Using Modified Bacterial Toxins To Deliver Vaccine Antigens

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1 Using Modified Bacterial Toxins To Deliver Vaccine Antigens Researchers are using toxins to deliver epitopes in candidate vaccines that specifically stimulate protective cytotoxic T lymphocyte responses Christine A. Shaw and Michael N. Starnbach Christine A. Shaw is a graduate student and Michael N. Starnbach is an Associate Professor in the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Mass. he intracellular niche of viruses and T other microbes can protect them against many elements of the immune response. Once inside host cells, these organisms are hidden from effectors of humoral immunity, and therefore T cell responses are often necessary for recognition and clearance of the pathogen. Microbial proteins in the cytosol of host cells activate CD8 cytotoxic T lymphocytes (CTL). Once activated, CTL lyse infected cells and secrete cytokines that stimulate other immune cells at the site of infection. Because CTL are key components in protecting against intracellular microbes, researchers are interested in developing safe and effective vaccines that specifically stimulate protective CTL. The challenge in designing vaccines that stimulate CTL is to deliver antigens not only into the bloodstream of the recipient, but also into the cytosol of host cells. Our lab and others have taken a cue from the microbial world, harnessing the inherent ability of bacterial toxins to enter mammalian cells and using modified versions of these toxins as vaccines. Presentation of MHC Class I Epitopes to CTL In the cytoplasm of mammalian host cells, the multicatalytic proteasome and other proteases are constantly degrading proteins into peptides. These peptides are transported into the endoplasmic reticulum (ER) by the heterodimeric transporter associated with antigen processing (TAP) complex. Once in the ER lumen, short peptides of 8 10 amino acids bind nascent major histocompatibility complex (MHC) class I (MHC-I) molecules, and the resulting complexes are transported via the secretory pathway to the cell surface. These complexes are surveyed by circulating CTL. In uninfected cells, the peptides presented by the MHC-I molecules are derived from normal cellular self proteins and thus trigger no response by circulating CTL. However, if there is foreign protein present in the cytoplasm of host cells, it too will be degraded and presented by MHC-I molecules. Then, T cell receptors (TCRs) present on circulating CTL will recognize these foreign peptide- MHC-I complexes. Following antigen recognition, these CTL become activated, proliferate, and differentiate into effector and memory populations. Effector cells not only lyse infected target cells, thereby depriving the organism of its replicative niche, but also secrete cytokines such as interferon- that aid in pathogen clearance by recruiting and activating other immune cells. The cells that differentiate into memory CTL remain present in the host long after clearance of the pathogen. Memory cells expand more quickly upon reexposure to that pathogen, allowing more efficient clearance. Because CTL immunity is generated predominantly to proteins present in the cytosol of host cells, vaccines designed to stimulate this T cell population must be engineered for cytosolic delivery of pathogen-derived proteins. We have been pursuing the use of modified bacterial toxins as carriers of heterologous antigens. The inherent ability of these toxins to bind to host cells and translocate into the cytosol allows for cytosolic processing of attached proteins, stimulating CTL specific for the pathogen from which the protein is derived. 384 Y ASM News / Volume 69, Number 8, 2003

2 Anthrax Toxin Can Deliver CTL Antigens into Host Cells The work in our lab using anthrax toxin (AT) from Bacillus anthracis as a vaccine delivery strategy has resulted from a productive collaboration with a departmental colleague, R. John Collier, whose research career has focused on the biochemistry of bacterial toxins. AT consists of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). Pair-wise combinations of PA and either LF or EF generate functional toxin molecules. As the first step in cellular entry, PA binds to surface-expressed anthrax toxin receptor (ATR) and is cleaved by a furin-like protease to generate an activated form of PA (PA 63 ). PA 63 oligomerizes on the cell surface and then can bind to LF or EF. The toxin complex then enters the cell via receptor-mediated endocytosis. When the endosome is acidified, a heptameric PA pore facilitates translocation of the catalytic LF or EF molecules into the cytosol. Work by both John Collier and Steve Leppla at the National Institutes of Health, Bethesda, Md., has shown that a protein fragment containing the N-terminal 255 amino acids of LF (LFn) retains PA binding and translocation activities, but is entirely devoid of the catalytic LF activity. We are focusing on the ability of PA to deliver into the host cell recombinant proteins consisting of LFn fused to CTL epitopes from a variety of bacterial and viral pathogens. Immunodominant CTL epitopes from a variety of proteins, including ovalbumin (OVA), listeriolysin O (LLO) from Listeria monocytogenes, nucleoprotein (NP) from lymphocytic choriomeningitis virus (LCMV), as well as several antigens from HIV, were genetically fused to LFn and expressed as recombinant fusion proteins