Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Jun 20.
Published in final edited form as: Virology. 2008 Apr 10;376(1):183–190. doi: 10.1016/j.virol.2008.02.033

An SV40 VP1-Derived Epitope Recognized by CD8+ T Cells Is Naturally Processed and Presented by HLA-A*0201 and Cross-Reactive with Human Polyomavirus Determinants

Hephzibah Rani S Tagaram a, Alan M Watson b, Francois A Lemonnier c, Kevin Staveley-O’Carroll a,b, Satvir S Tevethia b, Todd D Schell b,*
PMCID: PMC2464359  NIHMSID: NIHMS53812  PMID: 18402997

Abstract

The CD8+T cell responses directed toward the VP1 antigens of human polyomaviruses JC and BK recently were shown to be cross-reactive. Two HLA-A*0201-restricted determinants from each virus have been defined and include JC p100–108 (ILMWEAVTL) and BK p108–116 (LLMWEAVTV) as well as JC p36–44 (SITEVECFL) and BK p44–52 (AITEVECFL). We asked whether VP1 from the related SV40 contains similar HLA-A*0201-restricted determinants. In this study, we demonstrate that CD8+ T cells specific for SV40 VP1 p110–118 (ILMWEAVTV), but not p46–54 (SFTEVECFL), can be induced in HLA-A*0201-transgenic mice and that these CD8+ T cells cross-react with the corresponding determinants from JC and BK virus. The SV40 p110 determinant was found to be processed and presented in SV40-infected cells. These results indicate that the JCp36/BKp44 determinants are distinctive for the human polyomaviruses while the JCp100/BKp108/SVp110 determinants are shared by all three viruses, providing a target for CD8+ T cell cross-reactivity.

Keywords: CD8+ T cells, SV40, HHDII transgenic mouse, immunization, T cell epitope

Introduction

The polyomaviruses JC and BK infect a high proportion of the human population, with prevalence reported up to 60% seropositivity for JC virus and up to 90% seropositivity for BK virus by adulthood (Dorries et al., 1994; Padgett and Walker, 1973; Taguchi et al., 1982). These viruses, acquired during childhood and adolescence, persist for the lifetime of the host but are only associated with human disease following the onset of immune suppression, implicating the immune response in lifelong control of these viruses. JC virus is the causative agent of progressive multifocal leukoencephalopathy (PML) (Padgett et al., 1971), a neurological disorder resulting from demyelination following destruction of JC virus-infected oligodendrocytes within the brains of AIDS patients (Antinori et al., 2001). Increased levels of BK virus in the kidneys is associated with nephropathy in renal transplant patients undergoing immunsuppressive therapy, often resulting in kidney failure (Randhawa and Brennan, 2006). In addition, high level BK viremia has been observed in association with late-onset hemorrhagic cystis in immunosuppressed bone marrow transplant patients (Arthur et al., 1986; Bedi et al., 1995; Childs et al., 1998).

The cellular immune response directed against the human polyomaviruses has recently been characterized. Koralnik and colleagues first defined the HLA-A*0201-restricted CD8+ T cell response toward determinants in the VP1 antigen of JC virus in PML patients, finding a positive correlation between the presence of VP1-reactive CD8+ T cells and prolonged survival (Du Pasquier et al., 2001; Koralnik et al., 2001). HLA-A*0201-restricted determinants corresponding to JC VP1 residues 100–108 (p100) (Koralnik et al., 2002) and 36–44 (p36) (Du Pasquier et al., 2003) were defined using CD8+ T cells isolated from PML patients as well as healthy controls (Du Pasquier et al., 2004). Subsequent studies demonstrated the presence of HLA-A*0201-restricted determinants in the VP1 antigen of BK virus, corresponding to VP1 residues 108–116 (p108) and 44–52 (p44), using both healthy subjects and kidney transplant recipients undergoing immunosuppressive therapy (Krymskaya et al., 2005). However, CD8+ T cells recognizing the BK VP1 determinants p108 and p44 were found to cross-react with the corresponding determinants from JC VP1 (Koralnik et al., 2002; Krymskaya et al., 2005). Thus, cross-reactive CD8+ T cells initiated by one polyomavirus may provide some level of existing immunity toward related polyomaviruses.

The closely related Simian virus 40 (SV40) has recently come under scrutiny regarding its possible link with several human cancers. SV40 was discovered in the early 1960’s as an accidental contaminant of formalin-inactivated (Salk) polio vaccines grown in rhesus monkey kidney cells (Sweet and Hilleman, 1960). The virus was originally characterized for its ability to induce tumors in rodents (Eddy et al., 1962; Girardi et al., 1962) and to transform multiple cell types in vitro due to expression of the viral early region gene products, including the oncoprotein large T antigen (Butel et al., 1972). DNA sequences specific to SV40 have been reported to be present in a proportion of several human cancers including mesothelioma, non-Hodgkins lymphoma, pediatric brain tumors and osteosarcomas [reviewed in (Engels, 2005; Vilchez and Butel, 2004)]. Multiple laboratories have detected SV40 sequences in such human tumors using PCR-based approaches, in some cases confirming their authenticity by DNA sequence analysis. This issue remains controversial, however, due to publication of some results which failed to detect SV40 sequences in these same types of tumors and due to the absence of high level SV40-specific serological responses in tumor patients [reviewed in (Shah, 2007)]. The prevalence of the closely related human polyomaviruses, JC and BK, in the human population has caused concern that both PCR and serological results could be tainted by contamination or cross-reactivity, respectlvely. In particular, the low level serological response to SV40 detected in humans has been attributed to cross-reactivity of BK and JC virus-induced antibodies toward SV40 (Viscidi et al., 2003). This is not surprising given the high degree of homology among these polyomaviruses at both the nucleotide (Frisque, 1983) and amino acid level (Mann and Carroll, 1984).

