Safety and immunogenicity of novel recombinant BCG and modified vaccinia virus Ankara vaccines in neonate rhesus macaques.
ABSTRACT Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA(401) or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA(401) induced high frequencies of BCG-specific IFN-gamma-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-gamma responses. MVA.HIVA elicited HIV-1-specific IFN-gamma responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.
- SourceAvailable from: Ofer Levy
Article: Ontogeny of early life immunity.[Show abstract] [Hide abstract]
ABSTRACT: The human immune system comprises cellular and molecular components designed to coordinately prevent infection while avoiding potentially harmful inflammation and autoimmunity. Immunity varies with age, reflecting unique age-dependent challenges including fetal gestation, the neonatal phase, and infancy. Here, we review novel mechanistic insights into early life immunity, with an emphasis on emerging models of human immune ontogeny, which may inform age-specific translational development of novel anti-infectives, immunomodulators, and vaccines.Trends in immunology. 05/2014;
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ABSTRACT: Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 107 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/106 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.PLoS ONE 01/2014; 9(7):e103314. · 3.53 Impact Factor
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ABSTRACT: Introduction: Effective vaccines are the best solution for stopping the spread of HIV/AIDS and other infectious diseases. Their development and in-depth understanding of pathogen-host interactions rely on technological advances. Areas covered: Rational vaccine development can be effectively approached by conceptual separation of, on one hand, design of immunogens from improving their presentation to the immune system and, on the other, induction of antibodies from induction of killer CD8(+) T cells. The biggest roadblock for many vaccines is the pathogens' variability. This is best tackled by focusing both antibodies and T cells on the functionally most conserved regions of proteins common to many variants, including escape mutants. For vectored vaccines, these 'universal' subunit immunogens are most efficiently delivered using heterologous prime-boost regimens, which can be further optimised by adjuvantation and route of delivery. Expert opinion: Development of vaccines against human diseases has many features in common. Acceleration of vaccine discovery depends on basic research and new technologies. Novel strategies should be safely, but rapidly tested in humans. While out-of-the-box thinking is important, vaccine success largely depends on incremental advances best achieved through small, systematic, iterative clinical studies. Failures are inevitable, but the end rewards are huge. The future will be exciting.Expert opinion on biological therapy 02/2014; · 3.22 Impact Factor
JOURNAL OF VIROLOGY, Aug. 2010, p. 7815–7821
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 15
Safety and Immunogenicity of Novel Recombinant BCG and Modified
Vaccinia Virus Ankara Vaccines in Neonate Rhesus Macaques?
Maximillian Rosario,1John Fulkerson,2Shamit Soneji,3Joe Parker,1Eung-Jun Im,1†
Nicola Borthwick,1Anne Bridgeman,1Charles Bourne,2Joan Joseph,4
Jerald C. Sadoff,2and Toma ´s ˇ Hanke1*
MRC Human Immunology Unit1and MRC Human Haematology Unit,3Weatherall Institute of Molecular Medicine, University of
Oxford, The John Radcliffe, Oxford OX3 9DS, United Kingdom; Aeras Global TB Vaccine Foundation, 1405 Research Blvd.,
Rockville, Maryland 208502; and 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
Received 6 April 2010/Accepted 12 May 2010
Although major inroads into making antiretroviral therapy available in resource-poor countries have been
made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect
infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus
Calmette-Gue ´rin (BCG) is given to most infants at birth, and its recombinant form could be used to prime
HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immuno-
gen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine
BCG.HIVA401or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus
Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe,
giving local reactions consistent with the expected response at the injection site. No systemic adverse events or
gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA401induced high frequencies of
BCG-specific IFN-?-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detect-
able HIV-1-specific IFN-? responses. MVA.HIVA elicited HIV-1-specific IFN-? responses in all eight animals,
but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were
lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other
recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn
monkeys. These results inform further infant vaccine development and provide comparative data for two
human infant vaccine trials of MVA.HIVA.
