PUBLICATIONS
 
The Aduro technology has been featured on the cover of Nature Medicine and Infection and Immunity.
A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: Phase 1 studies of safety and immune induction.
Le D.T., D. Brockstedt, R. Nir-Paz, J. Hampl, S. Mathur, J. Nemunaitis, D.H. Sterman, R. Hassan, E.R. Lutz, B. Moyer, M. Giedlin, J. L. Louis, E. Sugar, A. Pons, A.L. Cox, J. Levine, A. L. Murphy, P.B. Illei, T. Dubensky, J. Eiden, E.M. Jaffee, and D. Laheru.
Clin Cancer Res. 2011 Dec 6. [Epub ahead of print]
Abstract:
PURPOSE: Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ΔactA/ΔinlB). Uptake by phagocytes in the liver results in local inflammatory responses, and activation and recruitment of NK and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ΔactA/ΔinlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T cell responses against mesothelin-expressing murine tumors. These two Phase 1 studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively.
EXPERIMENTAL DESIGN: A single intravenous injection of ANZ-100 was evaluated in a dose escalation study in subjects with liver metastases. Nine subjects received 1x10(6), 3x10(7), or 3x10(8) colony forming units [cfu]. CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung, pancreatic or ovarian cancers. 17 subjects received up to 4 doses of 1x10(8), 3x10(8), 1x10(9), or 1x10(10) cfu.
RESULTS: A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple infusions of CRS-207 were well tolerated up to 1x10(9) cfu, the determined maximum tolerated dose. Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/chemokine levels and NK cell activation. In the CRS-207 study, Listeriolysin O and mesothelin-specific T cell responses were detected and 37% of subjects lived > 15 months.
CONCLUSIONS: ANZ-100 and CRS-207 administration was safe and resulted in immune activation.
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Tumor-associated antigen expressing Listeria monocytogenes induces effective primary and memory T-cell responses against hepatic colorectal cancer metastases.
Olino, K., S. Wada, B. H. Edil, X. Pan, K. Meckel, W. Weber, J. Slansky, K. Tamada, P. Lauer, D. Brockstedt, D. Pardoll, R. Schulick, and K. Yoshimura.
Ann Surg Oncol. 2011 Oct 7. [Epub ahead of print].
Abstract: PURPOSE: Despite advances in therapy for the treatment of metastatic colorectal cancer, many patients die of hepatic disease. Current immunotherapeutic strategies are likely limited by inhibitory signals from the tumor. To successfully eliminate tumor deposits within an organ, an appropriate immunologic milieu to amplify antitumor responses must be developed.
METHODS: We used a murine model utilizing the CT26 colon cancer cell line to analyze primary and memory tumor-specific T-cell responses induced by an attenuated actin A and internalin B deleted immunodominant tumor-associated antigen expressing strain of Listeria monocytogenes for the treatment of metastatic colorectal cancer.
RESULTS: Treatment of mice bearing established hepatic metastases with this L. monocytogenes strain led to the generation of a strong initial tumor-specific cytotoxic CD8(+) T-cell response that successfully treated 90% of animals. Tumor antigen-specific central and effector memory T cells were also generated and protected against tumor rechallenge. These cell populations, when measured before and after tumor rechallenge, showed a marked expansion of antigen-specific effector CD8(+) effector memory T cells. This strain of L. monocytogenes was able to down-modulate the expression of the immune checkpoint molecule, PD-1, within the tumor microenvironment but had variable effects on CTLA-4 expression.
CONCLUSIONS: This L. monocytogenes strain generated a highly effective antitumor T-cell response, providing a basis for the development of this vaccine platform in patients with liver metastases.
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Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity.
Sauer, J. D., S. Pereyre, K. A. Archer, T. P. Burke, B. Hanson, P. Lauer, and D. A. Portnoy.
Proc Natl Acad Sci U S A. 2011 Jul 26;108(30):12419-24. Epub 2011 Jul 11.
Abstract: Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes, strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes. Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.
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Dynamic imaging of the effector immune response to listeria infection in vivo.
Waite, J. C., I. Leiner, P. Lauer, C.S. Rae, G. Barbet, H. Zheng, D.A. Portnoy, E. G. Pamer, and M.L. Dustin.
PLoS Pathog. 2011 Mar;7(3):e1001326. Epub 2011 Mar 24.
