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The Journal of Immunology, 2006, 176: 3391-3401.
Copyright © 2006 by The American Association of Immunologists

The BZLF1 Homolog of an Epstein-Barr-Related {gamma}-Herpesvirus Is a Frequent Target of the CTL Response in Persistently Infected Rhesus Macaques1

Mark H. Fogg*, Deirdre Garry{dagger}, Amany Awad{dagger}, Fred Wang* and Amitinder Kaur2,{dagger}

* Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and {dagger} Department of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, MA 01772


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although CD8+ T lymphocytes targeting lytic infection proteins dominate the immune response to acute and persistent EBV infection, their role in immune control of EBV replication is not known. Rhesus lymphocryptovirus (rhLCV) is a {gamma}-herpesvirus closely related to EBV, which establishes persistent infection in rhesus macaques. In this study, we investigated cellular immune responses to the rhLCV BZLF1 (rhBZLF1) homolog in a cohort of rhLCV-seropositive rhesus macaques. rhBZLF1-specific IFN-{gamma} ELISPOT responses ranging between 56 and 3070 spot-forming cells/106 PBMC were detected in 36 of 57 (63%) rhesus macaques and were largely mediated by CD8+ T lymphocytes. The prevalence and magnitude of ELISPOT responses were greater in adult (5–15 years of age) rather than juvenile macaques (<5 years of age), suggesting that rhBZLF1-specific CTL increase over time following early primary infection. A highly immunogenic region in the carboxyl terminus of the rhBZLF1 protein containing overlapping CTL epitopes restricted by Mamu-A*01 and other as yet unidentified MHC class I alleles was identified. The presence of a robust CD8+ T lymphocyte response targeting this lytic infection protein in both rhesus macaques and humans suggests that these CTL may be important for immune control of EBV-related {gamma}-herpesvirus infection. These data underscore the utility of the rhLCV-macaque model for studies of EBV pathogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epstein-Barr virus infection is widely prevalent among humans and is associated with a variety of malignancies including nasopharyngeal carcinoma, Burkitt lymphoma, and posttransplant lymphoproliferative disease. EBV preferentially infects B lymphocytes and persists in vivo in a subset of latently infected B lymphocytes that only express latent infection viral proteins, thus comprising a stable reservoir of infected cells (1). Reactivation of EBV resulting in a switch from latent infection to lytic infection occurs periodically and leads to intermittent, asymptomatic, virus shedding in the oropharynx of immunocompetent hosts (1). EBV induces a robust humoral and cellular immune response that persists for the life of the host. Several lines of evidence suggest that cellular immune responses to EBV, while unable to prevent the establishment of viral persistence, are nevertheless essential for maintaining control of virus-induced lymphoproliferation. The increased incidence of EBV-related tumors such as B cell lymphomas in immunosuppressed individuals and the resolution of posttransplant lymphoproliferative disease following adoptive transfer of in vitro expanded EBV-specific T lymphocytes provides strong support for a critical role of T cell immunity in maintaining asymptomatic EBV infection (1, 2).

EBV-infected humans mount a robust CD8+ T lymphocyte response to both latent and lytic EBV proteins. Early studies using EBV-immortalized B cell lines as Ag-presenting and target cells led to the frequent and almost exclusive detection of CTL responses to the latent proteins (3, 4, 5). Because EBV-immortalized B cells express the full range of latent proteins and only a few express lytic proteins, their use as APCs or target cells not surprisingly led to an underestimation of the contribution of lytic proteins to the EBV-specific T lymphocyte response. With the use of peptides and vaccinia recombinants as Ags, it soon became apparent that EBV lytic proteins are a prominent target of the host cellular immune response. Two immediate early gene products, BZLF1 and BRLF1, and six early gene products, BMLF1, BMRF1, BHLF1, BHRF1, BALF2, and BALF5, have been shown to be frequent CTL targets in several studies (6, 7, 8, 9, 10). BZLF1, the key protein in initiating the cascade of lytic genes resulting in lytic replication, is a frequent target of EBV-specific CTL in humans with asymptomatic, persistent EBV infection, often exceeding the response to latent proteins (7, 8, 11, 12). An unusually high frequency of BZLF1-specific CTL can be observed in patients with acute infectious mononucleosis (AIM)3 (9, 13, 14). However, the importance of lytic protein-specific CTL for controlling primary and persistent EBV infection, or preventing EBV-associated malignancies is not known.

A B cell immortalizing {gamma}-herpesvirus (Cercopithicine herpesvirus 15, rhesus lymphocryptovirus (rhLCV)) homologous to EBV at both the genomic and protein level (15) naturally infects rhesus macaques (16). Similar to EBV infection in humans, rhLCV infection is ubiquitous among rhesus macaques (17), and establishes a life-long asymptomatic persistent infection in B cells associated with periodic reactivation and shedding of virus in oral secretions. Immunosuppression can result in the development of rhLCV-associated B cell lymphomas and epithelial cell lesions similar to oral hairy leukoplakia (18). The ability to reproduce primary infection, persistent infection, and lymphomagenesis after experimental infection of rhLCV naive rhesus macaques provides an animal model system for EBV infection in which the role of cellular immune responses to lytic infection Ags might be experimentally tested (18, 19). There is currently little published data on cellular immune responses to rhLCV in rhesus macaques (20). Because a large portion of the CD8+ T lymphocyte response in EBV-infected humans is directed toward the BZLF1 protein, we asked whether this immediate early lytic gene was also a dominant target for the immune response in rhLCV-infected rhesus macaques.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Rhesus macaques (Macaca mulatta) enrolled in the study were housed at the New England Primate Research Center in accordance with institutional and federal guidelines of animal care (21). Fifty-four rhLCV-seropositive rhesus macaques in the conventional colony with naturally acquired rhLCV infection and three rhLCV-naive rhesus macaques in the specific pathogen-free colony that seroconverted following oral inoculation with rhLCV 24–36 mo before evaluation were included in the study. Six rhLCV-seronegative rhesus macaques in the specific pathogen-free colony were used as negative controls.

