Vaccine 30 (2012) 5761–5769
Contents lists available at SciVerse ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin
fusion vaccine with improved safety and immune response夽,夽夽
David N. Taylor a,∗ , John J. Treanor b , Eric A. Sheldon c , Casey Johnson d , Scott Umlauf a , Langzhou Song a ,
Uma Kavita a , Ge Liu a , Lynda Tussey a , Karen Ozer e , Thomas Hofstaetter a , Alan Shaw a
a
VaxInnate Corporation, 3 Cedar Brook Drive, Cranbury, NJ 08512, United States
University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, United States
c
Miami Research Associates, 6141 Sunset Drive, Suite 501, Miami, FL 33143, United States
d
Johnson County Clin-Trials, Lenexa, KS 66219, United States
e
CYTEL, Inc., 675 Massachusetts Avenue, Cambridge, MA 02139, United States
b
a r t i c l e
i n f o
Article history:
Received 20 January 2012
Received in revised form 22 June 2012
Accepted 29 June 2012
Available online 11 July 2012
Keywords:
Influenza vaccine
Flagellin adjuvant
Recombinant vaccine
Influenza prevention
Vaccine
a b s t r a c t
Background: We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs
consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand,
flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin
in VAX128B and was fused in both positions in VAX128C.
Methods: In a dose escalation trial, 112 healthy subjects 18–49 and 100 adults ≥65 years old were enrolled
in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses
ranging from 0.5 to 20 ␮g. VAX128C was selected for second study performed in 100 subjects 18–64
years old comparing 1.25 and 2.5 ␮g doses. All subjects were followed for safety and sera collected preand post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and
cytokine levels were also measured.
Conclusions: In the first study high HAI titers and high seroconversion and seroprotection rates were
observed at doses ≥2.5 ␮g in adults 18–49. In adults ≥65 years, the vaccines doses of ≥4 ␮g were required
to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A
was tested up to 8 ␮g, VAX128B to 16 ␮g and VAX128C to 20 ␮g. Dose escalation for VAX128A was stopped
at 8 ␮g because one subject had temperature 101.6 ◦ F associated with a high CRP response, VAX128B was
stopped at 16 ␮g because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not
stopped before reaching the 20 ␮g dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 ␮g. The peak GMT was 385 (95%CI 272–546), 79% (71–87%) seroconversion
and 92% (84–96%) seroprotection.
Discussion: Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The worldwide need for seasonal and pandemic influenza vaccines has increased interest in the development of innovative
technologies for influenza vaccine production [1]. In addition to
夽 This study was presented in part at the 14th Annual Conference on Vaccine
Research, May 16, 2011, Baltimore, MD (abstract S-6).
夽夽 The study was approved by the Institutional Review boards at Miami Research
Associates, Miami, FL and Johnson County Clin Trials, Lenexa, KS. All subjects gave
written informed consent prior to participation. The trial was registered on ClinicalTrials.gov number NCT01172054.
∗ Corresponding author. Tel.: +1 609 860 2289; fax: +1 609 860 2290;
mobile: +1 919 349 6109.
E-mail address: [email protected] (D.N. Taylor).
0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.06.086
improvements in vaccine production efficiency, enhancement of
the immunopotency of influenza vaccines is required to meet seasonal and pandemic needs on a global scale. A vigorous immune
response to the hemagglutinin (HA) outer surface glycoprotein is
widely considered to represent an important surrogate of protection against influenza infection and associated disease.
We developed a novel H1N1 pandemic influenza vaccine
designated, VAX128, based on A/California/7/2009. VAX128 is
a recombinantly produced vaccine that activates the immune
response by coupling a potent immune stimulator, the bacterial protein flagellin (STF2), to the globular head domain of the
influenza HA antigen [2]. The globular head domain of HA stably
refolds to form the conformationally sensitive antigenic sites that
span the majority of the neutralizing epitopes in HA. This vaccine
elicits a potent virus-specific neutralizing antibody response and
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D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
Fig. 1. Ribbon diagram and schematic representation of the flagellin-HA globular head fusion vaccine candidates for influenza A/California/07/2009. Indicated are the flagellin
domains D0, D1, D2, and D3 as well as HA1 (open circle) of Influenza A/California/07/2009.
the prokaryotic expression system allows for very efficient manufacture. VAX128 is highly protective in mice [3].
Previously, we tested VAX125, a monovalent seasonal prototype based on the hemagglutinin of H1N1 Solomon Islands, in
Phase I studies in healthy young adult and elderly subjects [4,5].
VAX125 was highly immunogenic at a dose of 1 ␮g. Although the
vaccine was generally well tolerated, there were a few instances
of flu-like symptoms (malaise and fever) in the first 24 h following vaccination at doses of 3–8 ␮g. These symptoms were
associated with elevated C-reactive protein (CRP) responses suggesting that they were mediated by cytokine production. Although
VAX125 was safe and immunogenic in the 1–2 ␮g dose level, it
was important to ensure that the amount of flagellin in a multivalent vaccine would be well tolerated. In a previous study in
mice, we observed that the A/Vietnam/1203/2004 H5 HA, a poor
immunogen, was highly immunogenic when substituted for the D3
domain of flagellin [6]. For VAX128 we produced similar modifications in the attachment site(s) of the HA to the flagellin moiety
and found that modification of the attachment site could influence reactogenicity in mice without impacting immunogenicity
[3]. In the current study we performed a head-to-head comparison of the safety and immunogenicity of three VAX128 constructs
first in the rabbit model and then in humans where we evaluated the safety profiles, CRP and cytokine response to doses up to
20 ␮g.
2. Methods
2.1. Vaccine constructs
VAX128A, VAX128B, and VAX128C differ in the site of attachment of the H1 A/CA07/2009 HA subunit to flagellin (Fig. 1). In
VAX128A the HA globular head is fused to the C-terminal end
of flagellin; in VAX128B the HA globular head is moved from
the C-terminus to replace domain three (D3) of flagellin, and in
VAX128C the HA globular head is fused to the C-terminal end of
flagellin and a second copy replaces D3. The basic cloning, production and analytical assays for the different construct formats are
very similar. Flagellin accounts for over 60% of the vaccine composition in VAX128A and VAX128B and 46% in VAX128C. F147 buffer
(10 mM Tris, 150 mM NaCl, 10 mM l-histidine, 0.5% (v/v) ethanol,
5% trehalose (w/v), 0.1 mM EDTA, 0.02% (w/v) polysorbate-80, pH
7.0) is the vaccine buffer and was used as the control substance in
the rabbit studies.
2.2. Rabbit model
Before evaluating the safety and immunogenicity in humans,
a dose ranging study of VAX128A, VAX128B and VAX128C was
carried out in the rabbit model. VAX125 was added as an active
comparator because it was constructed similarly to VAX128A and
has a known profile in both pre-clinical and clinical studies [4,5].
Groups of 4–6 New Zealand White rabbits were immunized with
doses of 0.5, 1.5, 5 or 15 ␮g delivered intramuscular (IM) on days
0 and 21. Sera collected before immunization on day 0 and 7 days
after the booster immunization were evaluated for HAI antibody
titers. Rabbit body temperature was measured during the 24 h postimmunization and CRP was measured one day after the first dose.
Body temperature and CRP elevations were compared to the F147
buffer control.
2.3. Human subjects
Subjects were healthy as ascertained by medical history,
screening physical examination and screening laboratory analysis. Subjects with active medical conditions, abnormal screening
laboratory tests (CBC and differential, and AST and ALT), allergies
to vaccines, influenza vaccination within the previous 6 months,
or receipt of other vaccines within 30 days were excluded from
participation. For subjects ≥65 years old, mental status was based
on the results of the Short Portable Mental Status Questionnaire
(SPMSQ) and frailty was defined by the Canadian Study of Health
and Aging Clinical Frailty Scale (CSHA-CFS). Subjects with more
than mild dementia or mild frailty as determined by these two tests
were also excluded.
2.4. Study design
We performed two studies to evaluate the VAX128 vaccine
constructs in humans. Study 1 was designed as a dose escalation
study. A total of 112 healthy young adults 18–49 years old and 100
community-living adults who were ≥65 years of age enrolled, with
D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
5763
Table 1
Number of subjects enrolled at each dose level by VAX128 vaccine construct A, B or C or placebo.
Dose (␮g)
Age 18–49 years
Age ≥65 years
Age 18–64
VAX128
VAX128
VAX128C
A
0
0.5
1.25
2.5
4
8
12
16
20
3
3
12
12
6
0
0
0
Total
36
B
C
Plac.
Total
A
3
3
3
3
3
3
12
0
3
3
3
3
3
3
12
6
10
0
0
0
0
0
0
0
0
10
9
9
18
18
12
6
24
6
0
0
3
3
3
21
0
0
0
30
36
10
112
30
B
C
Plac.
0
3
3
3
6
3
12
0
0
3
3
3
3
3
3
12
10
0
0
0
0
0
0
0
0
Total
10
0
9
9
9
30
6
15
12
30
30
10
100
Total
49
51
100
VAX128 (STF2.HA1 A/CA/07/09), Plac. = F147 dilution buffer placebo.
healthy young adults enrolled at one study center (Miami, FL) and
healthy subjects ≥65 years enrolled at a second center (Lenexa,
KS). VAX128A, B or C vaccine was administered according to a
protocol-specified dose escalation schedule to assess the safety and
immunogenicity of the three vaccine constructs at doses of 0.5 ␮g,
1.25 ␮g, 2.5 ␮g, 4.0 ␮g, 8.0 ␮g, 12.0 ␮g, 16.0 ␮g, or 20.0 ␮g, delivered as a single-dose IM vaccination on day 0. The study design was
the same for subjects ≥65 years except dosing started at 1.25 ␮g.
Each dosing group was divided into 3 subjects for each vaccine
plus one placebo for a total of 10 subjects. Dose escalation in both
age groups took place if the Safety Monitoring Committee (SMC)
concluded that the preceding lower dose was well tolerated based
on assessments at the day 1 clinic visit, including the safety laboratory data. Each week 10 subjects were immunized, 3 received
VAX128A, 3 VAX128B and 3 VAX128C and 1 placebo. The SMC
evaluated the safety in an unblinded fashion and made recommendations that included stopping, increasing the dose or repeating
the dose. Stopping rules were predefined in the protocol. There
were several instances where the association of symptoms with
a certain dose could not be clearly determined so the dose was
repeated. This led to some unevenness in the group sizes as shown
in Table 1.
