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Vaccines that can coordinately induce multi-epitope T cell–mediated immunity, T helper functions, and immunologic memory may offer effective tools for cancer immunotherapy. Here, we report the development of a new class of recombinant protein cancer vaccines that deliver different CD8+ and CD4+ T-cell epitopes presented by MHC class I and class II alleles, respectively. In these vaccines, the recombinant protein is fused with Z12, a novel cell-penetrating peptide that promotes efficient protein loading into the antigen-processing machinery of dendritic cells. Z12 elicited an integrated and multi-epitopic immune response with persistent effector T cells. Therapy with Z12-formulated vaccines prolonged survival in three robust tumor models, with the longest survival in an orthotopic model of aggressive brain cancer. Analysis of the tumor sites showed antigen-specific T-cell accumulation with favorable modulation of the balance of the immune infiltrate. Taken together, the results offered a preclinical proof of concept for the use of Z12-formulated vaccines as a versatile platform for the development of effective cancer vaccines. Cancer Res; 75(15); 3020–31. ©2015 AACR.

Legend for Supplementary Figures S1-S7.

Peptides and proteins used in the study, with all the minimal epitopes including 4 flanking residues (S1); Purified proteins were analyzed by Coomassie blue and Western Blot (S2); Z12 transports efficiently its cargo into the cytoplasm of DC (S3); Choice of adjuvant influences proportions of effector and memory phenotype antigen-specific CD8+ T cells induced after Z12-OVALP vaccination (S4); Persistance of antigen-specific cytotoxic T cells after vaccination (S5); Balance of brain tumor infiltrating lymphocytes is altered by Z12-OVALP vaccination (S6); Effector cytokine expression by brain tumor infiltrating CD8+ T cells is augmented by Z13-MultiE2 vaccination (S7).

Mycobacterium tuberculosis (Mtb) represents a major burden to global health, and refined vaccines are needed. Replication-deficient lymphocytic choriomeningitis virus (rLCMV)-based vaccine vectors against cytomegalovirus have proven safe for human use and elicited robust T cell responses in a large proportion of vaccine recipients. Here, we developed an rLCMV vaccine expressing the Mtb antigens TB10.4 and Ag85B. In mice, rLCMV elicited high frequencies of polyfunctional Mtb-specific CD8 and CD4 T cell responses. CD8 but not CD4 T cells were efficiently boosted upon vector re-vaccination. High-frequency responses were also observed in neonatally vaccinated mice, and co-administration of rLCMV with Expanded Program of Immunization (EPI) vaccines did not result in substantial reciprocal interference. Importantly, rLCMV immunization significantly reduced the lung Mtb burden upon aerosol challenge, resulting in improved lung ventilation. Protection was associated with increased CD8 T cell recruitment but reduced CD4 T cell infiltration upon Mtb challenge. When combining rLCMV with BCG vaccination in a heterologous prime-boost regimen, responses to the rLCMV-encoded Mtb antigens were further augmented, but protection was not significantly different from rLCMV or BCG vaccination alone. This work suggests that rLCMV may show utility for neonatal and/or adult vaccination efforts against pulmonary tuberculosis.

