Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals.

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Efficient stimulation of T cell responses by human IFN-alpha-induced dendritic cells does not require Toll-like receptor triggering.

Dendritic cells (DC) can be activated by proinflammatory cytokines or upon toll-like receptor (TLR) triggering. These stimuli induce specific patterns of phenotypic modulation and gene expression profiles. We investigated whether TLR triggering represents an indispensable requirement for the induction of T cell responses by human DC generated upon culture of monocytes in the presence of granulocyte macrophage colony-stimulating factor and interferon-alpha (IFN-DC). As model stimulator we chose imidazoquinolone (3M-001), a synthetic TLR7 agonist used in the treatment of skin infections and tumors and as experimental adjuvant. At difference with DC generated upon culture of monocytes in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL-4) (IL-4-DC), IFN-DC display a semimature phenotype. Furthermore, IFN-DC, but not IL-4-DC are able to induce CD4+ and CD8+ T cell responses, in steady state, for example, in the absence of TLR triggering. 3M-001 treatment induces up-regulation of the surface expression of costimulatory molecules and "de novo" production of IL-12 and IL-6 in IFN-DC. However, TLR7 triggering fails to significantly enhance the capacity of IFN-DC to induce antigen-specific cytotoxic T lymphocytes and to stimulate allogeneic CD4+ T cells. These data indicate that TLR engagement and IL-12 production do not represent indispensable prerequisites for optimal antigen-presenting cell function in IFN-DC, qualifying these cells as powerful cellular reagents of potential use in active specific immunotherapy.

Heterologous prime-boost immunotherapy of melanoma patients with Influenza virosomes, and recombinant Vaccinia virus encoding 5 melanoma epitopes and 3 co-stimulatory molecules. A multi-centre phase I/II open labeled clinical trial.

To the exception of early stages of disease, the morbidity and mortality of melanoma is considerable, with no acknowledged therapeutic options beyond surgery. Immunotherapy of melanoma has achieved some success, but further refinements are urgently needed in order to realize its potential. This paper describes a multi-centre phase I/II open labeled, controlled clinical trial investigating 2 innovative immunotherapeutic reagents. Two successive groups of 20 resected AJCC stages IIb-IV melanoma patients will be treated, first with melanoma epitopes included into Influenza virosomes (group 1), and second with a heterologous prime-boost protocol priming with a recombinant Vaccinia virus, and boosting with Influenza virosomes (group 2). Five melanoma epitopes from three different melanoma differentiation antigens were included into Influenza virosomes, that cross-stimulate CD4+ T cells and are endowed with high adjuvant capacity in the generation of CTL. The same five melanoma epitopes, two co-stimulatory molecules CD80 and CD86, and the CD40 ligand, a marker known to play a crucial role in CTL generation and memory maintenance were encoded in a recombinant Vaccinia virus. GM-CSF will be administered as a supporting cytokine. Both Influenza virosomes and octo-recombinant Vaccinia virus are innovative and original constructs assessed for the first time in human. Immunotherapy foresees 12 weekly immunizations for each group. Toxicity and adverse events will be monitored clinically. Immunological efficacy will be assessed dynamically by ex-vivo multimer analysis, Elispot, and quantitative real-time PCR for up to 3 months following completion of immunotherapy schedule. Disease free survival will be assessed by 4-monthly serial clinic visits, including physical and FDG-PET examinations, for a follow-up time of 2 years. Quality of life will be assessed with a dedicated FACT-BRM 4 questionnaire.

Clinical applications of virosomes in cancer immunotherapy.

Cancer immunotherapy is increasingly accepted as a treatment option for advanced stage disease. The identification of tumour-associated antigens in 1991 has prompted the development of antigen-specific immunotherapeutic strategies for a variety of cancers. Many of them result in some immunological responses in cancer patients; however, clinical results were not observed concomitantly with immunological responses; therefore, further improvements in the field of immunotherapy are urgently needed. Virosomes are lipidic envelopes devoid of genetic information, but which retain the antigenic profile and fusogenic properties from their viral origin. Virosomes are versatile antigen carriers and can be engineered to perform various tasks in cancer immunotherapy. Preclinical data have fostered the development of innovative clinical protocols. Hence, immunopotentiating reconstituted influenza virosomes will be assessed in breast and melanoma immunotherapy, and may contribute to the development of clinically effective cancer vaccines and ultimately improve patient outcomes. The objective of this review is to provide an overview of the potential clinical applications of virosomes as innovative and potentially effective reagents in active specific cancer immunotherapy.

Advanced liposomal vectors as cancer vaccines in melanoma immunotherapy.

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.

Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8+ T cells induced by TCR triggering or homeostatic cytokines.

Production of indoleamine 2,3-dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L-kynurenine (L-kyn) and 3-hydroxyanthranilic acid (3-HAA) on survival and antigen-dependent and independent proliferation of CD8(+) cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8(+) T cells. Non toxic concentrations of L-kyn or 3-HAA did not significantly inhibit antigen-specific CTL responses. However, 3-HAA, but not L-kyn markedly inhibited antigen-independent proliferation of CD8(+) T cells induced by common receptor gamma-chain cytokines IL-2, -7 and -15. Our data suggest that CD8(+) T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3-HAA profoundly affects homeostatic proliferation of CD8(+) T cells.

Efficient induction of tumoricidal cytotoxic T lymphocytes by HLA-A0201 restricted, melanoma associated, L(27)Melan-A/MART-1(26-35) peptide encapsulated into virosomes in vitro.

Cancer immunotherapy requires the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) specific for tumor associated antigens (TAA). While a number of TAA have been identified, there is an urgent need for the development of adjuvants capable of stimulating CTL responsiveness. Previously, we reported the capacity of immunopotentiating reconstituted influenza virosomes (IRIV) to enhance CTL responses specific for synthetic peptides simultaneously added to cultures in soluble form. This effect was based on IRIV mediated activation of CD4(+) T cells. Here we investigated the "in vitro" immunogenicity of a novel virosome formulation coupling in a single reagent the adjuvant power of IRIV to the capacity of liposomes to efficiently encapsulate synthetic peptides. As a model epitope we chose L(27)Melan-A/Mart-1(26-35) HLA-A0201 restricted peptide from a melanoma-associated antigen widely used in tumor immunotherapy. The reagent thus developed induced the proliferation of CD4(+) T cells characterized by a T helper 1 cytokine profile and CXCR3 expression. Most importantly, it significantly enhanced the generation of L(27)Melan-A/Mart-1(26-35) specific CTL, as compared to soluble peptides, in particular at low nominal epitope concentrations (<1 microg/ml). These effector cells were able to efficiently kill HBL melanoma cells expressing Melan-A/MART-1 and HLA-A0201. The adjuvant effects observed were also detectable in the absence of CD4(+) T cells. Taken together our results suggest that this highly immunogenic antigenic formulation might qualify for clinical use in active, antigen-specific, melanoma immunotherapy.

Selective responsiveness to common gamma chain cytokines in peripheral blood-derived cytotoxic T lymphocytes induced by Melan-A/MART-1(27-35)targeted active specific immunotherapy.

We have comparatively evaluated the proliferative response of CTL induced in metastatic melanoma patients upon immunization against Melan-A/MART-1(27-35) tumor associated antigen (TAA) to IL-2, IL-7 or IL-15 cytokines, sharing a receptor common gamma-chain (c gamma-c cytokines). Twenty-eight CTL clones were generated from CD8+ T cells obtained from 3 patients during the contraction phase of immune response following a successful vaccine mediated expansion of specific effectors. All clones were able to kill tumor cell lines expressing HLA-A0201 and Melan-A/MART-1, and displayed phenotypic characteristics of effector/memory (CD45RA-/CCR7-) or CD45RA+/CCR7- effector cells in intermediate to late developmental stage (CD28-/CD276+/-) CTL. Proliferative responses could be elicited or enhanced by IL-2 and IL-15, but not IL-7, in the absence or in the presence of T-cell receptor (TCR) triggering, respectively. Accordingly, only IL-2 and IL-15 were able to promote the survival of the CTL clones under investigation. While all clones expressed high amounts of receptor c gamma-c (CD132), lower, but detectable, expression of IL-7 receptor alpha chain was also observed. CD8+ cells from one of the patients treated were obtained 6 months after the last vaccine boost and were cultured in the presence of Melan-A/MART-1(27-35) and each of the 3 cytokines under investigation. Consistent with data from CTL clones, expansion of Melan-A/MART-1(27-35) tetramer positive cells was only observed in the presence of IL-2 or IL-15 but not IL-7. Instead, when CD8+ cells from the same patient were sampled shortly (14 days) after an additional vaccination only IL-2 was able to promote the expansion of Melan-A/MART-1(27-35) tetramer positive cells. Taken together these data suggest a selective responsiveness of TAA-specific CTL to different c gamma-c cytokines.

Degradation of the tumor antigen epitope gp100(280-288) by fibroblast-associated enzymes abolishes specific immunorecognition.

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.

Influenza virosomes enhance class I restricted CTL induction through CD4+ T cell activation.

Immunopotentiating reconstituted influenza virosomes (IRIV) are one of the few adjuvants currently licensed for human use. While their adjuvant capacity in the induction of humoral responses is clearly documented, few data exist on their effects on T cell immune response. Here we addressed IRIV adjuvance in the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) in vitro. Lymphocyte stimulation with IM(58-66) and IRIV resulted in marked expansion of specific CTL as compared to cultures performed in the presence of either antigen alone or antigen and control liposomes (L). Studies addressing underlying adjuvant mechanisms demonstrated that IRIV activated CD4/CD45RO+ T cells, induced a cytokine profile consistent with T helper 1 (Th1) stimulation and increased the percentage of CD4+ T cells expressing CXCR3. Furthermore, supernatants from IRIV stimulated PBMC cultures promoted dendritic cell maturation. Most importantly, IRIV mediated CTL adjuvance required the presence of live CD4+ T cells. Powerful adjuvant effects of IRIV were also observed in the induction of CTL specific for the melanoma associated Melan-A/MART-1(27-35), HLA-A0201 restricted epitope. Taken together these findings indicate that IRIV are endowed with a high adjuvant capacity for HLA class I restricted CTL induction, largely attributable to their ability to antigenically stimulate CD4+ T cells.

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.