JOURNAL OF VIROLOGY, June 2010, p. 5898–5908
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 12
Novel Recombinant Mycobacterium bovis BCG, Ovine Atadenovirus,
and Modified Vaccinia Virus Ankara Vaccines Combine To Induce
Robust Human Immunodeficiency Virus-Specific CD4 and
CD8 T-Cell Responses in Rhesus Macaques?
Maximillian Rosario,1Richard Hopkins,1John Fulkerson,2Nicola Borthwick,1Ma ´ire F. Quigley,3
Joan Joseph,4Daniel C. Douek,3Hui Yee Greenaway,5Vanessa Venturi,5Emma Gostick,6
David A. Price,3,6Gerald W. Both,7Jerald C. Sadoff,2and Toma ´s ˇ Hanke1*
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe,
Oxford OX3 9DS, United Kingdom1; Aeras Global TB Vaccine Foundation, 1405 Research Blvd., Rockville, Maryland 208502;
Vaccine Research Centre, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 208923; Catalan HIV Vaccine Research and Development Center, AIDS Research Unit, Infectious Diseases Department,
Hospital Clinic, August Pi i Sunyer Biomedical Research Institute, School of Medicine, University of Barcelona,
170 08036 Barcelona, Spain4; Computational Biology Unit, Centre for Vascular Research, University of
New South Wales, Kensington, New South Wales 2052, Australia5; Department of Medical Biochemistry and
Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom6; and
Biotech Equity Partners Pty., Ltd., Riverside Life Sciences Building, 11 Julius Ave.,
North Ryde, New South Wales 2113, Australia7
Received 13 December 2009/Accepted 30 March 2010
Mycobacterium bovis bacillus Calmette-Gue ´rin (BCG), which elicits a degree of protective immunity against
tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has
been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety
record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed
a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described
safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo
T-cell assays, BCG.HIVA401as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in
BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401was used as a priming com-
ponent in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-
vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell re-
sponses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation
in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited
into the memory pool. These desirable features are thought to be associated with good control of HIV-1
infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein
derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel
vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401and OAdV.HIVA in nonhuman
primates. These results strongly support their further exploration.
Vaccine strategies must balance safety with immunogenicity.
Recombinant attenuated subunit vaccines are generally re-
garded as safe, but not sufficiently immunogenic as stand-alone
vaccines (17). Heterologous prime-boost regimens employing
diverse attenuated viruses or bacteria as vectors delivering a
common, often T cell-based, immunogen have been shown to
induce stronger responses than multiple repeated dosings of
the same vaccine modalities (19, 22, 39, 54). This is because
heterologous regimens allow boosting of pathogen insert-spe-
cific responses while avoiding the accumulation of antivector
immunity, which can significantly decrease vaccine “take” (1,
41). Results of the STEP study, which used a candidate single-
vector human immunodeficiency virus type 1 (HIV-1) vaccine
(6, 17, 41), have highlighted the need for novel alternative
vaccine vectors and strategies. Such alternatives could comple-
ment the limited mainstream vectors and provide additional
safety and immunogenicity through increased flexibility, for
example, through the availability of personalized vaccination
regimens based on preexisting immune status and/or respon-
siveness to vaccination.
Mycobacterium bovis bacillus Calmette-Gue ´rin (BCG) re-
mains the world’s most widely used vaccine, with over three
billion doses administered since its deployment in 1920s. It is
the only licensed vaccine against tuberculosis and is adminis-
tered at birth as part of the WHO Expanded Programme on
Immunization (EPI). Due to its many attractive features, BCG
or related mycobacterial vectors have also been explored in the
context of vaccines against a number of infectious agents such
as Leishmania, Borrelia burgdorferi, Streptococcus pneumoniae,
Bordetella pertussis, malaria, cottontail rabbit papillomavirus,
* Corresponding author. Mailing address: MRC Human Immunol-
ogy Unit, Weatherall Institute of Molecular Medicine, The John Rad-
cliffe, Oxford OX3 9DS, United Kingdom. Phone: 44 1865 222355.
Fax: 44 1865 222502. E-mail: firstname.lastname@example.org.
?Published ahead of print on 7 April 2010.
measles virus, and indeed human and simian immunodefi-
ciency viruses (34). Many of these vaccines showed immuno-
genicity and protection in murine models, and some were also
immunogenic in nonhuman primates (8, 56, 67, 68). In human
adults, recombinant BCG (rBCG) vaccines alone failed to pro-
vide consistent protection against Lyme disease (13). In addi-
tion to adult applications, we have suggested the use of rBCG
expressing an HIV-1-derived immunogen as the priming com-
ponent of a vaccine platform against mother-to-child transmis-
sion of HIV-1 through infected breast milk (32), where it
would be critical to elicit a protective HIV-1-specific response
as soon as possible after birth.
