First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus–Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens
First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus-Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens
Julien Nyombayire 2
Omu Anzala 1
Brian Gazzard 0
Etienne Karita 2
Philip Bergin 5
Peter Hayes 5
Jakub Kopycinski 5
Gloria Omosa-Manyonyi 1
Akil Jackson 0
Jean Bizimana 2
Bashir Farah 1
Eddy Sayeed 4
Christopher L. Parks 4
Tetsuro Matano 6
Len Dally 3
Burc Barin 3
Harriet Park 4
Jill Gilmour 5
Angela Lombardo 4
Jean-Louis Excler 4
Patricia Fast 4
Dagna S. Laufer 4
Josephine H. Cox 4
0 Chelsea and Westminster Healthcare NHS Foundation Trust
1 Kenya AIDS Vaccine Initiative Institute of Clinical Research , Nairobi
2 Projet San Francisco , Kigali , Rwanda
3 Emmes Corporation , Rockville, Maryland
4 International AIDS Vaccine Initiative , New York , New York
5 Human Immunology Laboratory, International AIDS Vaccine Initiative , London , United Kingdom
6 National Institute of Infectious Diseases , Tokyo , Japan
Background. We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)-vectored, human immunodeficiency virus type 1 (HIV-1) vaccine. Methods. Sixty-five HIV-1-uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 primeboost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35-vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Results. All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot-determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeVGag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8+ T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic. Conclusions. SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime-boost sequence appears to determine which arm of the immune response is stimulated. Clinical Trials Registration. NCT01705990.
points may be critical for an HIV preventive vaccine. Although
mucosally administered vaccines have been tested and licensed
for other diseases [9–12], mucosal administration of an HIV
preventive vaccine has seldom been evaluated . Sendai
virus (SeV) is a nonsegmented negative-sense RNA virus in
the Paramyxoviridae family that can infect the upper
respiratory tract [14–17]. As a live viral vector that is not pathogenic in
humans, SeV offers several properties important for a successful
vaccine: it does not integrate into the host genome, it replicates
only in the cytoplasm without DNA intermediates or a nuclear
phase, and it does not undergo genetic recombination. SeV is
genetically and antigenically related to hPIV-1 [18–21]. A live
nonrecombinant SeV vaccine against human parainfluenza
virus type 1 (hPIV-1) administered intranasally in adults and
young children was safe and immunogenic [22, 23]. SeV
antibodies cross-reactive with hPIV-1 antibodies are present in
most people .
Intranasal delivery of a vaccine could induce a first line of
defense at mucosal points of entry and induce effective systemic
immune responses [12, 25, 26]. Nonhuman primate studies
with SeV bearing simian immunodeficiency virus (SIV) genes
demonstrated protection against SIV challenge and evidence
that SeV vectors may boost responses primed by other HIV-1
vaccines [27–29]. Intranasal administration and heterologous
prime-boost administration were shown to reduce effects of
preexisting immunity [29, 30].
In this study, we report the first-in-human safety and
immunogenicity evaluation of a replication-competent SeV-vectored
HIV-1 vaccine administered intranasally; the vaccine was
administered intranasally at a lower dose (SL) or higher dose
(SH) of SeV vector encoding clade A HIV-1 Gag (SeV-Gag),
given alone or as a heterologous prime-boost with a
nonreplicating adenovirus (Ad) serotype 35 HIV-1 vaccine containing
genes HIV-1 encoding Gag, reverse transcriptase, integrase,
and Nef (Ad35-GRIN) administered intramuscularly. The
Ad35-GRIN was selected for these prime-boost regimens
because it has well-known safety profile and robust
immunogenicity in both US and African populations [4, 7, 8, 31].
Volunteers and Study Design
This study was a multicenter, randomized, placebo-controlled,
dose-escalation trial that was double blinded with respect to
vaccine or placebo but not regimen. The doses were based on
preclinical data [28, 29] and a nonrecombinant live SeV vaccine
study in humans ; the initial group was administered a lower
dose for safety. The study was conducted at Projet San Francisco
(Kigali, Rwanda), the Kenya AIDS Vaccine Initiative Institute of
Clinical Research (Nairobi, Kenya), and the St Stephen’s AIDS
Trust (London, United Kingdom). The objectives were to
evaluate the safety and immunogenicity of 4 different 2-dose
regimens (administered at 0 and 4 months) that comprised
SeV-Gag administered at 2 × 107 (SL) or 2 × 108 (SH) cell
infectious units and Ad35-GRIN vaccine administered at 1 × 1010
viral particles. Volunteers and clinical/laboratory personnel
were blind to allocation between active vaccine and placebo.