Subjects remained at the study site for 4 h following vaccination
and were also evaluated at clinic visits on study days 1, 7, 14, and
28 following vaccination. Participants maintained a memory aide
beginning on the day of vaccination and for 6 days thereafter to
record solicited local and systemic reactions graded as none, mild,
moderate, or severe. Adverse reactions were assessed by study participants using a 4 point scale (0–3). Solicited local reactions were
redness, swelling or induration, pain and ecchymosis. Solicited systemic reactions were fever, headache, joint pain, fatigue, muscle
aches, shivering (chills) and increased sweating. Long term safety
follow-up occurred by telephone on days 180 and 360. Serum Creactive protein was measured pre-vaccination and one day after
vaccination and cytokine levels were measured pre-vaccination
and 2 h after vaccination. Immune response was evaluated on days
0, 14 and 28.
VAX128C was selected for further testing based on assessment
of reactogenicity and immunogenicity in the first study. In the second study 100 healthy adults 18–64 years old were enrolled at both
centers and randomized to receive 1.25 ␮g or 2.5 ␮g of VAX128C;
there was no placebo group. Approximately 25% of the population
in each dose group was 50–64 years old. Safety assessment was
performed with the same methods used in the first study. Laboratory was also the same except that a day 1 visit was not required
and CRP was not obtained. Cytokine profiles were still performed
prior to vaccination and 2 h after. Immune response was evaluated
on days 0 and 21.
2.5. Laboratory assays
2.5.1. Clinical chemistries
Laboratory analysis included CBC, BUN, creatinine, urinalysis
and liver function tests. C reactive protein (CRP) assays were performed on Day 0 and day 1 in the first study.
2.5.2. Cytokine bead array
Cytokines were measured on plasma collected pre-vaccination
and 2 h post-vaccination using the Cytokine Bead Array (CBA, Becton Dickenson), according to manufacturer’s instructions, and read
using flow cytometry. Cytokines assessed included IL-1␤, IL-6, IL-8,
IL-10, IL12p70, and TNF-␣.
2.5.3. HAI assay
Serum samples were assessed for antibody to the California 07
H1 antigen by microtiter hemagglutination inhibition (HAI) using
standard methods [7]. Egg-grown influenza A/California/04/09 was
obtained from the CDC, Atlanta, GA, and expanded in eggs for use
as antigen in the assay. Serum samples were treated with receptordestroying enzyme (Denka Seiken, Tokyo, Japan); prior to testing
and tested in serial 2-fold dilutions beginning at an initial dilution
of 1:4. Serum samples with no reactivity at 1:4 were assigned a
value of 1:2.
2.5.4. Flagellin (STF2)-specific IgG ELISA
Sera were tested for flagellin-specific IgG antibody by ELISA as
previously described [4].
2.6. Statistical analysis
Statistical analyses were performed at the two-sided significance level of ˛ = 0.05 unless otherwise stated. Serology endpoints
were analyzed using log10 transformed data, back transformed to
the original scale for presentation of geometric mean titers and
mean fold increases. All subjects were included in the descriptive statistics and safety and tolerability analyses. Chi-square or
Fisher’s exact tests for categorical data and analysis of variance
for continuous data was employed. Frequency of vaccination site
abnormalities; incidence of local and systemic adverse events (AEs)
and their relationship to the study drug; and changes in clinical laboratory results, vital signs, and physical examination findings were
the primary safety measures. Rates of reactions were compared
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D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
(0.5 ␮g) VAX128C was significantly more immunogenic than either
VAX128A or VAX128B.
For VAX125 and VAX128A body temperature and serum CRP
were affected in a dose-dependent fashion (Fig. 3). VAX128B
and VAX128C did not show temperature elevation at any dose.
VAX128B and VAX128C showed low CRP levels comparable to baseline levels at 0.5, 1.5 and 5 ␮g. VAX128B, but not VAX128C, showed
a CRP increase at 15 ␮g. The association between the body temperature and CRP elevation with VAX125 and VAX128A suggests that
the elevated body temperature may occur as a result of cytokine
release for which CRP is a marker.
3.2. Vaccine safety in humans
Fig. 2. Humoral immune responses to VAX128A, VAX128B and VAX128C in rabbits.
Four to six rabbits per group were given two immunizations with vaccine at indicated doses on days 0 and 21. Serum was collected 7 days after the booster dose for
HAI testing. Results show the geometric mean (GMT) ± 95% CI. At the lowest dose
VAX128C was significantly more immunogenic than either VAX128A or VAX128B,
p < 0.05, at the highest dose VAX128C was significantly more immunogenic than
VAX128A, p < 0.01 in 2-way ANOVA with Bonferroni correction.
using a Fisher’s Exact test on proportions and a two-sided 95% confidence interval. In the rabbit model a two-way ANOVA analysis
with a Bonferroni adjustment for multiple comparisons, was performed to determine if there was a significant difference between
different vaccines or between different doses of the same vaccine.
This analysis was used to determine if the difference among different vaccines at each dose level was significantly different, adjusting
for multiplicity. All programs for data output and analyses were
written in SAS® version 9.1 (SAS Institute, Cary, NC).
3. Results
3.1. Vaccine immunogenicity and safety in rabbits
All three of the VAX128 vaccine constructs were immunogenic in the rabbit model with ≥4-fold increases in HAI antibody
titer observed 7 days post the booster immunization at all dose
levels (Fig. 2). At the highest dose (15 ␮g) VAX128C was significantly more immunogenic than VAX128A and at the lowest dose
A total of 312 subjects were enrolled in two studies. In the first
study 112 adults 18–49 years old and 100 adults ≥65 years old were
enrolled in a dose escalating trial to compare three VAX128 vaccine
constructs, designated A, B and C (Table 1). The dose ranged from
0.5 ␮g to 20 ␮g. Subjects in first study were enrolled from July to
October 2010. The young adults were enrolled at one site in Miami,
FL and the elderly were enrolled at a site in Lenexa, KS. In a second
study two dose levels were selected from the first study based on
the safety and immunogenicity profile and subjects aged 18–64
years were randomized to 1.25 or 2.5 ␮g. The second study was
conducted in November 2010 with the two sites from the first study
enrolling approximately equally. The mean age of the young adults
was 31.5 years and the 73.1 years for the elderly. Young adults were
equally divided between genders and the elderly were 64% women.
