Control of advanced choroid plexus tumors in SV40 T antigen transgenic mice following priming of donor CD8(+) T lymphocytes by the endogenous tumor antigen.

Mouse models in which tumors arise spontaneously due to the transgenic expression of an oncoprotein provide an opportunity to test approaches that target the immune-mediated control of tumor progression. In this report we investigated the role of SV40 Tag-specific CD8(+) T cells in the control of advanced choroid plexus tumor progression using large tumor Ag (Tag) transgenic mice. Since mice of the SV11 line are tolerant to the immunodominant SV40 Tag-derived CTL epitopes, mice with advanced stage tumors were reconstituted with naive C57BL/6 spleen cells following a low dose of gamma-irradiation. This led to the priming of CTLs specific for the H2-K(b)-restricted epitope IV by the endogenous Tag and a significant increase in the life span of Tag transgenic mice. Epitope IV-specific CD8(+) T cells accumulated and persisted in the brains and tumors of SV11 mice, as determined by analysis with epitope-specific MHC class I tetramers. Brain-infiltrating epitope IV-specific T cells were capable of producing IFN-gamma as well as lysing syngeneic Tag-transformed cells in vitro. In addition, the adoptive transfer of spleen cells from Tag-immune C57BL/6 mice resulted in a dramatic increase in the control of tumor progression in SV11 mice and was associated with the accumulation of CD8(+) T cells specific for multiple Tag epitopes in the brain. These results indicate that the control of advanced stage spontaneous choroid plexus tumors is associated with the induction of a strong and persistent CD8(+) T cell response to Tag.

Cytotoxic T lymphocytes from HLA-A2.1 transgenic mice define a potential human epitope from simian virus 40 large T antigen.

Recent reports have documented the presence of SV40 large T antigen (T ag) sequences in a number of human tumors and raised the question of whether cellular immunity to T ag is elicited in such individuals. We used HLA-A2.1 transgenic C57BL/6 mice to identify an epitope from T ag recognized by CD8+ CTLs when presented by this human MHC class I molecule. Immunization of HLA-A2.1 transgenic mice with syngeneic T ag-transformed cells resulted in the induction of HLA-A2.1-restricted, T ag-specific CTLs. The target epitope, residues 281-289 (KCDDVLLLL) of T ag, was identified using both cell lines expressing T ag variants and synthetic T ag peptides. Peptide 281-289 bound stably to HLA-A2.1 molecules, effectively sensitized target cells for CTL lysis, and was efficiently processed from endogenous T ag in cells of both mouse and human origin. CTLs were not cross-reactive on the human BK or JC virus T ags. Thus, SV40 T ag 281-289 represents a potential specific CTL recognition epitope for humans.

Lymphotoxin alpha-/- mice develop functionally impaired CD8+ T cell responses and fail to contain virus infection of the central nervous system.

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8(+) T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin alpha-deficient mice develop CD8(+) T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8(+) T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTalpha possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.

Sequential loss of cytotoxic T lymphocyte responses to simian virus 40 large T antigen epitopes in T antigen transgenic mice developing osteosarcomas.

The role of CTL tolerance in tumor immunity to SV40 large T antigen (T ag)-induced tumors was studied using T ag transgenic mice of the line 501 (H2b). 501 mice express SV40 T ag under the influence of the alpha-amylase promoter, which leads to the development of osteogenic osteosarcomas late in life and eventual death between 12 and 17 months of age. We determined the ability of 501 mice to respond to the four H2b-restricted T ag CTL epitopes, which include epitope I (T ag 206-215), epitope II/III (T ag 223-231), the immunorecessive epitope V (T ag 489-497), restricted by H2-Db, and epitope IV (T ag 404-411), restricted by H2-Kb. We demonstrate that 501 mice are partially tolerant to the H2b-restricted T ag epitopes. Immunization of 4-month-old 501 mice with T ag-transformed syngeneic cell lines or a recombinant vaccinia virus expressing full-length T ag elicited CTL responses against the H2-Kb-restricted T ag epitope IV only. In contrast, immunization of 4-month-old 501 mice with recombinant vaccinia viruses expressing individual T ag epitopes as minigenes elicited CTLs against epitopes I, IV, and V, but not against epitope II/III. Complete tolerance to epitopes I, IV, and V developed in 501 mice, but the age when tolerance was detected varied for each epitope. Tolerance to epitope I occurred by 6 months of age and was accelerated in the absence of CD4+ T cells. Tolerance to the immunorecessive epitope V was observed in 12-month-old 501 mice but was independent of the presence of osteosarcomas. In contrast, CTLs specific for epitope IV were detected in mice from 3 to 14 months of age but not in mice that had developed osteosarcomas. Analysis of epitope IV-specific CD8+ cells derived from 3-month-old 501 mice with H2-Kb/epitope IV tetramers revealed decreased numbers of epitope IV-specific CD8+ cells in 501 mice relative to C57BL/6 mice, with a further decrease in older 501 mice. Tumor progression resulted in loss of H2-Kb/epitope IV tetramer staining CD8+ cells. Thus, progression to tolerance to individual T ag CTL epitopes in 501 mice is epitope dependent.

