Evaluation of DNA vaccination with recombinant adenoviruses using bioluminescence imaging of antigen expression: impact of application routes and delivery with dendritic cells

Recombinant DNA vaccines are able to induce strong CD8+ T cell mediated immunity and have become increasingly attractive for the prevention and treatment of infectious diseases and cancer. Dendritic cells (DC), which critically control cellular immune responses, have been transduced with antigen ex vivo and used as 'nature's adjuvant' to enhance vaccine efficacy. The impact of the application route on the in vivo distribution of antigen and the stimulation of CD8+ T cells have been subjects of considerable debate. Here we report the construction of vectors expressing a fusion protein between EGFP, the H2-K(b)-binding peptide OVA(aa257-264) and green click beetle luciferase as a model antigen which allows for simultaneous quantitative assessment of antigen expression using fluorescence and bioluminescence imaging in correlation with CD8+ T cell stimulation in vivo. We applied this construct to evaluate DNA vaccination with recombinant adenoviral vectors, assess the impact of using cultured DC for vaccine delivery and investigate different application routes. Antigen expression was non-invasively followed in vivo by visualizing bioluminescence with an ultrasensitive CCD camera. CD8+ T cell stimulation was detected with H2-K(b)-OVA(aa257-264) tetramers. We found that intravenous injection of adenovirus-transduced DC stimulated the strongest OVA(aa257-264)-specific cytotoxic T-lymphocyte (CTL) responses although it delivered two orders of magnitude less antigen in vivo when compared to direct injection of recombinant adenovirus. We believe that our experimental approach has the potential to facilitate translational development of improved genetic immunization strategies targeting DC directly in vivo.     

A persistent virus vector confers superior anti-tumor immunity, compared with a non-persistent vector

Active vaccination strategies using viral vectors often give disappointing protection from tumor development, and usually require multiple immunizations. These approaches normally use viruses that cause acute infections, as they provoke potent CD8 T cell responses. Persistent virus vectors have not been used in this setting due to the perception that exhaustion of the T cell response occurs and would lead to poor anti-tumor protection. However, such exhaustion generally only occurs in high-load virus infections, whereas T cell function is intact in lower-load persistent infections. In fact, CD8 T cell responses in these infections, which are adapted for long-term immune surveillance, have properties that may make them more desirable for long-term anti-tumor immunity. In this report, we show that a persistent gammaherpesvirus vector provides superior protection against melanoma, relative to a non-persistent mutant of the same virus. These data suggest that vaccine vectors derived from persistent viruses may perform better than those from acute viruses at mediating anti-tumor protection.     

Engineered lentivector targeting of dendritic cells for in vivo immunization

We report a method of inducing antigen production in dendritic cells by in vivo targeting with lentiviral vectors that specifically bind to the dendritic cell-surface protein DC-SIGN. To target dendritic cells, we enveloped the lentivector with a viral glycoprotein from Sindbis virus engineered to be DC-SIGN-specific. In vitro, this lentivector specifically transduced dendritic cells and induced dendritic cell maturation. A high frequency (up to 12%) of ovalbumin (OVA)-specific CD8(+) T cells and a significant antibody response were observed 2 weeks after injection of a targeted lentiviral vector encoding an OVA transgene into naive mice. This approach also protected against the growth of OVA-expressing E.G7 tumors and induced regression of established tumors. Thus, lentiviral vectors targeting dendritic cells provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens.     

Efficient priming of CD4+ and CD8+ T cells by DNA vaccination depends on appropriate targeting of sufficient levels of immunologically relevant antigen to appropriate processing pathways