Previous investigations in mice revealed crossreactivity of SV40 T antigen-induced CD8+ T cells toward the T antigens of BK and JC virus (Tevethia et al., 1998). In the current report, we investigated whether SV40 VP1 contains determinants similar to the known HLA-A*0201- restricted JC and BK determinants in VP1 and whether CD8+ T cells responsive toward SV40 VP1 are cross-reactive with the corresponding human polyomavirus determinants. Using line HHDII mice, which express the human HLA-A*0201 molecule, we assessed a) the immunogenicity of the heterologous VP1 peptide determinants from SV40, b) the cross-reactivity of SV40-responsive CD8+ T cells toward JC and BK VP1 determinants and vice versa and c) whether the SV40 VP1 determinants are naturally processed and presented from SV40-infected cells.

Results

SV40 peptide p110, but not peptide p46, binds to HLA-A*0201

SV40 VP1 peptides that correspond to the known epitopes from JC and BK virus VP1 include peptide 110–118 (SVp110) and peptide 46–54 (SVp46). The sequences are shown in figure 1A for comparison with those from the human polyomaviruses. SVp110 differs from the corresponding JC and BK peptides by only a single amino acid with conservative changes of leu⇒val at position 9 compared to JCp100 and leu⇒ile at position 1 compared to BKp108. However, SVp46 varies from the corresponding JC and BK peptides at both positions 1 and 2, with the critical anchor residue at position 2 changed from Ile to a bulky phe residue, which is not favored for peptides that bind to HLA-A*0201 (Rammensee et al., 1995).

Figure 1.

Figure 1

Open in a new tab

Sequence and peptide/MHC stability of HLA-A*0201-restricted polyomavirus VP1 determinants. A: Comparison of human polyomavirus VP1 determinants and the corresponding sequences from SV40 VP1. Residues with underlines vary from the JC virus sequence. B: VP1 peptide-induced stabilization of HLA-A*0201 surface expression. Peptides were incubated with T2 cells at the indicated concentrations overnight at 37°C and then stained for expression of HLA-A*0201. The control H-2Kb-binding peptide gB498–505 from Herpes simplex virus was used as a negative control.

Using the TAP-deficient T2 cell line, we determined the ability of titrated amounts of each VP1 peptide to stabilize HLA-A*0201 molecules on the cell surface following overnight incubation. The results demonstrate that the efficiency of SVp110 peptide to stabilize HLA-A*0201 was similar to that achieved with JCp100 and BKp108 peptides (Fig. 1B). In contrast, no HLA-A*0201 expression was detected following incubation of T2 cells with SVp46 while JCp36 and BKp44 peptides induced detectable HLA-A*0201 surface expression at concentrations of 10 µM and above. These results indicate that the SVp110 peptide, but not the SVp46 peptide, binds efficiently to HLA-A*0201. Lack of efficient binding to HLA-A*0201 by SVp46 is likely due to the absence of the isoleucine residue at position 2, predicted to be critical for binding to HLA-A*0201.

The SVp110 peptide is immunogenic in HHDII mice

To determine whether the SV40 VP1 peptides were immunogenic, line HHDII mice were immunized with either SVp110 or SVp46. After a booster immunization, splenocytes were restimulated in vitro with peptide pulsed irradiated syngeneic DCs and then analyzed after six days by intracellular cytokine staining (ICS) for the presence of CD8+ T cells responsive toward either the immunizing peptide or the corresponding peptides from BK and JC VP1. The results demonstrate that immunization with SVp110, but not SVp46, yielded CD8+ T cells capable of producing IFN-γ in response to the immunizing peptide (Fig. 2). The finding that SVp46 failed to induce a detectable CD8+ T cell response is not surprising given the poor binding of this peptide to HLA-A*0201. CD8+ T cells induced by immunization with SVp110 also responded to JCp100 (49.3%) and BKp108 (49.8%) at nearly identical frequencies as achieved with SVp110 (49.6%), suggesting that these T cells are cross-reactive, at least at the high concentration of peptide tested in this assay (Fig. 2).

Figure 2.

Figure 2

Open in a new tab

The SVp110 determinant is immunogenic in HHDII mice. Groups of 3 HHDII mice were immunized subcutaneously with 100 µg of peptides SVp110 or SVp46 emulsified in IFA. Mice received a booster immunization after two weeks and spleens were harvested after a further 7 days. Spleen cells were restimulated in vitro with syngeneic DCs pulsed with the immunizing peptide and cultured for 6 days. Responder cells were tested for their ability to produce IFN-γ following stimulation with the indicated peptides and stained by ICS. The percentage of CD8+ T cells that produced IFN-γ is indicated in each dot plot. This experiment was performed three times using three mice per group and achieved similar results.

Cross-reactivity among VP1-responsive CD8+ T cells

Since such a large proportion of the CD8+ T cells responded to SVp110 following one week of culture, we asked whether these cells could be detected directly ex vivo following immunization of HHDII mice with this peptide. In addition, groups of HHDII mice were immunized with the JCp100 and BKp108 peptides for comparison. As shown in figure 3A, CD8+ T cells responsive to each of the three peptides were detected in 3 of 3 mice with an average frequency of 5.6% for SVp110, 2.45% for JCp100 and 3.64% for BKp108. Thus, HHDII mice develop a robust response to SVp110 as well as to the corresponding peptides from JC and BK VP1 following a prime/boost immunization.

Figure 3.

Figure 3

Open in a new tab

CD8+ T cells responsive to SVp110 are detected directly ex vivo and are highly cross-reactive with determinants from the human polyomaviruses. A: Groups of three HHDII mice were immunized with the indicated VP1 peptides as described in figure 2 and received a booster immunization after one week. After a further 7 days, spleen cells were harvested and subjected to ICS for IFN-γ following a 6 hour stimulation with the same peptide used for immunization or the Gag p17 peptide derived from HIV. The percentage of CD8+ T cells that produced IFN-γ is indicated in each dot plot. B. Splenocytes from A were restimulated in vitro for 6 days with syngeneic DCs pulsed with the immunizing peptide and then tested by ICS for IFN-γ production following stimulation for 6 hours with titrated amounts of each of the indicated peptides. Data are plotted as the percentage of CD8+ T cells that produced IFN-γ at a given peptide concentration. This experiment was performed twice using three mice per group and gave similar results to those shown.

To determine the efficiency of cross-recognition among the VP1-responsive CD8+ T cells, splenocytes were expanded in vitro for six days with the immunizing peptide and the responder cells tested for their ability to produce IFN-γ following stimulation with titrated amounts (100 to 0.1 nM) of each of the heterologous peptides by ICS. CD8+ T cells induced by immunization with SVp110, recognized all three peptides with similar efficiencies, although there was a slight decrease (5 to 10-fold) in the efficiency of cross-recognition of JCp100 by these T cells (Fig. 3B). For 2 of 3 cultures derived from JCp100 immunized mice, the response to all three peptides was similar, with T cells from the remaining culture having an increased sensitivity to JCp100 relative to the other two peptides. For T cells induced by immunization with BKp108, the response to the immunizing peptide was consistently most efficient, with the response toward SVp110 reduced about 5-fold and that to JCp100 about 10-fold. Thus, CD8+ T cells induced by immunization with SVp100 are cross-reactive with the JC and BK corresponding determinants and vice versa, although the efficiency is reduced in some cases toward the corresponding peptides from the related viruses.

Vaccinia virus encoded SVp110 also induces cross-reactive CD8+ T cells

Our results demonstrate that peptide-induced CD8+ T cells are cross-reactive toward the heterologous VP1 determinants. To rule out the possibility that the observed CD8+ T cell cross-reactivity is a consequence of immunization with synthetic peptides, we engineered recombinant vaccinia viruses (rVV) that express the VP1 determinants as minigenes preceded by an endoplasmic reticulum targeting sequence (ES). This method induces potent CD8+ T cell responses which can be detected directly ex-vivo (Fu et al., 1998). Groups of HDDII mice were immunized with 107 PFU of rVV-ES-JCp100, rVV-ES-BKp108, rVV-ES-SVp110 or the control rVV-ES-SVT281 encoding the known HLA-A*0201-restricted epitope from SV40 T antigen (Schell et al., 2001). Splenocytes were analyzed by ICS after 10 days (Figure 4). All mice demonstrated a detectable ex-vivo response toward the determinant used for immunization and, as demonstrated with peptide immunized mice, a similar proportion of cells responded to the heterologous peptides. We consistently observed that mice immunized with rVV-ES-JCp100 had similar responses toward all three corresponding VP1 epitopes while mice immunized with rVV-ES-BKp108 and rVV-ES-SVp110 responded more efficiently toward both BKp108 and SVp110 peptides than toward JCp100 – a trend also observed following peptide immunization (see Fig. 3B). Splenocytes from rVV-ES-SVT281 immunized mice responded only to peptide SVT281, indicating that the vaccinia virus immunizations were specific for the indicated minigenes. These data demonstrate that de novo production of JC, BK, and SV40 VP1 epitopes in HHDII mice also recruits CD8+ T cells that are generally cross-reactive toward the polyomavirus determinants.

Figure 4.

Figure 4

Open in a new tab

Endogenously expressed VP1 determinants induce cross-reactive CD8+ T cell populations. HHDII mice were immunized with rVVs expressing ES-JCp100, ES-BKp108, ES-SVp110 or ES-SVT281. Ten days following immunization, spleens were harvested and evaluated by ICS for IFN-γ production following stimulation with the indicated peptides. The percentage of CD8+T cells responsive to each peptide is indicated in the dot plots. Data are representative of two independent experiments with three mice per group.

The SV40 VP1 p110 determinant is naturally processed and presented in SV40 infected cells

To determine whether the SVp110 determinant is naturally processed and presented from VP1 following SV40 infection, the ability of CD8+ T cells derived from SVp110 immunized mice to lyse SV40 infected TC7 monkey cells was determined. Since TC7 cells do not express the proper MHC molecule, SV40 infected TC7 cells were co-infected with rVV-A2.1, expressing HLA-A*0201, or rVV-Kd, expressing the mouse H-2Kd molecule. As shown in figure 5, SVp110- induced T cells efficiently lysed SV40-infected target cells co-expressing HLA-A*0201 in a dose-dependent manner above the background level of lysis obtained on the same cells co-expressing H-2Kd (Fig. 5). As a positive control, CD8+ T cells specific for the known HLA-A* 0201-restricted epitope T281 from SV40 T antigen efficiently lysed SV40 infected target cells co-expressing HLA-A*0201, but not cells co-expressing H-2Kd. These results indicate that the SVp110 determinant is naturally processed and presented from VP1 protein in the context of HLA-A*0201 and, therefore, represents a target epitope on SV40-infected cells.

Figure 5.

