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.
Mojca Skoberne, Alice Yewdall, Keith S. Bahjat, Emmanuelle Godefroy, Peter Lauer, Edward Lemmens, Weiqun Liu, Will Luckett, Meredith Leong, Thomas W. Dubensky, Dirk G. Brockstedt, Nina Bhardwaj
Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific CD8+ T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product–specific CD8+ T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector–induced CD8+ T cells proliferated poorly, produced low levels of IFN-γ in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the CD8+ T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection.
Shih-Wen Lin, Scott E. Hensley, Nia Tatsis, Marcio O. Lasaro, Hildegund C.J. Ertl
Initial immune responses to allergens may occur before birth, thereby modulating the subsequent development of atopy. This paradigm remains controversial, however, due to the inability to identify antigen-specific T cells in cord blood. The advent of MHC tetramers has revolutionized the detection of antigen-specific T cells. Tetramer staining of cord blood after CMV infection has demonstrated that effective CD8+ antigen-specific immune responses can follow intrauterine viral infections. We hypothesized that sensitization to antigens occurs in utero in humans. We studied cord blood B and T cell immune responses following vaccination against influenza during pregnancy. Anti-Fluzone and anti-matrix protein IgM antibodies were detected in 38.5% (27 of 70) and 40.0% (28 of 70), respectively, of cord blood specimens. Using MHC tetramers, HA-specific CD4+ T cells were detected among 25.0% (3 of 12) and 42.9% (6 of 14) of cord blood specimens possessing DRB1*0101 and DRB1*0401 HLA types, respectively, and were detected even when the DRB1 HLA type was inherited from the father. Matrix protein–specific CD8+ T cells were detected among 10.0% (2 of 20) of HLA-A*0201+ newborns. These results suggest that B and T cell immune responses occur in the fetus following vaccination against influenza and have important implications for determining when immune responses to environmental exposures begin.
Deepa Rastogi, Chaodong Wang, Xia Mao, Cynthia Lendor, Paul B. Rothman, Rachel L. Miller
Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimuriumvaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium–NY-ESO-1) through a type III protein secretion system. The S. typhimurium–NY-ESO-1 construct elicited NY-ESO-1–specific CD8+ and CD4+ T cells from peripheral blood lymphocytes ofcancer patients in vitro. Oral administration of S. typhimurium–NY-ESO-1 to mice resulted in the regression of established NY-ESO-1–expressing tumors. Intratumoral inoculation of S. typhimurium–NY-ESO-1 to NY-ESO-1–negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1–specific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimuriumtype III secretion system is a promising novel strategy for cancer vaccine development.
Hiroyoshi Nishikawa, Eiichi Sato, Gabriel Briones, Li-Mei Chen, Mitsutoshi Matsuo, Yasuhiro Nagata, Gerd Ritter, Elke Jäger, Hideki Nomura, Shigeto Kondo, Isao Tawara, Takuma Kato, Hiroshi Shiku, Lloyd J. Old, Jorge E. Galán, Sacha Gnjatic
Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose–escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-γ. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma.
Howard L. Kaufman, Gail DeRaffele, Josephine Mitcham, Dorota Moroziewicz, Seth M. Cohen, Karl S. Hurst-Wicker, Ken Cheung, David S. Lee, Joseph Divito, Magalese Voulo, Julie Donovan, Kate Dolan, Kelledy Manson, Dennis Panicali, Ena Wang, Heidi Hörig, Francesco M. Marincola
Current models of T cell memory implicate a critical role for IL-7 in the effector-to-memory transition, raising the possibility that IL-7 therapy might enhance vaccine responses. IL-7 has not been studied, to our knowledge, before now for adjuvant activity. We administered recombinant human IL-7 (rhIL-7) to mice during immunization against the male antigen HY and compared these results with those obtained from mice immunized with rhIL-2 and rhIL-15. Administration of rhIL-7 or rhIL-15, but not rhIL-2, increased effector cells directed against these dominant antigens and dramatically enhanced CD8+ effectors to subdominant antigens. The mechanisms by which the cytokines augmented effector pool generation were multifactorial and included rhIL-7–mediated costimulation and rhIL-15–mediated augmentation of the proliferative burst. The contraction phase of the antigen-specific response was exaggerated in cytokine-treated mice; however, CD8+ memory pools in rhIL-7– or rhIL-15–treated groups demonstrated superior long-term survival resulting in quantitative advantages that remained long after the cytokines were discontinued, as demonstrated by improved survival after challenge with an HY-expressing tumor undertaken several weeks after cytokine cessation. These results confirm the adjuvant activity of rhIL-15 and demonstrate that rhIL-7 also serves as a potent vaccine adjuvant that broadens immunity by augmenting responses to subdominant antigens and improving the survival of the CD8+ T cell memory pool.
