www.thelancet.com Vol 368 August 5, 2006 511
A preventive vaccine is the best long-term strategy to
control the HIV-1 pandemic. The Global HIV Vaccine
Enterprise1 has outlined a scientifi c strategic plan for
HIV vaccine development.2 Key features of this plan
include defi ning fundamental scientifi c barriers to the
development of an eff ective preventive vaccine, creating
vaccine centres charged with advancing the rational
design and development of vaccines, and establishing
standard preclinical and clinical laboratory assessments
of candidate vaccines. To date, most eff orts have focused
on the development of vaccines for adults. However,
infants, children, and adolescents constitute major risk
groups for HIV infection, and morbidity and mortality
are particularly high in infants infected with the virus.
An eff ective neonatal vaccine could prevent transmission
of the virus via breastmilk and provide the basis for
lifetime immunity. Here, we discuss the rationale for the
development of HIV vaccines for infants and outline
important scientifi c and logistical considerations for their
Rationale for paediatric HIV vaccines
Rising incidence and prevalence of infection
Since the beginning of the HIV pandemic 25 years ago,
an estimated 60 million individuals have been infected
with the virus; 39 million now live with the infection and
almost 5 million acquire the virus every year.3 HIV
infection disproportionately aff ects women and young
people, with half of all new infections arising in
individuals aged 15–24 years.
Heterosexual transmission remains the predominant
mode of transmission worldwide, and about half of all
newly infected individuals are female, particularly those
in their early to middle teenage years.3 The rising
incidence and prevalence of HIV in women of child-
bearing age have greatly increased the number of children
infected. In 2003, an estimated 2·2 million children had
HIV. About 2 million children are born to women
infected with HIV every year, and every one of them is at
risk of infection; 740 000 children younger than age
15 years were newly infected in 2004, most through
mother-to-child transmission (MTCT). This fi gure
represents almost 2000 new infections per day (15% of all
new infections). More than 90% of paediatric infections
occur in sub-Saharan Africa, where their increase has
reversed advances in child mortality achieved through
immunisation and public-health programmes over the
latter decades of the 20th century.
Limitations of antiretroviral effi cacy in reducing MTCT
Maternal or perinatal antiretroviral prophylaxis regimens
can greatly reduce MTCT of HIV.4 In a landmark study
(Paediatric AIDS Clinical Trial Group 076),5 treatment of
American and European pregnant women and their
infants with zidovudine resulted in a transmission rate
of 8% compared with a transmission rate of 26% in the
placebo group (67% reduction in MTCT). By use of a
combination of antiretroviral therapies, overall MTCT
rates have dropped to below 2% in most of the developed
Similarly, the shortest possible antiretroviral regimen,
consisting of a single dose of nevirapine administered to
a woman during delivery followed by a single dose of
nevirapine administered to the baby shortly after birth,
greatly reduced the risk of intrapartum HIV transmission
in Africa.8 However, even this regimen has been diffi cult
to implement widely in resource-poor settings; despite
an off er of corporate donation of nevirapine to prevention
of MTCT (PMTCT) programmes, many women who
Lancet 2006; 368: 511–21
University of Massachusetts
Medical School, Program in
373 Plantation Street, Suite
318, Worcester, MA 01605,
USA (Prof K Luzuriaga MD,
Prof J L Sullivan MD); Centre for
Institute of Child Health,
(Prof M-L Newell PhD); INSERM
U593, Institut de Santé
Publique, Epidémiologie et
Université Victor Segalen,
(Prof F Dabis MD); and
International AIDS Vaccine
Initiative, New Delhi, India
(J-L Excler MD)
Dr Katherine Luzuriaga
Vaccines to prevent transmission of HIV-1 via breastmilk:
scientifi c and logistical priorities
Katherine Luzuriaga, Marie-Louise Newell, Francois Dabis, Jean-Louis Excler, John L Sullivan
Mother-to-child transmission (MTCT) of HIV-1 is the major mode of paediatric infection. The rapidly increasing
incidence of MTCT worldwide has resulted in an urgent need for preventive strategies. Antiretroviral regimens can
prevent intrapartum HIV transmission; however, these regimens do not prevent HIV transmission through
breastfeeding. Furthermore, children who escape MTCT are again at risk of infection when they become sexually
active as adolescents. An infant vaccine regimen, begun at birth, would hence be a more attractive strategy and might
also provide the basis for lifetime protection. Unique features of MTCT and paediatric HIV disease could be helpful
in understanding correlates of immune protection and could facilitate rapid assessment of vaccine effi cacy. Thus,
there is compelling rationale to develop safe, eff ective HIV vaccines for use in infants and children. Here, we discuss
the scientifi c and logistical challenges for the development of paediatric HIV vaccines; available vaccines and
completed or planned paediatric vaccine trials are also discussed.
Search strategy and selection criteria
We identifi ed data for this review by searching PubMed for
relevant articles published in English until November, 2005,
using the following terms: “HIV vaccines“, alone or in
combination with “child“; and “modifi ed vaccinia Ankara“,
“poxvirus vaccines“, or “DNA vaccines“ in combination with
“HIV“. We also identifi ed numerous articles through searches
of our personal fi les.
www.thelancet.com Vol 368 August 5, 2006
could benefi t from its use probably do not receive the
drug because the necessary infrastructure and manpower
to deliver the intervention are lacking. Furthermore,
fi ndings of studies9–11 indicate the rapid selection of
viruses with resistance mutations after the use of one or
two antiretroviral drugs in the setting of incomplete
control of maternal viral replication. These data raise
concerns that the usefulness of the drug most commonly
available in resource-limited settings to prevent MTCT
might be reduced with more widespread use.