MHC typing

DNA was extracted from PBMC or B-lymphoblastoid cell lines using the QIAamp DNA blood kit (Qiagen) and subjected to PCR with rhesus macaque MHC class I sequence-specific primers essentially as described previously (22, 23). Primers specific for 21 MHC class I haplotypes were used: Mamu-A*01, A*02, A*03, A*04, A*05, A*06, A*07, A*08, A*11, A*13, NA4, NA7, B*01, B*03, B*04, B*07, B*12, B*17, NB2, NB4, and NB5 (22, 23, 24). Internal control primers specific for Mamu-DR were included in all reactions as an internal control (22). Mamu-A*13-positive animals were subtyped by sequencing of PCR products from PCR lacking internal control primers.

rhLCV serology

Serologic assays were performed essentially as described previously (17). Briefly, a peptide corresponding to aa 147–170 of the rhLCV small virus capsid Ag was resuspended at 2.5 µg/ml in bicarbonate buffer (50 mM; pH 9.6) and used to coat wells of an Immulon I 96-well microtiter plate (Dynatech Laboratories) overnight at 4°C. Unbound peptides were removed by washing with PBS containing 0.1% Tween 20 (Sigma-Aldrich) and blocked for 2 h at room temperature with PBS/BSA (PBS, 0.1% Tween 20, 3% BSA; Sigma-Aldrich). Serum was diluted 1/100 in PBS/BSA and added to the 96-well plates. After 1 h at room temperature, wells were washed and incubated with HRP-conjugated goat anti-human IgG Ab (Jackson ImmunoResearch Laboratories) in PBS/BSA for 1 h. Peroxidase activity was measured using o-phenylenediamine dihydrochloride tablets (Sigma-Aldrich). After 30 min at room temperature, absorbance was measured at 450 nm using a Bio-Rad microplate reader. Absorbance values three times above the mean obtained from triplicate wells containing the secondary Ab but no sera were considered positive.

Peptides

A pool of 60 15-aa long peptides overlapping by 11 aa that spanned the length of the published rhLCV BZLF1 (rhBZLF1) protein sequence (accession no. AAK95436) were used to measure the total rhBZLF1-specific cellular immune response. Shorter 8- to 11-aa long peptides were used for fine epitope mapping. Peptides were synthesized by F-moc chemistry at the Massachusetts General Hospital peptide core facility (Charlestown, MA) using an automated peptide synthesizer (MBS 396; Advanced Chemtech). Individual lyophilized peptides were resuspended at 100 mg/ml in 100% DMSO (Sigma-Aldrich). The final concentration of individual peptides used for stimulation in pools or otherwise was 1–2 µg/ml, and the final DMSO concentration was kept at <0.5%.

Vaccinia recombinants

Recombinant vaccinia viruses expressing the rhBZLF1 and rhLCV latent infection proteins, rhEBNA-1, -2, -3A, -3B, -3C, and -LP, truncated rhLMP1 (aa 212–589), and rhLMP2A (aa 1–309) were generated as described previously (25).

Antibodies

mAbs used for costimulation in intracellular cytokine staining (ICS) assays consisted of purified, azide-free anti-human CD28 mAb (clone 28.2; BD Pharmingen) and anti-human CD49d mAb (clone 9F10; BD Pharmingen) cross-linked with affinity-purified F(ab')2 of goat anti-mouse IgG (H+L) (Kirkegaard & Perry Laboratories). Fluorochrome-conjugated anti-human mAbs for multicolor flow cytometry were obtained from BD Pharmingen and included CD3 (clone SP34) FITC or PE, CD4 (clone L200) FITC, CD8 (clone SK1) PerCP, CD69 (clone FN50) PE, IFN-{gamma} (clone 4S.B3) allophycocyanin, and TNF-{alpha} (clone MAb11) allophycocyanin.

Isolation of PBMC

PBMC were isolated from heparinized blood by density gradient centrifugation over Ficoll-Hypaque (ICN Biomedicals) and suspended at 2 x 106 cells/ml in RPMI 1640 medium (Invitrogen Life Technologies) containing 2 mM L-glutamine (Invitrogen Life Technologies), 10 mM HEPES (Invitrogen Life Technologies), and supplemented with 10% FBS (Sigma-Aldrich) and 50 IU/ml each of penicillin and streptomycin (Invitrogen Life Technologies), henceforth referred to as R-10 medium.

Generation of B-lymphoblastoid cell lines

Autologous and MHC class I-matched B lymphoblastoid cell lines were derived by immortalizing B cells from the peripheral blood of rhesus macaques using Cercopithicine herpesvirus 12 (baboon LCV; baLCV) (26). This closely related but antigenically distinct lymphocryptovirus (LCV) has the ability to immortalize B cells from rhesus macaques in vitro and allows the generation of autologous and MHC class I-matched immortalized B cells with little or no recognition by CTL specific for rhLCV proteins. In contrast, immortalized B cells generated using rhLCV are recognized and lysed by rhLCV-specific CTL. PBMC were incubated at 37°C in a 5% CO2 incubator with supernatant of baLCV-producing B cells (S594 cell line) in the presence of 1 µg/ml cyclosporin. Transformed cells were propagated in RPMI 1640 medium containing 2 mM L-glutamine and 10 mM HEPES and supplemented with 20% FBS and 50 IU/ml each of penicillin and streptomycin.

Fractionation of lymphocytes

CD4+ or CD8+ T lymphocytes were selectively depleted by immunomagnetic sorting (StemCell Technologies). Briefly, 1 x 107 lymphocytes were incubated with 20 µl of tetrameric Abs anti-dextran/anti-CD4 or anti-dextran/anti-CD8 in 1 ml of PBS for 15 min at room temperature. Thereafter, cells were incubated with 60 µl of dextran-coated magnetic nanoparticle colloid for 15 min and subsequently run through a 0.3-inch high-gradient magnetic column of stainless steel mesh. The flow-through consisting of the negatively selected cell fraction was used in additional experiments. A small fraction of cells was analyzed by flow cytometry to assess the efficiency of depletion. In all instances, the negatively selected cells contained <1% of the depleted cell population.

Generation of effector PBMC

rhBZLF1-specific CTL were expanded by in vitro peptide stimulation. One-third of autologous PBMC pulsed with individual peptides at 10–100 µg/ml in R-10 medium for 90 min at 37°C were used as stimulator cells. Stimulator cells were washed and resuspended with the remaining two-thirds of autologous PBMC in R-10 medium and cultured in 24-well plates (Costar/Fisher Scientific) for 14 days. A total of 5–10 IU/ml recombinant human IL-2 (donated by M. Gately, Hoffman-LaRoche, Nutley, NJ) was added to the cultures 4–7 days after stimulation and twice a week thereafter. CTL assays were performed 14 days after Ag-specific stimulation.

