Vaccine 30 (2012) 2556–2563

Contents lists available at SciVerse ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Comparison of safety and immunogenicity of two doses of investigational

hepatitis B virus surface antigen co-administered with an immunostimulatory
phosphorothioate oligodeoxyribonucleotide and three doses of a licensed
hepatitis B vaccine in healthy adults 18–55 years of age!,!!,!,!!
Scott A. Halperin a,∗ , Brian Ward b , Curtis Cooper c , Gerald Predy d , Francisco Diaz-Mitoma e ,
Marc Dionne f , Joanne Embree g , Allison McGeer h , Paul Zickler i , Karl-Heinz Moltz j , René Martz k ,
Ingo Meyer l , Shelly McNeil a , Joanne M. Langley a , Eduardo Martins m , William L. Heyward m ,
J. Tyler Martin m
a
Canadian Center for Vaccinology, Dalhousie University, IWK Health Centre, and Capital Health, Halifax, Canada
b
McGill University Health Centre-Vaccine Study Centre, Montreal, Canada
c
Ottawa Hospital, Ottawa, Canada
d
Alberta Health Services, Edmonton, Canada
e
Northern Ontario Research Institute, Sudbury, Canada
f
Institut national de santé publique du Québec, Beauport, Canada
g
University of Manitoba, Winnipeg, Canada
h
Mount Sinai Hospital, Toronto, Canada
i
TASC Research Services, Surrey, Canada
j
APEX GmbH, München, Germany
k
Clinical Research Hamburg, Hamburg, Germany
l
Momentum Pharma Services, Hamburg, Germany
m
Dynavax Technologies Corporation, Berkeley, United States

a r t i c l e i n f o a b s t r a c t

Article history: Background: The currently licensed aluminum-hydroxide-adjuvanted hepatitis B vaccines require three
Received 19 September 2011 doses over a 6-month period to achieve high rates of protection in adults. We compared tolerability and
Received in revised form immunogenicity of two doses of an investigational hepatitis B vaccine using hepatitis B surface antigen
23 December 2011
adjuvanted with an immunostimulatory phosphorothioate oligodeoxyribonucleotide (HBV-ISS) to three
Accepted 28 January 2012
doses of a licensed alum-adjuvanted vaccine (HBV-Eng).
Available online 9 February 2012
Methods: In this randomized, observer-blind study, healthy adults received two doses of HBV-ISS at 0
and 4 weeks or three doses of HBV-Eng at 0, 4, and 24 weeks. The primary immunogenicity endpoint was
Keywords:
Hepatitis B vaccine
the seroprotection rate (antibody ≥ 10 mIU/mL) 8 weeks after the second dose of HBV-ISS compared to 4
Immunostimulatory sequences weeks after the third dose of HBV-Eng.
Randomized trial Results: A total of 2415 participants were randomized in a ratio of 3:1 to HBV-ISS (n = 1809) and HBV-Eng
(n = 606). The percentage of subjects exhibiting a seroprotective immune response at the primary time
point was significantly higher (95.1%) for HBV-ISS than for HBV-Eng (81.1%). Superiority of the seropro-
tective rates for HBV-ISS was demonstrated at all time points measured. Geometric mean concentrations

! Additional investigators and study sites: Ben Lasko, Manna Research, Toronto, Canada; Randy Hart, White Hills Medical Clinic, St. John’s, Canada; Dennis O’Keefe,
Commonwealth Medical Clinic, Mount Pearl, Canada; Martyn Chilvers, Sarnia Institute of Clinical Research, Sarnia, Canada; Randy MacKinnon, Island Clinical Trials,
Charlottetown, Canada; Isabelle Schenkenberger, Klinische Forschung Berlin, Berlin, Germany; Regina Sennewald, Pharos GmbH, Ulm, Germany; Werner Weber,
Pharos GmbH, Ulm, Germany; Gabriele Illies, SMO.MD GmbH, Magdeburg, Germany; and Olga Maus, Zentrum für Therapiestudien der Innomed GmbH, Leipzig, Germany.
!! Presented in part at the Digestive Disease Week Meeting, Chicago, Illinois, May 30–June 4, 2009 and the 48th Annual Meeting of the Infectious Diseases
Society of America, Vancouver, British Columbia, Canada, October 21–24, 2010.
! Drs. Martin, Heyward, and Martins were employees of Dynavax Technologies during the planning, implementation, and/or analysis of the study (Dr. Martins’
current address is Gilead Sciences Inc, Foster City, CA). All other authors had no financial interest in the vaccine or its manufacturer but received research funding to
undertake the study.
!! The study was funded by Dynavax Technologies Corporation.
∗ Corresponding author at: Canadian Center for Vaccinology, Dalhousie University, IWK Health Centre, 5850/5980 University Avenue, Halifax, Nova Scotia, B3K6R8,
Canada. Tel.: +1 902 470 8141; fax: +1 902 470 7232.
E-mail address: scott.halperin@dal.ca (S.A. Halperin).

