Meeting report - first discoidin domain receptors meeting.

For the first time, a meeting dedicated to the tyrosine kinase receptors DDR1 and DDR2 took place in Bordeaux, a famous and historical city in the south of France. Over the course of 3 days, the meeting allowed 60 participants from 11 different countries to exchange ideas and their new findings about these unique collagen receptors, focusing on their role in various physiological and pathological conditions and addressing their mechanisms of regulation and signalling. The involvement of these receptors in different pathologies was also considered, with emphasis on cancer development and potential therapeutic applications. Here, we summarize the key elements of this meeting.

A DNA telomerase vaccine for canine cancer immunotherapy.

Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 µg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.

Strong antigen-specific T-cell immunity induced by a recombinant human TERT measles virus vaccine and amplified by a DNA/viral vector prime boost in IFNAR/CD46 mice.

Cancer immunotherapy is seeing an increasing focus on vaccination with tumor-associated antigens (TAAs). Human telomerase (hTERT) is a TAA expressed by most tumors to overcome telomere shortening. Tolerance to hTERT can be easily broken both naturally and experimentally and hTERT DNA vaccine candidates have been introduced in clinical trials. DNA prime/boost strategies have been widely developed to immunize efficiently against infectious diseases. We explored the use of a recombinant measles virus (MV) hTERT vector to boost DNA priming as recombinant live attenuated measles virus has an impressive safety and efficacy record. Here, we show that a MV-TERT vector can rapidly and strongly boost DNA hTERT priming in MV susceptible IFNAR/CD46 mouse models. The cellular immune responses were Th1 polarized. No humoral responses were elicited. The 4 kb hTERT transgene did not impact MV replication or induction of cell-mediated responses. These findings validate the MV-TERT vector to boost cell-mediated responses following DNA priming in humans.

Anticancer DNA vaccine based on human telomerase reverse transcriptase generates a strong and specific T cell immune response.

Human telomerase reverse transcriptase (hTERT) is overexpressed in more than 85% of human cancers regardless of their cellular origin. As immunological tolerance to hTERT can be overcome not only spontaneously but also by vaccination, it represents a relevant universal tumor associated antigen (TAA). Indeed, hTERT specific cytotoxic T lymphocyte (CTL) precursors are present within the peripheral T-cell repertoire. Consequently, hTERT vaccine represents an attractive candidate for antitumor immunotherapy. Here, an optimized DNA plasmid encoding an inactivated form of hTERT, named INVAC-1, was designed in order to trigger cellular immunity against tumors. Intradermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse models elicited broad hTERT specific cellular immune responses including high CD4+ Th1 effector and memory CD8+ T­cells. Furthermore, therapeutic INVAC­1 immunization in a HLA-A2 spontaneous and aggressive mouse sarcoma model slows tumor growth and increases survival rate of 50% of tumor-bearing mice. These results emphasize that INVAC-1 based immunotherapy represents a relevant cancer vaccine candidate.

Long-term maintenance of skin immune system in a NOD-Scid IL2rγ(null) mouse model transplanted with human skin.

We developed a NOD-Scid IL2rγ(null) mouse model transplanted with human skin that brings fundamental insight on in vivo cellular mechanisms of intradermal immunization and antigen presentation by dermal dendritic and epidermal Langerhans cells for skin T-cell immunity. Indeed, T-cell immunity is a crucial checkpoint for the induction of in vivo rapid control of skin infection. With the long-term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin immune response but also to test new compounds and devices for cutaneous routes of vaccination, as well as new therapeutics approach for skin diseases, allergies or infections.

Optimization of a gene electrotransfer procedure for efficient intradermal immunization with an hTERT-based DNA vaccine in mice.

