Vaccine xxx (2006) xxx–xxx
A review of vaccine research and development: The human
immunodeficiency virus (HIV)?
Marc P. Girarda,∗, Saladin K. Osmanovb,1, Marie Paule Kienyb,1
aUniversity Paris 7, 39 rue Seignemartin, FR 69008 Lyon, France
bInitiative for Vaccine Research, World Health Organization, 20 Avenue Appia, CH-1211Geneva 27, Switzerland
Received 19 November 2005; received in revised form 10 February 2006; accepted 13 February 2006
Since the discovery of AIDS in 1981, the global spread of HIV has reached pandemic proportions, representing a global developmental and
public health threat. The development of a safe, globally effective and affordable HIV vaccine offers the best hope for the future control of
the pandemic. Significant progress has been made over the past years in the areas of basic virology, immunology, pathogenesis of HIV/AIDS
and the development of antiretroviral drugs. However, the development of an HIV vaccine faces formidable scientific challenges related to
the high genetic variability of the virus, the lack of immune correlates of protection, limitations with the existing animal models and logistical
problems associated with the conduct of multiple clinical trials. More than 35 vaccine candidates have been tested in Phase I/II clinical
trials, involving more than 10,000 volunteers, and two Phase III trials have been completed, themselves involving more than 7500 volunteers.
Multiple vaccine concepts and vaccination strategies have been tested, including DNA vaccines, subunit vaccines, live vectored recombinant
vaccines and various prime-boost vaccine combinations. This article reviews the state of the art in HIV vaccine development, summarizes the
results obtained so far and discusses the challenges to be met in the development of the various vaccine candidates.
© 2006 World Health Organization. Published by Elsevier Ltd. All rights reserved.
Keywords: Acquired immunodeficiency syndrome; AIDS; Human immunodeficiency virus; HIV-1; Simian immunodeficiency virus; SIV; Vaccines
Disease burden ......................................................................................................
HIV vaccine reseach: challenges and difficulties ........................................................................
4.1. Why is it so difficult to develop an HIV vaccine? .................................................................
5.1. Live attenuated vaccines........................................................................................
5.2. Inactivated vaccines............................................................................................
5.3. Virus-like particles (VLP) ......................................................................................
5.4.Subunit vaccines ..............................................................................................
?The authors alone are responsible for the views expressed in this publication, which does not necessarily reflect the views of the World Health
∗Corresponding author. Tel.: +33 478 748 531.
E-mailaddresses:firstname.lastname@example.org (M.P. Girard), email@example.com (S.K. Osmanov), firstname.lastname@example.org (M.P. Kieny).
1Tel.: +41 22 791 35 91.
0264-410X/$ – see front matter © 2006 World Health Organization. Published by Elsevier Ltd. All rights reserved.
JVAC-6044; No. of Pages 20
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
Naked DNA and live recombinant vaccines.......................................................................
The development of prime-boost combinations ...................................................................
Fusion proteins and peptides....................................................................................
Envelope-based subunit vaccines........................................................................
Non-structural protein subunit vaccines ..................................................................
The Acquired Immunodeficiency Syndrome (AIDS)
emerged in the human population in the summer of 1981.
There now is convincing evidence that AIDS is a zoonosis
and that its etiological agent, the human immunodeficiency
the middle of the 19th century [1,2] and probably arose and
spread among humans as a result of increased unsafe injec-
the virus to adapt to humans through serial passages. Today,
HIV/AIDS is the leading cause of death in sub-Saharan
Africa and the fourth biggest killer in the world. An esti-
mated 14,000 people/day (5 million persons/year, including
600,000 children less than 15 years of age) become infected
with HIV, with more than 95% of them living in underdevel-
oped regions of the world . The number of HIV infections
is equally distributed between men and women, but infection
rates in young women in today’s Africa are close to three
times higher than those among young men, reflecting the
degree to which gender inequities are driving the epidemic,
independence, education and access to health information
and services, and have difficulty avoiding exposure to the
The development of a safe, effective, easy to administer
and affordable AIDS vaccine is urgently needed. The first
Phase I trial of an HIV vaccine was conducted in the USA
in 1987. Since then, more than 35 candidate vaccines have
been tested in over 65 Phase I/II clinical trials, involving
more than 10,000 healthy human volunteers in more than 10
countries [4,270]. Two Phase III trials have been carried to
completion  and a third one is in progress . However,
we still are years away from an effective HIV vaccine, due
to multiple hurdles and challenges. The development of a
safe and effective vaccine is hampered by the high genetic
correlates of protection [9,10], the difficulty of generating
broadly neutralizing antibodies , the absence of relevant
and predictive animal models and the complexities related to
the preparation and conduct of multiple large-scale clinical
trials, especially in developing countries [12,13].
This article reviews the current progress in HIV/AIDS
vaccine research and development and the many challenges
that still need to be met in the field [14–17].
2. Disease burden
At the end of 2005, the global number of adults
and children living with HIV/AIDS was estimated by
WHO/UNAIDS to have reached 40.3 million with an esti-
from the disease .
Sub-Saharan Africa remains the hardest-hit region in the
world, with at least 25 million infected people, accounting
for 60% of the people living with HIV/AIDS and 77% of
AIDS deaths in the world. The overwhelming majority of
HIV infections in the region stems from heterosexual trans-
mission . In many African countries, the overall HIV
prevalence in the adult population can be greater than 10%,
with figures reaching up to 38.8% in some areas. Among
the most severely hit countries are South Africa, with more
than 5.6 million infected people and a prevalence of 30%
among 21–29-year-old adults, especially women, together
with Botswana, Mozambique, Zimbabwe, and Tanzania.
The highest infection rates are found among commercial
sex workers, truck drivers and seasonal migrant workers.
Sub-Saharan Africa also is home to an estimated 500,000
HIV-infected infants who became infected prior to the intro-
duction of mother-to-child prevention programmes based
on the use of antiretroviral drugs. In addition, sub-Saharan
Africa faces numerous wars and civil conflicts, producing
large numbers of refugees who are at heightened risk of con-
Botswana, Swaziland or Zimbabwe, where a quarter to more
than a third of the general population is infected with HIV-1,
has decreased from 65 years in 1985–1990 to 37 years in
A remarkable success story in the fight against AIDS was
achieved in Uganda, which faced the onset of a severe HIV
epidemic in the mid 1980s. Through voluntary HIV coun-
selling, expanded treatment of STDs, awareness campaigns
and community mobilization encouraging delayed initiation
infection has declined significantly since 1992—from nearly
30 to 11.2% in prenatal clinic settings in Kampala and from
13 to 5.9% in clinics outside major urban areas. Another
important HIV prevention strategy could be adult male cir-
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
Uganda to obtain more data on the potential protective effect
of circumcision on female-to-male transmission.
The estimated number of people living today with HIV
in Asia and the Pacific region is more than 8.3 million, but
the accuracy of the figure is questionable in view of the fast
of all new infections worldwide by the end of the decade.
Increasing sex trade, use of illicit drugs, and high rates of
nerability of the populations in the region. Gender inequities
also play an important role in the epidemic, as young girls
are frequently steered towards commercial sex work by their
families. Substantial transmission also occurs among men
who have sex with men, with prevalence rates of 14–20%
reported in certain male homosexual communities.
The estimated number of adults and children living with
HIV in Latin America and the Caribbean at the end of 2005
was 2.1 million. While in some countries HIV infections
remain concentrated mainly among men who have sex with
men and injecting drug users, other countries are experienc-
ing increasing rates of heterosexual transmission.
The Eastern European countries continue to experience
tions, most of which occur among injecting drug users. The
number of people living with HIV/AIDS in the region is esti-
are living in Russia alone, where commercial sex workers,
injection drug users and prisoners are major victims of the
tuberculosis and HIV epidemics .
ment (HAART) in industrialized countries has considerably
reduced disease progression to AIDS and transformed HIV
infection from a lethal disease to an effectively manageable
chronic disease. AIDS among infants, which a decade ago
took the lives of thousands of babies per year may be on
the verge of elimination. Thus, in New York city, the num-
ber of babies born with HIV fell from 321 in 1990 to 5
in 2003, mostly due to education, counselling, testing and
antiviral treatment of the mother during pregnancy. How-
ever, successes achieved in treatment and care are not being
matched by progress in prevention, as some 75,000 individu-
als become infected with HIV every year in industrialized
countries, where an estimated 1.6 million people are liv-
ing with HIV/AIDS, and where a new wave of rising HIV
infection rates is emerging, particularly in the young and
The HIV, together with the simian, the feline, and the
tively), the Visna virus of sheep, the caprine arthritis-
encephalitis virus (CAEV) and the equine infectious anemia
virus (EIAV), belongs to the genus Lentivirus in the family
Retroviridae. These enveloped RNA viruses produce char-
responsible for the transformation of the viral RNA genome
into a proviral DNA copy that integrates into the host cell
chromosome. The provirus is eventually transcribed into a
set of mRNAs that encode the viral proteins and progeny
to SIVcpz, a commensal virus in chimpanzees, and probably
arose as the result of a single transmission event from chim-
panzees to humans , whereas HIV-2 is closely related to
a commensal virus in sooty mangabey monkeys. HIV-1 is
further subdivided into three groups, M (“major”), O (“out-
strains responsible for the global AIDS pandemic belong to
group M, which has evolved in humans to form at least 10
genetic subtypes, also known as clades, designated by let-
ters from A to K . HIV-1 subtype B predominates in
industrialized countries as well as in Latin America and the
Caribbean. Subtypes A and D are more common in Central
Africa. Subtype C accounts for the majority of infections in
southern Africa, eastern Africa and India. Genetic subtypes,
in turn, have diversified further. Even within a given subtype,
antibodies that are specific for isolates from one patient typ-
ically do not recognize isolates from other patients. Over the
course of the epidemic and as a result of frequent dual and
superinfections, HIV strains can recombine and form mosaic
viruses, some of which, called “Circulating Recombinant
CRF01 AE, which predominates in south-eastern Asia, and
CRF07 BCand08 BC,whichareprevalentinChina.Amino
acid sequence of the viral envelope glycoprotein shows up
to 25–35% divergence between different subtypes and up to
20% divergence within any given subtype, which constitutes
a formidable challenge to vaccine development.
The genome of HIV is a single-stranded positive sense
ical retrovirus proteins Gag, further cleaved into M (matrix),
C (capsid) and N (nucleocapsid), Pol, cleaved into protease,
reverse transcriptase and integrase, and Env, a 160kD glyco-
a transmembrane gp41 subunit that together form trimeric
spikes on the surface of the virion. In addition, the HIV
genome encodes a variety of non-structural proteins, such as
the transactivator protein Tat , the splice regulator pro-
tein Rev  and accessory proteins such as Nef [26,27], Vif
, Vpr  and Vpu.
The mature virion contains a cone-shaped protein cap-
sid, which encapsidates two strands of the genomic RNA,
the replication enzymes, tRNAs and cellular proteins such as
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
cyclophilin A and Tsg101. The capsid is enclosed within a
lipid envelope, which is studded with the viral glycoprotein
spikes and cellular proteins, including MHC class II anti-
gens and ICAM-1 , and which lies on top of a layer of
embedded myristyl groups. However, the actual structure of
the virion still remains speculative .
The envelope glycoprotein comprises two subunits, the
external gp120 subunit which binds to the CD4 receptor and
CCR-5 or CXCR-4 coreceptors on the surface of target cells
trimeric organization, and plays a major role in fusion of the
virus and host cell membranes through its hydrophobic N-
terminal fusion peptide and a fusion active hairpin structure
involving two heptad repeats that can fold into a six-helix
coiled-coil bundle [33–36]. Neutralizing human monoclonal
tification of several neutralization epitopes on gp120 ,
some of which are carbohydrate-specific [38,39] and oth-
ers overlap the CD4 receptor- or coreceptor-binding sites
[40–43], but all of them appear to be little accessible to the
cognate antibodies due to hindrance by the many glycosy-
lation motifs on the molecule  as well as by the hyper-
variable loops [45,46], which act as antigenic decoys .
have been described, with corresponding epitopes located at
the base of the gp41 ectodomain [48–53]. A note of caution
these monoclonal antibodies were reactive with the human
phospholipid cardiolipin [54,55].
4. HIV vaccine reseach: challenges and difficulties
4.1. Why is it so difficult to develop an HIV vaccine?
Natural infection with HIV does not result in virus clear-
ance by the host immune system and the development of
natural immunity to re-infection. In spite of intense and
sustained immune responses by both the humoral and cell-
mediated defences, HIV is able to resist eradication and
continues depleting CD4+ T cells, which eventually leads
to clinical progression to AIDS. There even is evidence that
superinfection with a second HIV isolate can readily occur
in HIV-infected persons, leading to the emergence of recom-
binant virus variants and generating increased virus diversity
between different SIV strains in SIV-infected monkeys .
HIV-1 integrates as a latent proviral DNA into the genome
of long-lived memory CD4+ T cells, which provide a persis-
tent reservoir of the virus that escapes immune surveillance
[60–62]. It has been calculated that it would take up to 60
years to eradicate a reservoir of as few as 1× 10E5 latently
infected cells. The window of opportunity for an HIV vac-
cine is therefore narrowly limited to the very early stages of
infection, before the virus can seed the lymphoid organs in
mucosal tissues [63,64].
HIV also has developed multiple mechanisms to circum-
regulate the major histocompatibility complex (MHC) class
I molecules and by doing so to minimize its recognition by
it to evade immune responses through the emergence of viral
Another difficulty with the development of an effective
HIV vaccine stems from the fact that the virus envelope
binding sites in crypts that are masked by the hypervariable
loops of the molecule and by glycan residues [45,70,71].
makes it hard to generate broadly cross-reactive neutraliz-
ing antibodies against primary virus isolates from patients
[11,15,72–75]. The complete lack of efficacy of antibody
responses raised by monomeric gp120 vaccines in protection
against HIV infection has been proven beyond any doubt in
Although neutralizing antibodies administered passively
rapid and constant selection of neutralizing antibody-escape
trary to laboratory-adapted virus strains, which use CXCR-4
ing antibodies targeted to the hypervariable V3 loop [87,88],
primary virus isolates, which use CCR-5 as a coreceptor
(“R5” strains), are difficult to neutralize, which casts doubt
on the possibility for a vaccine to elicit protection against
infection by the induction of neutralizing antibodies alone.