The participants were healthy HIV-negative adults 18–50
years of age engaging in behavior at low risk for HIV-1
infection; all women were nonpregnant and used an effective
method of contraception until 4 months after the last vaccination
(detailed inclusion/exclusion criteria are in Supplementary
Materials). The respective local governmental ethics and regulatory
bodies for each clinical research center approved the study.
Written informed consent was obtained from each volunteer
prior to undertaking any study procedure. The study was
conducted in accordance with International Conference on
Harmonization’s good clinical practice and good clinical laboratory
practice guidelines .
The study design is presented in Table 1 and in the
Consolidated Standards of Reporting Trials diagram (Supplementary
Figure 1). Volunteers in part I received low-dose SeV-Gag
vaccine followed by Ad35-GRIN vaccine (SLA) or placebo.
Following review of safety data from part I by an independent safety
review board, a different set of volunteers was randomly
assigned to participate in part II. Volunteers in part II received
either the higher dose of SeV-Gag as a prime followed by
Ad35-GRIN vaccine (SHA); an Ad35-GRIN prime given
intramuscularly, followed by the higher-dose SeV-Gag boost given
intranasally (ASH); prime-boost with the higher-dose
SeVGag given intranasally (SHSH); or placebo.
Each group had 16 volunteers: 12 vaccine recipients and 4
placebo recipients. Enrollment of an additional volunteer was
allowed yielding a sample size of 65. Local and systemic
reactogenicity were reported for days 0 through 14 following each
vaccination, adverse events (AEs) were reported through month 1
following the second study vaccination, and serious adverse
events (SAEs) were reported through the final study visit.
Hematologic and biochemical parameters were assessed at 4 time
points after vaccination (Supplementary Materials).
Reactogenicity and AEs were assessed using an adapted version of the
Division of AIDS Table for Grading the Severity of Adult and
Pediatric Adverse Events, version 1.0.
The SeV-Gag vaccine is based on a replication-competent
vector derived from the SeV Z strain  with HIV-1 subtype A
gag inserted in the 3′ terminal region of the virus genome
, upstream of the nucleoprotein gene. SeV-Gag vaccine
and placebo were administered by syringe; the head was tilted
back, and 100 µL was instilled into each nostril of the volunteer
over approximately 3 minutes to allow absorption.
The Ad35-GRIN vaccine is a recombinant, replication-defective
Ad35 vaccine; it has been previously tested in 4 clinical trials
[4, 7, 8, 31] and a recently completed trial in Kenya . The
Abbreviations: Ad35-GRIN, adenovirus 35–vectored vaccine encoding Gag, reverse transcriptase, integrase, and Nef; ASH, Ad35-GRIN prime followed by SeV-Gag boost; HIV-1, human
immunodeficiency virus type 1; SHA, higher-dose SeV-Gag prime and Ad35-GRIN boost; SHSH, higher-dose SeV-Gag prime and boost; SLA, lower-dose SeV-Gag prime and Ad35-GRIN
boost; SeV-Gag, Sendai virus–vectored vaccine encoding HIV-1 Gag.
a Data are 1 × 107 or 1 × 108 cell infectious units/100 µL per nostril (for SeV-Gag) or 1 × 1010 viral particles (for Ad35-GRIN).
b Overenrollment was allowed per protocol; one additional volunteer, identified post unblinding as a placebo recipient, was enrolled.
Table 1. Study Immunization Regimens and Schedule
Ad35-GRIN vaccine and placebo were both administered
intramuscularly in 0.5 mL. The gag in SeV-Gag and Ad35-GRIN
were fully homologous with regard to amino acid sequence.