Background KISIMATM platform allows the development of protein-based cancer vaccines able to induce a potent, tumor-specific CD8 and CD4 T cells response. While the cell penetrating peptide and peptide agonist for Toll like receptor (TLR)-2 and TLR-4 confer, respectively, the cell delivery and self-adjuvanticity properties, the multiantigenic domain allows the targeting of different cancer antigens, resulting in anti-tumoral efficacy in different murine models. Oncolytic viruses exert their therapeutic effects by a prolonged oncolytic action and the associated intratumoral inflammation as well as general immune activation. Arming oncolytic virus with tumor associated antigens can additionally enhance the tumor-specific T cell portion and therefore positively affect the balance of antitumor versus antiviral immune responses. The protein vaccine KISIMATM and the recombinant oncolytic virus VSV-GP-TAA (vesicular stomatitis virus pseudotyped with LCMV GP expressing tumor-associated antigens) are both promising vaccine candidates that offer a new cancer vaccination opportunity when combined in heterologous prime-boost regimen. Materials and Methods Mice were vaccinated with subcutaneous (s.c.) injection of KISIMA-TAA vaccine and/or with intravenous injection of VSV-GP-TAA in different settings. Immunogenicity was assessed by measuring the peripheral antigen-specific response. Anti-tumoral efficacy as well as in depth monitoring of TILs and tumor microenvironment modulation were assessed following therapeutic vaccination in different tumor models. Additionally, transcriptome and immunohistochemistry analyses of the TC-1 tumor have been performed. Combination of heterologous prime-boost with checkpoint blockade PD-1 therapy has been assessed. Results Priming with KISIMA-TAA followed by VSV-GP-TAA boost induced a large pool of polyfunctional and persistent antigen-specific cytotoxic T cells in the periphery as well as within the tumor in several tumor models. Frequencies of antigen specific T cells are significantly higher than the respective homologous vaccinations. Additionally, transcriptome analysis of a cold tumor model revealed profound changes in the tumor microenvironment upon heterologous vaccination, including a strong upregulation of gene signatures of several pro-inflammatory cytokines and chemokines required for antitumor immunity along with dendritic and T cell trafficking and activation. This was corroborated by flow-cytometric analysis of tumor-infiltrating leukocytes showing massive CD8+ and CD4+ T cell infiltration as well as repolarization of M2-like macrophages towards M1-phenotype. The presence of the CD8+ T cells within the tumor core was confirmed by immunohistochemistry analysis. Moreover, combining heterologous vaccination with checkpoint blockade further improved its therapeutic efficacy and the number of long-term survivors. Conclusions The KISIMA/VSV-GP heterologous prime-boost approach holds great promise for patients with primary or acquired resistance to checkpoint blockade due to its ability to induce tumor-specific T cell, improve T cell infiltration and increase tumor inflammation, even in tumors with limited permissivity for the oncolytic virus. Disclosure Information E. Belnoue: A. Employment (full or part-time); Significant; AMAL Therapeutics SA. K. Das: None. M. Rossi: A. Employment (full or part-time); Significant; AMAL Therapeutics SA. T. Hofer: None. S. Danklmaier: None. T. Nolden: A. Employment (full or part-time); Significant; Viratherapeutics GmbH. L. Schreiber: None. K. Angerer: None. J. Kimpel: None. S. Hoegler: None. L. Kenner: None. D. von Laer: None. K. Elbers: A. Employment (full or part-time); Significant; Viratherapeutics GmbH. G. Wollmann: None. M. Derouazi: A. Employment (full or part-time); Significant; AMAL Therapeutics SA.

Novel immunopreventive strategies are emerging that show great promise for conferring long-term protection to individuals at high risk of developing colorectal cancer. The KISIMA vaccine platform utilizes a chimeric protein comprising: 1) a selected tumor antigen, 2) a cell-penetrating peptide to improve antigen delivery and epitope presentation, and 3) a TLR2/4 agonist to serve as a self-adjuvant. This study examines the ability of a KISIMA vaccine against achaete-scute family bHLH transcription factor 2 (Ascl2), an early colon cancer antigen, to reduce colon tumor formation by stimulating an anti-tumor immune response. Vaccine administrations were well-tolerated and led to circulating antibodies and antigen-specific T cells in a mouse model of colorectal cancer. To assess preventive efficacy, the vaccine was administered to mice either alone or in combination with the immune checkpoint inhibitor anti-PD-1. When delivered to animals prior to colon tumor formation, the combination strategy significantly reduced the development of colon microadenomas and adenomas, as compared to vehicle-treated controls. This response was accompanied by an increase in the intraepithelial density of CD3+ T lymphocytes. Together, these data indicate that the KISIMA-Ascl2 vaccine shows great potential to be a safe and potent immunopreventive intervention for individuals at high risk of developing colorectal cancer.

Background: KISIMA࣪ is a vaccine platform based on a single chimeric fusion protein, containing a proprietary cell-penetrating peptide (CPP) for antigen delivery, a proprietary Toll-like receptor (TLR)-peptide agonist with self-adjuvant properties and a modulable multi-antigenic domain (Mad). ATP128 vaccine targets 3 antigens: carcinoembryonic antigen (CEA), Survivin and Achaete-scute complex homolog 2 (ASCL2); it is used in combination with a PD-1 inhibitor in the treatment of MSS/MMR proficient stage IV colorectal cancer (CRC) patients, after first line of standard of care therapy or as perioperative administration in patients with resectable liver metastases. Method: KISIMA-01 (NCT04046445) is an open-label, multi-center Phase 1b trial to investigate the safety, tolerability and immunogenicity of AT128 alone or in combination with the anti-PD-1 antibody ezabenlimab in patients with stage IV CRC. ATP128 is given SC q2w for the first 3 immunizations (prime) and q4w for the last 3 immunizations (boost). Ezabenlimab is administered q3w starting with the first ATP128 administration. Blood and tissue samples are collected before, during and after ATP128 treatment to monitor the induction of a tumor associated antigen-specific immune response (ELISpot) and immune-related changes in the peripheral blood and in the tumor microenvironment by immunohistochemistry (IHC) and flow cytometry. Results: In more than 50% of evaluated patients treated with ATP128 alone or with ezabenlimab, a cellular immune response against at least one out of three antigens was observed as determined by IFN-γ ELISpot analyses of patient PBMCs after the 3rd vaccination. Analysis of liver metastases by IHC indicated that evaluated patients were positive for all 3 antigens in ATP128. Furthermore, for the patients with paired biopsies, a significant increase in CD8 T cells infiltration into the tumor parenchyma was observed after 3 vaccine administrations, along with a significantly higher proportion of CD45RO expressing memory cells within the CD4 population as compared to baseline. Tumor infiltrating lymphocytes (TILs) flow cytometry analysis comparing untreated (historical controls) and ATP128/ezabenlimab-treated patients showed a similar quantity of the different infiltrated subsets but an improved quality of infiltrated T cells, indicated by an increase (more than 2-fold) in proportion of central memory T cells and an impressive decrease of the proportion of cells positive for exhaustion markers expression in KISIMA-01 patients. Conclusions: Analyses indicate induction of ATP128-specific immune response in the peripheral circulation and increased infiltration of TILs into liver metastases with an improved quality of T cells. Citation Format: Heinz-Josef Lenz, Hanz Prenen, Eric Van Cutsem, Thibaud Kössler, Jean-François Mayol, Francesca Trapani, Matthieu Tihy, Laura Rubbia-Brandt, Christian Toso, Thomas Bogenrieder, Elodie Belnoue, Madiha Derouazi, Scott Kopetz. ATP128 vaccine with ezabenlimab promotes antigen-specific immune responses in stage IV colorectal cancer in the KISIMA-01 phase 1b trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT507.

Novel adjuvants, such as Toll-like receptors (TLRs) agonists, are needed for the development of new formulations able to circumvent limitations of current vaccines. Among TLRs, TLR7/8 agonists represent promising candidates, as they are well described to enhance antigen-specific antibody responses and skew immunity toward T helper (TH) 1 responses. We find here that the incorporation of the synthetic TLR7/8 ligand 3M-052 in a cationic DOEPC-based liposome formulation shifts immunity toward TH1 responses and elicits strong and long-lasting germinal center and follicular T helper cell responses in adult mice. This reflects the prolonged recruitment of innate cells toward the site of immunization and homing of activated antigen-loaded monocytes and monocyte-derived dendritic cells toward draining lymph nodes. We further show that this adjuvanticity is independent of type I IFN but NF-κB-dependent. Overall, our data identify TLR7/8 agonists incorporated in liposomes as promising and effective adjuvants to enhance TH1 and germinal center responses.