To compare vectors and heterologous prime-boost regimens
directly, we have advocated and pioneered the development of
a panel of vaccine modalities delivering the same shared im-
munogen (18). Our first such model immunogen is called
HIVA (21). This is a T-cell immunogen comprising HIV-1
consensus clade A Gag and a string of partially overlapping
immunodominant CD8 T-cell epitopes originating from Gag,
Pol, Nef, and Env, which has already been tested extensively in
human volunteers (20). To facilitate iterative preclinical im-
provements of the HIVA vaccines, epitopes recognized by
murine (58) and rhesus macaque (44) CD8 T cells were also
incorporated. Furthermore, we have formulated HIVA into
various vaccine modalities, including plasmid DNA (21), mod-
ified vaccinia virus Ankara (MVA) (21), human adenovirus
serotype 5 (HAdV-5) (5), Semliki Forest virus replicons (18,
49), recombinant lysine auxotroph BCG strain Pasteur (32),
and baculovirus-expressed and purified, bluetongue virus-de-
rived chimeric NS1 tubules (37); the immunogenicity of these
vectors has been compared directly and in heterologous com-
binations. More recently, we reported on the immunogenicity
of a novel and promising vaccine vector derived from ovine
atadenovirus type 7 (OAdV) (5); OAdV is the prototype mem-
ber of the genus Atadenovirus, which is structurally and bio-
logically distinct from Mastadenovirus (e.g., HAdV-5) (2, 50).
Importantly, no immunity to OAdV has so far been detected in
human sera (26). In mice, OAdV.HIVA induced strong poly-
functional HIVA-specific T cell responses with distinct kinetics
from those induced by HAdV5.HIVA and displayed demon-
strable single-dose efficacy against a surrogate virus challenge
(5). OAdV is approved for use in a phase I human clinical trial
(http://clinicaltrials.gov identifier no. NCT00625430). All of
the vectors/modalities we explore are perceived to be safe and
acceptable for use in humans.
Here, as a step toward translating our results into human
volunteers, we constructed a novel vaccine designated
BCG.HIVA401vectored by AERAS-401, a Danish 1331 strain
of BCG with improved immunogenicity and safety (57), and
demonstrated priming of T cells to the HIVA transgene prod-
uct in rhesus macaques. These BCG.HIVA401-primed HIV-1-
specific CD4 and CD8 T-cell responses were readily boosted
with MVA.HIVA and OAdV.HIVA vaccines to elicit broad
and robust HIV-1-specific T cell responses.
MATERIALS AND METHODS
BCG.HIVA401vaccine construction and preparation. Escherichia coli strains
were grown at 37°C on tryptic soy agar (EMD) or in tryptic soy broth (Teknova)
with gentle agitation. Kanamycin was used at a final concentration of 50 ?g/ml,
and hygromycin was used at a final concentration of 150 ?g/ml. The M. bovis
endosomalytic rBCG strain AERAS-401 (57) and derivatives were cultured at
37°C on 7H10 agar (BD Biosciences) or in Middlebrook 7H9 medium supple-
mented with 10% oleic acid-albumin-dextrose-catalase (OADC) enrichment
(BD Biosciences) plus 0.05% (vol/vol) tyloxapol (Sigma). For antigen expression
studies, strains were cultured in the above growth medium without OADC
enrichment and supplemented with 3% glucose.
An expression cassette encoding the HIVA immunogen linked to the M. bovis
Ag85B promoter and a 19-kDa signal peptide was generated by PCR from
plasmid pJH222.HIVA (32), using primers 85BpromL4 (5?-GCGGATTAATA
CGGAAATGAGACGACTTTGCGCC-3?) and HIVAR4 (5?-GCGGATTAAT
AAGCTTCCTCTAGATGCATGCTCGAGCG-3?). The cassette was ligated
into the integrative shuttle plasmid pCB02, which uses the mycobacteriophage
L5 integrase (38), and electroporated into rBCG strain AERAS-401 as described
previously (40). Integrants were selected on 7H10 plus hygromycin and con-
firmed by PCR; PCR-positive rBCG.HIVA401isolates were grown in protein-
free 7H9 medium and analyzed by Western blotting for HIVA expression.
Briefly, cells were pelleted by centrifugation and culture supernatants were
concentrated 100?. The resultant fractions were separated by discontinuous
SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and
probed with anti-Pk tag antibodies (Serotec).
Preparation of MVA.HIVA virus stock. Construction of MVA.HIVA was
described previously (21). Working vaccine stocks were grown in chicken embryo
fibroblast cells using Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and glu-
tamine, and then purified on a 36% sucrose cushion; the titer was determined,
and the stocks were stored at ?80°C until use.
Preparation of OAdV.HIVA virus stock. Construction of OAdV.HIVA was
described previously (5). OAdV.HIVA was grown on permissive CSL503 ovine
fetal lung cells, and the titer was determined according to published procedures
(3). In macaques, OAdV.HIVA is expected to be an attenuated nonreplicating
virus as it is in all nonovine cell types that have been tested (2).
Peptides and preparation of peptide pools. HIVA-derived 15-mer peptides
overlapping by 11 amino acid residues were kindly provided by the International
AIDS Vaccine Initiative and employed as described previously (46). Peptides
were synthesized, purified by high-performance liquid chromatography and con-
firmed to be ?80% pure using mass spectroscopy (Sigma-Genosys). Individual
peptides were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to yield a
stock of 40 mg/ml and stored at ?80°C. Peptides were either mixed into pools 1
to 4, each containing 20 to 23 individual peptides, corresponding to the Gag p24
and p17 regions of HIVA or combined into one pool of 90 peptides called pool
90. Pool 5 corresponded to the C-terminal polyepitope region of HIVA. Working
pool stocks were prepared by combining 20 ?l of each peptide and adding
phosphate-buffered saline (PBS) to a final volume of 5 ml; stocks were then
sterile filtered, aliquoted, and stored at 4°C for up to 1 week before use. Purified
protein derivative (PPD) RT49 was purchased from Statens Serum Institute,
Denmark, and used at a concentration of 20 ?g/ml in assay wells.