Perinatal antiretroviral regimens do not prevent
transmission of HIV through breastfeeding, although
randomised trials12 of other regimens that might are
underway. A vaccine regimen started at birth would not
only prevent MTCT, but might also provide the basis for
lifetime protection against HIV infection. Use of existing
global infrastructures for the delivery of routine
childhood vaccines would enhance the feasibility of this
Assessment of vaccine effi cacy
Findings of clinical trials indicating antiretroviral
treatment effi cacy in PMTCT provided proof-of-concept
regarding the feasibility of preventing HIV transmission
between individuals.13 Trials of HIV vaccines in
breastfeeding infants might similarly yield evidence of
vaccine effi cacy or correlates of immune protection more
rapidly than vaccine trials in adults. Natural history
studies of MTCT and interventional trials of PMTCT
have organised appropriate cohorts and defi ned
transmission and incidence rates. At-risk mother-infant
pairs can be easily identifi ed through voluntary
counselling and testing in antenatal clinics; many centres
have proven their ability to enrol and follow up mothers
and their infants in PMTCT trials. Within these research
settings, an infant can be diagnosed with HIV within
days or weeks of infection. Finally, if a vaccine does not
provide sterilising immunity, the high, early viral loads
and rapid tempo of disease progression in infants14–16
could allow rapid assessment of vaccine effi cacy in
lowering viral load or altering disease outcome after
infection. Concurrent assessment of vaccine-induced
immune responses could lead to the identifi cation of
potential correlates of immune protection. In this respect,
a parallel can be drawn with data from a randomised
controlled trial17 in Mozambique of a malaria protein
subunit vaccine (RTS, S/ASO2A, GlaxoSmithKline
Biologicals, Rixensart, Belgium), which showed a 30%
reduction in clinical episodes and a 58% reduction in
episodes of severe malaria in a cohort of vaccinated
children aged 1–5 years.
There is, therefore, compelling rationale for the rapid
development of safe, eff ective HIV vaccines for use in
newborn children. But what are the scientifi c and
operational research issues associated with their
Pathogenesis of MTCT and vaccine
Transmission of HIV via breastmilk
In the absence of antiretroviral therapy, 25–30% of
women infected with HIV transmit the virus to their
infants. MTCT can occur during gestation, during
delivery, or postpartum through breastfeeding. In non-
breastfed populations, 25–30% of infected infants have
detectable provirus in their peripheral blood lymphocytes
at birth, suggesting that they were infected in utero.18 In
the remaining 70–75%, HIV RNA or provirus is not
detected in the peripheral blood at birth, but becomes
detectable within weeks of birth, compatible with the
intrapartum transmission of HIV. In breastfed
populations, breastfeeding probably accounts for a third
to half of all infections.19,20
In many areas—eg, the USA, Europe, Brazil, and
Thailand—the availability, aff ordability, safety, and
cultural acceptability of formula feeding have contributed
to the feasibility of using formula to prevent HIV
transmission through breastmilk. However, in resource-
limited settings, breastfeeding remains crucial for infant
health and survival.21,22 In the regions with the highest
HIV incidence and prevalence, the high cost of formula,
poor hygienic conditions, and lack of potable water and
refrigeration make formula feeding impractical and
Although data from some studies19,23 suggest that trans-
mission via breastmilk commonly occurs within the fi rst
months of life, fi ndings of a recent meta-analysis20
indicate a constant risk of transmission (8·9 trans-
missions per 100 child-years of breastfeeding) between
age 1 month and 18 months. The probability of trans-
mission has been estimated at 0·00064 per L ingested
and 0·00028 per day of breastfeeding; the latter is
roughly equivalent to the probability of HIV transmission
per unprotected sex act between adults.24 Low CD4-cell
counts and high plasma HIV RNA copy numbers are
associated with the highest risk of breastmilk
transmission.20,25 Rousseau and colleagues26 have shown
that every log10 increase in breast milk cell-associated
virus is associated with a three-fold increase in the risk
of MTCT. However, oral transmission of cell-free simian
immunodefi ciency virus (SIV) has also been documented
in infant macaques.27
Viral pathogenesis of early paediatric infection
Clinical manifestations of HIV infection in children
diff er greatly from those in adults. Studies from the
USA, Europe,28,29 and Africa30 have documented faster
rates of disease progression and death in infants than in
adults. HIV-associated morbidity and mortality seem to
be particularly high in African cohorts; results of a
pooled analysis30 revealed that about a third of children
with HIV infection die by their fi rst birthday and more
than half by the age of 2 years, with children infected in
utero or intrapartum having the fastest progression.
www.thelancet.com Vol 368 August 5, 2006 513
Although the precise mechanisms of MTCT are
unknown, intrapartum transmission probably results
from a discrete exposure to virus in maternal blood or
cervicovaginal secretions across infant mucosal surfaces,
whereas breastmilk transmission is thought to result
from many low-dose exposures across the gastrointestinal
tract. The relative contribution of cell-free versus cell-
associated viruses in intrapartum and breastmilk
transmission remains unclear. Models of transmission
suggest that HIV fi rst infects submucosal macrophages
or dendritic cells and that these cells then transmit the
virus to CD4-positive T cells. These cells constitute the
short-lived, productively infected population of cells that
produce the majority of plasma virus in the absence of
therapy31,32 and might constitute an important latent viral
reservoir in individuals on suppressive antiretroviral
therapy.33,34 Viruses isolated in early vertical infection
most often use the CCR5 co-receptor, although occasional
use of other receptors has been reported.