In view of all these problems, recent vaccine approaches
have focused on the induction of cellular immune responses
[14,15,23,89–91]. Evidence for the role of CD8+ T cells in
the control of virus replication includes temporal correlation
decline of primary viremia [92,93], the fact that several HLA
class I alleles (HLA-B57, HLA B-27, HLA-B63) are associ-
CTL escape viral mutants during primary infection [95–98]
SIV after experimental depletion of CD8+ T cells [99–101].
The induction of a cellular immune response against HIV,
especially a CD8+ CTL response, although not being able to
provide sterilizing immunity and protection from infection,
should hopefully enable vaccinees to control virus replica-
tion following infection, reduce their virus load, slow down
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
their progression towards disease, and reduce the probability
of secondary transmission of the virus.
4.2. Animal models
A significant obstacle in HIV vaccine research has been
the difficulty in developing an appropriate animal model.
The only animals susceptible to experimental infection with
Macaca nemestrina. Both maintain low levels of persis-
tent virus load and do not develop clinical manifestations
of AIDS. African monkeys are the natural hosts to a vari-
ety of SIVs (SIVagm, SIVsm, SIVsyk, SIVcol, etc.), but
do not appear to develop a clinical disease following infec-
tion with these viruses . In contrast, Asian monkeys,
to SIVmac infection and progressively develop an immun-
odeficiency syndrome, which fully mimics human AIDS
. Plasma virus levels during primary and chronic SIV-
mac251 infection in macaques parallel those observed in
progressing to disease slowly, as in HIV-1 infected human
loads and behaving as fast progressors [104,105]. As in
HIV-1-infected humans, the cellular immune responses to
SIVmac during primary and chronic infection differ sig-
nificantly , and evidence of immune escape is readily
immune system, especially the memory CD4+ CCR-5+ T
cells in the gut-associated lymphoid tissue (GALT), is the
major site of viral replication and of CD4+ T-cell depletion
in SIV-infected macaques [107–110] as in HIV-1-infected
individuals [111–114]. The SIV/macaque model is currently,
for studies on potential protective immune responses against
Another animal model used in HIV vaccine research is
based on the use of SIV/HIV hybrid viruses (SHIVs) that
were engineered to carry the env gene from an HIV-1 iso-
late in the context of an SIV genome. These viruses can
replicate in rhesus macaques, and after serial passages in the
SHIV variants that are capable of wiping out the circulating
CD4+ T-cell population of the animal within a few weeks
drome within a year. However, the relevance to HIV of these
pathogenic SHIVs, such as SHIV 89.6P [117–119], is being
questioned [120,121]. They exhibit a “X4” cellular tropism
and, paradoxically, turn out to be much easier to contain by
probably are a more appropriate and predictive model than
“X4” SHIVs, but they have not yet been widely used in vac-
cine protection experiments.
The monkey models suffer from the fact that experi-
ments to evaluate vaccine efficacy require challenging the
vaccinated animals with a huge dose of virus, usually in
the range of 103–105TCID50, equivalent to up to 5×107
SIV RNA copies/ml. Such a high dose of virus is required
to achieve100% infection of control animals in the placebo
group after a single exposure. However, high doses of virus
where concentrations of less than 103to a few 105HIV RNA
copies/ml of seminal plasma have been reported [126–128].
It has recently been observed that repeated low dose (10–30
TCID50) mucosal challenge of monkeys with SIV results
in the same viral and immunological kinetics of infection as
lenge might change the results of preclinical vaccine efficacy
studies in the future (see as an example, ).
Of note is the fact that the challenge virus used in animal
model experiments is nearly always homologous to the vac-
cine, giving good chances of success that are unrealistic in
5.1. Live attenuated vaccines
The observation that nef-deleted mutants of SIV could
confer protection against challenge with pathogenic SIV in
rhesus macaques [130–132] served as a model in favor of
a live attenuated HIV vaccine approach. However, the SIV
infection. It does not protect the vaccinated monkeys against
superinfection with wild-type virus, although it does protect
the animals from progression to AIDS. SIV ?nef still may
cause AIDS, especially when administered orally to infant
monkeys [133,134]. Additional deletions or mutations of the
virus can result in further attenuation but at the expense of
its protective efficacy [135–137]. In view of such safety con-
but efforts are currently being made by IAVI, among others,
to try and explore systematically the nature of the protec-
tive immune responses generated with live attenuated SIV
vaccines in macaques .
5.2. Inactivated vaccines
The difficulty of inactivating HIV-1 with formalin with-
out losing or destroying the antigenicity of the viral envelope
has been a deterrent to the development of whole inacti-
sublethal doses of formalin followed by heat inactivation
at 62◦C . The resulting killed virus preparation was
shown to induce in mice and non-human primates modest
erologous primary isolates of HIV in a variety of infectivity
assays . Another way to inactivate HIV or SIV infectiv-
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
(aldithriol-2)  or by alkylation with N-ethyleneimine
(NEM). Both compounds have been found to completely
inactivate the infectivity of HIV-1 and SIV, while keeping
the envelope glycoprotein spikes intact and functional .
The immunogenicity of the resulting preparations was stud-
ied in the SIV/macaque model : vaccinated monkeys
were not protected against infection with wild-type virus but
experienced decreased viremia after challenge and showed
no significant depletion of CD4+ T cells.
5.3. Virus-like particles (VLP)
When coexpressed in cells, for example using either a
baculovirus or a vaccinia virus expression system, the Gag
and Env proteins of HIV or SIV spontaneously assemble
to form pseudo-virions, i.e. virus-like particles (VLPs) that
only contain envelope and core proteins. SIV VLPs have
been tested as immunogens in non-human primate models
either after priming with vaccinia virus recombinants or by
the nasal route using the cholera toxin B subunit (CTB) as
a mucosal adjuvant [146,147]. The administration of VLP
vaccines via mucosal surfaces might be a promising strategy
to elicit mucosal and systemic anti-HIV immune responses
and found to elicit low but significant titers of neutralizing
antibodies against both homologous and heterologous pri-
mary HIV-1 isolates when administered in the presence of
a block copolymer adjuvant or of alum combined with CpG
cle for HIV genes. Expression of the structural proteins of
Kunjin virus, an arbovirus, was used to prepare VLPs and
HIV-1 gag gene . The resulting VLPs were found to
elicit strong CTL and antibody responses to HIV Gag.
Still another approach has been to induce systemic and
mucosal neutralizing antibodies against HIV by immuniza-
VLPs. The ELDKWA amino acid sequence (2F5 epitope)
capsid protein and the resulting chimeric protein was used to
5.4. Subunit vaccines
5.4.1. Envelope-based subunit vaccines
Initial trials of HIV-1 Env-based subunit vaccines showed
that soluble recombinant envelope glycoproteins gp120 or
gp140 (gp120 prolonged with the ectodomain of gp41) were
well-tolerated and elicited neutralizing antibodies to the
homologous vaccine strain, but not to heterologous primary
virus isolates [76,152,153]. These vaccines elicited steriliz-
ing immunity against homologous virus challenge in animal
models [87,88,154]. Two gp120 subunit vaccines were fur-
ther evaluated in a Phase II trial in the USA , and one
such vaccine, based on monomeric gp120 added with alum
Phase III efficacy trials. The first trial involved 5000 volun-
teers at risk (mostly men who had sex with men) in the USA,
Canada and the Netherlands, who were immunized every 6
months with a 300?g mixture of two subtype B gp120s. The
second trial involved 2500 volunteers in Thailand (mostly
injection drug users), who were immunized with a mixture
of a subtype E (CRF AE) and a subtype B gp120s. None
of these trials showed a statistically significant reduction of
HIV infection in the vaccinees in spite of continuous booster
immunizations during the 36 months of the study. A reduc-
tion of the number of HIV infections was observed in certain
ethnic subgroups in the first trial, correlating with a higher
level of anti-gp120 antibody, but the numbers were too small
to provide statistical confidence .
The subtype E/B gp120 vaccine from the trial in Thai-
land is presently being used for booster immunizations in
a prime-boost Phase III trial, which was launched in late
2003 in Thailand in collaboration between the Ministry of
Health of Thailand, WRAIR, Sanofi-Pasteur and VaxGen.
This community-based trial, which includes 16,000 volun-
teers and is meant to last for 4 years, involves a priming
immunization with a recombinant canarypox virus (ALVAC)
vaccine that expresses CRF AE gp120 and subtype B Gag,
Pol and Nef antigens followed by boosts with the gp120 sub-
unit vaccine .
Other envelope-based subunit vaccine approaches aimed
at eliciting HIV neutralizing antibodies are at an early
clinical stage of evaluation. These include trimeric gp140
molecules stabilized by the addition of heterologous trimer-
ization domains at the C-terminus of the gp41 ectodomain
[156,157], and similar trimers in which the gp120 moiety
was deleted of the variable V2 loop, in order to expose neu-
tralization epitopes overlapping the CD4-binding site. This
approach is developed by Chiron [158–160]. Immunization
of rabbits and macaques with a DNA vaccine encoding a V2-
deleted gp140 from a South African subtype C HIV-1 strain,
followed by boosting with oligomeric V2-deleted gp140 in
MF59 adjuvant elicited high titers of Env-binding antibod-
ies and low level heterologous neutralizing antibodies .
Gp140-GCN4 trimeric immunogens were found to induce
a modest but significant heterologous neutralization activ-
ity, which could be efficiently increased by emulsification in
adjuvants AS01B, AS02A or AS03 from GSK .
Gp140 trimers internally stabilized by an intermolecu-
lar disulfide bond between gp120 and gp41 (SOS gp140),
have been developed with an expectation to induce both
ing with DNA encoding a membrane-bound form of the
SOS gp140 protein followed by repeated immunization with
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
the soluble trimers resulted in high titer antibodies active
against neutralization-sensitive HIV-1 strains, but even the
most potent of the sera had a quite limited ability to cross-
neutralize primary heterologous HIV-1 strains .
Still another approach has been to covalently couple
monomeric gp120 or oligomeric gp140 molecules to solu-
ble CD4 or to synthetic mimetics of the CD4 receptor in
order to induce the conformational changes that are sup-
posed to take place in the glycoprotein spike at the time of
virus entry, resulting in the exposure of neutralization epi-
topes that overlap the coreceptor-binding site. This approach
land , which also engineered single chain derivatives of
CD4 molecule . The single chain fusion proteins were
shown to elicit a broadly neutralizing antibody response in
the induction of anti-CD4 antibodies. In contrast, covalently
linked gp120-CD4 complexes were reported to elicit broadly
neutralizing antibodies in macaques that were not related to
anti-CD4 antibodies . However, this observation was
not confirmed in later experiments (Sadoff J, personal com-
munication). Replacing the CD4 binding sequences by the
appropriate synthetic mimetic might be key to this problem
but higher affinity mimetics need to be developed .
Engineering hyperglycosylated derivatives of gp120 was
epitopes on the molecule that form part of the CD4-binding
site . This approach is under development.
Induction of fusion-blocking antibodies by immuniza-
tion with recombinant oligomeric gp41 molecules is another
promising new approach that still is at an early preclinical
stage of development [170,171].
Finally, IAVI’s Neutralizing Antibody Consortium has
undertaken to solve the crystal structure of the complexes
formed between a broadly HIV-neutralizing human mono-
clonal antibody and the viral envelope glycoprotein spikes to
try to unravel new clues for vaccine design .
It has been generally recognized that the extremely high
level of genetic variability of HIV and its continuous evo-
lution over time within one single individual, as well as
between different population groups and diverse geographi-
cal regions represents a major challenge for the development
of HIV vaccines capable of eliciting broadly reactive neu-
tralizing antibodies against globally prevalent HIV strains
. To address this challenge, multiple parallel vaccine
strategies are being explored, such as the use of cocktails of
envelope immunogens derived from globally prevalent HIV
strains [55,174], that of multivalent DNA or live vectored
vaccines incorporating envelope genes from various clades,
as well as combinations of live-vectored vaccines as a prime
followed by vaccination with a subunit recombinant vaccine
the problem of the broad genetic diversity of HIV have been
opened by the development of synthetic genes derived from
theoretically defined consensus or ancestral HIV envelope
sequences, which have been expressed in an immunogenic
form [177–179]. The problem still, however, remains basi-
cally unsolved .
5.4.2. Non-structural protein subunit vaccines
that are expressed very early in the virus life cycle might
lead to rapid elimination of infected cells has prompted the
development of subunit vaccines based on the viral trans-
activator Tat [180–182]. Immunization with Tat protected
macaques against SHIV infection [183–185] or resulted in
attenuated virus replication in the animals . However,
immunization with different forms of the Tat protein did not
demonstrate any efficacy against virus challenge [187,188]
and a recent study using an adenovirus type 5 (Ad5)-HIV Tat
recombinant vaccine elicited no protection in rhesus mon-
keys against a SHIV challenge, in spite of demonstrable
Tat subunit vaccine developed by Neovax in collaboration
with Sanofi Pasteur, has been evaluated in a Phase I trial and
should eventually enter Phase II trials. A Phase I clinical trial
of another subunit Tat vaccine was carried out in Italy on
HIV seropositive and HIV negative volunteers , show-
ing that the vaccine was well-tolerated and immunogenic.
Phase II trials are planned to begin presently in seronegative
volunteers at risk as well as in HIV-infected volunteers. At
based on the use of a trimeric gp140 molecule deleted of the
V2 loop (gp140 ?V2 SF162) that will be mixed with either
Tat or Nef .
Different vaccine approaches using the Tat antigen have
also been developed such as a Tat-adenyl cyclase fusion pro-
tein  or a Tat–Nef fusion protein that was combined
together with a recombinant gp120 subunit vaccine in the
AS02A adjuvant (an oil-in-water emulsion with monophos-
phoryl lipid A and the saponin derivative QS21). This com-
strain . The Tat–Nef fusion protein, which is developed
clinical trial using MVA as a vector and was found to induce
strong CD4+ T cell and antibody responses.