Abstract: Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites.
Interplay between CD8α dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses.
Kapadia, D., A. Sadikovic, Y. Vanloubbeeck, D. Brockstedt, and L. Fong.
PLoS One. 2011 Apr 29;6(4):e19376.
Abstract: During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.
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Priming and activation of human ovarian and breast cancer-specific CD8+ T cells by polyvalent Listeria monocytogenes-based vaccines.
Sinnathamby, G., P. Lauer, J. Zerfass, B. Hanson, A. Karabudak, J. Krakover, A. A. Secord, T. M. Clay, M. A. Morse, T. W. Dubensky Jr, D. G. Brockstedt, R. Philip, and M. Giedlin. J Immunother. 2009 Oct;32(8):856-69
Abstract: Immunotherapeutic vaccine is potentially an effective strategy to combat cancer. Essential components of an effective vaccine must include antigens that are processed by the major histocompatibility complex class I pathway, presented by the tumor major histocompatibility complex molecules, and an effective antigen delivery platform that is capable of breaking self-tolerance. In this study, we characterized a set of ovarian cancer-specific T-cell epitopes delivered by live-attenuated recombinant Listeria monocytogenes (Lm DeltaactADeltainlB) as a vaccine vector. We present data that peptide-specific T cells recognize the human monocytic cell line THP-1 infected with recombinant Lm DeltaactADeltainlB encoding the epitopes. Furthermore, we demonstrate that recombinant L. monocytogenes (Lm)-infected antigen-presenting cells can prime and expand epitope-specific CD8 T cells in vitro and such CD8 T cells recognize not only peptide-loaded targets but also ovarian and breast tumor cells presenting endogenous epitopes. Finally, peptide-specific T cells generated using peripheral blood mononuclear cell from ovarian cancer patients recognize target cells infected with recombinant Lm DeltaactADeltainlB encoding the epitopes. Our results demonstrate that live-attenuated recombinant Lm can be used effectively as a vehicle to deliver cancer peptide antigens singly or as a multiepitope construct. Thus, the use of recombinant live-attenuated Lm strains encoding endogenously processed and presented tumor epitopes/antigens represents an attractive strategy for active cancer immunotherapy in a clinical setting.
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Suppression of cell-mediated immunity following recognition of phagosome-confined bacteria.
Bahjat, K. S., N. Meyer-Morse, E. E. Lemmens, J. A. Shugart, T. W. Dubensky, D. G. Brockstedt, and D. A. Portnoy.
PLoS Pathog. 2009 Sep;5(9):e1000568. Epub 2009 Sep 4.
Abstract: Listeria monocytogenes is a facultative intracellular pathogen capable of inducing a robust cell-mediated immune response to sub-lethal infection. The capacity of L. monocytogenes to escape from the phagosome and enter the host cell cytosol is paramount for the induction of long-lived CD8 T cell-mediated protective immunity. Here, we show that the impaired T cell response to L. monocytogenes confined within a phagosome is not merely a consequence of inefficient antigen presentation, but is the result of direct suppression of the adaptive response. This suppression limited not only the adaptive response to vacuole-confined L. monocytogenes, but negated the response to bacteria within the cytosol. Co-infection with phagosome-confined and cytosolic L. monocytogenes prevented the generation of acquired immunity and limited expansion of antigen-specific T cells relative to the cytosolic L. monocytogenes strain alone. Bacteria confined to a phagosome suppressed the production of pro-inflammatory cytokines and led to the rapid MyD88-dependent production of IL-10. Blockade of the IL-10 receptor or the absence of MyD88 during primary infection restored protective immunity. Our studies demonstrate that the presence of microbes within a phagosome can directly impact the innate and adaptive immune response by antagonizing the signaling pathways necessary for inflammation and the generation of protective CD8 T cells.