Laboratory Assessments for Safety and Immunogenicity
Hematologic and biochemical assays were conducted at the
clinical sites in Africa and at a third-party accredited laboratory
in the United Kingdom. Vaccine-induced seropositivity/
seroreactivity was assessed in each country (Supplementary
Materials). For detailed collection and immunogenicity testing
methods, see the Supplementary Materials. Briefly, peripheral
blood mononuclear cells (PBMCs) were processed and
cryopreserved at each clinical site. Mucosal fluids from nasal
swabs, parotid and transudated saliva, rectal secretions, and
cervicovaginal secretions in females were processed as previously
described [36, 37]. Colorectal biopsy specimens were pooled
and disaggregated by collagenase digestion to isolate mucosal
mononuclear cells within 6 hours of collection, and intracellular
cytokine staining (ICS) assays were performed after an
overnight rest as described elsewhere [38, 39]. T-cell responses
were assessed by qualified interferon-γ (IFN-γ) enzyme-linked
immunospot (ELISPOT) and ICS assays, using peptides
matched to Gag, and by a functional viral inhibition assay,
using a panel of 8 HIV-1 strains from subtypes A, B, C, and
D [7, 8, 31, 40, 41].
An enzyme-linked immunosorbent assay (ELISA) was used
with HIV-1 subtype B Gag p24 protein (BH10) to assess Gag
p24 binding antibodies in serum and mucosal samples [7, 31,
36]. Serum neutralizing antibodies (NAbs) against SeV were
assessed as described previously .
Samples for analysis of viral shedding were collected from the
middle turbinate region, saliva, and urine on days 2, 5, 6, 7, and
9 (±1 day) after the first vaccination with SeV-Gag or placebo in
the SLA, SHA, and SHSH groups as described in the
The statistical methods are described in the Supplementary
Demographic Characteristics and Participant Flow and Recruitment
The study was conducted between March 2013 and March
2015. Of 65 volunteers, 36 (55.4%) were enrolled in Rwanda,
21 (32.3%) were enrolled in Kenya, and 8 (12.3%) were enrolled
in the United Kingdom. Volunteers were first enrolled in group
SLA only in Rwanda, followed by competitive enrollment at all
sites for groups SHA, ASH, and SHSH. Twenty participants
(30.8%) were female, and the mean age was 31.3 years (range,
19–48 years; Supplementary Table 1). All volunteers completed
study vaccinations and visits per protocol (Supplementary
Vaccine Safety and Tolerability
All vaccination regimens were generally well tolerated
(Supplementary Figures 2A and 2B). There was no statistically
significant difference in the frequency of grade 2 or higher upper or
lower respiratory tract reactogenicity following any SeV-Gag
vaccination, compared with placebo (Supplementary Table 2).
All local reactogenicity events after Ad35-GRIN intramuscular
injection were graded as mild or moderate. The frequency of
grade 2 local pain, tenderness, erythema, and swelling following
Ad35-GRIN vaccination was similar in the vaccine and placebo
groups (Supplementary Table 3). Most systemic reactogenicity
(chills, malaise, myalgia, headache, nausea, vomiting, and
fever) was grade 1 or 2. The overall frequency of any grade 2
or higher systemic reactogenicity following any vaccination
was similar in vaccine and placebo groups (Supplementary
Tables 2 and 3). One volunteer (in the ASH group) reported grade
3 malaise on day 2 after Ad35 vaccination and grade 3 chills,
malaise, and myalgia on day 0 after SeV-Gag vaccination
(Supplementary Figure 2).
There was no difference between groups in the proportion of
volunteers with grade 2 or higher unsolicited AEs (P = .525;
data not shown). The proportions of volunteers with any
unsolicited respiratory AEs (cough, influenza-like illness, nasal
congestion, pneumonia, and/or rhinitis) within 4 weeks of
vaccination or at any time during the trial were not statistically
significant between volunteers receiving SeV-Gag vaccination
and placebo recipients (Supplementary Table 4). No
vaccinerelated SAE was reported, and no apparent pattern in clinical
AEs or AEs determined by laboratory analysis was observed.
No volunteers tested positive for vaccine-induced
seropositivity/seroreactivity at the end of the study.
Mucosal samples, including nasopharyngeal fluid, parotid
gland saliva, oral fluid (transudate), and cervicovaginal and
rectal secretions, were collected at 8 time points. Compliance was
excellent for nasal and oral sampling, good for cervicovaginal
sampling, but poor for rectal sampling (Supplementary
Viral shedding samples were collected from the middle
turbinate region, saliva, and urine on days 2, 5, 6, 7, and 9 after
first vaccination with SeV-Gag or placebo. Overall, 20% of all
samples (vaccine vs placebo, P = not significant) across all
groups and visits (141 of 702) were positive by the cell
infectious unit assay, which used immunostaining to detect cells
infected with SeV. The polyclonal SeV antiserum used in
this assay cross-reacts with hPIV-1, and a positive readout
in this assay is either SeV or hPIV-1. Further analysis by
PCR specific for SeV indicated that 12% of the cell infectious
unit–positive samples (17 of 141) were positive for SeV and
that all bore the intact HIV-1 gag insert. These 17 samples
were from nasal swabs from 15 of 36 volunteers (42%)
receiving only SeV-Gag. No SeV-Gag virus was detected in nasal
samples after day 4 or at any time in saliva or urine
(Supplementary Table 5).
