Investigation of combined kV/MV CBCT imaging with a high-DQE MV detector.

PURPOSE: Combined kV-MV cone-beam tomography (CBCT) imaging has been proposed for two potentially important image-guided radiotherapy applications: (a) scan time reduction (STR) and (b) metal artifact reduction (MAR). However, the feasibility of these techniques has been in question due to the low detective quantum efficiencies (DQEs) of commercially available electronic portal imagers (EPIDs). The goal of the work was to test whether a prototype high DQE MV detector can be used to generate acceptable quality pretreatment CBCT images at acceptable dose levels. METHODS: 6MV and 100 kVp projection data were acquired on a Truebeam system (Varian, Palo Alto, CA). The MV data were acquired using a prototype EPID containing two scintillators (a) a standard copper-gadolinium oxysulfide (Cu-GOS) screen having a zero-frequency DQE (DQE(0)) value of 1.4%, and (b) a prototype-focused cadmium tungstate (CWO) pixelated "strip" with a DQE(0) = 22%. The kV data were acquired using the standard onboard imager (DQE(0) = 70%). The angular spacing of the MV projections was 0.81° and the source output was 0.03 MU/projection while the kV projections were acquired with an angular spacing of 0.4° at 0.3 mAs/projection. Image quality was evaluated using (a) an 18-cm diameter electron density phantom (CIRS, Norfolk, VA) with nine contrast inserts and (b) the resolution section of the 20-cm diameter Catphan phantom (The Phantom Laboratory, Greenwich, NY). For the MAR studies, two opposing CIRS phantom inserts were replaced by steel rods. The reconstruction methods were based on combining MV and kV data into one sinogram. The MAR reconstruction utilized mostly kV raw data with only those rays corrupted by metal requiring replacement with MV data (total absorbed dose = 0.7 cGy). For the STR study, projections from partially overlapping 105°kV and MV acquisitions were combined to create a complete dataset that could have been acquired in 18 sec (absorbed dose = 2.5 cGy). MV-only (4.3 cGy) and kV-only (0.3 cGy) images were also reconstructed. RESULTS: The average signal-to-noise ratio (SNR) of the inserts in the MV-only CWO and GOS CIRS phantom images were 0.62× and 0.12× the SNR of the inserts in kV-only image, respectively. The limiting spatial resolutions in the MV-only GOS, MV-only CWO, and kV-only Catphan images were 3, 6, and 8 lp/cm, respectively. In the combined kV/CWO STR reconstruction, all contrast inserts were visible while only two were detectable in the kV/Cu-GOS image due to high levels of noise (average SNRs of kV/CWO and kV/GOS inserts were 0.97× and 0.18× the SNR of the kV-only inserts, respectively). In the kV-MV MAR reconstructions, streaking artifacts were substantially reduced with all inserts becoming clearly visible in the kV/CWO image while only two were visible in the kV/Cu-GOS image (average SNRs of the kV/CWO and kV/Cu-GOS CIRS with metal inserts were 0.94× and 0.35× the SNRs of the kV-only CIRS without metal inserts). CONCLUSIONS: We have demonstrated that a high-DQE MV detector can be applied to generating high-quality combined kV-MV images for SRT and MAR. Clinically acceptable doses were utilized.

Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

Molecular adjuvant HMGB1 enhances anti-influenza immunity during DNA vaccination.

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag.

DNA vaccination: antigen presentation and the induction of immunity.

DNA vaccination, or genetic immunization, is a novel vaccine technology that has great potential for reducing infectious disease and cancer-induced morbidity and mortality worldwide. Since their inception, DNA vaccines have been used to stimulate protective immunity against many infectious pathogens, malignancies, and autoimmune disorders in animal models. Plasmid DNA encoding a polypeptide protein antigen is introduced into a host where it enters host cells and serves as an epigenetic template for the high-efficiency translation of its antigen. An immune response, which is mediated by the cellular and/or humoral arms of the immune system and is specific for the plasmid-encoded antigen, ensues. It is thought that "professional" antigen-presenting cells play a dominant role in the induction of immunity by presenting vaccine peptides on MHC class I molecules, following direct transfection or "cross"-presentation, and MHC class II molecules after antigen capture and processing within the endocytic pathway. The correlates of immunity can be manipulated according to many immunization parameters, including the method of vaccine delivery, presence of genetic adjuvants, and vaccine regimen. DNA vaccines first advanced to the clinic five years ago, and the initial picture of their utility in humans is emerging. However, further analysis is required to determine their ultimate efficacy and safety in human beings. This technology has acquired a strong foothold in the field of experimental immunotherapy, and it is hoped that it will eventually represent the next generation of prophylactic and therapeutic vaccines.