Showing papers by "Trevor Douglas published in 2013"

Biomimetic Antigenic Nanoparticles Elicit Controlled Protective Immune Response to Influenza

Abstract: Here we present a biomimetic strategy toward nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus-like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multistrain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response.

P22 Viral Capsids as Nanocomposite High-Relaxivity MRI Contrast Agents

Abstract: Attachment of multiple chelated Gd3+ ions to the interior of bacteriophage P22 viral capsids affords nanoscale MRI contrast agents with extremely high relaxivity values. Highly fenestrated “wiffleball” morphology is unique to P22 and assures water exchange between the environment and interior cavity of the capsid. The cavity of P22 “wiffleball” was functionalized with a branched oligomer comprising multiple DTPA–Gd complexes resulting in an impressive payload of 1,900 Gd3+ ions inside each 64 nm capsid. High relaxivities of r1,ionic = 21.7 mM–1 s–1 and r1,particle = 41,300 mM–1 s–1 at 298 K, 0.65 T (28 MHz) are reported, with r1/r2 ratio of 0.80 and optimized rotational correlation time for this system. Specific design modifications are suggested for future improvements of viral capsid-based MRI contrast agents directed toward clinical translation.

Stabilizing viral nano-reactors for nerve-agent degradation

Abstract: Virus like particles, and other naturally occurring protein containers, have emerged as excellent building blocks for nanomaterials design and synthesis. Here we exploit a directed assembly and encapsulation approach to sequester multiple copies of a phosphotriesterase (PTE) enzyme within the capsid of bacteriophage P22. Phosphotriesterase, from Brevundimonas diminuta, is an intriguing enzyme as it is highly active against a wide range of harmful insecticides and nerve agents such as Soman and Sarin. However, difficulty in expressing large quantities of the active recombinant enzyme has limited efforts to scale-up its use. Additionally, as a mesophilic enzyme its low heat tolerance and susceptibility to proteolysis makes it a less than ideal candidate as a practical bioremediation tool. Through encapsulation of the PTE within the P22 capsid, we demonstrate a greatly enhanced thermal tolerance of the enzyme, maintaining 50% of its activity to 60 °C. Additionally, the P22 capsid confers protection to the enzyme from proteases, as well as stabilizing the enzyme against desiccation. Thus, our engineered P22 encapsulation system greatly enhances the stability of the mesophilic phosphotriesterase and results in a robust and active nanoparticle reactor.

Inducible Bronchus-Associated Lymphoid Tissue (iBALT) Synergizes with Local Lymph Nodes during Antiviral CD4+ T Cell Responses

Abstract: Background: Exposure of the lungs to an antigen or pathogen elicits the formation of lymphoid satellite islands termed inducible bronchus-associated lymphoid tissue (iBALT) However, little is known about how the presence of iBALT, induced by a stimulus unrelated to the subsequent challenge agent, influences systemic immunity in distal locations, whether it be independently, antagonistically, or synergistically Here, we determined the kinetics of the influenza-specific responses in the iBALT, tracheobronchial lymph node (TBLN), and spleen of mice with and without pre-formed iBALT Methods and Results: Mice with VLP-induced iBALT or no pre-formed iBALT were challenged with influenza We found that, as we have previously described, those mice whose lungs contained pre-formed iBALT were protected from morbidity, and furthermore, that these mice had increased dendritic cell, and alveolar macrophage accumulation in both the iBALT and TBLNs This translated to similarly accelerated kinetics and intens

Biophysical chemistry: unravelling capsid transformations.

Abstract: The interactions between a virus capsid and its cargo are essential for viral infection as well as in the design of synthetic virus-like particles Now a combination of analytical techniques has unravelled key steps in the transformation of a model virus and the release of its RNA cargo

High-Gd-Payload P22 protein cage nanoparticles for imaging vascular inflammation

Abstract: Methods 1) P22: The P22 protein cage (60 nm) is bioengineered with an internal polymer network with amine functional groups allowing incorporation of ~9100 Gd-DTPA molecules per cage via the amine groups (Figure 1: [1]). This provides a per cage relaxivity of 70000 mMs, superior to Gd-DTPA for the equivalent Gd concentration. 2) Atherosclerosis Models: Both ApoE-deficient (ApoE) and FVB mice were used. ApoE mice develop atherosclerosis enhanced by high-fat diet. FVB mice develop macrophage-rich carotid lesions with carotid ligation in combination with high-fat diet and diabetes induction [2]. 3) P22-polymer-Gd in vivo MR imaging: Mice were injected intravenously with P22-polymer-Gd (N=5, 20 μmol Gd/kg, one-fifth the typical clinical dose) or Magnevist (N=1, 20 μmol Gd/kg). Vascular MRA at 1T was performed (Aspect M2, 500 mT/m, 2500 T/m/s) using 3D-SPGR (TR/TE=12 ms/2.1 ms, slice thickness=1 mm, FOV=5 cm, matrix=128x128, FA=45). Vessel wall MRI at 3T was performed (Signa HDx, GE Healthcare, 50mT/m, 150 T/m/s) with a phased-array mouse coil (RAPID MR International), using a double inversion recovery fast spin echo sequence (TR/TE= 400 ms/15 ms, slice thickness=1mm, FOV=3 cm, matrix= 256x256) up to 24 hours after injection. 4) RGD-targeted P22 ex vivo fluorescence imaging: Molecular targeting of P22 was evaluated by attaching RGD peptides externally, which targets the aVb3 integrin, upregulated on activated macrophages. ApoE mice (N=4) were injected intravenously with RGDP22 or RGD P22 (labeled with Cy5.5, 4 nmol/mouse). Forty-eight hours later, ex vivo fluorescence imaging was performed using Maestro imaging system (Cri, Woburn, MA). Maximum plaque signal intensities were measured and compared.