DNA vaccination consists in administering an antigen-encoding plasmid in order to trigger a specific immune response. This specific vaccine strategy is of particular interest to fight against various infectious diseases and cancer. Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues. Here, a gene electrotransfer protocol into the skin has been optimized in mice for efficient intradermal immunization against the well-known telomerase tumor antigen. First, the luciferase reporter gene was used to evaluate gene electrotransfer efficiency into the skin as a function of the electrical parameters and electrodes, either non-invasive or invasive. In a second time, these parameters were tested for their potency to generate specific cellular CD8 immune responses against telomerase epitopes. These CD8 T-cells were fully functional as they secreted IFNγ and were endowed with specific cytotoxic activity towards target cells. This simple and optimized procedure for efficient gene electrotransfer into the skin using the telomerase antigen is to be used in cancer patients for the phase 1 clinical evaluation of a therapeutic cancer DNA vaccine called INVAC-1.

Neutrophils transport antigen from the dermis to the bone marrow, initiating a source of memory CD8+ T cells.

The bone marrow (BM) has been identified as a possible organ for T cell priming, yet the fundamental mechanisms of a polyclonal immune response in the BM remain unknown. We found that after intradermal injection of modified vaccinia Ankara virus, unexpected sources of newly primed polyclonal virus-specific CD8(+), but not CD4(+), T cells were localized in the BM and the draining lymph nodes (dLNs) prior to blood circulation. We identified neutrophils as the virus-carrier cells from the dermis to the BM. In both neutrophil-depleted and Ccr1(-/-) mice, virus-specific BM CD8(+) responses were lost. Myeloid antigen-presenting cells were required for BM CD8(+) T cell priming. A systems biology analysis of dLN and BM virus-specific CD8(+) T cells revealed distinct transcriptional and multifunctional profiles for cells primed in each organ. We provide direct evidence for how antigen is transported to the BM, providing a source of virus-specific memory CD8(+) T cells.

Universal cancer peptide-based therapeutic vaccine breaks tolerance against telomerase and eradicates established tumor.

PURPOSE: To evaluate CD4(+) helper functions and antitumor effect of promiscuous universal cancer peptides (UCP) derived from telomerase reverse transcriptase (TERT). EXPERIMENTAL DESIGN: To evaluate the widespread immunogenicity of UCPs in humans, spontaneous T-cell responses against UCPs were measured in various types of cancers using T-cell proliferation and ELISPOT assays. The humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice were used to study the CD4(+) helper effects of UCPs on antitumor CTL responses. UCP-based antitumor therapeutic vaccine was evaluated using HLA-A*0201-positive B16 melanoma that express TERT. RESULTS: The presence of a high number of UCP-specific CD4(+) T cells was found in the blood of patients with various types of cancer. These UCP-specific T cells mainly produce IFN-γ and TNF-α. In HLA transgenic mice, UCP vaccinations induced high avidity CD4(+) T(H)1 cells and activated dendritic cells that produced interleukin-12. UCP-based vaccination breaks self-tolerance against TERT and enhances primary and memory CTL responses. Furthermore, the use of UCP strongly improves the efficacy of therapeutic vaccination against established B16-HLA-A*0201 melanoma and promotes tumor infiltration by TERT-specific CD8(+) T cells. CONCLUSIONS: Our results showed that UCP-based vaccinations strongly stimulate antitumor immune responses and could be used to design efficient immunotherapies in multiple types of cancers.

Intradermal immunization triggers epidermal Langerhans cell mobilization required for CD8 T-cell immune responses.

The potential of the skin immune system for the generation of both powerful humoral and cellular immune responses is now well established. However, the mechanisms responsible for the efficacy of skin antigen-presenting cells (APCs) during intradermal (ID) vaccination still remain to be elucidated. We have previously demonstrated in clinical trials that preferential targeting of Langerhans cells (LCs) by transcutaneous immunization shapes the immune response toward vaccine-specific CD8 T cells. Others have shown that ID inoculation of a vaccine, which targets dermal APCs, mobilizes both the cellular and humoral arms of immunity. Here, we investigated the participation of epidermal LCs in response to ID immunization. When human or mouse skin was injected ID with a particle-based vaccine, we observed significant modifications in the morphology of epidermal LCs and their mobilization to the dermis. We further established that this LC recruitment after ID administration was essential for the induction of antigen-specific CD8 T cells, but was, however, dispensable for the generation of specific CD4 T cells and neutralizing antibodies. Thus, epidermal and dermal APCs shape the outcome of the immune responses to ID vaccination. Their combined potential provides new avenues for the development of vaccination strategies against infectious diseases.

