A novel adenovirus expressing Flt3 ligand enhances mucosal immunity by inducing mature nasopharyngeal-associated lymphoreticular tissue dendritic cell migration

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.     

Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice

Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.     

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.     

CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1

It has become evident that naturally occurring CD25(+) regulatory T cells (T(reg) cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if T(reg) cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25(+) T(reg) cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8(+) T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8(+) T-cell reactivity in T(reg) cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25(+) T(reg) cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8(+) T-cell memory pool. Moreover, in the challenge experiments, memory CD8(+) T cells generated with plasmid DNA in the absence of T(reg) cells cleared the virus more effectively compared with control groups. We conclude that CD25(+) T(reg) cells quantitatively as well as qualitatively affect the memory CD8(+) T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25(+) T(reg) cells and if it is possible to devise means of limiting T(reg) cell activity to enhance vaccine efficacy.     

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4⁺ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4⁺ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.     

Psychological stress exerts an adjuvant effect on skin dendritic cell functions in vivo

Psychological stress affects the pathophysiology of infectious, inflammatory, and autoimmune diseases. However, the mechanisms by which stress could modulate immune responses in vivo are poorly understood. In this study, we report that application of a psychological stress before immunization exerts an adjuvant effect on dendritic cell (DC), resulting in increased primary and memory Ag-specific T cell immune responses. Acute stress dramatically enhanced the skin delayed-type hypersensitivity reaction to haptens, which is mediated by CD8(+) CTLs. This effect was due to increased migration of skin DCs, resulting in augmented CD8(+) T cell priming in draining lymph nodes and enhanced recruitment of CD8(+) T cell effectors in the skin upon challenge. This adjuvant effect of stress was mediated by norepinephrine (NE), but not corticosteroids, as demonstrated by normalization of the skin delayed-type hypersensitivity reaction and DC migratory properties following selective depletion of NE. These results suggest that release of NE by sympathetic nerve termini during a psychological stress exerts an adjuvant effect on DC by promoting enhanced migration to lymph nodes, resulting in increased Ag-specific T cell responses. Our findings may open new ways in the treatment of inflammatory diseases, e.g., psoriasis, allergic contact dermatitis, and atopic dermatitis.     

Stable Antigen Is Most Effective for Eliciting CD8+ T-Cell Responses after DNA Vaccination and Infection with Recombinant Vaccinia Virus In Vivo

The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.     

Intestinal epithelial antigen induces mucosal CD8 T cell tolerance, activation, and inflammatory response

Intestinal autoimmune diseases are thought to be associated with a breakdown in tolerance, leading to mucosal lymphocyte activation perhaps as a result of encounter with bacterium-derived Ag. To study mucosal CD8(+) T cell activation, tolerance, and polarization of autoimmune reactivity to self-Ag, we developed a novel (Fabpl(4x at -132)-OVA) transgenic mouse model expressing a truncated form of OVA in intestinal epithelia of the terminal ileum and colon. We found that OVA-specific CD8(+) T cells were partially tolerant to intestinal epithelium-derived OVA, because oral infection with Listeria monocytogenes-encoding OVA did not elicit an endogenous OVA-specific MHC class I tetramer(+)CD8(+) T cell response and IFN-gamma-, IL-4-, and IL-5-secreting T cells were decreased in the Peyer's patches, mesenteric lymph nodes, and intestinal mucosa of transgenic mice. Adoptive transfer of OVA-specific CD8(+) (OT-I) T cells resulted in their preferential expansion in the Peyer's patches and mesenteric lymph nodes and subsequently in the epithelia and lamina propria but failed to cause mucosal inflammation. Thus, CFSE-labeled OT-I cells greatly proliferated in these tissues by 5 days posttransfer. Strikingly, OT-I cell-transferred Fabpl(4x at -132)-OVA transgenic mice underwent a transient weight loss and developed a CD8(+) T cell-mediated acute enterocolitis 5 days after oral L. monocytogenes-encoding OVA infection. These findings indicate that intestinal epithelium-derived "self-Ag" gains access to the mucosal immune system, leading to Ag-specific T cell activation and clonal deletion. However, when Ag is presented in the context of bacterial infection, the associated inflammatory signals drive Ag-specific CD8(+) T cells to mediate intestinal immunopathology.     

Concomitant gemcitabine therapy negatively affects DC vaccine-induced CD8+ T-cell and B-cell responses but improves clinical efficacy in a murine pancreatic carcinoma model

Background

Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model.

Materials and methods

Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8⁺ T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively.

Results

Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8⁺ T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8⁺ T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination.

Conclusions

Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.     

Protection from bacterial infection by a single vaccination with replication-deficient mutant herpes simplex virus type 1

Adaptive immune responses in which CD8(+) T cells recognize pathogen-derived peptides in the context of major histocompatibility complex class I molecules play a major role in the host defense against infection with intracellular pathogens. Cells infected with intracellular bacteria such as Listeria monocytogenes, Salmonella enterica serovar Typhimurium, or Mycobacterium tuberculosis are directly lysed by cytotoxic CD8(+) T cells. For this reason, current vaccines for intracellular pathogens, such as subunit vaccines or viable bacterial vaccines, aim to generate robust cytotoxic T-cell responses. In order to investigate the capacity of a herpes simplex virus type 1 (HSV-1) vector to induce strong cytotoxic effector cell responses and protection from infection with intracellular pathogens, we developed a replication-deficient, recombinant HSV-1 (rHSV-1) vaccine. We demonstrate in side-by-side comparison with DNA vaccination that rHSV-1 vaccination induces very strong CD8(+) effector T-cell responses. While both vaccines provided protection from infection with L. monocytogenes at low, but lethal doses, only rHSV-1 vaccines could protect from higher infectious doses; HSV-1 induced potent memory cytotoxic T lymphocytes that, upon challenge by pathogens, efficiently protected the animals. Despite the stimulation of relatively low humoral and CD4-T-cell responses, rHSV-1 vectors are strong candidates for future vaccine strategies that confer efficient protection from subsequent infection with intracellular bacteria.     

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

Oral or nasal antigen induces regulatory T cells that suppress arthritis and proliferation of arthritogenic T cells in joint draining lymph nodes

The propagation of mucosal tolerance as a therapeutic approach in autoimmune diseases remains a difficult goal to achieve, and therefore further mechanistic studies are necessary to develop potential clinical protocols to induce mucosal regulatory T cells (Tr cells). In this study we addressed whether oral or nasal proteoglycan induced functional Tr cells in the cartilage proteoglycan-induced chronic arthritis model. Both nasal and oral application of human proteoglycan before induction of disease suppressed arthritis severity and incidence. Tolerized mice showed enhanced numbers of IL-10 producing CD4(+) cells in the paw-draining lymph nodes. Furthermore, CD4(+) spleen cells displayed enhanced expression of molecules associated with Tr cells, such as IL-10, Foxp3, and TGF-beta. Transfer of CD4(+) spleen cells from mucosally tolerized donors into proteoglycan-immunized mice abolished arthritis and reduced humoral responses, indicative of Tr cells with the capacity to inhibit already induced immune responses. Tr cells were activated upon transfer, because enhanced proliferation was observed in the joint draining lymph nodes compared with activated T cells from nontolerized donors. Upon cotransfer with naive proteoglycan-specific T cells, mucosally induced Tr cells inhibited proliferation of these arthritogenic T cells in vivo. Herein we show that both oral and nasal Ag application induced Tr cells, which had a direct inhibitory effect on already established pathogenic B and T cell responses.     

A Vaccine That Co-Targets Tumor Cells and Cancer Associated Fibroblasts Results in Enhanced Antitumor Activity by Inducing Antigen Spreading

Dendritic cell (DC) vaccines targeting only cancer cells have produced limited antitumor activity in most clinical studies. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To co-target CAFs and tumor cells we developed a new compound DC vaccine that encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2). DC-shA20-FAP-TRP2 vaccination induced robust FAP- and TRP2-specific T-cell responses, resulting in greater antitumor activity in the B16 melanoma model in comparison to monovalent vaccines or a vaccine encoding antigens and a control shRNA. DC-shA20-FAP-TRP2 vaccination enhanced tumor infiltration of CD8-positive T cells, and induced antigen-spreading resulting in potent antitumor activity. Thus, co-targeting of tumor cells and CAFs results in the induction of broad-based tumor-specific T-cell responses and has the potential to improve current vaccine approaches for cancer.     

Enhanced immunoprotective potential of Mycobacterium tuberculosis Ag85 complex protein based vaccine against airway Mycobacterium tuberculosis challenge following intranasal administration

This study examined the role of intranasal vaccination with Mycobacterium tuberculosis antigen85 complex proteins formulated in dimethyldioctadecylammonium bromide against airway Mycobacterium tuberculosis challenge in mice. Intranasal vaccination with antigen85A and antigen85B induced a significantly higher level of interferon-gamma, interleukin-12 and interleukin-4 in cervical lymph nodes together with IgA and IgG, predominantly IgG2a isotype in nasal secretion over subcutaneous vaccination. Further, intranasal vaccination with antigen85A and antigen85B imparted protection comparable with that obtained from intranasal or subcutaneous Mycobacterium bovis bacillus Calmette-Guerin immunization. These results suggest that mucosal vaccination via the intranasal route is of importance in the development of vaccine for tuberculosis.     

Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.     

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.     

New insights in antigen processing and epitope selection: development of novel immunotherapeutic strategies for cancer, autoimmunity and infectious diseases

Current evidence suggests that MHC class II-restricted CD4+ T-cells play a crucial role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Antigens must be processed within endosomal and lysosomal compartments of antigen presenting cells (APC) before binding to MHC class II molecules for display to T-cells. Only a limited number of processed peptides termed immunodominant are selected for display by MHC class II molecules and prove capable of inducing strong T-cell responses. Thus processing reactions within APC are of central importance for the development of effective vaccines as they modulate the number of peptide: class II complexes by enhancing or disrupting epitope formation and display. Studies suggest that there are substantial gaps in our knowledge of how antigen processing and presentation by APC regulates epitope selection and immunodominance in disease situations. Here we describe new insights in antigen processing and epitope selection with relevance to immunotherapeutic strategies for cancer, autoimmunity and infectious diseases.     

Influencing the fates of CD4 T cells on the path to memory: lessons from influenza

In the face of emerging infectious diseases caused by rapidly evolving and highly virulent pathogens, such as influenza, we are challenged to develop innovative vaccine strategies that can induce lasting protection. Since CD4 T cells are needed to generate and maintain protective B-cell and CD8 T-cell immunity, and can also mediate additional protective mechanisms, vaccines should ideally elicit efficient CD4 T cell, in addition to CD8 T and B-cell responses. We outline here the process of CD4 T-cell differentiation from na?ve to effector and from effector to memory with an emphasis on how exposure to microbial products and variables in antigen presentation can impact the functional quality and heterogeneity of activation-based CD4 T-cell subsets in vitro and in vivo. We discuss the impact of different phases of antigen recognition, the inflammatory milieu, acute versus chronic antigen presentation, and the contribution of residual antigen depots on CD4 T-cell effector differentiation and the formation and maintenance of CD4 T-cell memory. We propose that novel vaccine strategies, which incorporate both microbial products and antigen targeting, may provide a flexible and long-lived memory CD4 T-cell pool.