Generation of CTL clones

Following in vitro stimulation of PBMC with peptide for 2 wk, CD4+ T lymphocytes were selectively depleted by immunomagnetic separation. CD8+-enriched effector T lymphocytes were plated in 96 replicates at 10, 3, and 1 cells/well into 96-well U-bottom plates (Costar) in R-10 medium containing 50 IU/ml IL-2 and 5 µg/ml Con A (Sigma-Aldrich) along with 2 x 105 cells/ml gamma-irradiated (100 Gy) autologous baLCV-immortalized B cells and 1 x 106 cells/ml gamma-irradiated (30 Gy) human feeder PBMC. Con A was removed after 3–4 days, and clones were subsequently maintained in culture with R-10 medium containing 50 IU/ml IL-2. Clones were restimulated with peptide-pulsed autologous baLCV-immortalized B cells and feeder PBMC every 2 wk.

Chromium release assay

Autologous or allogeneic baLCV-immortalized B cells labeled with 50 µCi of 51Cr (NEN/PerkinElmer) per 106 cells and pulsed or not with individual rhBZLF1 peptides were used as target cells. In CTL assays using vaccinia virus-infected target cells, baLCV-immortalized B cells were infected overnight with a recombinant vaccinia virus at a multiplicity of infection of 10 PFU/cell and then labeled with 51Cr. BaLCV-immortalized B cells infected with wild-type vaccinia strain NYCBH were used as controls. Target cells were dispensed at 104 cells/well in duplicate along with effectors (effector PBMC or CTL clones) at different E:T ratios into a 96-well U-bottom plate (Costar) and incubated for 5 h at 37°C in a 5% CO2 incubator. At the end of incubation, plates were spun at 1000 rpm for 10 min at 4°C, and supernatant was harvested onto a LumaPlate-96 (PerkinElmer) and allowed to dry overnight. Emitted radioactivity was measured in a 1450 MicroBeta Plus liquid scintillation counter (Wallac). Spontaneous release was measured from wells containing target cells alone, and maximum release from wells containing target cells and 5% Triton X-100 (Sigma-Aldrich). Percentage of specific lysis was calculated as [(test release – spontaneous release)/(maximum release – spontaneous release)] x 100. Spontaneous release was <25% in all assays.

IFN-{gamma} ELISPOT assay

ELISPOT assays were conducted using a monkey IFN-{gamma} ELISPOT kit (Mabtech). Briefly, PBMC resuspended in R-10 medium were placed at input numbers ranging between 3 x 105 and 5 x 105 cells/well in a 96-well microtiter plate (Millipore) coated with the anti-human IFN-{gamma} mAb (clone GZ-4). Following overnight stimulation with rhBZLF1 peptides or medium alone, cells were removed by extensive washing. Wells were serially incubated with biotinylated anti-human IFN-{gamma} mAb (clone 7-B6-1) followed by streptavidin/alkaline phosphatase. Spots were developed by the addition of alkaline phosphatase conjugate substrates, NBT, and 5-bromo-4-chloro-3-indolyl phosphate in color development buffer (Bio-Rad). Spots were counted with a KS ELISPOT Automated Reader (Carl Zeiss) using KS ELISPOT Software 4.5 (performed by Zellnet). The rhBZLF1-specific spot frequency was calculated after subtraction of the spot frequency in control medium wells. Spot frequencies greater than three SD above the mean response in rhLCV-seronegative rhesus macaques and >2-fold above background in negative control wells were considered positive. IFN-{gamma} ELISPOT assays using recombinant vaccinia viruses for comparison of rhBZLF1 responses with rhLCV latent proteins were performed as described previously (20).

ICS assay

ICS assays to detect the frequency of peptide-specific CD4+ or CD8+ T lymphocytes were performed as described previously (27). Briefly, PBMC were stimulated with rhBZLF1 peptides in the presence of 10 µg/ml costimulatory mAbs anti-CD28 and anti-CD49d, which had been cross-linked for optimal costimulation. PBMC were stimulated for 6 h in the presence of brefeldin A (Sigma-Aldrich) added at 10 µg/ml for the final 4 h of stimulation. At the end of stimulation, cells were surface stained with conjugated anti-CD3 and anti-CD8 mAb for 30 min at 4°C and sequentially fixed in 1x FACS Lysing Solution (BD Biosciences) followed by permeabilization with 1x FACS Permeabilizing Solution (BD Biosciences). Permeabilized cells were incubated with anti-CD69 and anti-IFN-{gamma} or anti-TNF-{alpha} mAbs for 30 min at 4°C, washed, and kept at 4°C overnight in 2% paraformaldehyde. Two hundred thousand events were collected on a FACSCalibur (BD Biosciences), and data were analyzed using CellQuest software (BD Biosciences) and FlowJo (Tree Star) software. rhBZLF1-specific CD8+ T lymphocytes were delineated by gating on CD3+CD8+ T lymphocytes that coexpressed CD69 and the cytokine of interest.

Tetramer synthesis and staining

Biotinylated monomers of the Mamu-A*01 class I molecule and the Mamu-A*01-restricted rhBZLF1 epitope aa 237–246 were synthesized by the National Institutes of Health Tetramer Core Facility at the Emory University Vaccine Center. Tetramers were made by incubation of biotinylated monomers with streptavidin-APC (Molecular Probes). Staining of PBMC with appropriately titered tetramer was performed by incubation at 37°C for 15 min, followed by surface staining with conjugated anti-CD3 and anti-CD8 mAb at 4°C for 30 min. Cells were then washed, fixed in 2% paraformaldehyde, and analyzed. Two hundred thousand lymphocyte events were collected on a FACSCalibur (BD Biosciences), and data were analyzed using CellQuest (BD Biosciences) and FlowJo (Tree Star) software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
T lymphocyte recognition of the lytic protein rhBZLF1 in rhesus macaques with persistent rhLCV infection

To determine whether the rhLCV lytic protein rhBZLF1 is a target of the rhLCV-specific cellular immune response in rhesus macaques, IFN-{gamma} ELISPOT responses to overlapping 15-aa peptides spanning the entire length of the rhBZLF1 protein were evaluated in 54 rhesus macaques with naturally acquired rhLCV infection and in three macaques with experimentally acquired rhLCV infection. Positive rhBZLF1-specific IFN-{gamma} ELISPOT responses (above 50 spot-forming cells (SFC)/106 PBMC) were observed in 35 of 54 (65%) naturally infected and one of three experimentally infected rhLCV-seropositive macaques (Fig. 1A). The rhBZLF1-specific response among all rhBZLF1 responders ranged between 56 and 3070 SFC/106 PBMC (mean, 370 SFC/106 PBMC). In nine naturally infected macaques tested at two or three time-points 1–8 mo apart, the rhBZLF1-specific ELISPOT response in individual animals varied <3-fold over time, suggesting a relatively stable response (data not shown).


Figure 1
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  		FIGURE 1. IFN-{gamma} ELISPOT responses to the rhLCV rhBZLF1 protein in rhesus macaques. PBMC from rhLCV-seropositive rhesus macaques with natural infection (n = 54) or experimental infection (n = 3), and rhLCV-seronegative rhesus macaques (n = 6) were stimulated overnight with peptides spanning the rhLCV rhBZLF1 protein. A, Spot frequencies plotted as a function of study group. B, Spot frequencies of naturally infected rhesus macaques plotted as a function of age. *, Statistically significant by Fisher’s protected least significant difference. C, Percentage of naturally infected rhesus macaques with a positive rhBZLF1 ELISPOT response plotted as a function of age. Spot frequencies after subtraction of the background in negative control wells are shown.

 
The cellular immune response to rhBZLF1 was directly compared with latent proteins in a subset of animals in whom the IFN-{gamma} ELISPOT response to rhLCV latent infection proteins has been previously studied (Table I and Ref.20). In these 11 animals, 63% (7 of 11) had detectable IFN-{gamma} ELISPOT responses to a rhBZLF1-vaccinia recombinant and 73% (8 of 11) had a positive IFN-{gamma} ELISPOT response to at least one of the rhLCV latent infection proteins expressed by recombinant vaccinia (Table I). rhBZLF1 responses were much more frequent than the response to any individual latent protein, with only rhEBNA-1-specific IFN-{gamma} ELISPOT responses (45% in this subset of 11 animals and 63% in a larger study of 23 animals) approaching anywhere near the frequency of rhBZLF1 responses (20). The average magnitude of the IFN-{gamma} ELISPOT response to rhBZLF1 (241 SFC/106 PBMC) was greater than that toward rhLCV latent proteins (115 SFC/106 PBMC). These data suggest that cellular immune responses to rhBZLF1 are more frequent and more robust compared with responses targeting latent proteins, a relationship that is similar to that in EBV-infected humans.


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  		Table I. Comparison of IFN-{gamma} ELISPOT responses to vaccinia recombinants expressing rhBZLF1 and rhLCV latent protein

 
The age of naturally rhLCV-infected macaques in this cohort ranged between 2.6 and 21 years. We compared the rhBZLF1 response in different age cohorts as a first approximation for changes in rhBZLF1 responses over time following primary LCV infection because virtually all rhesus macaques in a conventional colony acquire rhLCV infection by 1 year of age (17). When animals were divided into four sequential half-decade age cohorts, the two adult cohorts (5- to 10- and 10- to 15-year-old animals) had a similar magnitude (average ~200 SFC/106 PBMC) and frequency (~80%) of rhBZLF1-specific IFN-{gamma} ELISPOT responses (Fig. 1, B and C). In contrast, the magnitude and frequency of rhBZLF1 responses were lower in juvenile (<5 years old) and older macaques (>15 years old) compared with the adult macaques (Fig. 1, B and C). The lower rhBZLF1 responses in older macaques may be due to a waning of the rhLCV-specific immune response with age (Fig. 1B). The lower rhBZLF1 responses in the youngest cohort of macaques suggests that early primary rhLCV infection is not associated with peak rhBZLF1-specific T cell responses soon after infection. Instead, it appears that these lytic protein responses increase to a more robust steady-state level with age and exposure to periodic rhLCV reactivation.

rhBZLF1-specific IFN-{gamma} ELISPOT responses are predominantly mediated by cytotoxic CD8+ T lymphocytes

Because ELISPOT assays were performed on unfractionated PBMC stimulated with rhBZLF1 peptides, positive responses could be attributable to either or both CD4+ and CD8+ T lymphocytes. The T lymphocyte subset mediating the rhBZLF1-specific ELISPOT response was evaluated by the flow cytometric ICS assay in 11 animals (Table II). PBMC were stimulated in vitro for 6 h with a peptide pool containing all 60 rhBZLF1 peptides in the presence of costimulatory Abs and Brefeldin A and thereafter analyzed for coexpression of the recent activation marker CD69 along with the cytokines TNF-{alpha} or IFN-{gamma} (Fig. 2A). In the example shown, 2.04% of circulating CD8+ T lymphocytes but only 0.16% of CD4+ T lymphocytes responded specifically to therhBZLF1 peptides (Fig. 2A). The rhBZLF1-specific response was wholly or predominantly mediated by CD8+ T lymphocytes in 10 of 11 macaques (Table II), with the frequency of rhBZLF1-specific CD8+ T lymphocytes ranging between 0.11 and 2.04% (Fig. 2B). In one macaque (Mm161-85) with a relatively weak ELISPOT response (164 SFC/106 PBMC), the ICS assay did not reveal a positive signal above background (Table II). rhBZLF1-specific CD4+ T lymphocyte responses were either absent (8 of 10 macaques) or >3-fold lower in magnitude compared with the CD8+ T lymphocyte response (Fig. 2B).


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  		Table II. T lymphocyte subsets mediating recognition of rhBZLF1 in rhLCV-infected rhesus macaques

 

Figure 2
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  		FIGURE 2. rhBZLF1-specific IFN-{gamma} ELISPOT responses are predominantly mediated by CD8+ T lymphocytes. Representative data from replicate experiments shown. A, Representative analysis of ICS assay on unstimulated (top panel) and rhBZLF1-stimulated PBMC (bottom panel) from one rhesus macaque (Mm227-95). Lymphocytes gated on CD3+ T lymphocytes were further gated on CD8+ or CD8– (CD4+) T lymphocytes. Upper right quadrant of plots gated on CD8+ and CD4+ T lymphocytes show the frequency coexpressing CD69 and TNF-{alpha}. Frequencies ≥0.1% after subtraction of background obtained with unstimulated PBMC were considered to be positive. B, Frequency of rhBZLF1-specific CD4+ and CD8+ T lymphocytes in 10 animals. C, rhBZLF1-specific CD8-mediated CTL activity detected in peptide-stimulated PBMC. Effectors generated following 2 wk of in vitro peptide stimulation were tested against autologous baLCV-immortalized B cells either pulsed or not pulsed with rhBZLF1 peptides. Percentage of specific lysis shown after subtraction of lysis obtained with unpulsed baLCV-immortalized B cells. Percentage of lysis of unpulsed baLCV-immortalized B cells was <5%. Representative data on Mm309-98 is shown.

 
We then assessed the cytolytic ability of rhBZLF1-specific IFN-{gamma}-secreting CD8+ T lymphocytes by a classical 51Cr release assay using effector cells generated by stimulating PBMC in vitro for 10–14 days with rhBZLF1 peptides (Fig. 2C). Autologous target cells for the CTL assay consisted of rhesus B cells immortalized with the closely related but antigenically distinct LCV, which naturally infects baboons (baLCV). Latently infected baLCV-immortalized B cells are not recognized by rhLCV-specific CTL (M. Fogg and A. Kaur, unpublished data). Vigorous CTL activity against peptide-pulsed but not unpulsed autologous baLCV-immortalized B cells was detected in 5 of 5 animals tested (Fig. 2C and data not shown). The cytolytic activity was mediated by CD8+ T lymphocytes because depletion of CD8+ T lymphocytes but not CD4+ T lymphocytes abrogated lytic activity (Fig. 2C). rhBZLF1-specific CTL were also able to lyse autologous baLCV-immortalized B cells infected with recombinant vaccinia virus expressing rhBZLF1, but not control vaccinia, suggesting that they could recognize endogenously processed Ag (data not shown). In all, these data demonstrate that similar to humans, the rhBZLF1-specific cellular immune response in rhesus macaques is predominantly mediated by cytotoxic CD8+ T lymphocytes.

Identification of an immunodominant region of CTL recognition in the carboxyl terminus of the rhBZLF1 protein

In particular HLA haplotypes, the CD8+ T lymphocyte response to BZLF1 dominates the total EBV-specific T cell response and is directed toward a single immunodominant epitope (8, 11, 28). To determine whether a similar phenomenon occurs in rhLCV-infected rhesus macaques, the epitope specificity of the rhBZLF1-specific response was mapped in 11 macaques using sequential testing with rhBZLF1 peptide pools and individual peptides (Fig. 3 and Table II). Sixteen pools (A–P) of rhBZLF1 peptides each consisting of 7–8 peptides were made such that each peptide was represented in two unique pools (Fig. 3A). ELISPOT responses to peptide pools A–P narrowed identification of the T cell epitope(s) being recognized to one or more 15-aa peptides. Further testing with the potential individual peptides allowed confirmation of the epitope(s) specificity to the level of 15–19 aa (Table II and Fig. 3B). In the example shown with PBMC from Mm309-98, maximal IFN-{gamma} ELISPOT responses observed with peptide pools C, D, and P narrowed the dominant response to one or more epitopes contained within peptides nos. 59 and 60 (Fig. 3, A and B). Subsequent testing confirmed a positive response to both peptides, suggesting that either a single optimal epitope was located in the 11-aa overlapping region shared between peptides nos. 59 and 60, or that more than one epitope was targeted (Fig. 3B). Mm309-98 also demonstrated weak ELISPOT reactivity to pools A, H, and N, suggesting the presence of additional subdominant epitopes contained within peptides nos. 41 and 48 (Fig. 3). These responses were not further characterized. The ELISPOT response to peptides nos. 59 and 60 in Mm309-98 was shown to be mediated by CD8+ T lymphocytes by the ICS assay as well as by the ELISPOT and CTL assay performed on CD8-enriched and CD8-depleted PBMC (Fig. 2C and data not shown).


Figure 3
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  		FIGURE 3. Mapping epitope specificity of rhBZLF1-specific peptide responses by IFN-{gamma} ELISPOT. A, Illustration of peptide pool matrix. Each pool is denoted by an alphabet (A–P), whereas numbers shown in cells denote individual peptides. B, Representative IFN-{gamma} ELISPOT analysis on Mm309-98 showing sequential ELISPOT assays with the rhBZLF1 peptide pool matrix and individual peptides. The results were reproducible in replicate experiments.

 
A similar process of deconvolution in a further 10 animals revealed that the rhBZLF1-specific CD8+ T lymphocyte response in eight of the 11 macaques targeted epitopes located in peptide nos. 47–60 spanning the carboxyl 185–247 aa of the rhBZLF1 protein (Table II). In six of the eight macaques with diverse MHC class I background, the CD8+ T lymphocyte epitopes were located in the terminal 19 aa (aa 229–247) of the rhBZLF1 protein, suggesting the presence of more than one CTL epitope in this region restricted by different MHC class I alleles (Table III). These data suggest that CTL recognition of rhBZLF1 in a sizeable majority of rhesus macaques is narrowly focused to a common immunodominant region recognized in the context of different MHC class I alleles.


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  		Table III. MHC class I haplotype of rhesus macaques with CD8+ T lymphocyte epitopes within the carboxyl terminus of the rhLCV BZLF1 protein

 
Identification of a Mamu-A*01-restricted CD8+ T lymphocyte epitope within the carboxyl terminus of the rhBZLF1 protein

Of the six rhesus macaques that recognized a CTL epitope in the carboxyl-terminal 19 aa of the rhBZLF1 protein, the only known shared MHC class I allele was Mamu-B*12 (Table III). To determine whether the same CTL epitope was recognized in all the animals, we mapped the optimal epitope and determined its MHC restriction in one experimentally infected (Mm309-98) and one naturally infected (Mm395-93) rhesus macaque that shared the Mamu-B*12 and Mamu-A*01 MHC class I alleles (Table III).

Using a panel of 8- to 10-aa long overlapping peptides (nos. 58A–58M; Fig. 4A) covering the carboxyl-terminal 19 aa of the rhBZLF1 protein, PBMC from the experimentally infected rhesus macaque Mm309-98 responded to peptides nos. 58I, 58J, 58L, and 58M with spot frequencies that were comparable to the 11-aa peptide 60 (Fig. 4A, left panel). On peptide titration, the sensitizing peptide concentration required for half maximal response (SD50) was lowest with the 10-aa peptide 58L (aa 237–246), indicating that it was the minimal optimal epitope (Fig. 4A, right panel). These data were confirmed by CTL lines generated by in vitro stimulation of PBMC with peptide 60 (Fig. 4B). The 10-aa peptide 58L was recognized at an SD50 of <10–3 ng/ml (Fig. 4B, right panel), confirming it as the minimal optimal CTL epitope. Furthermore, peptide 58L-specific CTL were able to recognize target cells infected with recombinant vaccinia expressing rhBZLF1, suggesting that peptide 58L was endogenously processed and presented by infected APCs (data not shown). The region of the mapped CTL epitope 58L 237RTPDIIHEDL246 is closely homologous to the corresponding region in EBV BZLF1 and differs by only 2 aa (233RTPDVLHEDL242). Peptide 58L-specific CTL were able to recognize a variant peptide with the EBV BZLF1 sequence, albeit at a lower affinity (data not shown), suggesting that sequence variation of different rhLCV isolates in this region of BZLF1 would be unlikely to abrogate CTL recognition.


Figure 4
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  		FIGURE 4. Identification of a rhBZLF1-specific Mamu-A*01-restricted CTL epitope in one experimentally rhLCV-infected rhesus macaque (Mm309-98). A, IFN-{gamma} ELISPOT responses to overlapping 8- to 10-mer peptides spanning the carboxyl terminus 229–247 aa of the rhBZLF1 protein. Responses against 5 µg/ml peptide are shown in the left panel, and peptide titration of the positive responding peptides is shown in the right panel. B, CTL responses to the panel of overlapping peptides. Responses against 5 µg/ml peptide are shown in the left panel, and peptide titration of the positive responding peptides is shown in the right panel. Effector CTL were generated by in vitro stimulation of PBMC with peptide for 2 wk. Target cells consisted of peptide pulsed and unpulsed autologous baLCV-immortalized B cells. Specific lysis shown after subtraction of background lysis of unpulsed baLCV-immortalized B cells. Data from one of two experiments are shown. C, CTL recognition of the optimal epitope (peptide 58L) when presented on autologous baLCV-immortalized B cells or 721.221 cells expressing the Mamu-A*01 or Mamu-A*03 MHC class I allele. D, Representative Mamu-A*01/peptide 58L tetramer analysis of PBMC from a Mamu-A*01-positive (Mm309-98) and a Mamu-A*01-negative rhBZLF1 responder (Mm543-91) animals. Data shown on gated CD3+CD8+ T lymphocytes.

 
Based on the CTL recognition pattern of allogeneic targets pulsed with the optimal peptide 58L, it appeared that the CTL epitope recognized by Mm309-98 was presented by the Mamu-A*01 class I molecule (data not shown). To confirm MHC restriction, the MHC-null human B-lymphoblastoid cell line 721.221 (29) stably transfected with macaque MHC class I alleles was used as a target cell in CTL assays. Only autologous baLCV-immortalized B cells and Mamu-A*01-transfected 721.221 cells pulsed with peptide 58L were recognized by CTL from Mm309-98 (Fig. 4C), confirming the Mamu-A*01 restriction of this epitope. Further confirmation was obtained by synthesis of MHC tetramers of the Mamu-A*01 molecule bound to rhBZLF1 peptide 58L. CD8+ T lymphocytes binding the Mamu-A*01/peptide 58L tetramer were detected in the peripheral blood of Mm309-98 (Fig. 4D). Tetramer binding CD8+ T lymphocytes were not detected in a Mamu-A*01-negative rhBZLF1 responder macaque (Mm 543-91; Fig. 4D). Consistent with its Mamu-A*01 restriction, the optimal epitope 58L contained preferred residues for Mamu-A*01 binding at all three binding positions, namely the presence of proline in position 3, threonine at position 2, and leucine in the C terminus (30).

Identification of overlapping Mamu-A*01 and non-Mamu-A*01-restricted CTL epitopes in the carboxyl terminus of the rhBZLF1 protein

Only two of the six macaques recognizing CTL epitopes in the carboxyl-terminal 19 aa of the rhBZLF1 protein were Mamu-A*01 positive (Table III). This indicated that additional epitopes with a different MHC class I restriction were also present in this region. Unlike Mm309-98, the BZLF1-specific ELISPOT response in the Mamu-A*01-positive macaque Mm395-93 was not accounted for by recognition of peptide 58L (Fig. 5A). Instead, the 9-aa peptide 58F (residues 234–242) had the lowest SD50 in ELISPOT assays (Fig. 5B). In addition to 58F, the BZLF1-specific ELISPOT response in Mm395-93 also appeared to target other epitopes, because recognition of peptides such as 58J and 58K was unlikely to be explained by recognition of a CTL epitope in 58F (Fig. 5A).


Figure 5
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  		FIGURE 5. Identification of CTL epitopes in the carboxyl terminus of the rhBZLF1 protein in one naturally rhLCV-infected rhesus macaque (Mm395-93). A, IFN-{gamma} ELISPOT responses to overlapping 8- to 10-mer peptides spanning the carboxyl terminus 233–247 aa of the rhBZLF1 protein as described in Fig. 4. B, Peptide titration against responding peptides. ELISPOT assays were performed on PBMC stimulated overnight with the respective peptide. Representative data from one of three replicate experiments shown.

 
We used limiting dilution cloning of PBMC stimulated in vitro with the 15-aa peptide 58 to establish the epitope specificity of BZLF1-specific CTL recognition in Mm395-93. Sixteen active CTL clones survived repeated stimulation; 15 of them (representative clone 9 shown in Fig. 6A, left panel) had a peptide recognition pattern similar to that obtained with PBMC in ELISPOT assays (Fig. 5B). Thus, the 9-aa peptide 58F was recognized at an SD50 of <1 pg/ml and confirmed to be the optimal minimal epitope for the dominant BZLF1-specific CTL response in Mm395-93 (Fig. 6A, left panel). Dominant recognition of 58F was also observed in three other rhesus macaques (Mm105-93, Mm543-91, and Mm227-95) that were Mamu-A*01 negative (data not shown), indicating that the CTL epitope 58F was not presented by the Mamu-A*01 molecule. This was confirmed by the inability of clone 9 to recognize peptide 58F loaded on Mamu-A*01-positive target cells (Fig. 6B, left panel, and Table IV). Allogeneic targets that could present peptide 58F to clone 9 only shared the Mamu-B*12 MHC class I allele with Mm395-93 (Table IV). However, Mamu-B*12 was also present on two allogeneic target cell lines (Mm198-98 and Mm136-90) that were unable to present peptide 58F to clone 9 (Table IV). These data suggest the presence of a yet unidentified MHC class I allele common to Mm395-93, Mm105-93, Mm543-91, and Mm227-95 because all four macaques recognize peptide 58F, and the 58F-reactive T cell line (clone 9) was able to recognize peptide-pulsed target cells from these animals. Alternatively, we cannot exclude the possibility that subtypes of Mamu-B*12 with different peptide binding specificities are present. It is noteworthy that based on endogenous peptides eluted from Mamu-B*12 molecules, the predicted peptide binding motif of Mamu-B*12 (asparagine at P2) does not conform to the minimal rhBZLF1 epitope 58F (31). Finally, we cannot exclude the possibility of promiscuous recognition of the same epitope by more than one MHC class I allele.


Figure 6
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  		FIGURE 6. Identification of two overlapping CTL epitopes within the carboxyl terminus of the rhBZLF1 protein presented by different MHC class I alleles. Representative data on naturally rhLCV-infected rhesus macaque (Mm395-93) from one of at least two replicate experiments are shown. A, CTL analysis of two CTL clones with differing specificity for carboxyl-terminal rhBZLF1 peptides against autologous baLCV-immortalized B cells pulsed with individual rhBZLF1 peptides. B, CTL analysis of two CTL clones against autologous and allogeneic baLCV-immortalized B cells pulsed with optimal peptide for CTL activity. C, Mamu-A*01/peptide 58L tetramer analysis of two CTL clones.

 

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  		Table IV. MHC class I restriction of rhBZLF1-specific clones 9 and 39 derived from the naturally rhLCV-infected rhesus macaque Mm395–93

 
In contrast to the 15 clones represented by clone 9, the peptide recognition pattern of one clone from Mm395-93 (clone 39) was distinct and resembled that of Mm309-98 (Fig. 6A, right panel). Thus, the 10-aa peptide 58L was the optimal epitope (Fig. 6A, right panel). The ability to recognize peptide 58L on Mamu-A*01-positive but not Mamu-A*01-negative allogeneic target cells (Fig. 6B, right panel), as well as ability to bind Mamu-A*01/peptide 58L tetramers (Fig. 6C, right panel) confirmed that clone 39 recognized the same Mamu-A*01-restricted CTL epitope mapped in Mm309-98. These data demonstrate the presence in Mm395-93 of at least two different populations of rhBZLF1-specific CTL targeting epitopes in close proximity and provide further evidence for the immunogenicity of the carboxyl terminus of rhBZLF1 in LCV-infected rhesus macaques.

The Mamu-A*01-restricted rhBZLF1 epitope aa 237–246 is recognized in a subset of Mamu-A*01-positive rhLCV-seropositive macaques

From our analysis, two Mamu-A*01-positive animals recognized the Mamu-A*01-restricted peptide 58L. In one macaque (Mm309-98) it was the dominant response, whereas in the other macaque (Mm395-93) it was a subdominant response, suggesting heterogeneity in recognition of this epitope in Mamu-A*01-positive animals. Because Mamu-A*01 is a relatively common MHC class I allele in Indian rhesus macaques, we assessed recognition of the Mamu-A*01-restricted BZLF1 epitope in 16 randomly selected Mamu-A*01-positive rhLCV-seropositive rhesus macaques (Fig. 7). Consistent with the screening results of 54 naturally rhLCV-infected rhesus macaques with diverse MHC backgrounds, 12 of 16 Mamu-A*01-positive macaques (75%) responded to the rhBZLF1 whole peptide pool. Peptide 58L was recognized in only six rhesus macaques (37.5%). In four animals the ELISPOT response to peptide 58L was the dominant response, accounting for >70% of the total rhBZLF1 response, whereas in two macaques (Mm119-96 and Mm258-96) the response to peptide 58L accounted for <50% of the rhBZLF1 response (Fig. 7). From these data, it appears that the Mamu-A*01-restricted rhBZLF1 58L epitope is immunodominant in only a third of Mamu-A*01-positive rhesus macaques that respond to rhBZLF1.


Figure 7
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  		FIGURE 7. IFN-{gamma} ELISPOT responses to rhBZLF1 in Mamu-A*01-positive rhesus macaques. PBMC from 16 Mamu-A*01-positive, rhLCV-seropositive rhesus macaques were stimulated overnight with peptides spanning the rhLCV rhBZLF1 protein or peptide 58L containing the optimal Mamu-A*01 epitope. ELISPOT responses after subtraction of the background in negative control well are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that a significant portion of the host T cell response to an EBV-related {gamma}-herpesvirus in another species is directed toward the lytic protein homologue of EBV BZLF1. We show that the cellular immune response to rhBZLF1 is immunodominant in terms of its frequency in a MHC diverse population of macaques, as well as in the magnitude of the response in individual animals. First, we used overlapping peptides that spanned the rhBZLF1 protein and detected rhBZLF1-specific T lymphocytes in 63% of persistently infected macaques. T lymphocyte recognition was primarily mediated by MHC class I-restricted cytotoxic CD8+ T lymphocytes, with CD4+ T lymphocyte responses to rhBZLF1 being undetectable or significantly lower than the CD8+ T cell response in the same animal. Second, we found the magnitude of the rhBZLF1-specific IFN-{gamma} ELISPOT response in a given macaque to be higher than the response to individual latent infection proteins. By ICS, the rhBZLF1-specific CTL represented between 0.1 and 2.0% (average 0.5%) of the total CD8+ T cells. In addition, preliminary analysis of tetramer-positive cells in two Mamu-A*01-positive macaques detected 0.3 and 1.3% of CD8+ T lymphocytes that were Mamu-A*01/rhBZLF1 peptide 58L tetramer positive (data not shown).

BZLF1 is also considered to be an immunodominant target in humans, although the approach in these studies has been slightly different. A BZLF1 peptide library originally used to generate and screen CTL lines for BZLF1 activity detected BZLF1-specific CTL in 4 of 16 (25%) healthy EBV-seropositive donors (6). The response was mapped to two BZLF1 CTL epitopes, an HLA-B8-restricted epitope (RAK) and an HLA-Cw6-restricted epitope (RKC) (6). When these epitopes were used to determine the frequency of BZLF1 responders in a larger HLA-B8 and HLA-Cw6-positive population, BZLF1-specific CTL activity was detected in 67% (4 of 6) HLA-B8 and 60% (3 of 5) HLA-Cw6-positive individuals (6). Later studies on the prevalence of BZLF1-specific responses in persistent EBV infection have focused on recognition of these two epitopes. Epitope-specific responses were detected in 100% (6 of 6) of HLA-B8-positive individuals in two studies (11, 32) and in 50% (5 of 10) of HLA-Cw6 individuals (32). Studies that address whether BZLF1-specific responses are prevalent in a larger, more diverse HLA population are limited. Saulquin et al. (8) tested the EBV specificity of polyclonal T cell lines using a panel of COS cells cotransfected with cDNA expressing individual EBV proteins and 19 different HLA class I alleles, and found BZLF1 reactivity in 70% (7 of 10) of a HLA diverse group of healthy virus carriers. A dominant HLA-B8 response was still present (3 of 3 HLA-B8-positive individuals) but in addition, responses restricted through B14 (1 of 1 HLA-B14 positive individuals), B18 (3 of 3), and B44 (1 of 4) were also observed (8). Pudney et al. (10) generated T cell clones from 11 random individuals with AIM and showed that two of 11 patients had BZLF1-specific T cell clones, and both of these individuals were HLA-B8 positive. In terms of the magnitude of BZLF1 responses in a given individual, Tan et al. (11) used HLA-B8/RAK tetramers to show that 0.1–5.5% of CD8+ T cells are BZLF1 specific in persistently infected humans. In the same donors, the IFN-{gamma} ELISPOT response to the RAK peptide ranged between 250 and 2500 SFC/106 PBMC (11). Further studies in EBV-seropositive individuals have found an average of 693 SFC/106 PBMC in response to the RAK peptide and an average of 382 SFC/106 PBMC in response to the RKC peptide (C. Brander, personal communication). Thus, in humans the frequency and magnitude of BZLF1 responses are clearly dominant for the HLA-B8-restricted response to the RAK epitope, but there is limited data suggesting that this extends to other HLA backgrounds. Our study of 57 random macaques indicates that the frequency of rhBZLF1 responses in a diverse population is high and that the magnitude of responses in 10 random animals by the ICS assay (0.1–2.0%) and in two Mamu-A*01-positive macaques by tetramer analysis (0.3 and 1.3%; data not shown) was comparable to the magnitude of HLA-B8/RAK responses in persistently infected humans.

It was somewhat surprising to find that rhBZLF1 responses appear to increase over time to a set point in persistently infected hosts that is similar in both humans and macaques. At the surface, this appears different from humans where AIM patients frequently have extremely high BZLF1 responses, with up to 45% of peripheral blood CD8+ T cells being HLA-B8/RAK tetramer positive during the acute phase, which gradually decrease to steady-state levels that are similar in humans and macaques (13). A marked difference in the epidemiology of EBV infection in humans and rhLCV infection in captive rhesus macaques may be an important factor in the disparate kinetics of the macaque and human BZLF1 CTL response. Newborn and infant macaques are typically group housed with the parent and other adult macaques that increase the opportunities for rhLCV exposure early in life. Serologic testing reveals that virtually all captive rhesus macaques seroconvert by 1 year of age (17), so that primary infection is early and relatively synchronized, i.e., 10-year-old animals are all a similar number of years after primary rhLCV infection. These primary rhLCV infections early in life are usually clinically unremarkable, whereas experimental infection of adolescent macaques can occasionally result in a more remarkable clinical syndrome with atypical lymphocytosis, splenomegaly, and lymphadenopathy (19). In contrast, primary EBV infection is distributed throughout the first two decades of life and is not as synchronized as in captive macaques. Primary EBV infection that is delayed until adolescence is associated with an abnormally vigorous immune response and distinctive AIM clinical syndrome. Identifying AIM patients is the most frequently used method for recruiting human subjects with acute primary EBV infection. Thus, the extremely high BZLF1 responses observed in AIM patients may be a phenomenon tightly linked to the underlying immune abnormalities resulting in the AIM syndrome and may not necessarily reflect the immune response in the majority of humans who experience a much more benign acute syndrome with primary EBV infection. In support of this is the observation that neither lymphocytosis nor clonal T cell expansions are evident early in infection in asymptomatic EBV seroconverters (33).

rhBZLF1-specific CD8+ T lymphocytes demonstrated a marked predilection for recognition of CTL epitopes located in the carboxyl portion of the rhBZLF1 protein. In particular, more than one overlapping CTL epitope restricted by different MHC class I alleles was mapped to the terminal 19-aa residues (aa 229–247) of the rhBZLF1 protein. Although this region of BZLF1 has not been reported as a target for CTL recognition in humans, 4 of 6 described BZLF1 epitopes are contained within the carboxyl-terminal 72 residues (6, 8, 34, 35). Alignment of the carboxyl-terminal 72 aa of BZLF1 (aa 173–245) and rhBZLF1 (aa 176–247) proteins shows 84% sequence homology (overall BZLF1 homology is 71%). This region contains the DNA contact and multimerization domains (aa 175–195 and 196–245 of the BZLF1 protein, respectively), and all residues appear to be important for the DNA binding-dependent functions of BZLF1 (36). Due to the importance of these residues and sequence conservation between different rhLCVs, sequence variation is probably constrained by functional requirements, preventing the acquisition of mutations in this region. Focusing of the host T cell response to a highly conserved, functionally important region of the rhBZLF1 gene may be the host’s way of circumventing viral immune evasion.

In summary, our data indicates that the frequency and magnitude of the CTL response against rhBZLF1 in macaques is dominant. This may be similar to the BZLF1 CTL response in humans, although the human studies need to be expanded to populations with more diverse HLA backgrounds. There are significant differences in the kinetics of BZLF1-specific responses in our study compared with human studies. This may be due to fundamental differences in the epidemiology of rhLCV infection in macaques and humans and suggests that differences in BZLF1 responses may be associated with the nature and clinical presentation of primary LCV infection.


   Acknowledgments
 
We acknowledge Carolyn O’ Toole for help with manuscript preparation; Christian Brander and R. Paul Johnson for helpful discussion; David Watkins and William Rehrauer for advice on MHC typing; and the National Institutes of Health Tetramer Core facility for synthesis of the Mamu-A*01/rhBZLF1 peptide 58L tetramers.


   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 National Institutes of Health Grants P01 DE14388 (to F.W. and A.K.) and CA68057 (to F.W.) and a base grant to the New England Primate Research Center (United States Public Health Service P51RR00168). Back

2 Address correspondence and reprint requests to Dr. Amitinder Kaur, Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pinehill Drive, Southborough, MA 01772. E-mail address: amitinder_kaur{at}hms.harvard.edu Back

3 Abbreviations used in this paper: AIM, acute infectious mononucleosis; rhLCV, rhesus lymphocryptovirus; rhBZLF1, rhLCV BZLF1; ICS, intracellular cytokine staining; baLCV, baboon LCV; LCV, lymphocryptovirus; SFC, spot-forming cells; SD50, sensitizing peptide concentration required for half maximal response. Back

Received for publication August 11, 2005. Accepted for publication January 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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