Background KISIMATM platform allows the development of protein-based cancer vaccines able to induce a potent, tumor-specific CD8 and CD4 T cells response. While the cell penetrating peptide and the Anaxa portions confer, respectively, the cell delivery and self-adjuvanticity properties, the multiantigenic domain allows the targeting of different cancer antigens, resulting in anti-tumoral efficacy in different murine models.1 The first clinical candidate developed from KISIMATM is currently tested, together with anti-PD-1 blockade, in a phase I study in metastatic colorectal cancer patients. Stimulator of interferon genes agonists (STINGa) were shown to induce a potent type I interferon response in preclinical studies. The intratumoral administration of STINGa, to promote tumor inflammation, was shown to result in a protective spontaneous immune response in several murine tumor models. However, the encouraging preclinical results are not supported by recent clinical data, challenging the efficacy of unspecific monotherapy.As it is more and more clear that an effective cancer immunotherapy will require the combination of different treatment strategies, we investigate here the efficacy of combining KISIMATM cancer vaccine with STINGa treatment. Methods Mice were vaccinated with subcutaneous (s.c.) injection of KISIMATM vaccine combined with s.c. administration of STINGa. Safety and immunogenicity were assessed by measuring temperature, serum cytokines and the peripheral antigen-specific response. Anti-tumoral efficacy as well as in depth monitoring of TILs and tumor microenvironment modulation were assessed following therapeutic vaccination in a HPV16 E6 and E7 expressing TC-1 cold tumor model. Results Combination treatment was well tolerated and promoted the development of circulating antigen-specific CD8 T cells. In TC-1 tumor bearing mice, KISIMATM therapeutic vaccination resulted in the infiltration of both antigen-specific CD8 and CD4 T cells within the tumor, as well as a switch of tumor associated macrophages polarization toward the more inflammatory type 1. Combination therapy further increased the tumor microenvironment modulation induced by KISIMATM vaccine, promoting the polarization of inflammatory Thelper 1 CD4 T cells and increasing the effector function of antigen-specific CD8 T cells. The profound modulation of the tumor microenvironment induced by combination therapy enhanced the beneficial effect of KISIMATM vaccination, resulting in a prolonged tumor control. Conclusions Combination of KISIMATM cancer vaccine with systemic STINGa treatment induces the development of a potent, tumor-specific immune response resulting in a profound modulation of the TME. As check-point inhibitor (CPI) therapy is ineffective on poorly infiltrated tumors, combination with therapies able to highly enhance tumor infiltration by T cells could expand CPI indications. Ethics Approval The study was approved by the Canton of Geneva Ethic Board, under the license number GE165/19 Reference Belnoue E, et al. Targeting self and neo-epitopes with a modular self-adjuvanting cancer vaccine. JCI Insight 2019. 4:11.

Induction of a potent CD4 and CD8 T-cell response against tumor-specific and tumor-associated antigen is critical for eliminating tumor cells. Recent vaccination strategies have been hampered by an inefficacious and low amplitude immune response. Here we describe a self-adjuvanted chimeric protein vaccine platform to address these challenges, characterized by a multidomain construction incorporating (i) a cell penetrating peptide (CPP) allowing internalization of several multiantigenic Major Histocompatibility Complex (MHC)-restricted peptides within (ii) the multiantigenic domain (Mad) and (iii) a TLR2/4 agonist domain (TLRag). Functionality of the resulting chimeric protein is based on the combined effect of the above-mentioned three different domains for simultaneous activation of antigen presenting cells and antigen cross-presentation, leading to an efficacious multiantigenic and multiallelic cellular immune response. Helper and cytotoxic T-cell responses were observed against model-, neo- and self-antigens, and were highly potent in several murine tumor models. The safety and the immunogenicity of a human vaccine candidate designed for colorectal cancer treatment was demonstrated in a non-human primate model. This newly engineered therapeutic vaccine approach is promising for the treatment of poorly infiltrated tumors that do not respond to currently marketed immunotherapies.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.Molecular Therapy (2016); doi:10.1038/mt.2016.134.

Vaccines that can coordinately induce multi-epitope T cell–mediated immunity, T helper functions, and immunologic memory may offer effective tools for cancer immunotherapy. Here, we report the development of a new class of recombinant protein cancer vaccines that deliver different CD8+ and CD4+ T-cell epitopes presented by MHC class I and class II alleles, respectively. In these vaccines, the recombinant protein is fused with Z12, a novel cell-penetrating peptide that promotes efficient protein loading into the antigen-processing machinery of dendritic cells. Z12 elicited an integrated and multi-epitopic immune response with persistent effector T cells. Therapy with Z12-formulated vaccines prolonged survival in three robust tumor models, with the longest survival in an orthotopic model of aggressive brain cancer. Analysis of the tumor sites showed antigen-specific T-cell accumulation with favorable modulation of the balance of the immune infiltrate. Taken together, the results offered a preclinical proof of concept for the use of Z12-formulated vaccines as a versatile platform for the development of effective cancer vaccines. Cancer Res; 75(15); 3020–31. ©2015 AACR.