Over the fi rst few weeks of vertical infection, plasma
HIV RNA copy numbers
10⁵–10⁷ per mL.15,16,35,36 Peak and set-point plasma HIV
RNA concentrations are signifi cantly higher in
antiretroviral-naive Kenyan infants than in adults. In one
study,14 the average plasma HIV RNA set point in infants
who acquired HIV infection in utero or before age
2 months was almost 1 log higher than in infants who
acquired the infection through breastfeeding after
2 months of age. Most studies have shown that plasma
HIV RNA concentrations remain high in infants over the
fi rst 1–2 years of life.15,16 Gradual reductions in plasma
HIV RNA copy numbers have been noted thereafter in
vertically infected children up to age 5–6 years.37,38
Reductions in plasma HIV RNA after the initiation of
antiretroviral treatment are associated with clinical
benefi t.39,40 Collectively, these data suggest that viral
replication is an important determinant of paediatric
disease and that the long-term increase in plasma viral
load noted during the fi rst 2 years of life probably
contributes to the faster disease progression seen in
children than in adults.
Many factors could contribute to the early, long-term
increase in plasma HIV RNA concentrations, including
the kinetics of viral replication, the size of the host cell
pool permissive to viral replication, and delayed or
ineff ective virus-specifi c immune responses. Although
relative lymphocytosis and an increased CD4-cell pool
size are present throughout infancy and early childhood,
the kinetics of viral replication in infants appear to be
very similar to those described in adults,32 suggesting
that infants might have reduced eff ector mechanisms to
control viral replication.
HIV-specifi c immune responses in infants
Diff erences in the generation of innate or adaptive
immune responses are thought to account for age-related
diff erences in outcome after various viral infections.
Defective, antibody-dependent, cell-mediated cytotoxicity
(ADCC) of HIV-infected cells by neonatal natural killer
cells has been documented;41 however, this defect appears
to reverse by 1 month of age. After the decline in
concentrations of passively acquired maternal ADCC
antibodies, the active generation of HIV-envelope-specifi c
cytotoxic antibodies by infected infants is delayed.42 The
ineffi cient production of such antibodies to the highly
glycosylated envelope protein could indicate the relative
ineffi ciency of antibody production to T-independent
antigens in children younger than age 2 years. Protein
conjugation has been used successfully to convert
T-independent antigens to T-dependent antigens to elicit
antibodies to Streptococcus pneumoniae and Haemophilus
infl uenzae in infants.43
The development of HIV-specifi c binding antibodies in
children positive for HIV is well documented; there are
fewer studies characterising the development of
neutralising antibodies (particularly to autologous viral
strains). Reports44,45 of reduced antibody quality and titres
in infants immunised against or infected with viruses
other than HIV before age 6 months have raised concerns
about such infants ability to generate protective antibody
responses. However, adult and infant rotavirus-specifi c B
cells have similar variable gene repertoires, suggesting
that additional doses of antigen and improved antigen or
adjuvant quality could improve the generation of virus-
specifi c antibody responses in infants.46 The fact that the
administration of hepatitis B vaccine between birth and
6 months of age is more than 90% eff ective in preventing
MTCT of hepatitis B virus indicates that young infants
are capable of making antibodies protective against viral
challenge in vivo.
Less robust HIV-specifi c cellular immunity might also
preclude the eff ective containment of viral replication in
early infection. Although HIV-specifi c CD8-positive T-cell
responses have been detected in infected infants within
the fi rst months of life, they are of lower frequency and are
less broad than in adults with primary infection.47–50 Since
infants share at least three HLA class I alleles with their
mothers, MTCT of CD8-cell escape variants could
compromise the generation of early infant CD8-cell
responses restricted by shared HLA alleles.51–53 HIV-specifi c
CD8-positive T-cell responses increase in frequency and
breadth over the fi rst year of life.49,54 Although the detection
of HIV-specifi c CD8-positive T cells seems to be related to
a reduction in viral load during primary infection in
adults,55 early HIV-specifi c CD8-cell responses in infants
do not correlate with a reduction in viral load over the fi rst
year of life, raising the question of whether the
measurement of interferon γ secretion ex vivo accurately
indicates CD8-positive T-cell activity in vivo. We53 and
others52 have noted that some early CD8-cell responses in
infants are associated with the selection of CD8-positive
T-cell escape variants, suggesting that CD8-positive T cells
can exert selective pressures in vivo. Further characterisation
of the ex vivo functional properties of infants’ HIV-specifi c
www.thelancet.com Vol 368 August 5, 2006
CD8-positive cells and better understanding of the
associations between CD8-cell responses and viral
replication or evolution in infants would be helpful in the
development of a neonatal vaccine.
