Functional endogenous cytotoxic T lymphocytes are generated to multiple antigens co-expressed by progressing tumors; after intra-tumoral IL-2 therapy these effector cells eradicate established tumors

Tumors contain many antigens that may be recognized by the immune system. It is not known whether these antigens, and the epitopes within these antigens, can all be recognized by the anti-tumor immune response or if such responses are restricted to a few dominant epitopes. Effector function of endogenous cytotoxic T lymphocytes (CTL) generated during tumor progression has previously been assessed by indirect, ex vivo assays, which often focused on a single antigen. Therefore, we evaluated the endogenous in vivo CTL response to multiple neo tumor antigens using murine Lewis lung carcinoma tumor cells transfected with ovalbumin or a polyepitope construct. Both express multiple MHC class I-restricted epitopes. Ovalbumin contains a known hierarchy of epitopes for given MHC molecules, whilst the polyepitope expresses a number of dominant epitopes. We show that as tumors progress, potent effector CTL are generated in vivo that are restricted to dominant epitopes; we did not see the responses to subdominant or cryptic epitopes. Our data show that the CTL recognizing tumor antigens vary in their lytic capacity, as the CTL responding to two of the four epitopes were particularly potent killers. The presence of these effector CTLs did not prevent tumor growth. However, intra-tumoral IL-2 treatment altered the potency, but not the hierarchy, of these CTL such that they mediated tumor regression. These results have implications for immunotherapy protocols.     

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.     

Internal initiation sites of de novo RNA synthesis within the hepatitis C virus polypyrimidine tract

Using fluorescein isothiocyanate (FITC)-conjugated H-2K(b) CTL epitope (SIINFEKL) as a model system, we investigated the antigen delivery route by pH-sensitive liposomes in vivo. Fluorescence was initially detected in lymph nodes at 3 h after immunization, and its intensity reached a peak value in superticial inguinal lymph node at 9 h. No trace could be detected in spleen even with prolonged monitoring for up to 24 h. These results strongly suggest that the presentation of CTL-peptide antigen vehicled by pH-sensitive liposomes exclusively occurs in lymph nodes. In mice immunized with the H-2K(b) CTL epitope encapsulated pH-sensitive liposomes, peptide-specific CTL response was detected at day 3. The response was strongly augmented by the second immunization and persisted up to at least 45 days. These results suggest that pH-sensitive liposome formula functions as a potential adjuvant of peptide antigens and is useful for the induction of antigen specific CTLsv in vivo.     

Sequences that flank subdominant and cryptic epitopes influence the proteolytic generation of MHC class I-presented peptides

The proteasome has been shown to make the proper C-terminal cleavage for the generation of several immunodominant class I-presented peptides whereas aminopeptidases generate their proper N termini. In this study, we show that these two distinct proteolytic processes are also involved in generating a subdominant OVA peptide KVVRFDKL (K-L). Moreover, proteasome inhibitors did not enhance the presentation of any K-L construct, suggesting that destruction of this peptide by proteasomes, if any, does not limit its presentation. We have further examined in intact cells the influence of residues flanking this epitope on these proteolytic processes. When the N-terminal flanking residues of K-L are fused to an immunodominant OVA peptide SIINFEKL (S-L), the presentation of S-L is reduced as compared with a construct with its natural flanking sequence and was not inhibited (or enhanced) by proteasome inhibitors. Similarly, a reduction in presentation was observed when the C-terminal flanking residues of the subdominant epitope were attached to S-L. A detailed analysis revealed that the Pro at the P1' position of K-L was responsible for this reduction, and presentation of these C-terminally extended constructs was sensitive to proteasome inhibitor. The study suggests that both the N- and C-terminal flanks of the subdominant peptide are suboptimal for Ag presentation. Moreover, three of four C-terminal residues that flank other subdominant or cryptic epitopes in OVA reduced the presentation of S-L. Therefore, the residues that flank the C termini of several subdominant and cryptic epitopes are often suboptimal for cleavage and may contribute to the phenomenon of immunodominance.     