The initial cellular events and interactions that occur following DNA immunization are likely to be key to determining the character and magnitude of the resulting immune response, and as such, a better understanding of these events could ultimately lead to the design of more effective pathogen-appropriate DNA vaccines. Therefore, we have used a variety of sensitive cell-based techniques to study the induction of adaptive immunity in vivo. We examined the efficacy of induction of Ag-specific CD4(+) and CD8(+) T cell responses in vivo by the adoptive transfer of fluorescently labeled Ag-specific TCR transgenic T cells and have demonstrated how such approaches can be used to study the effect of simple DNA construct manipulations on immunological priming. OVA-specific CD8(+) and CD4(+) T cells were activated and divided in vivo following immunization with DNA constructs that targeted OVA expression to different subcellular locations; however, the kinetics and degree of cell proliferation were dependent on the cellular location of the expressed protein. DNA vectors encoding cell-associated OVA resulted in greater CD8(+) T cell division compared with other forms of OVA. In contrast, soluble secreted OVA targeted to the classical secretory pathway enhanced division of CD4(+) T cells. Furthermore, the inclusion of mammalian introns to enhance protein expression increased the ability of poorly immunogenic forms of Ag to activate naive T cells, indicating that not only the location, but also the amount of Ag expression, is important for efficient T cell priming following DNA injection.     

The adjuvant effects of Mycobacterium tuberculosis heat shock protein 70 result from the rapid and prolonged activation of antigen-specific CD8+ T cells in vivo

Heat shock protein 70 (hsp70) is a potent adjuvant that links innate and adaptive immune responses. To study how hsp70 activates naive CD8(+) T cells in vivo, we tracked Ag-specific CD8(+) T cells in mice immunized with a fusion protein containing chicken OVA linked to hsp70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). On a molar basis, OVA.TBhsp70 was several hundred times more effective than OVA peptide plus CFA in eliciting specific CD8(+) T cell responses. Immunization with OVA.TBhsp70 activated >90% of detectable OVA-specific CD8(+) T cells within 3 days and led to the persistence of cytotoxic effectors for at least 17 days. These studies demonstrate that the potent adjuvant effect of M. tuberculosis hsp70 results from the relatively complete, rapid, and durable activation of Ag-specific CD8(+) T cells.     

Different functional capacities of latent and lytic antigen-specific CD8 T cells in murine gammaherpesvirus infection

Gammaherpesviruses can persist in the host in the face of an aggressive immune response. T cells recognize Ags expressed in both the productive and latent phases of the virus life cycle, however little is known about their relative roles in the long-term control of the infection. In this study we used the murine gammaherpesvirus 68 model system to investigate the relative properties of CD8 T cells recognizing lytic and latent viral Ags. We report that the CD8 T cell response to lytic phase epitopes is maximal in the lungs of infected mice at approximately 10 days postinfection, and is of progressively lesser magnitude in the mediastinal lymph nodes and spleen. In contrast, the CD8 T cell response to the latent M2 protein is maximal at approximately 19 days postinfection and is most prominent in the spleen, then progressively less in the mediastinal lymph node and the lung. Latent and lytic Ag-specific CD8 T cells had markedly different cell surface phenotypes during chronic infection, with latent Ag-specific cells being predominantly CD62L(high) or CD43 (1B11)(high). Lytic Ag-specific T cells had significantly lower expression of these markers. Importantly, latent but not lytic Ag-specific T cells could kill target cells rapidly in vivo during the chronic infection. These two different sets of CD8 T cells also responded differentially to IL-7, a cytokine involved in T cell homeostasis and the maintenance of T cell memory. These data have important implications for our understanding of immunological control during chronic gammaherpesvirus infections.     

CD8+ T cell dysfunction and increase in murine gammaherpesvirus latent viral burden in the absence of 4-1BB ligand

Studies of costimulatory receptors belonging to the TNFR family have revealed their diverse roles in affecting different stages of the T cell response. The 4-1BB ligand (4-1BBL)/4-1BB pathway has emerged as a receptor-ligand pair that impacts not the initial priming, but later phases of the T cell response, such as sustaining clonal expansion and survival, maintaining memory CD8(+) T cells, and supporting secondary expansion upon Ag challenge. Although the role of this costimulatory pathway in CD8(+) T cell responses to acute viral infections has been well-studied, its role in controlling chronic viral infections in vivo is not known to date. Using the murine gammaherpesvirus-68 (MHV-68) model, we show that 4-1BBL-deficient mice lack control of MHV-68 during latency and show significantly increased latent viral loads. In contrast to acute influenza infection, the numbers of MHV-68-specific memory CD8(+) T cells were maintained during latency. However, the virus-specific CD8(+) T cells showed defects in function, including decreased cytolytic function and impaired secondary expansion. Thus, 4-1BBL deficiency significantly affects the function, but not the number, of virus-specific CD8(+) T cells during gammaherpesvirus latency, and its absence results in an increased viral burden. Our study suggests that the 4-1BB costimulatory pathway plays an important role in controlling chronic viral infections.     

