article addendum
Human Vaccines 6:4, 292-296; April 2010; © 2010 Landes Bioscience
Towards a rational approach to vaccine development Claude Leclerc1,2 Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, Paris, France; 2INSERM, U883, Paris, France
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From Great Explorers to Louis Pasteur
Key words: vaccine, cancer immunotherapy, dendritic cells, cytotoxic T cells, tumor Submitted: 02/17/10 Accepted: 02/17/10 Previously published online: www.landesbioscience.com/journals/ vaccines/article/11878 Correspondence to: Claude Leclerc; Email:
[email protected]
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I was lucky enough to know very early on what I wanted to do with my life and how to achieve it. As a very young girl, I hunted through public libraries to find books on René Caillié, Roald Amundsen, Robert Scott and others. I was fascinated by these adventurers and secretly dreamed of becoming one of them. As I grew older, however, I realized that the era of world explorers was over. Science, on the other hand, offered almost unlimited new territories to discover. By the time I was 11 and started high school, I had already decided to study science. My parents and teachers thus had to abandon their preferred option of my learning Latin and Greek, which were considered mandatory at the time for a good education. Some years later, I also turned down the suggestion of doing mathematics, the subject of choice for the best students. Instead, I chose biology as the major topic for my Bachelor’s degree in Experimental Sciences. In 1969, I joined the University of Sciences, Paris. Here, my reading of the biographies of Marie Curie and Louis Pasteur and several essays by Jean Rostand, a French biologist and science writer, allowed me to put the final touches on my life plan. Although I knew almost nothing about the Pasteur Institute, I decided that after my Master’s degree, I would pursue a PhD at this Institute. I kept this a secret, however, since it seemed to be an unachievable dream. Having obtained my Master’s after four years of study at university, I wrote a letter to one of my professors, Prof. Chapeville, for advice on laboratories that may offer me
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a PhD. I received an answer a few days later, which, even now, I consider to be a clear sign of destiny. Prof. Chapeville suggested that I contact Louis Chedid at the Pasteur Institute. I remember my first visit to Louis Chedid’s lab in September 1973 very clearly. At that time, I was a shy, young woman, only 22 years old. I would have never believed that in 2010 I would be a Professor at this Institute, leading a team in the Department of Immunology. It was, and still is, an extraordinary adventure, although I have never forgotten my childhood passion for faraway lands, high mountains and deserts. But, that is another story… The Immunologist’s Dirty Little Secret In 1989, C. Janeway called adjuvants “the immunologist’s dirty little secret.” I then realized how wide the gap still was between vaccinology and immunology and the lack of understanding of what, at that time, was called inflammation, the old term for innate immunity. This “secret” was partially understood as early as 1973, when adjuvants started to be seen in a new light. Research into adjuvants is ongoing. The discovery of MDP, however, was a first step in the rational discovery of new adjuvants. In 2010, almost 40 years later, it is clear that the discovery of innate immunity and the identification of microorganism-derived molecules stimulating the various pathways of the innate immune system revolutionized the development of adjuvants. When I joined Louis Chedid’s group to start my PhD in 1973, his main objective
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Portrait of a leading vaccinologist
article addendum
was to identify the minimal structure able to replace mycobacteria in Freund’s complete adjuvant (FCA). At that time, the most common adjuvants used in the laboratory were FCA and incomplete adjuvant (FIA). FCA cannot be used for clinical or even veterinary immunization. Mineral oil has been used for certain veterinary vaccines or in clinical influenza trials, but the use of FIA for human vaccines has since been abandoned. Thus, in the 70s, the development of new adjuvants able to potentiate antibody production and cellular immunity, and ideally devoid of side effects, was a major goal. Soon after my arrival, our laboratory, in collaboration with Edgar Lederer, showed that N-acetylmuramyl L Alanyl D isoglutamine (known as muramyl dipeptide or MDP), a synthetic copy of part of the repetitive structure that constitutes most bacterial peptidoglycans, can replace whole mycobacteria in FCA.1 Over the following years, several hundreds of MDP derivatives were synthesized. We performed a large analysis of