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Impact of preexisting vector-specific immunity on vaccine potency: characterization of Listeria monocytogenes-specific humoral and cellular immunity in humans and modeling studies using recombinant vaccines in mice. Leong, M. L., J. Hampl, W. Liu, S. Mathur, K. S. Bahjat, W. Luckett, T. W. Dubensky, Jr., and D. G. Brockstedt. Infect Immun. 2009 Sep;77(9):3958-68. Epub 2009 Jun 15. Abstract: Recombinant live-attenuated Listeria monocytogenes is currently being developed as a vaccine platform for treatment or prevention of malignant and infectious diseases. The effectiveness of complex biologic vaccines, such as recombinant viral and bacterial vectors, can be limited by either preexisting or vaccine-induced vector-specific immunity. We characterized the level of L. monocytogenes-specific cellular and humoral immunity present in more than 70 healthy adult subjects as a first step to understanding its possible impact on the efficacy of L. monocytogenes-based vaccines being evaluated in early-phase clinical trials. Significant L. monocytogenes-specific humoral immunity was not measured in humans, consistent with a lack of antibodies in mice immunized with wild-type L. monocytogenes. Cellular immune responses specific for listeriolysin O, a secreted bacterial protein required for potency of L. monocytogenes-derived vaccines, were detected in approximately 60% of human donors tested. In mice, while wild-type L. monocytogenes did not induce significant humoral immunity, attenuated L. monocytogenes vaccine strains induced high-titer L. monocytogenes-specific antibodies when given at high doses used for immunization. Passive transfer of L. monocytogenes-specific antiserum to naive mice had no impact on priming antigen-specific immunity in mice immunized with a recombinant L. monocytogenes vaccine. In mice with preexisting L. monocytogenes-specific immunity, priming of naive T cells was not prevented, and antigen-specific responses could be boosted by additional vaccinations. For the first time, our findings establish the level of L. monocytogenes-specific cellular immunity in healthy adults, and, together with modeling studies performed with mice, they support the scientific rationale for repeated L. monocytogenes vaccine immunization regimens to elicit a desired therapeutic effect.
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Priming and activation of human ovarian and breast cancer-specific CD8+ T cells by polyvalent Listeria monocytogenes-based vaccines.
Sinnathamby, G., P. Lauer, J. Zerfass, B. Hanson, A. Karabudak, J. Krakover, A. A. Secord, T. M. Clay, M. A. Morse, T. W. Dubensky, Jr., D. G. Brockstedt, R. Philip, and M. Giedlin.
J Immunother. 2009 Oct;32(8):856-69. Abstract: Immunotherapeutic vaccine is potentially an effective strategy to combat cancer. Essential components of an effective vaccine must include antigens that are processed by the major histocompatibility complex class I pathway, presented by the tumor major histocompatibility complex molecules, and an effective antigen delivery platform that is capable of breaking self-tolerance. In this study, we characterized a set of ovarian cancer-specific T-cell epitopes delivered by live-attenuated recombinant Listeria monocytogenes (Lm DeltaactADeltainlB) as a vaccine vector. We present data that peptide-specific T cells recognize the human monocytic cell line THP-1 infected with recombinant Lm DeltaactADeltainlB encoding the epitopes. Furthermore, we demonstrate that recombinant L. monocytogenes (Lm)-infected antigen-presenting cells can prime and expand epitope-specific CD8 T cells in vitro and such CD8 T cells recognize not only peptide-loaded targets but also ovarian and breast tumor cells presenting endogenous epitopes. Finally, peptide-specific T cells generated using peripheral blood mononuclear cell from ovarian cancer patients recognize target cells infected with recombinant Lm DeltaactADeltainlB encoding the epitopes. Our results demonstrate that live-attenuated recombinant Lm can be used effectively as a vehicle to deliver cancer peptide antigens singly or as a multiepitope construct. Thus, the use of recombinant live-attenuated Lm strains encoding endogenously processed and presented tumor epitopes/antigens represents an attractive strategy for active cancer immunotherapy in a clinical setting2.
Killed but metabolically active Bacillus anthracis vaccines induce broad and protective immunity against anthrax. Skoble, J., J. W. Beaber, Y. Gao, J. A. Lovchik, L. E. Sower, W. Liu, W. Luckett, J. W. Peterson, R. Calendar, D. A. Portnoy, C. R. Lyons, and T. W. Dubensky, Jr. Infect Immun. 2009 Apr;77(4):1649-63. Epub 2009 Jan 21. Abstract: Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.
KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity.
Skoberne, M., A. Yewdall, K. S. Bahjat, E. Godefroy, P. Lauer, E. Lemmens, W. Liu, W. Luckett, M. Leong, T. W. Dubensky, D. G. Brockstedt, and N. Bhardwaj. J Clin Invest. 2008 Dec;118(12):3990-4001. doi: 10.1172/JCI31350. Epub 2008 Nov 6.