CD8(+) cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met(+) CTLs). Phenotypic and functional analysis of c-Met(+) CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met(-) CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met(+) CTLs in cell-mediated cytotoxicity reactions in vitro and in vivo and abrogates T-cell responses against metastatic melanoma in vivo Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met(+) CTLs are a naturally occurring CD8(+) T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8(+) T-cell-mediated anti-tumor immunity.

Follicular Th (TFH) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5highPD-1high CD4+ TFH cells that exhibit TFH features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal TFH cells fail to expand and to acquire a full-blown GC TFH phenotype, as reflected by a higher ratio of GC TFH/non-GC CD4+ T cells in immunized adults than neonates (3.8 × 10−3 versus 2.2 × 10−3, p = 0.01). Following the adoptive transfer of naive adult OT-II CD4+ T cells, OT-II TFH cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4+ OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell–intrinsic factors. Postponing immunization to later in life increases the number of TFH cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC TFH and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the TFH cell development limits early life GC responses and that adjuvants/delivery systems supporting TFH differentiation may restore adultlike early life GC B cell responses.

According to commonly held concepts, plasma cell (PC) longevity in bone marrow (BM) depends upon their access to survival niches. These are thought to exist in nursery cell types, which support PCs by secreting PC survival factors. To better define PC survival niches and their functioning, we adoptively transferred traceable Blimp-1-GFP PCs into recipient mice lacking a proliferation-inducing ligand (APRIL), IL-6, or macrophage migration inhibitory factor. Transferred BMPCs were preferentially associated with Ly-6Chigh monocytes (normalized colocalization index: 9.84), eosinophils (4.29), and megakaryocytes (2.12). Although APRIL was essential for BMPC survival, PC recruitment into the proximity of nursery cells was unimpaired in APRIL-deficient mice, questioning the concept that the same factors account for attraction/retention of PCs as for their local survival. Rather, the order of colocalization with BMPCs (monocytes > eosinophils > megakaryocytes) reflected these cells’ relative expression of CXCR4, VLA-4, and LFA-1, the homing and adhesion molecules that direct/retain PCs in the BM. This suggests a scenario wherein the cellular composition of the BMPC niche is defined by a common pattern of attraction/retention on CXCL12-abundant reticular docking cells. Thereby, PCs are directed to associate in a functional BM niche with hematopoietic CXCR4+VLA-4+LFA-1+ nursery cells, which provide PC survival factors.

Cerebral malaria (CM) develops in a small proportion of persons infected with Plasmodium falciparum and accounts for a substantial proportion of the mortality due to this parasite. The actual pathogenic mechanisms are still poorly understood, and in humans investigations of experimental CM are unethical. Using an established Plasmodium berghei-mouse CM model, we have investigated the role of host immune cells at the pathological site, the brain. We report in this study the detailed quantification and characterization of cells, which migrated and sequestered to the brain of mice with CM. We demonstrated that CD8+ αβ T cells, which sequester in the brain at the time when neurological symptoms appear, were responsible for CM mortality. These observations suggest a mechanism which unifies disparate observations in humans.

Members of a multigene family in the rodent malaria parasite Plasmodium yoelii yoelii code for 235-kilodalton proteins (Py235) that are located in the merozoite apical complex, are implicated in virulence, and may determine red blood cell specificity. We show that distinct subsets of py235 genes are expressed in sporozoites and hepatic and erythrocytic stages. Antibodies to Py235 inhibited sporozoite invasion of hepatocytes. The switch in expression profile occurred immediately after transition from one stage to another. The results suggest that this differential expression is driven by strong biological requirements and provide evidence that hepatic and erythrocytic merozoites differ.