Fraia Melchionda, Terry J. Fry, Matthew J. Milliron, Melissa A. McKirdy, Yutaka Tagaya, Crystal L. Mackall
Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14–amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti–P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-γ–positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene β-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.
Stefan Worgall, Anja Krause, Michael Rivara, Kyung-Kim Hee, Enrico V. Vintayen, Neil R. Hackett, Peter W. Roelvink, Joseph T. Bruder, Thomas J. Wickham, Imre Kovesdi, Ronald G. Crystal
DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1α (MIP-1α) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, coadministration of these plasmid cytokines also markedly augmented DNA vaccine--–elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.
Shawn M. Sumida, Paul F. McKay, Diana M. Truitt, Michael G. Kishko, Janelle C. Arthur, Michael S. Seaman, Shawn S. Jackson, Darci A. Gorgone, Michelle A. Lifton, Norman L. Letvin, Dan H. Barouch
A lack of relevant animal models has hampered preclinical screening and critical evaluation of the efficacy of human vaccines in vivo. Carcinoembryonic antigen–A2Kb (CEA–A2Kb) double transgenic mice provide a biologically relevant model for preclinical screening and critical evaluation of human CEA vaccine efficacy in vivo, particularly because such animals are peripherally tolerant of CEA. We established the utility of this model by demonstrating that an oral DNA minigene vaccine induces effective HLA-A2–restricted, CEA-specific antitumor CTL responses. This finding is supported by three lines of evidence: (a) an effective HLA-A2–restricted, CEA691-specific CTL response; (b) specific in vitro killing of CEA-A2Kb transduced MC-38 colon carcinoma cells; and (c) protective immunity induced in vaccinated mice against challenges of these tumor cells. Importantly, peripheral T cell tolerance against CEA in CEA-A2Kb double transgenic mice was broken by the CEA691 (IMIGVLVGV) minigene vaccine. In conclusion, CEA-A2Kb double transgenic mice were demonstrated to be good candidates for in vivo testing of human CEA–based vaccines. This result suggests a potential for these vaccines in future human vaccine development. The feasibility of using nonmutated self-antigens as targets for therapeutic vaccinations was indicated, provided that such antigens are presented in an immunogenic context; that is, as a DNA minigene in a bacterial carrier system.
He Zhou, Yunping Luo, Masato Mizutani, Noriko Mizutani, Jürgen C. Becker, F. James Primus, Rong Xiang, Ralph A. Reisfeld
Intradermal vaccination by gene gun efficiently delivers DNA vaccines into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. DCs, however, have a limited life span, hindering their long-term ability to prime antigen-specific T cells. We reason that a strategy that prolongs the survival of DNA-transduced DCs will enhance priming of antigen-specific T cells and DNA vaccine potency. Here we show that codelivery of DNA encoding inhibitors of apoptosis (BCL-xL, BCL-2, XIAP, dominant negative caspase-9, or dominant negative caspase-8) with DNA encoding model antigens prolongs the survival of transduced DCs. More importantly, vaccinated mice exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors. Among these antiapoptotic factors, BCL-xL demonstrated the greatest enhancement in antigen-specific immune responses and antitumor effects. Thus, coadministration of DNA vaccines with DNA encoding antiapoptotic proteins represents an innovative approach to enhance DNA vaccine potency.
Tae Woo Kim, Chien-Fu Hung, Morris Ling, Jeremy Juang, Liangmei He, J. Marie Hardwick, Sharad Kumar, T.-C. Wu
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