Abstract: Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.
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Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria.
Schmidt, N. W., R. L. Podyminogin, N. S. Butler, V. P. Badovinac, B. J. Tucker, K. S. Bahjat, P. Lauer, A. Reyes-Sandoval, C. L. Hutchings, A. C. Moore, S. C. Gilbert, A. V. Hill, L. C. Bartholomay, and J. T. Harty.
Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14017-22. Epub 2008 Sep 9. Abstract: Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.
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Short-term genome evolution of Listeria monocytogenes in a non-controlled environment.
Orsi, R. H., M. L. Borowsky, P. Lauer, S. K. Young, C. Nusbaum, J. E. Galagan, B. W. Birren, R. A. Ivy, Q. Sun, L. M. Graves, B. Swaminathan, and M. Wiedmann. BMC Genomics. 2008 Nov 13;9:539.
Abstract:
BACKGROUND: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses.
RESULTS: The two L. monocytogenes isolates from 1988 and the two isolates from 2000 had highly similar genome backbone sequences with very few single nucleotide (nt) polymorphisms (1 - 8 SNPs/isolate; confirmed by re-sequencing). While no genome rearrangements were identified in the backbone genome of the four isolates, a 42 kb prophage inserted in the chromosomal comK gene showed evidence for major genome rearrangements. The human-food isolate pair from each 1988 and 2000 had identical prophage sequence; however, there were significant differences in the prophage sequences between the 1988 and 2000 isolates. Diversification of this prophage appears to have been caused by multiple homologous recombination events or possibly prophage replacement. In addition, only the 2000 human isolate contained a plasmid, suggesting plasmid loss or acquisition events. Surprisingly, besides the polymorphisms found in the comK prophage, a single SNP in the tRNA Thr-4 prophage represents the only SNP that differentiates the 1988 isolates from the 2000 isolates.
CONCLUSION: Our data support the hypothesis that the 2000 human listeriosis outbreak was caused by a L. monocytogenes strain that persisted in a food processing facility over 12 years and show that genome sequencing is a valuable and feasible tool for retrospective epidemiological analyses. Short-term evolution of L. monocytogenes in non-controlled environments appears to involve limited diversification beyond plasmid gain or loss and prophage diversification, highlighting the importance of phages in bacterial evolution.
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Promises and challenges for the development of Listeria monocytogenes-based immunotherapies.
Brockstedt, D.G., and T.W. Dubensky. Expert Rev Vaccines. 2008 Sep;7(7):1069-84.
Abstract: Active immunotherapy has shown great promise in preclinical models for the treatment of infectious and malignant disease. Yet, these promising results have not translated into approved therapies. One of the major deficits of active immunotherapies tested to date in advanced clinical studies has been their inability to stimulate both arms of the immune system appropriately. The interest in using recombinant bacteria as vaccine vectors for active immunotherapy derives in part from their ability to stimulate multiple innate immune pathways and, at the same time, to deliver antigen for presentation to the adaptive immune system. This review will focus on the development of live-attenuated and killed strains of the intracellular bacterium Listeria monocytogenes for treatment of chronic infections and cancer. Early clinical trials intended to demonstrate safety as well as proof of concept have recently been initiated in several indications. Advances in molecular engineering as well as successes and challenges for clinical development of L. monocytogenes-based vaccines will be discussed.
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Constitutive activation of the prfA regulon enhances the potency of live-attenuated and KBMA Listeria monocytogenes-based vaccines.
Lauer, P., B Hanson, E.E. Lemmens, W. Liu, W.S. Luckett, M.L. Leong, H.E. Allen, J. Skoble*, K.S. Bahjat, N.E. Freitag, D.G. Brockstedt, and T.W. Dubensky. Infect Immun. 2008 Aug;76(8):3742-53. Epub 2008 Jun 9.
