Harald G. Foellmer

Requirement for CD40 Ligand in Costimulation Induction, T Cell Activation, and Experimental Allergic Encephalomyelitis

The mechanism of CD40 ligand (CD40L)-mediated in vivo activation of CD4+ T cells was examined by investigation of the development of experimental allergic encephalomyelitis (EAE) in CD40L-deficient mice that carried a transgenic T cell receptor specific for myelin basic protein. These mice failed to develop EAE after priming with antigen, and CD4+ T cells remained quiescent and produced no interferon-γ (IFN-γ). T cells were primed to make IFN-γ and induce EAE by providing these mice with B7.1+ antigen-presenting cells (APCs). Thus, CD40L is required to induce costimulatory activity on APCs for in vivo activation of CD4+ T cells to produce IFN-γ and to evoke autoimmunity.

Low Molecular Weight Inhibitors in Corneal Ulcerationa

RICHARD E. GALARDYP MARIE E. CASSABONNE, C A R L A ” E GIESE, JAMES H. GILBERT, FRANCE LAPIERRE, HENRY LOPEZ, MARY E. SCHAEFER, ROBERT STACK, MICHAEL SULLIVAN, BRENT SUMMERS, ROB TRESSLER, DAVE TYRRELL, AND JENNIFER WEEC; SCOTT D. ALLEN AND JOHN J. CASTELLO@; JOHN P. BARLETTA AND GREGORY S. SCHULTZe; LEONARD0 A. FERNANDEZf; SUSAN FISHER AND TIAN-YI CUF; HARALD G. FOELLMERh; DAMIAN GROBELNY’; AND WALTER M. HOLLERANJ

A West Nile Virus Recombinant Protein Vaccine That Coactivates Innate and Adaptive Immunity

A chimeric protein West Nile virus (WNV) vaccine capable of delivering both innate and adaptive immune signals was designed by fusing a modified version of bacterial flagellin (STF2 Delta ) to the EIII domain of the WNV envelope protein. This fusion protein stimulated interleukin-8 production in a Toll-like receptor (TLR)-5-dependent fashion, confirming appropriate in vitro TLR5 bioactivity, and also retained critical WNV-E-specific conformation-dependent neutralizing epitopes as measured by enzyme-linked immunosorbent assay. When administered without adjuvant to C3H/HeN mice, the fusion protein elicited a strong WNV-E-specific immunoglobulin G antibody response that neutralized viral infectivity and conferred protection against a lethal WNV challenge. This potent EIII-specific immune response requires a direct linkage of EIII to STF2 Delta , given that a simple mixture of the 2 components failed to induce an antibody response or to provide protection against virus challenge. The presence of a functional TLR5 gene in vivo is also required--TLR5-deficient mice elicited only a minimal antigen-specific response. These results confirm that vaccines designed to coordinately regulate the innate and adaptive immune responses can induce protective immune responses without the need for potentially toxic adjuvants. They also support the further development of an effective WNV vaccine and novel monovalent and multivalent vaccines for related flaviviruses.

Differential Regulation and Function of the Fas/Fas Ligand System in Human Trophoblast Cells

Abstract Trophoblast rejection, which is characterized by increased apoptosis, is mediated by T helper (Th)-1, or proinflammatory, cytokines, whereas Th-2, or anti-inflammatory, cytokines confer immune protection and facilitate implantation. We investigated the role of both types of cytokines on the expression and function of the Fas/Fas ligand (FasL) apoptotic pathway in trophoblast cells. First-trimester human trophoblast primary-culture cells as well as A3 and HTR/8 trophoblast cell lines were treated with proinflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα) and with the anti-inflammatory cytokines interleukin (IL)-6 and IL-10. Sensitivity to Fas-mediated apoptosis was measured using an activating anti-Fas monoclonal antibody. Cell viability was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and CellTiter 96 assay. Fas/FasL mRNA and protein expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Trophoblast cells normally express FasL, but low levels of Fas, and they are resistant to Fas-mediated apoptosis. IFN-γ and TNFα promote Fas expression and sensitivity, whereas IL-6 and IL-10 increase the resistance of trophoblast cells to Fas-mediated apoptosis. Furthermore, IL-10 treatment activates FLICE-like inhibitory protein (FLIP), a downstream inhibitor of Fas apoptotic signaling. Although trophoblast cells express Fas, susceptibility to Fas does not necessarily correlate with its expression. In this study, we demonstrate that Th-2 cytokines increase the resistance of trophoblast cells to Fas-mediated apoptosis either by inhibiting Fas expression or by inducing FLIP activation. This “trophoblast-cytokine-Fas/FasL triad” determines the ability of the Fas/FasL system to regulate trophoblast viability and, consequently, the success or failure of pregnancy.