Use of neonatal macaque model
As outlined above, many questions remain about the
mechanisms of transmission of HIV in breastmilk, early
events in neonatal HIV infection, and correlates of
immune protection. The neonatal macaque model has
been and will probably continue to be helpful in
addressing these questions. For example, several studies56
in infant macaques have provided data that lend support
to the idea of a protective role of antibodies against HIV
transmission. In particular, a combination of monoclonal
postexposure prophylaxis against intravenous or mucosal
exposure to chimeric SIV-HIV in neonatal macaques.57
Although there are concerns that the SIV-HIV challenge
viruses used in these studies are highly neutralisation
sensitive and might not represent transmitted viruses,
these studies have provided compelling data in support of
clinical trials of monoclonal antibodies in human infants.
One particular infant macaque model,58,59 in which several
low doses of uncloned SIV are delivered in breastmilk,
seems to be especially promising in its simulation of
breastmilk transmission; further studies with this model
could provide useful insights into the mechanisms of
viral transmission, early events after transmission,
potential correlates of protection, and vaccine effi cacy.
Paediatric vaccine pipeline
The induction of long-lasting, antiviral immunity has
been achieved most successfully through the use of live
attenuated vaccines, probably because these vaccines can
emulate the natural infection process, resulting in
protective innate and adaptive (humoral and cell-mediated)
immunity. Early eff orts to develop a live attenuated HIV
vaccine were overshadowed by major safety concerns,
including the possibility of reversion to virulence by
mutation or recombination with superinfecting viruses
and chromosomal integration with the attendant risk of
malignant transformation.60,61 Eff orts to develop an HIV
vaccine have thus primarily focused on recombinant
protein vaccines, replication-defective recombinant viral
vectors, and DNA vaccines.
Recombinant protein vaccines
Monomeric recombinant envelope (gp120) vaccines,
designed primarily to elicit HIV-specifi c antibodies, were
among the fi rst HIV vaccines to enter adult and paediatric
clinical trials. In PACTG 230, infants were randomised
to receive one of two recombinant HIV protein vaccines
(Chiron rgp120 with MF59 adjuvant [Chiron Corporation,
Emeryville, CA, USA] or VaxGen rgp120 with alum
[VaxGen, Brisbane, CA, USA]) at 4, 8, 12, and 20 weeks of
age. Both vaccines were tolerated well. Antibody responses
were detected by ELISA at 24 weeks in 87% of infants
immunised with the Chiron vaccine and were signifi cantly
higher in vaccine recipients than in infants who received
placebo.62 Furthermore, similarly high concentrations of
HIV-envelope-specifi c antibodies were detected by
12 weeks of age in 63% of infants who received an
accelerated vaccination schedule (consisting of vaccine
administration at birth and at 2, 8, and 20 weeks),
suggesting that the vaccine elicited antibody responses
despite the presence of high titres of maternal antibodies.
Lymphoproliferative responses to HIV proteins were
detected more frequently in vaccine recipients (56%)
than in controls (14%).63 HIV-specifi c lymphoproliferative
responses were detected by 4 weeks of age in all of
11 infants who received an accelerated vaccine schedule.
The results of these phase I trials of recombinant protein
vaccines indicate that young infants can generate
antibody and lymphoproliferative
vaccination even in the presence of high titres of maternal
HIV envelope-specifi c antibodies and when immunised
with an accelerated vaccine schedule that would be
necessary to protect against MTCT. Neutralising-antibody
data have not been reported from these neonatal studies.
However, data from adult trials, showing that antibodies
induced by subunit vaccines do not neutralise primary
HIV isolates, reduced overall enthusiasm for these
vaccines and further trials of subunit vaccines alone have
not been done in infants. Indeed, the results of two
trials64,65 in adults of subunit vaccines indicate no
protective eff ect.
Replication-defective recombinant viral vaccines
The induction of neutralising antibodies is associated
with vaccine-induced protection against many viral
infections.66 Several lines of evidence suggest that virus-
specifi c CD8-positive T cells, including those specifi c for
HIV,55,67–69 are important in the control of viral replication.
The detection of HIV-specifi c CD8-positive T cells in
exposed, uninfected individuals, including infants,70–72 has
been cited as additional rationale for the development of
vaccines to elicit these; however, these responses have not
been consistently detected in cohorts exposed to HIV.54,73
Antigen-specifi c CD8-positive T cells are most effi ciently
induced after intracellular antigen expression, which
leads to antigen processing and presentation through the
MHC class I pathway. Replication-defective recombinant
viruses have thus emerged as highly versatile vaccine
delivery systems. However, limited replication of some of
these vectors—eg, canarypox—or the development of
antivector immunity—eg, adenovirus—have limited the
immunogenicity of these vectors when administered
alone. Immunisation strategies that combine priming
with a DNA or recombinant viral vaccine followed by
boosting with a heterologous vaccine seem to be
particularly eff ective in generating responses to CD4-
positive or CD8-positive T cells. Of the replication-
defective recombinant viral vaccines, only canarypox
www.thelancet.com Vol 368 August 5, 2006 515
vaccines have been assessed in human infants, but
modifi ed vaccinia Ankara (MVA), adenovirus, and adeno-
associated virus vaccines are under consideration.
Avian poxvirus-vectored vaccines have also been assessed
in infants. PACTG 326 was a phase I trial74 of two diff erent
canarypox vaccines (ALVAC vCP205 and vCP1452; sanofi
pasteur, Marcy-l’Etoile, France) administered to infants of
HIV-infected women at birth and at ages 4, 8, and
12 weeks. The vaccine regimens were well tolerated.
vCP205 administered alone did not result in measurable
serum antibody concentrations; HIV-specifi c lympho-
proliferative responses (stimulation index ≥3) were
detected at two or more timepoints in 44–56% of vaccine
recipients, but were not detected in any placebo recipients.
HIV-specifi c CD8-positive T-cell responses were detected
by cytolytic assays after in-vitro stimulation of peripheral-
blood mononuclear cells on at least one occasion in 44–62%
of vaccinees. However, detectable responses were of low
magnitude (median HIV-specifi c lysis at an eff ector-to-
target ratio of 25-to-1 was 13–23%) and were repeatedly
detected in only two (11%) of the 18 vaccine recipients. In
the second phase of this study, vCP1452 was administered
with a recombinant gp120 boost (AIDSVAX B/B, VaxGen),
with the goal of increasing HIV-specifi c antibody responses
to vaccination. HIV gp120-specifi c antibodies were detected
at 24 weeks after the last immunisation in all infants who
received the boosted regimen.75 HIV env-specifi c lympho-
proliferative responses (defi ned as stimulation index >5
detected at two or more timepoints) were detected in 75%
of the group who received ALVAC plus protein boost, but
were not detected in any of the infants who received
ALVAC alone or placebo.76 HIV-specifi c CD8-positive
T-cell responses were detected only rarely, even after in-
vitro stimulation. Altogether, the modest immunogenicity
of the canarypox vaccines in these studies limited
enthusiasm for additional studies in infants. In 2005,
however, ALVAC-based vaccines were reported58 to confer
partial protection against repeated oral challenge of infant
macaques with virulent SIVmac251, and infected monkeys
had lower viral loads than uninfected monkeys. A limited
phase I trial (HPTN 027) of another canarypox vaccine
(ALVAC vCP1521) administered with a recombinant gp120
protein boost is planned in Uganda.
Modifi ed vaccinia Ankara
MVA, a live, attenuated form of vaccinia, is used as a
human vaccine vector because of its restricted replication
in human cells, immunogenicity, lack of virulence in
animal models (including neonatal or T-cell-depleted
animals), and proven safety record in more than
120 000 adults and children as a smallpox vaccine.77
Recombinant MVA vaccines confer protection against
measles in infant macaque models.78 DNA-MVA-based
vaccine prime-boost regimens are highly immunogenic
and can result in a greater than 80% reduction in liver
parasite burden in people challenged with heterologous
Plasmodium falciparum.79 However, MVA-fowlpox-based
vaccine prime-boost combinations seem to be even more
immunogenic and have protected malaria-naive adults
against challenge with the liver stage of P falciparum.80,81
Administration of an MVA vaccine expressing a highly
conserved mycobacterial antigen (antigen 85A) after
previous (0·5–38 years; median 18 years) vaccination with
BCG resulted in potent boosting of antimycobacterial
T-cell responses.82 An MVA-SIV vaccine has provided
limited protection against repeated oral challenge with
SIVmac251 in neonatal macaques.58 However, DNA-MVA
prime-boost regimens did not result in higher frequencies
of memory antigen-specifi c CD8-positive T cells than DNA
vaccination alone in macaques.83 MVA-SIV constructs,
administered alone or in combination with DNA-HIV
vaccines, protected against challenge with an SIV-HIV
chimera (SHIV89.6P84,85). Although the results of a phase I
trial on one DNA-MVA HIV vaccine regimen in HIV-
uninfected individuals indicated
genicity, boosted CD8-positive T-cell responses were
detected in HIV-infected individuals on therapy.86–88
These data suggest that the immunogenicity and effi cacy
of MVA varies with the vaccine product, the vaccines used
in combination with MVA, and the sequence of vaccines
Several MVA-HIV products are in phase I trials in HIV-
uninfected adults, including products that express HIV
gene products of clade B (Therion Biologics, Cambridge,
MA, USA; trial sponsored by the HIV Vaccine Trials
Network), or clade C (Therion, Aaron Diamond AIDS
Research Center, New York, NY, USA; trials sponsored by
International AIDS Vaccine Initiative). A phase I trial of
multivalent MVA-HIV vaccines administered with
fowlpox-HIV boosts (Therion) in people aged 18–24 years,
infected with HIV, and on suppressive antiretroviral
therapy, has began to enrol patients through the Pediatric
AIDS Clinical Trials Group. Preliminary safety data from
these adult studies should be available by the end of 2006
and should facilitate planning for paediatric trials.
Another group of replication-defective recombinant viral
vaccines being studied are the adenovirus-based vaccines.
These have elicited potent cellular immune responses
when administered alone; a single dose of an adenovirus-
based vaccine expressing Ebola virus glycoprotein was
protective against viral challenge in a cynomolgus
macaque model.89 However, rapid development of
antivector immunity has been documented after even a
single dose in preclinical primate studies and has resulted
in their use in prime-boost regimens. DNA-adenovirus
prime-boost regimens seem to be especially promising
and are being assessed in adults.90,91 Adenovirus type 5
pre-existing antibodies could compromise the use of this
vector in developing countries, where natural infection
with adenovirus type 5 is highly prevalent. Since passively
www.thelancet.com Vol 368 August 5, 2006
transferred maternal antibodies could potentially
interfere with the usefulness of adenovirus type 5 as a
neonatal vaccine, seroprevalence studies are being done
in pregnant women and young infants. To overcome
interference with pre-existing antibodies to the vector,
the potential use of other adenovirus strains (adenovirus
35, 11, and chimpanzee) that less commonly cause
natural infection is also under investigation. An
association between acute primary adenovirus infection,
lymphoid hyperplasia, and intestinal intussusception has
been reported in children.92
Recombinant adeno-associated virus
undergone extensive assessment for use in gene-transfer
protocols and is under investigation as a vaccine vector.
The administration of rAAV-SIV vaccines to macaques
resulted in strong humoral and cell-mediated SIV-specifi c
immune responses and suppression of viral replication.93
An rAAV-2 serotype HIV subtype C vaccine is in phase I
adult clinical trials; serum antibodies to the AAV-2 serotype
were detected in 30% of European, Brazilian, or Japanese
children younger than age 10 years and in more than 60%
of adolescents or adults, but antibodies to other serotypes
were less commonly detected.94 Additional seroprevalence
surveys of AAV serotype 1 and 2 are underway in several
developing countries. The eff ects of passively acquired
antibodies on the immunogenicity of rAAV vaccines are
unknown, but would be a potentially important
consideration for their use in neonatal vaccine trials.
virus (rAAV) has
In 1990, Felgner and colleagues95 showed that naked
plasmid DNA injected intramuscularly could result in
gene expression with subsequent antigen processing and
presentation through HLA class I and class II pathways to
produce protective CD4-positive and CD8-positive immune
responses to the encoded protein. Since then, DNA
vaccines have been developed against various pathogens,
including HIV.96 Although DNA vaccines are immuno-
genic in mice and monkeys (including neonates), current
vaccines are poorly immunogenic when administered
alone to people. Strategies to improve the immunogenicity
of DNA vaccines have included optimising gene expression
through potent promoters, increasing plasmid doses,
formulating the vaccines to limit plasmid degradation and
to increase transfection effi ciency, co-administration of
immunomodulators, and manipulations to enhance cell-
surface expression or secretion of encoded proteins.
Heterologous prime-boost strategies have shown particular
promise in augmenting the immunogenicity of DNA
vaccines. DNA-vector prime-boost regimens have been
very eff ective in generating antibody responses in
macaques97 and both humoral and cell-mediated responses
in people.98 As outlined above, DNA-MVA prime-boost
regimens have been assessed in macaques and in people
as potential malaria and HIV vaccine strategies. DNA-
adenovirus prime-boosting strategies seem to be especially
immunogenic in macaques.91 Partial control of viral
replication after challenge with SIVmac239 was noted in
macaques immunised with another DNA-adenovirus
prime-boosting regimen;99 control of viral replication in
this study was associated with SIV-specifi c CD8-cell
responses and the eventual loss of control with CD8-cell
escape. Clinical trials of DNA-adenovirus prime-boost
regimens are underway in adults.
Basic science questions
The Global HIV/AIDS Vaccine Enterprise2 has outlined
several scientifi c priorities. These include: improved
understanding of the virological and immunological
properties of infecting viral strains; defi nition of immune
correlates of protection against infection or natural
disease; and improved understanding of how best to
design vaccines to elicit strong neutralising antibody and
responses mediated by CD4-positive or CD8-positive T
cells. Each of these scientifi c priorities and those listed in
the panel are highly relevant to the development of
neonatal HIV vaccines. These include continued defi nition
of key features of the pathogenesis of transmission of HIV
via breastmilk (timing, mechanisms) and elucidation of
whether infants can generate eff ective immune responses.
Virological and immunological characterisation of
infecting strains will be important, using samples from
infants in areas with early and mature HIV epidemics.
Study of cohorts in diff erent geographical regions would
also allow the assessment of potential pathogenic
diff erences between viral clades or recombinants and of
host genetic factors—eg, HLA alleles; toll-like receptor or
chemokine receptor polymorphisms; polymorphisms in
cellular factors important for HIV replication—bearing in
mind that antiretroviral treatment is now the universal
standard of care for children infected with HIV, as well as
for adults. MTCT provides a unique situation in which
donor–recipient pairs and the timing of transmission can
be easily ascertained. The genetic characterisation of
viruses from mother–infant pairs could be particularly
informative in the understanding of: whether selective
viral variants are transmitted; the diversity of the early
quasispecies; how vaccine sequences compare with
circulating strains; and the extent to which transmitted
Panel: Key questions pertinent to the development of neonatal HIV/AIDS vaccines
G What are the mechanisms of transmission of HIV via breastmilk?
G To what extent does cell-free versus cell-associated virus contribute to transmission?
G What are the characteristics of transmitted viruses?
G What are the correlates of immune protection?
G Can infants generate eff ective immune responses and how can we design
immunogens to elicit them?
G How does HIV sequence variability aff ect effi cacy of HIV-specifi c immune responses?
G How can we elicit responses that persist and protect over a lifetime?
www.thelancet.com Vol 368 August 5, 2006 517
variants change within individuals or populations over
time. Characterisation of changes in viral sequences in
individuals and populations, coupled with immune-
response variables, could provide important data about
the sensitivity of transmitted viruses to antibody
neutralisation or selective pressures of CD8 cells. The
assessment of genetic sequences over time for mutations
associated with CD8-positive T-cell escape and correlation
with plasma viral load would also provide important
evidence about the in-vivo activity of HIV-specifi c CD8-cell
responses in infants.