IFN-γ ELISPOT Findings
Few ELISPOT responses were detected after 1 or 2 vaccinations
with SeV-Gag alone; in contrast, responses were detected in the
heterologous regimens after the second vaccination (groups
SLA, SHA, and ASH; Figure 1 and Table 2). Two weeks after
the Ad35-GRIN boost, 12 of 12 volunteers in group SLA and
10 of 11 in group SHA demonstrated Gag-specific responses.
Five group ASH volunteers had positive Gag responses at both
time points, 1 group ASH volunteer had a positive Gag response
after the first vaccination only, and 1 group ASH volunteer had a
positive Gag response after the second vaccination only. The
proportions were similar in groups SLA (100%) and SHA
(90.9%; P = not significant). In the ASH group, 6 of 11
individuals (54.5%) had positive responses to the Gag peptide pool 2
weeks after the Ad35-GRIN prime (Table 2), and their
responses remained steady after the SeV-Gag boost (Supplementary
Table 6). In the SLA and SHA groups combined, the Gag
response rate was significantly higher 2 weeks after the second
vaccination (95.7%), compared with the rate in the ASH
group 2 weeks after either the Ad35-GRIN prime or the
SeVGag boost (54.5% for both; P = .008).
With respect to the magnitude of the response, the IFN-γ
ELISPOT responses to Gag 2 weeks after Ad35-GRIN receipt
were greater after SeV-Gag priming (in groups SLA and SHA
combined) than after the Ad35-GRIN prime (in the ASH
group), with geometric mean responses of 248 and 54 IFN-γ
spot-forming units (SFU)/106 PBMCs (P = .002; Table 2). The
geometric mean responses in the SLA and SHA groups 2 weeks
after Ad35-GRIN receipt were similar (275 and 222 IFN-γ SFU/
2 Weeks After the First and Second Vaccinations
2 Weeks After First Vaccination
Response Rate,a Geometric Mean
Subjects, No. (%) Responseb (95% CI)
0 (0) 3 (2–5)
0 (0) 2 (1–5)
0 (0) 3 (2–4)
6 (55) 54 (25–120)
0 (0) 3 (2–7)
0 (0) 2 (1–3)
Range of Positive
2 Weeks After Second Vaccination
Abbreviations: Ad35-GRIN, adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef; ASH, Ad35-GRIN prime followed by SeV-Gag boost; CI, confidence
interval; HIV-1, human immunodeficiency virus type 1; SA, SHA and SLA groups combined; SHA, higher-dose SeV-Gag prime and Ad35-GRIN boost; SHSH, higher-dose SeV-Gag prime and boost;
SLA, lower-dose SeV-Gag prime and Ad35-GRIN boost; SeV-Gag, Sendai virus–vectored vaccine encoding HIV-1 Gag.
a Excludes samples in which the prevaccination values for the Gag peptide pool were positive (ie, cross-reactive).
b Data are IFN-γ spot-forming units per million peripheral blood mononuclear cells.
106 PBMCs, respectively; P = .73). The response rate and
magnitude in group SLA waned over time but remained constant in
group SHA; the ASH group had lower but more-stable response
rates (Figure 1 and Supplementary Table 6).
Flow cytometry was used to characterize the phenotype and
polyfunctionality of the response. Low responses were detected
in group ASH 2 weeks after the Ad35-GRIN prime and 2 weeks
after the SeV-Gag boost (Figure 2 and Supplementary Table 7).
However, in groups SLA and SHA, 2 weeks after receipt of the
Ad35-GRIN boost, the median magnitude of the Gag-specific
CD4+ T-cells expressing IFN-γ, IL-2, or TNF-α was
significantly higher than after receipt of each dose of the ASH regimen
(P = .006 and P = .013, respectively). After prime and boost
(in the SLA group), the median number of Gag-specific CD4+
T-cells expressing IFN-γ, IL-2, or TNF-α increased
approximately 45-fold, from 0.005% to 0.225% (Figure 2A and
Supplementary Table 7). In contrast, 2 weeks after receipt of
the Ad35-GRIN boost in the SLA and SHA groups and 2
weeks after receipt of each dose of the ASH regimen, no
significant difference was observed in the magnitude of the
Gagspecific CD8+ T-cells (Figure 2B and Supplementary Table 7).
Gag-specific CD4+ polyfunctional T-cell responses
predominated over CD8+ T-cell responses in groups SLA and SHA, with
the majority of volunteers positive for all 3 cytokines tested
(Figure 2C). Gag CD8+ T-cell responses were highest in group SHA,
with the majority being either triple positive or double positive
for IFN-γ and TNF-α (Figure 2D). Fresh colorectal biopsy
mucosal mononuclear cells and PBMCs were tested in parallel by
ICS in 16 volunteers, including placebo recipients who
consented to the procedures. The mucosal mononuclear cells and
PBMC samples had responses to SEB and/or CMV as expected,
but only 1 vaccinee, in group SHA, had Gag-specific responses
in both PBMCs and mucosal mononuclear cells 2 weeks after
prime-boost (data not shown).
Viral Inhibition Assay
Viral inhibition was detected in 2 of 9 and 3 of 9 individuals
after SeV-Gag prime and in 11 of 12 and 9 of 9 individuals
after the Ad35-GRIN boost in groups SLA and SHA, respectively
(P = not significant). When the vaccination order was reversed,
in the ASH group, viral inhibition was detected after the
Ad35GRIN prime in 9 of 10 volunteers but in only 4 of 10 after the
SeV-Gag boost (Table 3 and Supplementary Figure 3A and 3B).
The viral inhibition response rate after the boost in groups SLA
and SHA combined was greater than in the ASH group (20 of 21
[95%] vs 4 of 10 [40%]; P = .0017, by the Fisher exact 2-tailed
In the SLA and SHA groups, the median log inhibition after
the SeV-Gag prime was 0.88 and 0.95, respectively, and it
increased significantly to 1.72 and 2.07 after the Ad35-GRIN
boost (P < .0001). In group ASH, the median log inhibition
after receipt of Ad35-GRIN alone was 1.29, decreasing to 0.85
after SeV-Gag receipt (Table 3). The magnitude of viral
inhibition after boost in the SLA and SHA groups combined (1.85) was
greater than after the Ad35-GRIN prime (1.29) and the
SeVGag boost (0.85) in group ASH (P < .0001 for each comparison).
A response to 2 viruses was detected after the second dose of
placebo in 1 volunteer. The breadth of viral inhibition was
assessed by the number of viruses inhibited out of a panel of 8
viruses from multiple HIV-1 clades. The greatest median
breadth was observed in groups SLA and SHA after the
Ad35GRIN boost (6 and 4, respectively), compared with a median
breadth of 3 after the Ad35-GRIN prime alone in the ASH
group (Figure 3 and Table 3). Viral inhibition was not assessed
in group SHSH.
Gag p24 Antibodies
Gag p24–specific IgG and IgA were measured in serum samples
at baseline, 2 and 16 weeks after the first vaccination, and 2, 16,
32, and 48 weeks after the second vaccination. In SLA, SHA, and
SHSH recipients, GMTs were negative at all time points (ie, they
were ≤100) after prime and boost. The GMTs were significantly
higher (P < .0001, by the Kruskal-Wallis test) in the ASH group
after the SeV boost than in the other groups (53, 53, 245, 50, and
59 in the SLA, SHA, ASH, SHSH, and placebo groups,
respectively; Figure 4). Two volunteers in group ASH were excluded from
this analysis owing to positive titers at baseline. ASH titers
decreased to a GMT of 72 at 48 weeks after SeV-Gag receipt. Gag
p24–specific IgA responses were detected sporadically and at
low titers (data not shown).
Most volunteers from all groups had a preexisting positive titer
of serum SeV NAbs, with similar magnitudes and rates in
vaccine and placebo recipients before and after vaccination
(Supplementary Figure 4). There was no correlation between
SeV NAb titers at baseline and the magnitude of Gag ELISPOT
responses 4 weeks after Ad35-GRIN receipt in groups SLA and
SHA and Gag-specific ELISA responses 2 weeks after the SeV
boost in group ASH (Supplementary Figure 5). Mucosal
secretions were not tested for SeV NAbs.
This was the first-in-human trial assessing safety and
immunogenicity of a replication-competent SeV-vectored HIV-1
vaccine; it was administered in prime-boost regimens with
Positive results, proportiona
Abbreviations: Ad35-GRIN, adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef; ASH, Ad35-GRIN prime followed by SeV-Gag boost; HIV-1, human
immunodeficiency virus type 1; SA, SHA and SLA groups combined; SHA, higher-dose SeV-Gag prime and Ad35-GRIN boost; SLA, lower-dose SeV-Gag prime and Ad35-GRIN boost; SeV-Gag,
Sendai virus–vectored vaccine encoding HIV-1 Gag.
a Data are no. positive/no. tested. Positive results correspond to any positive value per individual over all 8 virus isolates. Any virus isolate that was positive at baseline (which occurred in 3
individuals) was counted as negative. All other values are calculated using all results over all virus isolates per volunteer.
b At least 1 positive result after prime or boost. Other statistics include both prime and boost values.
c Breadth corresponds to the no. of positive virus isolates per individual.
Ad35-GRIN in healthy volunteers. SeV has been safely tested as
a nonrecombinant Jennerian vaccine against hPIV-1 in adult
and pediatric populations [22, 23]. Both SeV-vectored and
Ad35-vectored vaccines were well tolerated, and adverse events
were not significantly different from those in placebo recipients.
Mucosal sampling was well accepted in this study when it was
limited to saliva, nasal, or cervicovaginal fluids, but rectal fluid
collection and biopsy had more-limited acceptance, as in
previous studies [36, 37].
We postulated that delivery of SeV-Gag by the intranasal
route might induce mucosal immune responses and circumvent
preexisting immunity to SeV, allowing immune responses to the
vaccine insert, as shown previously in animal models [30, 42].
Despite preexisting SeV NAbs in all groups and a lack of
persistent SeV shedding indicative of replication, there was a clear
take of SeV-Gag, as indicated by much greater Gag-specific
T-cell and antibody responses in the heterologous regimens,
compared with either vaccine given once. Mucosal antibody
responses were weak and sporadic, and neither mucosal
application nor parenteral priming or boosting amplified mucosal
Remarkably, the vaccines induced very different immune
responses when given in a different order. As a homologous
regimen, 2 doses of SeV-Gag induced minimal humoral and
cellular immune responses. In contrast, SeV-Gag primed
Tcell responses for a subsequent boost by Ad35-GRIN, while
SeV-Gag boosted humoral responses after priming with
Ad35-GRIN. The strongest Gag-specific T-cell responses were
detected by ELISPOT and ICS assays after the SeV-Gag prime
and Ad35-GRIN boost, with no clear effect of the SeV-Gag
dose. Functional viral inhibition responses mediated by T
cells  with greater breadth, magnitude, and frequency
were also seen in groups SLA and SHA after the Ad35-GRIN
boost. The frequency and magnitude of Gag ELISPOT
responses in the SLA and SHA groups combined were equivalent to
those of Ad35-GRIN given twice intramuscularly, indicating
that SeV-Gag given intranasally provides as strong a prime as
an Ad vector given intramuscularly . A so-called hidden
prime has been postulated previously in studies of DNA
vaccines followed by Ad vector boosts [43, 44], in which plasmid
DNA vaccines with or without electroporation and/or
molecular adjuvants such as interleukin 12 or interleukin 15 elicit very
modest T-cell and antibody responses in humans, but prime for
anamnestic responses when Ad vectors are given as a boost [8,
45–49]. One dose of SeV-Gag appears to be equivalent to 3
doses of DNA vaccine (up to 8 mg) in terms of priming
responses and is perhaps more effective at priming than a highly
attenuated VSV-Gag delivered intramuscularly [6, 50, 51]. The
ICS studies demonstrated that SeV-Gag stimulated CD4+
T cells, which may have provided help for the development of
CD8+ T cells . It was not possible in this study to determine
how SeV-Gag provides this potent T-cell priming; its ability to
infect mucosal cells after intranasal delivery may be important.
A direct comparison of 2 routes of delivery could help to resolve
In contrast, Gag-specific antibody responses were detected
only when Ad35-GRIN was given first and boosted by
SeVGag in group ASH. Previous studies have shown low-to-negligible
Gag-specific antibody responses after administration of a single
dose of Ad35-GRIN . After priming with Ad35-GRIN and
boosting with SeV-Gag, the Gag ELISA responses were
equivalent to those seen with Ad35-GRIN given twice intramuscularly,
indicating that SeV-Gag given intranasally provides as strong a
boost as an Ad vector given intramuscularly .
In summary, intranasal delivery of SeV is feasible, safe, and
immunogenic in the presence of preexisting systemic antivector
antibodies; neither antibody nor T-cell responses were
equivalent at the 2 SeV-Gag doses tested. The type of response elicited
was determined by the order of vaccines in the heterologous
regimen, as SeV-Gag priming mainly induced cellular
immunity whereas SeV-Gag boosting mainly induced serum humoral
immune responses against Gag. Mucosal antibody responses
were weak and sporadic, and only 1 participant had a mucosal
T-cell response. Whether mucosal antibodies are necessary or
sufficient for protection against sexual transmission of HIV is
unknown; if they are important, a more powerful immunogen
or different regimen will be needed. Further studies to elucidate
the mechanism of this antibody–T cell shift may be warranted.
These data suggest that intranasal delivery of a viral vector
capable of limited replication as part of a heterologous
primeboost regimen may be a valuable way to stimulate immune
responses, even in the presence of preexisting antivector
antibodies. SeV-Gag was shown to prime for T-cell responses
and to boost antibody responses, but in this configuration the
SeV-Gag by itself is not sufficiently immunogenic for further
STUDY GROUP MEMBERS
The S001 Study Team includes the following individuals: Rosine
Ingabire, Gina Ouattara, Alan Steele, Anne Gumbe, Kundai
Chinyenze, Sabrina Welsh, Carl Verlinde, Deborah King,
Cynthia Bishop, Paramesh Chetty, Lorna Clark, Mumtaz Booley,
Devika Zachariah, Kristen Syvertsen, Kamaal Anas, Marloes
Naarding, Emmanuel Cormier, Jim Ackland, and Mamoru
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 Tanya Scharton-Kersten (International
AIDS Vaccine Initiative [IAVI]), for advice on regulatory aspects for
the close out of the trial; Keiko Watanabe (IAVI), for helping keep the
US-Japanese collaboration working smoothly; Laura Sharpe, John Brennan,
Brendan McAtarsney, and Helen Coutinho (Human Immunology
Laboratory), for their laboratory expertise; John Coleman (IAVI Design and
Development laboratory), for advice on development of PCR assays; and Akihiro
Iida, Hitoshi Iwasaki, Tomohiro Kobayashi, and Toshiaki Tabata
(DNAVEC/ID Pharma), for their expertise in helping develop the SeV-Gag.
Disclaimer. The views expressed in this publication do not necessarily
represent the position of the Japanese government. The findings,
interpretations, and conclusions expressed in this work are those of the author(s)
and do not necessarily reflect the views of The World Bank, its board of
executive directors, or the governments they represent. The contents are
the responsibility of the International AIDS Vaccine Initiative and do not
necessarily reflect the views of the US Agency for International
Development or the US government.
Financial support. This work was supported by the United States
Agency for International Development (USAID), the Ministry of Finance
of Japan in partnership with the World Bank; the Bill and Melinda Gates
Foundation; the Ministry of Foreign Affairs of Denmark; Irish Aid; the
Ministry of Finance of Japan, in partnership with the World Bank; the Ministry
of Foreign Affairs of the Netherlands; the Norwegian Agency for
Development Cooperation and the United Kingdom Department for International
Development. The full list of IAVI donors is available at: http://www.iavi.org.
Potential conflicts of interest. E. S., C. L. P., J. H. C., D. S. L., A. L., T. H.,
H. H., and M. I. are named inventors on patent applications
99-2031-IFWPCT covering SeV HIV-1 vaccine vectors. E. S., C. L. P., J. H. C., A. L., T. H.,
H. H., and M. I. are named inventors on patent applications
99-2040-IFWPCT covering Sendai HIV-1 vaccine vectors. E. S., C. L. P., H. P., J. G., A. L.,
J.-L. E., P. F., D. S. L., J. H. C., K. C., S. W., C. V., P. C., M. B., D. Z., K. S.,
K. A., and E. C. are or were at the time of the study employees of IAVI, which
has development rights for the Ad35-GRIN product ( patent no. US
8,119,144 B2). T. H., H. H., and M. I. are employees of ID Pharma (Tsukuba,
Japan), which is developing SeV vaccine vectors for HIV-1 and other
diseases. 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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