Gregory Mullen

Leucine-rich repeats and pathogen recognition in Toll-like receptors

Toll-like receptors (TLRs) are the major cell-surface initiators of inflammatory responses to pathogens. They bind a wide variety of pathogenic substances through their ectodomains (ECDs). Here, we ask: what is the structural basis for this interaction? Toll-like receptor ECDs comprise 19-25 tandem copies of a motif known as the leucine-rich repeat (LRR). No X-ray structure of a TLR-ECD is currently available but there are several high-resolution LRR-containing proteins that can be used to model TLRs. We suggest that the basic framework of TLRs is a horseshoe-shaped solenoid that contains an extensive beta-sheet on its concave surface, and numerous ligand-binding insertions. Together, these insertions and the beta-sheet could provide a binding surface that is 10-fold greater in area than binding surfaces in antibodies and T-cell receptors.

Phase 1 Clinical Trial of Apical Membrane Antigen 1: an Asexual Blood-Stage Vaccine for Plasmodium falciparum Malaria

ABSTRACT Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naïve volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 μg, 20 μg, and 80 μg) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naïve humans, and our results support the further development of this vaccine.

A Randomized Controlled Phase 2 Trial of the Blood Stage AMA1-C1/Alhydrogel Malaria Vaccine in Children in Mali

A double blind, randomized, controlled Phase 2 clinical trial was conducted to assess the safety, immunogenicity, and biologic impact of the vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1), adjuvanted with Alhydrogel. Participants were healthy children 2-3 years old living in or near the village of Bancoumana, Mali. A total of 300 children received either the study vaccine or the comparator. No impact of vaccination was seen on the primary endpoint, the frequency of parasitemia measured as episodes >3000/microL/day at risk. There was a negative impact of vaccination on the hemoglobin level during clinical malaria, and mean incidence of hemoglobin <8.5 g/dL, in the direction of lower hemoglobin in the children who received AMA1-C1, although these differences were not significant after correction for multiple tests. These differences were not seen in the second year of transmission.

Phase 1 trial of AMA1-C1/Alhydrogel plus CPG 7909: an asexual blood-stage vaccine for Plasmodium falciparum malaria

Background Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. Methods A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 µg of AMA1-C1/Alhydrogel®+564 µg CPG 7909 (n = 15), 80 µg of AMA1-C1/Alhydrogel® (n = 30), or 80 µg of AMA1-C1/Alhydrogel+564 µg CPG 7909 (n = 30). Results Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 µg or 80 µg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 µg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. Conclusion/Significance The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. Trial Registration ClinicalTrials.gov NCT00344539

Cutting Edge: Histamine Inhibits IFN-α Release from Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (DC) are professional APC and a major source of type I IFN following viral infection. We previously showed that histamine alters the cytokine profiles of maturing monocyte-derived DC resulting in a change from Th1 to Th2 in their T cell polarizing function. In this study, we show that human plasmacytoid DC, activated by either CpG oligodeoxynucleotides or viral infection, also respond to histamine through H2 receptors, leading to a marked down-regulation of IFN-α and TNF-α and a moderate switch in their capacity to polarize naive T cells. Our findings provide an explanation for low levels of type I IFN frequently observed in atopic individuals.

Efficient bifunctional gallium-68 chelators for positron emission tomography: tris(hydroxypyridinone) ligands

A new tripodal tris(hydroxypyridinone) bifunctional chelator for gallium allows easy production of (68)Ga-labelled proteins rapidly under mild conditions in high yields at exceptionally high specific activity and low concentration.

The role of disulfide bonds in the assembly and function of MD-2

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDS/PAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-κB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

The TLR9 Ligand CpG Promotes the Acquisition of Plasmodium falciparum-Specific Memory B Cells in Malaria-Naive Individuals

Despite the central role of memory B cells (MBC) in protective immune responses, little is understood about how they are acquired in naive individuals in response to Ag exposure, and how this process is influenced by concurrent activation of the innate immune system’s TLR. In this longitudinal study of malaria-naive individuals, we examined the MBC response to two candidate malaria vaccines administered with or without CpG, a TLR9 ligand. We show that the acquisition of MBC is a dynamic process in which the vaccine-specific MBC pool rapidly expands and then contracts, and that CpG enhances the kinetics, magnitude, and longevity of this response. We observed that the percentage of vaccine-specific MBC present at the time of reimmunization predicts vaccine-specific Ab levels 14 days later; and that at steady-state, there is a positive correlation between vaccine-specific MBC and Ab levels. An examination of the total circulating MBC and plasma cell pools also suggests that MBC differentiate into plasma cells through polyclonal activation, independent of Ag specificity. These results provide important insights into the human MBC response, which can inform the development of vaccines against malaria and other pathogens that disrupt immunological memory.