5.5. Naked DNA and live recombinant vaccines
The majority of recent HIV vaccine studies have aimed
to develop T-cell-stimulating vaccines that induce a HIV-
specific CD8+ CTL response, whose role in control of virus
load and evolution of disease has been well-documented in
viral loads, thus resulting in lower probability of virus trans-
mission to seronegative partners. A variety of vaccines were
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
developed based on this strategy, including naked DNA vac-
already are in advanced stages of clinical trials (For recent
reviews, see [17,194]).
Results from preclinical studies on plasmid DNA vac-
be observed in macaques [195,196] as well as in the HIV-
1/chimpanzee model . Immunogenicity of the vaccines
IL-2 or GM-CSF [198,199]. Naked DNA vaccines express-
ing the HIV-1 gag gene and either IL-12 or IL-15, which
the USA, Brazil and Thailand. DNA vaccines were actually
found to be most useful as priming vaccines in prime-boost
against pathogenic SHIV challenges and shown to result in
reduction in virus load and protection against disease and
death in the vaccinated animals . The same approach
was, however, definitely less successful in protection against
SIV challenge [122,201,202].
The first live recombinant HIV vaccines that were devel-
oped were based on the use of vaccinia virus as a vec-
tor. Safety considerations prompted the search for other,
non-replicative poxvirus vectors. A canarypox virus vector
(ALVAC) was developed by Sanofi-Pasteur and intensively
evaluated in multiple Phase I/II trials [203–205]. It now is
being tested in a Phase III trial in Thailand (see above). The
modified vaccinia virus Ankara (MVA) vector was tested in
multiple prime-boost vaccine protection studies in macaques
trials in human volunteers in the USA and in several African
countries, most often as a boost in combination with a DNA
vaccine priming [23,207]. A subtype C multigenic env, gag,
tat–rev, nef MVA recombinant developed by Therion in col-
another MVA recombinant that expresses subtype A,E env,
gag, pol and was developed by the NIH is in Phase I trial in
been tested in Phase I trials in Australia . It is currently
three vectors were used to express a variety of HIV antigens,
of the poxvirus-based HIV vaccines in humans has usually
been relatively modest, with less than 35% of the vaccinees
scoring positive for T-cell responses at any point in time, as
by the disappointing results of recent IAVI-sponsored DNA
prime-MVA boost studies in the UK and in Africa.
Replication-defective adenovirus type 5 (Ad5) represents
volunteers. More than 50% of the volunteers showed sig-
nificant, long-lasting HIV-1-specific CD8+ T-cell responses
to HIV-1 peptides, including secretions of IFN-?, IL-2, and
TNF-?. A trivalent recombinant Ad5-gag/pol/nef vaccine
has now been engineered and tested in human volunteers. A
multi-center Phase II trial of this trivalent candidate vaccine
nizations at 0, 4 and 26 weeks (NIAID and Merck). The trial
is taking place in several centers in North America, Peru,
Brazil, the Caribbean Islands and Australia, with final results
expected in 2008.
Another non-replicative adenovirus vector is being devel-
oped by the NIH Vaccine Research Center (VRC), together
with a DNA-based vaccine. These are multicomponent vac-
cines, which express the Env glycoprotein from clades A,
B and C and the Gag, Pol and Nef proteins from clade B,
and are designed for use in a prime-boost regimen strategy
. The DNA vaccine was initially tested in Phase I trials
in the USA, where it showed good immunogenicity, and is
Makarere University and WRAIR. The VRC Ad5 recombi-
study in the USA and is now being tested on those volunteers
who were earlier primed with the DNA vaccine. A Phase I
DNA-Ad5 prime-boost trial was recently started in the USA,
Brazil and South Africa and will presently be extended to
East Africa in collaboration with IAVI.
There is little doubt that the best results so far, in terms
of percent human responders and levels and duration of T-
cell responses to HIV-1 Gag in human volunteers, have been
lem of frequently pre-existing anti-vector immunity in the
human population, especially in developing countries, which
may dampen the immune response to the HIV transgenes
. This has prompted the development by Merck, Cru-
cell and Transgene, in collaboration with IAVI, of candidate
(Ad6, Ad35, Ad11, or Ad24) to replace the Ad5 vector in
future HIV vaccine trials [214,215]. Like Ad5, these vectors
readily multiply to large yields in PRC-6 cells. Nonreplica-
tive chimpanzee adenoviruses (AdC68, AdC6 and AdC7),
against which humans do not have neutralizing antibodies,
also are being explored as novel vectors by GSK and IAVI.
In addition, as the major adenovirus neutralization epitopes
are carried by the penton spike on the adenovirus virion, the
development of adenovirus chimeras that escape anti-Ad5
pre-existing immunity has been achieved by replacing the
fiber gene of an Ad5 vector by that from a rare Ad subtype.
Adenovirus chimeras such as Ad5/Ad11 or Ad5/Ad35 have
been constructed and successfully tested in animal models
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
A different vaccine strategy using adenovirus vectors
also has been developed by the NIH, employing replication-
only in the E3 region . Ad4hr-, Ad5hr-, or Ad7hr-HIV
protein were found to protect chimpanzees against challenge
with a primary, heterologous HIV-1 isolate [218,219]. Pro-
tective efficacy was also demonstrated in the SIV/macaque
Ad5hr-SIV gag priming followed by SIV gp120 boosting
binant vector-based HIV vaccines is long [17,194]. Some of
the most promising vector candidates include the measles
virus (MV), vesicular stomatitis virus (VSV), Sendai virus
(SeV) and Venezuelan equine encephalitis virus (VEEV).
The Schwarz attenuated MV strain, which has a long stand-
ing safety and efficacy record as a live attenuated measles
vaccine, was engineered to express the HIV gp160 molecule
deleted of the V3 loop. The recombinant was found to
elicit neutralizing antibodies with broad specificity after a
single immunization in mice . Recombinant MV-HIV
vaccines were also successfully tested in a SHIV/macaque
model. Their development is currently planned in collabora-
tion between the Pasteur Institute and GSK.
AIDS vaccines based on an attenuated VSV vector
expressing the Gag and Env proteins were found to provide
complete protection against T-cell loss and disease progres-
sion in the SHIV/macaque model, with all the vaccinated
animals having low or undetectable virus load levels for up
to 14 months after challenge . VSV, which is developed
as a vector for HIV by Wyeth, is a particularly attractive vec-
tor as it can efficiently be administered by a mucosal route,
neurovirulence of VSV was circumvented in a new genera-
and truncating the G protein cytoplasmic tail . Not only
is this new vector devoid of detectable neurovirulence by the
intracerebral route in mice, ferrets and monkeys, but it also
appears to be more immunogenic that its wild-type parent
strain. A VSV vector capable of only a single cycle of repli-
cation was recently generated and found to be equivalent to
a replication-competent VSV vector in generating high-level
primary and memory CD8+ T-cell responses as well as anti-
body responses to Env in mice .
Another vector of interest for the construction of live
recombinant HIV vaccines is SeV, a paramyxovirus from
mice which is devoid of pathogenicity for primates .
A second-generation vector was engineered by deletion of
the fusion glycoprotein (F) gene, making the virus non-
replicative. Macaques which were primed with a DNA-Gag
vaccine then boosted with a single immunization with a
SeV?F-HIV-1 Gag recombinant fully controlled virus repli-
cation and CD4+ T-cell loss after challenge with pathogenic
during 1 year follow-up [226,274].
The alphavirus replicon particle vectors have been devel-
oped using Sindbis virus, Semliki Forest virus (SFV), and
Venezuelan equine encephalitis virus (VEEV). The vec-
tor RNA codes for its replicase and contains cis-active
replication sequences, which allow its self-amplification in
the cytoplasm of infected host cells. VEEV targets imma-
ture dendritic cells, which play a major role in present-
ing antigens to the immune system, and has a natural
lymphotropism that may be ideal for vaccine strategies.
The VEE replicon particles were shown to induce potent
and protective immune responses in primates [227,228].
A replication-incompetent vector that was engineered to
encode the subtype C HIV gag gene was tested in a
Phase I trial in the USA, South Africa and Botswana. This
SFV particles expressing HIV-1 gp140 efficiently primed
immune responses as measured after a single boost with
purified trimeric gp140 protein, resulting in a Th1-biased
immune response and neutralizing antibodies against HIV-
A number of other vectors has also been used to prepare
live recombinant HIV vaccines, including, among others:
- Adenovirus-associated virus [229,230], which is devel-
oped by Targeted Genetics and IAVI and was tested in
Phase I trials in Germany, Belgium and India. This can-
didate vaccine recently entered Phase II evaluation in
South Africa, later to be followed by trials in Uganda and
- BCG, which was developed as a vector for HIV by the
Japanese NIID and already underwent a Phase I trial in
based vectors may have potential as an HIV vaccine when
administered in combination with a recombinant poxvirus
vector-based vaccine in a “prime-boost” vaccine strategy
- Attenuated Salmonella, that are used as vectors for the
development of oral vaccines at the University of Mary-
Poliovirus attenuated Sabin strains from the oral polio
vaccine (OPV) also have been engineered to express short
sequences of the HIV genome and could be used as an oral
vaccine , but the future of this approach remains uncer-
tain in view of the probable adverse impact of pre-existing
immunity in the human population and the fact that OPV is
progressively abandoned, whenever possible, in favor of the
inactivated polio vaccine (IPV) in the poliomyelitis eradica-
Another picornavirus, rhinovirus, recently has been engi-
neered to express the 2F5 epitope on its surface. Intranasal
tralizing antibodies, which, if confirmed, would represent a
remarkable achievement .
of these various vector-based vaccine approaches.
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
5.6. The development of prime-boost combinations
The experience gained so far with the first generation
of candidate HIV vaccines has been that many were mod-
estly immunogenic and only induced short-lived immune
responses. One of the strategies experienced over the last
decade to increase their immunogenicity was to combine
these vaccines in “prime-boost” vaccination regimens, ini-
tially using DNA vaccine candidates for priming followed
by live viral vectored vaccines for boosting [23,175,235].
Another type of prime-boost vaccine regimen was developed
using two different live recombinant vectors expressing the
same antigens, such as an Ad5 vector followed by a poxvirus
vector , or two successive adenovirus vectors, such as
and FPV, whose combination has been tested in Phase I trials
in the USA and Brazil by Therion in collaboration with the
NIAID. Heterologous adenovirus prime-boost vaccine reg-
imens using successively Ad11 and Ad35 vectors, or the
same vectors in reverse order, were found to elicit in mice
higher frequency immune responses than homologous regi-
mens , emphasizing the advantage of multiple vector-
Ad5-poxvirus (MVA or ALVAC) prime-boost regimen were
significantly greater than those elicited by homologous reg-
imens with the individual vectors. However, the increased
immunogenicity observed in monkeys was not confirmed in
human volunteers. Combinations of different vectors in het-
erologous prime-boost regimens are likely to be developed
in the future to circumvent the problem of anti-vector immu-
nity, which follows immunization with any live recombinant
Another prime-boost strategy has been developed in the
hope to strengthen both the humoral and cellular immune
responses to vaccination, by combining a T-cell stimulating
nant vectored vaccine with a subunit vaccine, especially one
based on envelope proteins (gp120, gp140 or gp41) .
There is no strong evidence, however, that such a dual vacci-
nation regimen resulted in a significantly higher antibody or
For any prime-boost strategy to be commercially feasi-
ance the increased costs and complexities associated with
developing two vaccines, including potential regulatory and
licensing problems, as well as logistical hurdles with the
delivery of the vaccines in the field.
5.7. Fusion proteins and peptides
Multiepitopic combinations of peptides, fusion proteins
and long lipopeptides are at an early stage of clinical devel-
vector-based recombinant vaccines. Vaccine constructs that
express a series of minimal epitopes arranged in a string-like
fashion have been explored as a potential strategy to gener-
ate diverse CTL responses and bypass the natural hierarchy
of epitope bias [238,239]. Multi-epitope DNA immunogens
can efficiently prime for broadly reactive CTL responses
[89,240,241], but do not necessarily overcome hierarchies
of epitope dominance . A multiepitope DNA vaccine
developed by Epimmune is currently undergoing a Phase I
trial in the USA and Peru and will be followed by booster
immunization with a multiepitope recombinant fusion pro-
Induction of persistent HIV Gag-specific CD8+ CTL
responses was evaluated in a Phase I trial involving immu-
nization with a fusion protein comprising the HIV p24Gag
protein and detoxified Bacillus anthracis lethal factor to
target antigen-presenting cells (Avant Therapeutics and
Synthetic lipopeptides containing MHC class I-restricted
T-cell epitopes were found to induce strong CD8+ T-cell
responses against HIV in mice, non-human primates and
with sequences corresponding to that of CTL epitope-rich
regions in the HIV-1 Gag and Nef proteins were tested in
Phase I trials and were shown to induce strong, multiepitopic
CD4+ and CD8+ T-cell responses. Parallel Phase II trials
were started in 2004 in the USA and in France under the
sponsorship of NIAID and ANRS, respectively, to study the
efficiency of lipopeptides as priming or boosting immuno-
of a severe neurological side effect in one of the US volun-
teers. The trial now has resumed in France.
6. Concluding remarks
The history of the HIV pandemic is now well into its
third decade. Tremendous progress has been made in our
understanding of the complex interaction between HIV and
the host immune system that has laid ground for the devel-
opment of new, potent antiviral drugs able to control virus
replication. However, many basic questions that bear on the
feasibility of developing an HIV vaccine still remain unan-
swered, including identification of protective immune mech-
anisms, addressing the high variability of the virus and its
ability to evade immune responses and eliciting potent virus-
HIV-1 isolates [10,11,14–16,247,248].