Close to 2.3 million of children globally are infected with
human immunodeficiency virus type 1 (HIV-1). The majority
of neonatal infections occur in utero or intrapartum and, in the
absence of preventative interventions, up to 29% of infants
breast-fed by infected mothers acquire HIV-1 (6). Further-
more, HIV-1-infected children face a worse prognosis than
adults in that, without antiretroviral treatment (ART), 25% of
perinatally infected children progress to AIDS within 1 year
(10), and the median time to AIDS for the remaining children
is less than 7 years (2). It is now clearly established that ma-
ternal and extended infant ART can substantially reduce trans-
mission of HIV-1 through breast-feeding (23). However, in a
resource-poor setting, many logistical barriers to implementa-
tion of the ART-based prevention of mother-to-child-trans-
mission (PMTCT) remain (23). Because nutrition and hygiene
makes breast milk an important determinant of infant survival
(22, 28), formula feeding as a protective measure against
HIV-1 acquisition is recommended only if it is AFASS (ac-
ceptable, feasible, affordable, sustainable, and safe). Unfortu-
nately, AFASS it is still not for majority of infected mothers in
sub-Saharan Africa. Also, mixed bottle and breast feeding is
associated with a 10-fold increase in HIV-1 transmission rela-
tive to exclusive breast-feeding (4). Thus, an effective infant
vaccine against HIV-1 infection is the best and safest solution
for PMTCT of HIV-1 with the added practical option of pro-
Neonatal immunity is immature compared to the adult im-
mune system (25). The differences include naivety of the im-
mune cells, a tendency to develop Th2 responses (5) and an-
tigen-presenting cells with inefficient cytokine production (35).
For example, human cord blood T cells proliferated poorly and
produced low levels of interleukin-2 (IL-2) and gamma inter-
feron (IFN-?) when endogenous antigen-presenting cells pre-
sented the antigen (35, 44). Also, infant myeloid dendritic cells
are less efficient in priming Th1 responses because of their
decreased responsiveness to Toll-like receptor stimulation,
lower levels of surface costimulatory molecules, and lower
production of IL-12 (8, 27). In several infections, qualitative
and quantitative differences between human newborn and
adult responses were detected (1, 9, 26, 37). In contrast, other
studies of infants reported proliferation as well as IL-2 and
IFN-? production by T cells equal to that of adults following
T-cell receptor-independent activation (21, 46). These latter
observations indicate that neonate T cells are not intrinsically
* 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 (0) 1865 222355.
Fax: 44 (0) 1865 222502. E-mail: email@example.com.
† Present address: Division of Viral Pathogenesis, Beth Israel Dea-
coness Medical Center, Harvard Medical School, Boston, MA 02215.
?Published ahead of print on 19 May 2010.
“locked” into an immature phenotype but, given the correct
stimuli, they can develop mature immune responses (25). The
requirement for specific stimuli will likely differ for different
pathogens and vaccine vectors.
Mycobacterium bovis bacillus Calmette-Gue ´rin (BCG) is
commonly delivered at birth as an antituberculosis vaccine as a
part of the WHO Expanded Programme on Immunization
(EPI). It has been reported by several studies to promote an
adultlike Th1 response in newborns (16, 24, 34, 43), although
it was also suggested that delaying the BCG delivery to 10
weeks of age benefits the quantity and quality of BCG-induced
CD4 T-cell responses (20). BCG and related mycobacterial
vectors have been explored as vaccines against other infectious
agents, including human and simian immunodeficiency viruses
(19), and in adult animals showed immunogenicity and protec-
tion (3, 36, 39, 47, 48). The only clinical study of recombinant
BCG (rBCG) in adults failed to provide consistent efficacy (7).
We have suggested the use of rBCG expressing an HIV-1-
derived immunogen as the priming component of a heterolo-
gous vaccine platform for PMTCT of HIV-1 through infected
breast milk (18), where it is critical to prime HIV-1-specific
responses as soon as possible after birth. These responses
could be boosted a few weeks later or shortly after the already
busy EPI by heterologous vaccines delivering the same HIV-
1-derived immunogen. To this extent, we constructed the novel
candidate vaccine BCG.HIVA401(36) by inserting a gene cod-
ing for the HIV-1 clade A-derived immunogen HIVA (14) into
recombinant BCG strain AREAS-401 (40). AERAS-401 is a
newly developed strain that displayed enhanced safety (40) and
immunogenicity (11, 15) in murine models relative to its pa-
rental BCG vaccine strain Danish SSI-1331. Increased safety
represents an important feature should the BCG.HIVA401vac-
cine be deployed in babies born to HIV-1-infected mothers.
