Acceptance testing and clinical assessment of the in-vivo dose measurement EPIgray system / 中国医学装备

Objective:To assess the accuracy of dose reconstruction of the in-vivo dose verification EPIgray system in intensity-modulated radiation therapy(IMRT)and volumetric-modulated arc therapy(VMAT)through acceptance testing,and to explore the preliminarily clinical application of that.Methods:A total of 37 patients with cancer at various parts were selected for clinical testing.Referring to the suggestions of the acceptance manual of manufacturer and the American Association of Physicists in Medicine(AAPM)TG-119 report,the square field,strip field and intensity-modulated plan were adopted to test the precision of dose reconstruction of EPIgray system on phantom.The recognition ability of the system for error was researched through changed the thickness of homogeneous phantom and the skin distance of exposure source.A total of 37 patients with cancer at different parts who underwent VMAT were selected to conduct clinical test,and then,the consistence between the dose reconstruction of EPIgray system and the counted dose of treatment plan system were further analyzed.Results:In the tests of square field,EPIgray dose reconstruction demonstrated excellent linearity and higher accuracy.On the phantoms with different thicknesses of fields with different sizes,the highest precision of dose reconstruction of central axis of field was(0.10±0.39)%.The all precisions of dose reconstruction were within 3.0%besides the built region of dose and field edges.In the tests of IMRT and VMAT plan,the deviation of dose reconstruction was<5.0%.With the increasing of the complexity of plan and the heterogeneity of phantom,there was a slight decrease in the reconstruction precision,but all deviations of dose reconstruction were within the range of allowable deviations.In clinical testing of 37 patients,the average reconstruction deviation of the prescription dose point was(-0.6±4.8)%,and the average deviation of sampling points within the range of target area was(-2.1±2.7)%.The reconstruction deviations outside of field and that with large dose gradient were larger.Conclusion:The dose reconstructions of in-vivo dose verification system EPIgray has better consistency with treatment planning system in calculating dose,and the precision of dose reconstruction can meet the requirement of clinical application.

Production of human papillomavirus type 16 virus-like particles and its immunogenicity / 病毒学报

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.

Establishment and application of human papillomavirus type 16 pseudovirions neutralization assay / 生物工程学报

Human papillomaviruses (HPV) are causally associated with cervical cancer and genital warts. Lack of permissive and productive cell cultures for HPV has hindered the study of HPV and evaluation of virus-neutralizing antibodies. So generation of infectious virions in vitro is highly desirable. In this report, we got high titer infectious HPV16 pseudovirions by calcium phosphate co-transfection of codon optimized HPV16 capsid genes L1 and L2 and reporter plasmids into 293FT cell line. Electron micrograph indicated that the pseudovirions were morphologically similar with the intact HPV16 virions. To evaluate the feasibility of using the pseudovirions to identify neutralizing monoclonal antibodies (mAbs), pseudovirions were incubated with 2-fold gradient dilution of the well identified mAbs V5, E70, U4 and D9 and then used to infect 293FT cells preplated in 96-well tissue culture plate. The infection of pseudovirions could be inhibited by neutralizing mAbs V5, E70 and U4 that recognize surface conformational epitopes on L1 VLP, but not by mAb D9 that is reactive to a linear epitope buried in VLP, which indicated that the pseudovirions could be used to evaluate the neutralization efficiency of mono- and polyclonal antibodies. The pseudovirions were then employed to identify neutralizing mAbs from 18 mAbs generated previously in our lab, 8 of which were conformational and 10 were linear. PD1 and 3D10, both of which recognized conformational epitopes on L1 VLP, had obviously strong neutralizing efficiency, with the neutralizing titer reached 81,920 and 20,480 respectively, while none of the linear mAbs were neutralizing, which reflected that rare linear mAbs have neutralization activity. The mechanism of PD1 and 3D10 block the infection of HPV16 pseudovirions need to be further studied. The technologies about generation of HPV16 pseudovirions and screening neutralizing mAb in our report are economical and efficient, can be easily used in large scale. They pave the way for rapid and precise evaluation of the protection efficiency of our prophylactic HPV vaccine being developed now.