Shuguang Wu

HIV gp41 C-terminal Heptad Repeat Contains Multifunctional Domains RELATION TO MECHANISMS OF ACTION OF ANTI-HIV PEPTIDES

T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623–658), C36 (aa 628–663), CHR-3 (aa 633–668), T20 (aa 638–673), and CHR-5 (aa 643–678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.

ADS-J1 Inhibits Human Immunodeficiency Virus Type 1 Entry by Interacting with the gp41 Pocket Region and Blocking Fusion-Active gp41 Core Formation

ABSTRACT We previously identified a small-molecule anti-human immunodeficiency virus type 1 (anti-HIV-1) compound, ADS-J1, using a computer-aided molecular docking technique for primary screening and a sandwich enzyme-linked immunosorbent assay (ELISA) as a secondary screening method. In the present study, we demonstrated that ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay. Further mechanism studies confirmed that ADS-J1 does not block gp120-CD4 binding and exhibits a marginal interaction with the HIV-1 coreceptor CXCR4. However, ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D peptide, PIE7. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding of ADS-J1 to NHR. These results suggest that ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry.

Maleic anhydride-modified chicken ovalbumin as an effective and inexpensive anti-HIV microbicide candidate for prevention of HIV sexual transmission

BackgroundPrevious studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, β-lactoglobulin (β-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission.ResultsMaleic anhydride (ML), succinic anhydride (SU) and HP at different conditions and variable pH values were used for modification of proteins. All the anhydrate-modified globulin-like proteins showed potent anti-HIV activity, which is correlated with the percentage of modified lysine and arginine residues in the modified protein. We selected maleic anhydride-modified ovalbumin (ML-OVA) for further study because OVA is easier to obtain than β-LG, and ML is safer than HP. Furthermore, ML-OVA exhibited broad antiviral activities against HIV-1, HIV-2, SHIV and SIV. This modified protein has no or low in vitro cytotoxicity to human T cells and vaginal epithelial cells. It is resistant to trypsin hydrolysis, possibly because the lysine and arginine residues in OVA are modified by ML. Mechanism studies suggest that ML-OVA inhibits HIV-1 entry by targeting gp120 on HIV-1 virions and also the CD4 receptor on the host cells.ConclusionML-OVA is a potent HIV fusion/entry inhibitor with the potential to be developed as an effective, safe and inexpensive anti-HIV microbicide.

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

Four-way ligation for construction of a mammalian cell-based full-length antibody display library

A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.

Simultaneous expression of displayed and secreted antibodies for antibody screen.

The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS) analysis, as well as in conditioned medium in a secreted version for function analysis.

MO-DE-207A-11: Sparse-View CT Reconstruction Via a Novel Non-Local Means Method

PURPOSEnSparse-view computed tomography (CT) reconstruction is an effective strategy to reduce the radiation dose delivered to patients. Due to its insufficiency of measurements, traditional non-local means (NLM) based reconstruction methods often lead to over-smoothness in image edges. To address this problem, an adaptive NLM reconstruction method based on rotational invariance (RIANLM) is proposed.nnnMETHODSnThe method consists of four steps: 1) Initializing parameters; 2) Algebraic reconstruction technique (ART) reconstruction using raw projection data; 3) Positivity constraint of the image reconstructed by ART; 4) Update reconstructed image by using RIANLM filtering. In RIANLM, a novel similarity metric that is rotational invariance is proposed and used to calculate the distance between two patches. In this way, any patch with similar structure but different orientation to the reference patch would win a relatively large weight to avoid over-smoothed image. Moreover, the parameter h in RIANLM which controls the decay of the weights is adaptive to avoid over-smoothness, while it in NLM is not adaptive during the whole reconstruction process. The proposed method is named as ART-RIANLM and validated on Shepp-Logan phantom and clinical projection data.nnnRESULTSnIn our experiments, the searching neighborhood size is set to 15 by 15 and the similarity window is set to 3 by 3. For the simulated case with a resolution of 256 by 256 Shepp-Logan phantom, the ART-RIANLM produces higher SNR (35.38dB<24.00dB) and lower MAE (0.0006<0.0023) reconstructed image than ART-NLM. The visual inspection demonstrated that the proposed method could suppress artifacts or noises more effectively and preserve image edges better. Similar results were found for clinical data case.nnnCONCLUSIONnA novel ART-RIANLM method for sparse-view CT reconstruction is presented with superior image. Compared to the conventional ART-NLM method, the SNR and MAE from ART-RIANLM increases 47% and decreases 74%, respectively.