We showed that BCG.HIVA401in a heterologous combination
with recombinant modified vaccinia virus Ankara MVA.HIVA
and recombinant ovine atadenovirus OAdV.HIVA induced
robust polyfunctional HIV-1-specific T-cell responses in adult
macaques (36). Here, we assess the safety and immunogenicity
of the BCG.HIVA prime-MVA.HIVA boost regimen in new-
born rhesus macaques.
MATERIALS AND METHODS
BCG.HIVA401was described previously (36). The parental M. bovis endosoma-
lytic rBCG strain AERAS-401 (40) and BCG.HIVA401were grown at 37°C in
protein-free 7H9 medium or in Middlebrook 7H9 medium supplemented with
10% OADC enrichment (BD Biosciences) plus 0.05% (vol/vol) tyloxapol
(Sigma), and BCG.HIVA401was analyzed by Western blotting for HIVA expres-
sion. The titers and viabilities of the frozen stocks were determined on 7H10 agar
(BD Biosciences) plates by serial dilution.
Preparation of MVA.HIVA virus stock. Construction of MVA.HIVA was
described previously (14). Working vaccine stocks were grown in chicken embryo
fibroblast cells by using Dulbecco modified Eagle medium supplemented with
10% fetal bovine serum, penicillin-streptomycin, and glutamine and then puri-
fied on a 36% sucrose cushion, titered, and stored at ?80°C until use.
Rhesus macaques, vaccination, and isolation of lymphocytes. Eight 5- to
22-day-old Indian rhesus macaques (Macaca mulatta) from a breeding colony in
the United Kingdom were housed and treated strictly in accordance with UK
Home Office Guidelines. Four macaques per group were vaccinated with 107
CFU of parental AERAS-401 or BCG.HIVA401intradermally (i.d.), followed by
two intramuscular (i.m.) injections of 5 ? 107PFU of MVA.HIVA 11 and 14
weeks later. A 1-ml portion of peripheral blood was removed on each bleed from
the femoral vein and yielded between 0.8 ? 106to 1.7 ? 106peripheral blood
vaccine construction and preparation. Construction of
mononuclear cells (PBMC) following Lymphoprep cushion centrifugation (Ny-
comed Pharma). At necropsy, specific lymph nodes were dissected and pressed
through a mesh strainer to release residing lymphocytes. These isolated cells
were used fresh.
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 used as described previously (31). Briefly, peptides
were synthesized, purified by high-performance liquid chromatography and con-
firmed to be ?80% pure by using mass spectroscopy (Sigma-Genosys). Individ-
ual peptides were dissolved in dimethyl sulfoxide (Sigma-Aldrich) to yield a stock
of 40 mg/ml and stored at ?80°C. Peptides were combined into five pools, each
containing 20 to 23 individual peptides. Pools 1 to 4 corresponded to the Gag
p24/p17 regions of HIVA, while pool 5 corresponded to the C-terminal poly-
epitope region of HIVA. Working pool stocks were prepared by combining 20 ?l
of each peptide stock and adding phosphate-buffered saline to a final volume of
5 ml; stocks were then sterile filtered, divided into aliquots, and stored at 4°C for
up to 1 week before use. Purified protein derivative (PPD) RT49 was purchased
from the Statens Serum Institute (Denmark) and used at a concentration of 20
?g/ml in assay wells.
IFN-? ELISPOT assay. The frequencies of cells that released IFN-? upon
restimulation with HIVA-derived peptides or PPD RT49 were assessed by using
an enzyme-linked immunospot (ELISPOT) assay. The procedures and reagents
of a commercially available kit (Mabtech) were used throughout. Briefly, 200,000
PBMC were added per well, and 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 (NBT-BCIP), and quantified by spot count using the AID
ELISPOT reader system (Autoimmun Diagnostika). All assays were carried out
in duplicate, and the background counts were subtracted. For data analysis,
control counts from each neonate were pooled to form the control group. For
each peptide pair, a Wilcoxon test was performed, and P values were corrected
within each animal by using the Benjamini-Hochberg correction. Any P value
that was ?0.05 postcorrection was deemed significantly different from the con-
trol pool. Statistical software was used (http://www.r-project.org).