0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2012.01.087
 S.A. Halperin et al. / Vaccine 30 (2012) 2556–2563
were also significantly higher in the HBV-ISS group at all time points measured except at week 28
(24 weeks post-second dose of HBV-ISS and 4 weeks post-third dose HBV-ISS) at which time the anti-
body concentrations were similar. Both vaccines were welltolerated although injection-site reactions
were reported at a higher rate in HBV-ISS recipients. A short, two-dose regimen of HBV-ISS induced a
superior antibody response than a three-dose regimen of a licensed hepatitis B vaccine and was well
tolerated.
1. Introduction
Hepatitis B virus (HBV) infection has a worldwide distribu-
tion and can cause a serious, potentially fatal liver infection. HBV
can be transmitted vertically from mother to newborn, horizon-
tally amongst family members, through sexual contacts, and via
exchange of blood (injection drug use, blood transfusion) [1].
Disease severity ranges from subclinical to severe hepatitis with
hepatic failure and death [1]. After acute infection, 1–10% of adults
and 30–90% of newborns become chronic HBV carriers and some
progress to hepatic cirrhosis and hepatocellular carcinoma [2]. The
World Health Organization estimates that over 2 billion people
have been infected with HBV worldwide and over 350 million peo-
ple are chronic carriers. Approximately 4.5 million new cases occur
and 620,000 people die of acute or chronic HBV infections each year
[3–6].
An effective vaccine against HBV has been available for 30
years and is recommended for universal vaccination [7]. The first
generation HBV vaccines involved isolating hepatitis B surface anti-
gen (HBsAg) from plasma of chronically infected patients [8]. By
1986, safe and effective recombinant, yeast-derived HBsAg vac-
cines were developed [9]. Universal implementation of hepatitis
B vaccine programs led to a dramatic decrease in new cases of
HBV infection and a subsequent decrease in hepatocellular car-
cinoma [10]. For maximum immunogenicity, three doses of the
alum-adjuvanted vaccine must be given with a minimum inter-
val of 1 month between the first two doses and at least 4 months
between the second and third doses [11–13]. Other approved dos-
ing schedules that achieve adequate rates of seroprotection include
a 2, 4, 6 months schedule for infants, an accelerated adult schedule
(0, 1, 2 months with a fourth dose at 12 months), and a two-dose
schedule (0, 6 months) for adolescents [14–16]. Completion of a
vaccination schedule over 6 months can pose problems for ado-
lescents, individuals with predictably poor rates of compliance,
and individuals who need rapid protection (travelers to high-risk
areas, health care workers) [3,14,17,18]. Despite their success, the
current licensed vaccines are less immunogenic in older adults
≥40 years of age, males, smokers, and the obese (persons with
body mass index ≥30 kg/m2 ) [19–21]. As well, certain individu-
als respond poorly or not at all to vaccination even after repeated
vaccination courses (so called hyporesponders and nonresponders)
[22].
Immunostimulatory sequences (ISS) are bacterial DNA or syn-
thetic oligonucleotides [23,24] that stimulate the innate immune
system through toll-like receptor-9 (TLR-9). Activation of the
TLR-9 pathway results in the production of interferon gamma
and inducers of interferon gamma (interferon alpha and beta,
interleukin-12, interleukin-18) that drive the adaptive immune
response toward a T-helper cell 1 phenotype [25–35]. 1018 ISS is a
22-mer phosporothioate oligodeoxyribonucleotide that has broad
immunostimulatory activity in vitro and in vivo in multiple species,
including humans [36]. 1018 ISS administered with HBsAg was pre-
viously demonstrated to be immunogenic and well tolerated in
healthy adults in a dose-ranging phase 1 study [37] and in young
adults in a phase 2 study using a 0-, 8-week schedule [38]. The
purpose of this study was to compare the tolerability and immuno-
genicity of recombinant HBsAg adjuvanted with 1018 ISS with an
2557
© 2012 Elsevier Ltd. All rights reserved.
alum-adjuvanted, licensed hepatitis B vaccine in healthy adoles-
cents and adults.
2. Methods
2.1. Vaccines
The experimental vaccine (HBV-ISS) consisted of 3 mg 1018
ISS (sequence 5# -TGACTGTGAACGTTCGAGATGA-3# ) in combination
with 20 !g of yeast-derived recombinant HBsAg (manufactured
for Dynavax Technologies by Rentschler Biotechnologie GmbH,
Laupheim, Germany) per 0.5 mL dose; a single lot was used for
the study. The control, comparator vaccine (HBV-Eng) contained
20 !g of recombinant HBsAg vaccine per 1.0 mL with 0.5 mg of
aluminum hydroxide adjuvant (Engerix-B® , GlaxoSmithKline, Rix-
ensart, Belgium). To preserve the blind, sterile saline placebo was
given as a third dose to participants allocated to the HBV-ISS group
so that both groups received injections at 0, 4, and 24 weeks.
2.2. Study population
Healthy adolescents between 11 and 18 years of age in Canada
and adults between 18 and 55 years of age in Canada and Germany
were eligible to participate if they had no history of HBV infec-
tion or immunization and were seronegative for HBsAg, antibody
to HBsAg (anti-HBsAg), and antibody to hepatitis B core antigen
(anti-HBcAg). Individuals were excluded if they were pregnant
or breast-feeding, were unwilling to use effective contraception
during the study, had an autoimmune disease or any clinically sig-
nificant acute or debilitating chronic illness, were at high risk for
recent exposure to hepatitis B, hepatitis C, or human immunode-
ficiency virus, had received blood products or immunoglobulins in
the previous 3 months, had sensitivity to any component of any
of the vaccines, had ever received an injection of DNA plasmids or
oligonucleotides, were immunosuppressed or receiving systemic
corticosteroids, or had received any vaccines in the previous month.
2.3. Study design and procedures
The study was a participant-and observer-blinded, random-
ized, controlled, multicenter study. Participants provided written
informed consent prior to any study procedure. The study was
approved by the Research Ethics Boards of all participating institu-
tions in Canada and by a central Research Ethics Board in Germany.
Participants were randomly allocated to HBV-ISS or HBV-Eng by
a center-specific, computer-generated list produced by a contract
research organization. Randomization was in a 3:1 ratio (HBV-
ISS:HBV-Eng) with a block size of 4 stratified by age (11–39 years
vs. 40–55 years). Participants randomized to the HBV-Eng group
received three doses at 0, 4, and 24 weeks; participants random-
ized to receive HBV-ISS received two doses at 0 and 4 weeks and
placebo at 24 weeks.
Vaccines were given as intramuscular injections into the del-
toid muscle with a 1–1½ in. (2 in. in obese participants), 25-gauge
needle, alternating the arm with each injection. Participants were
monitored by study staff for 30 min after each injection for
2558 S.A. Halperin et al. / Vaccine 30 (2012) 2556–2563
immediate reactions. Participants measured their oral temperature
daily with a supplied digital thermometer and assessed selected
injection-site reactions (erythema, swelling, and pain) and sys-
temic adverse events daily (headache, fatigue, malaise, and fever);