The effect of a recombinant hybrid human interferon alpha (IFN-alpha) (which cross-reacts with murine cells) on C57BL/6 mice infected with Plasmodium yoelii sporozoites or parasitized erythrocytes was determined. IFN-alpha did not inhibit the development of the parasite in the liver, but it did reduce the blood parasite load and the hepatosplenomegaly induced by the infection in mice injected with blood-stage parasites. The extent of anemia in IFN-alpha-treated and control mice was similar, despite the lower parasite load in the IFN-alpha-treated mice. The reduced blood parasite load in IFN-alpha-treated mice was associated with reduced erythropoiesis and reticulocytosis. As reticulocytes are the preferred target cells for the strain of P yoelii used (P yoelii yoelii 265 BY), it was postulated that the inhibition of reticulocytosis in IFN-alpha-treated mice was causally related to the observed decreased blood parasite load. This was supported by the finding that IFN-alpha inhibited a different strain of P yoelii (17X clone A), which also displays a tropism for reticulocytes, but not a line of Plasmodium vinckei petteri, which infects only mature red blood cells. As human malaria species also display different tropism for reticulocytes, these findings could be relevant for people coinfected with multiple Plasmodium species or strains or coinfected with Plasmodium and virus. (Blood. 2001;97:3966-3971)

IFN-gamma has been implicated in the pathogenesis of experimental cerebral malaria (ECM). We have used mice lacking the alpha chain of the IFN-gamma receptor (KO mice) to define its role in the pathogenesis of ECM. Infected KO mice did not develop ECM and showed no leukocyte or parasite sequestration in the brain, and no hemorrhages. The resistance of KO mice to ECM was associated with the absence of any increases of TNF-alpha and ICAM-1 proteins in the brain, which are both essential for ECM. Wild-type (WT) mice which do not develop ECM, despite increased local production of TNF-alpha protein, showed no leukocyte accumulation in the brain and this was correlated with the absence of ICAM-1 protein from brain microvessels. KO mice infected with 106 parasitized erythrocytes (PE) of Plasmodium berghei ANKA (PbA) did not develop ECM, but they had high parasitemia and died earlier than WT mice which did not develop ECM. However, KO mice did not develop higher parasitemia than WT mice when both groups were infected with a lower dose (5x10(5) PE) of PbA-infected red blood cells. This indicates that different doses of PE may trigger different IFN-gamma responses and that there may be a threshold concentration for protection against parasitemia.

INFANT MORTALITY FROM VIRAL INFECTION REMAINS A MAJOR GLOBAL HEALTH CONCERN: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8(+) T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC), which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8) is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.

Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4+ and CD8+ T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-γ, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R–dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Viral infections in human infants frequently follow a protracted course, with higher viral loads and delayed viral clearance compared to viral infections in older children. To identify the mechanisms responsible for this protracted pattern of infection, we developed an infant infection murine model using the well-characterized lymphocytic choriomeningitis virus (LCMV) WE strain in 2-week-old BALB/c mice. In contrast to adult mice, in which viral clearance occurred as expected 8 days after infection, LCMV titers persisted for several weeks after infection of infant mice. LCMV-specific effector CD8 + T cells were elicited in infant mice and fully functional on day 7 but rapidly waned and could not be recovered from day 12 onwards. We show here that this results from the failure of LCMV-specific CD8 + T cells to expand and the absence of protective LCMV-specific memory CD8 + T cells. Under these early life conditions, viral control and clearance are eventually achieved only through LCMV-specific B cells that contribute to protect infant mice from early death or chronic infection.

Therapeutic cancer vaccination is an attractive treatment modality for cancer, but with limitations using existing whole-cell, peptide, or protein vaccines. We propose that a cell-penetrating peptide (CPP)-based vaccine delivering multi-epitopic antigens into antigen-presenting cells (APCs) offers great potential to induce an integrated antitumor immune response and robust, sustained therapeutic effect.

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4+ and CD8+ T cells were, however, responsible for the protection through the induction of IFN-γ and NO.

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8+ cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8+ T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-γ-secreting CD8+ splenocytes. Neonatal CD8+ T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c+ dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c+ DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4+ T cell help. The effective induction of neonatal CD8+ T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c+ DC may mature sufficiently to stimulate naive CD8+ neonatal T cells, even in the absence of strong maturation signals.