Vaccination regimens. Eight neonate rhesus macaques be-
tween 5 and 22 days old were allocated into groups 1 or 2
receiving either empty parental BCG strain AREAS-401 or
BCG.HIVA401, respectively, the latter of which expresses im-
munogen HIVA derived from consensus African HIV-1 clade
A structural proteins p24 and p17 of the Gag capsid coupled to
a string of partially overlapping HLA class I-restricted epitopes
(14) (Fig. 1A). The animals were then given two doses of
MVA.HIVA 11 and 14 weeks later (Fig. 1B). There were two
reasons for comparing the empty and recombinant BCG
strains. First, in our parallel murine experiments, it is difficult
to detect any HIV-1-specific CD8 T-cell responses induced by
BCG.HIVA alone: the BCG.HIVA priming was indicated only
indirectly by increased HIV-1-specific T-cell responses after
the MVA.HIVA boost (18; unpublished data). Therefore,
group 1 received empty parental BCG as a control for the
BCG.HIVA401prime. Second, two PedVacc clinical trials in
Africa test MVA.HIVA delivered to regular BCG-vaccinated
human neonates born to either healthy or HIV-1-positive
mothers (NCT00982579 and NCT00981695), which serve as
the first stage toward possible future trials testing the heterol-
ogous rBCG-rMVA boost regimen. The present experiment
provided an opportunity for a parallel evaluation of the same
regimen in nonhuman primates. We believe it is important to
run human and macaque studies in parallel to accumulate
comparative data for these two systems.
The BCG.HIVA401prime-MVA.HIVA boost regimen was
safe. The first aim of the present study was to assess the safety
of the novel recombinant BCG.HIVA401vaccine in neonate
7816ROSARIO ET AL. J. VIROL.
rhesus macaques. Thus, neonates macaques were injected with
107CFU i.d. of either AERAS-401 or BCG.HIVA401, and the
injection sites were closely monitored and documented. For
rigorous safety assessment, this dose was 10-fold higher than
that of BCG commonly used after birth for human babies as an
antituberculosis vaccine. All eight macaques developed a
slightly raised erythematous round lesion with induration by
week 1 similar to that observed in human neonates, which
resolved by week 4 (Fig. 2). In these small groups of neonates,
BCG.HIVA401caused inflammatory reactions indistinguish-
able from the parental BCG strain. The priming vaccination
was followed by two doses of 5 ? 107PFU of MVA.HIVA
i.m., which is same dose and route as that used in the two
PedVacc clinical trials. No adverse reactions were observed
from MVA.HIVA vaccination. No abnormal gross pathology
was observed in any of the eight neonate macaques at necropsy
(week 23). For gross pathological examination, animals were
laid supine. A midline incision from sternum to pubic symphy-
sis was made followed by lateral incisions to the anterior shoul-
der and thigh. Upon opening, the mediastinum appeared nor-
mal with no evidence of lung or cardiac pathology. The organs
of the peritoneum, kidneys, and adrenals were unremarkable
with no lymphadenopathy. Overall, treatment with both the
BCG.HIVA401and the MVA.HIVA vaccines was not associ-
ated with any systemic toxicological changes, and the findings
of local inflammation at the injection sites are considered to be
consistent with a predicted response to the vaccine adminis-
Neonate responses to PPD were strong and similar in both
groups receiving the parental AERAS-401 and BCG.HIVA401.
Next, we evaluated the BCG-induced response in peripheral
blood to PPD as a measure of the anti-M. tuberculosis re-
FIG. 1. HIVA immunogen and experimental design. (A) Sche-
BCG.HIVA401and MVA.HIVA. The immunogen HIVA consists of
consensus HIV-1 clade A Gag p24-p17 domains and a string of par-
tially overlapping epitopes (Ep). The polyepitope region contains
epitopes derived from Gag, which are not present in the p24-p17
domains, Pol, Nef, and Env (14). To facilitate the preclinical develop-
ment of the HIVA vaccines, the polyepitope region also includes
immunodominant Mamu-A*01-restricted epitope CTPYDINQM de-
rived from SIV Gag (CM9; residues 181 to 189) (red) (29). In
BCG.HIVA401(top), the HIVA transcription is driven by the myco-
bacterial 85B antigen promoter, and nucleotides coding for the 19-kDa
protein signal peptide (SP) are attached to the HIVA open reading
frame. In MVA.HIVA (bottom), the gene transcription is controlled
by vaccinia virus promoter P7.5 (14). (B) Experimental design. *, The
specific ages of neonates at the time of AERAS-401/BCG.HIVA401
vaccination were as follows: N3 and N8, 5 days; N1 and N2, 6 days; N4
and N7, 9 days; N6, 20 days; and N5, 22 days.