The experience with current viral vaccines suggests that
an effective vaccine or vaccine regimen against HIV infec-
tion might need to induce both neutralizing antibodies and
cell-mediated immunity. One of the challenges for HIV vac-
cine research, therefore, is to understand how to design
envelope immunogens to effectively elicit long-lasting high
titer broadly neutralizing antibodies. Meanwhile, the atten-
tion of the AIDS vaccine field has focused on the induc-
tion of HIV-specific cellular immune responses, including
CTL, based on the hypothesis that such a response would
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
hopefully enable the vaccinated persons to better control
their virus load following infection, slow down or even pre-
clude their progression to AIDS and decrease the probability
of secondary transmission. This hypothesis has been con-
firmed in the SHIV/macaque model using a variety of T-
where virus control following vaccination and challenge
often appears to be short-lived, ultimately leading to vaccine
These vaccines, therefore, urgently need to be tested in
clinical trials on human volunteers at risk . Of note is the
technical challenge associated with the design and follow-
up of large Phase III trials of vaccines that are not meant to
relation between viral loads at early times after infection and
speed of evolution of the disease are well documented in nat-
populations remains to be established. In addition, a number
of questions remains unanswered at present, like the nature
of the best end points for long-term follow-up of volunteers
in Phase III clinical trials, and the time during which volun-
teers will need to be monitored. These technical questions
are accompanied by a number of ethical questions relating to
standard of care and duration of the sponsors’ responsibility
for providing this care. The population-wide effects of vac-
cines that do not prevent infection but only reduce viral load
levels in vaccinees are largely unknown, but a few studies
based upon mathematical models suggest that even a 1-log
reduction in viral load, which is a reasonable expectation
with the currently available candidate vaccines, would have
20 years after introduction of the vaccine .
ment of HIV vaccines is the lack of scientific knowledge on
the nature and level of the immune responses that would be
required to achieve protection against HIV infection and/or
development of the disease. The most sensible strategies
help target the antigens to the antigen-presenting cells, espe-
DC-specific monoclonal antibody  has yielded impres-
sive results in mice, enhancing the efficiency and kinetics of
approach is now being studied in nonhuman primates.
Another player in the immune response, which proba-
bly has not received enough attention so far, is the CD4+
effector T cells, which seem to play a critical role in the pro-
tection provided by the live attenuated SIV?nef vaccine in
macaques (Johnson P, personal communication), and which
also were implicated as a correlate of protection in long-
term non-progressors [251–254]. The CD4+ T-cell response
to vaccines might be an important correlate of potential vac-
cine efficacy that should more systemically be taken into
to develop HIV vaccines that stimulate the mucosal immune
system so as to block the major virus transmission route. The
first cellular targets of HIV-1 are CD4+ CCR-5+ memory T
cells that are mostly found in the mucosal lymphoid tissue
of the GALT, where the early phase of HIV infection and
replication take place [110–114,255]. The need, therefore, is
to develop vaccines capable of raising an immune barrier at
the site of the genital, rectal and intestinal mucosa to effi-
ciently prevent HIV-1 infection. At this time, however, the
design of such vaccines remains a major challenge .
HIV-1-specific mucosal CTLs could be efficiently induced
in the intestinal mucosa by intrarectal administration of a
synthetic peptide vaccine incorporating a detoxified E. coli
heat-labile toxin, LT(R192G). Following a SHIV challenge,
the mucosally immunized monkeys readily cleared the virus
to undetectable levels both in blood and in the intestine
and did not experience any CD4+ T-cell depletion .
Mucosal HIV vaccine delivery should be considered among
the most effective immunization strategies for the induc-
tion of mucosal CTL that could provide early protection
against HIV replication and amplification [258,259]. Immu-
a VSV vectored vaccine expressing SIV antigens Gag and
Env resulted in significant protection against SIV infection
. Alphavirus replicons represent another type of recom-
binant vaccine that can be administered efficiently by the
mucosal route . The administration of a HIV-1 VLP
vaccine by the mucosal route is under study [147,148]. The
monkeys using tonsillar sprays of SIV vaccines [261,262].
Generally speaking, the nasal route of immunization is prob-
ably the most advantageous one, both from the point of view
of public acceptance and for its potential efficacy, but the use
mucosally targeted immunogens also has been attempted in
plants. A chimeric protein was expressed in plants that com-
bines the GM1 ganglioside-binding B subunit of the cholera
toxin (CTB) fused to a peptide (P1) which spans the 2F5
and 4E10 epitopes and the galactosyl ceramide-binding site
at the C-terminus of the ectodomain of HIV-1 gp41 .
The CTB-P1 fusion protein was found to induce vaginal and
intestinal antibody responses after intranasal immunization
in mice. This might pave the way to a new generation of
mucosally administered HIV vaccines.
Most of the efforts to develop and evaluate HIV vaccines
are borne by the National Institute of Allergy and Infectious
Diseases (NIAID), the Centers for Disease Control (CDC)
and the Walter Reed Army Institute for Research (WRAIR)
Agency (ANRS) in France, the International AIDS Vaccine
Initiative (IAVI) in New York, the European Union, initia-
tives at WHO and UNAIDS, and the African AIDS Vaccine
Programme (AAVP). The recent commitment of the Bill and
Melinda Gates Foundation has resulted in the foundation of
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
a Global HIV Vaccine Enterprise . The creation of a
Center for HIV/AIDS Immunology (CHAVI), which could
receive more than US$300 million from the NIAID over
the next 6 years, is part of this novel effort to speed up and
cine Trial Network (HVTN) established by NIAID in 2000,
with 25 clinical sites on four continents, represents a major
resource for clinical HIV vaccine research. The European
Union has similarly created the European and Developing
Countries Clinical Trials Partnership (EDCTP) with the aim
ing the efficacy of new drugs, microbicides, and vaccines.
Strong efforts are indeed needed to harmonize vaccine trials
and help to define the next clinical trial step in a ‘learning
by doing’ procedure based on continuous vaccine improve-
ment in iterative clinical trial processes . Developing
countries are essential partners in this international effort.
There is little doubt that the development of a safe, effec-
HIV/AIDS research and a formidable scientific and public
health challenge at the dawn of this century.
The efficient editorial help of Olga Assossou is gratefully
 Gao F, Bailes E, Robertson DL, Chen Y, Rodenburg CM, Michael
SF, et al. Origin of HIV-1 in the chimpanzee Pan troglodytes
troglodytes. Nature 1999;397(6718):436–41.
 Korber B, Muldoon M, Theiler J, Gao F, Gupta R, Lapedes A,
et al. Timing the ancestor of the HIV-1 pandemic strains. Science
 Joint United Nations Programme on HIV/AIDS (UNAIDS). Report
on the global AIDS epidemic, Geneva Switzerland, December
 Girard M, Mastro TD, Koff WC. Human Immunodeficiency Virus.
In: Plotkin SA, Orenstein WA, editors. Vaccines. Philadelphia:
Saunders; 2004. p. 1219–58.
 Cohen J. Public health. AIDS vaccine trial produces disappointment
and confusion. Science 2003;299(5611):1290–1.
 McNeil JG, Johnston MI, Birx DL, Tramont EC. Policy rebuttal.
HIV vaccine trial justified. Science 2004;303(5660):961.
 McCutchan FE, Viputtigul K, de Souza MS, Carr JK, Markowitz
LE, Buapunth P, et al. Diversity of envelope glycoprotein from
human immunodeficiency virus type 1 of recent seroconverters in
Thailand. AIDS Res Hum Retroviruses 2000;16(8): 801–5.
 Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C,
Detours V. Evolutionary and immunological implications of con-
temporary HIV-1 variation. Br Med Bull 2001;58:19–42.
 Lifson JD, Rossio JL, Arnaout R, Li L, Parks TL, Schneider DK,
et al. Containment of simian immunodeficiency virus infection:
cellular immune responses and protection from rechallenge fol-
lowing transient postinoculation antiretroviral treatment. J Virol
 Desrosiers RC. Prospects for an AIDS vaccine. Nat Med 2004;
 Burton DR, Desrosiers RC, Doms RW, Koff WC, Kwong PD,
Moore JP, et al. HIV vaccine design and the neutralizing antibody
problem. Nat Immunol 2004;5(3):233–6.
 Excler JL, Beyrer C. Human immunodeficiency virus vaccine
development in developing countries: are efficacy trials feasible? J
Hum Virol 2000;3(4):193–214.
 Esparza J, Osmanov S, Pattou-Markovic C, Toure C, Chang ML,
Nixon S. Past, present and future of HIV vaccine trials in devel-
oping countries. Vaccine 2002;20(15):1897–8.
 Emini EA, Koff WC. AIDS/HIV. Developing an AIDS vaccine:
need, uncertainty, hope. Science 2004;304(5679):1913–4.
 Garber DA, Silvestri G, Feinberg MB. Prospects for an AIDS
vaccine: three big questions, no easy answers. Lancet Infect Dis
 Lemckert AA, Goudsmit J, Barouch DH. Challenges in the search
for an HIV vaccine. Eur J Epidemiol 2004;19(6):513–6.
 Excler JL. AIDS vaccine development: perspectives, challenges and
hopes. Ind J Med Res 2005;121(4):568–81.
 Buve A, Bishikwabo-Nsarhaza K, Mutangadura G. The spread
and effect of HIV-1 infection in sub-Saharan Africa. Lancet
 Drobniewski FA, Balabanova YM, Ruddy MC, Graham C,
Kuznetzov SI, Gusarova GI, et al. Tuberculosis, HIV seropreva-
lence and intravenous drug abuse in prisoners. Eur Respir J
 Tatt ID, Barlow KL, Nicoll A, Clewley JP. The public health sig-
nificance of HIV-1 subtypes. AIDS 2001;15(Suppl. 5):S59–71.
 McCutchan FE. In: Thompson RCA, editor. The Molecular Epi-
demiology of Infectious Diseases. London: Holder, A; 2000. p.
 McCutchan FE. Understanding the genetic diversity of HIV-1.
AIDS 2000;14(Suppl. 3):S31–44.
 Robinson HL. New hope for an AIDS vaccine. Nat Rev Immunol
 Goldstein G. HIV-1 Tat protein as a potential AIDS vaccine. Nat
 Pomerantz RJ, Seshamma T, Trono D. Efficient replication of
human immunodeficiency virus type 1 requires a threshold level of
Rev: potential implications for latency. J Virol 1992;66(3):1809–13.
 Renkema GH, Manninen A, Saksela K. Human immunodefi-
ciency virus type 1 Nef selectively associates with a catalytically
active subpopulation of p21-activated kinase 2 (PAK2) indepen-
dently of PAK2 binding to Nck or beta-PIX. J Virol 2001;75(5):
 James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD,
Bond VC. Extracellular Nef protein targets CD4+ T cells for
apoptosis by interacting with CXCR4 surface receptors. J Virol
 Yu X, Yu Y, Liu B, Luo K, Kong W, Mao P, et al. Induc-
tion of APOBEC3G ubiquitination and degradation by an HIV-1
Vif–Cul5–SCF complex. Science 2003;302(5647):1056–60.
 Lahti AL, Manninen A, Saksela K. Regulation of T cell activation
by HIV-1 accessory proteins: Vpr acts via distinct mechanisms
to cooperate with Nef in NFAT-directed gene expression and to
promote transactivation by CREB. Virology 2003;310(1):190–6.
 Tardif MR, Tremblay MJ. Presence of host ICAM-1 in human
immunodeficiency virus type 1 virions increases productive infec-
tion of CD4+ T lymphocytes by favoring cytosolic delivery of viral
material. J Virol 2003;77(22):12299–309.
 Kuznetsov YG, Victoria JG, Robinson Jr WE, McPherson A.
Atomic force microscopy investigation of human immunode-
ficiency virus (HIV) and HIV-infected lymphocytes. J Virol
 Brelot A, Alizon M. HIV-1 entry and how to block it. AIDS
 Chan DC, Kim PS. HIV entry and its inhibition. Cell 1998;93(5):
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
 Eckert DM, Kim PS. Mechanisms of viral membrane fusion and
its inhibition. Annu Rev Biochem 2001;70:777–810.
 Markovic I, Clouse KA. Recent advances in understanding the
molecular mechanisms of HIV-1 entry and fusion: revisiting current
targets and considering new options for therapeutic intervention.
Curr HIV Res 2004;2(3):223–34.
 Desmezieres E, Gupta N, Vassell R, He Y, Peden K, Sirota L, et al.
Human immunodeficiency virus (HIV) gp41 escape mutants: cross-
resistance to peptide inhibitors of HIV fusion and altered receptor
activation of gp120. J Virol 2005;79(8):4774–81.
 Zolla-Pazner S. Identifying epitopes of HIV-1 that induce protective
antibodies. Nat Rev Immunol 2004;4(3):199–210.
 Kessler II JA, McKenna PM, Emini EA, Chan CP, Patel MD, Gupta
SK, et al. Recombinant human monoclonal antibody IgG1b12 neu-
tralizes diverse human immunodeficiency virus type 1 primary
isolates. AIDS Res Hum Retroviruses 1997;13(7):575–82.
 Scanlan CN, Pantophlet R, Wormald MR, Ollmann Saphire E, Stan-
field R, Wilson IA, et al. The broadly neutralizing anti-human
immunodeficiency virus type 1 antibody 2G12 recognizes a cluster
of alpha1→2 mannose residues on the outer face of gp120. J Virol
 D’Souza MP, Milman G, Bradac JA, McPhee D, Hanson CV,
Hendry RM. Neutralization of primary HIV-1 isolates by anti-
envelope monoclonal antibodies. AIDS 1995;9(8):867–74.
 Ugolini S, Mondor I, Parren PW, Burton DR, Tilley SA, Klasse
PJ, et al. Inhibition of virus attachment to CD4+ target cells is
a major mechanism of T cell line-adapted HIV-1 neutralization. J
Exp Med 1997;186(8):1287–98.
 Zwick MB, Bonnycastle LL, Menendez A, Irving MB, Barbas
III CF, Parren PW, et al. Identification and characterization of
a peptide that specifically binds the human, broadly neutralizing
anti-human immunodeficiency virus type 1 antibody b12. J Virol
 Xiang SH, Doka N, Choudhary RK, Sodroski J, Robinson
JE. Characterization of CD4-induced epitopes on the HIV
type 1 gp120 envelope glycoprotein recognized by neutraliz-
ing human monoclonal antibodies. AIDS Res Hum Retroviruses
 Koch M, Pancera M, Kwong PD, Kolchinsky P, Grundner C, Wang
L, et al. Structure-based, targeted deglycosylation of HIV-1 gp120
and effects on neutralization sensitivity and antibody recognition.