Nearly half of the young adults were Hispanic and nearly all of the
elderly population was white, non-Hispanic; reflecting the race,
ethnicity of Miami and Lenexa, respectively.
3.2.1. Young adults
The study design allowed the dose to be increased until the onset
of reactogenicity up to a maximum dose of 20 ␮g, the highest dose
that could be formulated to a 0.5 ml injection volume. Dose escalation continued until grade 3 (severe) systemic adverse events were
observed. Among subjects 18–49 years old, VAX128A was tested
up to 8 ␮g. One of 12 subjects who received 4 ␮g developed severe
fatigue, joint pain, muscle aches, chills and sweats. These symptoms were not associated with CRP or cytokine elevations. One of
6 subjects who received VAX128A at 8 ␮g had a fever of 101.6 ◦ F
(moderate), with mild arm pain, headache, muscle aches and chills
Fig. 3. Effect of VAX125, VAX128B, VAX128B and VAX128C on body temperature and serum C reactive protein (CRP) in rabbits. Groups of 4–6 rabbits were injected i.m. with
the indicated dose of VAX125 [4], VAX128B, VAX128B and VAX128C. Temperature was monitored for 10 h after vaccination. Sera were collected for CRP 24 h after vaccination
and CRP was measured using a commercial ELISA (Immunology Consultants Laboratory, Newberg, OR). Data are presented as group means ± SEM.
D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
5765
Table 2a
Number (%) of subjects 18–49 with local and systemic symptoms in study 1, n = 112.
VAX128A
Dose ranges
No. of subjects
Local symptoms (n, %)
None
Mild
Moderate
Severe
Systemic symptoms
None
Mild
Moderate
Severe
VAX128B
VAX128C
Placebo
0.5–1.25
6
2.5
12
4
12
8
6
0.5–12
18
16
12
0.5–12
18
16–20
18
0
10
0
3 (50)
2 (33)
1 (17)
2 (17)
3 (33)
7 (58)
0
3 (25)
4 (33)
4 (33)
1 (8)
0
3 (50)
2 (33)
1 (17)
3 (17)
7 (39)
8 (44)
0
2 (17)
7 (58)
3 (25)
0
4 (22)
9 (50)
4 (22)
1 (6)
2 (11)
3 (17)
10 (56)
3 (17)
8 (80)
2 (20)
0
0
3 (50)
3 (50)
0
0
9 (75)
1 (8)
2 (17)
0
11 (92)
0
0
1 (8)
3 (50)
2 (33)
1 (17)
0
11 (61)
6 (33)
1 (6)
0
9 (75)
1 (8)
1 (8)
1 (8)
12 (67)
3 (17)
3 (17)
0
11 (61)
3 (17)
3 (17)
1 (6)
6 (60)
2 (20)
2 (20)
0
that was associated with a 10-fold rise in CRP but no significant
increase in cytokines. VAX128B was tested up to 16 ␮g. At 16 ␮g,
one of 12 subjects had a severe headache and severe chills associated with a high CRP and cytokine response. A number of other
subjects who received high doses of VAX128B had ≥4-fold increase
in IL-6 without manifesting symptoms. VAX128C was tested up
to 20 ␮g. One of 12 subjects who received 16 ␮g reported severe
sweating associated with mild fatigue, muscle aches and chills.
None of six subjects who received 20 ␮g had severe systemic symptoms or elevations in CRP or cytokines. One subjects reported severe
arm pain associated with mild fever of 101.1 ◦ F. None of the 18 subjects who received VAX128C at 16 or 20 ␮g had significant rises CRP
or cytokines.
Local and systemic reactogenicity for young adults are shown
in Table 2a. The VAX128B and C subjects have been divided into a
low dose group ranging from 0.5 to 12 ␮g and a high dose group
ranging from 16 to 20 ␮g. Both constructs were well tolerated in
the low dose range where the distribution of systemic symptoms
was similar to subjects receiving placebo.
3.2.2. Elderly adults
Among subjects ≥65 years old, VAX128A was well tolerated
by all of 30 subjects who received doses ranging from 0.5 to 8 ␮g
(Table 2b). VAX128B was tested at doses up to 16 ␮g. One subject
who received 8 ␮g had severe chills with a 23-fold increase in CRP
and a 32-fold increase in IL-6. Another subject who received 16 ␮g
had moderate fatigue, joint pain, muscle aches and chills associated with an 82-fold increase in CRP and a 222-fold increase in
IL-6. VAX128C was tested up to 20 ␮g. VAX128C was well tolerated
at all of the lower doses. One of 12 subjects who received the 20 ␮g
dose had severe chills and moderate fatigue and muscle aches. This
subject also had a 20-fold increase in CRP, but little increase in
cytokines.
3.3. SAE and long term follow-up
Long term follow up for SAEs and new chronic conditions was
performed at day 180 and day 360 after vaccination. No subject in
either study experience a study product-related SAE.
3.3.1. CRP and cytokine response
CRP levels in subjects receiving all three constructs at the 0.5 ␮g
dose were similar to placebo (Table 3). There was a stepwise
increase in mean CRP level post vaccination and fold increase in
each dose group. At doses of 4–8 ␮g and 12–20 VAX128B show
higher responses in both CRP and IL-6 than VAX128A or VAX128C.
A total of 15 (4.8%) of 312 subjects who received VAX128A, B or
C had a 4-fold or greater increase in IL-6 (Table 4). In young adults
9 (8%) of 112 subjects had 4-fold increases in IL-6. One of these 9
subjects had severe systemic symptoms and the other 8 subjects
had either none or mild systemic symptoms. The subjects with the
highest cytokine responses received the 16 ␮g dose of VAX128B.
Elevations in CRP and IL-6 were significantly associated. In the
young adult cohort, 9 (8.4%) of 107 subjects had a ≥4-fold elevation
IL-6 and 8 of those 9 subjects had either a post-immunization of CRP
of ≥40 mg/L or a 40-fold increase in CRP. In contrast, only 2 of 98
subjects with a less than 4-fold increase IL-6 had a CRP elevation of
comparable magnitude, p < 0.0001, Fisher’s exact test, two-tailed.
Two subjects who received VAX128B at 16 ␮g had elevated
WBCs and one subject had elevated liver function tests on day 1.
The subject with the highest IL-6 value in Table 4 had a 10-fold
increase in SGOT and SGPT on day 1 that was normal on day 7. This
subject also had a WBC of 14,800 on day 1 that was normal by day 7.
The subject with an IL-6 of 832 also had an elevated WBC of 16,200
on day 1 with normal LFTs. No other subject in either the young
adult or elderly group had a chemistry or hematology laboratory
test abnormality.
Table 2b
Number (%) of subjects ≥65 with local and systemic symptoms in study 1, n = 100.
VAX128A
Dose ranges (␮g)
Local symptoms (n, %)
None
Mild
Moderate
Severe
Systemic symptoms
None
Mild
Moderate
Severe
VAX128B
VAX128C
Placebo
1.25–4
n=9
8
n = 21
1.25–12
n = 18
16
n = 12
1.25–12
n = 15
16–20
n = 15
0
n = 10
3 (33)
3 (33)
3 (33)
0
6 (29)
12 (57)
3 (14)
0
6 (33)
10 (56)
2 (11)
0
2 (17)
4 (33)
6 (50)
0
2 (20)
11 (67)
2 (13)
0
3 (20)
5 (33)
7 (47)
0
10 (100)
0
0
0
8 (89)
1 (11)
0
0
14 (67)
3 (14)
4 (19)
0
13 (72)
3 (17)
1 (6)
1 (6)
6 (50)
4 (33)
2 (17)
0
13 (87)
2 (13)
0
0
11 (73)
3 (20)
0
1 (7)
8 (80)
2 (20)
0
0
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D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
Table 3
CRP and IL-6 response by dose group among subjects 18–49 years old who received a single dose of VAX128A, B or C.
Dose 0.5 ␮g
No. of subjects
Mean CRP
At day 0
At day 1
Fold increase
CRP at day 1
Value ≥40
Fold inc. ≥40
Either
IL-6 fold increase
≥4-fold
≥10-fold
Dose 1.25–2.5 ␮g
Dose 4–8 ␮g
Dose 12–20 ␮g
Placebo
10
A
3
B
3
C
3
A
15
B
6
C
6
A
18
B
6
C
6
1.7
1.9
1.1
1.7
1.6
0.9
1.9
2.2
1.1
1.3
1.4
1.1
1.8
7.2
3.5
1.7
6.6
3.9
1.1
6.8
6.3
1.7
9.1
4.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1 (6)
0
1 (6)
1 (17)
3 (50)
4 (67)
0
0
0
4 (27)
1 (7)
5 (33)
0
1 (5)
1 (5)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2 (33)
1 (17)
0
0
5 (33)
3 (20)
2 (10)
1 (5)
1.0
12.8
13.1
B
15
1.3
7.3
5.8
C
21
1.7
13.7
8.1
1.7
7.2
4.2
Table 4
Cytokine response for subjects with fold rise of IL-6 among subjects who received VAX128 [15 (4.8%) of 312 subjects had a 4-fold or greater increase in IL-6].
VAX128 VAX128 Dose (␮g)
Study
construct
Age group
CRPDay 0
CRPDay 1
CRP fold
IL-6
Pre
IL-6
2h
IL-6
fold
TNF
Pre
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
18–49
18–49
18–49
18–49
18–49
18–49
18–49
18–49
18–49
≥65
≥65
≥65
≥65
18–49
18–49
0.1
17
0.4
2
13
2
3
1
2
2
1
1
4
ND
ND
9
51
35
20
100
44
68
24
59
50
22
99
76
ND
ND
90
3
87
13
7
22
23
47
33
23
44
82
20
ND
ND
2
2
1
2
2
2
2
2
3
4
2
3
2
1.8
1.6
13
132
31
8
20
16
832
1772
7488
135
16
604
29
7.9
6.4
6
85
24
4
8
8
389
1094
2463
32
8
222
13
4
4
0
0
0
2
1
1
0
2
3
1
0
0
0
1
0
B
B
C
C
B
B
B
B
B
B
B
B
C
C
C
4
8
12
16
16
16
16
16
16
8
16
16
20
2.5
2.5
3.3.2. Safety among subjects 18–64 years in study 2
VAX128C was selected for further study at doses of 1.25 and
2.5 ␮g. Per protocol there were 12 subjects (∼25%) 50–64 years of
age in each dose group. The rest of the subjects were in the 18–49
year age group. The two dose groups were matched according to
age and race-ethnicity. Both doses were well tolerated (Table 5). No
local symptoms were reported in 16%, mild to moderate symptoms
in 81%. Three subjects described severe arm pain at the site of injection. Two were in the 1.25 ␮g group and 1 was in 2.5 ␮g group. There
were no severe and few moderate systemic symptoms. There was
no difference in the proportion of mild or moderate local reactions
by dose.
TNF
2h
6
26
2
1
12
2
250
68
835
6
4
136
5
1
0
IL-10
Pre
IL-10
2h
2
2
1
2
2
2
1
1
2
2
2
2
2
1
1
9
30
17
4
5
15
33
237
148
33
6
112
4
3
3
IL-8
Pre
10
5
5
6
11
10
11
6
12
5
14
12
15
8
8
IL-8
2h
12
40
24
12
20
24
460
835
3285
18
17
150
20
6
7
3.4. Immunogenicity
3.4.1. HAI antibody response
Antibody responses were measured at day 0 and day 14 postvaccination. In study 1, there were no differences in HAI immune
responses by vaccine construct. In the young adult group for doses
ranging from 1.25 ␮g to 4 ␮g; the peak GMT (∼300), the GMT
fold increase (>10 fold), seroconversion and seroprotection rates
(∼89%) were very similar for each construct (data not shown). We
then pooled the immune response data for all three constructs
to compare the immune responses of the young adults compared
to the elderly. The dose-related trend in these immune response
Table 5
Number (%) of subjects 18–64 years old with local and systemic symptoms by severity grade after a dose of 1.25 (n = 49) or 2.5 ␮g (n = 51) of VAX128C.
Symptom severity
1.25 ␮g dose group
None
Mild
Mod
Severe
2.5 ␮g dose group
None
Mild
Mod
Severe
Arm pain
Headache
Fatigue
Joint pain
Muscle ache
Chills or shivering
Inc. sweating
Temperature
10 (20)
25 (51)
12 (24)
2 (4)
42 (86)
5 (10)
2 (4)
0
44 (90)
2 (4)
3 (6)
0
45 (92)
3 (6)
1 (2)
0
46 (94)
2 (4)
1 (2)
0
46 (94)
3 (6)
0
0
46 (94)
2 (4)
1 (2)
0
49 (100)
0
0
0
7 (14)
27 (53)
16 (31)
1 (2)
45 (88)
3 (6)
3 (6)
0
45 (88)
4 (8)
2 (4)
0
50 (98)
1 (2)
0
0
49 (96)
2 (4)
0
0
49 (96)
1 (2)
1 (2)
0
49 (96)
1 (2)
1 (2)
0
51 (100)
0
0
0
D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
HAI Mean Fold Rise
2048
128
1024
64
Mean Fold Rise
GM HAI titer (CA07)
HAI GMT
512
256
128
64
32
32
16
8
4
2
16
1
8
0.5
1.25
2.5
4
5767
8
1.25
12 16 20
2.5
Dose group
Age 18-49
Age ≥ 65
Age 18-49
8
12 16 20
Age ≥ 65
Seroprotection
Seroconversion
100
100
80
80
Percent
Percent
4
Dose group
60
40
20
60
40
20
0
0
1.25
2.5
4
8
12 16 20
Dose group
1.25
2.5
4
8
12 16 20
Dose group
Fig. 4. Comparison of immune response by dose 14 days after immunization among 112 subjects 18–49 years old and 100 subjects ≥65 years old who received a single IM
dose of VAX128 (CA07). Note: responses to VAX128A-C have been pooled.
parameters is seen for young and elderly adults in Fig. 4. In the
young adults the GMT and GM fold rise levels plateaued at a dose
of 1.25 ␮g and higher. Similarly, seroconversion and seroprotection
rates of 80% were observed at the 1.25 ␮g dose. In elderly adults
there was a more gradual increase GM fold rise and seroconversion rate throughout the dose range. There were no differences in
seroconversion and seroprotection rates by construct in either the
young adults or elderly vaccine groups.
In study 2, both the 1.25 ␮g and 2.5 ␮g doses of VAX128C were
highly immunogenic in 100 adults 18–64 years (Table 6). The GMT
was higher in the 2.5 ␮g group, but the mean fold increase, seroconversion and seroprotection rates were very similar for each dose
group. Overall, the peak reciprocal titer was 385 and the mean fold
increase was 19 with a 92% seroprotection and 79% seroconversion.
The lower limit of the 95% confidence interval for SC and SP was 71%
and 86%, respectively. There was no significant difference in results
by dose group (Table 6) or by age group (data not shown).
3.4.2. Flagellin IgG ELISA
All 3 constructs produce an antibody response to flagellin that
is similar in magnitude. Both young adults and seniors produced
antibodies to flagellin. There did not appear to be a difference in
the magnitude of response in the two age groups (Table 7).
4. Discussion
In both the rabbit model and in humans we found that replacing the HA antigen in the D3 position of flagellin in VAX128B and
VAX128C produced a vaccine that was at least as immunogenic and
better tolerated at higher doses than the C terminal fusion construct (VAX128A). These data demonstrate that the HA globular
head can be stably expressed in a prokaryotic (E. coli) system positioned in either the C terminal or D3 position of flagellin or both.