FIG. 2. Local reaction observed on infant macaques at the site of AERAS-401/BCG.HIVA401administration. Neonatal rhesus macaques were
given 107CFU i.d. of either AERAS-401 or BCG.HIVA401, i.e., 10? the human infant dose. Pictures of the injection sites were taken at 1 and
4 weeks after the vaccine injection.
VOL. 84, 2010IMMUNOGENICITY OF rBCG-rMVA IN NEONATE MACAQUES7817
sponse, mostly mediated by CD4?T cells by using an ex vivo
IFN-? ELISPOT assay. The PPD responses peaked for both
groups at 4 weeks postvaccination and declined thereafter (Fig.
3). On several occasions, the number of IFN-? spot-forming
units (SFU) was too high to be counted accurately and was
assigned a best estimate frequency of 2,000 SFU/106PBMC.
Therefore, median frequencies are used for the description of
these results. At peak, medians of 2,000 (all four animals) and
1,387 (range, 710 to 2,000) SFU/106PBMC were detected after
the AERAS-401 and BCG.HIVA401administrations, respec-
tively. The PPD responses induced by these two BCG strains
were similar and reached a statistically significant difference
(P ? 0.015) only at the last time point at week 23, enumerating
medians of 25 (range, 0 to 45) versus 167 (range, 110 to 326)
SFU/106PBMC for AERAS-401 and BCG.HIVA401, respec-
tively. This is not considered likely to be of any immunological
sequelae. The PPD-specific responses were not boosted by the
MVA.HIVA vaccinations. Thus, both parental strain AERAS-
401 and vaccine BCG.HIVA401alone elicited a strong BCG-
specific response in recipient neonate macaques.
Weak HIV-1-specific T-cell responses were only detected
after the MVA.HIVA boost. HIV-1-specific responses in the
circulating blood induced in groups 1 and 2 of neonate ma-
caques were determined prior to and throughout the immuni-
zation schedule in a standardized IFN-? ELISPOT assay using
five pools of 15-mer peptides overlapping by 11 amino acids
spanning the entire HIVA immunogen sequence (32). The
same HIVA peptide pools were used in previous nonhuman
primate studies (17, 33, 36, 45) and clinical trials (reviewed in
reference 13) of the HIVA vaccines. Here, a single adminis-
tration of BCG.HIVA401into neonates did not elicit any HIV-
1-specific responses detectable in an ex vivo IFN-? ELISPOT
assay 4 and 8 weeks later. The first HIV-1-specific T-cell re-
sponses were detected in all eight animals 1 week after the first
MVA.HIVA boost (week 12). With the exception of animal
N6, the responses of which totaled 837 SFU/106PBMC, the
frequencies detected in the other seven neonates were between
20 (N2) and 160 (N3) SFU/106PMBC in total (Fig. 4). It is
noteworthy that animals N6 and N7 had enlarged lymph nodes
in the left axilla at day 28, which resolved by day 56. Taking
advantage of five HIVA peptide pools, we estimated that the
neonates responded to at least a median of 2.5 (range, 1 to 5)
epitopes. Because the blood volumes taken from macaque
neonates were limited (1 ml per bleed), we did not perform any
tests to discriminate between CD4?and CD8?T cell-medi-
ated responses. The second MVA.HIVA administration
boosted IFN-? responses to single peptide pools in three ani-
mals (N3, N4, and N5) (weeks 15 or 19). This is likely due to
the anti-vector antibody and T-cell responses induced by the
first rMVA immunization, which dampens the insert-specific
T-cell stimulations. All animals were screened for the immu-
nodominant allele Mamu-A*01, and neonates N3, N5, and N8
were found to be positive. This did not influence the magnitude
of the vaccine-induced response. Overall, given the animal
numbers used in the present study, we did not observe any
benefit of BCG.HIVA401priming, which would be reflected in
augmented responses detected after the MVA.HIVA boost,
i.e., there was no difference between the group 1 and 2 elicited
HIV-1-specific IFN-? responses.