Abstract: Recombinant vaccines derived from the facultative intracellular bacterium Listeria monocytogenes are presently undergoing early-stage clinical evaluation in oncology treatment settings. This effort has been stimulated in part due to preclinical results that illustrate potent activation of innate and adaptive immune effectors by L. monocytogenes vaccines, combined with efficacy in rigorous animal models of malignant and infectious disease. Here, we evaluated the immunologic potency of a panel of isogenic vaccine strains that varied only in prfA. PrfA is an intracellularly activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in appropriately engineered vaccine strains. Mutant strains with PrfA locked into a constitutively active state are known as PrfA* mutants. We assessed the impacts of three PrfA* mutants, G145S, G155S, and Y63C, on the immunologic potencies of live-attenuated and photochemically inactivated nucleotide excision repair mutant (killed but metabolically active [KBMA]) vaccines. While PrfA* substantially increased Ag expression in strains grown in broth culture, Ag expression levels were equivalent in infected macrophage and dendritic cell lines, conditions that more closely parallel those in the immunized host. However, only the prfA(G155S) allele conferred significantly enhanced vaccine potency to KBMA vaccines. In the KBMA vaccine background, we show that PrfA*(G155S) enhanced functional cellular immunity following an intravenous or intramuscular prime-boost immunization regimen. These results form the basis of a rationale for including the prfA(G155S) allele in future live-attenuated or KBMA L. monocytogenes vaccines advanced to the clinical setting.
Listeria monocytogenes multidrug resistance transporters activate a cytosolic surveillance pathway of innate immunity.
Crimmins, G.T. A.A. Herskovits, K. Rehder, K.E. Sivick, P. Lauer, T.W. Dubensky, and D.A. Portnoy. Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10191-6. Epub 2008 Jul 16.
Abstract: To gain insight into the interaction of intracellular pathogens with host innate immune pathways, we performed an unbiased genetic screen of Listeria monocytogenes mutants that induced an enhanced or diminished host innate immune response. Here, we show that the major facilitator superfamily of bacterial multidrug resistance transporters (MDRs) controlled the magnitude of a host cytosolic surveillance pathway, leading to the production of several cytokines, including type I IFN. Mutations mapping to repressors of MDRs resulted in ectopic expression of their cognate transporters, leading to host responses that were increased up to 20-fold over wild-type bacteria, and a 20-fold decrease in bacterial growth in vivo. Mutation of one of the MDRs, MdrM, led to a 3-fold reduction in the IFN-beta response to L. monocytogenes infection, indicating a pivotal role for MdrM in activation of the host cytosolic surveillance system. Bacterial MDRs had previously been associated with resistance to antibiotics and other toxic compounds. This report links bacterial MDRs and host immunity. Understanding the mechanisms through which live pathogens activate innate immune signaling pathways should lead to the discovery of adjuvants, vaccines, and perhaps new classes of therapeutics. Indeed, we show that the mutants identified in this screen induced vastly altered type I IFN response in vivo as well.
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Activation of immature hepatic NK cells as immunotherapy for liver metastatic disease.
Bahjat, K.S., R.A. Prell, H.E. Allen, W. Liu, E.E. Lemmens, M.L. Leong, D.A. Portnoy, T.W. Dubensky, D.G. Brockstedt, and M.A. Giedlin. J Immunol. 2007 Dec 1;179(11):7376-84. Abstract: NK cells can identify and eliminate emerging tumors due to altered expression of activating and inhibitory ligands on aberrant cells, a process that is greatly enhanced following NK cell activation. As a principal site of both tumor metastases and immature NK cells, the liver represents a unique anatomic location in which activation of the innate immune system could provide substantial therapeutic benefit. We describe here the NK cell-dependent destruction of a primary hepatic tumor following infection with an attenuated intracellular bacterium derived from Listeria monocytogenes. NK cell-mediated immunity correlated with the ordered migration and maturation of NK cells within the liver. Cytolytic activity was partially dependent on NKG2D-mediated tumor cell recognition, but surprisingly was still effective in the absence of type I IFN. Significantly, NK cell-mediated destruction of a primary hepatic tumor in infected mice led to long-lived CD4- and CD8 T cell-dependent tumor-specific adaptive immunity. These findings establish that activation and differentiation of immature NK cells using complex microbial stimuli can elicit potent anti-tumor activity within the liver, promote cross-presentation of tumor-derived Ags leading to long-lived systemic anti-tumor immunity, and suggests a paradigm for clinical intervention of liver metastatic carcinoma.
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Role of PD-1 and its ligand, B7-H1, in early fate decisions of CD8 T cells.
