A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

A prime-boost strategy using virus-like particles pseudotyped for HCV proteins triggers broadly neutralizing antibodies in macaques.

Chronic hepatitis C virus (HCV) infection, with its cohort of life-threatening complications, affects more than 200 million persons worldwide and has a prevalence of more than 10% in certain countries. Preventive and therapeutic vaccines against HCV are thus much needed. Neutralizing antibodies (NAbs) are the foundation for successful disease prevention for most established vaccines. However, for viruses that cause chronic infection such as HIV or HCV, induction of broad NAbs from recombinant vaccines has remained elusive. We developed a vaccine platform specifically aimed at inducing NAbs based on pseudotyped virus-like particles (VLPs) made with retroviral Gag. We report that VLPs pseudotyped with E2 and/or E1 HCV envelope glycoproteins induced high-titer anti-E2 and/or anti-E1 antibodies, as well as NAbs, in both mouse and macaque. The NAbs, which were raised against HCV 1a, cross-neutralized the five other genotypes tested (1b, 2a, 2b, 4, and 5). Thus, the described VLP platform, which can be pseudotyped with a vast array of virus envelope glycoproteins, represents a new approach to viral vaccine development.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

The class 6 semaphorin SEMA6A is induced by interferon-gamma and defines an activation status of langerhans cells observed in pathological situations.

Originally implicated in axon guidance, semaphorins represent a large family of molecules that are now known to be expressed in the immune system. Among different semaphorins tested by reverse transcriptase-polymerase chain reaction in human immune cells, the expression of class 6 transmembrane semaphorin SEMA6A was restricted to dendritic cells (DCs). Using in-house generated monoclonal antibodies, SEMA6A expression appeared further restricted to Langerhans cells (LCs). In vivo, SEMA6A mRNA was expressed in freshly isolated skin LCs but SEMA6A protein was not detectable on normal skin and tonsillar epithelium. Of interest, SEMA6A protein was strongly expressed on skin and bone LCs and on LCs in draining lymph nodes from patients with LC histiocytosis or dermatopathic lymphadenitis, respectively, representing two inflammatory conditions in which LCs display an immature DC-LAMP(low), CD83(low), and CCR7+ phenotype. SEMA6A expression was low in resting LCs generated in vitro and was enhanced by interferon (IFN)-gamma but not by interleukin-4, interleukin-10, IFN-alpha/beta, or lipopolysaccharide. Most IFN-gamma-induced SEMA6A-positive cells remained immature with low CD83 and DC-LAMP/CD208 expression, but they expressed CCR7 and responded to macrophage inflammatory protein-3beta (MIP-3beta/CCL19). The expression of SEMA6A, for which the ligand and function remain unknown, may therefore identify an alternative IFN-gamma-dependent activation status of LCs in vivo.

Virus overrides the propensity of human CD40L-activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN-{gamma} and Th1 chemokine production.

Depending on the activation status, plasmacytoid dendritic cells (PDC) and myeloid DC have the ability to induce CD4 T cell development toward T helper cell type 1 (Th1) or Th2 pathways. Thus, we tested whether different activation signals could also have an impact on the profile of chemokines produced by human PDC. Signals that induce human PDC to promote a type 1 response (i.e., viruses) and a type 2 response [i.e., CD40 ligand (CD40L)] also induced PDC isolated from tonsils to secrete chemokines preferentially attracting Th1 cells [such as interferon-gamma (IFN-gamma)-inducible protein (IP)-10/CXC chemokine ligand 10 (CXCL10) and macrophage inflammatory protein-1beta/CC chemokine ligand 4 (CCL4)] or Th2 cells (such as thymus and activation-regulated chemokine/CCL17 and monocyte-derived chemokine/CCL22), respectively. Activated natural killer cells were preferentially recruited by supernatants of virus-activated PDC, and supernatants of CD40L-activated PDC attracted memory CD4(+) T cells, particularly the CD4(+)CD45RO(+)CD25(+) T cells described for their regulatory activities. It is striking that CD40L and virus synergized to trigger the production of IFN-gamma by PDC, which induces another Th1-attracting chemokine monokine-induced by IFN-gamma/CXCL9 and cooperates with endogenous type I IFN for IP-10/CXCL10 production. In conclusion, our studies reveal that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that virus converts the CD40L-induced Th2 chemokine patterns of PDC into a potent Th1 mediator profile through an autocrine loop of IFN-gamma.

A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells.

Dendritic cells (DC) produce interleukin-12 (IL-12) in response to Toll-like receptor (TLR) activation. Two major TLR signaling pathways participate in the response to pathogens: the nuclear factor-kappaB (NF-kappaB)-dependent pathway leading to inflammatory cytokine secretion including IL-12 and the interferon (IFN)-dependent pathway inducing type I IFN and IFN-regulated genes. Here we show that the two pathways cooperate and are likely both necessary for inducing an optimal response to pathogens. R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly(I:C) (TLR3 ligand) or lipopolysaccharide (LPS; TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-beta mRNA accumulation by mouse bone marrow-derived DC (BM-DC). Strikingly, IL-12p70 but not IL-12p40 secretion was strongly reduced in BM-DC from STAT1(-/-) and IFNAR(-/-) mice. STAT1 tyrosine-phosphorylation, IL-12p35, and IFN-beta mRNA accumulation were strongly inhibited in IFNAR(-/-) BM-DC activated with the TLR ligand combinations. Similar observation were obtained in human TLR8-expressing monocyte-derived DC (moDC) using neutralizing anti-IFNAR2 antibodies, although results also pointed to a possible involvement of IFN-lambda1 (also known as IL-29). This suggests that TLR engagement on DC induces endogenous IFNs that further synergize with the NF-kappaB pathway for optimal IL-12p70 secretion. Moreover, analysis of interferon regulatory factors (IRF) regulation in moDC suggests a role for IRF7/8 in mediating IRF3-independent type I IFN and possibly IL-12p35 synthesis in response to TLR7/8.

Human dendritic cells express neuronal Eph receptor tyrosine kinases: role of EphA2 in regulating adhesion to fibronectin.

Eph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+ progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a beta1 integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking.