HBV surface antigen glycan isomer(HBsAgGi), correlates with HBV-DNA andHBV-RNA virions
K. ANGATA1, M. SOGABE1, H. NARIMATSU1, A. MURATA2, and T. GENDA2
1RCMG Inc. , 2Juntendo University Shizuoka Hospital, Department ofGastroenterology and Hepatology
ABSTRACT:Background: In HBV patients’ sera, infectious particles containing HBV-DNA (Dane particles) or HBV-RNA particles are envelopedwith hepatitis B virus surface antigen (HBsAg). Infectious particles contain L-, M-, and S-HBsAg, while non-infectious sub-viralparticles (SVPs) mainly contain S-HBsAg. Because SVPs present much more than infectious particles, S-HBsAg is the most abundantin all HBV particles. Thus, general HBsAg test using S-HBsAg antibodies cannot distinguish infectious and non-infectious particles.
Previously, we found that O-glycosylated M-HBsAg was enriched in HBV-DNA particles. In this study, we developed and examined a
new antibody to detect infectious particles, which should contain O-glycosylated M-HBsAg, called as HBsAg glycan isomer (HBsAgGi).
Methods and Results: Western analysis indicated that HBsAgGi antibody recognized M-HBs modified with O-glycan but not L-HBs
without O-glycan on the PreS2 of genotype C. Immunoprecipitation (IP) experiments confirmed that both HBV-DNA and HBV-RNAcontaining particles were immunoprecipitated by HBsAgGi antibody. HBsAgGi localized in ER to Golgi in MHBs-expressing cells. Intreatment naïve chronic hepatitis B patients, serum HBsAgGi level was significantly correlated with the HBV-DNA level (p=0.002,n=32). However, HBsAgGi level was not associated with HBV-DNA level after NA treatment, which is consistent with the IP results.
Conclusions: O-glycosylated M-HBsAg is unique to genotype C infectious virions. New HBsAgGi antibody recognizes infectious HBVvirions containing HBV-DNA or HBV-RNA, indicating that the infectious particles with HBsAgGi are generated through glycosylation
pathway. Furthermore, NA treatment altered HBV-DNA and HBV-RNA status but not much HBsAgGi, suggesting that HBsAgGi is anuseful monitor of infectious HBV particles during therapy.

INTRODUCTIONRESULTS•Upon HBV infection, non-infectious particles are dominantly
present than infectious particles containing HBV DNA (Daneparticles) in patients. In contrast to non-infectious particles
mainly containing S-HBsAg, Dane particles contain all L-, M-,and S-HBsAgs. General HBsAg test using antibodies for SHBsAg detect all HBV particles. To distinguish infectious
particles and non-infectious subviral particles, we generated MHBsAg specific antibody. Because a lectin recognizing Oglycans could enrich DNA-containing particle, O-glycosylatedM-HBsAg determined by mass spectrometry was targeted.
This study aimed to examine a new antibody recognizing Oglycosylated M-HBsAg [HBsAg glycan isomer (HBsAgGi)] if it
can detect infectious HBV particles containing HBV-DNAand/or HBV-RNA.

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•••
•

HBsAgGi antibody recognizes M-HBsAg modified withO-glycans in genotype C HBV virion
NHBsAg gene

HBV virion

L-HBs

HBsAg

NPre-S1ON

Pre-S2Pre-S1

N, N-glycosylation site
O, O-glycosylation site

SHBsAgGi is specific to genotype C and present in ER toGolgi in M-HBsAg-expressing cells

NNNPre-S2

PreS2 peptides with O-glycans generated a genotype Cspecific antibody.
HBsAgGi antibody recognized M-HBs but not L-HBs,
which is not modified with O-glycan on the PreS2 ofgenotype C.
Mutations in O-glycosylation site or removal of Oglycans resulted in decrease of the recognition byHBsAgGi antibody.
HBsAgGi localized in ER to Golgi in M-HBs expressingcells.
In treatment naïve CHB patients, serum HBsAgGi level
was higher in HBe-positive patients (EPH) than HBenegative patients (ENH) at baseline.
HBsAgGi levels were significantly correlated with the
HBV-DNA level (n=32, p=0.002).
HBsAgGi antibody can immunoprecipitate HBV-RNAcontaining particles. Percentile of HBV-RNA particles
would increase after NA therapy.

