Hepatitis B virus surface antigen glycan isomer (HBsAgGi) asa new glyco-biomarker to detect infectious HBV virions#1315-CK. ANGATA1, M. SOGABE1, H. NARIMATSU1, A. MURATA2 and T. GENDA2
1RCMG Inc. , 2Juntendo University Shizuoka Hospital, Department of Gastroenterology and Hepatology

CONTACTST GCT12121 SS 4

Pre-S2M-HBs

HBV DNA

HBV core

N100

Infectious particle
containing HBV DNA

Non-infectious particle(subviral particle, SVP)

PreS2kDa

S-HBsAgNM-HBsAg

O
PreS2(N)37
L-HBsAg

PreS1

PreS2

Samples

S25
20

HBsAgGiSS

Anti-PreS2

HBV-DNA (LC/mL)

HBsAgGi (ng/mL)

P=0.0132*

HBcrAg (logU/mL)

qHBsAg (IU/mL)

ENH

HBcrAg

1000001054100001031000
10210100

1010
110.1
0.1

8

P<0.0001****6420EPH

ENH

ENH

HBsAg
vs HBV
HBsAg
vs DNAHBV DNA
n=32, r n=32,= 0.3895= 0.0273)
r = (P0.3895(P = 0.0273)

y = 0.1492xy += 2.2783
0.1492x + 2.2783
R² = 0.2828R² = 0.2828

37T
14725

G

HBsAgGi

A450nmFlag

4.54.544gA
Anti-HBsAgGi
Anti-m IgG(AF488)

gB

gC

gD

3.5
32.5
CHO-K1+ M-HBsAg

CHO-K1
CHO-K1 Western + M-HBsAg

HBsAgGi, 0.1 µg/mL

4.0

kDa

HBsAgGi

2.0
1.0

Nuclearstaining

0.0
A-PS2

B-PS2

C-PS2

PS2

M-HBs

B-PS2

32.5

y = 0.1365xy += 2.6296
0.1365x + 2.6296
R² = 0.1517R² = 0.1517

554.5

4.5

A75Flag

Merged37

O-glycan

C-PS2
PS225

Scale bar 50 µm

REFERENCES
1.

Angata et al. O-glycosylated HBsAg peptide can induce specific antibody
neutralizing HBV infection. Biochim Biophys Acta Gen Subj 2022, 1866;:
130020.

2.

Murata A, et al. Serum O-glycosylated hepatitis B surface antigen levels inpatients with chronic hepatitis B during nucleos(t)ide analog therapy. BMC
Gastroenterol. 2022, 22: 270.

3.

Okumura T et al. Kinetics of serum O-glycosylated M-hepatitis B surface
antigen with hepatocellular carcinoma history and nucleos(t)ide analoguetherapy in hepatitis B patients. J Viral Hepat. 2023, 30:731.

32.5

6.0
4.0

8.0
6.0

10.0
8.0

32.520.0

10.0

3.5

2.0
0.0

4.0
2.0

6.0
4.0

8.0
6.0

10.0
8.0

HBV-DNA(logIU/mL)
HBV-DNA(logIU/mL)

HBsAgGi antibody can immunoprecipitated both HBV DNA- andHBV RNA-containing particles.

3750

3.5

4HBV-DNA(logIU/mL)
HBV-DNA(logIU/mL)

75

25

424.02.0

B

HBV DNA / 20 µL

HBV RNA 25 µL

60

S-HBs

ELISA coat antigen

A-PS23.5

W/O HBsAgGi2 antibody 20.0
2.00.050

3.050

S

139HBsAgGi

75SEPH

160ELISAHBsAgGi antibody recognizes M-HBs but not L-HBs, which is not modified
with O-glycan on the PreS2.

PreS20ENH

n=32, r n=32,= 0.5318= 0.0017)
r = (P0.5318(P = 0.0017)

HBsAgGi specific to genotype C localized in ER to Golgi
in M-HBsAg-expressing cells

To generate HBsAgGi antibodies, PreS2 glycopeptides modified with Oglycans were generated. To analyze the HBsAgGi antibody, Westernblotting, immunostaining, and ELISA were performed. To characterizetarget particles of HBsAgGi, immunoprecipitated (IP) particles byHBsAgGi antibody was analyzed by qPCR and reverse-transcription PCR
for quantifying HBV DNA and HBV RNA, respectively. Furthermore, anew HBsAgGi ELISA system measured sera of chronic hepatitis B (CHB)
patients before and after nucleos(t)ide analog (NA) treatment toinvestigate clinical utility of the HBsAgGi.