 Kwong PD, Doyle ML, Casper DJ, Cicala C, Leavitt SA,
Majeed S, et al. HIV-1 evades antibody-mediated neutralization
through conformational masking of receptor-binding sites. Nature
 Labrijn AF, Poignard P, Raja A, Zwick MB, Delgado K, Franti
M, et al. Access of antibody molecules to the conserved core-
ceptor binding site on glycoprotein gp120 is sterically restricted
on primary human immunodeficiency virus type 1. J Virol
 Huang CC, Tang M, Zhang MY, Majeed S, Montabana E, Stanfield
RL, et al. Structure of a V3-containing HIV-1 gp120 core. Science
 Trkola A, Pomales AB, Yuan H, Korber B, Maddon PJ, Allaway
GP, et al. Cross-clade neutralization of primary isolates of human
immunodeficiency virus type 1 by human monoclonal antibodies
and tetrameric CD4-IgG. J Virol 1995;69(11):6609–17.
 Parker CE, Deterding LJ, Hager-Braun C, Binley JM, Schulke
N, Katinger H, et al. Fine definition of the epitope on the
gp41 glycoprotein of human immunodeficiency virus type 1 for
the neutralizing monoclonal antibody 2F5. J Virol 2001;75(22):
 Zwick MB, Labrijn AF, Wang M, Spenlehauer C, Saphire EO,
Binley JM, et al. Broadly neutralizing antibodies targeted to the
membrane-proximal external region of human immunodeficiency
virus type 1 glycoprotein gp41. J Virol 2001;75(22):10892–905.
 Barbato G, Bianchi E, Ingallinella P, Hurni WH, Miller MD, Cilib-
erto G, et al. Structural analysis of the epitope of the anti-HIV
antibody 2F5 sheds light into its mechanism of neutralization and
HIV fusion. J Mol Biol 2003;330(5):1101–15.
 Ofek G, Tang M, Sambor A, Katinger H, Mascola JR, Wyatt R, et
al. Structure and mechanistic analysis of the anti-human immun-
odeficiency virus type 1 antibody 2F5 in complex with its gp41
epitope. J Virol 2004;78(19):10724–37.
 Cardoso RM, Zwick MB, Stanfield RL, Kunert R, Binley JM,
Katinger H, et al. Broadly neutralizing anti-HIV antibody 4E10
recognizes a helical conformation of a highly conserved fusion-
associated motif in gp41. Immunity 2005;22(2):163–73.
 Haynes BF, Fleming J, St Clair EW, Katinger H, Stiegler G, Kunert
R, et al. Cardiolipin polyspecific autoreactivity in two broadly neu-
tralizing HIV-1 antibodies. Science 2005;308(5730):1906–8.
 Nabel GJ. Immunology. Close to the edge: neutralizing the HIV-1
envelope. Science 2005;308(5730):1878–9.
 Wang B, Lal RB, Dwyer DE, Miranda-Saksena M, Boadle R, Cun-
ningham AL, et al. Molecular and biological interactions between
two HIV-1 strains from a coinfected patient reveal the first evidence
in favor of viral synergism. Virology 2000;274(1):105–19.
 Altfeld M, Allen TM, Yu XG, Johnston MN, Agrawal D, Kor-
ber BT, et al. HIV-1 superinfection despite broad CD8+ T-cell
responses containing replication of the primary virus. Nature
 McCutchan FE, Hoelscher M, Tovanabutra S, Piyasirisilp S,
Sanders-Buell E, Ramos G, et al. In-depth analysis of a heterosexu-
ally acquired human immunodeficiency virus type 1 superinfection:
evolution, temporal fluctuation, and intercompartment dynamics
from the seronegative window period through 30 months postin-
fection. J Virol 2005;79(18):11693–704.
 Kim EY, Busch M, Abel K, Fritts L, Bustamante P, Stanton J,
et al. Retroviral recombination in vivo: viral replication patterns
and genetic structure of simian immunodeficiency virus (SIV)
populations in rhesus macaques after simultaneous or sequen-
tial intravaginal inoculation with SIVmac239Deltavpx/Deltavpr and
SIVmac239Deltanef. J Virol 2005;79(8):4886–95.
 Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K,
Pierson T, et al. Latent infection of CD4+ T cells provides a mecha-
nism for lifelong persistence of HIV-1, even in patients on effective
combination therapy. Nat Med 1999;5(5):512–7.
 Blankson JN, Persaud D, Siliciano RF. The challenge of viral reser-
voirs in HIV-1 infection. Annu Rev Med 2002;53:557–93.
 Peterlin BM, Trono D. Hide, shield and strike back: how
HIV-infected cells avoid immune eradication. Nat Rev Immunol
 Miller CJ, Li Q, Abel K, Kim EY, Ma ZM, Wietgrefe S, et al.
Propagation and dissemination of infection after vaginal transmis-
sion of simian immunodeficiency virus. J Virol 2005;79(14):9217–
 Gallo RC. The end or the beginning of the drive to an HIV-
preventive vaccine: a view from over 20 years. Lancet 2005;
 Evans DT, Desrosiers RC. Immune evasion strategies of the primate
lentiviruses. Immunol Rev 2001;183:141–58.
 O’Connor D, Friedrich T, Hughes A, Allen TM, Watkins D. Under-
standing cytotoxic T-lymphocyte escape during simian immunode-
ficiency virus infection. Immunol Rev 2001;183:115–26.
 Goulder PJ, Watkins DI. HIV and SIV CTL escape: implications
for vaccine design. Nat Rev Immunol 2004;4(8):630–40.
 Feeney ME, Tang Y, Pfafferott K, Roosevelt KA, Draenert
R, Trocha A, et al. HIV-1 viral escape in infancy followed
by emergence of a variant-specific CTL response. J Immunol
 Richman DD, Wrin T, Little SJ, Petropoulos CJ. Rapid evolution
of the neutralizing antibody response to HIV type 1 infection. Proc
Natl Acad Sci U S A 2003;100(7):4144–9.
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
 Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X,
et al. Antibody neutralization and escape by HIV-1. Nature
 Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: fusogens,
antigens, and immunogens. Science 1998;280(5371):1884–8.
 Parren PW, Moore JP, Burton DR, Sattentau QJ. The neutralizing
antibody response to HIV-1: viral evasion and escape from humoral
immunity. AIDS 1999;13(Suppl. A):S137–62.
 Calarota SA, Weiner DB. Present status of human HIV vaccine
development. AIDS 2003;17(Suppl. 4):S73–84.
 Gorny MK, Revesz K, Williams C, Volsky B, Louder MK,
Anyangwe CA, et al. The v3 loop is accessible on the surface of
most human immunodeficiency virus type 1 primary isolates and
serves as a neutralization epitope. J Virol 2004;78(5):2394–404.
 Yang X, Tomov V, Kurteva S, Wang L, Ren X, Gorny MK, et
al. Characterization of the outer domain of the gp120 glycoprotein
from human immunodeficiency virus type 1. J Virol 2004;78(23):
 Mascola JR, Snyder SW, Weislow OS, Belay SM, Belshe RB,
Schwartz DH, et al. Immunization with envelope subunit vaccine
products elicits neutralizing antibodies against laboratory-adapted
but not primary isolates of human immunodeficiency virus type 1.
The National Institute of Allergy and Infectious Diseases AIDS
Vaccine Evaluation Group. J Infect Dis 1996;173(2):340–8.
 Shibata R, Igarashi T, Haigwood N, Buckler-White A, Ogert R,
Ross W, et al. Neutralizing antibody directed against the HIV-1
envelope glycoprotein can completely block HIV-1/SIV chimeric
virus infections of macaque monkeys. Nat Med 1999;5(2):204–10.
 Mascola JR, Lewis MG, Stiegler G, Harris D, VanCott TC, Hayes
D, et al. Protection of Macaques against pathogenic simian/human
immunodeficiency virus 89.6PD by passive transfer of neutralizing
antibodies. J Virol 1999;73(5):4009–18.
 Mascola JR, Stiegler G, VanCott TC, Katinger H, Carpenter CB,
Hanson CE, et al. Protection of macaques against vaginal transmis-
sion of a pathogenic HIV-1/SIV chimeric virus by passive infusion
of neutralizing antibodies. Nat Med 2000;6(2):207–10.
 Baba TW, Liska V, Hofmann-Lehmann R, Vlasak J, Xu W, Ayehu-
nie S, et al. Human neutralizing monoclonal antibodies of the IgG1
subtype protect against mucosal simian-human immunodeficiency
virus infection. Nat Med 2000;6(2):200–6.
 Parren PW, Marx PA, Hessell AJ, Luckay A, Harouse J, Cheng-
Mayer C, et al. Antibody protects macaques against vaginal chal-
lenge with a pathogenic R5 simian/human immunodeficiency virus
at serum levels giving complete neutralization in vitro. J Virol
 Letvin NL, Robinson S, Rohne D, Axthelm MK, Fanton JW, Bilska
M, et al. Vaccine-elicited V3 loop-specific antibodies in rhesus
monkeys and control of a simian-human immunodeficiency virus
expressing a primary patient human immunodeficiency virus type
1 isolate envelope. J Virol 2001;75(9):4165–75.
 Nishimura Y, Igarashi T, Haigwood NL, Sadjadpour R, Donau OK,
Buckler C, et al. Transfer of neutralizing IgG to macaques 6h
but not 24h after SHIV infection confers sterilizing protection:
implications for HIV-1 vaccine development. Proc Natl Acad Sci
U S A 2003;100(25):15131–6.
 Nishimura Y, Igarashi T, Haigwood N, Sadjadpour R, Plishka
RJ, Buckler-White A, et al. Determination of a statistically valid
neutralization titer in plasma that confers protection against simian-
human immunodeficiency virus challenge following passive trans-
fer of high-titered neutralizing antibodies. J Virol 2002;76(5):
 Ferrantelli F, Rasmussen RA, Hofmann-Lehmann R, Xu W,
McClure HM, Ruprecht RM. Do not underestimate the power of
antibodies—lessons from adoptive transfer of antibodies against
HIV. Vaccine 2002;20(Suppl. 4):A61–5.
 Trkola A, Kuster H, Rusert P, Joos B, Fischer M, Leemann C, et
al. Delay of HIV-1 rebound after cessation of antiretroviral therapy
through passive transfer of human neutralizing antibodies. Nat Med
 Berman PW, Gregory TJ, Riddle L, Nakamura GR, Champe MA,
Porter JP, et al. Protection of chimpanzees from infection by HIV-
1 after vaccination with recombinant glycoprotein gp120 but not
gp160. Nature 1990;345(6276):622–5.
 Girard M, Meignier B, Barre-Sinoussi F, Kieny MP, Matthews T,
Muchmore E, et al. Vaccine-induced protection of chimpanzees
against infection by a heterologous human immunodeficiency virus
type 1. J Virol 1995;69(10):6239–48.
 McMichael A, Hanke T. The quest for an AIDS vaccine: is
the CD8+ T-cell approach feasible? Nat Rev Immunol 2002;2(4):
 McMichael AJ, Rowland-Jones SL. Cellular immune responses to
HIV. Nature 2001;410(6831):980–7.
 Letvin NL. Strategies for an HIV vaccine. J Clin Invest 2002;
 Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky
W, et al. Temporal association of cellular immune responses with
the initial control of viremia in primary human immunodeficiency
virus type 1 syndrome. J Virol 1994;68(7):4650–5.
 Kuroda MJ, Schmitz JE, Charini WA, Nickerson CE, Lifton MA,
Lord CI, et al. Emergence of CTL coincides with clearance of virus
during primary simian immunodeficiency virus infection in rhesus
monkeys. J Immunol 1999;162(9):5127–33.
 Frahm N, Adams S, Kiepiela P, Linde CH, Hewitt HS, Lichter-
feld M, et al. HLA-B63 presents HLA-B57/B58-restricted cytotoxic
T-lymphocyte epitopes and is associated with low human immun-
odeficiency virus load. J Virol 2005;79(16):10218–25.
 Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers
H, et al. Antiviral pressure exerted by HIV-1-specific cytotoxic
T lymphocytes (CTLs) during primary infection demonstrated by
rapid selection of CTL escape virus. Nat Med 1997;3(2):205–
 Evans DT, O’Connor DH, Jing P, Dzuris JL, Sidney J, da Silva
J, et al. Virus-specific cytotoxic T-lymphocyte responses select for
amino-acid variation in simian immunodeficiency virus Env and
Nef. Nat Med 1999;5(11):1270–6.
 Allen TM, O’Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel
TU, et al. Tat-specific cytotoxic T lymphocytes select for SIV
escape variants during resolution of primary viraemia. Nature
 Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S,
Peyerl FW, et al. Eventual AIDS vaccine failure in a rhesus
monkey by viral escape from cytotoxic T lymphocytes. Nature
 Matano T, Shibata R, Siemon C, Connors M, Lane HC, Martin
MA. Administration of an anti-CD8 monoclonal antibody inter-
feres with the clearance of chimeric simian/human immunodefi-
ciency virus during primary infections of rhesus macaques. J Virol
 Schmitz JE, Kuroda MJ, Santra S, Sasseville VG, Simon
MA, Lifton MA, et al. Control of viremia in simian immun-
odeficiency virus infection by CD8+ lymphocytes. Science
 Lifson JD, Rossio JL, Piatak Jr M, Parks T, Li L, Kiser R, et
al. Role of CD8(+) lymphocytes in control of simian immunodefi-
ciency virus infection and resistance to rechallenge after transient
early antiretroviral treatment. J Virol 2001;75(21):10187–99.
 Peeters M, Courgnaud V, Abela B, Auzel P, Pourrut X, Bibollet-
Ruche F, et al. Risk to human health from a plethora of simian
immunodeficiency viruses in primate bushmeat. Emerg Infect Dis
 Heeney JL. Primate models for AIDS vaccine development. AIDS
 Hel Z, Venzon D, Poudyal M, Tsai WP, Giuliani L, Woodward R,
et al. Viremia control following antiretroviral treatment and thera-
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
peutic immunization during primary SIV251 infection of macaques.
Nat Med 2000;6(10):1140–6.
 Parker RA, Regan MM, Reimann KA. Variability of viral load
in plasma of rhesus monkeys inoculated with simian immunodefi-
ciency virus or simian-human immunodeficiency virus: implications
for using nonhuman primate AIDS models to test vaccines and
therapeutics. J Virol 2001;75(22):11234–8.
 Goulder PJ, Altfeld MA, Rosenberg ES, Nguyen T, Tang Y,
Eldridge RL, et al. Substantial differences in specificity of HIV-
specific cytotoxic T cells in acute and chronic HIV infection. J
Exp Med 2001;193(2):181–94.
 Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR,
Knight HL, et al. Gastrointestinal tract as a major site of CD4+
T cell depletion and viral replication in SIV infection. Science
 Veazey RS, Tham IC, Mansfield KG, DeMaria M, Forand AE,
Shvetz DE, et al. Identifying the target cell in primary simian
immunodeficiency virus (SIV) infection: highly activated memory
CD4(+) T cells are rapidly eliminated in early SIV infection in
vivo. J Virol 2000;74(1):57–64.
 Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer
M. Massive infection and loss of memory CD4+ T cells in multiple
tissues during acute SIV infection. Nature 2005;434(7037):1093–7.
 Li Q, Duan L, Estes JD, Ma ZM, Rourke T, Wang Y, et al. Peak
SIV replication in resting memory CD4+ T cells depletes gut lam-
ina propria CD4+ T cells. Nature 2005;434(7037):1148–52.
 Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil
A, et al. Severe CD4+ T-cell depletion in gut lymphoid tissue
during primary human immunodeficiency virus type 1 infection and
substantial delay in restoration following highly active antiretroviral
therapy. J Virol 2003;77(21):11708–17.
 Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beil-
man GJ, et al. CD4+ T cell depletion during all stages of HIV
disease occurs predominantly in the gastrointestinal tract. J Exp
 Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A,
Hogan C, et al. Primary HIV-1 infection is associated with prefer-
ential depletion of CD4+ T lymphocytes from effector sites in the
gastrointestinal tract. J Exp Med 2004;200(6):761–70.
 Veazey RS, Lackner AA. HIV swiftly guts the immune system.
Nat Med 2005;11(5):469–70.
 Hel Z, Nacsa J, Tryniszewska E, Tsai WP, Parks RW, Montefiori
DC, et al. Containment of simian immunodeficiency virus infection
in vaccinated macaques: correlation with the magnitude of virus-
specific pre- and postchallenge CD4+ and CD8+ T cell responses.
J Immunol 2002;169(9):4778–87.
 Franchini G, Nacsa J, Hel Z, Tryniszewska E. Immune interven-
tion strategies for HIV-1 infection of humans in the SIV macaque
model. Vaccine 2002;20(Suppl. 4):A52–60.
 Reimann KA, Li JT, Veazey R, Halloran M, Park IW, Karlsson GB,
et al. A chimeric simian/human immunodeficiency virus expressing
a primary patient human immunodeficiency virus type 1 isolate
env causes an AIDS-like disease after in vivo passage in rhesus
monkeys. J Virol 1996;70(10):6922–8.
 Reimann KA, Li JT, Voss G, Lekutis C, Tenner-Racz K, Racz P, et
al. An env gene derived from a primary human immunodeficiency
virus type 1 isolate confers high in vivo replicative capacity to a
chimeric simian/human immunodeficiency virus in rhesus monkeys.
J Virol 1996;70(5):3198–206.
 Joag SV, Adany I, Li Z, Foresman L, Pinson DM, Wang C, et
al. Animal model of mucosally transmitted human immunodefi-
ciency virus type 1 disease: intravaginal and oral deposition of
simian/human immunodeficiency virus in macaques results in sys-
temic infection, elimination of CD4+ T cells, and AIDS. J Virol
 Feinberg MB, Moore JP. AIDS vaccine models: challenging chal-
lenge viruses. Nat Med 2002;8(3):207–10.
 Staprans SI, Feinberg MB. The roles of nonhuman primates in
the preclinical evaluation of candidate AIDS vaccines. Expert Rev
Vaccines 2004;3(Suppl. 4):S5–32.
 Horton H, Vogel TU, Carter DK, Vielhuber K, Fuller DH, Ship-
ley T, et al. Immunization of rhesus macaques with a DNA
prime/modified vaccinia virus Ankara boost regimen induces broad
simian immunodeficiency virus (SIV)-specific T-cell responses and
reduces initial viral replication but does not prevent disease pro-
gression following challenge with pathogenic SIVmac239. J Virol
 Vogel TU, Reynolds MR, Fuller DH, Vielhuber K, Shipley T,
Fuller JT, et al. Multispecific vaccine-induced mucosal cytotoxic T
lymphocytes reduce acute-phase viral replication but fail in long-
term control of simian immunodeficiency virus SIVmac239. J Virol
 Harouse JM, Gettie A, Eshetu T, Tan RC, Bohm R, Blanchard J, et
al. Mucosal transmission and induction of simian AIDS by CCR5-
specific simian/human immunodeficiency virus SHIV(SF162P3). J
 HsuM, HarouseJM, Gettie
J, Cheng-Mayer C. Increased mucosal transmission but not
enhanced pathogenicity of the CCR5-tropic, simian AIDS-inducing
simian/human immunodeficiency virus SHIV(SF162P3) maps to
envelope gp120. J Virol 2003;77(2):989–98.
 Vernazza PL, Eron JJ, Fiscus SA, Cohen MS. Sexual transmission
of HIV: infectiousness and prevention. AIDS 1999;13(2):155–66.
 Chakraborty H, Sen PK, Helms RW, Vernazza PL, Fiscus SA,
Eron JJ, et al. Viral burden in genital secretions determines male-
to-female sexual transmission of HIV-1: a probabilistic empiric
model. AIDS 2001;15(5):621–7.
 Gray RH, Wawer MJ, Brookmeyer R, Sewankambo NK, Serwadda
D, Wabwire-Mangen F, et al. Probability of HIV-1 transmission per
coital act in monogamous, heterosexual, HIV-1-discordant couples
in Rakai, Uganda. Lancet 2001;357(9263):1149–53.
 McDermott AB, Mitchen J, Piaskowski S, De Souza I, Yant LJ,
Stephany J, et al. Repeated low-dose mucosal simian immunod-
eficiency virus SIVmac239 challenge results in the same viral
and immunological kinetics as high-dose challenge: a model for
the evaluation of vaccine efficacy in nonhuman primates. J Virol
 Daniel MD, Kirchhoff F, Czajak SC, Sehgal PK, Desrosiers RC.
Protective effects of a live attenuated SIV vaccine with a deletion
in the nef gene. Science 1992;258(5090):1938–41.
 Whatmore AM, Cook N, Hall GA, Sharpe S, Rud EW, Cranage
MP. Repair and evolution of nef in vivo modulates simian immun-
odeficiency virus virulence. J Virol 1995;69(8):5117–23.
 Wyand MS, Manson K, Montefiori DC, Lifson JD, John-
son RP, Desrosiers RC. Protection by live, attenuated simian
immunodeficiency virus against heterologous challenge. J Virol
 Baba TW, Liska V, Khimani AH, Ray NB, Dailey PJ, Penninck
D, et al. Live attenuated, multiply deleted simian immunodefi-
ciency virus causes AIDS in infant and adult macaques. Nat Med
 Hofmann-Lehmann R, Vlasak J, Williams AL, Chenine AL,
McClure HM, Anderson DC, et al. Live attenuated, nef-deleted
SIV is pathogenic in most adult macaques after prolonged obser-
vation. AIDS 2003;17(2):157–66.
 Johnson RP, Lifson JD, Czajak SC, Cole KS, Manson KH, Glick-
man R, et al. Highly attenuated vaccine strains of simian immun-
odeficiency virus protect against vaginal challenge: inverse rela-
tionship of degree of protection with level of attenuation. J Virol
 Guan Y, Whitney JB, Detorio M, Wainberg MA. Construction
and in vitro properties of a series of attenuated simian immun-
odeficiency viruses with all accessory genes deleted. J Virol
A, BucknerC, Blanchard
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
 Blancou P, Chenciner N, Ho Tsong Fang R, Monceaux V, Cumont
MC, Guetard D, et al. Simian immunodeficiency virus promoter
exchange results in a highly attenuated strain that protects against
uncloned challenge virus. J Virol 2004;78(3):1080–92.
 Jekle A, Keppler OT, De Clercq E, Schols D, Weinstein M, Gold-
smith MA. In vivo evolution of human immunodeficiency virus
type 1 toward increased pathogenicity through CXCR4-mediated
killing of uninfected CD4 T cells. J Virol 2003;77(10):5846–54.
 Whitney JB, Ruprecht RM. Live attenuated HIV vaccines: pitfalls
and prospects. Curr Opin Infect Dis 2004;17(1):17–26.
 Koff WC, Johnson PR, Watkins DI, Burton DR, Lifson JD,
Hasenkrug KJ, et al. HIV vaccine design: insights from live atten-
uated SIV vaccines. Nat Immunol 2006;7(1):19–23.
 Poon B, Hsu JF, Gudeman V, Chen IS, Grovit-Ferbas K.
human immunodeficiency virus type 1 env can be used to induce
high-titer neutralizing antibody responses. J Virol 2005;79(16):
 Poon B, Safrit JT, McClure H, Kitchen C, Hsu JF, Gude-
man V, et al. Induction of humoral immune responses following
vaccination with envelope-containing, formaldehyde-treated, ther-
mally inactivated human immunodeficiency virus type 1. J Virol
 Arthur LO, Bess Jr JW, Chertova EN, Rossio JL, Esser MT, Ben-
veniste RE, et al. Chemical inactivation of retroviral infectivity by
targeting nucleocapsid protein zinc fingers: a candidate SIV vac-
cine. AIDS Res Hum Retroviruses 1998;14(Suppl. 3):S311–9.
 Rossio JL, Esser MT, Suryanarayana K, Schneider DK, Bess
Jr JW, Vasquez GM, et al. Inactivation of human immunode-
ficiency virus type 1 infectivity with preservation of conforma-
tional and functional integrity of virion surface proteins. J Virol
 Lifson JD, Rossio JL, Piatak Jr M, Bess Jr J, Chertova E, Schneider
DK, et al. Evaluation of the safety, immunogenicity, and protective
efficacy of whole inactivated simian immunodeficiency virus (SIV)
vaccines with conformationally and functionally intact envelope
glycoproteins. AIDS Res Hum Retroviruses 2004;20(7):772–87.
 Kang SM, Compans RW. Enhancement of mucosal immunization
with virus-like particles of simian immunodeficiency virus. J Virol
 Kang SM, Yao Q, Guo L, Compans RW. Mucosal immunization
with virus-like particles of simian immunodeficiency virus conju-
gated with cholera toxin subunit B. J Virol 2003;77(18):9823–30.
 Doan LX, Li M, Chen C, Yao Q. Virus-like particles as HIV-1
vaccines. Rev Med Virol 2005;15(2):75–88.
 Hammonds J, Chen X, Fouts T, DeVico A, Montefiori D, Spearman
P. Induction of neutralizing antibodies against human immunode-
ficiency virus type 1 primary isolates by Gag-Env pseudovirion
immunization. J Virol 2005;79(23):14804–14.
 Harvey TJ, Anraku I, Linedale R, Harrich D, Mackenzie J, Suhrbier
A, et al. Kunjin virus replicon vectors for human immunodeficiency
virus vaccine development. J Virol 2003;77(14):7796–803.
 Zhang H, Huang Y, Fayad R, Spear GT, Qiao L. Induction
of mucosal and systemic neutralizing antibodies against human
immunodeficiency virus type 1 (HIV-1) by oral immunization with
bovine Papillomavirus-HIV-1 gp41 chimeric virus-like particles. J
 Graham BS, Wright PF. Candidate AIDS vaccines. N Engl J Med
 Jeffs SA, Goriup S, Kebble B, Crane D, Bolgiano B, Sattentau Q,
et al. Expression and characterisation of recombinant oligomeric
envelope glycoproteins derived from primary isolates of HIV-1.
 Stott EJ, Almond N, Kent K, Walker B, Hull R, Rose J, et al.
Evaluation of a candidate human immunodeficiency virus type 1
(HIV-1) vaccine in macaques: effect of vaccination with HIV-1
gp120 on subsequent challenge with heterologous simian immun-
virions with increased
odeficiency virus-HIV-1 chimeric virus. J Gen Virol 1998;79(Part
 Graham BS, McElrath MJ, Connor RI, Schwartz DH, Gorse
GJ, Keefer MC, et al. Analysis of intercurrent human immun-
odeficiency virus type 1 infections in phase I and II trials of
candidate AIDS vaccines. AIDS Vaccine Evaluation Group, and
the Correlates of HIV Immune Protection Group. J Infect Dis
 Yang X, Lee J, Mahony EM, Kwong PD, Wyatt R, Sodroski J.
Highly stable trimers formed by human immunodeficiency virus
type 1 envelope glycoproteins fused with the trimeric motif of T4
bacteriophage fibritin. J Virol 2002;76(9):4634–42.
 Forsell MN, Li Y, Sundback M, Svehla K, Liljestrom P, Mas-
cola JR, et al. Biochemical and immunogenic characterization
of soluble human immunodeficiency virus type 1 envelope gly-
coprotein trimers expressed by semliki forest virus. J Virol
 Barnett AL, Cunningham JM. Receptor binding transforms the
surface subunit of the mammalian C-type retrovirus envelope
protein from an inhibitor to an activator of fusion. J Virol
 Srivastava IK, Stamatatos L, Kan E, Vajdy M, Lian Y, Hilt S, et
al. Purification, characterization, and immunogenicity of a soluble
trimeric envelope protein containing a partial deletion of the V2
loop derived from SF162, an R5-tropic human immunodeficiency
virus type 1 isolate. J Virol 2003;77(20):11244–59.
 Srivastava IK, VanDorsten K, Vojtech L, Barnett SW, Stamatatos
L. Changes in the immunogenic properties of soluble gp140
human immunodeficiency virus envelope constructs upon partial
deletion of the second hypervariable region. J Virol 2003;77(4):
 Lian Y, Srivastava I, Gomez-Roman VR, Zur Megede J, Sun Y,
Kan E, et al. Evaluation of envelope vaccines derived from the
South African subtype C human immunodeficiency virus type 1
TV1 strain. J Virol 2005;79(21):13338–49.
 Li Y, Svehla K, Mathy NL, Voss G, Mascola JR, Wyatt
R. Characterization of antibody responses elicited by human
immunodeficiency virus type 1 primary isolate trimeric and
monomeric envelope glycoproteins in selected adjuvants. J Virol
 Binley JM, Sanders RW, Clas B, Schuelke N, Master A, Guo Y, et
al. A recombinant human immunodeficiency virus type 1 envelope
glycoprotein complex stabilized by an intermolecular disulfide bond
between the gp120 and gp41 subunits is an antigenic mimic of the
trimeric virion-associated structure. J Virol 2000;74(2):627–43.
