Antibody Levels and Protection after Hepatitis B Vaccination: Results
of a 15-Year Follow-up
Brian J. McMahon, MD; Dana L. Bruden, MS; Kenneth M. Petersen, MD; Lisa R. Bulkow, MS; Alan J. Parkinson, PhD; Omana Nainan, PhD;
Marina Khristova, PhD, DSc; Carolyn Zanis, BS; Helen Peters, BS; and Harold S. Margolis, MD
Background: The duration of protection afforded by hepatitis B
vaccination is unknown.
Objective: To determine antibody persistence and protection
from hepatitis B virus (HBV) infection.
Design: Prospective cohort study.
Setting: 15 villages in southwest Alaska.
Participants: 1578 Alaska Natives vaccinated at age 6 months
Intervention: During 1981–1982, participants received 3 doses
of plasma-derived hepatitis B vaccine. This cohort was followed
annually over the first 11 years, and 841 (53%) persons were
tested at 15 years.
Measurements: Antibody to hepatitis B surface antigen (anti-
HBs), markers of HBV infection, and testing to identify HBV
Results: Levels of anti-HBs in the cohort decreased from a geo-
metric mean concentration of 822 mIU/mL after vaccination to 27
mIU/mL at 15 years. Initial anti-HBs level, older age at vaccina-
tion, and male sex were associated with persistence of higher
anti-HBs levels at 15 years when analyzed by a longitudinal linear
mixed model. After adjustment for initial anti-HBs level and sex,
those vaccinated at age 6 months to 4 years had the lowest
anti-HBs level at 15 years. Asymptomatic breakthrough infections
were detected in 16 participants and occurred more frequently in
persons who did not respond to vaccination than those who
responded (P ? 0.01). Among infected persons with viremia, 2
were infected with wild-type HBV and 4 had HBV surface glyco-
protein variants, generally accompanied by wild-type HBV.
Limitations: The loss of participants to follow-up at 15 years
was 47%. However, characteristics of persons tested were similar
to those of persons lost to follow-up.
Conclusions: Hepatitis B vaccination strongly protected against
infection for at least 15 years in all age groups. Antibody levels
decreased the most among persons immunized at 4 years of age
Ann Intern Med. 2005;142:333-341.
For author affiliations, see end of text.
countries and has been shown to be effective in reducing
the rate of chronic hepatitis B virus (HBV) infection (1, 2).
Protection has been demonstrated in persons and popula-
tions vaccinated for 5 to 10 years, and rates of asymptom-
atic breakthrough HBV infection have been extremely low
(3–9). However, the duration of protection afforded by
hepatitis B vaccination beyond 10 years and the possible
need for booster doses of this vaccine are unknown.
Alaska Natives have a high prevalence of chronic HBV
infection, primarily acquired during early childhood (10).
Between November 1981 and May 1982, Alaska Natives
residing in 15 villages in southwest Alaska were enrolled in
a cohort study to ascertain the immunogenicity and long-
term effectiveness of hepatitis B vaccination (11–14). We
report data on the persistence of antibodies to hepatitis B
surface antigen (anti-HBs), incidence of HBV infection,
and the genetic characteristics of the HBV isolates in per-
sons with breakthrough infections 15 years after initial vac-
cination of this cohort.
niversal vaccination of infants with hepatitis B vaccine
is included in the immunization programs of most
Participants and Data Collection
A total of 1578 Alaska Natives who were serologically
negative for hepatitis B surface antigen (HBsAg) and anti-
body to hepatitis B core antigen (anti-HBc) were vacci-
nated on a 0-, 1-, and 6-month schedule with 3 doses of
plasma-derived hepatitis B vaccine (Heptavax, Merck &
Co., Inc., West Point, Pennsylvania) beginning in 1981
(11). Persons younger than 20 years of age received the
10-?g dose, and adults received the 20-?g dose. Of the
1578 persons vaccinated, 1436 (91%) were tested for an
anti-HBs response 6 months after the last vaccine dose.