Figure 5

Open in a new tab

The SV40 epitope recognized by SVp110-induced CD8+ T cells is naturally processed and presented from SV40 infected cells. HHDII CD8+ T cell lines induced by peptide immunization with SVp110 (A) or SVT281 (B) and maintained by in vitro restimulation were tested for their ability to lyse SV40-infected (48 hrs) TC7 monkey cells co-infected with either rVV-A2.1 or rVV-Kd in a 4-hour 51Cr-release assay. The effector:target ratio (E:T) is indicated.

Discussion

We have identified a naturally processed and presented epitope from the SV40 major capsid protein VP1, which is recognized by HLA-A*0201-restricted CD8+ T cells in line HHDII transgenic mice. This determinant, SVp110, shares extensive sequence homology with the previously identified determinants from BK and JC virus VP1, such that CD8+ T cells induced by one determinant are cross-reactive with the corresponding determinants from the other two viruses. In contrast, SVp46 failed to induce a detectable CD8+ T cell response in HHDII mice, most likely due to the presence of a phenylalanine residue at the position 2 anchor position which likely disrupts binding to HLA-A*0201. Thus, only one of two known VP1 determinants are common between SV40 and the human polyomaviruses.

In humans, cross-reactivity of CD8+ T cells toward VP1-derived BK and JC determinants was first demonstrated by Krymskaya et al. (Krymskaya et al., 2005) in both healthy individuals and kidney transplant recipients with active BK virus infections and later by Chen et al. (Chen et al., 2006) in kidney transplant recipients with polyomavirus nephropathy. Using co-staining with MHC tetramers specific for JCp100 and BKp108, both studies revealed extensive cross-reactivity of in vitro stimulated human CD8+ T cells toward the corresponding determinants. Chen et al. (Chen et al., 2006) additionally demonstrated cross-reactivity of in vitro cultured CD8+ T cells toward the JCp36 and BKp44 determinants using the same approach. Of note in both studies, a minor population of CD8+ T cells with single specificity was apparent from the MHC tetramer staining, suggesting that some CD8+ T cell clones may be able to distinguish between the epitopes from different polyomaviruses. In our study, peptide titration assays revealed some differences in the avidity of CD8+ T cells toward heterologous determinants by bulk CD8+ T cell populations, with the most efficient response targeted against the immunizing peptide. Favored recognition of the immunizing peptide might indicate either the presence of some CD8+ T cells with specificity only for the immunizing peptide or the recruitment of CD8+ T cell clones with increased avidity for the immunizing peptide. We favor the second possibility since differences in the frequency of CD8+ T cells responding toward heterologous peptides was most noticeable at limiting peptide concentrations.

Krymskaya et al. (Krymskaya et al., 2005) immunized HHDII mice with a recombinant vaccinia virus expressing the BK virus full length VP1 protein. Subsequent analysis revealed that the responding CD8+ T cells in HHDII mice were cross-reactive with JCp100, although fewer cells responded to the JCp100 peptide compared to the BKp108 peptide. We additionally show here that CD8+ T cells derived from HHDII mice immunized with either BKp108 or JCp100 peptide or vaccinia virus encoded minigenes are largely cross-reactive toward the SVp110 peptide and that mice immunized against JCp100 cross-react with both BKp108 and SVp110. Thus, the HHDII T cell repertoire mimics the cross-reactive nature of the human T cell repertoire in response to the polyomavirus VP1 determinants.

Although we found that the SVp46 peptide was not immunogenic in HHDII mice, a recent study by Li et al. (Li et al., 2006) showed that human CD8+ T cells expanded from PBMCs of healthy individuals by in vitro stimulation with BKp44 peptide cross-react on both JCp36 and SVp46 in an intracellular cytokine staining assay. This apparent discrepancy might be explained by the use of high concentrations of peptides during the in vitro assay. Indeed, we found in a preliminary analysis that HHDII mice develop CD8+ T cells to both BKp44 and JCp36 peptide immunization and that the responding T cells produce IFN-γ in response to 1 µM SV40 p46 (data not shown). Whether the responding JCp36- and BKp44-induced CD8+ T cells can recognize reduced concentrations of the SVp46 peptide remains to be determined. Overall, our data demonstrating that the SVp46 peptide fails to induce stabilization of HLA-A*0201 or induce a detectable T cell response in HHDII mice suggests that inefficient binding of this peptide to MHC class I molecules may limit its immunogenicity and antigenicity in vivo.

The significance of CD8+ T cell cross-reactivity toward VP1 determinants in humans has not been elucidated, but studies of heterologous virus immunity in experimental models indicate that preferential expansion of cross-reactive T cells by heterologous viruses can skew immunodominance patterns toward cross-reactive epitopes and away from virus-specific responses (Brehm et al., 2002). In fact, attrition of memory T cells targeting virus-specific responses was observed in some cases (Selin et al., 1999; Selin et al., 1996). In the case of persistent polyomavirus infections, cross-reactive immunity may preferentially enhance immunity against multiple polyomaviruses but may limit the development of specific virus immunity that would serve as a signature for active virus infection, an issue raised during previous studies of CD8+ T cell responses to BK and JC viruses (Chen et al., 2006; Krymskaya et al., 2005).