Mouse immunizations and isolation of splenocytes. Groups of 5 female
BALB/c mice, 5 to 6 weeks of age, were injected with the following doses of
vaccines: 106CFU of BCG.HIVA401intraperitoneally (i.p.), 106PFU of
MVA.HIVA intramuscularly (i.m.), and/or 107infectious units (IU) of OAdV.
HIVA i.m. The interval between BCG.HIVA401prime and virus boosts was 12
weeks. Mice were sacrificed 4 weeks after the last immunization, the spleens were
then removed and pressed individually through a cell strainer (Falcon) using a
2-ml syringe with a rubber plunger. The splenocytes were washed twice and
suspended in 10 ml of lymphocyte medium (RPMI 1640 supplemented with 10%
FBS, penicillin/streptomycin, 20 mM HEPES, and 15 mM 2-mercaptoethanol).
All mouse procedures and care conformed strictly to United Kingdom Home
Measurement of supernatant IFN-? by Bio-Plex. To measure supernatant
gamma interferon (IFN-?), mouse splenocytes at 2 ? 106cells in 100 ?l of R10
(RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin) were
added to each well of a 96-well round-bottomed plate (Costar), pulsed with the
H-peptide at 2 ?g/ml or medium alone and incubated at 37°C in 5% CO2for
24 h. Plates were then spun at 330 ? g for 5 min, and the supernatant was
removed and stored at ?80°C until use. Upon thawing, the supernatant samples
were spun at 17,000 ? g for 10 min and the cytokine assay was performed using
a Bio-Plex mouse cytokine 23-Plex panel (1 ? 96-well) kit (Bio-Rad) according
to the manufacturer’s protocol.
Rhesus macaques, vaccination, and isolation of PBMC. Eight adult female
Indian rhesus macaques (Macaca mulatta) from a United Kingdom breeding
colony were housed and treated strictly in accordance with United Kingdom
Home Office Guidelines. Four macaques per group were vaccinated with either
BBMO or BBOM, where “B” represents 107CFU of BCG.HIVA401intrader-
VOL. 84, 2010IMMUNOGENICITY OF rBCG-rMVA-rOAdV REGIMENS IN MACAQUES5899
mally (i.d.), “M” represents 5 ? 107PFU of MVA.HIVA i.m., and “O” repre-
sents 1010IU of OAdV.HIVA i.m. Vaccines were delivered at intervals of 4
weeks. Phlebotomy was performed from the femoral vein. Peripheral blood
mononuclear cells (PBMC) were isolated using Lymphoprep cushion centrifu-
gation (Nycomed Pharma) and either used fresh or cryopreserved in liquid
nitrogen until use as described previously (30).
IFN-? ELISPOT assay. The frequencies of cells that released IFN-? upon
restimulation with HIVA-derived peptides or purified protein derivative (PPD)
RT49 (Statens Serum Institute, Denmark) were assessed using an enzyme-linked
immunospot (ELISPOT) assay. The procedures and reagents of a commercially
available kit (Mabtech) were used throughout. Briefly, the released IFN-? was
captured by monoclonal antibody (MAb) GZ-4 immobilized on the bottom of
the assay wells, visualized using a combination of the secondary MAb 7-B6-1
coupled to an enzyme and a chromogenic substrate (Bio-Rad), and quantified by
spot counting using the AID ELISPOT reader system (Autoimmun Diagnos-
tika). All assays were carried out in duplicate, and the background counts were
subtracted from the experimental counts.
In vitro culture of macaque peripheral blood mononuclear cells. Frozen
PBMC were thawed and restimulated with 2 ?g/ml peptides in R10 containing
500 U/ml human interleukin-2 (IL-2) and incubated in 5% CO2at 37°C. On day
3, 500 U/ml IL-2 was added, 2 ?g/ml of peptides was again added on day 7, and
500 U/ml IL-2 was added on day 10. On day 14, the cells were washed and
analyzed in an IFN-? ELISPOT assay.
CD4 cell depletion. Magnetic bead (Dynal) depletion of CD4 cells was carried
out according to the manufacturer’s instructions. Briefly, no fewer than 107
PBMC were washed with PBS and spun slowly at 4°C for 30 min with the
magnetic beads. Cells in the supernatant were harvested for analysis by IFN-?
CFSE proliferation assay. Frozen PBMC were thawed, resuspended in R10,
and incubated overnight at 37°C in 5% CO2. Cells were counted and placed at
2 ? 106in 96-well round-bottomed plates. Carboxyfluorescein succinimidyl ester
(CFSE; Invitrogen) was diluted in PBS at 1:2,000; washed cells were labeled at
this concentration for 8 min at 37°C and then washed again. Mock or peptide
pool 90 was added at 2 ?g/ml, and the cells were incubated in R10 with HEPES
buffer at 37°C in 5% CO2for 5 days. After incubation, cells were stained with
anti-CD3-allophycocyanin (APC) (BD) and anti-CD8-PerCP (peridinin chloro-
phyll protein) (BD). At least 5,000 events were acquired for each condition using
a FACS Calibur flow cytometer and analyzed with Summit (Dako) software.
Results are expressed with the following formula: proliferation index (PI) ?