Transcutaneous and intradermal vaccination.

Most vaccines are administered by intramuscular (i.m.) or subcutaneous (s.c.) routes, however, intradermal (i.d.) and transcutaneous (t.c.) techniques are regaining popularity. We will discuss in this review several factors that strongly justify the use of the cutaneous tissues and development of alternative methods for vaccination. That includes (1) our improved knowledge of skin physiology and better understanding of the barrier role of the horny layer, (2) the rationalization for targeting the different cutaneous layers, i.e. the epidermis, dermis, or hypodermis, (3) our advances in knowledge of the skin immune system, especially the plasticity of antigen-presenting cells (APCs) (i.e Langerhans cells (LC), dermal dendritic cells (DDC) and dermal macrophages) in the induction of immune responses, (4) the reduction of antigenic dose for some modes of cutaneous administration, (5) the increased need in needlefree vaccination strategies for developing countries to cope with blood contamination issues. Progress in skin immunization methods and better understanding of skin immunity allow proposing innovative and efficient vaccination strategies against infectious diseases.

Targeting of HIV-p24 particle-based vaccine into differential skin layers induces distinct arms of the immune responses.

Skin routes of immunization such as subcutaneous (SC), intradermal (ID) and transcutaneous (TC) administration are utilized for vaccination against various pathogens, without understanding their potential impact on the outcome of immune responses. We demonstrated that SC immunization induced HIV-1 p24 specific IgG in absence of antigen-specific CD8 T cells, whereas the ID route induced both cellular and humoral responses. Interestingly, TC application through empty hair follicular ducts, targeting epidermal Langerhans Cells (LCs), induced major CD8 effector cells, in the absence of IgG. However, high levels of mucosal IgA, were localized in the stratified epithelium of the vagina after TC prime. We propose that re-directing the immune responses by targeting differential skin immunization routes, offers enormous potential for innovative vaccination strategies, especially against HIV.

Lentiviral vector-based prime/boost vaccination against AIDS: pilot study shows protection against Simian immunodeficiency virus SIVmac251 challenge in macaques.

AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified, and long-lasting cellular immune responses against human immunodeficiency virus (HIV). Lentiviral vectors have demonstrated efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce nondividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive vesicular stomatitis virus serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, simian immunodeficiency virus (SIV) GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intrarectal challenge with SIVmac251, as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log(10) fold reduction) and by the full preservation of the CD28(+) CD95(+) memory CD4(+) T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 postchallenge. A not-optimized SIV GAG antigen was chosen to show the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection.

Nanoparticle-based targeting of vaccine compounds to skin antigen-presenting cells by hair follicles and their transport in mice.

Particle-based drug delivery systems target active compounds to the hair follicle and may result in a better penetration and higher efficiency of compound uptake by skin resident cells. As previously proposed, such delivery systems could be important tools for vaccine delivery. In this study, we investigated the penetration of solid fluorescent 40 or 200 nm polystyrene nanoparticles (NPs) as well as virus particles in murine skin to further investigate the efficacy of transcutaneously (TC) applied particulate vaccine delivery route. We demonstrated that 40 and 200 nm NPs and modified vaccinia Ankara (MVA) expressing the green-fluorescent protein penetrated deeply into hair follicles and were internalized by perifollicular antigen-presenting cells (APCs). Fibered-based confocal microscopy analyses allowed visualizing in vivo particle penetration along the follicular duct, diffusion into the surrounding tissue, uptake by APCs and transport to the draining lymph nodes. The application of small particles, such as ovalbumin coding DNA or MVA, induced both humoral and cellular immune responses. Furthermore, TC applied MVA induced protection against vaccinia virus challenge. Our results strengthen the concept of TC targeting of cutaneous APCs by hair follicles and will contribute to the development of advanced vaccination protocols using NPs or viral vectors.