all unsolicited adverse events were collected and tabulated by body
system. Adverse events were either measured (fever, erythema,
and swelling) or categorized as Grade 1 (mild, no interference
with activity), Grade 2 (moderate, some interference with activ-
ity), or Grade 3 (severe, significant interference or preventing
daily activity). Erythema and swelling were defined as Grade 0
(<25 mm), Grade 1 (25–50 mm), Grade 2 (51–100 mm), and Grade
3 (>100 mm). Fever was defined as Grade 0 (<38 ◦ C), Grade 1
(≥38 ◦ C and <38.5 ◦ C), Grade 2 (≥38.5 ◦ C and <39.5 ◦ C), and Grade
3 (≥39.5 ◦ C). Adverse events were recorded by participants in a
standardized diary for 7 days postvaccination and were collected
by study personnel at weeks 0 and 4 (time of the first two vac-
cinations), 12, 24 (time of third vaccination), and 28; telephone
reminders were performed 3 days after each injection.
Blood was collected by venipuncture before and 4 weeks after
dose 1 (at the time of dose 2); 4 and 8 weeks post-dose 2 (weeks
8 and 12); and before and 4 weeks post dose 3 (weeks 24 and
28). Anti-HBsAg was measured at each of these time points by
the hepatitis B enhanced chemiluminescence immunoassay (Hep
B ECi, Ortho Clinical Diagnostics, Raritan, NJ, USA); all testing was
done in a blinded fashion on code-labeled, matched pre- and post-
immunization sera. All anti-HBsAg levels were reported as mIU/mL.
Anti-nuclear antibodies and anti-single-stranded and anti-double-
stranded DNA were measured before the first injection and at week
28 (4 weeks post dose 3).
2.4. Data analysis and statistical considerations
The primary outcome for the study was the seroprotection
rate (SPR; proportion of participants with an anti-HBsAg level
≥10 mIU/mL) measured 4 weeks post-dose 3 (week 28) in partici-
pants who received HBV-Eng on a 0-, 4-, 24-week schedule and 8
weeks post-dose 2 (week 12) in participants who received HBV-ISS
on a 0-, 4-week schedule (time of maximum response based on ear-
lier studies). The hypothesis tested was that the SPR post-dose 2 for
HBV-ISS would be noninferior to the SPR post-dose 3 for HBV-Eng.
Noninferiority was declared if the lower limit of the 95% confidence
interval for the difference in SPR between HBV-ISS and HBV-Eng
was above −10%; superiority was declared if the lower limit of the
95% confidence interval for the difference in SPR between HBV-ISS
and HBV-Eng was above 0%. Additional outcomes included com-
parisons of SPR 4 weeks after the three-dose HBV-Eng schedule
and the two-dose HBV-ISS schedule, SPR at each visit when speci-
mens were obtained, and geometric mean antibody concentrations
(GMCs) using the same comparisons as with SPR.
Safety outcomes (solicited and unsolicited adverse events)
and laboratory measures of autoimmunity (antinuclear antibod-
ies [ANA] and anti-double-stranded DNA [anti-ds-DNA]) were also
compared. Adverse events were tabulated by day and severity; the
maximum severity reported was used for each time period. The
proportion of participants having an adverse event was calculated
by vaccine group, observation period, and severity.
The analysis population for immunogenicity outcomes was the
per-protocol population defined as participants who met the eligi-
bility criteria, did not violate the protocol in a substantial manner,
received all protocol-specified study injections, had anti-HBsAg
measurements and all injections within the specified day ranges,
and had an anti-HBsAg measurement at the time of their primary
endpoint. The intent-to-treat population was defined as partici-
pants who received at least one study injection and had at least one
post-baseline anti-HBsAg level. The safety population was defined
as enrolled participants who received at least one study injection
and had any post-baseline safety data. When treatment groups
were compared based on categorical variables, a contingency table
analysis was used with the Cochran–Mantel–Haenszel technique
to control for categorical age. Continuous variables (except for age)
were tested with an analysis of variance (ANOVA) model contain-
ing fixed effects for treatment and age. The model for age contained
only treatment as a fixed effect. All data analyses were performed
using Statistical Analysis Systems (SAS Version 8.2 or later, SAS
Institute, Cary, NC, USA).
A sample size of 1176 in the HBV-ISS group and 392 in the HBV-
Eng group (total 1568 participants) was calculated for the primary
immunogenicity outcome to have 90% power to demonstrate non-
inferiority in SPR following immunization with HBV-ISS compared
to HBV-Eng. A total sample size of 2400 (1800 HBV-ISS, 600 HBV-
Eng) was planned to ensure sufficient safety data on the HBV-ISS
cohort.
3. Results
3.1. Demographics and participant disposition
A total of 2910 individuals were screened for participation in the
study between 12 December 2006 and 12 July 2007 (Fig. 1). Of those,
482 were excluded because of pre-existing serologic markers for
HBV infection or immunization, failure to return for enrollment, or
withdrawal of consent. A total of 2428 participants were random-
ized. Although individuals between 11 and 18 years of age were
eligible to be enrolled in Canada, only 13 of the 2428 participants
enrolled were in this age cohort because most individuals of that
age had already been immunized with a hepatitis B vaccine as part
of universal immunization programs. For this reason, the analysis
focused only on the 2415 participants 18–55 years of age. The mean
age of participants was 39.9 years in the HBV-ISS group (n = 1809)
and 39.8 years in the HBV-Eng group (n = 606) (Table 1). A history
of a medical or surgical condition or event was reported by 82.1% of
HBV-ISS participants and 80.4% of HBV-Eng participants; the dis-
tribution of conditions by organ system was similar between the
two groups.
A total of 96.6% of randomized participants between 18 and 55
years of age completed the study, 96.5% (1746/1809) in the HBV-ISS
group and 97.0% (588/606) in the HBV-Eng group. Of the 63 partici-
pants who did not complete the study in the HBV-ISS group, loss to
follow up (n = 30) and withdrawal of consent (n = 18) were the most
common reasons. Other reasons included noncompliance with the
protocol (n = 3), protocol violation (n = 2), pregnancy (n = 6), with-
drawal by the sponsor because the participant was planning to be
out of the country for an extended period (n = 1), and seizure dis-
order (n = 1); there were two withdrawals related to an adverse
event. There were 18 participants in the HBV-Eng group who did
not complete the study; reasons included loss to follow up (n = 10),
non-compliance (n = 2), withdrawal of consent (n = 2), pregnancy
(n = 1), withdrawal by investigator (n = 1), and an adverse event
(n = 2).
3.2. Adverse events
The safety analysis was performed on all 2415 enrolled and ran-
domized participants. Pain at the injection site was reported by
35.3–38.6% of HBV-ISS recipients after one of the two doses and
21.6–33.7% of HBV-Eng recipients after one of the three doses; pain
was reported in progressively fewer vaccinees with subsequent
doses after either vaccine (Table 2). Severe pain was reported by
1.7% or fewer vaccine recipients after any dose. Injection-site ery-
thema and swelling was infrequent, reported by fewer than 5% of
 S.A. Halperin et al. / Vaccine 30 (2012) 2556–2563 2559

Fig. 1. Participant disposition.

either vaccine group; severe erythema or swelling was reported by solicited adverse events were no different than those reported by
0.3% or fewer recipients of either vaccine at any dose. the HBV-ISS group after their saline placebo injection (dose 3).
There were no differences in systemic adverse events between Severe systemic adverse events were infrequent; 1.2% or fewer par-
the vaccine groups (Table 2). After the second dose, rates of these ticipants reported severe fatigue, 1.3% or fewer severe headache,

Table 1
Summary of participant demographics and baseline characteristics.

Characteristic HBV-ISS (n = 1809) HBV-Eng (n = 606) Total (n = 2415)

Age, years
Mean (range) 39.9 (18–55) 39.8 (18–55) 39.9 (18–55)
18–39, n (%) 818 (45.2) 275 (45.4) 1093 (45.3)
40–55, n (%) 991 (54.8) 331 (54.6) 1322 (54.7)
Gender, n (%)
Male 852 (47.1) 262 (43.2) 1114 (46.1)
Female 957 (52.9) 344 (56.8) 1301 (53.9)
Race, n (%)
White 1690 (93.4) 556 (91.7) 2246 (93.0)
Black 39 (2.2) 20 (3.3) 59 (2.4)
Asian 43 (2.4) 22 (3.6) 65 (2.7)
First Nations or Inuit 16 (0.9) 3 (0.5) 19 (0.8)
Native Hawaiian or Pacific Islander 1 (0.1) 0 (0.0) 1 (0.0)
Other 20 (1.1) 5 (0.8) 25 (1.0)
Weight (kg)
n 1806 605 2411
Mean (range) 80.30 (43.0–173.0) 80.79 (39.3–179.0) 80.42 (39.3–179.0)
Height (cm)
n 1807 605 2412
Mean (range) 171.00 (139.7–228.6) 170.76 (147.0–196.0) 170.94 (139.7–228.6)
Body mass index
n 1806 605 2411
Mean (range) 27.41 (15.0–58.1) 27.62 (16.4–63.2) 27.46 (15.0–63.2)
Smoker, n (%)
Yes 654 (36.2) 224 (37.0) 878 (36.4)
No 1155 (63.8) 382 (63.0) 1537 (63.6)
2560 S.A. Halperin et al. / Vaccine 30 (2012) 2556–2563

Table 2
Summary of solicited reactions within 7 days of each injection.

Reaction First injection Second injection Third injection

0 weeks 4 weeks 24 weeks

HBV-ISS HBV-Eng HBV-ISS HBV-Eng Placebo HBV-Eng

n = 1809 % (95% CI) n = 606 % (95% CI) n = 1797 % (95% CI) n = 604 % (95% CI) n = 1768 % (95% CI) n = 598 % (95% CI)

Injection site 40.3 (38.0–42.6) 34.2 (30.4–38.1) 36.2 (34.0–38.5) 25.5 (22.1–29.2) 7.6 (6.4–9.0) 21.9 (18.7–25.4)
Pain 38.6 (36.3–40.9) 33.7 (29.9–37.6) 35.3 (33.1–37.6) 25.2 (21.8–28.8) 7.2 (6.1–8.5) 21.6 (18.3–25.1)
Milda 34.2 (32.0–36.4) 29.4 (25.8–33.2) 28.0 (26.0–30.2) 20.7 (17.5–24.1) 5.5 (4.5–6.7) 17.1 (14.1–20.3)
Moderateb 4.1 (3.3–5.2) 4.1 (2.7–6.0) 6.4 (5.3–7.6) 3.6 (2.3–5.5) 0.5 (0.2–1.0) 2.8 (1.7–4.5)
Severec 0.3 (0.1–0.6) 0.2 (0.0–0.9) 0.9 (0.5–1.4) 0.8 (0.7–1.9) 1.2 (0.8–1.9) 1.7 (0.8–3.1)
Redness 4.1 (3.3–5.2) 0.5 (0.1–1.4) 2.9 (2.2–3.8) 1.0 (0.4–2.2) 0.3 (0.1–0.7) 0.7 (0.2–1.7)
≥2.5 cm to ≤5.0 cm 4.0 (3.1–5.0) 0.5 (0.1–1.4) 2.6 (1.9–3.5) 0.7 (0.2–1.7) 0.3 (0.1–0.7) 0.5 (0.1–1.4)
≥5.1 cm to ≤10.0 cm 0.2 (0.0–0.5) 0.0 (0.0–0.6) 0.2 (0.1–0.6) 0.0 (0.0–0.6) 0.0 (0.0–0.2) 0.0 (0.0–0.6)
>10.0 cm 0.0 (0.0–0.2) 0.0 (0.0–0.6) 0.1 (0.0–0.4) 0.3 (0.0–1.2) 0.0 (0.0–0.2) 0.2 (0.0–0.9)
Swelling 2.3 (1.6–3.1) 0.7 (0.2–1.7) 1.5 (1.0–2.2) 0.5 (0.1–1.4) 0.2 (0.0–0.5) 0.5 (0.1–1.5)
≥2.5 cm to ≤5.0 cm 2.2 (1.5–2.9) 0.7 (0.2–1.7) 1.2 (0.7–1.8) 0.2 (0.0–0.9) 0.1 (0.0–0.4) 0.3 (0.0–1.2)
≥5.1 cm to ≤10.0 cm 0.1 (0.0–0.4) 0.0 (0.0–0.6) 0.2 (0.1–0.6) 0.0 (0.0–0.6) 0.1 (0.0–0.3) 0.0 (0.0–0.6)
10.0 cm 0.0 (0.0–0.2) 0.0 (0.0–0.6) 0.1 (0.0–0.4) 0.3 (0.0–1.2) 0.0 (0.0–0.2) 0.2 (0.0–0.9)
Systemic 26.9 (24.9–29.0) 29.2 (25.6–33.0) 21.3 (19.4–23.2) 18.5 (15.5–21.9) 12.8 (11.31–14.5) 14.5 (11.8–17.6)
Fatigue 17.4 (15.7–19.2) 16.7 (13.8–19.9) 13.8 (12.2–15.4) 12.1 (9.6–15.0) 7.9 (6.6–9.2) 10.0 (7.7–12.7)
Mild 11.9 (10.4–13.5) 10.4 (8.1–13.1) 9.4 (8.1–10.8) 7.0 (5.1–9.3) 4.7 (3.8–5.8) 5.0 (3.4–7.1)
Moderate 4.4 (3.5–5.4) 5.6 (3.9–7.8) 3.7 (2.9–4.6) 4.1 (2.7–6.0) 2.6 (1.9–3.4) 4.3 (2.9–6.3)
Severe 1.2 (0.7–1.8) 0.7 (0.2–1.7) 0.7 (0.4–1.2) 1.0 (0.4–2.1) 0.6 (0.3–1.0) 0.7 (0.2–1.7)
Headache 16.8 (15.1–18.6) 19.3 (16.2–22.7) 12.7 (11.2–14.4) 12.4 (9.9–15.3) 9.0 (7.7–10.4) 9.5 (7.3–12.2)
Mild 10.6 (9.2–12.1) 11.6 (9.1–14.4) 8.1 (6.9–9.4) 7.8 (5.8–10.2) 5.2 (4.2–6.3) 5.4 (3.7–7.5)
Moderate 5.2 (4.2–6.3) 6.4 (4.6–8.7) 3.7 (2.9–4.7) 4.0 (2.6–5.8) 3.0 (2.3–3.9) 3.5 (2.2–5.3)
Severe 1.0 (0.6–1.6) 1.3 (0.6–2.6) 0.9 (0.6–1.5) 0.7 (0.2–1.7) 0.8 (0.4–1.3) 0.7 (0.2–1.7)
Malaise 9.2 (7.9–10.6) 8.9 (6.8–11.5) 7.6 (6.4–8.9) 6.4 (4.6–8.7) 4.3 (3.4–5.4) 6.4 (4.5–8.6)
Mild 5.5 (4.5–6.7) 5.3 (3.6–7.4) 5.0 (4.0–6.1) 4.3 (2.8–6.2) 2.3 (1.7–3.1) 3.2 (1.9–4.9)
Moderate 3.1 (2.3–4.0) 2.8 (1.6–4.4) 2.0 (1.4–2.8) 1.7 (0.8–3.0) 1.4 (0.9–2.1) 2.7 (1.5–4.3)
Severe 0.6 (0.3–1.0) 0.8 (0.3–1.9) 0.7 (0.3–1.2) 0.5 (0.1–1.4) 0.6 (0.3–1.0) 0.5 (0.1–1.5)

n = 1783 n = 597 n = 1763 n = 591 n = 1675 n = 561

Fever 1.1 (0.6–1.7) 1.8 (0.9–3.3) 1.4 (0.9–2.1) 1.9 (0.9–3.3) 1.3 (0.8–2.0) 1.8 (0.9–3.3)
≥38.0 ◦ C to <38.5 ◦ C 0.6 (0.3–1.0) 1.0 (0.4–2.2) 0.5 (0.2–1.0) 1.2 (0.5–2.4) 0.8 (0.4–1.3) 0.9 (0.3–2.1)
≥38.5 ◦ C to <39.5 ◦ C 0.4 (0.2–0.8) 0.7 (0.2–1.7) 0.8 (0.4–1.3) 0.3 (0.0–1.2) 0.5 (0.2–0.9) 0.7 (0.2–1.8)
≥39.5 ◦ C 0.1 (0.0–0.4) 0.2 (0.0–0.9) 0.1 (0.0–0.4) 0.3 (0.0–1.2) 0.1 (0.0–0.3) 0.2 (0.0–1.0)
a
No interference with activity.
b
Some interference with activity.
c
Significant interference, prevents daily activity.

and 0.8% or fewer severe malaise. Only one participant (HBV-Eng)
reported a fever of 40 ◦ C or higher.
Unsolicited adverse events were reported by 60.5% of HBV-
ISS recipients and 62.0% of HBV-Eng recipients (data not shown).
Intercurrent infections were the most commonly reported adverse
event, reported by 32.5% of HBV-ISS recipients and 30.7% of HBV-
Eng recipients. Musculoskeletal complaints were reported by 14.8%
and 14.0% of HBV-ISS and HBV-Eng recipients, respectively. Other
common reports included gastrointestinal complaints (11.0% vs.
14.2%), respiratory complaints (10.2% vs. 9.1%), and nervous system
disorders (dizziness, headache; 14.0% vs. 13.9%); there were no dif-
ferences between the vaccine groups for any unsolicited adverse
event.
Overall, 41 participants reported 47 serious adverse events
including 28 (1.5%) participants in the HBV-ISS group (33 events)
and 13 (2.1%) participants in the HBV-Eng group (14 events).
Pulmonary embolism (3 participants) and jaw fracture, breast can-
cer, depression, and pneumothorax (2 participants each) were
reported by more than one participant. An autoimmune illness
was reported by one recipient in the HBV-ISS group (Wegener’s
granulomatosis 171 days post second dose) and one recipient in
the HBV-Eng group (systemic vasculitis 127 days after the second
dose). Four participants discontinued participation as a result of an
adverse event. A 35-year-old woman discontinued after developing
Guillain–Barré Syndrome 111 days after the second dose of HBV-
ISS and 5 days after receipt of influenza vaccine. A 25-year-old male
discontinued after developing a pulmonary embolism 42 days after
the second dose of HBV-ISS. There were also two discontinuations
in the HBV-Eng group: a 49-year-old woman discontinued after
increasing ocular pressure and blurred vision related to glaucoma
4 days after the first dose and a 35-year-old woman discontinued
because of a flare of arthritis in her hands 9 days after the second
dose. No deaths occurred during the study.
There were no significant changes in ANA and anti-ds-DNA,
two markers of autoimmunity, in recipients of either vaccine. ANA
titers were considered “normal” at levels ≤1:160 IU/mL. At base-
line, 89.3% (1616/1804) of participants in the HBV-ISS group and
89.3% (541/605) of participants in the HBV-Eng group had nor-
mal ANA titers. Postvaccination, at 28 weeks, 91.9% and 91.4%
of participants in the HBV-ISS and HBV-Eng groups, respectively,
had normal ANA titers. From baseline to week 28, 31 (1.7%) par-
ticipants in the HBV-ISS group and 8 (1.3%) participants in the
HBV-Eng group had a twofold ANA increase, 10 (0.6%) and 6
(1.0%), respectively, had a fourfold increase; and 1 (0.1%) and 0
(0%), respectively, had an eightfold increase. At baseline, 6 (0.3%)
of 1799 participants in the HBV-ISS group and 3 (0.5%) of 602
participants in the HBV-Eng group had positive anti-ds-DNA
results. At week 28, 10 (0.6%) and 3 (0.5%) participants, respectively,
had positive anti-ds-DNA results. From baseline, 9 (0.5%) of HBV-
ISS participants and 3 (0.5%) of HBV-Eng participants went from a
negative to positive result, and 5 (0.3%) and 3 (0.5%), respectively,
went from positive to negative.
 S.A. Halperin et al. / Vaccine 30 (2012) 2556–2563
Fig. 2. Proportion of participants seroprotected (anti-HBsAg ≥ 10 mIU/mL) after
each dose of HBV-ISS (closed circles; n = 1548–1557) or HBV-Eng (closed squares;
n = 531–533). Error bars represent 95% confidence intervals.
3.3. Antibody response
Four weeks after the first vaccination, 23.6% (95% CI 21.5–25.8)
of HBV-ISS recipients and 3.95% (2.30–5.61) of HBV-Eng recipi-
ents had seroprotective antibody levels of ≥10 mIU/mL (Fig. 2).
At 8 weeks (4 weeks post-dose 2), 88.5% (86.9–90.1) of HBV-ISS
recipients and 26.4% (22.6–30.1) of HBV-Eng recipients had sero-
protective levels. At 24 weeks (20 weeks post-dose 2 and pre-dose
3 for the HBV-Eng group), 98.3% (97.6–98.9) of HBV-ISS participants
and 32.4% (28.4–36.4) of HBV-Eng participants had seroprotective
levels. At week 28 (24 weeks post-dose 2 and 4 weeks post-saline
placebo for the HBV-ISS recipients and 4 weeks post-dose 3 for the
HBV-Eng recipients), 97.9% (97.2–98.7) of the HBV-ISS recipients
and 81.1% (77.7–84.4) of the HBV-Eng recipients had seroprotec-
tive levels. For the primary immunogenicity outcome, the SPR at
week 12 (8 weeks postvaccination) in the HBV-ISS group (95.1%;
94.0–96.1) was noninferior and superior to that of the HBV-Eng
group at week 28 (4 weeks post-dose 3) (difference of HBV-ISS
minus HBV-Eng = 13.9%; 10.6–17.6). Noninferiority and superior-
ity of SPR were also demonstrated at all other serology time points.
