Vaccine 24 (2006) 4062–4081

Review

A review of vaccine research and development: The human

immunodeficiency virus (HIV)夽
Marc P. Girard a,∗ , Saladin K. Osmanov b,1 , Marie Paule Kieny b,1
a University Paris 7, 39 rue Seignemartin, FR 69008 Lyon, France
b Initiative 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
Available online 28 February 2006

Abstract

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

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4063
2. Disease burden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4063
3. Virology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4064
4. HIV vaccine reseach: challenges and difficulties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4065
4.1. Why is it so difficult to develop an HIV vaccine? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4065
4.2. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4066
5. Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4066
5.1. Live attenuated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4066
5.2. Inactivated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4066
5.3. Virus-like particles (VLP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4067
5.4. Subunit vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4067

夽 The authors alone are responsible for the views expressed in this publication, which does not necessarily reflect the views of the World Health

Organization.
∗ Corresponding author. Tel.: +33 478 748 531.

E-mail addresses: marc.girard36@wanadoo.fr (M.P. Girard), osmanovs@who.int (S.K. Osmanov), kienym@who.int (M.P. Kieny).
1 Tel.: +41 22 791 35 91.

0264-410X/$ – see front matter © 2006 World Health Organization. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2006.02.031
 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081 4063

5.4.1. Envelope-based subunit vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4067

5.4.2. Non-structural protein subunit vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4068
5.5. Naked DNA and live recombinant vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4068
5.6. The development of prime-boost combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4071
5.7. Fusion proteins and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4071
6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4071
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4073
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4073

1. Introduction 2. Disease burden

The Acquired Immunodeficiency Syndrome (AIDS) At the end of 2005, the global number of adults
emerged in the human population in the summer of 1981. and children living with HIV/AIDS was estimated by
There now is convincing evidence that AIDS is a zoonosis WHO/UNAIDS to have reached 40.3 million with an esti-
and that its etiological agent, the human immunodeficiency mated 2.9–3.5 million HIV-infected persons dying every year
virus (HIV), crossed the simian-human species barrier before from the disease [3].
the middle of the 19th century [1,2] and probably arose and Sub-Saharan Africa remains the hardest-hit region in the
spread among humans as a result of increased unsafe injec- world, with at least 25 million infected people, accounting
tion and transfusion practices in postcolonial Africa, enabling for 60% of the people living with HIV/AIDS and 77% of
the virus to adapt to humans through serial passages. Today, AIDS deaths in the world. The overwhelming majority of
HIV/AIDS is the leading cause of death in sub-Saharan HIV infections in the region stems from heterosexual trans-
Africa and the fourth biggest killer in the world. An esti- mission [18]. In many African countries, the overall HIV
mated 14,000 people/day (5 million persons/year, including prevalence in the adult population can be greater than 10%,
600,000 children less than 15 years of age) become infected with figures reaching up to 38.8% in some areas. Among
with HIV, with more than 95% of them living in underdevel- the most severely hit countries are South Africa, with more
oped regions of the world [3]. The number of HIV infections than 5.6 million infected people and a prevalence of 30%
is equally distributed between men and women, but infection among 21–29-year-old adults, especially women, together
rates in young women in today’s Africa are close to three with Botswana, Mozambique, Zimbabwe, and Tanzania.
times higher than those among young men, reflecting the The highest infection rates are found among commercial
degree to which gender inequities are driving the epidemic, sex workers, truck drivers and seasonal migrant workers.
as many women in developing countries lack socio-economic Sub-Saharan Africa also is home to an estimated 500,000
independence, education and access to health information HIV-infected infants who became infected prior to the intro-
and services, and have difficulty avoiding exposure to the duction of mother-to-child prevention programmes based
virus. on the use of antiretroviral drugs. In addition, sub-Saharan
The development of a safe, effective, easy to administer Africa faces numerous wars and civil conflicts, producing
and affordable AIDS vaccine is urgently needed. The first large numbers of refugees who are at heightened risk of con-
Phase I trial of an HIV vaccine was conducted in the USA tracting HIV. Life expectancy in South African countries like
in 1987. Since then, more than 35 candidate vaccines have Botswana, Swaziland or Zimbabwe, where a quarter to more
been tested in over 65 Phase I/II clinical trials, involving than a third of the general population is infected with HIV-1,
more than 10,000 healthy human volunteers in more than 10 has decreased from 65 years in 1985–1990 to 37 years in
countries [4,270]. Two Phase III trials have been carried to 2000–2005.
completion [5] and a third one is in progress [6]. However, A remarkable success story in the fight against AIDS was
we still are years away from an effective HIV vaccine, due achieved in Uganda, which faced the onset of a severe HIV
to multiple hurdles and challenges. The development of a epidemic in the mid 1980s. Through voluntary HIV coun-
safe and effective vaccine is hampered by the high genetic selling, expanded treatment of STDs, awareness campaigns
variability of the virus [7,8], the lack of knowledge of immune and community mobilization encouraging delayed initiation
correlates of protection [9,10], the difficulty of generating of sexual activity, monogamy and use of condoms, the level of
broadly neutralizing antibodies [11], the absence of relevant infection has declined significantly since 1992—from nearly
and predictive animal models and the complexities related to 30 to 11.2% in prenatal clinic settings in Kampala and from
the preparation and conduct of multiple large-scale clinical 13 to 5.9% in clinics outside major urban areas. Another
trials, especially in developing countries [12,13]. important HIV prevention strategy could be adult male cir-
This article reviews the current progress in HIV/AIDS cumcision, which was found in a recent study in South Africa
vaccine research and development and the many challenges to offer close to 65% protection rate from HIV infection. Two
that still need to be met in the field [14–17]. other randomized controlled trials are underway in Kenya and
4064 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
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
pace at which the epidemic is expanding. It has been projected
that if current trends continue, the region will contribute 40%
of all new infections worldwide by the end of the decade.
Increasing sex trade, use of illicit drugs, and high rates of
sexually transmitted infections contribute to an increased vul-
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
one of the sharpest increases in the number of new HIV infec-
tions, most of which occur among injecting drug users. The
number of people living with HIV/AIDS in the region is esti-
mated to be above 1.6 million, 70–80% of whom are less than
30 years of age. More than 300,000 HIV seropositive persons
are living in Russia alone, where commercial sex workers,
injection drug users and prisoners are major victims of the
tuberculosis and HIV epidemics [19].
The introduction of the Highly Active Antiretroviral Treat-
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
marginalized communities.
3. Virology
The HIV, together with the simian, the feline, and the
bovine immunodeficiency viruses (SIV, FIV, and BIV, respec-
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-
acteristically slow, progressive infections. Lentivirus replica-
tion depends on the presence of an active reverse transcriptase
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
genomic RNA.
Two types of HIV have been described: HIV-1 and HIV-2,
the latter being less virulent, less transmissible and geograph-
ically limited to West Africa. HIV-1 is phylogenetically close
to SIVcpz, a commensal virus in chimpanzees, and probably
arose as the result of a single transmission event from chim-
panzees to humans [1], whereas HIV-2 is closely related to
SIVmac, the etiological agent of simian AIDS, and to SIVsm,
a commensal virus in sooty mangabey monkeys. HIV-1 is
further subdivided into three groups, M (“major”), O (“out-
lier”), and N (“non M/non O”). The vast majority of the HIV-1
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 [20]. 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
Forms” (CRF), can gain epidemiological dominance [20–23].
The major CRFs are CRF02 AG, prevalent in western Africa,
CRF01 AE, which predominates in south-eastern Asia, and
CRF07 BC and 08 BC, which are prevalent in China. 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
RNA molecule, about 9.5 kb in length, which encodes the typ-
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 160 kD glyco-
protein eventually cleaved into an external gp120 subunit and
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 [24], the splice regulator pro-
tein Rev [25] and accessory proteins such as Nef [26,27], Vif
[28], Vpr [29] 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 24 (2006) 4062–4081
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 [30], and which lies on top of a layer of
matrix protein attached to the inside of the membrane through
embedded myristyl groups. However, the actual structure of
the virion still remains speculative [31].
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
(for a review, see [32]), and the gp41 transmembrane subunit,
which anchors the spikes in the viral envelope, maintains their
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
antibodies, such as IgG b12 and 2G12, have allowed the iden-
tification of several neutralization epitopes on gp120 [37],
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 [44] as well as by the hyper-
variable loops [45,46], which act as antigenic decoys [47].
Two monoclonal fusion-blocking antibodies (2F5, 4E10) also
have been described, with corresponding epitopes located at
the base of the gp41 ectodomain [48–53]. A note of caution
has been raised by the results of one study, which showed that
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
[56–58]. Similarly, recombination was shown to readily occur
between different SIV strains in SIV-infected monkeys [59].
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
4065
infection, before the virus can seed the lymphoid organs in
mucosal tissues [63,64].
HIV also has developed multiple mechanisms to circum-
vent the host immune responses including its ability to down-
regulate the major histocompatibility complex (MHC) class
I molecules and by doing so to minimize its recognition by
CTL, as well as its high genetic evolution rates, which allows
it to evade immune responses through the emergence of viral
CTL [3,65–68] and neutralizing antibody [69,70] escape vari-
ants.
Another difficulty with the development of an effective
HIV vaccine stems from the fact that the virus envelope
glycoprotein conceals its conserved receptor- and coreceptor-
binding sites in crypts that are masked by the hypervariable
loops of the molecule and by glycan residues [45,70,71].
Neutralizing antibodies induced in response to gp120 are pri-
marily targeted to the hypervariable loops of the molecule and
only rarely do they recognize the receptor binding sites, which
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
the world’s first two Phase III clinical trials of AIDS vaccines
[5,76].
Although neutralizing antibodies administered passively
to non-human primates can provide protection against exper-
imental SHIV infection [77–85], proof of their protective role
in infected humans remains circumstantial [86]. The best evi-
dence for a role of HIV-1 neutralizing antibodies in vivo is the
rapid and constant selection of neutralizing antibody-escape
variants during the course of infection [69,70]. However, con-
trary to laboratory-adapted virus strains, which use CXCR-4
as a coreceptor (“X4” strains), and against which protection in
chimpanzees could readily be achieved by inducing neutraliz-
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
between the appearance of HIV-specific CD8+ T cells and the
decline of primary viremia [92,93], the fact that several HLA
class I alleles (HLA-B57, HLA B-27, HLA-B63) are associ-
ated with slow disease progression [94], the early selection of
CTL escape viral mutants during primary infection [95–98]
and the rapid increase of viral loads in macaques infected with
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
4066 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
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
HIV are chimpanzees, Pan troglodytes, and pigtail macaques,
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 [102]. In contrast, Asian monkeys,
especially rhesus monkeys from India, are highly susceptible
to SIVmac infection and progressively develop an immun-
odeficiency syndrome, which fully mimics human AIDS
[103]. Plasma virus levels during primary and chronic SIV-
mac251 infection in macaques parallel those observed in
humans, some animals containing viremia spontaneously and
progressing to disease slowly, as in HIV-1 infected human
long-term non-progressors, and others maintaining high virus
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 [106], and evidence of immune escape is readily
documented [96–98]. Perhaps most importantly, the mucosal
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,
therefore, considered to be the most appropriate animal model
for studies on potential protective immune responses against
HIV [115,116].
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
animal eventually lead to the emergence of highly pathogenic
SHIV variants that are capable of wiping out the circulating
CD4+ T-cell population of the animal within a few weeks
after infection and generating a lethal immunodeficiency syn-
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
vaccine-induced immune responses than “R5” viruses such as
SIV [122,123]. SHIV strains with an “R5” tropism [124,125]
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 –105 TCID50, 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
do not correspond to the natural exposures to HIV in humans,
where concentrations of less than 103 to a few 105 HIV 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
high dose challenges [129]. Such a new type of mucosal chal-
lenge might change the results of preclinical vaccine efficacy
studies in the future (see as an example, [271]).
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
humans.
5. Vaccines
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

