Identification of CD4+ T cell epitopes in C. burnetii antigens targeted by antibody responses

Coxiella burnetii is an obligate intracellular gram-negative bacterium that causes acute Q fever and chronic infections in humans. A killed, whole cell vaccine is efficacious, but vaccination can result in severe local or systemic adverse reactions. Although T cell responses are considered pivotal for vaccine derived protective immunity, the epitope targets of CD4(+) T cell responses in C. burnetii vaccination have not been elucidated. Since mapping CD4(+) epitopes in a genome with over 2,000 ORFs is resource intensive, we focused on 7 antigens that were known to be targeted by antibody responses. 117 candidate peptides were selected from these antigens based on bioinformatics predictions of binding to the murine MHC class II molecule H-2 IA(b). We screened these peptides for recognition by IFN-γ producing CD4(+) T cell in phase I C. burnetii whole cell vaccine (PI-WCV) vaccinated C57BL/6 mice and identified 8 distinct epitopes from four different proteins. The identified epitope targets account for 8% of the total vaccination induced IFN-γ producing CD4(+) T cells. Given that less than 0.4% of the antigens contained in C. burnetii were screened, this suggests that prioritizing antigens targeted by antibody responses is an efficient strategy to identify at least a subset of CD4(+) targets in large pathogens. Finally, we examined the nature of linkage between CD4(+) T cell and antibody responses in PI-WCV vaccinated mice. We found a surprisingly non-uniform pattern in the help provided by epitope specific CD4(+) T cells for antibody production, which can be specific for the epitope source antigen as well as non-specific. This suggests that a complete map of CD4(+) response targets in PI-WCV vaccinated mice will likely include antigens against which no antibody responses are made.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.     

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.     

DNA vaccine with discontinuous T‐cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi‐epitope Mycobacterium tuberculosis vaccine

DNA‐based vaccine is a promising candidate for immunization and induction of a T‐cell‐focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi‐functional T response against multi‐TB antigens, a multi‐epitope DNA vaccine and a ‘protein backbone grafting’ design method is adopted to graft five discontinuous T‐cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T‐cell epitopes derived from ESAT‐6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope‐scaffold construct was significantly more potent to induce IFN‐γ⁺ T response to five T‐cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi‐functional‐CD4⁺ T response. It also enhanced granzyme B⁺ CTL and IL‐2⁺ CD8⁺ T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi‐epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.     

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.     

Epitope cross-reactivity frequently differs between central and effector memory HIV-specific CD8+ T cells

HIV diversity may limit the breadth of vaccine coverage due to epitope sequence differences between strains. Although amino acid substitutions within CD8(+) T cell HIV epitopes can result in complete or partial abrogation of responses, this has primarily been demonstrated in effector CD8(+) T cells. In an HIV-infected Kenyan cohort, we demonstrate that the cross-reactivity of HIV epitope variants differs dramatically between overnight IFN-gamma and longer-term proliferation assays. For most epitopes, particular variants (not the index peptide) were preferred in proliferation in the absence of corresponding overnight IFN-gamma responses and in the absence of the variant in the HIV quasispecies. Most proliferating CD8(+) T cells were polyfunctional via cytokine analyses. A trend to positive correlation was observed between proliferation (but not IFN-gamma) and CD4 counts. We present findings relevant to the assessment of HIV vaccine candidates and toward a better understanding of how viral diversity is tolerated by central and effector memory CD8(+) T cells.     

Antigen targeting to CD11b allows efficient presentation of CD4+ and CD8+ T cell epitopes and in vivo Th1-polarized T cell priming

Bordetella pertussis adenylate cyclase (CyaA) is an invasive bacterial toxin that delivers its N-terminal catalytic domain into the cytosol of eukaryotic cells bearing the alpha(M)beta(2) integrin (CD11b/CD18), such as myeloid dendritic cells. This allows use of engineered CyaA for targeted delivery of CD8(+) T cell epitopes into the MHC class I pathway of APC and induction of robust and protective cytotoxic responses. In this study, we demonstrate that CyaA can efficiently codeliver both a CD8(+) T cell epitope (OVA(257-264)) and a CD4(+) T cell epitope (MalE(100-114)) into, respectively, the conventional cytosolic or endocytic routes of processing of murine bone marrow-derived dendritic cells. Upon CyaA delivery, a strong potentiation of the MalE(100-114) CD4(+) T cell epitope presentation is observed as compared with the MalE protein, which depends on CyaA interaction with its CD11b receptor and its subsequent clathrin-mediated endocytosis. In vivo, CyaA induces strong and specific Th1 CD4(+) and CD8(+) T cell responses against, respectively, the MalE(100-114) and OVA(257-264) epitopes. These results underscore the potency of CyaA for design of new vaccines.     

Human CD4+ T cell epitopes from vaccinia virus induced by vaccination or infection

Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4(+) T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4(+) T cell responses have been poorly characterized, and CD4(+) T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens.     

Human cytotoxic CD4+ T cells recognize HLA-DR1-restricted epitopes on vaccinia virus proteins A24R and D1R conserved among poxviruses

We previously demonstrated that vaccinia virus (VV)-specific CD4(+) cytolytic T cells can persist for >50 years after immunization against smallpox in the absence of re-exposure to VV. Nevertheless, there have been few studies focusing on CD4(+) T cell responses to smallpox vaccination. To ensure successful vaccination, a candidate vaccine should contain immunodominant CD4(+) T cell epitopes as well as CD8(+) T and B cell epitopes. In the present study, we established cytotoxic CD4(+) T cell lines from VV-immune donors, which recognize epitopes in VV proteins D1R and A24R in association with HLA-DR1 Ags. Comparisons of sequences between different members of the poxvirus family show that both epitopes are completely conserved among VV, variola viruses, and most mammalian poxviruses, including monkeypox, cowpox, and ectromelia. The CD4(+) T cell lines lysed VV-infected, Ag- and peptide-pulsed targets, and the lysis was inhibited by concanamycin A. We also detected these peptide-specific cytolytic and IFN-gamma-producing CD4(+) T cells in short-term bulk cultures of PBMC from each of the three VV-immune donors tested. These are the first VV-specific CD4(+) T cell epitopes identified in humans restricted by one of the most common MHC class II molecules, HLA-DR1, and this information may be useful in analyzing CD4(+) T cell responses to pre-existing or new generation VV vaccines against smallpox.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Subdominant/cryptic CD8 T cell epitopes contribute to resistance against experimental infection with a human protozoan parasite

During adaptive immune response, pathogen-specific CD8(+) T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8(+) T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8(+) T cells of H-2(a) infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8(+) T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8(+) T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.     

CD4(+) T cells induced by a DNA vaccine: immunological consequences of epitope-specific lysosomal targeting

Our previous studies have shown that targeting DNA vaccine-encoded major histocompatibility complex class I epitopes to the proteasome enhanced CD8(+) T-cell induction and protection against lymphocytic choriomeningitis virus (LCMV) challenge. Here, we expand these studies to evaluate CD4(+) T-cell responses induced by DNA immunization and describe a system for targeting proteins and minigenes to lysosomes. Full-length proteins can be targeted to the lysosomal compartment by covalent attachment to the 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II). Using minigenes encoding defined T-helper epitopes from lymphocytic choriomeningitis virus, we show that the CD4(+) T-cell response induced by the NP(309-328) epitope of LCMV was greatly enhanced by addition of the LIMP-II tail. However, the immunological consequence of lysosomal targeting is not invariably positive; the CD4(+) T-cell response induced by the GP(61-80) epitope was almost abolished when attached to the LIMP-II tail. We identify the mechanism which underlies this marked difference in outcome. The GP(61-80) epitope is highly susceptible to cleavage by cathepsin D, an aspartic endopeptidase found almost exclusively in lysosomes. We show, using mass spectrometry, that the GP(61-80) peptide is cleaved between residues F(74) and K(75) and that this destroys its ability to stimulate virus-specific CD4(+) T cells. Thus, the immunological result of lysosomal targeting varies, depending upon the primary sequence of the encoded antigen. We analyze the effects of CD4(+) T-cell priming on the virus-specific antibody and CD8(+) T-cell responses which are mounted after virus infection and show that neither response appears to be accelerated or enhanced. Finally, we evaluate the protective benefits of CD4(+) T-cell vaccination in the LCMV model system; in contrast to DNA vaccine-induced CD8(+) T cells, which can confer solid protection against LCMV challenge, DNA vaccine-mediated priming of CD4(+) T cells does not appear to enhance the vaccinee's ability to combat viral challenge.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Frequency of epitope-specific naive CD4(+) T cells correlates with immunodominance in the human memory repertoire

The frequency of epitope-specific naive CD4(+) T cells in humans has not been extensively examined. In this study, a systematic approach was used to examine the frequency of CD4(+) T cells that recognize the protective Ag of Bacillus anthracis in both anthrax vaccine-adsorbed vaccinees and nonvaccinees with HLA-DRB1*01:01 haplotypes. Three epitopes were identified that had distinct degrees of immunodominance in subjects that had received the vaccine. Average naive precursor frequencies of T cells specific for these different epitopes in the human repertoire ranged from 0.2 to 10 per million naive CD4(+) T cells, which is comparable to precursor frequencies observed in the murine repertoire. Frequencies of protective Ag-specific T cells were two orders of magnitude higher in immunized subjects than in nonvaccinees. The frequencies of epitope-specific memory CD4(+) T cells in vaccinees were directly correlated with the frequencies of precursors in the naive repertoire. At the level of TCR usage, at least one preferred Vβ in the naive repertoire was present in the memory repertoire. These findings implicate naive frequencies as a crucial factor in shaping the epitope specificity of memory CD4(+) T cell responses.     

Cytotoxic CD4+ T cell responses to EBV contrast with CD8 responses in breadth of lytic cycle antigen choice and in lytic cycle recognition

EBV, a B lymphotropic herpesvirus, encodes two immediate early (IE)-, >30 early (E)-, and >30 late (L)-phase proteins during its replication (lytic) cycle. Despite this, lytic Ag-induced CD8 responses are strongly skewed toward IE and a few E proteins only, all expressed before HLA I presentation is blocked in lytically infected cells. For comparison, we examined CD4(+) T cell responses to eight IE, E, or L proteins, screening 14 virus-immune donors to overlapping peptide pools in IFN-γ ELISPOT assays, and established CD4(+) T cell clones against 12 defined epitopes for target-recognition assays. We found that the lytic Ag-specific CD4(+) T cell response differs radically from its CD8 counterpart in that it is widely distributed across IE, E, and L Ag targets, often with multiple reactivities detectable per donor and with IE, E, or L epitope responses being numerically dominant, and that all CD4(+) T cell clones, whether IE, E, or L epitope-specific, show strong recognition of EBV-transformed B cell lines, despite the lines containing only a small fraction of lytically infected cells. Efficient recognition occurs because lytic Ags are released into the culture and are acquired and processed by neighboring latently infected cells. These findings suggested that lytic Ag-specific CD4 responses are driven by a different route of Ag display than drives CD8 responses and that such CD4 effectors could be therapeutically useful against EBV-driven lymphoproliferative disease lesions, which contain similarly small fractions of EBV-transformed cells entering the lytic cycle.     

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.