National Electrical Manufacturers Association NU-4 Performance Evaluation of the PET Component of the NanoPET/CT Preclinical PET/CT Scanner

The NanoPET/CT represents the latest generation of commercial preclinical PET/CT systems. This article presents a performance evaluation of the PET component of the system according to the National Electrical Manufacturers Association (NEMA) NU-4 2008 standard. Methods: The NanoPET/CT consists of 12 lutetium yttrium orthosilicate:cerium modular detectors forming 1 ring, with 9.5-cm axial coverage and a 16-cm animal port. Each detector crystal is 1.12 × 1.12 × 13 mm, and 1 module contains 81 × 39 of these crystals. An optical light guide transmits the scintillation light to the flat-panel multianode position-sensitive photomultiplier tubes. Analog-to-digital converter cards and a field-programmable gate array–based data-collecting card provide the readout. Spatial resolution, sensitivity, counting rate capabilities, and image quality were evaluated in accordance with the NEMA NU-4 standard. Energy and temporal resolution measurements and a mouse imaging study were performed in addition to the standard. Results: Energy resolution was 19% at 511 keV. The spatial resolution, measured as full width at half maximum on single-slice rebinning/filtered backprojection–reconstructed images, approached 1 mm on the axis and remained below 2.5 mm in the central 5-cm transaxial region both in the axial center and at one-quarter field of view. The maximum absolute sensitivity for a point source at the center of the field of view was 7.7%. The maximum noise equivalent counting rates were 430 kcps at 36 MBq and 130 kcps at 27 MBq for the mouse- and rat-sized phantoms, respectively. The uniformity and recovery coefficients were measured with the image-quality phantom, giving good-quality images. In a mouse study with an 18F-labeled thyroid-specific tracer, the 2 lobes of the thyroid were clearly distinguishable, despite the small size of this organ. The flexible readout system allowed experiments to be performed in an efficient manner, and the system remained stable throughout. Conclusion: The large number of detector crystals, arranged with a fine pitch, results in excellent spatial resolution, which is the best reported for currently available commercial systems. The absolute sensitivity is high over the field of view. Combined with the excellent image quality, these features make the NanoPET/CT a powerful tool for preclinical research.

Phase 1 trial of the Plasmodium falciparum blood stage vaccine MSP142-C1/alhydrogel with and without CPG 7909 in malaria naïve adults.

Background Merozoite surface protein 142 (MSP142) is a leading blood stage malaria vaccine candidate. In order to induce immune responses that cover the major antigenic polymorphisms, FVO and 3D7 recombinant proteins of MSP142 were mixed (MSP142-C1). To improve the level of antibody response, MSP142-C1 was formulated with Alhydrogel plus the novel adjuvant CPG 7909. Methods A Phase 1 clinical trial was conducted in healthy malaria-naïve adults at the Center for Immunization Research in Washington, D.C., to evaluate the safety and immunogenicity of MSP142-C1/Alhydrogel +/− CPG 7909. Sixty volunteers were enrolled in dose escalating cohorts and randomized to receive three vaccinations of either 40 or 160 µg protein adsorbed to Alhydrogel +/− 560 µg CPG 7909 at 0, 1 and 2 months. Results Vaccinations were well tolerated, with only one related adverse event graded as severe (Grade 3 injection site erythema) and all other vaccine related adverse events graded as either mild or moderate. Local adverse events were more frequent and severe in the groups receiving CPG. The addition of CPG enhanced anti-MSP142 antibody responses following vaccination by up to 49-fold two weeks after second immunization and 8-fold two weeks after the third immunization when compared to MSP142-C1/Alhydrogel alone (p<0.0001). After the third immunization, functionality of the antibody was tested by an in vitro growth inhibition assay. Inhibition was a function of antibody titer, with an average of 3% (range −2 to 10%) in the non CPG groups versus 14% (3 to 32%) in the CPG groups. Conclusion/Significance The favorable safety profile and high antibody responses induced with MSP142-C1/Alhydrogel + CPG 7909 are encouraging. MSP142-C1/Alhydrogel is being combined with other blood stage antigens and will be taken forward in a formulation adjuvanted with CPG 7909. Trial Registration ClinicalTrials.gov Identifier: NCT00320658