(sum of the cells in all generations)/(computed number of original parent cells
theoretically present at the start of the experiment). Animal M7 provided suffi-
cient cells for a positive concanavalin A (ConA; 5 ?g/ml) control and negative
Flow cytometric cell sorting and phenotypic analysis. Soluble fluorochrome-
labeled CM9/Mamu-A*01-APC tetramers were produced and used as previously
described previously (52). For phenotypic analyses of tetramer-reactive cells, the
following directly conjugated MAbs were used: anti-CD3-H7-APC, anti-CD4-
QD655, anti-CD8-Pacific Blue, anti-CD28-Texas Red-phycoerythrin (PE), anti-
CD45RA-fluorescein isothiocyanate (FITC), and anti-CD95-Cy5-PE. Dead cells
were excluded using LIVE/DEAD fixable Aqua dead cell stain kit (Invitrogen).
Flow cytometric cell sorting was conducted with a modified FACSAria (BD) at
70 lb/in2; postsort purity was consistently ?98%. In all cases, electronic com-
pensation was performed with antibody-capture beads (BD) stained separately
with individual MAbs present in the experimental samples.
Clonotype analysis. Molecular analysis of all expressed T-cell receptor ? locus
(TRB) gene products in sorted tetramer-labeled CD8 T-cell populations was
conducted using a template switch-anchored reverse transcription (RT)-PCR as
described previously (12, 52). Sequences were aligned using IMGT nomenclature
based on the extraction and characterization of macaque TR genes (15). Public
sequences were assigned with reference to an extensive database derived from
previous studies (29, 51, 52).
Construction and characterization of the next-generation
BCG.HIVA401vaccine. Construction of novel BCG strain
AERAS-401 [BCG1331?ureC::?pfoA(G137Q)], the starting
parental strain in this study, was described previously (57) and
was based on the observation that AERAS-401 conferred bet-
ter protection against model Mycobacterium tuberculosis chal-
lenge of mice than the commonly used BCG vaccine strain
SSI-1331 (BCG1331). Briefly, AERAS-401 was generated by
two modifications aimed at increasing strain immunogenicity.
First, BCG1331was transformed with a variant form of perfrin-
golysin O (pfoA), which is normally secreted by Clostridium
perfringens and associated with lysis of the endosome compart-
ment (33). The PfoA(G137Q) protein carries a single-amino-
acid substitution, G137Q, which results in the loss of the pfoA
toxicity to mammalian cells attributed to its short half-life in
the host cell cytosol, yet retains the ability to mediate bacterial
escape from a vacuole (33). This endosome lysis has also been
shown to confer a general trend toward increased survival of
immunodeficient SCID mice inoculated with AERAS-401 rel-
ative to BCG1331(57), which is an important safety feature for
vaccines that are to be deployed in populations with a high
incidence of HIV-1. Second, the pfoA(G137Q) gene was in-
serted into and deleted the ureC locus, the product of which
normally plays a role in the capacity of mycobacteria to block
the acidification of the early phagosome. A trend toward
enhanced efficacy was also observed for the ureC deletion
Here, to construct recombinant BCG expressing immuno-
gen HIVA, the HIVA open reading frame (21) was linked to
the M. bovis Ag85B promoter and oligonucleotide coding for
19-kDa protein signal peptide (Fig. 1A), as described previ-
ously (32); the whole HIVA expression cassette was then stably
inserted into the attB chromosomal locus of parental AERAS-
401 using an allelic exchange plasmid pCB02 (Fig. 1B) fol-
lowed by removal of the cointegrated antibiotic marker used
for initial recombinant selection. Thus, the resulting strain,
designated BCG.HIVA401, was markerless, fully compatible
with good laboratory practice and suitable for production of
master seed bacterial stock for manufacture of a clinical vac-
cine lot. The HIVA protein was readily detectable in the bac-
terial pellet after total cell lysate protein separation by SDS-
PAGE and staining with Coomassie brilliant blue (Fig. 1C). In
addition, HIVA released into the culture supernatant was de-
tected in Western blots using a MAb against the C-terminal Pk
tag (Fig. 1D).
BCG.HIVA401primes CD8 T-cell responses in mice. Before
committing to more labor-intensive and expensive experi-
ments in nonhuman primates, T-cell immunogenicity of the
novel BCG.HIVA401vaccine was confirmed in BALB/c
mice. Animals were injected either with BCG.HIVA401i.p.
(B), MVA.HIVA i.m. (M) or OAdV.HIVA i.m. (O) vac-
cines alone or in simple BO or BM prime-boost combina-
tions separated by an interval of 12 weeks. Mice were sac-
rificed 4 weeks after the boost. The immunogenicity readout
was focused on the immunodominant H-2Dd-restricted
epitope RGPGRAFVTI derived from HIV-1 Env (59), here
designated H, which was added for this very purpose to the
C terminus of HIVA (Fig. 1A). While no H-specific produc-
tion of IFN-? by immune splenocytes was detected after
administration of BCG.HIVA401alone, priming with this
vaccine increased responses to heterologous boosts by
MVA.HIVA and OAdV.HIVA relative to the viral vaccines
administered alone (Fig. 2). These results concur with our
published data on the immunogenicity of BCG.HIVA222
derived from the Pasteur strain in BALB/c mice (32) and
BCG.HIVA401on murine CD8 T cells.
5900ROSARIO ET AL.J. VIROL.
BCG.HIV401induces strong PPD-specific responses in ma-
caques. Next, the immunogenicity of BCG.HIVA401was as-
sessed in nonhuman primates. Eight rhesus macaques were
divided into 2 groups of 4 and vaccinated using either BBMO
or BBOM regimens delivering BCG.HIVA401i.d. and both
MVA.HIVA and OAdV.HIVA i.m. With the possible use of
rBCG as a prime for infant vaccination against breast milk
transmission in mind, the first three immunizations were
spaced at 4-week intervals. This is an accelerated regimen
compared to previous macaque schedules involving rBCG,
which used intervals of 18 to 23 weeks between individual
immunizations (8, 62). The first administration of 107CFU of
BCG.HIVA401was reactogenic at the site of injection, but the
lesions resolved spontaneously within 3 to 4 weeks. Adverse
effects after the second dosing were very mild. As a measure-
ment of BCG-specific immune responses and an indication of
BCG.HIVA401vaccine take, PBMC isolated at various time
points during the immunization schedule were assessed for
PPD reactivity in an IFN-? ELISPOT assay. In all macaques,
strong responses to PDD were detected; these peaked at a
median of 4,134 (range, 3,465 to ?4,500, which verges on the
threshold of reliable counting) spot-forming units (SFU)/106
PBMC at around week 7 (i.e., 3 weeks after the second
BCG.HIVA401administration) and declined gradually there-
after (Fig. 3). BCG-specific responses were not boosted by
either of the subsequent MVA.HIVA or OAdV.HIVA vac-
cines. Thus, the BCG.HIVA401vaccine alone elicited a strong
and boostable BCG-specific response in recipient macaques.
BCG.HIV401primes robust HIV-1-specific CD8 T-cell re-
sponses in rhesus macaques. Induction of HIV-1-specific re-
sponses was initially determined in IFN-? ELISPOT assays
using peptide pools 1 to 4, comprising 15-mers overlapping by
11 amino acids spanning the Gag p24/p17 region, and pool 5,
corresponding to the polyepitope domain of HIVA (47); these
are the same peptides that were used in previously published
macaque and clinical studies (14, 20, 30, 63). Pool 5 contained
the immunodominant Mamu-A*01-restricted epitope CTPYD
INQM derived from simian immunodeficiency virus (SIV) Gag
(CM9; residues 181 to 189) (44); macaques M3, M5, M6, and
M8 were Mamu-A*01?. Following the first BCG.HIVA401im-
munization, small but definite HIV-1-specific responses were
detected in the region of 60 SFU/106PBMC; these increased
FIG. 1. Expression of the HIVA immunogen from BCG.HIVA401. (A) A schematic representation of the HIVA insert shows the mycobacterial
85B antigen promoter, the 19-kDa signal peptide (SP), consensus clade A Gag p24 and p17 domains, and a string of partially overlapping epitopes
(Epitopes). The polyepitope region contains epitopes derived from Gag, which are not present in the p24 and p17 domains, Pol, Nef, and Env (21).
To facilitate the preclinical development of the HIVA vaccines, epitopes recognized by CD8 T cells from rhesus macaques in the context of
Mamu-A*01 (red) and mice in the context of H-2Dd(blue), and MAb SV5-Pk (24) (black) were coupled to the C terminus. (B) Schematic
depiction of E. coli-mycobacterium shuttle plasmid pCB02 with the following functional regions: res, ?-? resolvase binding site; M13, sequence
derived from phagemid M13; Ori, E. coli origin of replication; lacZ-?, LacZ-? complementation fragment, which facilitates blue/white screening
in E. coli; Int, mycobacteriophage L5 integrase (38); and 19-kDa SP, 19-kDa protein signal peptide. (C) Coomassie brilliant blue-stained gel of the
parental AERAS-401 and BCG.HIVA401whole-cell pellet lysate proteins separated by SDS-PAGE. Mrmarkers are shown on the left, and HIVA
is indicated (arrow). (D) Western blot of culture supernatants using anti-Pk tag MAb to detect the presence of the HIVA immunogen (arrow).
FIG. 2. Vaccine immunogenicity in BALB/c mice. Groups of
BALB/c mice were immunized as indicated at intervals of 12 weeks
and sacrificed 4 weeks after the last immunization. Splenocyes were
isolated and stimulated with peptide H or mock pulsed for 24 h.
Secretion of IFN-? into the supernatants was determined by using a
Luminex assay. B, BCG.HIVA401; M, MVA.HIVA; O, OAdV.HIVA;
n, no treatment. Results are shown as means ? standard deviations
(SD) (n ? 5). Statistical significance is indicated and was determined
by Student’s t test. (Analysis of variance was not appropriate as the
groups had vastly different SD.)
VOL. 84, 2010 IMMUNOGENICITY OF rBCG-rMVA-rOAdV REGIMENS IN MACAQUES5901
to a mean of 258 SFU/106PBMC after the second dose of
BCG.HIVA401(Fig. 4A). Macaques M1 to M4 and M5 to M8
then received MO and OM heretologous virus vaccine boosts,
respectively. HIV-1-specific T-cell responses increased only
modestly following the first recombinant virus administration.