nef mutant establishes a life-long, persistent low-grade viral
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-
cerns, this approach has not been actively pursued [138,139],
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 [140].
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-
vated HIV vaccines. The obstacle was circumvented by using
sublethal doses of formalin followed by heat inactivation
at 62 ◦ C [141]. The resulting killed virus preparation was
shown to induce in mice and non-human primates modest
but significant titers of antibodies capable of neutralizing het-
erologous primary isolates of HIV in a variety of infectivity
assays [142]. Another way to inactivate HIV or SIV infectiv-
ity has been to target the two nucleocapsid protein zinc finger
domains, which bind Zn ions in tetrahedral coordination com-
 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
plexes that are essential for virus infectivity. These complexes
can be disrupted by mild oxidation with 2,2 -dithiodipyridine
(aldithriol-2) [143] 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 [144].
The immunogenicity of the resulting preparations was stud-
ied in the SIV/macaque model [145]: 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
[148]. Gag–Env pseudovirions were also tested in guinea pigs
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
oligonucleotides [149].
Heterologous VLPs have also been used as a delivery vehi-
cle for HIV genes. Expression of the structural proteins of
Kunjin virus, an arbovirus, was used to prepare VLPs and
encapsidate recombinant Kunjin RNA replicons encoding the
HIV-1 gag gene [150]. 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-
tion with bovine papillomavirus (BPV)-HIV-1 gp41 chimeric
VLPs. The ELDKWA amino acid sequence (2F5 epitope)
from HIV-1 gp41 was fused to the N-terminus of the BPV L1
capsid protein and the resulting chimeric protein was used to
generate chimeric VLPs. The latter elicited gp41-specific IgG
after intra-muscular immunization and both systemic IgG and
intestinal secretory IgA after oral immunization in mice. HIV
neutralization activity could be detected in some of the mouse
sera [151].
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
4067
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 [155], and one
such vaccine, based on monomeric gp120 added with alum
(VaxGen), was tested in two double-blind, placebo-controlled
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 [5].
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 [6].
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 [161].
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 [162].
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
neutralizing and fusion-blocking antibodies [163,164]. Prim-
ing with DNA encoding a membrane-bound form of the
SOS gp140 protein followed by repeated immunization with
4068 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
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 [165].
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
has been developed by Merck and by the University of Mary-
land [166], which also engineered single chain derivatives of
gp120 covalently linked to the first two domains of the human
CD4 molecule [167]. The single chain fusion proteins were
shown to elicit a broadly neutralizing antibody response in
guinea pigs, but this property may have primarily been due to
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 [168]. 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 [64].
Engineering hyperglycosylated derivatives of gp120 was
attempted in order to target the immune response to conserved
epitopes on the molecule that form part of the CD4-binding
site [169]. 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 [172].
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
[173]. 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
as a boost [175,176]. In addition, novel avenues to circumvent
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 [11].
5.4.2. Non-structural protein subunit vaccines
The hope that immune responses directed against antigens
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 [186]. 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-specific T cell and antibody responses [189]. A detoxified
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 [190], 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
the same time, the AIDS Vaccine Integrated Project (AVIP), a
European consortium, is developing a novel vaccine approach
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 [191].
Different vaccine approaches using the Tat antigen have
also been developed such as a Tat-adenyl cyclase fusion pro-
tein [192] 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-
bined vaccine was able to prevent the development of AIDS in
rhesus monkeys following challenge with a pathogenic SHIV
strain [193]. The Tat–Nef fusion protein, which is developed
by GSK, was recently tested in human volunteers in a Phase I
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
the monkey model (see above). Although vaccines that stimu-
late the cellular arm of the immune response are not expected
to provide protection against infection, they should allow vac-
cinees who get infected to control virus replication and reduce
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 24 (2006) 4062–4081
developed based on this strategy, including naked DNA vac-