the relationship between structure and immunostimulant activity. Our results showed that one or several of the immunopharmacological properties, such as pyrogenic activity, of the original MDP molecule could be dissociated by small chemical modifications. At this time, cellular immunology was becoming a very exciting discipline, although considerable confusion remained. I had a particular interest in the mechanisms underlying the activity of these adjuvant molecules. However, the tools available to identify or purify immune cells or to analyze immune responses were still very limited. Despite this, we were able to identify macrophages, purified by their capacity to adhere to plastic dishes, as the major target of MDP.2 In particular, I demonstrated that low concentrations of MDP activated peritoneal exudate macrophages of mice. I then attempted to identify the MDP receptor at the surface of macrophages using monoclonal anti-MDP antibodies to block macrophages activation. To my surprise, the addition of these antibodies to cell cultures rendered the MDP conjugate highly effective in activating macrophages, at MDP concentrations a thousand times smaller than had previously been described.3 This synergistic effect was not observed with
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About Dr. Leclerc Dr. Claude Leclerc received her Master’s degrees in Biochemistry and Immunology in 1973 and 1974, followed by her PhD in Immunology in 1981 all from the University of Paris VII (Paris, France). Starting as Assistant Professor at the Pasteur Institut (Paris) in 1981, Claude Leclerc is today Professor and Head of the Immune Regulation and Vaccinology Unit in the Department of Immunology of the Pasteur Institut. She is also the Director of U883 INSERM and exerts various responsibilities in the administration of research in France. Claude Leclerc has worked in vaccinology for 35 years and has contributed to the development of synthetic adjuvants, synthetic peptide vaccines and several delivery systems. In particular, her laboratory has established the adenylate cyclase antigen delivery system targeting dendritic cells to elicit immune responses (CyaA). Based on this vector, she has recently developed therapeutic vaccines against cervical cancer as well as against melanoma, which will enter clinical trials in 2010. She has also developed a fully synthetic glycopeptidic cancer vaccine, in preclinical development. Her research has resulted in over 260 publications in renowned scientific journals including Nature Medicine, Nature Reviews Immunology, Immunity and The Journal of Experimental Medicine. She holds 24 patents, one of which enabled the creation of the biopharmaceutical company BT Pharma (Toulouse, France). As co-inventor of BT Pharma’s CyaA technology platform, Dr. Leclerc serves on the company’s Scientific Advisory Board. For her research in immunology, Dr. Leclerc has received numerous awards and honours, such as the Cancer Paris Distinction by the League against Cancer in 2004. She regularly organizes, chairs and speaks at national and international meetings in the fields of Immunology and Vaccinology. In addition to her research, Dr. Leclerc has been the Director of the General Immunology Course at the Pasteur Institute for many years, and is actively involved in postgraduate and undergraduate teaching.
a control monoclonal antibody of different specificity or with the monoclonal anti-MDP antibody F(ab)2 fragment. Therefore, in 1986, I suggested that MDP does not activate macrophages by binding to cell-surface receptors, but activates target cells by intracellular mechanisms.4 These findings were only confirmed 20 years later at the Pasteur Institute by Dana Philpott and Philippe Sansonetti, who demonstrated that Nod2 is a general sensor of peptidoglycan through intracellular
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recognition of muramyl dipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria.5 The discovery of MDP suppressive activity was my second major contribution to the field of adjuvants. I first observed that, for in vitro immunization performed with a low cell density, addition of MDP enhanced the response. By contrast, addition of MDP to cultures of high cell density induced a very marked suppression of the B-cell response to sheep erythrocytes,
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a widely used antigen at that time.6 Following these observations, I demonstrated that relatively high doses of MDP, injected before the antigen, also suppresses the antibody and cytotoxic T-cell responses of mice in vivo.7 Further investigation showed that the suppressive activity of MDP is mediated by the induction of suppressor T cells8 and inhibition of IL 2 production,9 and that MDP can prevent the development of an experimental model of auto-immune diabetes.10 The finding that such a small synthetic molecule may have both stimulant and suppressive properties was intriguing. The mechanisms underlying MDP suppressive activity, however, remain unknown. At this time, the study of suppressor T cells had become very unpopular, and continuing these investigations would be difficult, particularly given the increased pressure to publish once I became independent in 1987. Driven by his active pursuit of the clinical development of MDP derivatives, Louis Chedid left the Pasteur Institute in 1987 to join the University of South Florida in Tampa, Florida and to create a company. Given the limited feasibility of creating a biotechnology company in France at this time, his choice was certainly the right one. Nevertheless, MDP was never released onto the market, possibly due to the fact that MDP is effective in enhancing antibody responses but not for stimulating cytotoxic T cells. When Louis Chedid left France, I was strongly convinced that the future of vaccines relied on fundamental immunology. Since 1981, I had a permanent position as Assistant Professor at the Pasteur Institute, facilitating my decision to stay in the Institute. The General Director of the Institute, Prof. Raymond Dedonder, offered me the team leader position over the remaining members of Chedid’s laboratory. However, I was reluctant to take on such a big, heterogeneous group of people and to work in direct competition with Louis Chedid. It was only later that I fully realized the honor of this offer and the confidence that the Board of Pasteur Institute had placed in me. I needed to find a way of fulfilling my own scientific objectives, and a solution seemed far from simple. In the 1980s and 90s in France, it was highly unusual to become independent at 35 years
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old without “inheriting” a position following the retirement of the head of lab. However, at this point, the Department of Immunology at Pasteur decided to create, for the first time, a “Young Investigator Lab.” This opportunity was offered to young scientists to support the development of their own independent, small team over a three year period. The rest was simple. I responded to the call for proposals, was selected and joined the Department of Immunology in 1987. My “Young Investigator Laboratory” became a permanent Unit of Research in 1992. Since then, I have continued to build up a team, which today has five permanent senior scientists, with the aim of closing the gap between fundamental immunology and vaccinology. Since 2000, this Young Investigator Laboratory scheme, which offers independence to young investigators early in their careers, has become an important part of Pasteur Institute policy as a means to attract talented scientists. I was extremely lucky to benefit from such an opportunity as early as 1987. I will not list here all the projects that we have undertaken over the years; instead, I will provide two examples that demonstrate how fundamental immunology in my group has been a driving force for vaccine development. From Synthetic Adjuvants to Fully Synthetic Vaccines In the 80s, it became well established that chemically synthesized peptides representing selected regions of antigenic structures can induce antibodies, which bind to the native molecule. It was also shown that synthetic vaccines containing short peptide sequences could be prepared to induce a protective antibody response. However, at this time, a humoral response against such peptides could only be obtained by coupling these peptides to high molecular weight carrier proteins, such as tetanus toxoid. In particular, the induction of antibody responses against haptenic determinants (now called B-cell epitopes) was shown to be mediated through the cognate interaction between hapten-specific B cells and carrier-specific helper T cells. I therefore decided to construct fully synthetic vaccines by combining different synthetic B- and T-cell determinants
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within the same structure, with the idea that linking a single B-cell epitope to a single T-cell epitope would be sufficient to trigger antibody responses. I first analyzed the ability of B and T Iymphoytes to recognize several synthetic peptides. One of these peptides, a streptococcal peptide, S-34, was recognized by specific T cells in primed animals, triggering their proliferation. Its copolymerization with a synthetic hepatitis B virus (HBV) surface peptide, H(99-121), recognized only by B cells, allowed the construction of a bifunctional synthetic vaccine. Whereas the free HBV peptide was not immunogenic, the bifunctional synthetic construct containing both B and T cell determinants, in which HBV copolymerized with S-34, was able to elicit anti-H(99-121) peptide antibodies. This study demonstrated for the first time that fully synthetic vaccines can be constructed by appropriate selection and organization of B and T determinants.11 This was particularly important for me, since it confirmed my hypothesis that the rational design of a vaccine was becoming increasingly feasible, with improved understanding of fundamental immunology. We recently extended this approach to design a fully synthetic carbohydrate vaccine. A hallmark of cancer cells is altered glycosylation resulting in the abnormal expression of carbohydrate chains, such as Tn, sialyl-Tn and T antigens in many carcinomas (breast, colon, prostate and ovary), or gangliosides (GM2, GD2 or GD3) associated with melanomas. T and Tn carbohydrate epitopes are found on a variety of epithelial cells derived from breast or colon cancers, for example. To date, the development of immunogens to stimulate an immune response to such saccharidic antigens, including those of bacterial origin, is mostly based on the conjugation of carbohydrates to carrier proteins. In collaboration with Danièle Cantacuzène and Sylvie Bay, chemists at the Pasteur Institute, and Richard Lo-Man, one of my first PhD students, we developed an alternative strategy and designed a fully synthetic immunogen that does not require a protein carrier. This immunogen, called multiple antigenic glycopeptide (MAG), is based on a dendrimeric lysine core with four arms. Each arm is linked to a peptide backbone containing a pan-DR T-cell epitope, derived
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from tetanus toxin CD4+ T-cell epitope, with a trimeric saccharidic Tn residue at the N-terminal end of the peptide. This structure offers several advantages: the carbohydrate content is much higher than in traditional protein conjugates, the core matrix is non-immunogenic and it has a well-defined chemical structure. We showed that this fully synthetic MAG can induce anti-Tn IgG antibodies that recognize human tumor cell lines in mice and in non-human primates.12,13 Moreover, therapeutic immunization with this synthetic immunogen increased survival in tumor-bearing mice. The MAG-Tn3 molecule represents the first example of a fully synthetic glycosidic vaccine with potential immunotherapeutic activity against cancer. As such, we have embarked on a new adventure, with planning of a Phase I/II clinical trial of this vaccine candidate currently underway. Dendritic Cells: Reaching the Center of the Immunological World The series of seminal papers published between 1973 and 1979 by Ralf Steinman and Zanvil Cohn, with the general title: “Identification of a novel cell type in peripheral lymphoid organs of mice,”14-18 remain clear in my mind. The discovery of dendritic cells was certainly a major event in the history of immunology. I was already convinced that antigen had to be delivered to antigen presenting cells (APC) to trigger efficient T-cell responses and was trying to develop vaccine strategies based on this idea. I spent several years testing various approaches based on recombinant bacteria, BCG or salmonella, in collaboration with several world-class microbiologists of the Pasteur Institute, in particular Maurice Hofnung and Brigitte Gicquel. Although these studies were fairly successful, the approaches lacked specificity in the targeting of APCs. The discovery of dendritic cells and their unique properties led to a major change in the focus of my work. Indeed, I hypothesized that subunit vaccines, combining antigen delivery to APCs with appropriate DC maturation signals, may be as effective as live vectors. I again tested various strategies, in which antigen or peptides were coupled to inert
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or virus-like particles for their targeted delivery to APCs. The results obtained were much closer to my objective, but I was still not fully satisfied with the specificity of these approaches. Then, the talented microbiologists, Agnès Ullman and two of her students, Peter Sebo and Daniel Ladant, visited me to discuss the B. pertussis adenylate cyclase (CyaA), a molecule that they had studied extensively. CyaA is one of the major toxins produced by Bordetella pertussis. It can enter eukaryotic target cells where it is activated by endogenous calmodulin (CaM) to produce supraphysiological levels of intracellular cAMP, thus impairing cellular functions.19 The catalytic activity of this toxin can be completely suppressed by a single mutation, rendering the molecule non-toxic. CyaA is endowed with a unique mechanism of entry into eukaryotic cells involving the direct translocation of the catalytic domain, located within the first 400 amino acids, across the plasma membrane of the target cells into the cytosol. Agnès Ullmann and her colleagues suggested that this translocating property of CyaA could be used to deliver antigen to the cytosol of APCs. At that time, CyaA was thought to bind to all cells, including erythrocytes, and we presumed could also interact with immune cells. Being of clear interest to us, we and several members of the lab, in particular Catherine Fayolle, began to investigate the capacity of recombinant CyaA molecules carrying CD8+ T-cell epitopes to stimulate specific CTL responses. We identified various permissive sites within the catalytic domain of CyaA, which accepted the insertion of foreign sequences without affecting its ability to enter into eukaryotic cells. These experiments proved to be very successful. Indeed, using a CyaA carrying a single CTL epitope from the LCMV virus, we were able to fully protect mice against a challenge with a virulent strain of LCMV, which normally kills mice within one week.20 The efficiency of this approach seemed surprising. I was unable to understand how a few micrograms of a molecule binding to all cells, including erythrocytes, induced such strong T-cell responses. The work of Pierre Guermonprez, a talented PhD student, provided an explanation. Pierre had already started his thesis on
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another subject, but decided he would like to work on the mechanisms underlying the CyaA-induced T-cell responses. I eventually agreed to his decision, and despite being in the middle of his PhD, Pierre started on what we anticipated to be a very fruitful project. Indeed, Pierre demonstrated that CyaA binds specifically to the murine and human C11b/CD18 (Mac1) molecules and is therefore targeted to CD11b+ dendritic cells.21 When injected into mice, recombinant CyaA indeed binds very efficiently to CD11b+ CD8α- dendritic cells.22 Invasion of dendritic cells by CyaA carrying CTL epitopes thus allows very efficient delivery into the cytosol, leading to the presentation of these epitopes by MHC class I and class II molecules, as we discovered in later work. These remarkable properties explain the strong CTL responses stimulated by CyaA, independently of CD4+ helper T cells and CD40 signaling. The cellular specificity and invasive nature of CyaA were particularly suitable for the rational design of immunogens for T-cell priming. We established a longstanding, and ongoing, collaboration with Daniel Ladant and Peter Sebo to construct numerous recombinant CyaA molecules carrying different heterologous viral and/ or tumoral T-cell epitopes. We have repeatedly demonstrated the potency of the CyaA vector to stimulate strong and specific Th1 and CTL responses in animals and induce protective immunity against viral and tumoral challenges, as well as therapeutic efficacy for transplanted tumors. In particular, Gilles Dadaglio in our lab, in collaboration with Benoit Van Den Eynde at the Ludwig Institute in Belgium, showed the capacity of a recombinant CyaA molecule carrying an HLA-A2-restricted melanoma epitope to induce strong anti-melanoma CTL responses in HLA-A2 transgenic mice.23 Based on these preclinical results, the planning of a Phase I/II clinical trial of this molecule is now underway. In collaboration with the company BT Pharma,24 and using the same vector to target dendritic cells, we also demonstrated the potency of a therapeutic vaccine candidate against human papillomavirus (HPV) associated with cervical cancer. BT Pharma is currently planning a Phase I/II clinical trial of recombinant CyaA
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vaccines, carrying the E7 protein from HPV 16 and 18. Bringing these vaccine candidates from their development in the lab to clinical development is a significant achievement. However, it is also only the first step of a long and complex process. It has become increasingly clear to me over these last few years that efficient therapeutic vaccination against cancers will not only require the induction of efficient immune responses, but also elucidation of the cross-talk between the tumor and the host. Indeed, we have shown that the therapeutic efficacy of recombinant CyaA carrying the E7 protein from HPV 16 declines progressively as the tumor grows, reaching a non-significant value by the time it reaches 10 mm.25 Combining therapeutic vaccines with strategies to overcome these suppressive mechanisms could open up new perspectives in the treatment of cancer. This represents a major, and exciting, scientific challenge, but also offers hope in the development of better treatments for patients. References 1. Chedid L, Audibert F, Lefrancier P, Choay J, Lederer E. 1976. Modulation of the immune response by a synthetic adjuvant and analogs. Proc Natl Acad Sci USA 73:2472-2475. 2. Fevrier M, Birrien JL, Leclerc C, Chedid L, Liacopoulos P. 1978. The macrophage, target cell of the synthetic adjuvant muramyl dipeptide. Eur J Immunol 8:558-562. 3. Leclerc C, Bahr GM, Chedid L. 1984. Marked enhancement of macrophage activation induced by synthetic muramyl dipeptide (MDP) conjugate using monoclonal anti-MDP antibodies. Cell Immunol 86:269-277.
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4. Leclerc C, Chedid L. 1986. Are there receptors for MDP at the macrophage surface? . International J Immunother 2:109-114. 5. Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ. 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278:8869-8872. 6. Leclerc C, Juy D, Chedid L. 1979. Inhibitory and stimulatory effects of a synthetic glycopeptide (MDP) on the in vitro PFC response: factors affecting the response. Cell Immunol 42:336-343. 7. Leclerc C, Juy D, Bourgeois E, Chedid L. 1979. In vivo regulation of humoral and cellular immune responses of mice by a synthetic adjuvant, N-acetyl-muramyl-Lalanyl-D-isoglutamine, muramyl dipeptide for MDP. Cell Immunol 45:199-206. 8. Leclerc C, Bourgeois E, Chedid L. 1982. Demonstration of muramyl dipeptide (MDP)-induced T suppressor cells responsible for MDP immunosuppressive activity. Eur J Immunol 12:249-252. 9. Leclerc C, Morin A, Deriaud E, Chedid L. 1984. Inhibition of human IL 2 production by MDP and derivatives. J Immunol 133:1996-2000. 10. Leclerc C, Deriaud E, Schutze MP, Chedid L. 1988. Prevention of low dose streptozotocin induced diabetes by muramyl dipeptide. Int J Immunopharmacol 10:293-298. 11. Leclerc C, Przewlocki G, Schutze MP, Chedid L. 1987. A synthetic vaccine constructed by copolymerization of B and T cell determinants. Eur J Immunol 17:269273. 12. Lo-Man R, Bay S, Vichier-Guerre S, Deriaud E, Cantacuzene D, Leclerc C. 1999. A fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy. Cancer Res 59:1520-1524. 13. Lo-Man R, Vichier-Guerre S, Perraut R, Deriaud E, Huteau V, BenMohamed L, et al. 2004. A fully synthetic therapeutic vaccine candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-specific antibodies in nonhuman primates. Cancer Res 64:4987-4994. 14. Steinman RM, Cohn ZA. 1973. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 137:1142-1162. 15. Steinman RM, Cohn ZA. 1974. Identification of a novel cell type in peripheral lymphoid organs of mice. II. Functional properties in vitro. J Exp Med 139:380397.
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16. Steinman RM, Lustig DS, Cohn ZA. 1974. Identification of a novel cell type in peripheral lymphoid organs of mice. 3. Functional properties in vivo. J Exp Med 139:1431-1445. 17. Steinman RM, Adams JC, Cohn ZA. 1975. Identification of a novel cell type in peripheral lymphoid organs of mice. IV. Identification and distribution in mouse spleen. J Exp Med 141:804-820. 18. Steinman RM, Kaplan G, Witmer MD, Cohn ZA. 1979. Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro. J Exp Med 149:1-16. 19. Ladant D, Ullmann A. 1999. Bordetella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol 7:172-176. 20. Saron MF, Fayolle C, Sebo P, Ladant D, Ullmann A, Leclerc C. 1997. Anti-viral protection conferred by recombinant adenylate cyclase toxins from Bordetella pertussis carrying a CD8+ T cell epitope from lymphocytic choriomeningitis virus. Proc Natl Acad Sci USA 94:3314-3319. 21. Guermonprez P, Khelef N, Blouin E, Rieu P, Ricciardi-Castagnoli P, Guiso N, et al. 2001. The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18). J Exp Med 193:1035-1044. 22. Guermonprez P, Fayolle C, Rojas MJ, Rescigno M, Ladant D, Leclerc C. 2002. In vivo receptor-mediated delivery of a recombinant invasive bacterial toxoid to CD11c + CD8 alpha -CD11bhigh dendritic cells. Eur J Immunol 32:3071-3081. 23. Dadaglio G, Morel S, Bauche C, Moukrim Z, Lemonnier FA, Van Den Eynde BJ, et al. 2003. Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes. Int Immunol 15:1423-1430. 24. Preville X, Ladant D, Timmerman B, Leclerc C. 2005. Eradication of established tumors by vaccination with recombinant Bordetella pertussis adenylate cyclase carrying the human papillomavirus 16 E7 oncoprotein. Cancer Res 65:641-649. 25. Berraondo P, Nouze C, Preville X, Ladant D, Leclerc C. 2007. Eradication of large tumors in mice by a tritherapy targeting the innate, adaptive, and regulatory components of the immune system. Cancer Res 67:8847-8855.
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