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Vaccine 31 (2013) 5506–5517
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Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Review
Obstacles to ideal anti-HIV antibody-dependent cellular cytotoxicity
responses
Leia H. Wren, Ivan Stratov, Stephen J. Kent, Matthew S. Parsons ∗
Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC, Australia
a r t i c l e
i n f o
Article history:
Received 30 June 2013
Received in revised form 11 August 2013
Accepted 13 August 2013
Available online 24 August 2013
Keywords:
HIV
ADCC
NK cells
a b s t r a c t
A safe and effective vaccine against HIV is a global health priority. Large-scale phase III clinical vaccine
trials based on neutralizing antibodies and cytotoxic T-lymphocytes have failed to provide protection,
highlighting the lack of understanding of basic immune correlates of protection against HIV. The partial
success of the RV144 vaccine trial, however, sparked an intense research effort to identify and describe the
protective potential of non-neutralizing antibodies. Correlates of protection analyses have identified antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) as potentially important. Despite
the attractiveness of utilizing ADCC antibodies for HIV vaccine design, it is important to note that effective
ADCC responses are contingent on many factors. As discussed in this review, these factors are important
considerations for determining the feasibility of designing an optimal ADCC antibody-inducing vaccine
construct. Important determinants of ADCC responses include characteristics of the antibody, such as isotype and subclass, antigen-specificity, titer, durability and glycosylation of the constant region. Second,
ADCC immune responses are highly contingent on the natural killer (NK) cell effectors. This review will
describe the current state of knowledge regarding the ontogeny of NK cells, highlighting the continuous
“education” they undergo that determines their functional potential upon stimulation. Other important
NK cell factors, such as constant region receptor polymorphisms, cellular exhaustion, and the effects of
the cytokine milieu on cellular function, will also be covered. Finally, an exciting, but yet untested, role
for NK cell-mediated ADCC lies in its potential ability to eliminate latently infected cells, which harbor
the viral reservoir. The review will address the potential of a two-pronged attack, where latently infected
cells are induced to express HIV antigens and then eliminated by NK cells via an ADCC mechanism, with
the goal of inducing a cure.
© 2013 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5507
Antibody approaches to HIV vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5507
ADCC antibodies against HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5507
Ab factors determining ADCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5507
4.1.
Ab isotype and subclass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5507
4.2.
Titer and clonal nature of Ab response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5508
4.3.
Ab Fc glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5508
4.4.
Galactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5508
4.5.
Fucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5508
4.6.
Sialic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5508
4.7.
Ideal ADCC Abs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5509
NK cell factors determining ADCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5509
5.1.
“Education” of NK cells to enhance their ability to mediate ADCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5509
5.2.
Receptor repertoire and regulation of activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5511
5.3.
NK cell “exhaustion” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5511
5.4.
The influence of FcR polymorphisms on ADCC immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5512
∗ Corresponding author. Tel.: +61 3 83449939; fax: +61 3 8344 3846.
E-mail address: [email protected] (M.S. Parsons).
0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.08.035
L.H. Wren et al. / Vaccine 31 (2013) 5506–5517
6.
7.
8.
9.
5507
Exogenous factors that influence ADCC immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5512
6.1.
Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5512
Potential integration of factors to eradicate latent HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5512
Potential integration of factors for vaccination against HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5513
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5514
Conflict of interest statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5514
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5514
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5514
1. Introduction
Human immunodeficiency virus (HIV) infection is a global
health concern. There are currently over 30 million HIV-infected
people, and United Nations AIDS (UNAIDS) reports over two million new infections annually [1]. Due to these staggering numbers,
there is an urgent need for novel prophylactic vaccine constructs
and innovative therapies for eliminating viral reservoirs from HIVinfected people. This review will discuss the evidence behind
designing antibody (Ab)-based vaccines/therapies against HIV. The
difficulties in eliciting anti-HIV neutralizing Abs and the protective
potential of non-neutralizing Abs will be addressed. The evidence
identifying Ab-dependent cellular cytotoxicity (ADCC) as an important non-neutralizing Ab-triggered effector function against HIV, as
well as the factors that determine the strength of ADCC responses
will be considered. Lastly, the review will integrate the many factors influencing ADCC for the design of therapeutics to eliminate
reactivated latent HIV or for the design of prophylactic vaccines.
2. Antibody approaches to HIV vaccines
The most promising research direction for designing an effective vaccine against HIV has been broadly neutralizing antibodies
(BnAbs). These Abs are capable of neutralizing a wide array of
viral isolates, both within and across HIV subtypes [2]. The production of Abs with this breadth of neutralization potential occurs in
approximately 20% of infected individuals [3]. A growing number
of monoclonal prototypes for these BnAbs have been discovered,
purified and mass-produced from a subset of such HIV-infected
individuals. These Abs are directed against an array of epitopes
on the HIV envelope (Env), which consists of homotrimers of
the non-covalently associated gp120 and gp41, including the CD4
binding-site of gp120 [4], the glycan shield [5], the membrane
proximal external region of gp41 [6] and epitopes dependent
on trimer formation [2]. The efficacy of BnAbs in preventing
infection has been demonstrated in primate models utilizing the
chimeric simian-human immunodeficiency virus (SHIV) [7–10].