 Sanders RW, Vesanen M, Schuelke N, Master A, Schiffner L,
Kalyanaraman R, et al. Stabilization of the soluble, cleaved,
trimeric form of the envelope glycoprotein complex of human
immunodeficiency virus type 1. J Virol 2002;76(17):8875–89.
 Beddows S, Schulke N, Kirschner M, Barnes K, Franti M,
Michael E, et al. Evaluating the immunogenicity of a disulfide-
stabilized, cleaved, trimeric form of the envelope glycoprotein
complex of human immunodeficiency virus type 1. J Virol
 Devico A, Silver A, Thronton AM, Sarngadharan MG, Pal R. Cova-
lently crosslinked complexes of human immunodeficiency virus
type 1 (HIV-1) gp120 and CD4 receptor elicit a neutralizing
immune response that includes antibodies selective for primary
virus isolates. Virology 1996;218(1):258–63.
 Fouts TR, Tuskan R, Godfrey K, Reitz M, Hone D, Lewis GK, et
al. Expression and characterization of a single-chain polypeptide
analogue of the human immunodeficiency virus type 1 gp120-CD4
receptor complex. J Virol 2000;74(24):11427–36.
 Fouts T, Godfrey K, Bobb K, Montefiori D, Hanson CV, Kalya-
naraman VS, et al. Crosslinked HIV-1 envelope-CD4 receptor com-
plexes elicit broadly cross-reactive neutralizing antibodies in rhesus
macaques. Proc Natl Acad Sci U S A 2002;99(18):11842–7.
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
 Pantophlet R, Wilson IA, Burton DR. Hyperglycosylated mutants
of human immunodeficiency virus (HIV) type 1 monomeric
gp120 as novel antigens for HIV vaccine design. J Virol
 McGaughey GB, Barbato G, Bianchi E, Freidinger RM, Garsky
VM, Hurni WM, et al. Progress towards the development of a
HIV-1 gp41-directed vaccine. Curr HIV Res 2004;2(2):193–204.
 Lenz O, Dittmar MT, Wagner A, Ferko B, Vorauer-Uhl K, Stiegler
G, et al. Trimeric membrane-anchored gp41 inhibits HIV mem-
brane fusion. J Biol Chem 2005;280(6):4095–101.
 Pantophlet R, Burton DR. GP120: target for neutralizing HIV-1
antibodies. Annu Rev Immunol 2006.
 Kuiken CL, Foley B, Freed E, Hahn BH, McCutchan FE, Mellors
JW, et al. HIV Sequence Compendium 2002, Theoretical Biology
and Biophysics Group. Los Alamos, NM: Los Alamos Laboratory;
 Hurwitz JL, Slobod KS, Lockey TD, Wang S, Chou TH, Lu S.
Application of the polyvalent approach to HIV-1 vaccine develop-
ment. Curr Drug Targets Infect Disord 2005;5(2):143–56.
 Mascola JR, Sambor A, Beaudry K, Santra S, Welcher B, Louder
MK, et al. Neutralizing antibodies elicited by immunization of
monkeys with DNA plasmids and recombinant adenoviral vectors
expressing human immunodeficiency virus type 1 proteins. J Virol
 Seaman MS, Xu L, Beaudry K, Martin KL, Beddall MH, Miura A,
et al. Multiclade human immunodeficiency virus type 1 envelope
immunogens elicit broad cellular and humoral immunity in rhesus
monkeys. J Virol 2005;79(5):2956–63.
 Gao F, Weaver EA, Lu Z, Li Y, Liao HX, Ma B, et al. Antigenic-
ity and immunogenicity of a synthetic human immunodeficiency
virus type 1 group m consensus envelope glycoprotein. J Virol
 Doria-Rose NA, Learn GH, Rodrigo AG, Nickle DC, Li F, Maha-
lanabis M, et al. Human immunodeficiency virus type 1 subtype B
ancestral envelope protein is functional and elicits neutralizing anti-
bodies in rabbits similar to those elicited by a circulating subtype
B envelope. J Virol 2005;79(17):11214–24.
 Thomson SA, Jaramillo AB, Shoobridge M, Dunstan KJ, Everett
B, Ranasinghe C, et al. Development of a synthetic consensus
sequence scrambled antigen HIV-1 vaccine designed for global use.
 Gallo RC. Tat as one key to HIV-induced immune pathogenesis
and Tat (correction of Pat) toxoid as an important component of a
vaccine. Proc Natl Acad Sci U S A 1999;96(15):8324–6.
 Ferrantelli F, Cafaro A, Ensoli B. Nonstructural HIV proteins as
targets for prophylactic or therapeutic vaccines. Curr Opin Biotech-
 Rezza G, Fiorelli V, Dorrucci M, Ciccozzi M, Tripiciano A,
Scoglio A, et al. The presence of anti-Tat antibodies is predic-
tive of long-term nonprogression to AIDS or severe immunodefi-
ciency: findings in a cohort of HIV-1 seroconverters. J Infect Dis
 Cafaro A, Caputo A, Fracasso C, Maggiorella MT, Goletti D,
Baroncelli S, et al. Control of SHIV-89.6P-infection of cynomol-
gus monkeys by HIV-1 Tat protein vaccine. Nat Med 1999;5(6):
 Osterhaus AD, van Baalen CA, Gruters RA, Schutten M, Siebelink
CH, Hulskotte EG, et al. Vaccination with Rev and Tat against
AIDS. Vaccine 1999;17(20–21):2713–4.
 Maggiorella MT, Baroncelli S, Michelini Z, Fanales-Belasio E,
Moretti S, Sernicola L, et al. Long-term protection against
SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus mon-
keys. Vaccine 2004;22(25–26):3258–69.
 Pauza CD, Trivedi P, Wallace M, Ruckwardt TJ, Le Buanec H,
Lu W, et al. Vaccination with tat toxoid attenuates disease in
simian/HIV-challenged macaques. Proc Natl Acad Sci U S A
 Silvera P, Richardson MW, Greenhouse J, Yalley-Ogunro J, Shaw
N, Mirchandani J, et al. Outcome of simian-human immunodefi-
ciency virus strain 89.6p challenge following vaccination of rhesus
macaques with human immunodeficiency virus Tat protein. J Virol
 Allen TM, Mortara L, Mothe BR, Liebl M, Jing P, Calore B, et al.
Tat-vaccinated macaques do not control simian immunodeficiency
virus SIVmac239 replication. J Virol 2002;76(8):4108–12.
 Liang X, Casimiro DR, Schleif WA, Wang F, Davies ME, Zhang
ZQ, et al. Vectored Gag and Env but not Tat show efficacy against
simian-human immunodeficiency virus 89.6P challenge in Mamu-
A*01-negative rhesus monkeys. J Virol 2005;79(19):12321–31.
 Caputo A, Gavioli R, Ensoli B. Recent advances in the development
of HIV-1 Tat-based vaccines. Curr HIV Res 2004;2(4):357–76.
 Ensoli B. Rational vaccine strategies against AIDS: background
and rationale. Microbes Infect 2005;7(14):1445–52.
 Mascarell L, Fayolle C, Bauche C, Ladant D, Leclerc C. Induction
of neutralizing antibodies and Th1-polarized and CD4-independent
CD8+ T-cell responses following delivery of human immunodefi-
ciency virus type 1 Tat protein by recombinant adenylate cyclase
of Bordetella pertussis. J Virol 2005;79(15):9872–84.
 Voss G, Manson K, Montefiori D, Watkins DI, Heeney J, Wyand
M, et al. Prevention of disease induced by a partially heterologous
AIDS virus in rhesus monkeys by using an adjuvanted multicom-
ponent protein vaccine. J Virol 2003;77(2):1049–58.
 Sauter SL, Rahman A, Muralidhar G. Non-replicating viral vector-
based AIDS vaccines: interplay between viral vectors and the
immune system. Curr HIV Res 2005;3(2):157–81.
 Letvin NL, Montefiori DC, Yasutomi Y, Perry HC, Davies ME,
Lekutis C, et al. Potent, protective anti-HIV immune responses
generated by bimodal HIV envelope DNA plus protein vaccination.
Proc Natl Acad Sci U S A 1997;94(17):9378–83.
 Barouch DH, Craiu A, Kuroda MJ, Schmitz JE, Zheng XX,
Santra S, et al. Augmentation of immune responses to HIV-1 and
simian immunodeficiency virus DNA vaccines by IL-2/Ig plas-
mid administration in rhesus monkeys. Proc Natl Acad Sci U S
 Boyer JD, Ugen KE, Wang B, Agadjanyan M, Gilbert L, Bagarazzi
ML, et al. Protection of chimpanzees from high-dose heterolo-
gous HIV-1 challenge by DNA vaccination. Nat Med 1997;3(5):
 Calarota SA, Weiner DB. Approaches for the design and evalua-
tion of HIV-1 DNA vaccines. Expert Rev Vaccines 2004;3(Suppl.
 Giri M, Ugen KE, Weiner DB. DNA vaccines against human
immunodeficiency virus type 1 in the past decade. Clin Microbiol
 Nabel GJ. HIV vaccine strategies. Vaccine 2002;20(15):1945–7.
 Casimiro DR, Wang F, Schleif WA, Liang X, Zhang ZQ, Tobery
TW, et al. Attenuation of simian immunodeficiency virus SIV-
mac239 infection by prophylactic immunization with dna and
recombinant adenoviral vaccine vectors expressing Gag. J Virol
 McDermott AB, O’Connor DH, Fuenger S, Piaskowski S, Martin
S, Loffredo J, et al. Cytotoxic T-lymphocyte escape does not always
explain the transient control of simian immunodeficiency virus SIV-
mac239 viremia in adenovirus-boosted and DNA-primed Mamu-
A*01-positive rhesus macaques. J Virol 2005;79(24):15556–66.
 Pialoux G, Excler JL, Riviere Y, Gonzalez-Canali G, Feuillie V,
Coulaud P, et al. A prime-boost approach to HIV preventive vaccine
using a recombinant canarypox virus expressing glycoprotein 160
(MN) followed by a recombinant glycoprotein 160 (MN/LAI). The
AGIS Group, and l’Agence Nationale de Recherche sur le SIDA.
AIDS Res Hum Retroviruses 1995;11(3):373–81.
 Belshe RB, Stevens C, Gorse GJ, Buchbinder S, Weinhold K, Shep-
pard H, et al. Safety and immunogenicity of a canarypox-vectored
human immunodeficiency virus Type 1 vaccine with or without
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
gp120: a phase 2 study in higher- and lower-risk volunteers. J
Infect Dis 2001;183(9):1343–52.
 Paris R, Bejrachandra S, Karnasuta C, Chandanayingyong D,
Kunachiwa W, Leetrakool N, et al. HLA class I serotypes and
cytotoxic T-lymphocyte responses among human immunodeficiency
virus-1-uninfected Thai volunteers immunized with ALVAC-HIV in
combination with monomeric gp120 or oligomeric gp160 protein
boosting. Tissue Antigens 2004;64(3):251–6.
 Amara RR, Villinger F, Altman JD, Lydy SL, O’Neil SP, Staprans
SI, et al. Control of a mucosal challenge and prevention of AIDS by
a multiprotein DNA/MVA vaccine. Science 2001;292(5514):69–74.
 Im EJ, Hanke T. MVA as a vector for vaccines against HIV-1.
Expert Rev Vaccines 2004;3(Suppl. 4):S89–97.
 Kent SJ, Zhao A, Best SJ, Chandler JD, Boyle DB, Ramshaw
IA. Enhanced T-cell immunogenicity and protective efficacy of a
human immunodeficiency virus type 1 vaccine regimen consisting
of consecutive priming with DNA and boosting with recombinant
fowlpox virus. J Virol 1998;72(12):10180–8.
 Shiver JW, Fu TM, Chen L, Casimiro DR, Davies ME, Evans
RK, et al. Replication-incompetent adenoviral vaccine vector
elicits effective anti-immunodeficiency-virus immunity. Nature
 Casimiro DR, Chen L, Fu TM, Evans RK, Caulfield MJ, Davies
ME, et al. Comparative immunogenicity in rhesus monkeys of DNA
plasmid, recombinant vaccinia virus, and replication-defective ade-
novirus vectors expressing a human immunodeficiency virus type
1 gag gene. J Virol 2003;77(11):6305–13.
 Shiver JW, Emini EA. Recent advances in the development of HIV-
1 vaccines using replication-incompetent adenovirus vectors. Annu
Rev Med 2004;55:355–72.
 Santra S, Seaman MS, Xu L, Barouch DH, Lord CI, Lifton MA,
et al. Replication-defective adenovirus serotype 5 vectors elicit
durable cellular and humoral immune responses in nonhuman pri-
mates. J Virol 2005;79(10):6516–22.
 Kostense S, Koudstaal W, Sprangers M, Weverling GJ, Penders
G, Helmus N, et al. Adenovirus types 5 and 35 seroprevalence
in AIDS risk groups supports type 35 as a vaccine vector. AIDS
 Vogels R, Zuijdgeest D, van Rijnsoever R, Hartkoorn E, Damen I,
de Bethune MP, et al. Replication-deficient human adenovirus type
35 vectors for gene transfer and vaccination: efficient human cell
infection and bypass of preexisting adenovirus immunity. J Virol
 Barouch DH, Pau MG, Custers JH, Koudstaal W, Kostense S,
Havenga MJ, et al. Immunogenicity of recombinant adenovirus
serotype 35 vaccine in the presence of pre-existing anti-Ad5 immu-
nity. J Immunol 2004;172(10):6290–7.
 Nanda A, Lynch DM, Goudsmit J, Lemckert AA, Ewald BA,
Sumida SM, et al. Immunogenicity of recombinant fiber-chimeric
adenovirus serotype 35 vector-based vaccines in mice and rhesus
monkeys. J Virol 2005;79(22):14161–8.
 Patterson LJ, Malkevitch N, Pinczewski J, Venzon D, Lou Y, Peng
B, et al. Potent, persistent induction and modulation of cellular
immune responses in rhesus macaques primed with Ad5hr-simian
immunodeficiency virus (SIV) env/rev, gag, and/or nef vaccines
and boosted with SIV gp120. J Virol 2003;77(16):8607–20.