in Escherichia coli. When added to target cells along with PA, the LFn-epitope fusion proteins enter the host cell cytosol, where the proteins are processed and presented by MHC-I molecules. Cell-surface display of these peptide-mhc-i complexes sensitizes cells for in vitro lysis by epitope-specific CTL. Not only do LFn-epitope fusions sensitize cells for CTL lysis in vitro, but they also stimulate CTL in vivo. For example, NP specific CTL (subscripts refer to the amino acid numbers of an antigenic epitope derived from that protein) can be cultured from the spleens of mice after a single intraperitoneal injection of picomolar quantities of the LFn-NP fusion protein and PA. Importantly, epitope-specific CTL can be cultured from mouse spleens up to one year after the animals were immunized, suggesting the presence of long-lasting memory CTL. The CTL that are primed in mice following immunization with AT-epitope fusions protect the animals following a subsequent bacterial or viral challenge. For example, mice immunized with LFn-LLO plus PA contain, on average, 20- to 30-fold fewer bacteria in their spleens and livers two days after an L. monocytogenes challenge, compared to control mice. Similarly, most mice vaccinated with LFn- NP plus PA had serum viral titers after LCMV challenge that were below the level of detection within 14 days, whereas unimmunized mice had titers of between PFU/ml of serum. These results have encouraged us to continue pursuing toxin fusions as an approach for delivering antigens. Several additional studies reveal the versatility of LFn plus PA as an intracellular delivery tool. For instance, injecting a mouse with a single fusion containing tandem epitopes from two different organisms (LLO from L. monocytogenes and NP from LCMV) stimulates sets of CTL that are specific for each of these pathogens. Multiple epitopes from one pathogen can also be fused to a single LFn molecule, by including either several minimal epitopes or one longer protein sequence. Stimulating CTL against several epitopes in a single fusion may provide an additive protective effect. This approach may be needed when applied to outbred human populations with multiple MHC-I alleles that may bind different peptides within any given protein. With the incorporation of multiple epitopes it becomes more likely that a peptide or peptides within the fusion protein will bind more of the MHC-I alleles present in the human population. Using this strategy a larger proportion of the population can be protected. We have demonstrated this concept by immunizing mice with a single LFn fusion containing two tandem NP epitopes presented by two different murine MHC-I alleles. This fusion protein stimulates NP-specific CTL in different strains of mice, where each of the strains expresses MHC-I molecules that can present only Volume 69, Number 8, 2003 / ASM News Y 385

3 one of the two epitopes. LFn can also mediate the cytosolic delivery of longer peptides or even whole proteins, not only minimal, epitopelength peptides. This was originally shown by fusing LFn to the catalytic domains of other toxins, such as diphtheria toxin and shiga toxin (ST). As a strategy for vaccination, AT fusions have been used to deliver a 287-amino-acid fragment of LCMV NP, as well as HIV gp120 and p24. In all three cases, the fusion protein targets cells for lysis by epitope-specific CTL. Not only do longer fusion proteins have the potential to deliver multiple epitopes, they also eliminate the need to identify precise peptides recognized by the responding CTL. Several Toxins of the AB Type Can Deliver Antigens A number of other bacterial toxins of the AB class, including Bordetella pertussis adenylate cyclase toxin (ACT), pertussis toxin (PT), Pseudomonas exotoxin A (PE), and ST translocate their toxic catalytic domains into host cytosol. As we have done with AT, others have usurped the ability of these toxins to translocate, using them to deliver epitopes into host cells where they can be processed, presented, and used to stimulate CTL. However, the toxinmediated epitope delivery systems developed to date vary significantly in their design and in the mechanism by which their antigenic cargo is delivered to the host MHC-I processing and presentation pathway. To ensure safety, a toxin s enzymatic activity must be inactivated before it is used to deliver epitopes into host cells. Methods of inactivating these molecules to produce safe vaccines include deleting the catalytic portion, removing the entire active domain, and mutating key residues to block or reduce enzymatic activity. The toxins used in these immunization strategies differ in how amenable they are to inserted heterologous sequence. For instance, when an epitope is fused to the amino or carboxyl terminus of the toxin molecule (such as those fusions generated to AT, PE, and ST), relatively large numbers of foreign amino acids are accommodated. However, there may be a size restriction when the heterologous sequence is inserted into permissive sites in the middle of the toxin molecule (such as in the fusions generated to ACT and PT), because the foreign sequence may interfere with natural toxin folding or action. Using a toxin-mediated delivery system that accommodates longer peptide sequences or entire antigenic proteins, instead of minimal epitopes, may prove advantageous. However, as toxin fusions become larger, increases in immunogenicity due to the inclusion of multiple epitopes