In order to understand natural resistance to filariasis, we comparedLitomosoides sigmodontisprimary infection of C57BL/6 mice, which eliminate the worms before patency, and BALB/c mice, in which worms complete their development and produce microfilariae. Our analysis over the first month of infection monitoredmigration of the infective larvae from the lymph nodes to the pleural cavity, where the worms settle. Although immune responses from the mouse strains differed from the outset, the duration of lymphatic migration (4 days) and filarial recovery rates were similar, thus confirming that the proportion of larvae that develop in the host species upon infection is not influenced by host genetic variability. The majority of worms reached the adult stage in both mouse strains; however, worm growth and molting were retarded in resistant C57BL/6 mice. Surprisingly, the only immune responses detected at 60 h postinfection occurred in the susceptible mice and only upon stimulation of cells from lymph nodes draining the inoculation site with infective larva extract: massive production of interleukin-6 (IL-6) and IL-5 (the latter cytokine was previously suspected to have an effect onL. sigmodontisgrowth). However, between days 10 and 30 postinfection, extraordinarily high levels of type 1 and type 2 cytokines and expansion of pleural leukocyte infiltration were seen in the resistant C57BL/6 mice, explaining the destruction of worms later. Our results suggest that events early in the infection determine susceptibility or resistance to subsequent microfilarial production and a parasite strategy to use specific immune responses to its own benefit.

Infection withPlasmodium bergheiANKA induces cerebral malaria in susceptible mice. Brain-sequestered CD8+T cells are responsible for this pathology. We have evaluated the role of CCR2, a chemokine receptor expressed on CD8+T cells. Infected CCR2-deficient mice were as susceptible to cerebral malaria as wild-type mice were, and CD8+T-cell migration to the brain was not abolished.

Infection of susceptible mouse strains with Plasmodium berghei ANKA (PbA) is a valuable experimental model of cerebral malaria (CM). Two major pathologic features of CM are the intravascular sequestration of infected erythrocytes and leukocytes inside brain microvessels. We have recently shown that only the CD8+ T-cell subset of these brain-sequestered leukocytes is critical for progression to CM. Chemokine receptor–5 (CCR5) is an important regulator of leukocyte trafficking in the brain in response to fungal and viral infection. Therefore, we investigated whether CCR5 plays a role in the pathogenesis of experimental CM. Approximately 70% to 85% of wild-type and CCR5+/- mice infected with PbA developed CM, whereas only about 20% of PbA-infected CCR5-deficient mice exhibited the characteristic neurologic signs of CM. The brains of wild-type mice with CM showed significant increases in CCR5+ leukocytes, particularly CCR5+ CD8+ T cells, as well as increases in T-helper 1 (Th1) cytokine production. The few PbA-infected CCR5-deficient mice that developed CM exhibited a similar increase in CD8+ T cells. Significant leukocyte accumulation in the brain and Th1 cytokine production did not occur in PbA-infected CCR5-deficient mice that did not develop CM. Moreover, experiments using bone marrow (BM)–chimeric mice showed that a reduced but significant proportion of deficient mice grafted with CCR5+ BM develop CM, indicating that CCR5 expression on a radiation-resistant brain cell population is necessary for CM to occur. Taken together, these results suggest that CCR5 is an important factor in the development of experimental CM.

Item does not contain fulltext Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.

Introduction Cytopathic viruses causing acute infections in immunologically mature hosts often follow a prolonged course in early life, characterized by high viral titers maintained for several weeks. Accordingly, lymphocytic choriomeningitis virus (LCMV, WE strain) runs an acute course in adults but a protracted course in infant BALB/c mice, in which LCMV-specific CD8 + and CD4 + T cells fail to expand and control infection. To identify whether these defects were due to a dysfunction of innate immunity in infant mice, we analyzed early events of viral infection. Methods Type I IFN production was assessed early after LCMV infection of infant (2-week-old) and adult mice by ELISA and real-time PCR. We compared viral titers (by plaque assay) and T cell responses (by FACS) of LCMV-infected infant/adult wild-type and infant/adult IFNAR −/− mice. Activation and function of plasmacytoid and conventional dendritic cells were analyzed by FACS and real-time PCR. The effect of supplementing recombinant IFN-alpha early after infection on viral control and T cell responses was investigated in infant mice. Results Our data showed an insufficient immediate-early IFN-alpha production in infant mice, which fails to support early viral control and the expansion of LCMV-specific T cells: disrupting IFNAR signaling in adult mice mimicked a protracted LCMV infection. Plasmacytoid dendritic cells (pDCs) which are the main source of type I IFNs in LCMV infection failed to acquire an activated phenotype in infant mice and displayed defective function in vivo upon LCMV infection reflected by the low expression of the central pDC transcription factor E2-2 and related genes (TLR7/9, IRF-7 and IRF-8), MyD88 and NF-KappaB. In contrast, the in vitro function of early life pDCs was normal suggesting an in vivo negative regulation of infant pDCs. Direct evidence of the contribution of type I IFNs for early life infection control was demonstrated by given exogenous IFN-alpha which restored virus control and CTL functionality in infant mice. Conclusion In this study, we identify an age-specific down-regulation of multiple factors which are critical for the activation and function of pDCs and demonstrate that it is orchestrated by the E2-2 pDC regulator. We show that this down-regulation prevents the immediate-early burst of type I IFN and the early viral control and permanently impairs CD4 + and CD8 + T cell responses. The results suggest that early life pDC responses are tightly regulated in vivo , possibly to avoid potentially harmful inflammatory or autoimmune reactions. This concept offers novel opportunities for prevention and therapy of infectious complications in early life.