However, the second viral boost resulted in a substantial en-
hancement of the HIV-1-specific T-cell response, which
reached mean frequencies of 1,305 and 1,194 SFU/106PBMC
for the BBMO and BBOM groups, respectively. Overall, there
were no notable differences between the two regimens, al-
though one time point following each of the first and second
heterologous boosts reached a statistically significant differ-
ence (P ? 0.02) in favor of OAdV.HIVA (Fig. 4A). These data
suggest that BCG.HIVA410primes T cell responses that can be
boosted to robust levels by heterologous virus-vectored vac-
Both BBMO and BBOM regimens induce broadly specific
anti-HIV-1 responses. The observation that vaccine-induced
T-cell responses to several of the 5 HIVA-derived peptide
pools provided the first indication that multiple epitopes were
targeted (Fig. 4B). To substantiate this, HIVA-specific T-cell
responses were deconvoluted at three time points to define
specificity at the level of individual 15-mer peptides. For the
peak responses (week 16), these peptides were employed in an
ex vivo IFN-? ELISPOT assay. To increase sensitivity, PBMC
isolated after BB (week 7) and BBMO/BBOM (week 17) vac-
cinations were first expanded in vitro prior to individual pep-
tide stimulation as described previously (14). These analyses
demonstrated that the BBMO/BBOM vaccine regimens in-
duced predominantly HIV-1 Gag-specific T cells that recog-
nized a median of 4 (range of 1 to 7) individual epitopes (Table
1). The protective Mamu-A*01 allele conferred no advantage
or disadvantage in terms of either the magnitude or the
breadth of the T-cell response. Thus, the HIVA vaccines used
in the BBMO/BBOM regimens elicited T-cell responses spe-
cific for multiple HIV-1 epitopes.
HIVA elicits T-cell responses that readily proliferate and
are oligoclonal. Proliferation in response to recall antigens is
the key feature of immunological memory. We therefore ex-
amined the ability of HIVA vaccine-induced T-cell responses
to undergo antigen-specific proliferation in vitro. Both the
BBMO and BBOM vaccine regimens elicited HIV-1-specific
T-cells that readily proliferated when stimulated with pool 90
(mix of pools 1 to 4) peptides (Fig. 5). There was no statistically
significant difference in the proliferative capacity of the HIV-
1-specific T cells induced by the two regimens.
It is established in the pathogenic SIVmac239 challenge
model that CD8 T-cell responses to the Mamu-A*01-restricted
SIV Gag-derived epitope CM9 are protective (7, 23, 45). Fur-
thermore, the early mobilization of public clonotypes within
this response has been identified as a molecular signature of
protection that predicts post-primary set point virus load (51).
We therefore determined the patterns of CM9-specific CD8 T
cell clonotype recruitment in two Mamu-A*01?macaques
(M3 and M5) following the BBMO and BBOM regimens
(week 18), respectively, using a template-switch-anchored RT-
In addition, we conducted a polychromatic flow cytometric anal-
ysis of CM9-specific CD8 T-cell phenotype. The CD3?CD4?
CD8?CD95?T cells that bound the CM9/Mamu-A*01 tetramer
in macaque M3 were predominantly CD28?CD45RA?. In con-
trast, the corresponding cells in macaque M5 displayed a hetero-
geneous phenotype (Fig. 6A). In terms of TCR usage, the CM9-
specific CD8 T-cell population in macaque M3 exhibited an
oligoclonal and highly skewed repertoire, whereas the corre-
sponding population in macaque M5 was more diverse and con-
tained two public clonotypes (Fig. 6B). These data demonstrate
directly the effective recruitment of CM9-specific CD8 T-cell
clonotypes into several compartments of the vaccine-induced
There is an increasing body of data to indicate that priming
with rBCG in heterologous regimens that boost with attenu-
ated viral or protein modalities represents an effective strategy
for the induction of passenger immunogen-specific T-cell re-
sponses (4, 8, 9, 32, 42, 56). Here, by using a more immuno-
genic and safer rBCG (57) in prime-boost regimens with
rMVA and novel rOAdV (5) vaccines, we demonstrated (i)
enhanced T-cell induction in mice by using regimens that incor-
porated BCG.HIVA401priming (with BCG.HIVA401priming for
increased responses not formally observed in rhesus macaques);
(ii) the induction in rhesus macaques of high-frequency HIV-1-
specific T-cell responses, which recognized multiple HIV-1 Gag-
derived epitopes; and (iii) the effective recruitment in rhesus
macaques of insert-specific T-cell clonotypes into diverse com-
partments of the memory pool. These are desirable features of
HIV-1 vaccine-induced T cells.
This is the first immunogenicity study of BCG.HIVA401and,
indeed, of the parental AERAS-401 BCG strain in nonhuman
primates. BCG.HIVA401alone induced strong T-cell re-
sponses to PPD, yet only weak responses specific for the HIV-1
transgene product HIVA (Fig. 3 and 4). This might represent
a positive feature of BCG-vectored vaccine usage during the
priming phase of more complex regimens because the nature
of priming events can dramatically influence the differentiation
and fate of the elicited CD8 T cells (60). Indeed, it was re-
ported previously that recombinant mycobacterium-induced T
lymphocytes that were skewed toward durable antigen-specific
FIG. 3. PPD-specific T-cell responses elicited by BCG.HIVA401.
Two groups of 4 rhesus macaques were immunized using either
BBMO (black) or BBOM (gray) regimen, where “B” represents
BCG.HIVA401, “M” represents MVA.HIVA, and “O” represents
OAdV.HIVA. Vaccination time points are indicated below the graph.
Responses to BCG were determined in an ex vivo IFN-? ELISPOT
assay using PPD as the antigen. The panel shows median responses for
the two groups after subtracting the mock-stimulated background. nd,
not done. The peak responses reached an overall median of 4,134
(range, 3,465 to ?4,500 at the threshold of reliable counting) SFU/106
PBMC at week 7.
5902 ROSARIO ET AL.J. VIROL.
memory CD8 T cells and rapidly expanded by heterologous
virus-vectored vaccines sharing the same immunogen (28, 60).