Patients with discordant responses to antiretroviral therapy have impaired killing of HIV-infected T cells

In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication. Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.     

An optimized CD8+ T-cell response controls productive and latent gammaherpesvirus infection

Strategies to prime CD8(+) T cells against Murine gammaherpesvirus 68 (gammaHV68; MHV68) latency have, to date, resulted in only limited effects. While early forms of latency (<21 days) were significantly reduced, effects were not seen at later times, indicating loss of control by the primed CD8(+) T cells. In the present study, we evaluated CD8(+) T cells in an optimized system, consisting of OTI T-cell-receptor (TCR) transgenic mice, which generate clonal CD8(+) T cells specific for K(b)-SIINFEKL of OVA, and a recombinant gammaHV68 that expresses OVA (gammaHV68.OVA). Our aim was to test whether this optimized system would result in more effective control not only of acute infection but also of later forms of latent infection than was seen with previous strategies. First, we show that OTI CD8(+) T cells effectively controlled acute replication of gammaHV68.OVA in liver, lung, and spleen at 8 and 16 days after infection of OTI/RAG mice, which lack expression of B and CD4(+) T cells. However, we found that, despite eliminating detectable acute replication, the OTI CD8(+) T cells did not prevent the establishment of latency in the OTI/RAG mice. We next evaluated the effectiveness of OTI T cells in OTI/B6 animals, which express B cells--a major site of latency in wild-type mice--and CD4(+) T cells. In OTI/B6 mice OTI CD8(+) T cells not only reduced the frequency of cells that reactivate from latency and the frequency of cells bearing the viral genome at 16 days after infection (similar to what has been reported before) but also were effective at reducing latency at 42 days after infection. Together, these data show that CD8(+) T cells are sufficient, in the absence of B cells and CD4(+) T cells, for effective control of acute replication. The data also demonstrate for the first time that a strong CD8(+) T-cell response can limit long-term latent infection.     

Adaptation of CD8 T Cell Responses to Changing HIV-1 Sequences in a Cohort of HIV-1 Infected Individuals Not Selected for a Certain HLA Allele

HIV evades CD8 T cell mediated pressure by viral escape mutations in targeted CD8 T cell epitopes. A viral escape mutation can lead to a decline of the respective CD8 T cell response. Our question was what happened after the decline of a CD8 T cell response and - in the case of viral escape – if a new CD8 T cell response towards the mutated antigen could be generated in a population not selected for certain HLA alleles. We studied 19 antiretroviral-naïve HIV-1 infected individuals with different disease courses longitudinally. A median number of 12 (range 2-24) CD8 T cell responses towards Gag and Nef were detected per study subject. A total of 30 declining CD8 T cell responses were studied in detail and viral sequence analyses showed amino acid changes in 25 (83%) of these. Peptide titration assays and definition of optimal CD8 T cell epitopes revealed 12 viral escape mutations with one de-novo response (8%). The de-novo response, however, showed less effector functions than the original CD8 T cell response. In addition we identified 4 shifts in immunodominance. For one further shift in immunodominance, the mutations occurred outside the optimal epitope and might represent processing changes. Interestingly, four adaptations to the virus (the de-novo response and 3 shifts in immunodominance) occurred in the group of chronically infected progressors. None of the subjects with adaptation to the changing virus carried the HLA alleles B57, B*58:01 or B27. Our results show that CD8 T cell responses adapt to the mutations of HIV. However it was limited to only 20% (5 out of 25) of the epitopes with viral sequence changes in a cohort not expressing protective HLA alleles.     

