N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.
OBJECTIVE
HBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients.


METHODS
Two hundred sixteen patients with HBsAg and anti-HBs were studied, with one hundred eighty-two HBV carriers without anti-HBs as a control group. Recombinant HBsAg bearing the most frequent N-glycosylation mutations were expressed in CHO and HuH7 cells. After confirming N-glycosylation at the most frequent sites (129 and 131), together with inserted mutations, functional analysis were performed to study antigenicity and secretion capacity.


RESULTS
One hundred twenty-three patients had the wild-type HBs gene sequence, 93 patients (43%) had mutants in the major hydrophilic region (MHR), and 47 of the 93 patients had additional N-glycosylation mutations, which were transmitted horizontally to at least 2 patients, one of whom was efficiently vaccinated. The frequency of N-glycosylation mutation in the case group was much higher than that of the control group (47/216 vs. 1/182). Compared with wild-type HBsAg, HBsAg mutants reacted weakly with anti-HBs using a chemiluminescent microparticle enzyme immunoassay. Native gel analysis of secreted virion in supernatants of transfected HuH7 cells indicated that mutants had better virion enveloping and secretion capacity than wild-type HBV.


CONCLUSIONS
Our results suggest that specific HBsAg MHR N-glycosylation mutations are implicated in HBV immune escape in a high endemic area.
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
De-Min
DM
Yu
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China; The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, People's Republic of China.
Xin-Hua
XH
Li
INSERM, U1052, 151 cours Albert Thomas, 69424 Lyon cedex 03, France.
Vannary
V
Mom
Wu Xi Hospital of Infectious Diseases, People's Republic of China.
Zhong-Hua
ZH
Lu
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Xiang-Wei
XW
Liao
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Yue
Y
Han
INSERM, U1052, 151 cours Albert Thomas, 69424 Lyon cedex 03, France.
Christian
C
Pichoud
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Qi-Ming
QM
Gong
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Dong-Hua
DH
Zhang
Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, People's Republic of China.
Yan
Y
Zhang
INSERM, U1052, 151 cours Albert Thomas, 69424 Lyon cedex 03, France; University Paris 13, Groupe des Hôpitaux Universitaires Paris Seine Saint Denis, Paris, France.
Paul
P
Deny
INSERM, U1052, 151 cours Albert Thomas, 69424 Lyon cedex 03, France; Lyon University and Hospices Civils de Lyon, Lyon, France. Electronic address: fabien.zoulim@inserm.fr.
Fabien
F
Zoulim
Department of Infectious Disease, Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China. Electronic address: zhangxinxin@rjh.com.cn.
Xin-Xin
XX
Zhang
D000293:Adolescent; D000328:Adult; D000368:Aged; D005260:Female; D006031:Glycosylation; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000276:immunology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057131:Immune Evasion; D008297:Male; D008875:Middle Aged; D009154:Mutation
D006514:Hepatitis B Surface Antigens
AHB; ALT; Acute Hepatitis B; CHB; CMIA; Chronic Hepatitis B; HBV; HBsAg; HCC; Immune escape; LC; MHR; Major hydrophilic region; Mutation; N-glycosylation; PCR; Polymerase chain reaction; aa; amino acid; anti-HBs; anti-HBs antibodies; aspartate aminotransferase; chemiluminescent microparticle enzyme immunoassay; hepatitis B virus; hepatitis B virus surface antigen; hepatocellular carcinoma; liver cirrhosis; major hydrophilic region; rHBsAg; recombinant hepatitis B virus surface antigen