Coincident Hepatitis B Surface Antigen and

Antibodies of Different Subtypes in Human

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of November 7, 2018.
Serum
Edward Tabor, Robert J. Gerety, Linda A. Smallwood and
Lewellys F. Barker
J Immunol 1977; 118:369-370; ;
http://www.jimmunol.org/content/118/1/369

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THE JOURNALOF IMMUNOLOGY
Copyright © 1977 by The Williams & Wilkins Co.
COMMUNICATIONS
Coincident Hepatitis B Surface Antigen and Antibodies of Different
Subtypes in Human Serum
E D W A R D T A B O R , 1 R O B E R T J. G E R E T Y , L I N D A A. S M A L L W O O D , AND L E W E L L Y S F. B A R K E R
From the Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
Evidence from a number of sources indicates a protective
role of antibodies to hepatitis B surface antigen (anti-HBs) ~in
resistance to infection with hepatitis B virus (HBV) (1-3).
However, the importance of residual immunity against each
subtype of hepatitis B surface antigen (HB~Ag) in protection
against reinfection has not been entirely clarified, although
there is some definite evidence of cross-protection in chimpan-
zees (4, 5). In addition, simultaneous infections with more than
one of the major antigenic subtypes of HBV have been reported
(6-8). The finding of anti-HB~ in the serum of some patients
with HB~Ag has raised the possibility that anti-HB~ may
correlate with partial but not complete protection against HBV
infection (9-12). This report describes coincident HB~Ag and
anti-HB~ with different subtype specificities in human sera,
and may provide a clue to lack of complete protection against
HBV in some persons after earlier HBV infection.
MATERIALS AND METHODS
Analysis of the subtype specificities of HBsAg and anti-HB.,
was made on three individuals' sera which gave positive
reactions by solid phase radioimmunoassay (RIA) for both
HB~Ag and anti-HB~. Serum No. 1 is from a chronic HB,Ag
carrier employed in a virology laboratory (kindly provided by
Dr. S. Krugman, New York University Medical School). Serum
No. 2 is from a healthy villager from Zambia (kindly provided
by Dr. A. Bayley, University Teaching Hospital, Lusaka,
Zambia), and serum No. 3 is from a patient with Kaposi's
sarcoma (kindly provided by Dr. C. Vogel, Jackson Memorial
Medical Center, Miami, Fla.).
The HB,Ag subtypes on all three sera were tested by a direct
solid phase RIA method with beads coated with subtype-
specific guinea pig antisera and subtype-specific 12SI-radiola-
beled guinea pig antisera (13), (reagents kindly provided by Dr.
R. Foemmel, Abbott Laboratories, North Chicago, Ill.). HBsAg
subtypes were also determined by an indirect RIA method
based on removal by test sera of activity of anti-d or anti-y in
guinea pig antibody made subtype specific by adsorption with
the appropriate HB~Ag subtype (14).
Anti-HB, specificity was determined by adsorption with
Submitted for publication August 12, 1976.
Address Reprint Requests to: Edward Tabor, M.D., Division of
Blood and Blood Products, Bureau of Biologics, 8800 Reckville Pike,
Bethesda, MD 20014.
2Abbreviations used in this paper: anti-HBs, antibodies to hepatitis
B surface antigen; HBV, hepatitis B virus; HB.Ag, hepatitis B surface
antigen.
369
Vol. 118, No. 1, January 1977
Printed in U.S.A.
purified HBsAG, subtypes adw and ayw, followed by testing
the adsorbed sera for anti-HB~ in the solid-phase RIA test for
anti-HBs (14). Anti.HB~ specificity was also tested on serum
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No. 1 by passive hemagglutination (PHA) with red cells coated
with HB,Ag, subtypes adw or ayw (4, 15, 16), and after neu-
tralization with whole serum containing HBsAg subtype adw
or ayw. Each sample was tested with positive and negative
controls, and after adsorption with normal human serum.
RESULTS
The neutralization data are shown in Table I. The summary
of subtyping results and methods applied to each specimen are
shown in Table II.
HB~Ag subtype. Direct subtyping showed that sera No. 1
and No. 2 reacted on beads coated with anti-d, but not beads
coated with anti-y, and therefore contain HBsAg with the d
determinant. Serum No. 3 showed reactivity mainly on the
beads coated with anti-y. Serum No. 3 showed reactivity mainly
on the beads coated with anti-y, and therefore contains HB~Ag
with the y determinant. These results were confirmed by in-
direct subtyping for sample 3, since this sample removed
greater than 50% of the anti-y but not the anti-d activity. Sera
Nos. 1 and 2 could not be subtyped by the indirect method, al-
though results for serum No. 2 suggest a subtype identical to
that found by the direct method.
Anti-HB~ subtype. Adsorption of sera Nos. 1 and 2 with
HB~Ag subtype ayw and serum No. 3 with HB~Ag subtype adw
completely removed the anti-HB~ activity from these sera, but
anti-HB, activity remained after adsorption of sera Nos. I and
2 with HBsAg subtype adw and serum No. 3 with HB~Ag
subtype ayw. This demonstrated that sera Nos. I and 2 contain
anti-y and serum No. 3 contains anti-d. Antibody to the
common determinant of all HB~Ag subtypes (anti-a) was not
detected in any sample (data not shown).
DISCUSSION
The association of anti-HBs with protection against HBV
infection is based on the observation that people and chimpan-
zees with circulating anti-HBs resist reinfection when they are
exposed to known infectious materials (1-3). Previous reports
of simultaneous HBsAg and anti-HB~ in serum (9-12) were
presumed to be the result of the humoral response to the
circulating HBsAg and were supported by reported disease
manifestations associated with circulating HB~Ag-anti-HB~
immune complexes, including periarteritis (17), chronic active
hepatitis (18), and glomerulonephritis (19).
This report describes three individuals infected with HBV,
370 E. TABOR, R, J. GERETY, L. A. SMALLWOOD, AND L. F. BARKER [VOL.118

TABLE I were studied for subtypes of HB~Ag and anti-HBs. In each case
Subtyping o[ HB,Ag and anti-HB, by RIA anti-HB.~ was found to be directed to a different subtype than
HB,AgSubtyping HB,AgSubtyping Anti-HB~Subtyping that of the circulating HBsAg, indicating that reinfection (or
(Direct)" (Indirect ~~ (Indirect)~ simultaneous infection) with a second subtype occurred.
Anti-d Anti-y
after after Sample
Serum Sample Sample adsorption adsorption Sample after REFERENCES
No. on anti-d on anti-y with with after adsorp-
coated c o a t e d s a m p l e s a m p l e adsorption tion
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TABLE II
Summary o[ subtypes and methods used
Serum No. Subtype of HB~Ag Subtype of Anti-HB~
(Methodt (Method)
1 ad a anti.y0, c
2 ad ~ anti-y °
3 ay"" a anti.d o
a HB,Ag by RIA, direct.
0anti-HBs by RIA after adsorption.
PHA direct and indirect, on ad and ay coated red blood cells, and
after adsorption.
HB~Ag by RIA, indirect, after adsorption.
in whom HB,Ag of one subtype and anti-HB~ with a different
subtype specificity were detected in the same serum sample.
The presence of HBsAg and anti-HB~ of different subtype
specificities probably resulted from infection with both of the
implicated subtypes. It is not possible to say whether: 1) the
first infection was self-limited and led to anti-HB8 develop-
ment before the second HBV infection, 2) the chronic carrier
state resulted from the first infection and superinfection with
the second subtype developed subsequently, or 3) infection
with both subtypes occurred at the same time with recovery
and development of an anti-HB~ response against one subtype
and development of the chronic carrier state with the other
subtype. The most important implication of these observations
is that anti-HB~ acquisition may not always indicate complete
protection against HBV infection. Although chimpanzees re-
cently recovered from infection with one HBV subtype have
been shown to resist reinfection with a different subtype (4, 5),
this study suggests the use of experimental HBV vaccines to
produce active immunization against one or multiple subtypes
will be needed to determine the extent of cross-protection, and
to determine whether a multivalent vaccine will be necessary
in geographic regions where more than one HB,Ag subtype is
endemic.
SUMMARY
Three patients with simultaneously detectable hepatitis B
surface antigen (HB~Ag) and antibody (anti-HBs) in their sera
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