An optimized CD4 T-cell response can control productive and latent gammaherpesvirus infection

CD4 T cells are important for control of infection with murine gammaherpesvirus 68 (gamma HV68), but it is not known whether CD4 T cells function via provision of help to other lymphocyte subsets, such as B cells and CD8 T cells, or have an independent antiviral function. Moreover, under conditions of natural infection, the CD4 T-cell response is not sufficient to eliminate infection. To determine the functional capacities of CD4 T cells under optimal or near-optimal conditions and to determine whether CD4 T cells can control gamma HV68 infection in the absence of CD8 T cells or B cells, we studied the effect of ovalbumin (OVA)-specific CD4 T cells on infection with a recombinant gamma HV68 that expresses OVA. OVA-specific CD4 T cells limited acute gamma HV68 replication and prolonged the life of infected T-cell receptor-transgenic RAG (DO.11.10/RAG) mice, demonstrating CD4 T-cell antiviral activity, independent of CD8 T cells and B cells. Despite CD4 T-cell-mediated control of acute infection, latent infection was established in DO.11.10/RAG mice. However, OVA-specific CD4 T cells reduced the frequency of latently infected cells both early (16 days postinfection) and late (42 days postinfection) after infection of mice containing CD8 T cells and B cells (DO.11.10 mice). These results show that OVA-specific CD4 T cells have B-cell and CD8 T-cell-independent antiviral functions in the control of acute infection and can, in the absence of preexisting CD8 T-cell or B-cell immunity, inhibit the establishment of gammaherpesvirus latency.     

Modified vaccinia virus Ankara-based vaccine vectors induce apoptosis in dendritic cells draining from the skin via both the extrinsic and intrinsic caspase pathways, preventing efficient antigen presentation

Dendritic cells (DC) are potent antigen-presenting cells and central to the induction of immune responses following infection or vaccination. The collection of DC migrating from peripheral tissues by cannulation of the afferent lymphatic vessels provides DC which can be used directly ex vivo without extensive in vitro manipulations. We have previously used bovine migrating DC to show that recombinant human adenovirus 5 vectors efficiently transduce afferent lymph migrating DEC-205(+) CD11c(+) CD8(-) DC (ALDC). We have also shown that recombinant modified vaccinia virus Ankara (MVA) infects ALDC in vitro, causing downregulation of costimulatory molecules, apoptosis, and cell death. We now show that in the bovine system, modified vaccinia virus Ankara-induced apoptosis in DC draining from the skin occurs soon after virus binding via the caspase 8 pathway and is not associated with viral gene expression. We also show that after virus entry, the caspase 9 pathway cascade is initiated. The magnitude of T cell responses to mycobacterial antigen 85A (Ag85A) expressed by recombinant MVA-infected ALDC is increased by blocking caspase-induced apoptosis. Apoptotic bodies generated by recombinant MVA (rMVA)-Ag85A-infected ALDC and containing Ag85A were phagocytosed by noninfected migrating ALDC expressing SIRPα via actin-dependent phagocytosis, and these ALDC in turn presented antigen. However, the addition of fresh ALDC to MVA-infected cultures did not improve on the magnitude of the T cell responses; in contrast, these noninfected DC showed downregulation of major histocompatibility complex class II (MHC-II), CD40, CD80, and CD86. We also observed that MVA-infected ALDC promoted migration of DEC-205(+) SIRPα(+) CD21(+) DC as well as CD4(+) and CD8(+) T cells independently of caspase activation. These in vitro studies show that induction of apoptosis in DC by MVA vectors is detrimental to the subsequent induction of T cell responses.     