Higher SPRs were observed in younger participants (aged 18–39
years) and older participants (aged 40–55 years) injected with
HBV-ISS at each time point compared to participants injected with
HBV-Eng (Table 3). Higher SPRs were achieved by younger recipi-
ents compared to older recipients of both vaccines.
The magnitude of the immune response was also greater after
the first and second doses of HBV-ISS compared to HBV-Eng (Fig. 3).
In HBV-Eng recipients, a substantial rise in GMC did not occur until
after dose 3, when the GMC rose from 7.19 (6.31–8.20) mIU/mL
to 348.2 (265.9–455.9) mIU/mL. In contrast, in the HBV-ISS group,
the GMC rose from 5.5 (5.13–5.88) mIU/mL 4 weeks post-dose
1 to 81.5 (75.1–88.5) mIU/mL 4 weeks post-dose 2, to 136.9
(127.5–146.9) mIU/mL 8 weeks post-dose 2. The GMC continued to
rise in this group and at week 24 (20 weeks post-dose 2) was 342.5
(320.2–366.5) mIU/mL. Four weeks later, the GMC in the HBV-ISS
group (24 weeks post-dose 2) was 320.0 (298.2–343.3) mIU/mL,
similar to the GMC in the HBV-Eng group 4 weeks post-dose 3.
GMCs of participants in the younger age stratum were higher
than those in the older age group for both HBV-ISS and HBV-Eng
(Table 3). For all time points, the younger recipients of the HBV-
ISS vaccine had GMCs approximately twofold higher than the older
participants. Lower responses for the older participants were also
observed after the first two doses of HBV-Eng although the results
were less dramatic given the low GMCs of both age strata. How-
ever, post-dose 3 of HBV-Eng, younger participants achieved a GMC
threefold higher than the older cohort.
The magnitude of the immune response was also examined by
comparing the proportion of participants who exceeded a thresh-
old antibody level of ≥100 mIU/mL at each time point. Statistical
2561
Fig. 3. Geometric mean antibody concentrations after each dose of HBV-ISS (closed
circles; n = 1548–1557) or HBV-Eng (closed squares; n = 531–533). Error bars repre-
sent 95% confidence intervals.
superiority was demonstrated for the HBV-ISS group compared to
the HBV-Eng group at week 8 (50.2% vs. 9.0%), week 12 (63.7% vs.
6.6%), week 24 (83.5% vs. 9.8%), and week 28 (82.2% vs. 65.5%). Anti-
body levels ≥100 mIU/mL were superior in the HBV-ISS group for
both the younger and older cohorts (data not shown).
4. Discussion
This study was designed to test the primary hypothesis that the
SPR 8 weeks after two doses of the HBV-ISS vaccine would be nonin-
ferior to the SPR 4 weeks after three doses of the licensed HBV-Eng.
The established noninferiority criterion was met in the study at the
primary time point of maximal response of the HBV-ISS as well
as at all other times when antibody levels were measured. More-
over, the SPR of HBV-ISS was statistically superior at all time points
compared to the licensed HBV-Eng. The magnitude of the antibody
response to HBV-ISS, as measured by GMC, was also noninferior to
HBV-Eng at all time periods assessed. Statistical superiority of the
GMC after HBV-ISS compared to HBV-Eng was also demonstrated at
all time periods except for week 28 (24 weeks post-dose 2 of HBV-
ISS and 4 weeks post-dose 3 of HBV-Eng). Of note, the superiority
of the immune response to HBV-ISS was observed for both younger
(<40 years of age) and older (≥40 years of age) participants. Both
vaccines were well tolerated by participants. Mild injection-site
adverse events were more commonly reported by HBV-ISS recip-
ients than HBV-Eng recipients but systemic adverse events were
reported with equal frequency after either vaccine.
The findings of this study support the results of previous stud-
ies with the HBV-ISS vaccine. Seroprotection rates as high as 87.5%
after the first dose and 100% after the second dose given 2 months
apart were found in a phase 1 dose-ranging study [37]. In a phase
2 comparison of two doses of HBV-ISS given at weeks 0 and 8 with
three doses of a licensed hepatitis B vaccine given at weeks 0, 8,
and 24 [38], 79% of HBV-ISS and 12% of HBV-Eng recipients had a
protective antibody response 4 weeks post dose 1. By 1 week post
dose 2, 100% of HBV-ISS and 18% of HBV-Eng recipients had pro-
tective levels. Rates of seroprotection after HBV-Eng in the current
study and the previous phase 2 study are similar to those reported
in other studies of HBV-Eng [14].
The rapid induction of protective antibody levels after a two-
dose schedule at 0 and 4 weeks has a number of advantages,
particularly where rapid protection is required or where follow-up
for subsequent doses cannot be guaranteed. Three-dose hepatitis B
immunization schedules spread over 6 months are problematic for
adolescents who are less frequent users of the health care system
[14]; a licensed two-dose adolescent schedule decreases the num-
ber of injections but still requires that the doses be separated by 6
months. For individuals with high-risk behaviors, rapid induction
of immunity diminishes the period of time the individual remains
at risk for acquisition of hepatitis B [18]. Two-dose programs
2562 S.A. Halperin et al. / Vaccine 30 (2012) 2556–2563

Table 3
Summary of seroprotection and geometric mean concentrations (GMC) by week and age stratification.

Age stratification Week Seroprotection GMC

HBV-ISS % (95% CI) HBV-Eng % (95% CI) HBV-ISS % (95% CI) HBV-Eng % (95% CI)

18–39 0
4 30.5 (27.0, 34.0) 5.80 (2.74, 8.87) 6.93 (6.18, 7.78) 3.16 (2.81, 3.55)
8 94.8 (93.1, 96.5) 33.0 (26.9, 39.2) 125.1 (112.1, 139.5) 8.43 (6.63, 10.7)
12 98.7 (97.8, 99.5) 30.1 (24.1, 36.1) 212.0 (193.7, 232.1) 7.24 (5.81, 9.03)
24 99.6 (99.1, 100.0) 39.7 (33.3, 46.1) 512.0 (470.2, 557.6) 9.63 (7.66, 12.1)
28 99.3 (98.6, 99.9) 89.4 (85.4, 93.4) 475.7 (434.4, 520.9) 655.9 (443.3, 970.4)

40–55 0
4 18.5 (15.9, 21.0) 2.61 (0.82, 4.39) 4.60 (4.25, 4.99) 2.77 (2.59, 2.96)
8 83.7 (81.2, 86.1) 21.5 (16.9, 26.1) 58.6 (52.3, 65.7) 5.29 (4.51, 6.20)
12 92.3 (90.5, 94.0) 16.9 (12.7, 21.1) 97.8 (88.6, 108.0) 4.46 (3.91, 5.09)
24 97.3 (96.2, 98.3) 27.0 (22.1, 32.0) 251.3 (228.5, 276.4) 5.81 (5.00, 6.76)
28 96.9 (95.8, 98.1) 74.9 (70.1, 79.8) 236.1 (213.9, 260.5) 218.4 (152.2, 313.6)

would be less expensive to deliver than those requiring three doses
in terms of vaccine cost and human resources. Additionally, the
improved immunogenicity in the older cohort raises the potential
for improved seroprotection amongst other hypo-responders and
nonresponders [21,22]. Finally, noncompletion of travel-related
vaccination series is common because of insufficient planning prior
to travel; rapid protection after a two-dose course completed in 4
weeks would facilitate protection prior to travel.
A number of other vaccines and schedules with improved
immunogenicity have also recently become available. Three-dose
schedules of licensed vaccine given at 0, 1, and 2 months have been
advocated to achieve more rapid responses for travelers and health
care workers with a fourth dose given at 1 year but result in lower
GMC [4]. A hepatitis B vaccine with a novel adjuvant (AS04) com-
prising aluminum phosphate and 3-O-desacyl-4# -monophosphoryl
lipid A (MPA) also provides more rapid and greater levels of protec-
tion against HBV after a four-dose regimen than the licensed alum
adjuvanted vaccine in renal failure and dialysis patients but is not
licensed for use in healthy adults [39,40].
One limitation of the current study was our inability to enroll
sufficient adolescent participants. Although the intention was to
enroll adolescents and adults at study sites in Canada, the existence
of universal, school-based, adolescent HBV vaccination programs
with high coverage rates in Canada for several years prior to the
study left very few eligible adolescents. Thus, only 13 adolescents
were enrolled in the study and the analysis was undertaken with
participants from 18 to 55 years of age. This limits the general-
izability of the study to adolescents, and additional studies with
pre-adolescents and adolescents will likely be required. Given the
improved immunogenicity to all hepatitis B vaccines including
HBV-ISS in younger individuals, it is likely that adolescents will
have a robust immune response to HBV-ISS. However, as HBV-ISS
contains a novel adjuvant, studies in this age cohort to demonstrate
safety, tolerability, and duration of protection are needed. Although
hepatitis B vaccines are used widely in infants and younger chil-
dren, studies with HBV-ISS in these age cohorts will likely await
the results of longer term postlicensure safety studies in adults.
In summary, HBV-ISS was welltolerated and immunogenic in
healthy adults from 18 to 55 years of age and demonstrated
superior immunogenicity and earlier onset of protection with a
two-dose schedule over 4 weeks compared to three doses of a
licensed, alum-adjuvanted hepatitis B vaccine. Comparison of the
antibody responses 1 year after immunization and beyond with
the currently licensed alum-adjuvanted vaccines will help to fur-
ther characterize the long-term benefits of this novel hepatitis
B vaccine. Additional studies in adolescents, immunocompro-
mised hosts, dialysis and renal failure patients, hyporesponders
and nonresponders, and eventually young infants and children
may be useful to demonstrate further the full range of potential
benefits.
Acknowledgments
At the Canadian Center for Vaccinology, we thank Petra Rykers,
Donna MacKinnon-Cameron, Christina Wang, and Bruce Smith for
undertaking the independent analysis of the data and Paula Ogg
for preparing the tables and figures for publication. We thank the
nurses and research assistants at all of the study sites for their
careful attention to detail.
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