 Robert-Guroff M, Kaur H, Patterson LJ, Leno M, Conley AJ,
McKenna PM, et al. Vaccine protection against a heterologous,
non-syncytium-inducing, primary human immunodeficiency virus.
J Virol 1998;72(12):10275–80.
 Peng B, Wang LR, Gomez-Roman VR, Davis-Warren A, Mon-
tefiori DC, Kalyanaraman VS, et al. Replicating rather than non-
replicating adenovirus-human immunodeficiency virus recombinant
vaccines are better at eliciting potent cellular immunity and priming
high-titer antibodies. J Virol 2005;79(16):10200–9.
 Zhao J, Pinczewski J, Gomez-Roman VR, Venzon D, Kalyanara-
man VS, Markham PD, et al. Improved protection of rhesus
macaques against intrarectal simian immunodeficiency virus
SIV(mac251) challenge by a replication-competent Ad5hr-SIVenv/
rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regi-
men. J Virol 2003;77(15):8354–65.
 Lorin C, Mollet L, Delebecque F, Combredet C, Hurtrel B,
Charneau P, et al. A single injection of recombinant measles virus
vaccines expressing human immunodeficiency virus (HIV) type 1
clade B envelope glycoproteins induces neutralizing antibodies and
cellular immune responses to HIV. J Virol 2004;78(1):146–57.
 Rose NF, Marx PA, Luckay A, Nixon DF, Moretto WJ, Donahoe
SM, et al. An effective AIDS vaccine based on live attenuated
vesicular stomatitis virus recombinants. Cell 2001;106(5):539–49.
 Roberts A, Kretzschmar E, Perkins AS, Forman J, Price R,
Buonocore L, et al. Vaccination with a recombinant vesicular
stomatitis virus expressing an influenza virus hemagglutinin pro-
vides complete protection from influenza virus challenge. J Virol
 Publicover J, Ramsburg E, Rose JK. A single-cycle vaccine vector
based on vesicular stomatitis virus can induce immune responses
comparable to those generated by a replication-competent vector.
J Virol 2005;79(21):13231–8.
 Matano T, Kano M, Nakamura H, Takeda A, Nagai Y. Rapid
appearance of secondary immune responses and protection from
acute CD4 depletion after a highly pathogenic immunodeficiency
virus challenge in macaques vaccinated with a DNA prime/Sendai
virus vector boost regimen. J Virol 2001;75(23):11891–6.
 Takeda A, Igarashi H, Nakamura H, Kano M, Iida A, Hirata T,
et al. Protective efficacy of an AIDS vaccine, a single DNA prim-
ing followed by a single booster with a recombinant replication-
defective Sendai virus vector, in a macaque AIDS model. J Virol
 Perri S, Greer CE, Thudium K, Doe B, Legg H, Liu H, et al.
An alphavirus replicon particle chimera derived from venezuelan
equine encephalitis and sindbis viruses is a potent gene-based vac-
cine delivery vector. J Virol 2003;77(19):10394–403.
 Davis NL, Caley IJ, Brown KW, Betts MR, Irlbeck DM, McGrath
KM, et al. Vaccination of macaques against pathogenic simian
immunodeficiency virus with Venezuelan equine encephalitis virus
replicon particles. J Virol 2000;74(1):371–8.
 Liu XL, Clark KR, Johnson PR. Production of recombinant adeno-
associated virus vectors using a packaging cell line and a hybrid
recombinant adenovirus. Gene Ther 1999;6(2):293–9.
 Johnson PR, Schnepp BC, Connell MJ, Rohne D, Robinson S,
Krivulka GR, et al. Novel adeno-associated virus vector vaccine
restricts replication of simian immunodeficiency virus in macaques.
J Virol 2005;79(2):955–65.
 Someya K, Cecilia D, Ami Y, Nakasone T, Matsuo K, Burda S,
et al. Vaccination of rhesus macaques with recombinant Mycobac-
terium bovis bacillus Calmette-Guerin Env V3 elicits neutralizing
antibody-mediated protection against simian-human immunodefi-
ciency virus with a homologous but not a heterologous V3 motif.
J Virol 2005;79(3):1452–62.
 Ami Y, Izumi Y, Matsuo K, Someya K, Kanekiyo M, Horibata S, et
al. Priming-boosting vaccination with recombinant Mycobacterium
bovis bacillus Calmette-Guerin and a nonreplicating vaccinia virus
recombinant leads to long-lasting and effective immunity. J Virol
 Devico AL, Fouts TR, Shata MT, Kamin-Lewis R, Lewis GK,
Hone DM. Development of an oral prime-boost strategy to
elicit broadly neutralizing antibodies against HIV-1. Vaccine
 Crotty S, Miller CJ, Lohman BL, Neagu MR, Compton L,
Lu D, et al. Protection against simian immunodeficiency virus
vaginal challenge by using Sabin poliovirus vectors. J Virol
 Wu L, Kong WP, Nabel GJ. Enhanced breadth of CD4 T-cell
immunity by DNA prime and adenovirus boost immunization to
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
human immunodeficiency virus Env and Gag immunogens. J Virol
 Casimiro DR, Bett AJ, Fu TM, Davies ME, Tang A, Wilson
KA, et al. Heterologous human immunodeficiency virus type
1 priming-boosting immunization strategies involving replication-
defective adenovirus and poxvirus vaccine vectors. J Virol
 Lemckert AA, Sumida SM, Holterman L, Vogels R, Truitt DM,
Lynch DM, et al. Immunogenicity of heterologous prime-boost reg-
imens involving recombinant adenovirus serotype 11 (Ad11) and
Ad35 vaccine vectors in the presence of anti-ad5 immunity. J Virol
 Yewdell JW, Bennink JR. Immunodominance in major histocom-
patibility complex class I-restricted T lymphocyte responses. Annu
Rev Immunol 1999;17:51–88.
 Ishioka GY, Fikes J, Hermanson G, Livingston B, Crimi C, Qin M,
et al. Utilization of MHC class I transgenic mice for development
of minigene DNA vaccines encoding multiple HLA-restricted CTL
epitopes. J Immunol 1999;162(7):3915–25.
 Hanke T, Samuel RV, Blanchard TJ, Neumann VC, Allen TM,
Boyson JE, et al. Effective induction of simian immunodeficiency
virus-specific cytotoxic T lymphocytes in macaques by using a
multiepitope gene and DNA prime-modified vaccinia virus Ankara
boost vaccination regimen. J Virol 1999;73(9):7524–32.
 Allen TM, Vogel TU, Fuller DH, Mothe BR, Steffen S, Boyson
JE, et al. Induction of AIDS virus-specific CTL activity in fresh,
unstimulated peripheral blood lymphocytes from rhesus macaques
vaccinated with a DNA prime/modified vaccinia virus Ankara boost
regimen. J Immunol 2000;164(9):4968–78.
 Subbramanian RA, Kuroda MJ, Charini WA, Barouch DH,
Costantino C, Santra S, et al. Magnitude and diversity of cytotoxic-
T-lymphocyte responses elicited by multiepitope DNA vaccination
in rhesus monkeys. J Virol 2003;77(18):10113–8.
 McEvers K, Elrefaei M, Norris P, Deeks S, Martin J, Lu Y, et
al. Modified anthrax fusion proteins deliver HIV antigens through
MHC Class I and II pathways. Vaccine 2005;23(32):4128–35.
 Mortara L, Gras-Masse H, Rommens C, Venet A, Guillet JG,
Bourgault-Villada I. Type 1 CD4(+) T-cell help is required
for induction of antipeptide multispecific cytotoxic T lympho-
cytes by a lipopeptidic vaccine in rhesus macaques. J Virol
 Gahery-Segard H, Pialoux G, Charmeteau B, Sermet S, Poncelet
H, Raux M, et al. Multiepitopic B- and T-cell responses induced
in humans by a human immunodeficiency virus type 1 lipopeptide
vaccine. J Virol 2000;74(4):1694–703.
 Pialoux G, Gahery-Segard H, Sermet S, Poncelet H, Fournier
S, Gerard L, et al. Lipopeptides induce cell-mediated anti-
HIV immune responsesin
 Tonini T, Barnett S, Donnelly J, Rappuoli R. Current approaches to
developing a preventative HIV vaccine. Curr Opin Investig Drugs
 Srivastava IK, Ulmer JB, Barnett SW. Neutralizing antibody
responses to HIV: role in protective immunity and challenges for
vaccine design. Expert Rev Vaccines 2004;3(Suppl. 4):S33–52.
 Anderson R, Hanson M. Potential public health impact of imperfect
HIV type 1 vaccines. J Infect Dis 2005;191(Suppl. 1):S85–96.
 Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii S,
Soares H, et al. In vivo targeting of antigens to maturing dendritic
cells via the DEC-205 receptor improves T cell vaccination. J Exp
 Rosenberg ES, Billingsley JM, Caliendo AM, Boswell SL,
Sax PE, Kalams SA, et al. Vigorous HIV-1-specific CD4+
T cell responses associated with control of viremia. Science
 Imami N, Pires A, Hardy G, Wilson J, Gazzard B, Gotch F. A
balanced type 1/type 2 response is associated with long-term non-
progressive human immunodeficiency virus type 1 infection. J Virol
 Younes SA, Yassine-Diab B, Dumont AR, Boulassel MR,
Grossman Z, Routy JP, et al. HIV-1 viremia prevents the
establishment of interleukin 2-producing HIV-specific memory
CD4+ T cells endowed with proliferative capacity. J Exp Med
 Imami N, Pires A, Burton C. The challenge of developing
an effective HIV-I vaccine. Drug Dis Today:Ther Strategies
 Haase AT. Perils at mucosal front lines for HIV and SIV and their
hosts. Nat Rev Immunol 2005;5(10):783–92.
 Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat
Med 2005;11(Suppl. 4):S45–53.
 Belyakov IM, Hel Z, Kelsall B, Kuznetsov VA, Ahlers JD, Nacsa
J, et al. Mucosal AIDS vaccine reduces disease and viral load in
gut reservoir and blood after mucosal infection of macaques. Nat
 Belyakov IM, Ahlers JD, Berzofsky JA. Mucosal AIDS vac-
cines: current status and future directions. Expert Rev Vaccines
 Kuznetsov VA, Stepanov VS, Berzofsky JA, Belyakov IM. Assess-
ment of the relative therapeutic effects of vaccines on virus load
and immune responses in small groups at several time points:
efficacy of mucosal and subcutaneous polypeptide vaccines in rhe-
sus macaques exposed to SHIV. J Clin Virol 2004;31(Suppl. 1):
 Gupta S, Janani R, Bin Q, Luciw P, Greer C, Perri S, et
al. Characterization of human immunodeficiency virus Gag-
specific gamma interferon-expressing cells following protective
mucosal immunization with alphavirus replicon particles. J Virol
 Stahl-Hennig C, Steinman RM, Ten Haaft P, Uberla K, Stolte N,
Saeland S, et al. The simian immunodeficiency virus deltaNef vac-
cine, after application to the tonsils of Rhesus macaques, replicates
primarily within CD4(+) T cells and elicits a local perforin-positive
CD8(+) T-cell response. J Virol 2002;76(2):688–96.
 Tenner-Racz K, Stahl Hennig C, Uberla K, Stoiber H, Ignatius
R, Heeney J, et al. Early protection against pathogenic virus
infection at a mucosal challenge site after vaccination with atten-
uated simian immunodeficiency virus. Proc Natl Acad Sci U S A
 Jiang JQ, Patrick A, Moss RB, Rosenthal KL. CD8+ T-cell-
mediated cross-clade protection in the genital tract following
intranasal immunization with inactivated human immunodefi-
ciency virus antigen plus CpG oligodeoxynucleotides. J Virol
 Berzofsky JA, Ahlers JD, Janik J, Morris J, Oh S, Terabe M, et al.
Progress on new vaccine strategies against chronic viral infections.
J Clin Invest 2004;114(4):450–62.
 Mitragotri S. Immunization without needles. Nat Rev Immunol
 Matoba N, Magerus A, Geyer BC, Zhang Y, Muralidharan M,
Alfsen A, et al. A mucosally targeted subunit vaccine candidate
eliciting HIV-1 transcytosis-blocking Abs. Proc Natl Acad Sci U
S A 2004;101(37):13584–9.
 Klausner RD, Fauci AS, Corey L, Nabel GJ, Gayle H, Berkley
S, et al. Medicine. The need for a global HIV vaccine enterprise.
 Cohen J. Immunology. New virtual center aims to speed AIDS
vaccine progress. Science 2005;309(5734):541.
 Kaufmann SH, McMichael AJ. Annulling a dangerous liaison:
vaccination strategies against AIDS and tuberculosis. Nat Med
 Flores JE. The HIV-1 pipeline and global expansion of clini-
cal trials. Keystone meeting, Banff, Canada, 2005 [Abstract no
M.P. Girard et al. / Vaccine xxx (2006) xxx–xxx
 Ellenberger D, Otten R, Li B, Rodriguez IV, Sariol C, Martinez
M, et al. Evidence of protection after repeated mucosal SHIV chal-
lenges to HIV-1 vaccinated rhesus monkeys. AIDS Vaccine 2005
meeting, Montreal, Quebec, Canada, September 6–9, 2005 [abstract
 Clarke D, Johnson E, Nasar F, Coleman J, Coorer D, Calderon
P, et al. Assessment of rVSV/HIV vaccine vector neurovirulence
in murine, ferret and non-human primate models. AIDS Vaccine
2005, Montreal, Canada, 2005 [abstract no 13].
 Arnold G, Velasco P, Wrin T, Hughes S, Elsasser D, Ma X, et al.
Recombinant human rhinovirus displaying the HIV-1 gp41 ELD-
KWA epitope can elicit broad neutralization of HIV-1 primary iso-
lates. AIDS Vaccine 2005, Montreal, Canada, 2005 [abstract 500].
 Kawada M, Igarashi H, Takeda A, Tsukamoto T, Yamamoto H,
Dohki S, et al. Involvement of multiple epitope-specific cyto-
toxic T-lymphocyte responses in vaccine-based control of simian
immunodeficiency virus replication in rhesus macaques. J Virol