In vivo cross-presentation of a soluble protein antigen: kinetics, distribution, and generation of effector CTL recognizing dominant and subdominant epitopes

Cross-presentation of exogenous Ags via the MHC class I pathway is now recognized for its role in self-tolerance, tumor immunity, and vaccine development. However, little is known about the in vivo distribution and kinetics of cross-presented protein Ags, nor the subsequent development of CTL effector responses to dominant or subdominant epitopes. We examined the location and duration of cross-presented Ag by using 5,6-carboxy-succinimidyl-fluorescein ester-labeled T cells from class I-restricted Ag-specific TCR mice. Comparisons of results from an in vitro (51)Cr release CTL assay with an in vivo CTL assay provided physiologically relevant insights into the functional capacities of CTL specific for epitopes with differing affinities. These data demonstrate that efficient cross-presentation of a dominant class I-restricted Ag is dose related and remains largely localized, but not limited to the draining lymph nodes for up to 3 wk following a single injection of soluble protein. Within this period, dominant peptide-specific CTL are fully functional in vivo throughout the secondary lymphoid system. However, no in vivo responses are seen to a subdominant or cryptic epitope. Prolonging Ag cross-presentation via use of IFA promoted persisting in vivo dominant epitope-specific CTL activity and revealed dose-responsive precursor CTL to the subdominant, but not to a cryptic epitope. Analysis of functional in vivo CTL responses demonstrated that, in the presence of strong ongoing responses to the dominant peptide, lytic activity of CTL directed at weaker epitopes is undetectable.     

DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines

DNA vaccination offers a strategy to induce immune attack on cancer cells, but tumor Ags are often weak. Inclusion of a "foreign" protein increases immunogenicity, and we found previously that fusion of the fragment C (FrC) of tetanus toxin to the tumor Ag sequence promotes Ab and CD4(+) responses against B cell tumors. For CTL responses, use of the full two-domain FrC may be less helpful, because known immunogenic MHC class I-binding peptides in the second domain could compete with attached tumor-derived epitopes. Therefore, we removed the second domain, retaining the N-terminal domain, which contains a "universal" helper epitope. We investigated the ability to induce CTL responses of candidate peptides placed at the C terminus of this domain. As test peptides, we repositioned the two known CTL motifs from the second domain to this site. Strong CTL responses to each peptide were induced by the engineered construct, as compared with the native FrC construct. Induced CTLs were able to specifically kill tumor cells transfected with FrC as a surrogate tumor Ag both in vitro and in vivo. Further reduction of the domain to a short helper epitope generated only weak CTL responses against fused peptides, and synthetic peptides mixed with the plasmid containing the first domain were ineffective. The single FrC domain-peptide vaccine design also was able to induce high levels of CTLs against a known epitope from carcinoembryonic Ag. Response to peptide was suppressed if two FrC domains were present, consistent with immunodominance. These principles and designs may have relevance for cancer vaccines delivered via DNA.     

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.     

Variable Processing and Cross-presentation of HIV by Dendritic Cells and Macrophages Shapes CTL Immunodominance and Immune Escape

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.     

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.     

Redox modulation inhibits CD8 T cell effector function

The evolutionary preservation of reactive oxygen species in innate immunity underscores the important roles these constituents play in immune cell activity and as signaling intermediates. In an effort to exploit these pathways to achieve control of aberrant immune activation we demonstrate that modulation of redox status suppresses cell proliferation and production of IL-2, IFN-gamma, TNF-alpha, and IL-17 in two robust CD8 T-cell-dependent in vitro mouse models: (1) response to alloantigen in an mixed leukocyte reaction and (2) CD8 T cell receptor transgenic OT-1 response to cognate peptide (SIINFEKL). To correlate these findings with cytotoxic lymphocyte (CTL) function we performed cytotoxicity assays and found that redox modulation diminishes the ability of alloantigen-specific and antigen-specific OT-1 CTLs to kill their corresponding antigen-expressing target cells. To further examine the mechanisms of redox-mediated repression of CTL target cell lysis, we analyzed the expression of the effector molecules IFN-gamma, perforin, and granzyme B and the degranulation marker CD107a (LAMP-1). In both models, redox modulation reduced the expression of these effector components by at least fivefold. These results demonstrate that redox modulation quells the CD8 T cell response to alloantigen and the T cell receptor transgenic CD8 T cell response to its cognate antigen by inhibiting proliferation, proinflammatory cytokine synthesis, and CTL effector mechanisms.     

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.     

The role of T cell antagonism and original antigenic sin in genetic immunization

To counter highly mutable pathogens like HIV-1, a number of vaccines are being developed to deliver multiple mutant forms of viral Ags to provoke multivalent antiviral CTLs. However, it is uncertain whether such multiple mutant epitope vaccines will generate the diverse CTL responses desired or will instead create immune interference. To characterize the role of immune interference by mutant epitopes in this process, we have tested a "worst case" scenario in which the immunodominant epitope of OVA (SIINFEKL) and its in vitro TCR antagonist (SIINFEDL) have been used to genetically immunize C57BL/6 mice. We demonstrate here that sequential delivery of these mutant epitopes provokes original antigenic sin in CD8 T cells as demonstrated by attenuation of CTLs, intracellular IFN-gamma production, and MHC I peptide-tetramer staining. By contrast, simultaneous exposure of the immune system to this agonist/antagonist pair not only fails to generate T cell antagonism in vivo, but also avoids original antigenic sin. These observations suggest that simultaneous immunization with vaccines containing mutant epitopes, even T cell antagonists, can indeed generate a diverse array of T cell responses and that at least some immune interference can be avoided by delivering mutant Ags to the immune system simultaneously.     

