Development of assays to determine coreceptor usage and study of molecular determinants of HIV-1 CRF01_AE coreceptor switching

การพัฒนาวิธีการตรวจหาการใช้โครีเซปเตอร์ และศึกษาลักษณะเฉพาะทางโมเลกุลของเชื้อเอชไอวี-1 ซีอาร์เอฟศูนย์หนึ่งเออี ในการเปลี่ยนสถานะภาพของการใช้โครีเซปเตอร์

Name: Sayamon Hongjaisee

ThaSH: HIV (Viruses)

Classification :.DDC: 616.9792

ThaSH: Molecular biology

Abstract: Since CCR5 antagonist has been approved for treating HIV-1 infected patients whose harbor only CCR5-using viruses, the determination of coreceptor usage is strictly recommended prior to prescription to exclude the presence of CXCR4-using viruses or dual/mixed viruses. In Thailand, there was no access to assays to determine the coreceptor usage that limited the clinical use of coreceptor antagonists. Moreover, there was no data to support the phenotypic assay to determine the coreceptor usage in HIV-1 CRF01_AE, which is the predominant subtype circulating in Thailand and Southeast Asia. As regards, this study aimed to develop the phenotypic assay to determine the coreceptor usage in Thai clinical samples. The entire envelope glycoprotein was amplified in triplicate amplifications and generated the env-pseudotyped viruses to infect the indicator cell lines bearing specific coreceptors. The coreceptor usage was successfully determined in 18 treatment-experienced patients; 14 patients harbored only CCR5-using viruses, 3 patients harbored dual/mixed viruses, and one patient harbored only CXCR4-using viruses. Furthermore, the results of phenotypic assay were used as a reference to evaluate the performances of various genotypic predictors for the prediction of the coreceptor usage. The same env fragment used to produce pseudotyped virus was sequenced in V3 region by bulk sequencing and determined the coreceptor usage using j predictors. The best results in terms of concordance, sensitivity, and specificity were obtained using Geno2pheno[coreceptor] at FPR 5% (78%, 100%, and 71%, respectively). However, the genotypic predictors were interpreted with algorithms, which have been developed using mostly data of HIV-1 subtype B and may be inaccurate and inappropriate for non-B subtypes. At this point, the Support Vector Machine based on Logistic Model Trees (SVM/LMT) method, which based on HIV-1 CRF01_AE subtype training data was specifically developed and proposed for prediction of coreceptor usage in this subtype. SVM was used as a classifier to classify the class of coreceptor usage whereas LMT selected the most relevant positions on V3 loop, which could be differentiated between CCR5 and CXCR4. For the optimized method, using only nine positions obtained from LMT model, the predictive performance of SVM reached a predictive accuracy as high as 97.8%, whereas the specificity and sensitivity were as high as 97.7% and 97.9%, respectively, which can improve the classification of the coreceptor usage for HIV-1 CRF01_AE subtype and could be used as a prediction tool. However, the effect of these selected amino acid positions on coreceptor binding of CRF01_AE viruses is little known. In this regard, this study also aimed to describe the role of selected amino acid positions in V3 loop of HIV-1 CRF01_AE subtype on functional property and coreceptor interaction. HIV-1 CRF01_AE R5-env plasmids were used to construct the mutants at position 5, 7, 11, 12, 18, 27, and 32 within V3 loop using PCR-based site-directed mutagenesis, which were performed in both single and combinations mutations in 16 patterns. All mutant viruses containing these substitution amino acids were produced based on env-pseudotyped viruses and determined the coreceptor usage using phenotypic assay in U373 cells expressing either CCR5 or CXCR4. The infectivity of viruses containing single mutations at position 7, 11, 18, and 32 including combination mutation at position 5 and 32, 18 and 32 were significantly decreased in CCR5-expressing cells, which could be interrupt the interaction between V3 loop and CCR5 coreceptor. However, the amino acid substitutions either single or combination mutations did not confer to use CXCR4 as a coreceptor, excepting the arginine substitution at position 11. The viral infectivity of S11R mutant was significantly increased in CXCR4-expressing cells but it did not support for coreceptor switching. Although, alterations within V3 loop are necessary for k coreceptor binding, but still are not sufficient for switching. Other regions, besides V3, may maintain viral fitness to mediate CCR5 usage. In summary, the assays for determination of coreceptor usage have been developed in both phenotypic and genotypic assays. The genotypic assay was preferred due to its accessible, simple and suitable for laboratories especially in resource-limited countries. Moreover, understanding of V3 genetic determinants could be served as a foundation for future research into therapeutics and development of new drugs that specifically target CRF01_AE infections.

Chiang Mai University Library

Address: Chiang Mai

Email: cmulibref@cmu.ac.th

Name: Niwat Maneekarn

Role: Cairman

Name: Francis Barin

Role: Member

Name: Wasna Sirirungsi

Role: Member

Name: Martine Braibant

Role: Member

Name: Chonlaphat Sukasem

Role: Member

Name: Tanawan Samleerat

Role: Member

Created: 2016

Modified: 2020-01-07

Issued: 2020-01-07

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: Th 616.9792 S274D

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Biomedical Science

Grantor: Chiang Mai University

©copyrights Chiang Mai University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	biomed80516saho_full.pdf	6.06 MB

ใช้เวลา

0.026246 วินาที