Abrogation of macrophage migration inhibitory factor decreases West Nile virus lethality by limiting viral neuroinvasion

The flavivirus West Nile virus (WNV) is an emerging pathogen that causes life-threatening encephalitis in susceptible individuals. We investigated the role of the proinflammatory cytokine macrophage migration inhibitory factor (MIF), which is an upstream mediator of innate immunity, in WNV immunopathogenesis. We found that patients suffering from acute WNV infection presented with increased MIF levels in plasma and in cerebrospinal fluid. MIF expression also was induced in WNV-infected mice. Remarkably, abrogation of MIF action by 3 distinct approaches (antibody blockade, small molecule pharmacologic inhibition, and genetic deletion) rendered mice more resistant to WNV lethality. Mif(-/-) mice showed a reduced viral load and inflammatory response in the brain when compared with wild-type mice. Our results also indicate that MIF favors viral neuroinvasion by compromising the integrity of the blood-brain barrier. In conclusion, the data obtained from this study provide direct evidence for the involvement of MIF in viral pathogenesis and suggest that pharmacotherapeutic approaches targeting MIF may hold promise for the treatment of WNV encephalitis.

Protective and Therapeutic Capacity of Human Single-Chain Fv-Fc Fusion Proteins against West Nile Virus

ABSTRACT West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.

CD62L Is Required on Effector Cells for Local Interactions in the CNS to Cause Myelin Damage in Experimental Allergic Encephalomyelitis

Adhesion molecules are believed to facilitate infiltration of leukocytes into the CNS of mice with experimental allergic encephalomyelitis (EAE). The role of the adhesion molecule CD62L (L-selectin) in the immunopathology of EAE is not known. To study this, we crossed CD62L-deficient mice with myelin basic protein-specific TCR (MBP-TCR) transgenic mice. CD62L-deficient MBP-TCR transgenic mice failed to develop antigen-induced EAE, and, despite the presence of leukocyte infiltration, damage to myelin in the CNS was not seen. EAE could, however, be induced in CD62L-deficient mice upon adoptive transfer of wild-type macrophages. Our results suggest that CD62L is not required for activation of autoimmune CD4 T cells but is important for the final destructive function of effector cells in the CNS and support a novel mechanism whereby CD62L expressed on effector cells is important in mediating myelin damage.

Fusion Loop Peptide of the West Nile Virus Envelope Protein Is Essential for Pathogenesis and Is Recognized by a Therapeutic Cross-Reactive Human Monoclonal Antibody

West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.

Type V Collagens of the Human Placenta: Trimer α-Chain Composition, Ultrastructural Morphology and Peptide Analysis

The alpha-chain trimer composition of type V collagen preparations from human placental membrane and villi was determined by two-dimensional electrophoresis on a non-denaturing polyacrylamide gel system followed by electrophoresis in the presence of sodium dodecylsulfate. In preparations isolated from placental membranes pure type V collagen was found with the alpha-chain composition [alpha 1(V)]2 alpha 2(V). In preparations from placental villi two different collagen trimers could be identified with alpha-chain compositions [alpha 1(V)]2 alpha 2(V) and [alpha 3(V)]3. Two-dimensional peptide maps after chymotryptic digestion of the various alpha-chain revealed distinct patterns for alpha 1(V), alpha 2(V) and alpha 3(V) suggesting unique structures for all three alpha-chains. Shadowing of the two collagen preparations with carbon-platinum by the rotary shadowing technique allowed the visualization of the individual molecules. In the placental membrane preparations, a uniform species of molecules was present while in placental villi preparations the same elongated form of collagen was found together with larger aggregates presumably containing molecules with the alpha 3-chain component. These data are interpreted to indicate that so-called type V collagen, at least in preparations from placental villi, contain two distinct collagen molecules.