Cytotoxic T-lymphocyte epitope immunodominance in the control of choroid plexus tumors in simian virus 40 large T antigen transgenic mice.

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.

Immunogenicity of herpes simplex virus type 1 mutants containing deletions in one or more alpha-genes: ICP4, ICP27, ICP22, and ICP0.

Replication defective mutants of HSV have been proposed both as vaccine candidates and as vehicles for gene therapy because of their inability to produce infectious progeny. The immunogenicity of these HSV replication mutants, at both qualitative and quantitative levels, will directly determine their effectiveness for either of these applications. We have previously reported (Brehm et al., J. Virol., 71, 3534, 1997) that a replication defective mutant of HSV-1, which expresses a substantial level of viral genes without producing virus particles, is as efficient as wild-type HSV-1 in eliciting an HSV-specific cytotoxic T-lymphocyte (CTL) response. In this report, we have further evaluated the immunogenic potential of HSV-1-derived replication defective mutants by examining the generation of HSV-specific CTL following immunization with viruses that are severely restricted in viral gene expression due to mutations in one or more HSV alpha genes (ICP4, ICP27, ICP22, and ICP0). To measure the CTL responses induced by the HSV alpha-mutants, we have targeted two H-2Kb-restricted CTL epitopes: an epitope in a virion protein, gB (498-505), and an epitope in a nonvirion protein, ribonucleotide reductase (RR1 822-829). The HSV mutants used in this study are impaired in their ability to express gB while a majority of them still express RR1. Our findings demonstrate that a single immunization with these mutants is able to generate a strong CTL response not only to RR1 822-829, but also to gB498-505 despite their inability to express wild-type levels of gB. Furthermore, a single immunization with any individual mutant can also provide immune protection against HSV challenge. These results suggest that mutants which are restricted in gene expression may be used as effective immunogens in vivo.

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity.

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

A molecular basis for how a single TCR interfaces multiple ligands.

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.

Cytotoxic T lymphocyte recognition sequences as markers for distinguishing among tumour antigens encoded by SV40, BKV and JCV.

Simian virus 40 (SV40) has been shown to be associated with a number of human tumours. Two other human papova viruses, BKV and JCV, infect humans at a relatively high frequency and are activated upon immune suppression. The T antigens of both of these viruses share considerable homologies with the transforming protein T antigen of SV40. We have used SV40 T antigen specific cytotoxic T lymphocyte (CTL) clones to discriminate among the T antigens of SV40, BKV and JCV. These CTL clones directed to four distinct CTL epitopes serve as specific probes and can differentiate subtle alterations or deletions in the CTL epitopes relative to SV40 T antigen. Using this strategy, we have been able to authenticate three SV40 viruses isolated from humans as all four distinct CTL epitopes in the T antigens encoded by these three SV40 human isolates (SVCPC, SVMEN, and SVPML-1) were found to be identical to prototype SV40. We have further identified a 198 amino acid deletion T antigen variant of SVCPC. The finding of a deletion mutant in the SVCPC virus population suggests that the cellular immune response may play a role in the selection of antigenic loss variants.

Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection.

SV40-transformed mKSA cells (H-2d) readily induce progressively growing tumors in adult syngeneic BALB/c mice while expressing the full complement of H-2d MHC class I antigens. BALB/c mice previously immunized with SV40, soluble SV40 T antigen, or irradiated SV40-transformed syngeneic, allogeneic, or xenogeneic cells reject an mKSA tumor challenge even though these mice have been considered low- or nonresponders to T antigen due to difficulty in demonstrating SV40 T antigen-specific CTL. We have investigated the role of H-2d-restricted CTL in the rejection of SV40 tumors in BALB/c mice. Immunization of BALB/c mice with SV40 induced T antigen-specific CTL which were largely. H-2Ld-restricted. However, following repeated in vitro restimulation with mKSA cells, CTL emerged which recognized a subdominant H-2Kd-restricted epitope corresponding to T antigen residues 499-507. Immunization of BALB/c mice with a recombinant vaccinia virus expressing the T499-507 epitope provided partial protection against a challenge of syngeneic mKSA tumor cells and induced the generation of T499-507-specific CTL. These results indicate that a subdominant H-2Kd-restricted CTL epitope can participate in the rejection of SV40 tumors in BALB/c mice.

An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope.

An immunological hierarchy among three H-2Db-restricted cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and II/III are immunodominant, whereas determinant V is immunorecessive. To assess the immunogenicity of each determinant individually and define mechanisms that contribute to the immunorecessive nature of determinant V, we constructed a panel of recombinant vaccinia viruses (rVVs) expressing minigenes encoding these determinants in various polypeptide contexts. We found the following. (i) Immunization of mice with an rVV encoding full-length SV40 Tag resulted in priming for CTL responses to determinants I and II/III but not determinant V. (ii) rVVs encoding peptide I or II/III in the cytosol or targeted to the endoplasmic reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs encoding peptide V minigenes were antigenic and immunogenic if the peptide was targeted to the ER, expressed in the cytosol with short flanking sequences, or expressed from within a self-protein, murine dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V (preceded by a Met codon only) could be enhanced by using a potent inhibitor of the proteasome. (v) H-2Db-epitope V peptide complexes decayed more rapidly than complexes containing epitope I or II/III peptides. In brefeldin A blocking experiments, functional epitope V complexes were detected longer on targets expressing ER-targeted epitope V than on targets expressing forms of epitope V dependent on the transporter associated with antigen processing. Therefore, limited formation of relatively unstable cell surface H-2Db complexes most likely contributes to the immunorecessive nature of epitope V within SV40 Tag. Increasing the delivery of epitope V peptide to the major histocompatibility complex class I presentation pathway by ER targeting dramatically enhanced the immunogenicity of epitope V.

Immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1) induces a CD8+ cytotoxic T-lymphocyte response and confers a level of protection comparable to that of wild-type HSV-1.

Replication-deficient viruses provide an attractive alternative to conventional approaches used in the induction of antiviral immunity. We have quantitatively evaluated both the primary and memory cytotoxic T-lymphocyte (CTL) responses elicited by immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1). In addition, we have examined the potential role of these CTL in protection against HSV infection. Using bulk culture analysis and limiting-dilution analysis, we have shown that a replication-deficient virus, d301, generates a strong primary CTL response that is comparable to the response induced by the wild type-strain, KOS1.1. Furthermore, the CTL induced by d301 immunization recognized the immunodominant, H-2Kb-restricted, CTL recognition epitope gB498-505 to a level similar to that for CTL from KOS1.1-immunized mice. The memory CTL response evoked by d301 was strong and persistent, even though the frequencies of CTL were slightly lower than the frequencies of CTL induced by KOS1.1. Adoptive transfer studies indicated that both the CD8+ and the CD4+ T-cell responses generated by immunization with d301 and KOS1.1 were able to limit the extent of a cutaneous HSV infection to comparable levels. Overall, these results indicate that viral replication is not necessary to elicit a potent and durable HSV-specific immune response and suggest that replication-deficient viruses may be effective in eliciting protection against viral pathogens.

Induction and persistence of a cytotoxic T lymphocyte (CTL) response against a herpes simplex virus-specific CTL epitope expressed in a cellular protein.