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.     

Cytotoxic T-lymphocyte clones specific for an immunodominant epitope display discerning antagonistic response to naturally occurring Epstein-Barr virus variants.

In the present study we have identified Epstein-Barr virus isolates which encode variant sequences within an HLA B35-restricted immunodominant cytotoxic T-lymphocyte (CTL) epitope that act as natural antagonists and can inhibit CTL activity on the wild-type epitope. This effect can be demonstrated if the wild-type epitope is presented as a synthetic peptide or when processed from a full-length Epstein-Barr virus protein expressed by recombinant vaccinia constructs. However, this antagonistic effect was only selectively seen with some CTL clones, while a strong agonistic effect was evident for other clones in the presence of the same variant peptide. The data presented in this study strongly suggest that it is unlikely that the variant viruses can completely antagonize a virus-specific CTL response by this mechanism since the host immune response is capable of generating CTLs expressing a diverse array of T-cell receptors. Moreover, many of these CTLs can recognize the variant sequences as efficiently as wild-type epitope.     

Mapping and characterization of the primary and anamnestic H-2(d)-restricted cytotoxic T-lymphocyte response in mice against human metapneumovirus

Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus.     

Role of APC in the selection of immunodominant T cell epitopes

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.     

Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumor growth in C57BL/6 mice

Previously, we identified human papillomavirus type 16 (HPV-16) E5 as a tumor rejection antigen that can induce cytotoxic T lymphocytes (CTLs) to protect against tumor growth (D. W. Liu et al., J. Virol. 74:9083-9089, 2000). In the present study, we further mapped the CTL epitope of E5 protein by analyzing E5-specific CD8(+) gamma interferon-positive (IFN-gamma(+)) double-positive cells in C57BL/6 mice with flow cytometry. The results showed the region spanning amino acids 25 to 33 (VCLLIRPLL) contained the potential D(b)-restricted CTL epitope. Subsequently, to determine whether peptide E5 25-33-based vaccination could induce E5-specific CTL activity, syngeneic animals received E5 25-33 emulsified with either CpG oligodeoxynucleotide (CpG ODN 1826) or Freund's adjuvant, and the growth of the tumors was monitored. The results showed that although both adjuvants induced E5-specific CD8(+) IFN-gamma(+) T cells and eradicated E5-containing tumor growth, CpG ODN was found to stimulate stronger CTL response than Freund's adjuvant. We also compared the immune response of the effector/memory/recall phase induced by E5 25-33 peptide or by E5 protein that was synthesized in vivo by adenovirus-based E5 gene delivery. E5 25-33 peptide plus CpG ODN was shown to be a superior vaccine compared to the adenovirus-based E5 gene. Interestingly, their chronological patterns of immune response were similar, suggesting that E5 25-33 is a major CTL peptide of E5 protein.     

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5

Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.     

Interleukin-4 Diminishes CD8+ Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo

Although interleukin-4 (IL-4) has been implicated in respiratory syncytial virus (RSV)-enhanced disease, the mechanism by which it modulates immune responses to primary RSV infection remains unclear. We have developed a system to investigate the effect of IL-4 on RSV epitope-specific cytotoxic T-lymphocyte (CTL) effector function in vivo, using an H-2K(d)-restricted RSV M2 epitope. BALB/c mice were infected with recombinant vaccinia virus (rVV) constructed to express RSV M2 protein (vvM2) alone or coexpress M2 and IL-4 (vvM2/IL-4). Splenocytes were assessed for M2-specific CTL activity in a direct (51)Cr release assay and intracellular gamma interferon (IFN-gamma) production by fluorescence-activated cell sorting analysis. Mice infected with vvM2/IL-4 had less M2-specific primary CTL activity than those infected with vvM2. M2-specific CTL frequency, as measured by M2 peptide-induced intracellular IFN-gamma production, was diminished in the vvM2/IL-4 group, partially accounting for the reduction of CTL activity. Mice immunized with either construct were challenged intravenously with RSV 4 weeks postimmunization, and direct CTL were measured. These results demonstrate that local expression of IL-4, at the time of antigen presentation, diminishes the cytolytic activity of primary and memory CD8(+) RSV-specific CTL responses in vivo.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.