Passive transfer of BnAbs to rhesus macaques prior to challenge
with SHIV has repeatedly been demonstrated to provide sterilizing
immunity. Given this remarkable observation, much consideration
has been given to designing vaccines capable of eliciting anti-HIV
BnAbs through vaccination. Unfortunately, a BnAb-inducing vaccine has yet to be designed. It is generally thought that novel
vaccination constructs and strategies will be required to elicit Abs
with the extensive somatic hypermutation observed on BnAbs [11].
3. ADCC antibodies against HIV
Despite the lack of success in designing a BnAb-inducing vaccine
there has been a recent surge of interest in Abs capable of targeting HIV via neutralization-independent mechanisms. In particular,
there have been significant studies on Abs capable of soliciting the
help of cells of the innate immune system, such as natural killer
(NK) cells, to detect and respond to Ab bound to viral antigens
on infected cells via a mechanism termed ADCC. This interest has
mainly been driven by the results of the recent RV144 HIV vaccine
efficacy trial in Thailand, which revealed modest protection against
HIV infection in the absence of strong or broad neutralizing Ab or
cytotoxic T-lymphocyte responses [12]. The vaccine regimen, however, did induce Abs capable of activating NK cells for ADCC [13,14].
Furthermore, the presence of ADCC Abs in vaccinees was associated
with protection from infection, as long as the Ab carriers also had
low levels of anti-Env IgA [15], which has the potential to inhibit
ADCC by blocking access to Env epitopes by anti-viral IgG [16].
While the potential role of ADCC in the efficacy of the RV144
trial provides some of the most robust evidence that this immune
mechanism could be useful for prophylactic vaccination against
HIV, there is also a large body of literature demonstrating the potential of ADCC to target HIV and result in positive outcomes. Indeed,
Abs capable of mediating anti-HIV ADCC have been observed in
highly exposed seronegative individuals (HESN) [17], higher titers
of ADCC Abs have been reported in elite controllers [18], ADCC
Abs that target a wider array of antigens have also been observed
in slow progressors [19], and immunodeficiency virus-infected
macaques that have slow progressing infections sustain higher
ADCC responses than animals with more rapid disease progression
[20]. Furthermore, and perhaps most impressive, elegant experiments have revealed that some of the protection conferred by
passively transferred BnAbs may be a result of ADCC. Indeed, modification of the anti-CD4 binding site BnAb, b12, so it could not
initiate NK cell-mediated ADCC resulted in a diminished capability
of the Ab to protect macaques from SHIV infection [21].
Despite the assortment of positive data regarding the potential
of ADCC as a prophylactic anti-HIV immune response, many questions remain about how to most appropriately and successfully
induce and maintain potent ADCC-based immunity against HIV.
There are numerous factors that can influence the NK cell effector
functions elicited by anti-viral Abs. The ability of Abs to activate
NK cells for ADCC is determined by characteristics of the Abs themselves, exogenous factors that influence the functional potential
of NK cells, as well as several intrinsic features of NK cells. One
pertinent issue is that vaccines against HIV that induce anti-viral
Abs are notorious for their inability to maintain sufficient Ab titers
[22,23]. Indeed, Ab titers induced by the RV144 regimen dropped
off rapidly within 6 months of the last vaccination, an occurrence
that may be related to the reduction in efficacy over time [22,24].
Further concerns are raised by the prejudice of the ADCC functional
potential of NK cells by genetic determinants through an ontological process termed education [25–28]. As such, it is uncertain
that all individuals carry NK cells capable of mediating sufficient
Ab-dependent effector functions to provide protection against
incoming viral infections via this immune response. Nonetheless,
much research is being conducted to determine the factors necessary for the occurrence of an ideal ADCC response, which could
one day be implemented as ammunition against HIV.
4. Ab factors determining ADCC
4.1. Ab isotype and subclass
The mediation of ADCC by NK cells occurs through the ligation
of the low affinity receptor for the constant region (Fc) of IgG (i.e.,
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L.H. Wren et al. / Vaccine 31 (2013) 5506–5517
CD16 or Fc␥RIIIa) by antigen-bound Abs. The CD16 receptor recognizes subclasses within the IgG isotype with differing affinities
(i.e., IgG3 > IgG1 > IgG4 > IgG2) [29]. Consistent with these differing affinities, IgG1 and IgG3 Abs activate NK cells to mediate more
robust ADCC [30]. In terms of anti-HIV ADCC, IgG1 Abs are the most
potent inducers of ADCC [31].
