL/6 mice with LCMV Armstrong and determined LCMV-specific responses 8 days after infection. We have identified six responses after infection, therefore demonstrating that the repertoire of CD4⁺ T-cell responses is indeed broad in the context of H-2^b mice. A subset of these responses is stronger than the previously characterized response against the NP 309-328 epitope. Interestingly, similar to the H-2^d setting, nested CD8⁺ T-cell epitopes were identified in four of the CD4⁺ T-cell epitopes. Furthermore, we identified that the immunogenic regions of the previously identified GP 61-80 and NP 309-328 epitopes are GP 66-80 and NP 311-325, respectively. We further investigated whether these six responses were present during the memory phase of infection and whether a secondary challenge would provide a heightened immune response. We observed responses against GP 66-80 and NP 311-325 under these circumstances. Additionally, we were interested in determining what, if any, responses were present following persistent infection. We discovered that two CD4⁺ T-cell epitopes, located at GP 66-80 and GP 126-140, elicited responses. These findings illustrate the complexity of the CD4⁺ T-cell response repertoire and demonstrate the need to accurately characterize these responses.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Peptide synthesis. Fifteen-mer peptides overlapping by 11 amino acids for the entire proteome of LCMV strain Armstrong (GP, NCBI accession no. AAX49341; NP, NCBI accession no. AAX49342; L, NCBI accession no. AAX49344; Z, NCBI accession no. AAX49343) were synthesized (Pepscan, Lelystad, The Netherlands). The resultant 664 LCMV peptides for the entire proteome were divided into 83 pools, with eight peptides per pool for initial screening purposes. Specific peptides were subsequently tested individually at a concentration of 10 µg/ml.

Mice and infections. C57BL/6 (H-2^b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All studies were conducted at the California State University—San Marcos and the La Jolla Institute for Allergy and Immunology, in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care and according to Institutional Animal Care and Use Committee-approved animal protocols.

C57BL/6 (H-2^b) mice were infected with 2 x 10⁵ PFU of LCMV Armstrong intraperitoneally (i.p.). Mice were sacrificed by CO₂ inhalation, and spleens were harvested 8 days postinfection.

For LCMV clone 13 experiments, C57BL/6 mice were infected with 2 x 10⁶ PFU of LCMV clone 13 intravenously. Mice were sacrificed by CO₂ inhalation, and spleens were harvested 8 days postinfection.

ELISPOT assay. The gamma interferon (IFN-) ELISPOT assays were performed as previously described (30, 48). Briefly, mouse CD4⁺ T cells were isolated from the spleens of LCMV-infected mice with anti-CD4⁺ magnetic beads (Miltenyi Biotech, Inc., Auburn, CA). Purified CD4⁺ T cells (1 x 10⁵ to 2 x 10⁵) were cultured with 1 x 10⁵ to 2 x 10⁵ syngeneic splenocytes from uninfected mice and peptides (either peptide pools or individual peptides, tested at 10 µg/ml) in flat-bottom 96-well nitrocellulose plates (Immobilon-P membrane; Millipore, Bedford, MA), which had been precoated with 2 µg/ml anti-mouse IFN- monoclonal antibody (Mabtech, Inc., Mairemont, OH). After 20 h, plates were washed with phosphate-buffered saline (PBS)-0.5% Tween 20 and then incubated with 1 µg/ml biotinylated anti-mouse IFN- monoclonal antibody (Mabtech) for 3 h at 37°C. After additional washes with PBS-0.5% Tween, spots were developed by incubation with Vectastain ABC peroxidase (Vector Laboratories, Burlingame, CA) and then 3-amino-9-ethylcarbazole solution (Sigma-Aldrich, St. Louis, MO) and counted by computer-assisted image analysis (Zeiss KS ELISPOT reader).

Experimental values were expressed as the mean net spots per million CD4⁺ cells for each peptide pool or individual peptide. For the initial screening of the 83 pools, responses against each pool were considered positive if (i) the number of spot-forming cells (SFCs)/10⁶ CD4⁺ T cells exceeded the absolute value of the mean negative control wells (effectors plus antigen-presenting cells [APCs] without peptide) by twofold, (ii) the value exceeded 50 SFCs/10⁶ CD4⁺ cells, and (iii) these conditions were met in at least two replicate independent experiments. Positive pools were deconvoluted into their eight individual components and tested again, to determine which individual peptides were responsible for the pooled IFN- response. Responses against individual peptides were considered positive if they exceeded the threshold of the mean negative control wells (effectors plus APCs without peptide) by twofold and exceeded a threshold of 200 SFCs/10⁶ CD4⁺ cells.