Vaccine-elicited responses were detected in regional tissues.
It has been shown in the simian immunodeficiency virus (SIV)/
rhesus macaque model that after oral inoculation of newborn
FIG. 3. BCG-specific T-cell responses elicited by AERAS-401/
BCG.HIVA401administration to infant macaques. Two groups of four
infant rhesus macaques were immunized with 107CFU i.d. of either
empty parental AERAS-401 or BCG.HIVA401, and the responses elicited
to BCG were determined in an ex vivo IFN-? ELISPOT assay using PPD
as the antigen. The times of vaccinations are shown below in weeks. Bp,
parental AERAS-401; B, BCG.HIVA401; M, MVA.HIVA. The panel
shows median responses for AERAS-401 (gray bars) or BCG.HIVA401
(black bars); the mock-stimulated background yielded a vast majority of
wells with no spots.*, Statistically significant difference (P ? 0.015) in a
two-tail Student t test, which was not reached at any other time points. nd,
FIG. 4. HIV-1-specific T-cell responses elicited by HIVA. Two
groups of four infant rhesus macaques were immunized with 107CFU
i.d. of either empty parental AERAS-401 (Bp; panel A) or
BCG.HIVA401(B; panel B) at week 0 and boosted by 5 ? 107PFU of
MVA.HIVA i.m. at weeks 11 and 14 (M11 and M14). HIV-1-specific
T-cell responses were measured in an ex vivo IFN-? ELISPOT assay
using HIVA-derived peptide pools 1 to 5 (from light gray to black
bars). Note that Pool 5 contains Mamu-A*01-restricted epitope CM9.
For the ELISPOT data analysis, control counts from each neonate
were pooled to form the control group. For each peptide pair, a
Wilcoxon test was performed, and P values were corrected within each
animal using Benjamini-Hochberg correction. Any P value that was
?0.05 postcorrection was deemed significantly different from the con-
trol pool. Only significant values were represented in the graph, with
the exception of bars marked by “x”. Software was used for the statis-
tical analysis (http://www.r-project.org). *, Mamu-A*01?animals.
7818ROSARIO ET AL. J. VIROL.
animals, the virus spreads rapidly within days from the oral
mucosa to regional and peripheral lymph nodes (30). Thus, at
postmortem examination (week 23), lymphocytes from several
lymphoid organs of group 2 neonate macaques N5-N8 were
isolated and analyzed for vaccine-induced T cells in a standard
ex vivo IFN-? ELISPOT assay. Although weak PPD responses
between 15 and 45 SFU/106cells were detected in all animals
at least in one of the examined tissues (spleen and subman-
dibular, retropharyngeal, and axillary lymph nodes), HIV-1-
specific responses of 25 and 28 SFU/106cells were detected in
at two sites only in neonate N6 (Table 1), which responded
with the most vigorous IFN-? response in the PBMC (Fig. 4).
Note that at week 23, no HIV-1-specific responses were de-
tectable in the peripheral blood.
In the course of this work, we assessed the safety and im-
munogenicity of a heterologous prime-boost regimen consist-
ing of one dose of a novel BCG.HIVA401strain, followed by
two doses of MVA.HIVA vaccines in rhesus macaque neo-
nates, an immunologically, technically, and ethically challeng-
ing model. We showed that both vaccines were safe causing
only local reactions consistent with injection and induction of
immune responses. BCG.HIVA induced strong BCG-specific
responses but failed to elicit any directly or indirectly (through
enhancement of boost) detectable HIV-1-specific IFN-?-pro-
ducing T-cell responses. MVA.HIVA induced weak, but con-
sistent anti-HIV-1 responses. This is the first time that
AERAS-401 strain of BCG and both the BCG.HIVA401and
the MVA.HIVA vaccines have been tested in macaque neo-
Vaccination with the parental AREAS-401 and recombinant
BCG.HIVA401strains induced high frequencies of IFN-?-pro-
ducing PBMC responding to the mycobacterium PPD stimu-
lation, and these frequencies were similar for both strains.
However, cellular responses elicited by BCG.HIVA401specific
for the HIV-1-derived passenger immunogen HIVA were un-
detectable in ex vivo and cultured (not shown) IFN-?
ELISPOT assays. Although similar results were obtained in
adult mice, BCG.HIVA401induce low, but definite and boost-
able HIV-1-specific responses in adult rhesus macaques (36).
Thus, the T-cell immunogenicity already decreased by some-
what inefficient production and/or processing of rBCG-ex-
pressed HIVA observed in adult animals was further compro-
mised by the relative immaturity of the infant immune system.
In 10-week-old human neonates vaccinated with BCG at birth,
responses to BCG were mediated mainly by CD4?T cells,
displayed complex cytokine patterns, and suggested that rely-
ing on IFN-? production alone may underestimate the magni-
tude and complexity of infant response (38). Thus, the focus in
the present study on the standard IFN-? detection could have
also contributed to the failure to detect HIV-1-specific re-
sponses induced by BCG.HIVA401vaccination.
With the exception of macaque N6, HIV-1-specific T-cell
responses elicited by the MVA.HIVA vaccination were weak,
but detected after the first MVA.HIVA vaccination in all eight
neonates. The second MVA.HIVA administration reboosted
these responses in three of eight of these animals. This is again
in contrast to adult rhesus macaques, in which MVA.HIVA
induced strong HIV-1-specific T-cell responses (17, 33, 36, 45).
The immunogenic and protective properties of rMVAs in non-
human primate infants was investigated by Van Rompay et al.
(41, 42). In the first study, neonate rhesus macaques were
vaccinated with MVA-SIVgpe expressing SIV Gag, Pol, and
Env and challenged orally with pathogenic SIVmac251. The
vaccinees survived longer and displayed better antibody re-
sponses than members of the control group, while only two
vaccinees had detectable SIV-specific IFN-? responses after
the MVA.SIVgpe vaccination (42). In a subsequent study, de-
livering SIV Gag, Pol, and Env using both canarypox and MVA
vectors to neonate macaques prior to oral SIVmac251 chal-
lenge reduced SIVmac251 acquisition and decreased viremia
in infants that became infected (41). Here, the HIVA immuno-
gen is designed for humans and, as such, consists of HIV-1 Gag
protein coupled to a string of HLA-restricted epitopes. Be-
cause HIV-1 does not replicate in rhesus macaques, the neo-
nates in the present experiment were not challenged. In any
case, the protective efficacy in the SIV/macaque model has not
been validated by human protection, and its predictive value to
the vaccine’s performance in humans is at present unclear (12).
In contrast to vaccine efficacy, the SIV/macaque model has
been extremely useful for following the dissemination of infec-
tion after oral challenge. It was shown that the virus passes
from the oral mucosa to the submandibular and retropharyn-
geal lymph nodes, followed by the axillary lymph nodes and the
Peyer’s patches within 4 days. A key factor involved in the
PMTCT of HIV-1 is likely to be the local immunity at the port
of entry, where the vaccine-induced T cells may have to re-
spond. The results of the BCG.HIVA401neonate rhesus ma-
caque trial demonstrated that recall IFN-? ELISPOT re-
sponses to HIVA peptides were detected in cells derived from
the retropharyngeal and submandibular lymph nodes at nec-
ropsy (week 23) in the best responding animal N6. Indeed,
TABLE 1. BCG.HIVA401-MVA.HIVA regimen-induced tissue-
specific responses in neonate rhesus macaquesa
HIVA peptide pool
aResponses were determined in an ex vivo IFN-? ELISPOT assay. LN, lymph
node; ND, not done.
VOL. 84, 2010IMMUNOGENICITY OF rBCG-rMVA IN NEONATE MACAQUES 7819
stronger PPD-specific responses were detected more consis-
tently at the studied immunological sites of these neonates.
In conclusion, we tested two candidate HIV-1 vaccines,
BCG.HIVA401and MVA.HIVA, for the first time in rhesus
macaque infants. We found the vaccines safe, but less immu-
nogenic for T cells than when administered to adult animals.
These results inform further development of vaccines against
mother-to-child transmission of HIV-1 through breast milk
and complement two MVA.HIVA infant trials in Africa.
This study was supported by the Medical Research Council (United
Kingdom). T.H. is a Jenner investigator.
We thank Warren Kitchen and his team at the Defense Science and
Technology Laboratory (United Kingdom) for excellent animal care
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