PreS1

S S 149Loop 2omfr sgggg tsAsAsA sA ien
12B
B
B
S
S
B tee
HH-H
H pa
PrPr
SML-2

DMETHODS AND MATERIALSomfr sgggg tsAsAsA sA ien
12B
B
B
S
S
B tee
HH-H
H pa
PrPr
SML-

4

HBsAgGivs HBV
HBsAgGivs DNAHBV DNA

146 N

N

S

137K

S

S-HBs

13
SS 8675
50

107O

Pre-S2kDa

Loop 1S

HBsAgGi
N130 G

8HBsAgGi (log ng/mL)

Pre-S1T 116

EPH

HBsAgGi (log ng/mL)
HBsAg(log ng/mL)

HBsAg

NNNT

HBsAgGi (log ng/mL)

L-HBs

210100P<0.0001****

10The correlation of HBsAgGi with HBV DNA at baseline
in NA naïve CHB patients

Fold of original 10-3

HBV virion

31000
10PN
T

410000
10IP
Sup

70.0

50
4030
2010

Scale bar: 500 µm
Orig IPSup IP- Gi

Sup+ Gi

Portion of each fraction (%)

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

HBsAgGi (log ng/mL)

Pre-S2N

P=0.0041**

100000105EPH

T37AG145G R
130N
G130N N+14N
6Q 146Q

Pre-S1OHBV-DNA

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

THBsAg geneNHBsAgGi

PreS2 peptides with or without glycans generated genotype specificantibodies. For genotype C, HBsAgGi antibody recognized M-HBs but not
L-HBs that is not modified with O-glycan on the PreS2. Mutations in Oglycosylation site or removal of O-glycans resulted in decrease of therecognition by HBsAgGi antibody. HBsAgGi localized in ER to Golgi inMHBs-expressing cells, suggesting generation of HBsAgGi throughglycosylation pathway. Immunoprecipitation experiments confirmed thatboth HBV DNA- and HBV RNA-containing particles were collected byHBsAgGi antibody. In treatment naïve CHB patients, serum HBsAgGilevel was higher in HBe-positive patients (EPH) than HBe-negativepatients (ENH) at baseline. HBsAgGi levels were significantly correlatedwith the HBV DNA level (p=0.002, n=32).

Glycosylation of HBsAg: O-glycosylated PreS2 (HBsAgGi) presents onM-HBsAg in HBV virion.
NDistribution of HBsAgGi, HBV-DNA, qHBsAg and HBcrAg at baseline
in NA naïve EPH and ENH patients

WHepatitis B virus surface antigen (HBsAg) gene generates L-, M-, and SHBsAg. In HBV patients’ sera, non-infectious particles are muchdominant than infectious particles containing HBV DNA (Dane particles).
Dane particles contain all HBsAgs, but non-infectious particles mainlycontain S-HBsAg. Thus, general HBsAg test using antibodies recognizing
S-HBsAg does not always distinguish infectious and non-infectious
subviral particles. O-glycosylated M-HBsAg was determined by mass
spectrometry, because a lectin recognizing O-glycans could enrich DNAcontaining particle. This study aimed to develop a new marker tomeasure infectious particles which contain O-glycosylated M-HBsAg[HBsAg glycan isomer (HBsAgGi)].

PreS1

Kiyohiko Angata
RCMG Inc.
Email: angata@medglyco.com
Phone: +81-29-828-8010
Website: https://med-glycosci.com

RESULTSWTIn HBV patients’ sera, non-infectious particles
are much dominant than infectious particles
containing HBV DNA (Dane particles). Daneparticles contain all HBsAgs (L-, M- and SHBsAg), but non-infectious particles mainlycontain S-HBsAg. Thus, general HBsAg test
using antibodies recognizing S-HBsAg does
not always distinguish infectious and noninfectious HBV particles. It has been shownthat a lectin recognizing O-glycans could
enrich DNA-containing particles. This study
aimed to develop a new marker to measureinfectious particles which contain Oglycosylated M-HBsAg [HBsAg glycan isomer(HBsAgGi)].
First, we developed a new HBsAgGi antibody
recognizing M-HBs but not L-HBs that is not
modified with O-glycan on the PreS2.
Mutation analysis also confirmed thatHBsAgGi partly recognized O-glycans.
HBsAgGi localized in ER to Golgi, suggesting
generation of HBsAgGi depends on
glycosylation pathway.
Next, we developed ELISA system andmeasured HBsAgGi in sera of chronic
hepatitis B (CHB) patients before and after
nucleos(t)ide analog (NA) treatment toinvestigate clinical utility of the HBsAgGi.
Immunoprecipitation with HBsAgGi antibody
confirmed that both HBV DNA- and HBV RNAcontaining particles were collected by theantibody. At baseline, serum HBsAgGi level
was higher in HBe-positive patients than HBenegative patients. HBsAgGi levels weresignificantly correlated with the HBV DNA
level (p=0.002, n=32).
Taken together with above results, HBsAgGi
would be a new glyco-biomarker to monitor
viral kinetics in CHB patients during therapy.

INTRODUCTION

T37A
G145
G R
130NG130
N N+14N6Q 146Q

ABSTRACT

60.0
50.0
40.0
30.0
20.0
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CONCLUSIONSNew HBsAgGi antibodies recognize HBV particles in genotype specificmanner dependent on the presence of O-glycans in PreS2 domain.
HBsAgGi specifically presents in minor infectious fraction of HBV virions
containing HBV DNA or HBV RNA. Taken together with ELISA analysis ofpatients’ sera, HBsAgGi would be a new glyco-biomarker to monitor viralkinetics in CHB patients during therapy.

10.0