Our results raise the possibility that existing human polyomavirus-induced VP1- responsive memory CD8+ T cells could cross-react with SVp110 in potentially SV40 exposed humans. Because SV40 is thought to replicate poorly in most human cells compared to the human polyomaviruses (O'Neill et al., 1990), lack of robust virus replication coupled with the presence of both polyomavirus-induced cross-reactive antibodies and memory T cells targeting VP1 could mask an SV40-specific immune signature. In addition, our study suggests that T cells targeting the SVp110 determinant, but not SVp46 could expand by exposure to SV40. Interestingly, Chen et al. (Chen et al., 2006) found that CD8+ T cells specific for BKp108 were favored over CD8+ T cells specific for BKp44 in kidney transplant recipients with active BK virus infections, while healthy individuals had the reverse profile. Thus, one consequence of cross-reactive immunity toward the polyomaviruses could be that little to no SV40-specific responses would be detected in humans with pre-existing polyomavirus immunity, providing an alternate explanation for the absence of evidence for a strong virus-specific immune response to SV40 infection in humans.

While VP1-directed CD8+ T cells and antibodies could provide protection against SV40 infection, immunity directed against this late gene product would have little effect on tumors expressing SV40 T antigen. In fact, the VP1-targeted cross-reactive immune response might initially serve to limit responses toward any virus specific determinants in T antigen. A few studies have demonstrated the existence of HLA-restricted determinants in T antigen recognized by CD8+ T cells. Three HLA-A*0201-restricted determinants have been defined for SV40 T antigen encompassing residues 281–289 (Schell et al., 2001), 285–293 (Bright et al., 2002) and 577–585 (Velders et al., 2001) with CD8+ T cells specific for peptide 285–293 shown to be present in a mesothelioma patient (Bright et al., 2002). However, more studies are required to determine any association of T antigen-specific CD8+ T cell responses with individuals potentially exposed to SV40.

In summary, our results demonstrate that the SV40 VP1 determinant p110–118 is immunogenic in HHDII mice, is naturally processed and presented by HLA-A*0201 in virus infected cells and the responding CD8+ T cells are highly cross-reactive with the corresponding determinants from the human polyomaviruses.

Materials and methods

Mice

HHDII mice express a chimeric MHC class I molecule composed of the cytosolic, transmembrane and α3 domains of H-2Db and the α1 and α2 domains of HLA-A*0201 covalently linked to the human β2m light chain (Pascolo et al., 1997). In addition, these mice are deficient for expression of mouse β2m and H-2Db. HHDII mice were bred and maintained at the animal facility of the Milton S. Hershey Medical Center under specific pathogen free conditions. All experiments using mice were conducted in accordance with policies established by the NIH under a protocol approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University College of Medicine.

Peptides and viruses

Peptides were synthesized in the Macromolecular Synthesis Core Facility at The Pennsylvania State University College of Medicine by 9-fluroenylmethoxycarbonyl chemistry using an automated peptide synthesizer (9050 MilliGen PepSynthesizer). Peptides were solubilized in DMSO and diluted to the appropriate concentration with RPMI 1640. Amino acid sequences of the peptides used in the present study are shown in figure 1. Wild type SV40 strain VA 45–54 was propogated as previously described (Tevethia et al., 1974). The vaccinia virus recombinants rVV-A2.1, which encodes the human HLA-A*0201 heavy chain (O'Neil et al., 1993), and rVV-Kd, which encodes the murine H-2Kd heavy chain (Bacik et al., 1994) were generously provided by Drs. Jonathan Yewdell and Jack Bennink (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Vaccinia virus recombinants expressing the VP1 determinants JCp100 (rVV-ES-JCp100), BKp108 (rVV-ES-BKp108) and SVp110 (rVV-ES-SVp110) or SV40 T antigen determinant 281–289 (KCDDVLLLL; rVV-ES-SVT281) preceded by the endoplasmic reticulum insertion sequence from the adenovirus E19 protein were engineered and propagated as described previously (Fu et al., 1998).

Cell lines and Media

TC7 monkey kidney cells were maintained in Dulbecco’s Modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml kanamycin, 2 mM L-glutamine, 10 mM HEPES buffer, 0.075% (w/v) NaHCO3, and 5–10% FBS. TAP-deficient T2 cells (Salter et al., 1985) were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, and 25 µg/ml sodium pyruvate.

Peptide-induced stabilization of HLA-A*0201 molecules

Peptide-induced stabilization of HLA-A*0201 molecules on the surface of T2 cells was performed as previously described (Schell et al., 2001) and detected by flow cytometry after staining with mAb BB7.2 (ATCC HB 82) and secondary detection with goat anti-mouse IgG. The H-2Kb-binding gB498–505 (SSIEFARL) peptide from Herpes Simplex Virus glycoprotein B (Bonneau et al., 1993) was used as a negative control for these experiments.

Immunizations and in vitro culture of lymphocytes

HHDII mice were immunized subcutaneously at the base of the tail with 100 µg of specific VP1-derived peptide or SV40 T antigen peptide 281–289 (KCDDVLLLL) (Schell et al., 2001) and 160 µg of the HBV core helper peptide 128–140 emulsified in 100 µl of incomplete Freund’s adjuvant as described previously (Schell et al., 2001). A booster dose of the same peptide was administered two weeks later. Immunized mice were sacrificed and spleens harvested one week following the booster immunization. For immunization with rVVs, mice received 107 PFU of the indicated viruses by intravenous injection into the tail vein and spleen cells were harvested after 10 days for ex vivo analysis. For in vitro culture of lymphocytes, single cell suspensions of RBC-depleted splenocytes were co-cultured with peptide pulsed syngeneic bone marrow-derived dendritic cells (DCs). DC’s were isolated from the bone marrow and maintained in culture with GM-CSF-containing supernatant from X-63GM.1 cells (Norbury et al., 1997) essentially as described (Lutz et al., 1999). In brief, bone marrow was flushed from the femurs of HHDII mice with RPMI-1640. The resulting cells were cultured in 100 mm petri dishes at 4×106 cells/dish in 10 ml of complete RPMI-1640 medium supplemented with 10% heat inactivated FBS and 10% X-63GM.1 supernatant. On day 3, 10 ml of fresh media was added to the cultures. On days 6 and 9, 10 ml of supernatant was removed from each dish and any floating cells recovered by centrifugation, resuspended in 10 ml of fresh media and added back to the original dish. On day 11, the cells were harvested, frozen and stored at −80°C until use. Purity of CD11c+ cells at day 11 was between 70–80%. For T cell stimulation, DCs were resuspended at 1×106/ml in complete RPMI-1640 plus 100 nM specific peptide and incubated at 37°C with rocking for 1 hour followed by two washes to remove excess peptide. DCs were irradiated 3000 rads using a 60Co-source GammaCell irradiator (MDS Nordion). A total of 5×105 peptide pulsed DCs was co-cultured with 1×107 splenocytes from immunized HHDII mice in 4 ml of complete RPMI-1640 medium containing 10% FBS. T lymphocyte cultures were maintained in vitro by weekly passage with peptide pulsed DCs but with the addition of 5 U/ml recombinant human IL-2 (Amgen).