It should be noted that a small number of studies have demonstrated that NK cells also express receptors that bind the constant
regions of IgM and IgA [32,33]. Furthermore, the receptor for the
constant region of IgA triggers Ab-dependent signaling upon ligation [32]. The contribution of this receptor to NK cell-mediated
ADCC, however, is less understood/established than the contribution of CD16. In addition, IgA can compete with IgG for antigen
binding and reduce the observed ADCC [16]. Given the conflicting
data regarding the role of IgA in triggering NK cell-mediated ADCC
and the robust data regarding the ability of IgG to trigger NK
cell-mediated ADCC, future vaccine constructs may benefit from
inducing IgG-biased Ab responses. Furthermore, as IgG subclasses
that are poor inducers of ADCC (i.e., IgG2 & IgG4) could theoretically
also compete for antigen binding sites and inhibit ADCC mediated
by IgG subclasses that induce robust ADCC (i.e., IgG1 and IgG3), vaccine constructs designed to bias the Ab response towards IgG1 and
IgG3 production are desired.
the induced Ab titers waned [38]. Combined with the demonstration that vaccinated individuals developed polyclonal anti-Env Ab
responses directed against an array of epitopes [14], it appears as if
maintaining high titers of ADCC Abs and/or high ADCC responses,
which is associated with positive outcomes during chronic infection and childhood exposure to HIV through breast milk [18,35],
was also important in RV144-induced ADCC responses.
The notion that sustained high titers of ADCC-competent Abs
is important for immune-mediated protection from immunodeficiency virus infection has also been supported in primate studies.
Indeed, a recent report by Alpert et al. demonstrated that protection of macaques vaccinated with attenuated SIV was linked
with the level of Abs capable of mediating ADCC [36]. Furthermore, the level of ADCC Abs was higher and more sustained in
the macaques vaccinated with the attenuated virus compared to
macaques vaccinated with a non-persistent SIV vaccine construct.
These observations corroborate the previously mentioned association of lower efficacy of the RV144 vaccine with duration from
last immunization [24], which coincided with the waning of Ab
titers [22]. Cumulatively, these results highlight the importance of
the titer of ADCC-competent Abs for elicitation of NK cell-mediated
ADCC responses, and suggest that the efficacy of vaccines inducing
ADCC Abs is dependent upon sustaining high titers of these Abs
post immunization.
4.2. Titer and clonal nature of Ab response
4.3. Ab Fc glycosylation
Although most individuals infected with HIV carry at least some
Abs capable of activating NK cells through CD16 [34], positive
outcomes in the context of HIV infection are usually observed in
individuals with high titers of such Abs [18]. Assessments of the
role of ADCC in protection from disease progression and protection of infants from mother to child transmission via breastfeeding
have revealed that ADCC Ab titers and/or ADCC response magnitudes are directly associated with protective outcomes [18,35].
Corroborating this evidence is the demonstration that the titers
of ADCC-competent Env-binding IgG and/or magnitudes of ADCC
responses in primate models of HIV infection have been shown to
correlate with higher CD4+ T-cell counts and protection from infection [20,36]. Further, Smalls-Mantey et al. recently demonstrated
that the magnitude of ADCC observed is proportional to the quantity of anti-Env Ab bound to the surface of HIV ADCC target cells [37].
In conjunction with data suggesting high ADCC titers are desirable,
the work by Smalls-Mantey et al. suggests that it may be desirable
for these high titers to consist of a polyclonal Ab population. This
would allow the clustering of Abs directed against different Env
epitopes on the limiting number of viral spikes on the cell surface.
Indeed, experiments comparing purified polyclonal IgG to monoclonal anti-HIV Abs demonstrated the importance of polyclonal
anti-viral Ab responses for effective ADCC.
Coinciding with the evidence suggesting polyclonal ADCCcompetent Ab levels, important for the magnitude of ADCC
observed, are several features of the ADCC responses observed
in RV144 trial vaccine recipients. The vaccine regimen induced
ADCC-competent Abs against several Env epitopes [14]. The ADCC
responses were primarily directed to conformational epitopes recognized by the A32 monoclonal Ab to the C1 region of Env, but also
included non-A32 recognized epitopes. Furthermore, the titers of
Abs in the sera of these vaccine recipients declined quickly after
the final immunization [22]. This decrease in Ab titer with duration
since final immunization coincides with decreased vaccine efficacy
with time since last immunization [24]. Although the RV144 trial
was reported to have mediated 31% efficacy over a follow up of
42 months, if the trial had been powered to study efficacy at 12
months after the first immunization (6 months after the last immunization), 60% efficacy could have been reported [24]. It has been
speculated that the vaccine may have lost its protective capacity as
Further to variable region features, the glycosylation profile of
the constant region of an Ab can greatly alter the response elicited
by interaction with the innate immune system. Glycosylation of
asparagine 297 residues impacts the structure of the Ab, as well
as influences Fc-dependent effector functions [39]. The exact composition of the glycovariants, however, determines the particular
innate effector cell populations utilized and the inflammatory or
anti-inflammatory activity produced by the Ab. The sera IgG of
healthy individuals can contain between 30 and 40 of these glycovariants, and it has been demonstrated that dramatic alteration
of IgG glycosylation profiles is linked to disease activity in a number of autoimmune conditions, such as rheumatoid arthritis and
osteoarthritis [40–42]. The presence, absence, or quantity of three
major sugar residues (i.e., fucose, galactose and sialic acid) within
the glycans has been shown to alter ADCC activity.
4.4. Galactose
There have been conflicting reports regarding the effect of galactosylatation of Ab Fcs, with some groups reporting enhanced ADCC
activity while others not observing this effect [43–46].
4.5. Fucose
Fucosyation of Ab glycans has been linked to pro-inflammatory
Ab functions. Though it is the removal of fucose residues that has
been shown to increase the affinity of IgG for the activating receptor
Fc␥RIIIa and increase ADCC activity up to a 100-fold [46,47].
4.6. Sialic acid
The addition of certain sugar residues can hinder the ADCC function of Abs. For example, the addition of sialic acid to the glycan
residues confers an anti-inflammatory function to the Ab. Sialylation of IgGs hinders their Fc␥RIIIA binding and therefore ADCC
mediating activity [48].
A number of studies have explored glycoengineering of IgGs
for enhanced effector functions in the context of immunotherapy
[49,50]. Changes in serum IgG glycosylation is observed during
L.H. Wren et al. / Vaccine 31 (2013) 5506–5517
5509
Fig. 1. The ability of an Ab to activate NK cells for ADCC is determined by the characteristics of the variable and constant regions, as well as the glycosylation of the constant
region.
pregnancy, ageing, as well as during a number of disease states.
For this reason modulation of IgG glycosylation is thought to be an
active process [51,52]. The process of Ab glycosylation, however, is
poorly understood and the elicitation of Abs with specific glycosylation profiles by vaccination will require further research into the
control of this process.
4.7. Ideal ADCC Abs
Collectively, the reviewed literature constructs a biochemical
definition of the ideal ADCC Ab. As highlighted in Fig. 1, Abs best
suited to mediate ADCC: (1) are specific for antigens present on the
surface of putative target cells; (2) carry the constant region of the
IgG1 or IgG3 subclass [30,31]; (3) lack either sialic acid or fucose glycans [46–48]. Furthermore, ADCC Abs should induce more robust
responses when they are present as high titer polyclonal populations containing clones specific for numerous epitopes on the
antigen [37].
5. NK cell factors determining ADCC
5.1. “Education” of NK cells to enhance their ability to mediate
ADCC
The ability of NK cells to mediate effector functions upon
encountering putative target cells is determined through a twotier vetting process (Fig. 2). The first stage of this procedure, termed
education, is initiated during early NK cell ontogeny and continues
throughout the lifespan of the cell [53]. This education process
determines whether the cell will be conferred with the potential
to mediate effector functions, including direct and Ab-dependent
cytolysis/degranulation and secretion of cytokines [25–27,54,55].
During this process the NK cell, through its activating and inhibitory
receptors, interacts with the normal healthy self-environment.
In general, it has been demonstrated that NK cells that carry
inhibitory receptors for self-ligands are conferred with the ability
to mediate enhanced effector functions [25–27,54,55]. Alternatively, NK cells that carry activating receptors that recognize
self-ligands are less able to mediate effector functions [56]. Cumulatively, this is thought to regulate NK cells and prevent them from
amassing the ability to mediate anti-self immune responses. The
process, however, does equip NK cells to respond to malignant
or virus-infected cells, which often upregulate stress-induced
ligands for activating NK cell receptors and downregulate class I
major histocompatibility complexes (MHC-I or HLA-I) that serve
as ligands for many inhibitory NK cell receptors [57–60].
Evidence of the occurrence of NK cell education in vivo has been
provided by several studies in mice and humans. Indeed, mice that
lack MHC-I exhibit hypofunctional NK cells [61]. When such mice
are used to create transgenic strains, with single MHC-I genes, a
gain in NK cell functionality is observed, and this functionality is
restricted to the NK cell subset that expresses an inhibitory receptor
that interacts with the newly expressed MHC-I. A role for activating
NK cell receptors reducing the functional potential of NK cells has
also been illustrated in mice. Artificial overexpression of ligands
for activating receptors renders NK cells expressing these receptors
hypofunctional [62,63].
Similar to studies in mice, an abundance of evidence suggests
NK cell education is an important determinant of NK cell functional potential in humans. Numerous groups have shown that
human NK cell education is a result of a subset of receptors termed
killer immunoglobulin-like receptors (KIR) interacting with their
HLA-I ligands. Indeed, individuals that carry combinations of the
inhibitory KIR3DL1 and its HLA-Bw4 ligand, or combinations or
inhibitory KIR2DL1/2/3 receptors and their HLA-C ligands, have
been shown to have higher NK cell responsiveness to stimulation
with HLA-I-devoid target cells, as well as allogeneic or autologous ADCC target cells [25–28,54,55]. Furthermore, this function
is mostly observed in the NK cells expressing the inhibitory KIR,
which have been educated through interactions with their HLA-I
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Fig. 2. The regulation of NK cell functionality is a two-tier process. An ongoing ontological process termed education represents the first tier of this process. During education,
NK cells screen the cellular surfaces of normal healthy cells. If inhibitory NK cell receptors find cells expressing their ligands (top left), the NK cell is conferred (i.e., educated)
with the potential to mediate effector functions upon future interactions with putative target cells (i.e., tier two) that result in cumulative activating signals (top right). If
inhibitory NK cell receptors fail to find cells expressing their ligands (bottom left), the NK cell remains hypofunctional (i.e., uneducated) and incapable of mediating effector
functions upon future stimulations that result in cumulative activating signals (bottom right).
ligands. Also coinciding with the role of activating receptors on NK
cell education in mice, human NK cells expressing the activating
KIR2DS1 have been shown to be hypofunctional to in vitro stimulation when they are obtained from individuals that carry its HLA-C2
ligand [56].
The evidence discussed above may create the appearance that
NK cell function potential is regulated in an off/on manner through
the interaction of inhibitory and activating receptors with selfligands. The process of NK cell education, however, is a much
more complicated process, which involves the tuning of functional
potential on the basis of the cumulative signals received through
activating and inhibitory receptors [64–67]. Research in both mice
and humans support this notion. The isolation and in vitro stimulation of NK cells from both species has revealed that the NK
cells that are most likely to respond, and respond with the widest
array of effector functions, are those NK cells that carry numerous
inhibitory NK cell receptors for which the donor carries the MHCI ligands. Indeed, NK cells that receive low cumulative inhibitory
signals during education are conferred with the ability to mediate degranulation of cytotoxic granules. With increasing levels of
cumulative inhibitory signals NK cells gain the ability to produce
and secrete cytokines, such as IFN␥ and TNF␣.
An additional layer of complexity regarding the process of NK
cell education is that the functional potential of the NK cells is
not set during the initial interaction of the NK cells with the
self-environment, but that education is a continuous ontological
process throughout the lifespan of the cell. Indeed, elegant studies
in mice have demonstrated that the transfer of NK cells from MHCI-devoid mice into MHC-I-competent mice results in the conferral
of function to the cells that were beforehand hypofunctional [68].
Similarly, the transfer of NK cells from MHC-I-competent mice to
MHC-I-devoid mice results in the educated NK cells losing their
ability to respond to stimulation [69]. These observations likely also
have relevance during viral infections. Indeed, many viruses, such
as HIV, have the ability to downregulate HLA-I expression [57]. Furthermore, in areas of high viral replication, such as lymph nodes,
the repertoire of peptides presented on HLA-I molecules would be
altered. As KIR are involved in NK cell education and their interactions with HLA-I are influenced by the groove-bound peptide [70],
viral infections may have the potential to alter the NK cell education
process in areas of high viral replication that contain NK cells.
As mentioned previously, and of relevance to this review,
several research groups have demonstrated that NK cell education
is important for deciding the functional potential of NK cells to
mediate effector functions after stimulation by Ab-coated target
cells. Indeed, NK cells educated through KIR2DL1/2/3 and KIR3DL1
mediate higher effector functions after stimulation by non-self
ADCC target cells than do non-educated NK cells [25,27,28].
L.H. Wren et al. / Vaccine 31 (2013) 5506–5517
Furthermore, KIR3DL1 educated NK cells have been demonstrated
to mediate higher anti-HIV ADCC against autologous targets than
non-educated NK cells [26]. This observation suggests that ADCC
can overcome the inhibitory potential of KIR/HLA-I interactions
and utilize the functional potential conferred through the education process. It should be noted, however, that research on the
ability of educated NK cells to be activated for ADCC by cancer
monoclonal Ab therapies has revealed that KIR3DL1-expressing NK
cells mediate enhanced function in the absence of HLA-Bw4, but
they are inhibited in the presence of HLA-Bw4 [28]. The different
functionalities of KIR3DL1-expressing NK cells in the presence
of HLA-Bw4 in anti-HIV and anti-cancer ADCC are intriguing. As
ADCC against HIV usually involves the utilization of polyclonal Ab
mixtures, and is indeed more efficient in the presence of polyclonal
Abs, we hypothesize that the reason, KIR3DL1-expressing NK cells
are more easily inhibited in anti-cancer ADCC, is that the NK cells
are not maximally stimulated by the monoclonal Ab treatments.