cines and live vectored recombinant vaccines, some of which
already are in advanced stages of clinical trials (For recent
reviews, see [17,194]).
Results from preclinical studies on plasmid DNA vac-
cines in non-human primates were rather disappointing, even
though successful protection against a SHIV challenge could
be observed in macaques [195,196] as well as in the HIV-
1/chimpanzee model [197]. Immunogenicity of the vaccines
could be improved by modification of the promoter, the use of
synthetic genes with optimized codons, insertion of the trans-
gene into an alphavirus replicon, or coexpression of cytokines
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
were developed by Wyeth, are being tested in Phase I trials in
the USA, Brazil and Thailand. DNA vaccines were actually
found to be most useful as priming vaccines in prime-boost
strategies, using live recombinant vaccines for booster immu-
nization (see below). These strategies were tested in monkeys
against pathogenic SHIV challenges and shown to result in
reduction in virus load and protection against disease and
death in the vaccinated animals [200]. 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
[206] and is currently still undergoing evaluation in Phase I/II
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-
laboration with IAVI is thus undergoing trial in India, whereas
another MVA recombinant that expresses subtype A,E env,
gag, pol and was developed by the NIH is in Phase I trial in
the USA, soon to be followed by Thailand. The fowlpox virus
(FPV) also has been used as a vector for HIV vaccines and has
been tested in Phase I trials in Australia [208]. It is currently
being evaluated in combination with MVA in the USA. These
three vectors were used to express a variety of HIV antigens,
such as Gag, Env, Pol and Nef. However, the immunogenicity
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
determined by IFN-␥ ELISPOT assays. This was emphasized
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
one of the most promising live viral vectors for HIV vaccines
[209–211]. It was first used by Merck for the development of a
4069
pilot, monovalent Ad5-gag recombinant that was shown to be
highly immunogenic in mice, nonhuman primates and human
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
has been launched, involving some 1200 men and 400 women
at high-risk who will be followed for 3 years after three immu-
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
[175]. The DNA vaccine was initially tested in Phase I trials
in the USA, where it showed good immunogenicity, and is
undergoing a Phase I trial in Uganda in collaboration with the
Makarere University and WRAIR. The VRC Ad5 recombi-
nant candidate vaccine also has undergone testing in a Phase I
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
obtained with the Ad5 recombinant vaccines, either delivered
alone or as boosting immunogens after plasmid DNA priming
[212]. However, these vaccines are confronted with the prob-
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
[213]. This has prompted the development by Merck, Cru-
cell and Transgene, in collaboration with IAVI, of candidate
vaccines based on less prevalent human adenovirus serotypes
(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
[216].
4070 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
A different vaccine strategy using adenovirus vectors
also has been developed by the NIH, employing replication-
competent Ad recombinants in which the Ad vector is deleted
only in the E3 region [217]. Ad4hr-, Ad5hr-, or Ad7hr-HIV
recombinants together with boosts of subunit envelope glyco-
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
model, using a replication-competent Ad5hr-SIV env/rev and
Ad5hr-SIV gag priming followed by SIV gp120 boosting
[220].
The list of other virus vectors used to construct live recom-
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 [221]. 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 [222]. 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,
for example as a single intranasal vaccination [223]. Potential
neurovirulence of VSV was circumvented in a new genera-
tion vector by reshuffling the viral N gene in the VSV genome
and truncating the G protein cytoplasmic tail [272]. 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 [224].
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 [225].
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
SHIV 89.6P and remained with undetectable virus load levels
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
approach is developed by Alphavax in the USA. Recombinant
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-
1 [157].
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
Zambia.
- BCG, which was developed as a vector for HIV by the
Japanese NIID and already underwent a Phase I trial in
Thailand with an early construct [231]. Recombinant BCG-
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
[232].
- Attenuated Salmonella, that are used as vectors for the
development of oral vaccines at the University of Mary-
land [233].
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 [234], 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-
tion program.
Another picornavirus, rhinovirus, recently has been engi-
neered to express the 2F5 epitope on its surface. Intranasal
administration of the recombinant virus induced broadly neu-
tralizing antibodies, which, if confirmed, would represent a