Intracellular cytokine assays

After 6 days of culture, splenocytes were tested for IFN-γ production by intracellular cytokine staining (ICS). One million responder cells were stimulated with 1 µM of the indicated VP1 peptides, SVT281 peptide or the unrelated HLA-A*-0201-binding HIV gag p17 (SLYNTVATL) peptide plus 1 µg/ml of brefeldin A for 5 hours at 37°C. In some assays 10-fold dilutions of the indicated peptides were used. Cells were stained for CD8 and IFN-γ using the Cytofix/Cytoperm kit (Pharmagen) as described by the manufacturer. Cells were fixed with 2% paraformaldehyde and analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson) and the data analyzed using FlowJo software (Treestar).

Cytotoxicity assays

Cytotoxicity of the responding splenocyte cultures was assessed using a standard 51Cr-release assay as described previously (Schell et al., 1999). Briefly, monolayers of TC7 cells were infected with SV40 strain VA 45–54 (MOI of 10) for 48 hours and then superinfected with rVV-A2.1 or rVV-Kd (MOI of 1) for four hours in the presence of 200 µCi of sodium 51chromate. Cells from lymphocyte cultures were mixed with the infected TC7 cells at the indicated ratios and incubated for 4 hours at 37° C, 5% CO2 after which 100 µl of supernatant was collected and counted on a Cobra gamma-counter (Packard Instruments). Percent specific lysis was determined as described previously (Schell et al., 1999). All data represent the means of triplicate samples. Spontaneous release values for TC7 cells co-infected with SV40 and vaccinia virus recombinants was consistently around 30%.