CD8+ cytotoxic T-lymphocytes recognize small epitope peptides in association with MHC class I molecules expressed on the cell surface. In this study, we have determined whether an 8 amino acid viral CTL epitope, when expressed in a cellular protein, can be appropriately processed, presented, and recognized by the corresponding epitope-specific CTL and whether it is capable of inducing a CTL response in vivo. An H-2Kb-restricted CTL epitope from herpes simplex virus type 1 (HSV-1) glycoprotein B (gB epitope, residues 498-505) was cloned into the mouse dihydrofolate reductase protein (DHFR) at amino acid position 87. The recombinant DHFRs were expressed in vaccinia virus recombinants. To distinguish the recombinant DHFR proteins from the endogenous DHFR, an antibody epitope, recognized by monoclonal antibody PAb 901 and derived from simian virus 40 (SV40) T antigen was tagged to the C-termini of recombinant DHFR proteins. In vivo expression of recombinant DHFR was demonstrated by immunoprecipitation with the monoclonal antibody PAb 901. The H-2b cells infected with recombinant vaccinia virus expressing the recombinant DHFR were specifically lysed by gB epitope-specific CTL. Furthermore, the recombinant DHFR was functional in inducing a long lasting HSV gB epitope-specific CTL response upon immunization of C57BL/6 (B6) mice. These results indicate that a viral epitope expressed in a cellular protein can be efficiently processed, presented, and recognized by epitope-specific CTL and show that cellular proteins expressing CTL epitopes can be used for induction of CD8+ T lymphocyte responses.

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.

Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen.

Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association.

Polymorphism within the herpes simplex virus (HSV) ribonucleotide reductase large subunit (ICP6) confers type specificity for recognition by HSV type 1-specific cytotoxic T lymphocytes.

A panel of herpes simplex virus type 1 (HSV-1)-specific, CD8+, major histocompatibility complex class I (H-2Kb)-restricted cytotoxic T-lymphocyte (CTL) clones was derived from HSV-1-immunized C57BL/6 (H-2b) mice in order to identify the HSV-1 CTL recognition epitope(s) which confers type specificity. HSV-1 x HSV-2 intertypic recombinants were used to narrow the region encoding potential CTL recognition epitopes to within 0.51 to 0.58 map units of the HSV-1 genome. Using an inhibitor of viral DNA synthesis and an ICP6 deletion mutant, the large subunit of ribonucleotide reductase (ICP6, RR1) was identified as a target protein for these type-specific CTL. Potential CTL recognition epitopes within RR1 were located on the basis of the peptide motif predicted to bind to the MHC class I H-2Kb molecule. A peptide corresponding to residues 822 to 829 of RR1 was shown to confer susceptibility on H-2Kb-expressing target cells to lysis by the type 1-specific CTL. On the basis of a comparison of the HSV-1 RR1 epitope (residues 822 to 829) with the homologous sequence of HSV-2 RR1 (residues 828 to 836) and by the use of amino acid substitutions within synthetic peptides, we identified HSV-1 residue 828 as being largely responsible for the type specificity exhibited by HSV-1-specific CTL. This HSV-1 RR1 epitope, when expressed in recombinant simian virus 40 large T antigen in primary C57BL/6 cells, was recognized by the HSV-1 RR1-specific CTL clones. These results indicate that an early HSV protein with enzymatic activity provides a target for HSV-specific CTL and that type specificity is dictated largely by a single amino acid.

In vivo priming and activation of memory cytotoxic T-lymphocytes (CTL) by a chimeric simian virus 40 T antigen expressing an eight amino acid residue herpes simplex virus gB CTL epitope.

The simian virus 40 (SV40) large T antigen was used as an immunogenic vector to express a herpes simplex virus type 1 (HSV-1) glycoprotein B (gB), H-2Kb-restricted cytotoxic T lymphocyte (CTL) recognition epitope corresponding to amino acid residues 498-505. Immunization of naive, C57BL/6 mice with a cell line, B6/350gB, expressing the chimeric T antigen was able to induce the generation of gB498-505-specific CTL in both the lymph nodes and the spleen. Splenic-derived, gB498-505-specific memory CTL (CTLm) were detected in these mice for at least 6 months following immunization at a slightly lower frequency than in those mice immunized with infectious HSV-1. B6/350gB was also able to activate in vitro gB498-505-specific memory CTL obtained from mice previously challenged with HSV. Overall, these findings support the use of a chimeric T antigen as a vector in determining the immunogenic potential of individual CTL epitopes and to assess their potential contribution in inducing a protective immune response in vivo.

Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes.

Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.