remarkable achievement [273].
It is too early at this time to evaluate the chances of success
of these various vector-based vaccine approaches.
 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
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 [236], or two successive adenovirus vectors, such as
Ad11 and Ad35, or two succcessive poxviruses, such as MVA
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 [237], emphasizing the advantage of multiple vector-
based vaccines. In rhesus monkeys, responses arising from an
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
vaccine.
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
vaccine such as recombinant plasmid DNA or a live recombi-
nant vectored vaccine with a subunit vaccine, especially one
based on envelope proteins (gp120, gp140 or gp41) [203].
There is no strong evidence, however, that such a dual vacci-
nation regimen resulted in a significantly higher antibody or
T-cell response.
For any prime-boost strategy to be commercially feasi-
ble, the combined regimen needs to demonstrate significantly
greater efficacy over single modality vaccines in order to bal-
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-
opment, either alone or in prime-boost combinations with live
vector-based recombinant vaccines. Vaccine constructs that
express a series of minimal epitopes arranged in a string-like
4071
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 [242]. 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-
tein.
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
WRAIR) [243].
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
humans without additional adjuvant [244–246]. Lipopeptides
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-
gens, but the trials had to be interrupted due to the occurrence
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-
neutralizing antibodies with broad specificity against primary
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
4072 M.P. Girard et al. / Vaccine 24 (2006) 4062–4081
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-
cell-stimulating vaccines, but not in the SIV/macaque model,
where virus control following vaccination and challenge
often appears to be short-lived, ultimately leading to vaccine
failure [122,123,201,202,274].
These vaccines, therefore, urgently need to be tested in
clinical trials on human volunteers at risk [15]. 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
induce protection against infection. Indeed, although the cor-
relation between viral loads at early times after infection and
speed of evolution of the disease are well documented in nat-
ural infection, the translation of this correlation in vaccinated
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
a major impact and significantly reduce HIV mortality within
20 years after introduction of the vaccine [249].
Among the great many hurdles that are met in the develop-
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
adopted todate in HIV vaccine research therefore aim at elicit-
ing an effective response of both arms of the immune system.
One way to increase the immune response to HIV would be to
help target the antigens to the antigen-presenting cells, espe-
cially dendritic cells (DC). Coupling the antigen of choice to a
DC-specific monoclonal antibody [250] has yielded impres-
sive results in mice, enhancing the efficiency and kinetics of
the immune response as compared with soluble antigen. This
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
acount.
Last, but not least, it would seem of paramount importance
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 [256].
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 [257].
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-
nization of macaque monkeys by the nasal or oral route using
a VSV vectored vaccine expressing SIV antigens Gag and
Env resulted in significant protection against SIV infection
[222]. Alphavirus replicons represent another type of recom-
binant vaccine that can be administered efficiently by the
mucosal route [260]. The administration of a HIV-1 VLP
vaccine by the mucosal route is under study [147,148]. The
tonsillar route of immunization has been explored in macaque
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
of nonliving vaccines by the nasal route still raises a few prob-
lems including that of adjuvants [263–265] The production of
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 [266].
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)
in the USA, as well as by the French National AIDS Research
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 24 (2006) 4062–4081
a Global HIV Vaccine Enterprise [267]. 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
coordinate the search for an HIV vaccine [268]. The HIV Vac-
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
to help developing countries strengthen their capacity in test-
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 [269]. Developing
countries are essential partners in this international effort.
There is little doubt that the development of a safe, effec-
tive, and affordable HIV vaccine constitutes a top priority for
HIV/AIDS research and a formidable scientific and public
health challenge at the dawn of this century.
Acknowledgement
The efficient editorial help of Olga Assossou is gratefully
acknowledged.
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