Regardless, the presently available data suggests that NK cell
education is a determinant of NK cell ADCC functional potential,
and this phenomenon should be taken into consideration when
interpreting studies investigating HIV vaccines and therapies that
utilize ADCC responses.
5.2. Receptor repertoire and regulation of activation
The second tier of regulation for NK cell functionality, alluded to
in Fig. 2 and in the NK cell education section above, is the generation of a cumulative signal during the interaction of activating and
inhibitory NK cell receptors with ligands on the surface of putative
target cells. The activation of NK cells to mediate effector functions
requires that a cumulative positive signal be delivered from these
surface interactions [71]. Furthermore, the strength of this activating signal has been demonstrated to determine the exact effector
functions that are mediated by the NK cell [72]. Lower cumulative
activating signals induce NK cells to degranulate cytotoxic granules
and secrete chemokines (i.e., CCL3. CCL4, & CCL5). Larger cumulative activating signals allow NK cells to secrete cytokines, such as
IFN␥ and TNF␣.
The majority of research investigating the impact of cumulative
receptor signaling on NK cell activation has been conducted using
Ab-independent forms of stimulation. A role for activating and
inhibitory receptors other than CD16, however, has been observed
during the stimulation of NK cells via ADCC. Indeed, Bryceson
et al. recently demonstrated that ligation of co-stimulatory receptors, such as 2B4 and NKG2D, acts synergistically with ligation
of CD16 during NK cell activation [73]. Similarly, several investigators have demonstrated that inhibitory KIR receptors, such
as KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1, can inhibit ADCC
responses [28,74]. With regards to anti-HIV ADCC, KIR that interact with HLA-C and the HLA-E-binding inhibitory NKG2A receptor
dramatically reduce ADCC [74]. Interestingly, KIR3DL1-expressing
NK cells mediate anti-HIV ADCC against autologous target cells. As
each of these inhibitory receptors contributes to NK cell education, these results represent somewhat of a conundrum: Why only
some educated NK cells are capable of mediating ADCC against targets expressing the cognate antigen for their educating inhibitory
receptor? Although more research is needed to elucidate the reason
for these observations, perhaps the most likely factor at play here
is that the activating signal received through interaction with the
ADCC target cells is stronger than the inhibitory signals being mediated through KIR3DL1, but not those being transduced through the
inhibitory NKG2A or KIR2DL1/2/3 receptors. As such, the KIR3DL1expressing NK cells are able to utilize the functional advantage
conferred through the education process, whereas the function
conferred through NKG2A or KIR2DL1/2/3-mediated education is
inaccessible.
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Although many questions remain regarding the reasons for the
observed anti-HIV ADCC functionalities of NK cells with different
phenotypes, it is well established that the receptor repertoire of
NK cells greatly influences the ability of NK cells to respond to Abcoated target cells. As such, the NK cell receptor repertoire, and
the interpersonal differences in this repertoire, represents another
factor that is necessary to consider when designing vaccines and
immunotherapies utilizing ADCC.
5.3. NK cell “exhaustion”
Chronic viral infections such as HIV and hepatitis C virus (HCV),
as well as some malignancies, have been demonstrated to alter
the phenotype, functionality and subset distribution of NK cells.
These disease entities can decrease the prevalence of the highly
cytolytic CD56dimCD16+ NK cell subset and increase the frequency
of the less functional CD56− CD16+ subset [75,76]. Furthermore,
these illnesses have been associated with reductions in the expression of the natural cytotoxicity receptors (NCRs, i.e., NKp30, NKp44,
& NKp46) [77–79], which has been linked to decreased activation of
NK cells through these important receptors. The expression of CD16
is also reduced in patients with these conditions [80,81]. Lastly, NK
cells from these patients are less functional upon ex vivo stimulation
[79,82,83].
Although the exact mechanism(s) for NCR and CD16 expression alterations has not yet been determined, we hypothesize that
these phenotypes are reached as a result of chronic activation and
exhaustion. This hypothesis is supported by the demonstration that
in vitro activation of NK cells produces similar alterations in phenotype and functionality as are observed in chronic infection. Indeed,
activation by either MHC-I-devoid or ADCC target cells results in NK
cell downregulation of CD16 and CD56, reduces functionality upon
secondary stimulation, and reduces the expression of the activating
NCRs [84–87].
Collectively, these observations raise concerns about the desirability of continued NK cell activation during chronic viral
infections. A robust correlation between high ADCC titers and
slower HIV disease progression has been demonstrated [18]. This
observation is difficult to reconcile with the demonstrations that
activation of NK cells for direct or Ab-dependent effector functions
alters the cellular phenotype in manner similar to that observed
in progressive HIV infection and AIDS [75–77,79,81,84–87]. This
conundrum highlights the necessity for studies with more relevance regarding the in vivo role of ADCC in HIV disease progression.
It is necessary to carryout long-term studies evaluating the potential of autologous NK cells to mediate ADCC against autologous
infected targets in the presence of autologous Abs from patients
with different disease progression patterns. Given the complicated
interplay of activating and inhibitory receptors in educating NK
cells for ADCC, as well as determining the activation of the same
NK cells during interactions with putative target cells, it is notable
that many published studies are confounded by assessing ADCC
with cell lines or mixtures of Abs and effector cells from different donors [14,18,34]. Admittedly, it is difficult to disqualify a
role for NK cells and ADCC in slowing HIV disease progression,
given the observations of higher frequencies of function-conferring
KIR3DL1/HLA-Bw4 combinations in HIV-infected slow progressors
[55,88–90], as well as the demonstration of higher titers of ADCC
Abs in elite controllers [18]. We raise the hypothesis that anti-HIV
ADCC responses may be most beneficial during the early stages
of HIV infection when NK cells remain most functional. This may
“set the scene” for later disease-progression profiles. Indeed, ADCC
Abs are observed during early HIV infection and NK cells expressing KIR3DL1 are expanded at this time also [91]. Collaboratively,
these NK cells and Abs may eliminate infected cells and reduce
the viral load set point, ultimately reducing the level of continuous
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stimulation of NK cells required, as well as the general level of
immune activation.
Aside from the issues activation-induced NK cell exhaustion
raises about the role of ADCC and general NK cell activation in
protection from disease progression, this phenomenon is also an
important consideration for the goal of utilizing ADCC for therapeutic and prophylactic purposes. Indeed, if ADCC Abs were induced
by vaccines or injected in the format of an immunotherapy, the
efficacy of these Abs may be drastically reduced in individuals
carrying exhausted NK cells resulting from another chronic infection or malignancy. For individuals carrying a non-exhausted NK
cell repertoire, however, it is likely that these NK cells would be
fully capable of utilizing vaccine-induced anti-viral Abs to eliminate
desired cells via ADCC.
5.4. The influence of FcR polymorphisms on ADCC immunity
A critical component of the NK cell for its ADCC potential is the
CD16 or Fc␥RIIIa receptor. This is the low avidity Fc receptor, which
recognizes immobilized IgG bound to its cognate antigen. A very
important polymorphism has been observed in this receptor. This
polymorphism at position 158 determines the degree of expression
of the CD16 receptor, its affinity for antigen-bound IgG and the
magnitude of the ADCC responses elicited via signaling through the
receptor [92]. Individuals that carry a valine (V) at position 158 have
CD16 variants that are expressed at a higher surface density, bind
with a higher affinity to antigen-bound IgG and trigger higher ADCC
responses upon ligation; whereas, the opposite is true for each of
these observations for CD16 from individuals with a phenylalanine
(F) at position 158.
Most work characterizing the differences in CD16 polymorphisms at position 158 has been conducted in vitro; however,
polymorphisms at position 158 have also been demonstrated
to be of clinical relevance. Indeed, individuals carrying the
function-enhancing V158 polymorphism have better outcomes
upon initiation of monoclonal Ab cancer therapies [93]. Given this
clinical relevance, CD16 polymorphisms have also been hypothesized to be of importance for anti-HIV ADCC, and potentially of
relevance for understanding disease progression and/or vaccineinduced protection. Counterintuitive to the notion that ADCC is
important for protection from HIV infection and disease progression, individuals carrying the more highly functional V158
polymorphism have been shown to be more likely to contract HIV
infection and exhibit a progressive infection [94]. Similarly, and also
counterintuitive to the notion that ADCC is involved in protection
from HIV infection in vaccine recipients, low-risk VAX004 vaccinees homozygous for the V158 polymorphism have been shown
to be more likely to become HIV infected than those carrying the
F158 polymorphism [95]. These observations are concurrent with
the notion that continued high levels of ADCC in chronic viral infection may be undesirable, but are at odds with the view that ADCC
could be protective if ADCC-competent Abs are present prior to HIV
infection. It should be noted, however, that the CD16 V158 polymorphism has also been associated with higher rates of infection
in non-vaccinated individuals [94]. As these individuals most likely
do not have Abs capable of mediating ADCC against HIV-infected
target cells, it is highly unlikely that the CD16 polymorphism is
increasing the rate of infection in an Ab-dependent manner. Thus,
the relationship of the CD16 receptor to increased infection is most
likely explainable due to a non-Ab-dependent effector function
or through genetic linkage to some unidentified marker. As such,
the interpretation of the association of the CD16 V158 polymorphism with enhanced infection in the VAX004 trial remains open
to debate.