An IFN-gamma-IL-18 signaling loop accelerates memory CD8+ T cell proliferation

Rapid proliferation is one of the important features of memory CD8(+) T cells, ensuring rapid clearance of reinfection. Although several cytokines such as IL-15 and IL-7 regulate relatively slow homeostatic proliferation of memory T cells during the maintenance phase, it is unknown how memory T cells can proliferate more quickly than na?ve T cells upon antigen stimulation. To examine antigen-specific CD8(+) T cell proliferation in recall responses in vivo, we targeted a model antigen, ovalbumin(OVA), to DEC-205(+) dendritic cells (DCs) with a CD40 maturation stimulus. This led to the induction of functional memory CD8(+) T cells, which showed rapid proliferation and multiple cytokine production (IFN-gamma, IL-2, TNF-alpha) during the secondary challenge to DC-targeted antigen. Upon antigen-presentation, IL-18, an IFN-gamma-inducing factor, accumulated at the DC:T cell synapse. Surprisingly, IFN-gamma receptors were required to augment IL-18 production from DCs. Mice genetically deficient for IL-18 or IFN-gamma-receptor 1 also showed delayed expansion of memory CD8(+) T cells in vivo. These results indicate that a positive regulatory loop involving IFN-gamma and IL-18 signaling contributes to the accelerated memory CD8(+) T cell proliferation during a recall response to antigen presented by DCs.     

CD8+ CTL priming by exact peptide epitopes in incomplete Freund's adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8(+) T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA(257-264) peptide in IFA transiently activated CD8(+) effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA(323-339)) restored both secondary expansion and in vivo effector functions of CD8(+) T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8(+) T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8(+) T cell reactivity in vivo.     

Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity

Dendritic cell (DC)-based vaccines are being developed for treatment of patients with cancer, in part because DC are potent inducers of CD8(+) CTL. DC MHC class I:antigenic peptide complexes that are required for CTL elicitation are most often generated by incubating DC with peptides or by transfecting (or transducing) DC with cDNAs (or viral vectors) that encode protein Ags. The former approach is feasible when MHC class I Ags and relevant peptides are known. The latter approach may be hampered by inefficient DC transfection (transduction) and/or difficulties associated with preparation and use of viral vectors. Herein we demonstrate that a bacterial recombinant model tumor-associated Ag (OVA) that contains the HIV TAT protein transduction domain (PTD) was readily engineered and purified, efficiently transduced murine lymphocytes and DC, and was processed by proteasomes for MHC class I-restricted presentation to CTL. In addition, PTD-containing rOVA was processed and presented by DC to CD4 T cells as efficiently as native OVA or rOVA lacking the PTD. PTD-OVA-transduced DC induced CTL in vivo in a Th cell-independent fashion and vaccinated against OVA-expressing tumors. In contrast, rOVA lacking the PTD did not enter the DC MHC class I presentation pathway and DC treated with this protein did not prime OVA-specific CTL in vivo. Treatment of mice harboring clinically apparent OVA-expressing tumors with PTD-OVA-transduced DC resulted in tumor regression in some animals. This straightforward vaccination strategy may translate into DC-based treatments for patients with cancer and other serious diseases.     

Multiple dendritic cell populations activate CD4+ T cells after viral stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+) T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+) T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+) and CD8(+) T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+) and CD4(+) T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+) T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+) T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+) T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.     

Cell-associated ovalbumin is cross-presented much more efficiently than soluble ovalbumin in vivo

To better understand the antigenic requirements for cross-presentation, we compared the in vivo efficiency of presentation of cell-associated vs soluble OVA with the OT-I (CD8) and OT-II (CD4) TCR transgenic lines. Cross-presentation of cell-associated OVA was very efficient, requiring as little as 21 ng of OVA to activate OT-II cells and 100-fold less to activate OT-I cells. In contrast, soluble OVA was presented inefficiently, requiring at least 10,000 ng OVA for activation of either T cell subset. Thus, cell-associated OVA was presented 500-fold more efficiently than soluble OVA to CD4 T cells and 50,000-fold more efficiently to CD8 T cells. These data, which represent the first quantitative in vivo analysis of cross-presentation, show that cell-associated OVA is very efficiently presented via the class I pathway.     

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.     

Protective capacity of virus-specific T cell receptor-transduced CD8 T cells in vivo

The transfer of T cell receptor (TCR) genes by viral vectors represents a promising technique to generate antigen-specific T cells for adoptive immunotherapy. TCR-transduced T cells specific for infectious pathogens have been described, but their protective function in vivo has not yet been examined. Here, we demonstrate that CD8 T cells transduced with the P14 TCR specific for the gp33 epitope of lymphocytic choriomeningitis virus exhibit protective activities in both viral and bacterial infection models in mice.     

Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope

Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity.