Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Brachytherapy workflow for locally advanced cervical cancer: A survey of Canadian Medical Physicists.

PURPOSE: To report on brachytherapy (BT) workflows for image-based treatments of locally advanced cervical cancer (CC) in Canada. METHODS: Medical Physicists in every Canadian cancer center were contacted and those with a CC-BT program were emailed a 44-item electronic questionnaire surveying workflow patterns including: fractionation schedules, prescription, equipment, imaging, and treatment delivery. RESULTS: Of 47 centers contacted, all 34 who performed CC-BT participated in the survey. Brachytherapy boost, following external beam treatments, was delivered using high-dose-rate (HDR); one center also used pulsed-dose-rate. Intracavitary and/or interstitial treatments were done in 47% centers for 25-80% of their patients. All centers used image-based planning: CT (32%), CT planned with MRI for contouring (47%), MRI (18%), or cone beam CT (3%). For those performing volume-based planning (74%), the contours commonly included Clinical Target Volume (CTV)-High Risk (HR), CTV-Intermediate Risk, rectum, sigmoid, and bladder. The most common HDR dose-fractionation schedule was 7 [4.6 - 10] Gy in 4 [3 - 6] fractions with radiobiological dose prescriptions performed in 62% centers. Medical physics contribution was significant during most activities along the BT treatment pathway in all centers, especially in planning (88%), second checks (68%), and during treatment delivery (88%). CONCLUSIONS: Compared to previous surveys, there is an increasing trend in the use of image-based volumetric planning, interstitial procedures, and radiobiological dose prescription. Cervical cancer brachytherapy in Canada is becoming more streamlined with the use of international practice guidelines. Involvement of medical physicists is vital to all stages of CC-BT, including program implementation, routine quality control, dosimetry, and treatment delivery.

A two-step scheme for distortion rectification of magnetic resonance images.

The aim of this work is to demonstrate a complete, robust, and time-efficient method for distortion correction of magnetic resonance (MR) images. It is well known that MR images suffer from both machine-related spatial distortions [gradient nonlinearity and main field (B0) inhomogeneity] and patient-related spatial distortions (susceptibility and chemical shift artifacts), and growing interest in the area of MR-based radiotherapy treatment planning has put new requirements on the geometric accuracy of such images. The authors present a two-step method that combines a phantom-based reverse gradient technique for measurement of gradient nonlinearities and a patient-based phase difference mapping technique for measurement of B0 inhomogeneities, susceptibility, and chemical shift distortions. The phase difference mapping technique adds only minutes to the total patient scan time and can be used to correct a variety of images of the same patient and anatomy. The technique was tested on several different phantoms, each designed to isolate one type of distortion. The mean distortion was reduced to 0.2 +/- 0.1 mm in both gradient echo and spin echo images of a grid phantom. For the more difficult case of a highly distorted echo planar image, residual distortion was reduced to subvoxel dimensions. As a final step, the technique was implemented on patient images. The current technique is effective, time efficient, and robust and provides promise for preparing distortion-rectified MR images for use in MR-based treatment planning.

Characterization, prediction, and correction of geometric distortion in 3 T MR images.

The work presented herein describes our methods and results for predicting, measuring and correcting geometric distortions in a 3 T clinical magnetic resonance (MR) scanner for the purpose of image guidance in radiation treatment planning. Geometric inaccuracies due to both inhomogeneities in the background field and nonlinearities in the applied gradients were easily visualized on the MR images of a regularly structured three-dimensional (3D) grid phantom. From a computed tomography scan, the locations of just under 10 000 control points within the phantom were accurately determined in three dimensions using a MATLAB-based computer program. MR distortion was then determined by measuring the corresponding locations of the control points when the phantom was imaged using the MR scanner. Using a reversed gradient method, distortions due to gradient nonlinearities were separated from distortions due to inhomogeneities in the background B0 field. Because the various sources of machine-related distortions can be individually characterized, distortions present in other imaging sequences (for which 3D distortion cannot accurately be measured using phantom methods) can be predicted negating the need for individual distortion calculation for a variety of other imaging sequences. Distortions were found to be primarily caused by gradient nonlinearities and maximum image distortions were reported to be less than those previously found by other researchers at 1.5 T. Finally, the image slices were corrected for distortion in order to provide geometrically accurate phantom images.

A novel method for detection of IFN-lambda 3 binding to cells for quantifying IFN-lambda receptor expression.

Type III interferons (IFN-lambdas) are important antiviral cytokines that also modulate immune responses acting through a unique IFN-λR1/IL-10R2 heterodimeric receptor. Conflicting data has been reported for which cells express the IFN-λR1 subunit and directly respond to IFN-λs. In this study we developed a novel method to measure IFN-λ3 binding to IFN-λR1/IL-10R2 on the surface of cells and relate this to a functional readout of interferon stimulated gene (ISG) activity in various cell lines. We show that Huh7.5 hepatoma cells bind IFN-λ3 at the highest levels with the lowest Kd(app), translating to the highest induction of various ISGs. Raji and Jurkat cell lines, representing B and T cells, respectively, moderately bind IFN-λ3 and have lower ISG responses. U937 cells, representing monocytes, did not bind IFN-λ3 well and therefore, did not have any ISG induction. Importantly, knockdown of IFNLR1 in Huh7.5 cells decreased our binding signal proportionally and reduced ISG induction by up to 93%. IFN-λ3 responsiveness increased over time with maximal ISG responses seen at 24h for all but one gene. These data confirm our new IFN-λ3 binding assay can be used to quantify IFN-λ receptor surface expression on a variety of cell types and reflects IFN-λ3 responsiveness.