Antibody-producing plasma cells depend on their environment for survival, but the molecules involved in this process are still not well defined. Plasma cells are fully equipped to respond to a proliferation inducing ligand (APRIL) from the tumor necrosis factor (TNF) superfamily, by virtue of their constitutive expression of the B-cell maturation antigen (BCMA), as canonical receptor from the TNF receptor superfamily, and the heparan sulfate proteoglycan (HSPG), CD138, as co-receptor. Here, we report that APRIL promoted the in vitro survival of plasma cells by upregulating expression of several anti-apoptotic molecules, such as bcl-2, bcl-xL and mcl-1. We further observed an in situ localization for APRIL consistent with this pro-survival role, both in mucosa-associated lymphoid tissues (MALT) and the bone marrow. In upper MALT, the tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils, recruited from the blood, infiltrated the crypt epithelium. HSPG retained secreted APRIL in the sub-epithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. The requirement on an inflammatory reaction in niche establishment implies that plasma-cell survival in mucosa is associated to pathogen presence, and must be short as a consequence. We observed also APRIL in the bone marrow. In this latter organ, maturating granulocytes produced constitutively APRIL. Such constitutive expression of a plasma cell pro-survival explains, at least in part, why plasma-cell longevity in the bone marrow can be so long lasting. These in situ human observations were confirmed in vivo with APRIL-deficient mice.

Most types of cancer are difficult to eradicate, and some, like hepatocellular carcinoma, are almost always fatal. Among various interventions to improve the survival of patients with cancer, immunotherapy seems to hold some promises. However, it requires relevant animal models for preclinical development. In this study we report a new and relevant experimental model where liver tumors grow inside a nontumoral parenchyma of adult mice. This model is based on the intrasplenic injection in syngeneic recipient mice of hepatocytes from transgenic mice expressing SV40 large T oncogene specifically in the liver. Using this model where no apparent spontaneous cellular immune response was observed, immunization using a single injection of monoepitopic SV40 T Ag short peptide was sufficient to provoke liver tumor destruction, leading rapidly to complete remission. Tumor regression was associated with the induction of a long-lasting CD8+ T cell response, observed not only in the spleen but also, more importantly, in the tumoral liver. These results show the efficacy of peptide immunotherapy in the treatment of liver cancer.

Most C57BL/6 mice infected i.p. with Plasmodium berghei ANKA (PbA) die between 7 and 14 days with neurologic signs, and the remainder die later (>15 days) with severe anemia. Daily i.p. injections of a recombinant human IFN-α (active on mouse cells) prevented death by cerebral malaria (87% deaths in the control mice vs 6% in IFN-α-treated mice). The mechanisms of this IFN-α protective effect were multiple. IFN-α-treated, PbA-infected mice showed 1) a marked decrease in the number of PbA parasites in the blood mediated by IFN-γ, 2) less sequestered parasites in cerebral vessels, 3) reduced up-regulation of ICAM-1 expression in brain endothelial cells, 4) milder rise of blood levels of TNF, 5) increased levels of IFN-γ in the blood resulting from an increased production by splenic CD8+ T cells, and 6) fewer leukocytes (especially CD8+ T cells) sequestered in cerebral vessels. On the other hand, IFN-α treatment did not affect the marked anemia observed in PbA-infected mice. Survival time in IFN-α-treated mice was further increased by performing three blood transfusions over consecutive days.