Goldberg, M.V., C.J. Maris, E.L. Hipkiss, A.S. Flies, L. Zhen, R.M. Tuder, J.F. Grosso, T.J. Harris, D. Getnet, K.A. Whartenby, D.G. Brockstedt, T.W. Dubensky, L. Chen, D.M. Pardoll, and C.G. Drake.
Blood. 2007 Jul 1;110(1):186-92. Epub 2007 Mar 28. Abstract: Expression of the PD-1 receptor on T cells has been shown to provide an important inhibitory signal that down-modulates peripheral effector responses in normal tissues and tumors. Furthermore, PD-1 up-regulation on chronically activated T cells can maintain them in a partially reversible inactive state. The function of PD-1 in the very early stages of T-cell response to antigen in vivo has not been fully explored. In this study, we evaluate the role of PD-1 and its 2 B7 family ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), in early fate decisions of CD8 T cells. We show that CD8 T cells specific for influenza hemagglutinin (HA) expressed as a self-antigen become functionally tolerized and express high levels of surface PD-1 by the time of their first cell division. Blockade of PD-1 or B7-H1, but not B7-DC, at the time of self-antigen encounter mitigates tolerance induction and results in CD8 T-cell differentiation into functional cytolytic T lymphocytes (CTLs). These findings demonstrate that, in addition to modulating effector functions in the periphery, B7-H1:PD-1 interactions regulate early T-cell-fate decisions.
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Cytosolic entry controls CD8 + T cell potency during bacterial infection.
Bahjat, K.S., W. Liu, E.E. Lemmens, S.P. Schoenberger, D.A. Portnoy, T.W. Dubensky, and D. Brockstedt.
Infect Immun. 2006 Nov;74(11):6387-97. Epub 2006 Sep 5. Abstract: Interaction with host immunoreceptors during microbial infection directly impacts the magnitude of the ensuing innate immune response. How these signals affect the quality of the adaptive T-cell response remains poorly understood. Utilizing an engineered strain of the intracellular pathogen Listeria monocytogenes that infects cells but fails to escape from the phagosome, we demonstrate the induction of long-lived memory T cells that are capable of secondary expansion and effector function but are incapable of providing protective immunity. We demonstrate that microbial invasion of the cytosol is required for dendritic cell activation and integration of CD40 signaling, ultimately determining the ability of the elicited CD8+-T-cell pool to protect against lethal wild-type L. monocytogenes challenge. These results reveal a crucial role for phagosomal escape, not for delivery of antigen to the class I major histocompatibility complex pathway but for establishing the appropriate cellular context during CD8+-T-cell priming.
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Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity.
Chan, C.W., E. Crafton, H.N. Fan, J. Flook, K. Yoshimura, M. Skarica, D. Brockstedt, T.W. Dubensky, M.F. Stins, L.L. Lanier, D.M. Pardoll, and F. Housseau.
Nat Med. 2006 Feb;12(2):207-13. Epub 2006 Jan 29. Abstract: Natural killer (NK) cells and dendritic cells (DCs) are, respectively, central components of innate and adaptive immune responses. We describe here a third DC lineage, termed interferon-producing killer DCs (IKDCs), distinct from conventional DCs and plasmacytoid DCs and with the molecular expression profile of both NK cells and DCs. They produce substantial amounts of type I interferons (IFN) and interleukin (IL)-12 or IFN-gamma, depending on activation stimuli. Upon stimulation with CpG oligodeoxynucleotides, ligands for Toll-like receptor (TLR)-9, IKDCs kill typical NK target cells using NK-activating receptors. Their cytolytic capacity subsequently diminishes, associated with the loss of NKG2D receptor (also known as Klrk1) and its adaptors, Dap10 and Dap12. As cytotoxicity is lost, DC-like antigen-presenting activity is gained, associated with upregulation of surface major histocompatibility complex class II (MHC II) and costimulatory molecules, which formally distinguish them from classical NK cells. In vivo, splenic IKDCs preferentially show NK function and, upon systemic infection, migrate to lymph nodes, where they primarily show antigen-presenting cell activity. By virtue of their capacity to kill target cells, followed by antigen presentation, IKDCs provide a link between innate and adaptive immunity.
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Selective targeting of antitumor immune responses with engineered live-attenuated Listeria monocytogenes. K. Yoshimura, A. Jain, H.E. Allen, L.S. Laird, C.Y. Chia, S. Ravi, D.G. Brockstedt, M.A. Giedlin, K.S. Bahjat, M.L. Leong, J.E. Slansky, D.N. Cook, T.W. Dubensky, D.M. Pardoll, and R.D. Schulick.