From 1982 to 1992, serum specimens were obtained
annually and once again during 1996 from as many of the
1578 consenting participants as possible. The Institutional
Review Boards of the Alaska Area Native Health Service,
the Indian Health Service, the Centers for Disease Control
and Prevention, and the Yukon-Kuskokwim Health Cor-
poration and the Norton Sound Regional Alaska Native
health boards approved this study. All participants 18 years
of age and older and parents of children younger than 18
years of age had provided signed informed consent to par-
Editors’ Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Editorial comment . . . . . . . . . . . . . . . . . . . . . . . . . 384
Summary for Patients . . . . . . . . . . . . . . . . . . . . . . I-34
Conversion of tables into slides
1 March 2005 Annals of Internal Medicine Volume 142 • Number 5 333
ticipate in the study; children older than 7 years of age gave
verbal assent. The number of HBsAg-positive persons in
each village was obtained from a registry used to follow
patients with chronic HBV infection (15).
All serum specimens were tested for HBsAg, anti-HBs,
and anti-HBc by radioimmunoassay using commercial test
kits (Abbott Laboratories, Abbott Park, Illinois). At the
initial testing of the cohort for anti-HBs, results were re-
ported in sample ratio units. However, subsequent anti-
HBs results were reported in milli international units
(mIU) per mL using a World Health Organization refer-
ence standard (12–14). To ensure comparability of results
over time, all serum specimens from each participant with
sufficient volume (99.9% of all specimens collected during
the study) were retested to determine anti-HBs levels in
Detection of HBV DNA and Nucleic Acid Sequencing
Hepatitis B virus DNA was extracted from serum
specimens (50 ?L) of participants with serologic markers
of HBV infection by using commercially available reagents
(MasterPure Complete DNA and RNA Purification Kit,
Epicentre Technologies, Madison, Wisconsin), as described
previously (16, 17). The HBsAg genomic region was then
amplified by dilution cloning polymerase chain reaction by
using previously described primers and methods to identify
circulating variants of HBsAg (16, 17). The polymerase
chain reaction products were purified and the nucleic acid
sequence of the amplified region were determined by using
prism dye or dRhodamine terminator cycle reactions (Ap-
plied Biosystems, Foster City, California) and automated
sequencing (ABI Model 373 or 377, Applied Biosystems)
(18). Sequence data were further analyzed by Sequence
Navigator (ABI) and GCG software (19).
The initial anti-HBs level was measured 6 months af-
ter the third dose of vaccine and 1 year after the first dose
of vaccine. Participants with an initial anti-HBs level of at
least 10 mIU/mL were considered vaccine responders. An
anti-HBs level of 2 mIU/mL or greater was considered a
positive result on subsequent specimens. A booster re-
sponse was defined as a 2-fold or greater increase in anti-
HBs levels between serologic test results.
A definite HBV infection in a participant was defined
as 1) 2 or more consecutive serum specimens positive for
anti-HBc more than 1 year after the initial vaccine dose, 2)
a single positive anti-HBc result with a positive HBV DNA
result, or 3) any HBsAg-positive test result. A “possible”
HBV infection was defined as a single positive or 2 non-
consecutive positive anti-HBc results. Participants who de-
veloped anti-HBc were interviewed for history of icterus or
other clinical signs or symptoms compatible with acute
hepatitis, and village and hospital medical records were
reviewed for evidence of an illness compatible with viral
Among persons who inadvertently received additional
doses of hepatitis B vaccine during the follow-up period,
anti-HBs results after the additional vaccine dose or doses
were excluded from the analyses. Results for anti-HBs
among persons with definite HBV infections were ex-
cluded from analyses after anti-HBc appeared.
The primary outcomes in this study were the cumula-
tive number of persons with a definite HBV infection dur-
ing all follow-up years and the anti-HBs levels at the 15-
year follow-up. The definitions of age classes (0 to 4 years,
5 to 19 years, 20 to 49 years, ?50 years) were similar to
those in a previously published analysis of this cohort (14).
Although these data have been presented previously (11–
14), we have provided the proportion of persons initially
responding to vaccination. Quantitative anti-HBs levels are
presented as geometric mean concentrations (GMCs). In
bivariate analyses, analysis of variance was used to test the
15-year anti-HBs concentrations (log-transformed). Inci-
dence rates of definite HBV infection were compared by
using the Fisher exact test.
We analyzed factors associated with anti-HBs levels
over the 15 years after the first vaccine dose by using a
linear mixed model (PROC MIXED in SAS version 9.1,
SAS Institute, Inc., Cary, North Carolina) (19). We chose
a longitudinal mixed linear model because it makes infer-
ences by using information from the entire cohort collected
at all follow-up time points. Levels of anti-HBs were log-
transformed before analysis, and concentrations of 0
mIU/mL were assigned a value of 1.0 mIU/mL. Factors
considered in the model were time (entered as a continu-
Although administration of hepatitis B vaccine for infants
is routine practice in many countries, we do not know
whether the protection that this vaccine offers lasts be-
yond 10 years. Such information is essential to develop
policies about booster vaccination.
Of 841 Alaska Natives who received 3 doses of hepatitis B
vaccination during 1981–1982 and were followed for 15
years, 84% had protective levels of antibody to hepatitis B
surface antigen that indicated continued protection. The
greatest decline in antibody levels occurred in people who
received vaccine before 4 years of age. Definite asymp-
tomatic breakthrough infections occurred in 16 partici-
Only about half of the initial cohort of 1578 was available
for testing at 15 years.
Antibody Levels and Protection after Hepatitis B Vaccination
334 1 March 2005 Annals of Internal Medicine Volume 142 • Number 5
Appendix Table 1. Number of Missing Observations Due to Persons Who Dropped Out and Were Missing Intermittently in a Study of
the Immunogenicity of 3 Doses of Hepatitis B Vaccine Administered during 1981–1982*
Removed for Analysis, n†
* Anti-HBs ? antibody to hepatitis B surface antigen.
† Persons participated in the follow-up study; however, because they had received a fourth dose of vaccine or had evidence of definite hepatitis B virus infection
(seroconverted), the anti-HBs level was removed for analysis.
‡ Numbers in these columns sum to total number of participants (n ? 1578) for each row in the table.
§ Persons with missing (or removed) data from given follow-up year to the end of the study (counts are cumulative).
? Six months after third dose and 1 year after first dose of vaccine. A total of 1578 participants received hepatitis B virus vaccine, were enrolled in the study, and participated
in at least 1 follow-up blood draw.
Appendix Table 2. Comparison of Persons Who Participated at
15-Year Serum Draw (n ? 783) with Members of the Cohort
Who Did Not Participate (n ? 703), Excluding All Who Had
Evidence of Definite Hepatitis B Virus Infection or Who Had
Received an Additional Dose of Vaccine during the 15-Year
Level at 15
(n ? 783), n†
(n ? 703), n†‡
Age class at initial
vaccination, % (n)
Initial antibody GMC
11-year antibody GMC
Persons with antibody
level ? 10 mIU/mL at
863 (1191) 850 (1187)
95 (677/714)94 (602/640)
41 (44)39 (43)
74 (445/600)72 (215/300)
* Anti-HBs ? antibody to hepatitis B surface antigen; GMC ? geometric mean
† Does not include persons with evidence of definite breakthrough infection (n ?
16) or persons with ?4 doses of hepatitis B virus vaccine (n ? 76).
‡ Includes 121 persons who were deceased at the time of the 15-year follow-up.
§ Of the 783 participants and 703 nonparticipants, 714 and 640, respectively, had
an initial anti-HBs blood draw.
? Of the 783 participants and 703 nonparticipants, 600 and 300, respectively, had
an 11-year anti-HBs blood draw.
Appendix Table 3. Results from Linear Mixed Model of Levels
of Antibody to Hepatitis B Surface Antigen over 15 Years of
P ValueParameter Estimates
?0.0010.88 (0.86 to 0.90)
?0.2Female vs. male ?0.02
(?0.06 to 0.02)
0–4 y: 0.13 (0.06 to
5–19 y: 0.06 (0.01 to
?20 y: reference
?0.07 (?0.08 to ?0.06)
0.0032 (0.0028 to
?0.006 (?0.009 to
Female vs. male ?0.009
(?0.014 to ?0.005)
0–4 y: ?0.057 (?0.064
5–19 y: ?0.025 (?0.031
?20 y: reference
Age class at initial
Time ? initial
Time ? sex
Time ? age class‡ 203.0
* Anti-HBs ? antibody to hepatitis B surface antigen.
† Wald chi-square test for 0–4 y vs. 5–19 y ? 7.5 (P ? 0.02); for 5–19 y vs. ?20
y ? 4.9 (P ? 0.03).
‡ Wald chi-square test for 0–4 y vs. 5–19 y ? 85.7 (P ? 0.001); for 5–19 y vs.
?20 y ? 65.3 (P ? 0.001).
W-48 1 March 2005 Annals of Internal Medicine Volume 142 • Number 5