may be offset by a decrease in translocation efficiency. This balance has been demonstrated using ACT fused to one, two, three, or four tandem copies of an NP CTL epitope. Although two copies of the epitope stimulate a greater immune response than does one copy, the level of CTL stimulation decreases when a third or fourth epitope is added. This decrease in activity is most likely due to reduced translocation efficiency when the number of epitopes increases. Toxin Fusion Systems Differ in Delivery, Processing, and Presentation of Attached Antigen The type of cell targeted by toxin fusions depends on where toxin receptors are distributed. For example, AT utilizes the ubiquitously expressed anthrax toxin receptor (ATR), whereas other toxins such as ACT and ST enter via receptors expressed on particular subsets of host cells, mainly limited to antigen-presenting cells (APCs) such as macrophages and dendritic cells. This cell type restriction may be useful in naturally targeting toxin fusions to those cells most proficient in antigen presentation. Although all of these toxin-mediated delivery systems effectively stimulate CTL, they differ in whether they require all or only some of the components of the classical MHC-I processing pathway. The required components of the endogenous pathway can be deduced in vitro using drugs that inhibit different MHC-I processing and presentation steps or by using cells with mutations in genes encoding these processes. For instance, presentation of class I MHC-restricted epitopes is expected to be reduced when cells are treated with a proteasome inhibitor such as lactacystin, or a secretory pathway inhibitor such as brefeldin A. Epitope presentation should also be reduced in TAP-deficient cells in which peptide transport into the ER is blocked. The results of these types of experiments with 386 Y ASM News / Volume 69, Number 8, 2003

4 FIGURE 1 Model for anthrax toxin-mediated delivery of epitopes to stimulate cytotoxic T cells. (a) Toxin binding. Protective antigen (PA) binds to its cellular receptor, anthrax toxin receptor (ATR), expressed on host cells. Proteolytic cleavage of PA generates PA 63.PA 63 then oligomerizes and is able to bind a recombinant fusion protein containing the PA-binding domain of lethal factor (LFn) and a cytotoxic T-cell (CTL) epitope. (b) Cytoplasmic delivery. After LFn fusion protein binding, the entire complex is endocytosed via receptor-mediated endocytosis. Following endosome acidification, a heptameric PA pore mediates translocation of the LFn-epitope fusion protein into the host cytoplasm. (c) Epitope processing and presentation. Once in the cytosol, the fusion protein is processed by the proteasome into peptides. The peptides are then transported into the endoplasmic reticulum (ER) by the antigen-processing (TAP) complex, where they bind nascent MHC class I molecules (MHC-I). The resulting MHC-I:peptide complexes are transported to the cell surface via the secretory pathway. (d) CTL activation. Antigen-presenting cells (APC) that display a peptide epitope can be recognized by epitope-specific T cell receptors (TCR) on circulating CTL. This results in CTL activation and differentiation into memory and effector populations. Effector CTL lyse APC and secrete cytokines that activate other components of the immune response. Memory CTL remain in the host for extended periods of time and rapidly proliferate to provide effector functions following subsequent exposure to the antigen. Volume 69, Number 8, 2003 / ASM News Y 387

5 AT, ACT, and ST suggest that epitopes fused to these toxins are delivered to the cytosol, processed, and presented by the class I pathway. Epitopes fused to two other AB toxins appear to be presented by MHC-I molecules without requiring proteasome degradation, TAP transport, and secretory traffic. In these cases, processing and MHC-I presentation of toxin fusions seem to involve a nonconventional pathway. Presentation of PE-fused CTL epitopes, for instance, is independent of all three of these classical processing steps. This finding suggests that CTL epitopes are released by endosomal proteases prior to translocation, perhaps followed by peptide loading in endocytic compartments containing recycling MHC-I molecules. On the other hand, MHC-I presentation of PT-fused antigen is dependent on secretory traffic, but not on proteasome degradation or peptide transport into the ER. PT follows a retrograde transport pathway through the endosome to the Golgi apparatus and ER before translocation into the cytosol. Therefore, peptides fused to PT may be released by ER-resident proteases and subsequently bind nascent MHC-I molecules without ever entering the cytoplasm. In these two cases we do not understand why the fused CTL epitopes are not processed in a classical manner. Perhaps the toxin molecules do not actually reach the cytosol. Alternatively, the epitope may be efficiently clipped off before the toxin has a chance to translocate. Both classically and nonclassically processed antigens are capable of stimulating CTL. However, proteolysis of a fusion in the endosome or ER may generate a different CTL epitope profile than that generated by the proteasome during a natural intracellular infection with the epitopecontaining pathogen. Therefore, CTL stimulated by a nonclassically processed toxin-fusion may not recognize antigen presented by MHC-I molecules during a subsequent infection. Using Toxin Fusions To Vaccinate