This quality memory induction is possibly a consequence of the
strong CD4 T cell help that mycobacteria elicit (43, 61). Sim-
ilarly, despite the induction of specific T cells at or below the
limit of detection, DNA priming increased the consistency of
the subsequent MVA.HIVA boost to 100% in a group of 8
human volunteers (14).
The MO and OM boosts yielded T-cell responses of similar
overall frequencies. The first viral boost resulted in only a small
augmentation of HIV-1-specific T-cell responses, possibly due
to the accelerated regimen. Thus, after 4 weeks, mycobacteria
might still persist at high levels, thereby driving a cytokine
storm and ongoing activation of innate responses that could
decrease vaccine take. Nevertheless, the second viral vacci-
nation delivered a substantial T-cell boost and induced
broadly targeted responses capable of rapid expansion upon
antigenic reexposure. Small, but significant differences were
detected between the two heterologous boosts in macaques,
with OAdV.HIVA inducing higher frequencies of HIV-1-
specific T cells than MVA.HIVA (Fig. 4A). Greater T cell
induction by OAdV.HIVA over MVA.HIVA was also de-
tected in mice (Fig. 2). The overall T-cell immunogenicity of
our two regimens is similar to a recently published regimen
consisting of two rBCG primes boosted by an rHAdV-5
vaccine (8). Clearly, further experiments are necessary to
FIG. 4. HIV-1-specific T-cell responses elicited by HIVA. Macaques M1 to M4 and M5 to M8 were vaccinated using the BBMO or BBOM
regimen, respectively, where “B” represents BCG.HIVA401, “M” represents MVA.HIVA, and “O” represents OAdV.HIVA, at the time points
indicated. HIV-1-specific T-cell responses were measured in an ex vivo IFN-? ELISPOT assay using HIVA-derived peptide pools. Pool-stimulated
responses are shown after subtracting the mock-stimulated background. nd, not done due to lack of sample availability. Panel A shows the sum
of HIV-1-specific responses induced by the BBMO (black) and BBOM (gray) regimens. The results are shown as mean ? SD (n ? 4). Asterisks
indicate statistically significant (P ? 0.02) difference determined using Student’s t test. Panel B shows vaccine-elicited T-cell responses in individual
rhesus macaques to individual peptide pools. Pools 1 to 4 (four grades of gray) span the HIV-1 Gag p24 and p17 regions of HIVA; pool 5 (black)
corresponds to the polyepitope string and contains the Mamu-A*01-restricted epitope CM9. ¶, Mamu-A*01?animals.
VOL. 84, 2010 IMMUNOGENICITY OF rBCG-rMVA-rOAdV REGIMENS IN MACAQUES5903
optimize the timing, dosage, and administration routes of
the BCG.HIVA401, MVA.HIVA, and OAdV.HIVA vacci-
nation regimen. However, the availability of other HIVA
vaccine modalities provides the opportunity for further aug-
mentation of vaccine-induced HIV-1-specific T-cell fre-
Optimized rBCG is a very attractive priming component for
adult prophylactic HIV-1 vaccines. We have argued that rBCG
is also a logical means of preventing HIV-1 transmission from
infected mothers to breastfeeding infants (32), because BCG is
an integral component of the EPI given to infants at birth or
upon the first contact with a health care worker and is followed
TABLE 1. The breadth of HIVA vaccine-induced T-cell responses
T-cell response toa:
BB at wk 7 (cultured)
wk 16 (ex vivo) wk 17 (cultured)
29–30 QIGWMTSNPPIPVGDIYKR 7–8
aB, BCG.HIVA401; M, MVA.HIVA; and O, OAdV.HIVA. Underlined portions of sequences were previously identified Mamu-A*01-restricted epitopes (30, 44).
An “8” at the end of the peptide sequence indicates that a positive restimulation was obtained with CD4 cell-depleted PBMC.
bPeptides were 15-mers overlapping by 11 amino acids. Where two peptides are shown, both stimulated IFN-? responses, but the minimal epitope was not identified
and so the whole 19-amino-acid region is shown.
5904ROSARIO ET AL. J. VIROL.
by other EPI vaccines between 4 and 6 weeks later, depending
on the national guidelines. In immunocompromised individu-
als, BCG can cause disseminated disease and is, therefore, not
recommended for HIV-1-infected infants. Nevertheless, be-
cause of the risk of tuberculosis in impoverished countries,
WHO guidelines do recommend BCG vaccination for healthy
asymptomatic babies of unknown HIV-1 status (64). Thus,
rBCG expressing an HIV-1 immunogen would serve as a dual-
priming platform for vaccines against M. tuberculosis (42) and
HIV-1. In this respect, the demonstrably increased safety in
SCID mice of AERAS-401 relative to its parental BCG strain
SSI-1331 (57) is an important reassurance for such an ap-
proach. Here, we have tested the immunogenicity of BBMO
and BBOM regimens delivered 4 weeks apart with the view
that it is critical in infants to elicit protective HIV-1-specific
responses as soon after birth as possible to prevent breast milk
transmission, although in infants, only one dose of rBCG
would be used. While robust anti-HIV-1 T-cell responses were
induced by the 4-wk-gap regimen, these might be higher still if
more commonly used intervals between individual doses were
employed (8, 62).