SU-F-I-08: CT Image Ring Artifact Reduction Based On Prior Image

PURPOSEnIn computed tomography (CT) system, CT images with ring artifacts will be reconstructed when some adjacent bins of detector dont work. The ring artifacts severely degrade CT image quality. We present a useful CT ring artifacts reduction based on projection data correction, aiming at estimating the missing data of projection data accurately, thus removing the ring artifacts of CT images.nnnMETHODSnThe method consists of ten steps: 1) Identification of abnormal pixel line in projection sinogram; 2) Linear interpolation within the pixel line of projection sinogram; 3) FBP reconstruction using interpolated projection data; 4) Filtering FBP image using mean filter; 5) Forwarding projection of filtered FBP image; 6) Subtraction forwarded projection from original projection; 7) Linear interpolation of abnormal pixel line area in the subtraction projection; 8) Adding the interpolated subtraction projection on the forwarded projection; 9) FBP reconstruction using corrected projection data; 10) Return to step 4 until the pre-set iteration number is reached. The method is validated on simulated and real data to restore missing projection data and reconstruct ring artifact-free CT images.nnnRESULTSnWe have studied impact of amount of dead bins of CT detector on the accuracy of missing data estimation in projection sinogram. For the simulated case with a resolution of 256 by 256 Shepp-Logan phantom, three iterations are sufficient to restore projection data and reconstruct ring artifact-free images when the dead bins rating is under 30%. The dead-bin-induced artifacts are substantially reduced. More iteration number is needed to reconstruct satisfactory images while the rating of dead bins increases. Similar results were found for a real head phantom case.nnnCONCLUSIONnA practical CT image ring artifact correction scheme based on projection data is developed. This method can produce ring artifact-free CT images feasibly and effectively.

WE‐AB‐207A‐12: HLCC Based Quantitative Evaluation Method of Image Artifact in Dental CBCT

PURPOSEnImage artifacts are usually evaluated qualitatively via visual observation of the reconstructed images, which is susceptible to subjective factors due to the lack of an objective evaluation criterion. In this work, we propose a Helgason-Ludwig consistency condition (HLCC) based evaluation method to quantify the severity level of different image artifacts in dental CBCT.nnnMETHODSnOur evaluation method consists of four step: 1) Acquire Cone beam CT(CBCT) projection; 2) Convert 3D CBCT projection to fan-beam projection by extracting its central plane projection; 3) Convert fan-beam projection to parallel-beam projection utilizing sinogram-based rebinning algorithm or detail-based rebinning algorithm; 4) Obtain HLCC profile by integrating parallel-beam projection per view and calculate wave percentage and variance of the HLCC profile, which can be used to describe the severity level of image artifacts.nnnRESULTSnSeveral sets of dental CBCT projections containing only one type of artifact (i.e. geometry, scatter, beam hardening, lag and noise artifact), were simulated using gDRR, a GPU tool developed for efficient, accurate, and realistic simulation of CBCT Projections. These simulated CBCT projections were used to test our proposed method. HLCC profile wave percentage and variance induced by geometry distortion are about 3∼21 times and 16∼393 times as large as that of the artifact-free projection, respectively. The increase factor of wave percentage and variance are 6 and133 times for beam hardening, 19 and 1184 times for scatter, and 4 and16 times for lag artifacts, respectively. In contrast, for noisy projection the wave percentage, variance and inconsistency level are almost the same with those of the noise-free one.nnnCONCLUSIONnWe have proposed a quantitative evaluation method of image artifact based on HLCC theory. According to our simulation results, the severity of different artifact types is found to be in a following order: Scatter>Geometry>Beam hardening>Lag>Noise>Artifact-free in dental CBCT.

SU-G-206-04: A Method for Realizing Phantom Calibration and Geometry Calibration Accurately Based On a Geometry Evaluation Index

PURPOSEnFor traditional geometric calibration, the calibration accuracy relies on both accuracy of geometry phantom manufacture and accuracy of ball bearings (BB) location estimation. In this work, we have developed a method to perform phantom calibration and geometry calibration iteratively and accurately in a whole procedure.nnnMETHODSnWe have designed and manufactured a geometry phantom with BB and an evaluation phantom of a crystal ball contained in a cubic gel box. Our calibration method consists of five steps: 1) Estimate BB locations using spiral CT image, which are then used to initialize the particles in Particle Swarm Optimization (PSO) algorithm; 2) Perform geometric calibration; 3) Reconstruct the images of the evaluation phantom based on the current geometry calibration; 4) Evaluate the reconstructed images using a geometry evaluate index; 5) Update BB locations in PSO algorithm. Repeat step2)-5) until our stopping criteria is met. The edge of the crystal ball in the calibration phantom on CBCT images is detected by Hough transform to define two circular rings outside and inside the ball. The evaluation index used in step 4) is defined as the difference of the averaged image intensities of these two circular rings.nnnRESULTSnWe have demonstrated the feasibility and performance of our method on a benchtop CBCT system. It is observed that inaccurate BB locations lead to severe image distortion and relative small evaluation index. With our method, streak artifacts are reduced and the structure becomes sharper and clearer. The evaluation index increases fast within 10 iterations, and then becomes stable gradually.nnnCONCLUSIONnOur method can perform accurate phantom calibration and geometry calibration together in a whole procedure. It helps to mitigate the impact of the geometry phantom manufacture errors on the calibration, which could hence save the cost of the geometry phantom manufacture.