Acknowledgements

We thank Jeremy Haley for excellent technical assistance and Nate Schaffer for assistance in the Flow Cytometry Core Facility. This work was supported by research grants CA-25000 from the National Cancer Institute/National Institutes of Health, a grant from the Barsumian Trust and a grant from the Pennsylvania Tobacco Settlement. KSOC was supported by RO8 award #416-67HY from the National Institutes of Health.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Antinori A, Ammassari A, Giancola ML, Cingolani A, Grisetti S, Murri R, Alba L, Ciancio B, Soldani F, Larussa D, Ippolito G, De Luca A. Epidemiology and prognosis of AIDS-associated progressive multifocal leukoencephalopathy in the HAART era. J Neurovirol. 2001;7(4):323–328. doi: 10.1080/13550280152537184. [DOI] [PubMed] [Google Scholar]
  2. Arthur RR, Shah KV, Baust SJ, Santos GW, Saral R. Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants. N Engl J Med. 1986;315(4):230–234. doi: 10.1056/NEJM198607243150405. [DOI] [PubMed] [Google Scholar]
  3. Bacik I, Cox JH, Anderson R, Yewdell JW, Bennink JR. TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide. J Immunol. 1994;152(2):381–387. [PubMed] [Google Scholar]
  4. Bedi A, Miller CB, Hanson JL, Goodman S, Ambinder RF, Charache P, Arthur RR, Jones RJ. Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation. J Clin Oncol. 1995;13(5):1103–1109. doi: 10.1200/JCO.1995.13.5.1103. [DOI] [PubMed] [Google Scholar]
  5. Bonneau RH, Salvucci LA, Johnson DC, Tevethia SS. Epitope specificity of H-2Kb-restricted, HSV-1-, and HSV-2-cross-reactive cytotoxic T lymphocyte clones. Virology. 1993;195(1):62–70. doi: 10.1006/viro.1993.1346. [DOI] [PubMed] [Google Scholar]
  6. Brehm MA, Pinto AK, Daniels KA, Schneck JP, Welsh RM, Selin LK. T cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens. Nat Immunol. 2002;3(7):627–634. doi: 10.1038/ni806. [DOI] [PubMed] [Google Scholar]
  7. Bright RK, Kimchi ET, Shearer MH, Kennedy RC, Pass HI. SV40 Tag-specific cytotoxic T lymphocytes generated from the peripheral blood of malignant pleural mesothelioma patients. Cancer Immunol Immunother. 2002;50(12):682–690. doi: 10.1007/s00262-001-0240-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Butel JS, Tevethia SS, Melnick JL. Oncogenicity and cell transformation by papovavirus SV40: the role of the viral genome. Adv Cancer Res. 1972;15:1–55. doi: 10.1016/s0065-230x(08)60371-1. [DOI] [PubMed] [Google Scholar]
  9. Chen Y, Trofe J, Gordon J, Du Pasquier RA, Roy-Chaudhury P, Kuroda MJ, Woodle ES, Khalili K, Koralnik IJ. Interplay of cellular and humoral immune responses against BK virus in kidney transplant recipients with polyomavirus nephropathy. J Virol. 2006;80(7):3495–3505. doi: 10.1128/JVI.80.7.3495-3505.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Childs R, Sanchez C, Engler H, Preuss J, Rosenfeld S, Dunbar C, van Rhee F, Plante M, Phang S, Barrett AJ. High incidence of adeno- and polyomavirus-induced hemorrhagic cystitis in bone marrow allotransplantation for hematological malignancy following T cell depletion and cyclosporine. Bone Marrow Transplant. 1998;22(9):889–893. doi: 10.1038/sj.bmt.1701440. [DOI] [PubMed] [Google Scholar]
  11. Dorries K, Vogel E, Gunther S, Czub S. Infection of human polyomaviruses JC and BK in peripheral blood leukocytes from immunocompetent individuals. Virology. 1994;198(1):59–70. doi: 10.1006/viro.1994.1008. [DOI] [PubMed] [Google Scholar]
  12. Du Pasquier RA, Clark KW, Smith PS, Joseph JT, Mazullo JM, De Girolami U, Letvin NL, Koralnik IJ. JCV-specific cellular immune response correlates with a favorable clinical outcome in HIV-infected individuals with progressive multifocal leukoencephalopathy. J Neurovirol. 2001;7(4):318–322. doi: 10.1080/13550280152537175. [DOI] [PubMed] [Google Scholar]
  13. Du Pasquier RA, Kuroda MJ, Schmitz JE, Zheng Y, Martin K, Peyerl FW, Lifton M, Gorgone D, Autissier P, Letvin NL, Koralnik IJ. Low frequency of cytotoxic T lymphocytes against the novel HLA-A*0201-restricted JC virus epitope VP1(p36) in patients with proven or possible progressive multifocal leukoencephalopathy. J Virol. 2003;77(22):11918–11926. doi: 10.1128/JVI.77.22.11918-11926.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Du Pasquier RA, Kuroda MJ, Zheng Y, Jean-Jacques J, Letvin NL, Koralnik IJ. A prospective study demonstrates an association between JC virus-specific cytotoxic T lymphocytes and the early control of progressive multifocal leukoencephalopathy. Brain. 2004;127(Pt 9):1970–1978. doi: 10.1093/brain/awh215. [DOI] [PubMed] [Google Scholar]
  15. Eddy BE, Borman GS, Grubbs GE, Young RD. Identification of the oncogenic substance in rhesus monkey kidney cell culture as simian virus 40. Virology. 1962;17:65–75. doi: 10.1016/0042-6822(62)90082-x. [DOI] [PubMed] [Google Scholar]
  16. Engels EA. Does simian virus 40 cause non-Hodgkin lymphoma? A review of the laboratory and epidemiological evidence. Cancer Invest. 2005;23(6):529–536. doi: 10.1080/07357900500202820. [DOI] [PubMed] [Google Scholar]
  17. Frisque RJ. Nucleotide sequence of the region encompassing the JC virus origin of DNA replication. J Virol. 1983;46(1):170–176. doi: 10.1128/jvi.46.1.170-176.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fu TM, Mylin LM, Schell TD, Bacik I, Russ G, Yewdell JW, Bennink JR, Tevethia SS. An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope. J Virol. 1998;72(2):1469–1481. doi: 10.1128/jvi.72.2.1469-1481.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Girardi AJ, Sweet BH, Slotnick VB, Hilleman MR. Development of tumors in hamsters inoculated in the neonatal period with vacuolating virus, SV-40. Proc Soc Exp Biol Med. 1962;109:649–660. doi: 10.3181/00379727-109-27298. [DOI] [PubMed] [Google Scholar]
  20. Koralnik IJ, Du Pasquier RA, Kuroda MJ, Schmitz JE, Dang X, Zheng Y, Lifton M, Letvin NL. Association of prolonged survival in HLA-A2+ progressive multifocal leukoencephalopathy patients with a CTL response specific for a commonly recognized JC virus epitope. J Immunol. 2002;168(1):499–504. doi: 10.4049/jimmunol.168.1.499. [DOI] [PubMed] [Google Scholar]