SN

M-HBs

HBV core

Infectious particle
containing HBV DNA

NO

Pre-S2
HBV DNA

CHO-K1
CHO-K1 Western + M-HBsAg

ELISA

HBsAgGiSHBsAgGi
A450nmgA
HBsAgGi, 0.1 µg/mL

4.0

S-HBsNon-infectious particle(subviral particle, SVP)

gDkDa
75

HBsAgGi

2.0

SgC

50

3.0

NgB

Anti-HBsAgGi
Anti-m IgG(AF488)1.037

Nuclearstaining

0.0
A-PS2

B-PS2

C-PS2

PS2

M-HBs

25

S-HBs

75

ELISA coat antigen

METHODS AND MATERIALS

PreS1
PreS2

PreS2

N

M-HBsAg

OPreS2

g

kDa
75

S

S-HBsAg

g(N)

50

S37
PreS1

L-HBsAgPreS2SDistribution of HBsAgGi, HBV-DNA, qHBsAg and HBcrAg
at baseline in NA naïve EPH and ENH patients

HBsAgGikDaLoop 16420ENH

EPH

ENH

HBcrAgP=0.0132*

100000105410000
1031000
102100101010110.10.1

8P<0.0001****6420EPH

ENH

ENH

Correlation of HBsAgGi with HBV-DNA at baseline
in NA naïve CHB patients

Hn=32, r = 0.5318 (P = 0.0017)

138

S S 149139SS

HBsAgGi (log ng/mL)SS

13716037

T
147146 N

K25G
D

Loop 2HBsAgGi

Flag

HBV particles immunoprecipitated by HBsAgGi
antibody contain HBV-RNA.

43.5
32.5254.543.532.520.0

2.04.0

6.0

8.0

10.0

HBV-DNA (logIU/mL)

HBV-RNA % in
IP fraction

CONCLUSIONS

Percent (%)••••0W

n=32

y = 0.1492x + 2.2783
R² = 0.2828

4.575
50107

EPH

T37A
G
145G R
130NG
130N N+14N
6Q 146Q

TW
TTW

T

130 G

100210100

8HBsAgGi vs HBV DNA
C

12121 SS 4T 116

31000
10P<0.0001****

10PNT41000010EPH

Anti-PreS2T37AG
145G R
130NG130N N+14N
6Q 146Q

T G

P=0.0041**

100000105

qHBsAgMutations in antigenic loop in S-HBs did not affectthe binding of HBsAgGi antibody.
S

HBV-DNA
HBV-DNA (LC/mL)

HBsAgGi

25
20Samples

25

PS2

HBsAgGi (log ng/mL)

PreS1

omfrg nts
A
A
Asss
12sA eB
B
B
B tieS reS
H-H-H
H pa
Pr
PSM
Lgg

C-PS2

HBcrAg (logU/mL)

omfrg nts
AAAsss
12sA eB
B
B
B tieS reS
H-H-HH paPrPSMLg

37O-glycan

HBsAgGi (ng/mL)

HBsAgGi antibody specifically recognizes M-HBsAg
but not L-HBsAg

Flag

MergedB-PS2

qHBsAg (IU/mL)

To generate HBsAgGi antibodies, PreS2 glycopeptides
modified with O-glycans were syntehsized. To characterize theHBsAgGi antibody, western blotting, immunostaining, andELISA were performed. To investigate clinical utility of theHBsAgGi, sera of chronic hepatitis B (CHB) patients before
and after nucleos(t)ide analog (NA) treatment were analyzed
by a new HBsAgGi ELISA system and compared with other
HBV markers (qHBsAg, HBV-DNA, and HBcrAg). To
characterize target particles of HBsAgGi, immunoprecipitated(IP) particles by HBsAgGi antibody were analyzed by reversetranscription PCR for quantifying HBV-RNA.

g50

A-PS248W

HBsAgGi antibody recognizes HBV particles in genotype
specific manner dependent on the presence of Oglycans in PreS2 domain.
HBsAgGi levels were significantly correlated with theHBV-DNA level in CHB patients during therapy.
HBsAgGi-positive virions contain HBV-RNA.
Thus, HBsAgGi specifically presents in infectious fractionof HBV virions containing HBV-DNA or HBV-RNA.

0.0