Ovine atadenovirus (OAdV) is a novel and underexplored
vaccine vector, which is distinct from the more commonly used
mastadenoviruses (2, 50). Here, we demonstrated that OAdV
is capable of antigen presentation for the induction of effective
CD8 and CD4 T cell responses in heterologous prime-boost
regimens. In addition, OAdV avoids the problems and possible
risks associated with preexisting immunity to HAdV vectors,
such as HAdV-5, in target populations. This work, which is the
first demonstration of the safety and T-cell immunogenicity of
OAdV in nonhuman primates, justifies its further development
as a vaccine vector for HIV-1, other infections, and cancer.
The HIVA construct (21) has been an extremely useful
model immunogen both for clinical (14, 20, 65) and preclinical
(30, 31, 36, 48) vaccine development. The epitopes recognized
FIG. 5. Proliferative capacity of HIVA vaccine-induced HIV-1-spe-
cific T cells. PBMC were isolated after either the BBMO (black) or
BBOM (gray) regimen, where “B” represents BCG.HIVA401, “M” rep-
resents MVA.HIVA, and “O” represents OAdV.HIVA, at week 17 and
assessed for their proliferation upon antigenic reexposure following stim-
ulation with pool 90 (pools 1 to 4) peptides or mock stimulation. Events
were gated on CD3?CD8?cells, and the number of cells showing CFSE
dilution following peptide pool restimulation was determined on dot plots
with CD8 on the y axis. Representative examples of CFSE dot plots for
mean ? SD (n ? 4 and 3; with macaque M5 not tested due to lack of
sample availability) calculated after mock background subtraction.
FIG. 6. Immunophenotype and clonal composition of CM9-specific CD8 T-cell populations. (A) The phenotypic profiles, as defined by
expression of CD28 and CD45RA, of CM9/Mamu-A*01 tetramer-reactive CD8 T cells are shown for macaques M3 and M5 following BBMO or
BBOM vaccination (week 18), respectively, where “B” represents BCG.HIVA401, “M” represents MVA.HIVA, and “O” represents OAdV.HIVA.
Plots are gated on singlet, live, CD3?CD4?CD8?CD95?cells. The CM9/Mamu-A*01 tetramer-positive events, with their percentages indicated
in the top right corner, are shown as red dots superimposed on density plots showing the phenotype of all gated cells. (B) T-cell receptor ? (TRB)
CDR3 amino acid sequences, TRB variable (TRBV), and TRB joining (TRBJ) usage and the relative frequency (Freq.) of CD8 T cell clonotypes
specific for the CM9 epitope are shown for macaques M3 and M5 at week 18. Colored boxes in the CDR3 sequence column indicate public
clonotypes. Public clonotypes were defined on the basis of TRB amino acid sequences that were present in more than one macaque with reference
to an extensive database derived from previous studies (29, 51, 52).
VOL. 84, 2010 IMMUNOGENICITY OF rBCG-rMVA-rOAdV REGIMENS IN MACAQUES5905
in mice and Mamu-A*01?rhesus macaques continue to be
mapped, and the corresponding CD8 T-cell responses are be-
ing characterized in greater detail. However, there is no ap-
propriate challenge available for the vaccinated animals be-
cause HIVA is designed for humans and is, therefore, derived
from HIV-1 antigens; HIV-1 does not replicate in rhesus ma-
caques. As for human efficacy, broad Gag-specific responses
have been associated with good control of HIV-1 replication in
chronically infected patients (27, 35, 53), responses to HIVA
have been readily detected in exposed uninfected children in
Kenya (55), and it has been demonstrated that HIVA boosts
specific CD8 and CD4 T cell responses effectively in patients
infected with a variety of HIV-1 clades (10, 11, 66). Thus,
HIVA as a Gag-based immunogen concurs at several levels
with the emerging correlates of T-cell-mediated control of
In conclusion, although patients have benefited from highly
active antiretroviral treatment, the HIV-1 pandemic continues
unabated and an effective vaccine remains the best solution for
halting the spread of this virus in resource-poor areas. New,
safer, and more immunogenic vaccine vectors in combination
are necessary for the definition of an optimal strategy. Here,
we tested for the first time in nonhuman primates a unique
vector combination of two novel vaccines based on modified
BCG and sheep atadenovirus and demonstrated their potential
utility for further HIV-1 vaccine development.
This work was supported by the Medical Research Council (United
Kingdom); the Australian Research Council (ARC; DP0452362); the
Spanish Research Council; the Foundation for Research and Preven-
tion of AIDS in Spain (FIPSE 36338/02); the Fundacio ´n Mutua Mad-
rilen ˜a de Automo ´viles (second call for proposals); Fundacio BCN
SIDA 2002; and the Intramural Research Program of the Vaccine
Research Center, National Institute of Allergy and Infectious Dis-
eases, National Institutes of Health (United States). D.A.P. is a Med-
ical Research Council (United Kingdom) Senior Clinical Fellow, T.H.
is a Jenner Institute Investigator, V.V. is an ARC Future Fellow, and
M.F.Q. is a Marie Curie International Outgoing Research Fellow.
We thank Linda Lockett and Jan Shaw of CSIRO Molecular and
Health Technologies for producing purified OAdV.HIVA and for
assistance with statistical analysis, respectively, and Warren Kitchen
and his team at the Defense Science and Technology Laboratory
(United Kingdom) for excellent animal care and handling.
The authors have no conflict of interest, except for G.W.B., who is
an inventor of the ovine atadenovirus vector system and Chief Scien-
tific Officer of Biotech Equity Partners Pty., Ltd., which has licensed
the vector from the CSIRO.
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