  21. Koralnik IJ, Du Pasquier RA, Letvin NL. JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy. J Virol. 2001;75(7):3483–3487. doi: 10.1128/JVI.75.7.3483-3487.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krymskaya L, Sharma MC, Martinez J, Haq W, Huang EC, Limaye AP, Diamond DJ, Lacey SF. Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides. J Virol. 2005;79(17):11170–11178. doi: 10.1128/JVI.79.17.11170-11178.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li J, Melenhorst J, Hensel N, Rezvani K, Sconocchia G, Kilical Y, Hou J, Curfman B, Major E, Barrett AJ. T-cell responses to peptide fragments of the BK virus T antigen: implications for cross-reactivity of immune response to JC virus. J Gen Virol. 2006;87(Pt 10):2951–2960. doi: 10.1099/vir.0.82094-0. [DOI] [PubMed] [Google Scholar]
  24. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999;223(1):77–92. doi: 10.1016/s0022-1759(98)00204-x. [DOI] [PubMed] [Google Scholar]
  25. Mann RS, Carroll RB. Cross-reaction of BK virus large T antigen with monoclonal antibodies directed against SV40 large T antigen. Virology. 1984;138(2):379–385. doi: 10.1016/0042-6822(84)90365-9. [DOI] [PubMed] [Google Scholar]
  26. Norbury CC, Chambers BJ, Prescott AR, Ljunggren HG, Watts C. Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells. Eur J Immunol. 1997;27(1):280–288. doi: 10.1002/eji.1830270141. [DOI] [PubMed] [Google Scholar]
  27. O'Neil BH, Kawakami Y, Restifo NP, Bennink JR, Yewdell JW, Rosenberg SA. Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA. J Immunol. 1993;151(3):1410–1418. [PMC free article] [PubMed] [Google Scholar]
  28. O'Neill FJ, Xu XL, Miller TH. Host range determinant in the late region of SV40 and RF virus affecting growth in human cells. Intervirology. 1990;31(2–4):175–187. doi: 10.1159/000150152. [DOI] [PubMed] [Google Scholar]
  29. Padgett BL, Walker DL. Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy. J Infect Dis. 1973;127(4):467–470. doi: 10.1093/infdis/127.4.467. [DOI] [PubMed] [Google Scholar]
  30. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet. 1971;1(7712):1257–1260. doi: 10.1016/s0140-6736(71)91777-6. [DOI] [PubMed] [Google Scholar]
  31. Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Perarnau B. HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice. J Exp Med. 1997;185(12):2043–2051. doi: 10.1084/jem.185.12.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rammensee H-G, Friede T, Stevanovic S. MHC ligands and peptide motifs: first listing. Immunogenetics. 1995;41:178–228. doi: 10.1007/BF00172063. [DOI] [PubMed] [Google Scholar]
  33. Randhawa P, Brennan DC. BK virus infection in transplant recipients: an overview and update. Am J Transplant. 2006;6(9):2000–2005. doi: 10.1111/j.1600-6143.2006.01403.x. [DOI] [PubMed] [Google Scholar]
  34. Salter RD, Howell DN, Cresswell P. Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics. 1985;21(3):235–246. doi: 10.1007/BF00375376. [DOI] [PubMed] [Google Scholar]
  35. Schell TD, Lippolis JD, Tevethia SS. Cytotoxic T lymphocytes from HLA-A2.1 transgenic mice define a potential human epitope from simian virus 40 large T antigen. Cancer Res. 2001;61(3):873–879. [PubMed] [Google Scholar]
  36. Schell TD, Mylin LM, Georgoff I, Teresky AK, Levine AJ, Tevethia SS. Cytotoxic T-lymphocyte epitope immunodominance in the control of choroid plexus tumors in simian virus 40 large T antigen transgenic mice. J Virol. 1999;73(7):5981–5993. doi: 10.1128/jvi.73.7.5981-5993.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Selin LK, Lin MY, Kraemer KA, Pardoll DM, Schneck JP, Varga SM, Santolucito PA, Pinto AK, Welsh RM. Attrition of T cell memory: selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses. Immunity. 1999;11(6):733–742. doi: 10.1016/s1074-7613(00)80147-8. [DOI] [PubMed] [Google Scholar]
  38. Selin LK, Vergilis K, Welsh RM, Nahill SR. Reduction of otherwise remarkably stable virus-specific cytotoxic T lymphocyte memory by heterologous viral infections. J Exp Med. 1996;183(6):2489–2499. doi: 10.1084/jem.183.6.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shah KV. SV40 and human cancer: a review of recent data. Int J Cancer. 2007;120(2):215–223. doi: 10.1002/ijc.22425. [DOI] [PubMed] [Google Scholar]
  40. Sweet BH, Hilleman MR. The vacuolating virus, S.V. 40. Proc Soc Exp Biol Med. 1960;105:420–427. doi: 10.3181/00379727-105-26128. [DOI] [PubMed] [Google Scholar]
  41. Taguchi F, Kajioka J, Miyamura T. Prevalence rate and age of acquisition of antibodies against JC virus and BK virus in human sera. Microbiol Immunol. 1982;26(11):1057–1064. doi: 10.1111/j.1348-0421.1982.tb00254.x. [DOI] [PubMed] [Google Scholar]
  42. Tevethia MJ, Ripper LW, Tevethia SS. A simple qualitative spot complementation test for temperature-sensitive mutants of SV40. Intervirology. 1974;3(4):245–255. doi: 10.1159/000149761. [DOI] [PubMed] [Google Scholar]
  43. Tevethia SS, Mylin L, Newmaster R, Epler M, Lednicky JA, Butel JS, Tevethia MJ. Cytotoxic T lymphocyte recognition sequences as markers for distinguishing among tumour antigens encoded by SV40, BKV and JCV. Dev Biol Stand. 1998;94:329–339. [PubMed] [Google Scholar]
  44. Velders MP, Macedo MF, Provenzano M, Elmishad AG, Holzhutter HG, Carbone M, Kast WM. Human T cell responses to endogenously presented HLA-A*0201 restricted peptides of Simian virus 40 large T antigen. J Cell Biochem. 2001;82(1):155–162. doi: 10.1002/jcb.1148. [DOI] [PubMed] [Google Scholar]
  45. Vilchez RA, Butel JS. Emergent human pathogen simian virus 40 and its role in cancer. Clin Microbiol Rev. 2004;17(3):495–508. doi: 10.1128/CMR.17.3.495-508.2004. table of contents. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Viscidi RP, Rollison DE, Viscidi E, Clayman B, Rubalcaba E, Daniel R, Major EO, Shah KV. Serological cross-reactivities between antibodies to simian virus 40, BK virus, and JC virus assessed by virus-like-particle-based enzyme immunoassays. Clin Diagn Lab Immunol. 2003;10(2):278–285. doi: 10.1128/CDLI.10.2.278-285.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES