A Randomized, Controlled, Observer-Blinded Phase 1 Study of the Safety and Immunogenicity of a Respiratory Syncytial Virus Vaccine With or Without Alum Adjuvant
A Randomized, Controlled, Observer-Blinded Phase 1 Study of the Safety and Immunogenicity of a Respiratory Syncytial Virus Vaccine With or Without Alum Adjuvant
Joanne M. Langley 2
Naresh Aggarwal 1
Azhar Toma 0
Scott A. Halperin 2
Shelly A. McNeil 2
Laurence Fissette 3
Walthere Dewé 3
Maarten Leyssen 3
Jean-François Toussaint 3
Ilse Dieussaert 3
0 Manna Research , Toronto , Canada
1 Aggarwal and Associates Limited , Brampton
2 Canadian Center for Vaccinology, IWK Health Centre-Nova Scotia Health Authority-Dalhousie University , Halifax
3 Vaccine Discovery and Development, GSK Vaccines , Rixensart , Belgium
Background. Respiratory syncytial virus (RSV) is a leading cause of childhood bronchiolitis and pneumonia, particularly in early infancy. Immunization of pregnant women could boost preexisting immune responses, providing passive protection to newborns through placental transfer of anti-RSV antibody. Methods. In this first-in-humans clinical trial of a purified recombinant RSV protein F vaccine engineered to preferentially maintain prefusion conformation (RSV-PreF), 128 healthy men 18-44 years old were randomized to one dose of a RSV-PreF vaccine containing 10, 30, or 60 µg of RSV-PreF antigen, with or without alum adjuvant, or control, and followed for one year for safety and immunogenicity outcomes. Results. Injection site pain was the most common adverse event, reported by up to 81.3% of participants. The highest RSV neutralizing antibody responses were in the 30 µg RSV-PreF/alum, 60 µg RSV-PreF/alum, and 60 µg RSV-PreF/nonadjuvant groups. Responses were evident on day 7, and 30 days after vaccination these participants had RSV-A neutralizing antibody titers of ≥1:512, and >70% had titers of 1:1024, with titers increasing by 3.2-4.9 fold. Responses remained high on day 60 but waned on days 180 and 360. Conclusions. The RSV-PreF vaccine elicited rapid RSV neutralizing antibody responses in healthy young men, with an acceptable adverse event profile.
associated illness occurs in the first 6 months of life, and
maternal RSV vaccination has thus been identified as a potential
strategy to protect the infant . Since most women of child-bearing
age would have preexisting antibody from prior infection, a
RSV vaccine given during the third trimester of pregnancy
would be expected to boost preexisting antibody levels and
result in increased passage of anti-RSV antibodies through the
placental active-transport mechanism for immunoglobulin G
(IgG). Maternal immunization potentially could also protect
the infant through a reduced risk of infection transmission
from the mother and, possibly, from passive immunity
conferred through breast milk. RSV antibodies are known to be
transferred efficiently across the placenta , and high cord
blood RSV antibody levels are associated with a lower incidence
of severe RSV-associated LRTI [7, 8].
The RSV F surface glycoprotein, which is highly conserved
across A and B subgroup isolates and considered essential in
disease pathogenesis , is a target for passive immunization
with monoclonal antibodies , which reduce the risk of
RSV-associated hospitalization. There is evidence that the
prefusion conformation of the F glycoprotein, rather than the
postfusion form, is the main target of naturally induced anti-RSV
neutralizing antibody (nAb) in human serum  and, thus,
would be a preferred vaccine antigen. A specific epitope on the
prefusion conformation, site ø (zero), is thought to be one of the
major targets of RSV nAb  and results in potent neutralizing
activity in animal models . In this first-in-humans study,
the safety, reactogenicity, and immunogenicity of a RSV vaccine
for pregnant women, containing purified recombinant RSV
glycoprotein F engineered to preferentially maintain prefusion
conformation (RSV-PreF), was evaluated.
This was a randomized, controlled, observer-blinded,
firstin-humans, phase 1 clinical trial to evaluate the safety and
reactogenicity of a single dose of 1 of 6 formulations of an RSV
vaccine in 18–44-year-old healthy men at 3 sites in Canada. The
study was conducted in 2 sequential steps, with dose escalation in
step 2. The study was initiated on 22 July 2013, and day 360 visits
were concluded on 16 March 2015. The study (clinical trials
registration NCT01905215) was undertaken in compliance with
Good Clinical Practice guidelines, the Declaration of Helsinki,
and national regulatory requirements and was approved by
local or regional institutional review boards at each study site.
Eligible men were 18–44 years of age at the time of vaccination;
healthy, based on medical history and clinical examination; able
to comply with the protocol; and gave informed written
consent. Women were excluded from participation on the guidance
of the regulatory authority, which advised that later testing of
this novel product could occur in women of childbearing age.
Exclusionary criteria were immunocompromise, a family
history of immunodeficiency, autoimmune disease, a malignancy
within 5 years, a history of hypersensitivity to latex or any
vaccine component, acute illness or fever, participation in another
clinical study, receipt or intent to receive another vaccine 30
days previous to or after the study vaccine (with the exception
of influenza vaccine, which could be administered ≥15 days
before study vaccination), or receipt of either immunoglobulins or
blood products within 3 months or previous RSV vaccination or
any investigational product within 30 days. Any hematological
or biochemical value outside the normal range at the local
laboratory that was considered clinically significant by the
investigator was also considered exclusionary; participants could be
rescreened within 30 days.
The RSV-PreF antigen is prepared in Chinese hamster ovary
(CHO) cells, in which it is expressed as a soluble, secreted
protein. The PreF conformation was characterized by analytical
ultracentrifugation, electron microscopy, specific monoclonal
antibody (D25) testing, and human sera neutralization
inhibition (Blais et al, unpublished data). The study vaccines
contained 10, 30, or 60 µg of a recombinant PreF protein antigen,
in isotonic saline diluent, with or without alum adjuvant (500
µg of aluminum hydroxide). Vaccines were manufactured by
GlaxoSmithKline Vaccines (Rixensart, Belgium). All vaccines
were presented as a 0.5-mL single dose. The 3 alum-adjuvanted
vaccines were presented as liquid in 0.5-mL monodose vials.
These vaccines were turbid in appearance. The 3 PreF plain
vaccines were presented in a monodose vial as lyophilized antigen
that was reconstituted with diluent and was clear and colorless
after reconstitution. The placebo was lyophilized saccharose (20
mg) that was reconstituted in isotonic saline and was clear in
At the screening visit, after the consent process, medical history
was obtained, physical examination was performed, and blood
samples were obtained for safety analysis. Eligible participants
were then invited to attend visit 1 within 30 days of the
Study group assignment was allocated by an Internet-based
central randomization system. The randomization sequence
was generated using MATerial Excellence, a software program
developed for use in Statistical Analysis System (SAS®) (Cary,
North Carolina) by GSK (Rixensart, Belgium). A
randomization blocking scheme was used to ensure balance between
study groups in each step, and a minimization procedure
accounted for age category (18–32 or 33–44 years). Participants
and all study personnel, except for an unblinded nurse, whose
sole role was to prepare and administer study vaccines, were not
aware of treatment assignment.
In step 1, participants were randomly assigned as a ratio of
1:1:1:1:1 to receive a placebo injection or a study vaccine
containing either 10 or 30 µg of a recombinant PreF protein
antigen, in isotonic saline diluent, with or without alum adjuvant
(500 µg of aluminum hydroxide). In step 2, participants were
randomly assigned at a ratio of 1:1:1 to receive a study vaccine
containing 60 µg of a recombinant PreF protein antigen, in
isotonic saline diluent, with or without the alum adjuvant, or
placebo (Figure 1).
One dose of study vaccine was administered intramuscularly
in the deltoid region of the nondominant arm. Participants were
observed closely for 60 minutes following injection. During the
first 3 vaccination days in each step, a maximum of 10
participants were vaccinated each day, at least 60 minutes apart.
At the vaccination visit, participants were instructed to use a
diary card for recording any solicited injection site or general
adverse events (AEs) and any unsolicited AEs and concomitant
medications for 7 days and to bring the diary card to the next
visit. A second diary card was provided on day 7 to record, up to
day 30, unsolicited AEs and concomitant medications. At each
study visit, participants were asked whether there were any
changes in their health or AEs since the last visit. Participants
attended study sites at the screening visit and on days 0, 7, 30,
60, 180, and 360.
Safety and Reactogenicity
The primary outcome was the assessment of safety and
reactogenicity from vaccination up to day 60. A secondary outcome
was occurrence of AEs from days 60 to 360. These AEs were
solicited local AEs ( pain, redness, and swelling) and general
AEs ( fever, headache, gastrointestinal symptoms, and fatigue)
during the day of vaccination and for the following 6 days,
occurrence of any abnormal hematological (hemoglobin level,
white blood cell, lymphocyte, neutrophil, eosinophil, and
platelet count) or biochemical (alanine aminotransferase,
aspartate aminotransferase, and creatinine levels) findings at
days 0, 7, 30, 60, 180, and 360; any unsolicited AE during
the 30 days after vaccination; and the occurrence of any
serious adverse events (SAEs), AEs leading to study
withdrawal, or investigator-determined clinically significant AEs from
days 0 to 360.
The intensity grading scheme for solicited AEs is seen in
Supplementary Table 1. Unsolicited AEs were assigned to the
categories of mild (easily tolerated by the participant, causing
minimal discomfort, and not interfering with everyday
activities; grade 1), moderate (sufficiently discomforting to interfere
with normal everyday activities; grade 2), or severe ( preventing
normal, everyday activities; grade 3) by the investigator.
Grading of intensity of laboratory parameters was based on Food and
Drug Administration guidance .
Study holding rules were in place. All SAEs were reported to
an internal Safety Review Committee in addition to local,
institutional, and national authorities, as required.
Secondary objectives of the study were to evaluate humoral
immune responses to a single dose of vaccine 7, 30, and up to 60
days after vaccination and the persistence of responses from
days 60 to 360, using neutralizing titers against RSV serotypes
A and B and enzyme linked immunosorbent assay (ELISA)
antibody titers against RSV F at days 0, 7, 30, 60, 180, and 360. The
anti-F protein IgG ELISA is an indirect ELISA allowing the
detection and quantitation of specific IgG antibodies directed
against the RSV F protein in human serum samples; the antigen
used for the ELISA is the same PreF protein used for
immunization. Antigens are purified from a CHO cell expression system
and are coated by passive adsorption onto 96-well microplates.
After a washing and a blocking step, serial 2-fold dilutions of
test sera, controls, and a reference standard are incubated to
allow specific binding of antibodies directed against the F
protein antigens. Bound IgGs are detected by addition of a goat
anti-human IgG antibody conjugated to horseradish
peroxidase. After a washing step, the horseradish peroxidase substrate
solution (TMB/H2O2) is added, and a colored product develops
proportionally to the amount of anti-F protein IgG antibody
present in the test serum. The color is quantified by reading
the optical densities at 450–620 nm, using a spectrophotometer.
Antibody concentrations of individual serum and control
samples are determined after interpolation from the reference
standard curve, using a 4-parameter equation, and are expressed in
arbitrary ELISA units (ELU) per milliliter.
Palivizumab-competing antibodies (PCAs) were also evaluated at these time
points, based on the method of Glenn et al.
As a tertiary objective, the study sought to evaluate whether
the vaccine induced immune responses to host cell proteins.
These results are published separately.
Hematological and biochemical tests were performed at local
laboratories. Serologic assays were performed at GSK
Laboratories, Laval, Canada (Blais et al, unpublished data).
Sample Size and Statistical Analysis
This was a first-in-humans study, and therefore there were no
previous estimates of the frequency of AEs or of immune
responses. As the primary outcome was safety, the sample size
was based on the likely precision around estimates of the
percentage of participants in each vaccine group with symptoms
following vaccination. With 16 participants per group, the
lower limit (LL) of the 95% confidence interval (CI) on an
AE incidence of 25.0% would be 7.3%, and the upper limit
(UL) would be 52.4%. The corresponding UL and LL on the
95% CI for symptoms frequencies of 50.0% and 75.0% in a
group of 16 participants would be 24.7% and 75.3%, and
47.6% and 92.7%, respectively.