Most significantly we have demonstrated that the safety window
can be increased without impairing immune response.
We found that VAX128B and VAX128C have a better safety profile than VAX128A. In both VAX128B and VAX128C the D3 portion
of flagellin is replaced with the HA globular head. It is possible that
these constructs act as altered agonists that either quantitatively or
qualitatively change the TLR5 signaling such that the vaccines are
better tolerated. In preclinical studies we observed that the dose
response curve for cytokine production is lowest for VAX128C. The
clinical cytokine responses at doses of 16 ␮g or higher appeared
to differentiate VAX128C from VAX128B and it was for this reason
that VAX128C was selected for further testing. VAX128C has two
HA heads at either end of the molecule and a lower proportion of
flagellin per weight. In both VAX128B and VAX128C the D3 portion
of flagellin is replaced with the HA globular head. As shown in Fig. 1
the D3 region of flagellin is spatially close to the TLR5 binding site
located in the D1 portion of flagellin [9], while the HA globular head
attached to the C-terminal end of the protein at the end of the D0
portion of flagellin is distant from the binding site and less likely to
interfere with TLR5 activity at this location. It is also possible that
the D3 portion of flagellin contributes to the TLR binding [10].
VAX128A showed reactogenicity at 4 ␮g, but CRP elevations did
not occur until the 8 ␮g and then only associated with mild temperature elevation. VAX128B was well tolerated up to a dose of
16 ␮g and VAX128C was well tolerated at 20 ␮g, the highest dose
tested. The added safety is likely to be important for formulating the multivalent seasonal influenza vaccine. Assuming that the
other components are immunogenic at the 1–2 ␮g level similar to
VAX128, then it would be possible to produce a trivalent or quadrivalent vaccine that is well within the safety window. This may also
be important for producing vaccine for pandemic influenza strains
where higher doses may be required for optimum immunogenicity.
There was a good correlation in the safety and immunogenicity results found in the rabbit model and in the clinical studies.
5768
D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
Table 6
Serum HAI immune response among 97 healthy subjects 18–64 years old who received a single IM dose of 1.25 or 2.5 ␮g of VAX128C (CA07) in study 2.
VAX128C dose (␮g)
1.25 (n = 47)
2.5 (n = 50)
95% C.I.
GMT day 0
GMT day 21
Mean fold response
Seroconversiona (%)
Seroprotectionb (%)
a
b
16
306
20
79
89
Total (n = 97)
95% C.I.
11–23
187–502
12–33
67–90
80–98
26
478
19
80
94
15–43
290–789
11–31
69–91
87–100
95% C.I.
20
385
19
79
92
15–28
272–546
13–27
71–87
86–97
Percent with a 4-fold or greater increase in titer to a titer of 1:40 or greater.
Percent with a post vaccination HAI titer of 1:40 or greater.
The rabbit model allows us to test for both symptoms such as
weight loss, decreased food consumption and temperature elevation and also for CRP release which in humans appears to be
a very useful marker for cytokine release. CRP is an acute phase
reactant that rises after release of cytokines, primarily IL-6 [8].
The rabbit model can be used to screen the other components of
the multivalent seasonal vaccine as well as guide the ratio of the
monovalent components when formulating a multivalent vaccine.
In the clinical study we found that both CRP and cytokines, in particular, IL-6, TNF-␣ and IL-8 were useful biological markers for the
activation of the immune response by the flagellin portion of the
vaccine. CRP was a better marker of immune activation since an
increase was measurable even at low vaccine doses while cytokine
responses were not observed until the highest doses. CRP appears
to have a more prolonged peak that can be captured at a single time point while cytokine peaks may not always be captured
at a single timepoint 2 h after vaccination. The one subject who
received VAX128B at 16 ␮g and had severe systemic AEs also had
very elevated CRP and IL-6 values. There were 6 other subjects who
received VAX128B was also had 4-fold increases in IL-6 suggesting
that cytokine response may be a useful marker of potential reactogenicity. It is also worth noting that the two severe systemic AEs
observed with VAX128A and VAX128C were not associated with
high cytokine release.
We also demonstrated that VAX128 was well tolerated in adults
≥65 years at doses up to 20 ␮g and in the second study we found
VAX128 at doses of 1.25 or 2.5 ␮g was as immunogenic in subjects aged 50–64 as in subjects 18–49 years (data not shown). We
estimate that a dose 2–4 times the young adult dose would be
required in adults ≥65. This is similar to our previous studies in the
elderly with VAX125 [5]. These data suggest that stimulation of the
immune response via the innate immune system will be a successful approach for the elderly. Because of the significant morbidity
and mortality caused by influenza in elderly populations, prevention of influenza by vaccination is a major priority for public health.
Unlike egg- or cell-culture based vaccines, however, the manufacturing of recombinant vaccines in E. coli is not capacity constrained
and the higher dose required for the elderly and other immunocompromised subjects can easily be supplied, at lower cost than
the traditional vaccines.
Antibodies to flagellin have been observed in patients with
Crohn’s disease (CD) and it has been postulated that abnormal immune reactivity to intestinal microbial flora underlies CD
pathogenesis in genetically susceptible individuals [11]. Intestinal
bacteria appear to play a role in mucosal inflammation by triggering acquired and innate immune responses in the gut. Flagellin
as a TLR5 ligand is likely to be one of the mediators of mucosal
inflammation, although the evidence thus far is that the flagellin
of Salmonella and E. coli are not part of this association [12]. Our
vaccines are adjuvanted with Salmonella flagellin and are administered intramuscularly where they stimulate an immune response
most likely via the TLR5 activation of macrophages. The end result
is an immune response to the hemagglutinin portion of the vaccine
as well as to the flagellin portion. It is unlikely that the microgram quantities of flagellin given IM would increase the flagellin
load in the gut and it is also unlikely that the transient activation
of systemic immune system by vaccine flagellin would exacerbate
inflammatory bowel disease. These subjects were followed for one
year after vaccination and none developed a new chronic gastrointestinal condition.