ICCS for IFN-. ICCS assays were performed as previously described (30). Splenocytes from LCMV-infected mice were cultured in 96-well U-bottom plates (1 x 10⁶ cells per well) in complete RPMI (RPMI 1640 with 10% fetal bovine serum, 2 mM L-glutamine, 1 U/ml penicillin G, and 100 µg/ml streptomycin), at 37°C with 5% CO₂ for 1 to 2 h, in the presence of the indicated peptides (at a final concentration of 10 µg/ml). Brefeldin A (1 µg/ml) was added 2 h later, and the cells continued in culture for 6 to 10 h. Cells were harvested, washed with PBS containing 5% fetal bovine serum, and stored on ice. Cells were stained according to the BD Cytofix/Cytoperm Plus kit (BD Biosciences, San Jose, CA) with antibodies to CD4 or a combination of CD4 with CD62L and/or CD8 and anti-IFN- antibodies (BD Pharmingen, San Jose, CA). The cells were collected using a BD FACSCalibur, and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR). A CD4⁺/IFN-⁺ response was considered positive if it exceeded the mean background (response against irrelevant peptide) + 3 standard deviations (SDs), corresponding to 0.73%. A CD8⁺/IFN-⁺ response was considered positive if it exceeded the mean background (response against irrelevant peptide) + 3 SDs, corresponding to 0.59%.

IA^b-peptide binding assays. H-2^b class II MHC was purified, and peptide binding assays were performed, essentially as previously described (45). Briefly, mouse B-cell lymphoma LB27.4 cells were utilized as sources of murine IA^b. MHC molecules were purified by affinity chromatography using the anti-IA^b monoclonal antibody Y3JP. Quantitative peptide binding assays were based on the inhibition of binding of radiolabeled probe peptide ROIV (peptide 569.01, an artificial ligand with sequence YAHAAHAAHAAHAAHAA) to purified IA^b molecules. Assays were performed at pH 5.5 in PBS containing 1% digitonin and in the presence of a cocktail of protease inhibitors (45). MHC binding of the radiolabeled peptide was determined by capturing MHC-peptide complexes on antibody-coated Lumitrac 600 plates (Greiner Bio-one, Frickenhausen, Germany) and measuring bound cpm using the TopCount (Packard Instrument Co., Meriden, CT) microscintillation counter. The average 50% inhibitory concentration (IC₅₀) of ROIV (peptide 569.01) for IA^b was 28 nM.

Memory and secondary challenge of LCMV Armstrong. Two groups of six C57BL/6 mice were infected with 2 x 10⁵ PFU LCMV Armstrong as previously described. Three mice from each group were sacrificed by CO₂ inhalation 35 days postinfection and tested in ELISPOT assays as previously described. The three remaining mice from each group were infected with 2 x 10⁵ PFU LCMV Armstrong for a secondary challenge at day 35 of the original infection, as previously described. Eight days postinfection (or 43 days post-original infection), each group was sacrificed by CO₂ inhalation and tested in ELISPOT assays as described above. Uninfected C57BL/6 mice were used as controls.

Sequence analysis. Amino acid sequences of the GP and NP proteins were aligned using EMBOSS pairwise alignment algorithms (http://www.ebi.ac.uk/Tools/emboss/align/). Each sequence was individually compared to LCMV Armstrong. The following sequences were used: LCMV Armstrong 53b (NP 694851), LCMV clone 13 (DQ361066), LCMV WE (AAA46265), LCMV CHV1 (AAA82176), LCMV CHV2 (AAA82177), LCMV CHV3 (AAA82178), LCMV WE-HPI (CAC01231), LCMV CH-5692 (AAL01687), and LCMV CH-5871 (AAL01687). The sequences were derived from the Arenavirus Sequence Database (http://epitope.liai.org:8080/projects/arena/arena_home). Each CD4⁺ T-cell epitope was compared to the peptide sequences for sequence homology.