Safety analysis was conducted on the total vaccinated cohort,
defined as all participants who received study vaccine, and their
demographic characteristics were described. The percentage of
participants with at least 1 AE, with a severe AE or an SAE
following vaccine, were tabulated, with an exact 95% CI. The
number and percentage of participants with a hematologic or
biochemistry result below or above the local laboratory range
were tabulated. Unsolicited AEs were classified according to
the Medical Dictionary for Regulatory Activities (MeDRA®,
International Conference on Harmonization).
The immunogenicity analysis was performed on the
according-to-protocol cohorts for immunogenicity (on days 0, 7, 30,
and 60) and persistence (on days 180 and 360), which included
participants who did not develop an exclusionary medical
condition or receive an exclusionary concomitant medication
during the respective follow-up periods and for whom at least 1
appropriately timed anti-RSV serologic response was available.
For each anti-RSV assay, the percentage of participants, and
associated exact 95% CIs, with values above the cutoff were
Geographic ancestry, no. (%)
American Indian or
10 µg RSV-PreF
30 µg RSV-PreF
60 µg RSV-PreF
30.2 ± 8.7 30.8 ± 7.5 31.8 ± 7.3
29.0 (18.0– 32.5 (20.0– 32.0 (18.0–
45.0) 44.0) 45.0)
Abbreviations: n, number of participants; RSV-PreF, respiratory syncytial virus engineered to preferentially maintain a prefusion conformation; SD, standard deviation; y, years.
a Saccharose NaCl.
determined, as were geometric mean concentrations (GMCs),
geometric mean titers (GMTs), and their respective 95% CIs.
Reverse cumulative distribution curves were created using
data from before vaccination and 7, 30, 60, 180, and 360 days
after vaccination. The geometric mean of the fold increase
from baseline to days 7, 30, 60, 180, and 360 was calculated,
with respective 95% CIs, and the percentage of participants in
each study group with various fold increases was tabulated. For
the sake of this analysis, Vaccine Response (VR) was defined as
at least a 4-fold increase from pre-vaccination if pre-vaccination
NA titre <7 log2 (128), at least a 3-fold increase from
prevaccination if pre-vaccination NA titre in [7–8] log2, at least a
2.5-fold increase from pre-vaccination if pre-vaccination NA
titre in  log2, and at least 1-fold increase from
pre-vaccination if pre-vaccination NA titre >10 log2 (1024), and was
reported at Day 30, 60, 180 and 360.
Between-group GMT ratios at each time point after
vaccination were calculated using an analysis of covariance model on
the log10 transformation of the titers, including the vaccine
group as a fixed effect and the prevaccination titer as the
regressor. Seropositivity for antibody was determined as follows: anti–
RSV-PreF antibody concentration, ≥10 ELU/mL; anti–RSV-A
nAb titer, ≥8 ED60; anti–RSV-B nAb titer, ≥6 ED60; and
PCA, ≥3.34 µg/mL.
The total vaccinated cohort comprised 128 participants, and
121 completed the study. Participant flow is seen in Figure 1.
The according-to-protocol cohort was 119 for analysis of
immunogenicity and 114 and 110 for analysis of the persistence
of immune responses at days 180 and 360, respectively.
The median age of participants in the total vaccinated cohort
was 32 years, and the geographic heritage and/or ancestry of
60.9% was white–Caucasian/European. Age, heritage, and
ethnographic characteristics did not differ across study groups
participant each who received 10-alum or 30 µg of antigen
without alum (ie, the 30-plain group) reported swelling. The most
common systemic AE was fatigue and was reported in up to
43.8% of RSV F vaccine recipients, with 3 reporting severe
fatigue, compared with 11.8%–25.0% of controls (33). Fever
(body temperature, ≥37.5°C) occurred in 8 of 112 vaccine
participants (7.0%) and 1 of 16 controls (6.0%). One participant in
the 10-alum group had a temperature of >38.5°C; no participant
had a temperature of >39.5°C.
At least 1 unsolicited symptom was reported by 18.8%–37.5%
of participants, depending on study group. One
non–vaccinerelated SAE (traumatic left knee dislocation) was reported in
the 60-plain group. One medically attended visit, deemed by
the local investigator as unrelated to vaccination, was observed
in the 10-alum group. Use of antipyretic therapy in the 7 days
after vaccination ranged from 6.3% (in the 60-alum group) to
31.3% (in the 60-plain group) in vaccinated participants. No
placebo recipient took antipyretic medication.
Safety and Reactogenicity
Pain was reported by up to 81.3% of RSV-F vaccine recipients
(Figure 2), with a single participant in the group that received 60
µg of RSV-PreF plus alum (ie, the 60-alum group) reporting
severe (grade 3) pain. One participant reported redness, and 1
All study participants were RSV seropositive at baseline. The
GMCs, by study group, for anti–RSV-PreF antibody and
GMTs for anti–RSV-A and anti–RSV-B nAb up to day 360
are seen in Figures 3 and 4. In all vaccine groups, titers were
increased on days 7 and 30, but titers remained unchanged in
controls. The highest boost responses were seen in the 30-alum,
60-alum, or 60-plain groups, in which, 30 days after
vaccination, all participants had anti–RSV nAb titers of ≥1:512,
>70.0% had titers of ≥1024, and titers increased 3.2–4.9 fold.
Anti–RSV nAb titers for RSV-A and RSV-B strains were
similar. The highest Vaccine Response rates were observed on day
30 in the 30-alum and 60-alum groups (85.7% and 84.6%,
respectively; Table 2).
Responses by all immunogenicity measures decreased at day
60 and waned further at day 180. At day 360, anti–RSV-PreF
antibody concentrations (based on ELISA) and PCA
concentrations remained higher than at baseline, as did nAb responses, in
the 30-alum, 60-alum, and 60-plain groups. GMT kinetics are
seen in Figures 4 and 5.
In this first-in-humans study of a candidate subunit RSV vaccine
engineered to maintain the prefusion conformation of
glycoprotein F, which is thought to present the most potent epitope,
acceptable reactogenicity and rapid and robust immune responses
were observed. Temporary injection-site pain and fatigue, mostly
Table 2. Vaccine Response Rates at Days 30 to 360
Subjects With Response,
Subjects With Titer
>1:1024, No. (%)
Vaccine Response were defined as having increased by at least 4-fold from the
prevaccination neutralizing antibody (nAb) titer if the prevaccination titer was <7 log2 (ie,
<128), by at least 3-fold if the titer was 7–8 log2 (ie, 128–256), by at least 2.5-fold if the
titer was 10 log2 (ie, 1024), and by at least 1-fold if the titer was >10 log2 (ie, >1024).
Abbreviation: RSV-PreF, respiratory syncytial virus engineered to preferentially maintain a
mild in intensity, were the most common AEs in all study groups.
No clear differences were observed in terms of safety and
reactogenicity between the vaccine formulations, and no
vaccine-related SAEs or withdrawals occurred. It is reassuring that
postimmunization fever was uncommon and of low grade with
this vaccine intended for use in pregnant women, as fever in
pregnancy has been associated with adverse outcomes .
Immune responses to the RSV PreF vaccine, demonstrated by
detection of nAb to RSV-A and RSV-B subtypes, RSV F ELISA
antibody, and PCA to the F glycoprotein, were highest in the 2
alum-adjuvanted formulations (30-alum and 60-alum) and the
60-plain group. Notably, all vaccine preparations were
associated with robust immune responses. The nAb responses were
consistent across A and B subtypes. The robust PCA responses
confirm that the RSV F ELISA antibodies generated by the
vaccine are binding to epitope in the antigenic site of the F protein
of RSV-A that is associated with palivizumab efficacy. Further,
all measures of immunogenicity demonstrated responses to a
single dose of vaccine as early as 7 days after vaccination.
This early immune response suggests that this response was
amnestic in nature. Given the small sample size of this study, it is
difficult to determine which vaccine candidate is preferred for
further clinical development, although a trend for slightly
higher immune responses was observed in the higher-dose vaccine
groups. Furthermore, the added value of the alum in the vaccine
could not be determined through this study. Therefore, further
development of the RSV-PreF vaccine is underway in women of
childbearing age, using the higher doses of the RSV-PreF
vaccine with and without alum.
Although immune responses persisted, as expected, a decline
in immune responses was seen in all study groups between days
60 and 180 and, further, by day 360. The data also seem to
suggest that the magnitude of the decline in immune responses did
not vary according to antigen dose or the presence or absence of
alum adjuvant. One year after a single dose of vaccine, nAb
titers for the 60-alum, 30-alum, and 60-plain groups were higher
than at baseline, as were PreF ELISA-determined and PCA
It is not known whether these serologic measures correlate
with clinical protection. An immunologic correlate of clinical
protection would ideally be determined during efficacy trials.
While a trough level of 40 µg/mL of PCA was correlated with
a high level of protection against severe disease in infants who
received passive immunoprophylaxis , it is not yet known
what specific level of anti–RSV PreF nAb transplacentally
transferred to infants might correlate with protection from
significant RSV disease in early infancy. Every 2-fold increase in
anti-RSV nAb is associated with a significantly reduced risk
of RSV-associated hospitalization . Assessment of RSV
vaccine immunogenicity in adults must take into account prior
immune response, since adults are expected to have been
previously exposed and have baseline titers. We noted variation
in baseline titers, which makes assessment of vaccine
immunogenicity more complicated.
Given waning of antibody over the year after immunization,
timing of vaccination will need to be carefully considered.
Administration of a maternal RSV vaccine would need to be timed
to take advantage of the placental active-transport mechanism
that begins at gestation weeks 28 to 32, to occur late enough in
pregnancy to ensure that titers had not waned prior to delivery,
and to align with time points for other aspects of obstetric care.
Potentially, a vaccine delivered during the third trimester could
accomplish this goal.
A few RSV F vaccine candidates intended for maternal
immunization to protect infants are in preclinical or early phase
clinical development . In a phase 2 trial of a postfusion
RSV F vaccine in adults and women of childbearing age,
mean neutralizing antibody titers 28 days after a single
immunization increased across different formulations by a factor 2.0
to 3.9 (RSV-A) and 1.2 to 1.8 (RSV-B) . In that same study,
an acceptable safety profile was observed after a single and two
consecutive vaccine administrations. A phase 3 study of this
vaccine in pregnant women is underway (clinical trials
registration NCT02247726). The study reported here was not powered
to detect differences between groups in reactogenicity or
immunogenicity or to select the final formulation or schedule. Larger
studies in diverse populations of women of childbearing age,
before and during the RSV season, are ongoing to evaluate these
In summary, a phase 1 study of a prefusion RSV F vaccine
intended for maternal vaccination to protect young infants
demonstrated acceptable reactogenicity. One dose of all
formulations produced a brisk immune response.
Supplementary materials are available at http://jid.oxfordjournals.org.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Acknowledgments. We thank Dr Benjamin Lasko, Martine Douha,
Catena Lauria, Anne-Sophie Perraux, Marie-Pierre David, Marta Picciolato,
Nathalie Michelet, Laura Maria Scurtu, Julie Todoroff, and study
participants, for their contributions.
All authors participated in the design, implementation or analysis, or
interpretation of the study and in the development of this manuscript. All
authors had full access to the data and gave final approval before
submission. J. M. L. drafted the manuscript and was responsible for
submission of the publication.
J. M. L. holds the Canadian Institutes of Health
Research–GlaxoSmithKline Chair in Pediatric Vaccinology at Dalhousie University (Halifax,
Financial support. This trial was supported by GlaxoSmithKline
Biologicals SA. GlaxoSmithKline Biologicals SA was involved in all stages of
the study conduct and analysis. GlaxoSmithKline Biologicals SA paid for
open access of the article. The authors received no remuneration for the
development of the present manuscript.
Potential conflicts of interest. GlaxoSmithKline Biologicals was
involved in all stages of the study conduct and analysis and paid for open
access of the article, and the authors received no remuneration from
GlaxoSmithKline Biologicals for the development of the present
manuscript. J. M. L., S. A. H., and S. A. M. have received research funding
from GSK, Immunovaccine, Sanofi Pasteur, Pfizer, Pan Provincial Vaccine
Enterprise (Prevent), Novavax, the Public Health Agency of Canada, and the
Canadian Institutes of Health Research. M. L., L. F., W. D., I. D., and J.-F. T.
are employees of the GSK group of companies. W. D. and I. D. own stock/
share options or restricted shares in the GSK group of companies. J.-F. T. has
a patent application supporting the use of the GSK PreF antigen. All other
authors report no potential conflicts. All authors have submitted the ICMJE
Form for Disclosure of Potential Conflicts of Interest. Conflicts that the
editors consider relevant to the content of the manuscript have been disclosed.
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