Adaptive immune responses (particularly neutralising
antibodies) are probably the main correlates of immune
protection for most viral infections and available
vaccines.66 Innate responses might also contribute to early
resistance or control of infection. Fortunately, new assays
and techniques have been developed over the past decade
that allow precise measurement and characterisation of
innate and HIV-specifi c adaptive immune responses.
Many of these assays can accommodate the small volumes
of blood available from infants and will be helpful in
characterising HIV-specifi c immune responses after
natural infection or vaccination.50,54
Vaccine trial design
Over the next few years, we expect that phase I/II trials of
two or three prime-boost strategies will have been
completed in adults, paving the way for their use in
paediatric studies. The results of phase I trials will be
important in establishing the safety and immunogenicity
of these products in breastfed infants of HIV-infected
women. Since a substantial proportion of transmission
of HIV via breastmilk occurs within the fi rst 6 months,
phase I/II trials should be designed with the aim of
generating protection within 2–3 months of birth. The
administration of preventive HIV vaccines, at the same
time that routine childhood immunisations are given in
infancy, would enhance the feasibility of vaccine delivery;
once phase I/II studies have proven the vaccines to be
safe and eff ective in infants, studies to exclude adverse
interactions between HIV and other childhood vaccines
will be necessary. Effi cacy trials could proceed once
favourable safety and immunogenicity data from these
phase I/II trials are available.
The wide-scale implementation of highly active
antiretroviral therapy is expected not only to provide
virological and clinical benefi ts to individuals on therapy,
but also to reduce their risk of HIV transmission. Plasma
HIV RNA concentrations of less than 1700 copies per mL
or 1000 copies per mL have been associated with reduced
risk of sexual transmission and MTCT, respectively.100 The
introduction of perinatal antiretroviral regimens into
breastfeeding populations has reduced overall MTCT rates
from 25–40% to 8–20%.101 Several postnatal antiretroviral
regimens are being assessed for effi cacy in preventing
breastmilk HIV transmission, but even a reduction in
overall transmission rates to 5% would not preclude a
vaccine effi cacy trial in breastfed infants. On the
assumption that a vaccine would not be associated with an
increased risk of acquisition of infection, the estimated
sample size for a comparative study between prophylactic
antiretroviral and vaccine versus antiretroviral alone in
breastfed infants would require about 3500 HIV-exposed
infants to document a reduction in MTCT through
breastfeeding assessed at 6 months from 5% in infants
receiving antiretroviral therapy alone to 3% in the
antiretroviral plus vaccine group. However, to address the
possibility that a vaccine regimen might result in increased
transmission, a sample size of 4200 infants would be
necessary. Even the larger sample size is feasible through
multicentre collaborative trials.
Ethical and regulatory issues
The increasing availability of agents to prevent or treat
important paediatric clinical syndromes, as well as the
increasing appreciation that growth and maturation could
aff ect the pharmacokinetics, response to, or toxic eff ects
of drugs and biological agents, has led to the prevailing
view that paediatric clinical trials are necessary to ensure
equal access to novel agents and to continue to advance
clinical care of children. In the USA, the Institute of
Medicine recently convened a panel of experts to review
the ethical conduct of clinical research in children.102 The
US Congress, Food and Drug Administration (FDA), and
the National Institutes of Health (NIH) have collaborated
lately to increase paediatric research, and recent federal
statutes require manufacturers of drugs and biological
agents to undertake paediatric studies to ensure that new
products are appropriately labelled for children. Eff orts
are underway worldwide to increase paediatric research
eff orts and to defi ne guidelines for the inclusion of
children in research.
Children are vulnerable, both biologically and socially.
As such, any research study in children must incorporate
safeguards.103 US federal regulations and international
guidelines generally require that risks to participants be
reduced to a minimum and be reasonable in relation to
the expected benefi ts. Robust systems to protect research
participants provide a good foundation for protecting
participants in paediatric research. However, additional
resources should be committed to the development of
ethical and legal standards that specifi cally protect
children. The development of these standards and
paediatric research protocols depends on the participation
of a diverse group of individuals with expertise in the
scientifi c, psychosocial, and ethical aspects of paediatric
clinical care and research.
For any new drug or biological agent, preliminary safety
studies in adults are generally required before the initiation
of paediatric studies. Since some infants might already be
infected before vaccination—ie, those with in-utero
infection—early adult studies of vector-based vaccines
should include their assessment in immunocompromised
animals and HIV-infected (as well as uninfected) adults.
For the US Congress, FDA, and
NIH research and statutes see
www.thelancet.com Vol 368 August 5, 2006
The precise requirements for preclinical (in particular,
reproductive toxicology studies) and adult testing can vary
according to the product, but paediatric trials should not
be delayed until an agent has been fully assessed in adults.
Antiretroviral agents have successfully entered clinical
trials soon after preliminary adult studies opened, and
paediatric vaccine trials should ideally follow this model.
An essential point in the undertaking of paediatric HIV
vaccine trials is that all other preventive and therapeutic
measures that apply to the study population should be
off ered to trial participants. For example, standard-of-
care antiretroviral treatment should be off ered to
pregnant women who are HIV positive for their own
health and to prevent MTCT. Maternal access to
appropriate antiretrovirals during gestation should be
continued postpartum; standard-of-care drugs should
also be provided to infants diagnosed with HIV who
participate in vaccine trials. This approach might increase
the number of required participants, but vaccine studies
will remain eminently feasible.
Informed consent is the cornerstone of good research
practice. A successful neonatal vaccine regimen to prevent
transmission of HIV via breastmilk will probably depend
on the initiation of immunisations at birth and an
accelerated vaccine schedule over the fi rst 2–3 months of
life. Informed consent to begin treatment could be diffi cult
to obtain during labour or delivery. Completion of the
informed consent process before delivery has greatly
facilitated the enrolment of newborn children into previous
trials of antiretroviral treatment and HIV vaccines.
Liability concerns are often raised as a possible obstacle
to HIV vaccine trials in children. In the USA, for licensed
and recommended childhood vaccines, the Vaccine Injury
Compensation Act provides liability protection for vaccine
manufacturers and compensation for children who might
have been harmed by vaccination. There is no comparable
system for investigational vaccines, and most large
pharmaceutical companies self-insure, an approach that
could prove diffi cult for small biotech companies and
non-governmental organisations involved in the search
for an HIV vaccine. Governmental indemnifi cation
insurance might provide a means to remove this potential
The Global Vaccine Enterprise Scientifi c Strategic Plan
emphasises the need to establish a global system of
laboratories—vaccine immune monitoring centres—to
collaborate in characterising HIV-specifi c immune
responses in the context of natural infection or vaccine
trials. Priority activities for these laboratories include: the
development of a wide range of standardised assays to
allow comparison across and between natural history,
preclinical, and clinical studies; the development and
sharing of necessary reagents; and continuous quality
assurance. A set of so-called core or reference laboratories
will be charged with assay development, standardisation,
training, and quality assurance along with the undertaking
of specialised assays (particularly those requiring
specialised equipment). Satellite laboratories at clinical
study sites will process blood specimens, do basic
virological or immunological assessments, and ship
specimens for more specialised assessments to core
The development of a parallel but integrated
consortium of laboratories focused on studying the
pathogenesis of breastmilk HIV transmission and the
characterisation of infant immune responses to natural
infection or vaccines would greatly facilitate the
development of neonatal vaccines. Paediatric scientists
should be integrated into the laboratory decision-making
process of the Global Vaccine Enterprise. Wherever
possible, protocols, laboratory space, personnel, reagents,
and quality-assurance mechanisms should be shared.
However, paediatric investigators might need to develop
reagents uniquely suited for studies of neonates—eg,
panels of infecting viral strains. Furthermore, early
diagnosis of HIV infection in infants is crucial within
natural history and vaccine studies, but few assays are
available that can reliably diagnose young infants in
limited-resource settings. The development and
standardisation of diagnostic assays for use in fi eld
settings is important. Finally, paediatric investigators
will probably need to adapt or prioritise assays owing to
the limited blood volumes available from infants and
confi rm the usefulness of these assays. For example,
ELISPOT assays have been used in many paediatric
studies to detect antigen-specifi c T cells because of the
very small blood volumes needed for the assays. Available
data66 suggest that ELISPOT assays that measure
interferon γ secretion give a general indication of the
immunogenicity of a vaccine, but that the results do not
necessarily indicate protective
development and validation of assays that allow more
thorough assessment of responder-cell phenotype—eg,
expressing CD4 or CD8—and function than presently
possible are needed. Although multiparameter fl ow-
cytometry-based assays seem promising, the large blood
volumes and specialised equipment needed could hinder
their application to large-scale paediatric studies.
Emerging technologies—eg, chip-based MHC-peptide
arrays—could be especially useful for such high-
throughput paediatric studies.
The development of a safe and eff ective paediatric
preventive HIV vaccine would be an extremely important
advance and would have a major eff ect on control of the
HIV/AIDS pandemic. Unique features of MTCT and
paediatric HIV disease could be especially helpful in
elucidating correlates of immune protection and could
facilitate rapid assessment of vaccine effi cacy. Vaccine
responses primed during infancy could provide the basis
for lifetime immunity to HIV. We therefore propose that
For WHO guidelines on care,
treatment, and support for
women living with HIV/AIDS
and their children in resource-
constrained settings see
www.thelancet.com Vol 368 August 5, 2006 519
international vaccine initiatives, including the Global
Vaccine Enterprise, should include the development of
preventive paediatric HIV vaccines as a priority. Paediatric
scientists should be integrated into the leadership and
activities of the Global Vaccine Enterprise. Adult and
paediatric investigators should collaborate closely on
preclinical and clinical vaccine development, particularly
to ensure the timely order of studies in adults and
children. The development of a consortium of laboratories
focused on studying the pathogenesis of transmission of
HIV via breastmilk and the characterisation of infants’
immune responses to natural infection or vaccines would
greatly facilitate the development of neonatal vaccines.
This consortium of paediatric laboratories should be
integrated into the network of vaccine immune
monitoring centres; sharing of laboratory space,
personnel, equipment, protocols, reagents, and quality-
assurance programmes could provide economy of scale.
Confl ict of interest statement
We declare that we have no confl ict of interest.
We thank Patricia Fast, Executive Director, Medical Aff airs, International
AIDS Vaccine Initiative, for very helpful discussions. K Luzuriaga was
supported by funding from the US National Institutes of Health (grants
NIH/NIAID R01 AI032391 and NIH/NICHD K24 HD01489, which was
not involved in study design, data interpretation, writing of the Review,
or in the decision to submit this paper for publication. The Ghent
Working Group on HIV in Women and Children (KL, ML-N, FD, JLS are
members) also provided funding.
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