Cancer Res. 2006 Jan 15;66(2):1096-104.
Abstract: Improved immunization and ex vivo T-cell culture strategies can generate larger numbers and more potent tumor-specific effector cells than previously possible. Nonetheless, the capacity of these cells to eliminate established tumors is limited by their ability to efficiently enter tumor-bearing organs and mediate their effector function. In the current study, we show that the administration of an engineered organ-homing microbe selectively targets tumor-specific immune responses to metastases within that organ. Specifically, an attenuated Listeria monocytogenes strain, which preferentially infects the liver following systemic administration, dramatically enhances the activity of a cancer vaccine against liver metastases but not metastases in the lung. This enhanced activity results from both local recruitment of innate immune effectors as well as concentration and increased activation of vaccine-induced antitumor T cells within the liver. These findings show a general approach to focus systemic cancer immunotherapies to specific organs bearing tumor metastases by taking advantage of differential tropisms and the proinflammatory nature of microbes.
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Killed but metabolically active (KBMA) microbes: A new vaccine paradigm for eliciting effector T cell responses and protective immunity.
Brockstedt, D. G., M.A. Giedlin, K.S. Bahjat, M.L. Leong, W. Liu, W. Luckett, Y. Gao, P. Schnupf, D. Kapadia, G. Castro, A Sampson-Johannes, A. Stassinopoulos, H. G. Archie Bouwer, J.E. Hearst, D.A. Portnoy, D.N. Cook, and T.W. Dubensky, Jr.
Nat Med. 2005 Aug;11(8):853-60. Epub 2005 Jul 24. Abstract: We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.
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Listeria-based cancer vaccines that segregate immunogenicity from toxicity.
Brockstedt, D.G., M.A. Giedlin, M.L. Leong, K.S. Bahjat, Y. Gao, W. Luckett, W. Liu, D.N. Cook, D.A. Portnoy, and T.W. Dubensky Jr.
Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13832-7. Epub 2004 Sep 13. Abstract: The facultative intracellular bacterium Listeria monocytogenes is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a Listeria platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type Listeria. By selectively deleting two virulence factors, ActA (DeltaactA) and Internalin B (DeltainlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cell-to-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria DeltaactA/DeltainlB-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB/c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm(3)) with recombinant Listeria DeltaactA/DeltainlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria DeltaactA/DeltainlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.
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Listeria monocytogenes as a vaccine vector: Virulence attenuation or existing anti-vector immunity does not diminish therapeutic efficacy.
Starks, H., K.W. Bruhn, H. Shen, R.A. Barry, T.W. Dubensky, D. Brockstedt, D.J. Hinrichs, D.E. Higgins, J.F. Miller, M. Giedlin, and H. G. Archie Bouwer.
J Immunol. 2004 Jul 1;173(1):420-7. Abstract: The bacterium L. monocytogenes is a proposed vaccine carrier based upon the observation that this pathogen replicates within the intracytoplasmic environment facilitating delivery of Ag to the endogenous Ag processing and presentation pathway with subsequent stimulation of peptide specific MHC class I-restricted CD8(+) effector cells. In this report, we evaluate virulence-attenuated strains of Listeria monocytogenes as vaccine vectors and examine whether existing antivector (antiListerial) immunity limits or alters its efficacy as a therapeutic cancer vaccine. Following immunization with virulence-attenuated mutants, we found that the effectiveness of L. monocytogenes as a recombinant cancer vaccine remains intact. In addition, we found that antibiotic treatment initiated 24 or 36 h following therapeutic immunization with recombinant L. monocytogenes allows full development of the antitumor response. We also demonstrate that the vaccine vector potential of L. monocytogenes is not limited in animals with existing antiListerial immunity. For these latter studies, mice previously immunized with wild-type L. monocytogenes were infused with melanoma cells and then 5 days later challenged with recombinant tumor Ag expressing L. monocytogenes. Collectively, these results add additional support for the use of L. monocytogenes as a vaccine vector and underscore its potential to be used repeatedly for stimulation of recall responses concomitant with primary cell-mediated responses to newly delivered heterologous tumor-associated epitopes.
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