Humans Although all these toxin systems sensitize target cells for lysis by CTL in vitro, the ability to stimulate CTL in animals so far is limited to AT, PT, ST, and ACT. When toxins such as these are used in vivo as tools for immunization, it is important to determine whether the recipients have preexisting antibodies that are specific to the toxin being evaluated. For example, toxins themselves are often used as vaccines. The neutralizing antibodies resulting from such a vaccination inhibit toxin binding and therefore protect the immunized individual against the effects of bacterial infection. However, these antibodies might also render toxin-fusion immunization ineffective. Even in the absence of preexisting immunity, the toxin-fusion dose required to stimulate CTL may also stimulate a toxin-specific, neutralizing antibody response, limiting the efficiency of repeated immunizations with the same vector. Although any one toxin-fusion delivery strategy may face these problems, alternatives will likely be found among the other toxin-based systems under development. We have begun to address these types of issues for the AT-mediated epitope delivery system. Currently, most humans have not been vaccinated against B. anthracis, and therefore do not have high levels of neutralizing antibody specific for this toxin. Additionally, when LFn fusions and PA are administered to mice at the low dose required to stimulate CTL in vivo, there is a negligible antibody response to the toxin. Therefore in this case, prior immunization with an LFn-epitope fusion plus PA does not interfere with the priming of epitope-specific CTL during a subsequent LFn fusion-based immunization. Stimulating protective CTL specific for infectious disease agents is only one possible application of the toxin fusion proteins. If the fusion protein instead carries a tumor antigen, it can stimulate tumor-specific CTL in a mammalian host. Tumor-specific CTL can participate in cancer immunity by recognizing and lysing cancer cells. In one promising example of this application, mice were immunized with an ACT- OVA fusion protein. The OVA-specific CTL that were stimulated in the mice protected them against what would have been a lethal graft of OVA-transfected melanoma cells. For many of the pathogens for which there is no effective vaccine, it will be necessary to use methods that stimulate multiple arms of the adaptive immune response including CTL, helper T cells, and antibodies. Because of the technical challenges of stimulating CTL, researchers are likely to continue developing new methods to introduce antigens into the cytosol of cells. Anticipated efforts include the develop- 388 Y ASM News / Volume 69, Number 8, 2003

6 ment of new toxin fusion systems as well as recombinant bacterial and viral delivery vectors and DNA vaccines. The AT system we have developed is but one attractive approach to delivering antigens to the host cell cytosol as a means for stimulating CTL responses. LFn fusions can be easily produced using expression vectors in E. coli, appear safe for in vivo use, are processed by the endogenous MHC class I pathway, and stimulate protective, epitope-specific CTL in mice. We will continue to modify this system to better protect the host. For instance, incorporating additional CTL or helper T-cell epitopes may stimulate more CTL and therefore offer greater immune protection. Targeting toxin fusions to professional APCs may also stimulate CTL more effectively. SUGGESTED READING Ballard, J. D., R. J. Collier, and M. N. Starnbach Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. Proc. Natl. Acad. Sci. USA 93: Carbonetti, N. H., T. J. Irish, C. H. Chen, C. B. O Connell, G. A. Hadley, U. McNamara, R. G. Tuskan, and G. K. Lewis Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway. Infect. Immun. 67: Dadaglio, G., Z. Moukrim, R. Lo-Man, V. Sheshko, P. Sebo, and C. Leclerc Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis. Infect. Immun. 68: Doling, A. M., J. D. Ballard, H. Shen, K. M. Krishna, R. Ahmed, R. J. Collier, and M. N. Starnbach Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity. Infect. Immun. 67: Fayolle, C., D. Ladant, G. Karimova, A. Ullmann, and C. Leclerc Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J. Immunol. 162: Goletz, T. J., K. R. Klimpel, N. Arora, S. H. Leppla, J. M. Keith, and J. A. Berzofsky Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein. Proc. Natl. Acad. Sci. USA 94: Haicheur, N., E. Bismuth, S. Bosset, O. Adotevi, G. Warnier, V. Lacabanne, A. Regnault, C. Desaymard, S. Amigorena, P. Ricciardi-Castagnoli, B. Goud, W. H. Fridman, L. Johannes, and E. Tartour The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens. J. Immunol. 165: Lippolis, J. D., K. S. Denis-Mize, L. H. Brinckerhoff, C. L. Slingluff, D. R. Galloway, and V. H. Engelhard Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways. Cell. Immunol. 203: Lu, Y., R. Friedman, N. Kushner, A. Doling, L. Thomas, N. Touzjian, M. Starnbach, and J. Lieberman Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity. Proc. Natl. Acad. Sci. USA 97: Sebo, P., C. Fayolle, O. D Andria, D. Ladant, C. Leclerc, and A. Ullmann Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8 cytotoxic T cells. Infect. Immun. 63: Ulmer, J. B., J. J. Donnelly, and M. A. Liu Presentation of an exogenous antigen by major histocompatibility complex class I molecules. Eur. J. Immunol. 24: Volume 69, Number 8, 2003 / ASM News Y 389