Table 7
Day 14 flagellin antibody geometric mean titers (GMT) and mean fold rise by dose group.
Dose (␮g)
Number of subjectsa
Flagellin IgG GMT at day 14
VAX128A
VAX128B
Flagellin IgG fold rise at day 14
VAX128C
VAX128A
VAX128B
VAX128C
Young adults
0.5
1.25
2.5
4
8
12
16
20
3-3-3
3-3-3
11-3-3
12-3-3
6-3-3
0-3-3
0-11-12
0-0-6
30
146
44
120
202
31
36
96
61
56
130
157
17
29
44
22
47
68
82
135
32
154
61
92
157
34
27
59
264
52
140
122
17
50
51
31
38
42
48
50
Elderly
1.25
2.5
4
8
12
16
20
3-3-3
3-3-3
3-2-3
20-6-3
0-3-3
0-12-3
0-0-12
53
49
45
142
55
21
73
339
223
94
7
59
73
203
14
595
106
25
17
14
32
20
6
26
82
40
8
6
33
24
36
7
53
16
a
Number of subjects for VAX128 constructs A-B-C listed in order.
D.N. Taylor et al. / Vaccine 30 (2012) 5761–5769
The production of biological products in E. coli is straightforward and could be relatively easy to adapt to low resource
settings. Although significant issues of vaccine distribution and
delivery would remain, this could be a useful strategy for expanding influenza vaccination to these critically important settings. In
this study we have demonstrated that this approach can be used
with multiple strains and that improved safety can be achieved by
modifying the attachment of the HA head to flagellin.
Acknowledgements
We would like to thank Helena Petridis and Sonya Littlejohn
at VaxInnate for clinical trial coordination, our project coordinators Meredith Argulles at MRA and Monica Atwood at JCCT. We are
indebted to Arthi Krishnappa for performing the serological studies at VaxInnate and Theresa Fitzgerald for performing the HAI tests
and Xia Jin for supervising the CBA assays at University of Rochester.
Contributors: DT, AS and TH designed the clinical trials. ES and
CJ enrolled the study subjects and supervised the collection of clinical data. JT and UK supervised the serological studies. SU and LT
supervised the preclinical studies. KO performed the data and statistical analysis. Conflicts of interest statement: DT, UK, SU, LT, AS
and TH are employees of VaxInnate Corporation. JT is on the SAB of
Novartis and Immune Targeting Systems, and have received grant
support (payments to the URMC) from Protein Sciences, GlaxoSmithKline, Sanofi, Wyeth, and Ligocyte, in addition to VaxInnate.
Funding: VaxInnate Corporation funded the study.
5769
References
[1] Lambert LC, Fauci AS. Influenza vaccines for the future. N Engl J Med
2010;363:2036–44.
[2] Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, et al. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution
to global pandemic and seasonal needs. PLoS One 2008;3:e2257.
[3] Liu G, Tarbet B, Song L, Reiserova L, Weaver B, Chen Y, et al. Immunogenicity and
efficacy of flagellin-fused vaccine candidates targeting 2009 pandemic H1N1
influenza in mice. PLoS One 2011;6:e20928.
[4] Treanor JJ, Taylor DN, Tussey L, Hay C, Nolan C, Fitzgerald T, et al. Safety and
immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion
vaccine (VAX125) in healthy young adults. Vaccine 2010;28:8268–74.
[5] Taylor DN, Treanor JJ, Strout C, Johnson C, Fitzgerald T, Kavita U, et al. Induction
of a potent immune response in the elderly using the TLR-5 agonist, flagellin,
with a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125,
STF2.HA1 SI). Vaccine 2011;29:4897–902.
[6] Song L, Zhang Y, Yun NE, et al. Superior efficacy of a recombinant flagellin: H5N1
HA globular head vaccine is determined by the placement of the globular head
within flagellin. Vaccine 2009;27:5875–84.
[7] Dowdle WN, Kendal AP, Noble GR. Influenza viruses. In: Lenette EH, Schmidt NJ,
editors. Diagnostic procedures for viral, rickettsial, and chlamydial infections.
5th ed. Washington, DC: American Public Health Association; 1979. p. 603–5.
[8] Black S, Kushner I, Samols D. C-reactive protein. J Biol Chem
2004;279:48487–90.
[9] Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL,
et al. Toll-like receptor 5 recognizes a conserved site on flagellin required for
protofilament formation and bacterial motility. Nat Immunol 2003;4:1247–53.
[10] Andersen-Nissen E, Smith KD, Strobe KL, Barrett SL, Cookson BT, Logan SM, et al.
Evasion of toll-like receptor 5 by flagellated bacteria. Proc Natl Acad Sci USA
2005;102:9247–52.
[11] Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347:417–29.
[12] Lodes MJ, Cong Y, Elson CO, Mohamath R, Landers CJ, Targan SR, et al.
Bacterial flagellin is a dominant antigen in Crohn disease. J Clin Invest
2004;113:1296–306.