Statistical analyses. For each peptide or pool studied, three mice were used per experiment, and either the splenocytes were pooled (ELISPOT) or each mouse was tested independently (ICCS). All peptides and pools were tested in triplicate and in three or more independent experiments. The average and SD of the triplicate samples were then calculated. Responses among groups of mice are presented as means with SDs. Comparisons of mean immune responses were performed using one-sided t tests, including Fisher's exact test, with P values of <0.05 considered significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of LCMV-derived CD4⁺ T-cell epitopes. A set of 664 overlapping peptides spanning the entire LCMV Armstrong proteome was subdivided into 83 pools of eight peptides each, grouped in sequential order. To identify CD4⁺ T-cell epitopes, C57BL/6 mice were infected with 2 x 10⁵ PFU of LCMV Armstrong via i.p. inoculation and 8 days later splenic CD4⁺ T cells were enriched and stimulated with each peptide pool. CD4⁺ T-cell reactivity against each pool was determined in standard IFN- ELISPOT assays. A total of 17 positive pools from NP, GP, and L proteins were identified (data not shown).

To define specific epitopes, positive pools were decoded, and each of the eight peptides contained within a positive pool was tested. Responses greater than twice the mean negative control wells and exceeding 200 SFCs/10⁶ CD4⁺ cells were considered positive. Nine pools failed to yield significant responses against individual peptides (data not shown). In the remaining eight pools, 10 antigenic peptides were identified that met the positivity criteria. Figure 1 shows the positive responses observed against individual peptides from the original positive pools. The 10 responses detected against peptides were located in their source proteins at GP 6-20, GP 31-45, GP 61-75, GP 66-80, GP 126-140, GP 171-185, GP 186-200, NP 201-215, NP 236-250, and NP 311-325.

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FIG. 1. Identification of LCMV-derived CD4+ epitopes by ELISPOT. C57BL/6 (H-2b) mice were infected i.p. with 2 x 105 PFU of LCMV Armstrong. Eight days postinfection, CD4+ T-cells were purified and tested against each of the eight peptides contained in each positive pool at a concentration of 10 mg/ml per peptide. Responses against individual peptides were considered positive if they exceeded the threshold of double the mean negative control wells (effectors plus APCs without peptide) and exceeded a threshold of 200 SFCs/106 CD4+ cells. Each positive peptide is labeled by its proteomic location.

To verify that the IFN- responses observed in the ELISPOT assays were indeed due to CD4⁺ T-cells, we performed ICCS experiments. Mice were infected with LCMV, and 8 days later, splenocytes were isolated and incubated with each of the peptides and then stained for intracellular IFN- and CD4 and CD8 surface staining. The 10 peptides identified as positive in the ELISPOT assays, as described above, were tested (GP 6-20, GP 31-45, GP 61-75, GP 66-80, GP 126-140, GP 171-185, GP 186-200, NP 201-215, NP 236-250, and NP 311-325). In Fig. 2, the mean responses against each of the peptides are shown for both CD4⁺/IFN-⁺ (Fig. 2A) and CD8⁺/IFN-⁺ (Fig. 2B) cells. The percentage of CD4⁺ cells producing IFN- in response to each peptide was measured from LCMV Armstrong-infected animals. Each of the peptides was tested using at least six different mice. The percentage of CD4⁺/IFN-⁺ releasing cells from unstimulated mice was 0.16% ± 0.57% (mean + 3 SDs), and accordingly, we defined a value of 0.73% as a threshold value to consider IFN- responses against a given peptide as positive.

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FIG. 2. CD4+ versus CD8+ IFN- production as measured by intracellular cytokine staining. C57BL/6 (H-2b) mice were infected i.p. with 2 x 105 PFU of LCMV Armstrong. Eight days postinfection, splenocytes were purified and tested in ICCS assays measuring IFN- production and surface staining for CD4 (A) and CD8 (B) against 10 peptides identified as positive in the ELISPOT assays.

Out of the 10 peptides tested, 6 peptides identified in the ELISPOT assay scored positive for IFN- production by gated CD4⁺ cells in the ICCS assay (Fig. 2A). The percentage of CD4⁺/IFN-⁺ cells ranged from 0.25 to 3.04% against the peptide panel. The strongest response observed was against GP 66-80, followed by GP 126-140, GP 186-200, NP 311-325, GP 31-45, and GP 6-20. Interestingly, peptide GP 61-75, nested within the previously characterized GP 61-80 epitope, did not give a significant response in the ICCS, whereas the overlapping peptide, GP 66-80, elicited the strongest response.

Next, we assessed if the lack of response against some peptides in the ICCS assay could be explained by contaminating CD8⁺ cells. Figure 2B shows the IFN- production by CD8⁺ T cells in response to each of the 10 peptides. The response observed in unstimulated mice was 0.20% ± 0.39% (mean + 3 SD), yielding a threshold value of 0.59% to consider a response against a given peptide as positive. For the four peptides which did not yield significant IFN- production by CD4⁺ T cells, GP 61-75, GP 171-185, NP 201-215, and NP 236-250, three peptides yielded strong CD8⁺/IFN-⁺ staining: GP 171-185, NP 201-215, and NP 236-250. Out of the remaining six peptides, which had significant IFN- responses by CD4⁺ T cells, for two peptides, GP 6-20 and GP 186-200, the newly identified epitopes were not associated with CD8⁺/IFN-⁺ staining. However, each of the other four epitopes yielded CD8⁺/IFN-⁺ staining. GP 31-45 contains the previously identified MHC class I-restricted epitope, GP 33-41 (34, 42, 50, 54). Indeed, the GP 31-45 peptide elicited a CD8⁺ T-cell response, with 11.7% of the CD8⁺ cells producing IFN-, thus demonstrating that this region contains overlapping CD4⁺ and CD8⁺ T-cell epitopes. The NP 311-325 epitope contains peptide NP 314-322, predicted to bind to the MHC molecule K^b with an affinity of 774 nM (J. Sidney, personal communication). The NP 311-325 peptide elicited a CD8⁺ T-cell response with 0.66% of the CD8⁺ cells producing IFN-. GP 66-80 contains a recently identified epitope, GP 67-77, which is restricted by D^b (20, 27). GP 126-140 also elicited a CD8⁺/IFN- response consisting of 0.97%. Therefore, out of the six CD4⁺ epitopes, four of them also yielded CD8⁺/IFN-⁺ staining.

We were interested in determining whether the overlap between the set of CD4⁺ and CD8⁺ T-cell epitopes is statistically significant. To pursue this, each of the 664 15-mer peptides overlapping the LCMV proteome was analyzed for the presence or absence of a CD4⁺ T-cell response as well as whether it contained a nested CD8⁺ T-cell epitope in the setting of H-2^b. The Fisher exact test (one tailed) yielded a P value of 0.0033. We further examined if this overlap was due only to a preferred recognition of peptides from the GP and NP proteins. Limiting the same test to the 208 peptides overlapping GP and NP still gives a significant association of CD4 and CD8 response targets (P = 0.0093). This demonstrates that the overlap of the two sets of immune response targets is statistically significant.

MHC peptide binding affinity of identified epitopes. To determine the MHC binding affinity for each of the identified epitopes, we conducted MHC-peptide binding assays. H-2^b class II MHC was purified, and quantitative inhibition binding assays were performed for each peptide using IA^b, as described in Materials and Methods (45). The binding assays revealed that five out of the six peptides bound to IA^b with an affinity of less than 11,000 nM (Table 1). Although found to yield strong immunogenicity responses, as measured in both ELISPOT and ICCS assays, peptide GP 186-200 did not efficiently bind to IA^b. The greatest affinity to IA^b was detected against GP 66-80, which interestingly also elicited the strongest ICCS response. Table 1 summarizes the binding affinities as well as the IFN- levels detected both by ELISPOT and ICCS.

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TABLE 1. Binding affinities and IFN- levels detected by ELISPOT and ICCSa

Characterization of responses after chronic infection. To determine if responses were present after infection with clone 13, a virus which yields persistent infection, two groups of three mice per group were infected with 2 x 10⁶ PFU LCMV clone 13 intravenously. Eight days postinfection, the mice were sacrificed by CO₂ inhalation and tested in IFN- ELISPOT assays with the six peptides which resulted in responses after LCMV Armstrong infection. Responses greater than twice the mean of negative control wells and exceeding 200 SFCs/10⁶ CD4⁺ T cells were considered positive. Of the six characterized CD4⁺ epitopes identified above, responses were observed against two epitopes, GP 66-80 and GP 126-140. Figure 3 shows the responses observed against the individual peptides. We observed weak and not statistically significant responses against GP 31-45, GP 186-200, and NP 311-325. The epitope GP 6-20 was negative in two replicate experiments.

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FIG. 3. Identification of CD4+ responses after chronic infection. C57BL/6 (H-2b) mice were infected retroorbitally with 2 x 106 PFU of LCMV clone 13. Eight days postinfection, CD4+ T cells were purified and tested against each of the six epitopes at a concentration of 10 mg/ml per peptide. Responses against individual peptides were considered positive if they exceeded the threshold of double the mean negative control wells (effectors plus APCs without peptide) and exceeded a threshold of 200 SFCs/106 CD4+ cells. Each peptide is labeled by its proteomic location.

Magnitude of CD4⁺ T-cell epitopes in the memory stage and after secondary challenge with LCMV Armstrong. To determine the magnitude of CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection, C57BL/6 mice were infected with 2 x 10⁵ PFU LCMV Armstrong and tested in ELISPOT assays as described in Materials and Methods.

The responses observed after 35 days postinfection were from two of the six epitopes tested (GP 66-80 and NP 311-325). Both epitopes met the criteria established for a positive response (twice the mean for negative control wells and exceeding 200 SFCs/10⁶ CD4⁺ T cells). Furthermore, after secondary challenge with 2 x 10⁵ PFU LCMV Armstrong at 35 days of infection, we observed similar responses (Fig. 4). No response was detected with the uninfected control mice. After both infections, weak responses were observed for GP 31-45, GP 126-140, and GP 186-200. The GP 6-20 epitope was negative in two replicate experiments.

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FIG. 4. Identification of CD4+ responses during memory stage and secondary infection of LCMV Armstrong. C57BL/6 (H-2b) mice were infected i.p. with 2 x 105 PFU of LCMV Armstrong. Thirty-five days postinfection, three mice from each group were used in ELISPOT assays and three mice from each group were challenged with a second (2°) infection of LCMV Armstrong. Eight days postinfection, CD4+ T cells were purified and tested against each of the six epitopes at a concentration of 10 mg/ml per peptide. Responses against individual peptides were considered positive if they exceeded the threshold of double the mean negative control wells (effectors plus APCs without peptide) and exceeded a threshold of 200 SFCs/106 CD4+ cells. Each positive peptide is labeled by its proteomic location.

Sequence analysis of newly identified CD4⁺ T-cell epitopes. To determine whether there was sequence homology among the newly characterized CD4⁺ T-cell epitopes, sequences from several strains of LCMV were aligned to reveal any differences among the strains of LCMV using the sequence of the newly identified CD4⁺ T-cell epitopes. Table 2 shows the sequence alignments among various strains of LCMV. There were no amino acid changes from LCMV Armstrong to LCMV clone 13.

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TABLE 2. Sequence alignments of LCMV strains

There were several changes in the overlapping CD4/CD8 epitope GP 31-45, where there was a GS change at position 1 in seven of the nine strains compared. There was an IL change at position 13 in three strains. There were an FL change at position 14 in two strains and an AT change at position 15 in two strains. The other five epitopes had few changes among the different strains. This shows us that while the majority of the epitopes were conserved between the several different strains assessed, differences were observed in the immunodominant CD8⁺ epitope GP 31-45.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have identified a set of six LCMV-specific CD4⁺ T-cell epitopes recognized following acute infection with LCMV Armstrong of C57BL/6 mice, consisting of GP 6-20, GP 31-45, GP 66-80, GP 126-140, GP 186-200, and NP 311-325. Out of the six epitopes characterized in this study, four epitopes are novel (GP 6-20, GP 31-45, GP 126-140, and GP 186-200). The remaining epitopes have been previously characterized and reported in the literature as GP 61-80 and NP 309-328 (34-36). We have now detected the specific 15-mer regions within each of these peptides responsible for immunogenicity as GP 66-80 and NP 311-325, respectively. It is interesting to note that the GP 61-75 peptide did not elicit significant IFN- production in our ICCS assays. Also, one of the peptides did not efficiently bind to IA^b. It is possible that a region contained within this 15-mer peptide is responsible for binding to the MHC molecule and the entire peptide sequence hinders its binding. These studies emphasize the importance of detailed epitope identification.

Our lab recently identified nine CD4⁺ T-cell epitopes in the BALB/c (H-2^d) mouse system (30). To see if this breadth of responses was only unique to the H-2^d system or whether the repertoire of responses was not fully characterized in other MHC backgrounds, we were interested in identifying all CD4⁺ T-cell epitopes in the H-2^b system. Our data clearly show that a broad repertoire of class II responses exist after LCMV infection in both the H-2^d and H-2^b systems. The complexity of the helper responses has recently been elucidated for other viral infections. Richards et al. recently showed that a broad repertoire of influenza virus-specific CD4⁺ T-cell responses exist in the HLA-DR1 transgenic mouse system (39). Only considering the hemagglutinin protein, as many as 30 different peptides were recognized in these transgenic mice (39). It has also been shown that a broad repertoire of CD4⁺ T-cell responses can be detected after vaccinia virus infection, consisting of 14 responses restricted by IA^b (31). We can conclude from these data that the breadth of helper responses is quite complex not only against LCMV, but also against other viruses.

In LCMV infection of both H-2^d and H-2^b mice, with the exception of one CD4⁺ T-cell epitope derived from the Z protein in the H-2^d system, all epitopes identified to date derived from the NP and GP proteins (30). NP and GP proteins are expressed early and abundantly in infected cells, providing the major components of LCMV virions. Consistent with their abundance, these proteins play a significant role in the immunodominance of CD8⁺ T-cell responses against the virus (24, 38). We did not identify any CD4⁺ T-cell epitopes derived from the L protein, despite its large size (2,210 residues) (6, 40) and despite the fact that several CD8⁺ response targets were recently identified (24). Our findings correlate well with previous studies suggesting that CD4⁺ T-cell epitopes are preferentially associated with structurally stable regions of proteins (25, 41). However, given the breadth of responses targeted against different regions of the GP protein, our findings diverge from previous studies, which have shown that immunodominant responses cluster in limited regions of particular antigens (8, 10, 25, 37).

One of the strongest CD4⁺ T-cell responses identified in this study was directed against a region that contains a nested CD8⁺ T-cell epitope. Previous research has described the GP 33-41 epitope as an immunodominant CTL response in C57BL/6 mice (15, 42, 50, 54). Our studies identify GP 31-45, NP 311-325, GP 66-80, and GP 126-140 as regions containing overlapping CD4⁺ and CD8⁺ T-cell epitopes. We have previously identified a region of overlapping CD4⁺ and CD8⁺ epitopes against LCMV in the H-2^d system directed toward the NP 116-130 region (NP 116-130 CD4⁺ T-cell epitope and NP 118-126 CD8⁺ T-cell epitope) (30). Homann et al. reported the GP 67-77 region is dually restricted by MHC class I and II molecules in the H-2^b system, which we have confirmed in this study (20). In influenza virus infection of mice in the H-2^b system, it has recently been shown that an overlap exists between CD4 and CD8 T-cell epitopes, in PB2 91-105 and NP 311-325 (9). However, this is the first study illustrating extensive epitope sharing between CD4⁺ and CD8⁺ T-cells. It is tempting to speculate that the overlap of these two different cellular immune responses might be related to the immundominance of particular viral regions and may be influenced by proteasomal processing, MHC binding, and/or T-cell receptor repertoires.

The breadth of CD4⁺ T-cell responses has not been fully characterized following chronic infection of LCMV in H-2^b mice. This study identified two responses directed against the glycoprotein during persistent infection with LCMV clone 13. This is the first time a multiple set of CD4⁺ T-cell responses have been thoroughly characterized and a subset has been detected following clone 13 infection. In our previous study, CD4⁺ T-cell responses went undetectable after clone 13 infection in the H-2^d background (30).

We further identified responses that were present in the memory phase of infection. These responses were derived against the GP and NP peptides GP 66-80 and NP 311-325, respectively. These two responses were also present after secondary challenge with LCMV Armstrong. For the other weaker responses, it is possible that immunization with the peptide would result in stronger memory responses.

We found that there was sequence homology among the epitopes for the different strains of LCMV. This indicates that most of the epitopes are derived from conserved portions of the proteome. These conserved regions may therefore be valuable targets for vaccine development.

CD4⁺ T-cell responses have been shown to be instrumental in establishing and sustaining effective CD8⁺ T-cell responses in the setting of viral and bacterial infection (4, 21, 43, 44). In this study, we have characterized the profile of a successful set of helper responses against LCMV, which results in viral clearance. Our studies illustrate the complex nature of the CD4⁺ T-cell response against LCMV and the need for accurate characterization of immune responses.

 
This work was supported by 5SC3GM082343-02 (B.R.M.), HHSN266200400023C (A.S. and M.J.B.), AI50840 (M.J.B.), and NIH grant AI-065359 "Pacific Southwest Center For Biodefense and Emerging Infectious Diseases" (M.J.B.).

We thank Matthias von Herrath and Shane Crotty for input into the peptide design for the initial experiments and Stuart Perry for insight into data presentation.

 
* Corresponding author. Mailing address: Department of Biological Sciences, California State University, San Marcos, CA 92096. Phone: (760) 750-4637. Fax: (760) 750-3063. E-mail: bmothe{at}csusm.edu

Published ahead of print on 1 October 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, December 2008, p. 11734-11741, Vol. 82, No. 23
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