Notwithstanding the open issues of debate regarding the role
of CD16 polymorphisms in determining susceptibility to HIV
infection, the polymorphisms at position 158 are clearly important
for determining the expression and function of the receptor. As
such, analysis of these polymorphisms will be essential to take
into consideration for vaccine and therapeutic design.
6. Exogenous factors that influence ADCC immunity
6.1. Cytokines
Exogenous soluble factors, such as cytokines, have great potential to alter the nature of many immune responses. Indeed, a role for
many cytokines in adjusting the function of NK cells for direct and
Ab-dependent effector functions has been demonstrated. Several
independent investigations have illustrated the ability of IL-2, IL10, IL-12, IL-15, IL-18 and IL-22 to enhance the cytolytic potential
of NK cells [96]. Similarly, several groups have noted suppressive
roles for both IL-4 and TGF-␤.
Aside from demonstrations of the effects of cytokines on NK
cells in vitro, the plasma levels of these molecules in individuals with chronic viral infections have been linked to ex vivo NK
cell function. For example, heightened levels of TGF-␤ have been
linked with reduced functionality. Blockade of TGF-␤ has even
been shown to increase the function of NK cells isolated from
Hepatitis B virus (HBV)-infected patients [97].
Although the demonstration that plasma cytokines can influence NK cell functionality during chronic viral infection may be
important for understanding the immunopathology of chronic viral
infections, perhaps the most important potential of the effects
of cytokines on NK cell functionality is their ability to prime NK
cells for therapeutic purposes. Certainly, priming of NK cells with
cytokines, such as IL-2, has been shown to increase the functionality of NK cells and increase their efficacy for attacking cancer
cells when re-administered to animals or humans with malignancies [98]. Such ex vivo cytokine priming of NK cells could be of
much interest for immunotherapies directed at attacking reactivated latent HIV reservoirs.
7. Potential integration of factors to eradicate latent HIV
For years it has been demonstrated that HIV establishes a
chronic latent viral reservoir in resting memory CD4+ T-cells and
probably other cells that become infected, such as NK cells [99,100].
This reservoir remains stable throughout treatment of HIV infection
with combined antiretroviral therapy (cART), prevents the eradication of virus from the body and is the reason lifelong cART is
required for infected individuals. Recently, there have been several publications demonstrating latent HIV is reactivated by histone
deacetylase (HDAC) inhibitors in vitro and in vivo [101–103]. This
observation has raised hope that reactivated virus, and the cells
harboring this virus, can be eliminated, producing a functional cure
that would allow patients to live without the need for lifelong cART.
One of the most essential questions raised by this new possibility is
how to best eliminate the infected cells identified upon reactivation
of virus.
As activation of NK cells through CD16 induces the cytolysis of
target cells baring antigen-bound Abs [27], we propose that ADCC
could be utilized to eliminate the reactivated latently infected CD4+
T-cells [104]. Clinical studies involving the reactivation of latently
infected T-cells, however, have not induced the spontaneous clearance of reactivated infected cells [101], suggesting that the in vivo
ADCC responses available on cART at this time are insufficient to
clear these cells.
As explained in the above sections, several reasons exist that
may explain the inability of NK cells to respond sufficiently to clear
latently infected cells. First, although cART undoes some of the
functional exhaustion of NK cells in HIV-infected subjects [79,105],
L.H. Wren et al. / Vaccine 31 (2013) 5506–5517
NK cells from infected individuals on cART still exhibit more baseline activation than NK cells from uninfected individuals [106]. An
accumulation of too many exhausted NK cells could reduce the
ability of the NK cell pool to mediate sufficient ADCC responses
to clear reactivated infected cells. Second, NK cell education has
been demonstrated to be important for NK cell-mediated ADCC
[25–28]. With regards to anti-HIV ADCC education of NK cells
through KIR3DL1/HLA-Bw4 combinations seem to be of importance
[26]. Indeed, KIR3DL1-educated NK cells mediate robust ADCC
against autologous target cells; whereas, NK cells expressing the
education-competent KIR2DL1/2/3 or NKG2A receptors have been
demonstrated to be inhibited by their ligands that are present
on autologous HIV ADCC target cells [74]. Interestingly, KIR3DL1educated NK cells lose their functional advantage for ADCC in
HIV-infected individuals, regardless of cART [26]. As such, it is possible that individuals could have too few functional educated NK
cells after the reactivation of latent viral reservoirs, and reduced
ability of NK cells to clear reactivated infected cells.
Deficits in NK cell functional potential in chronically HIVinfected individuals potentially represent a major hurdle to
overcome for utilization of NK cell-mediated responses to clear
reactivated latently infected CD4+ T-cells. Implementation of in
vivo cytokine therapy, or re-administration of NK cells activated
ex vivo with cytokines, could represent methodologies to overcome these problems. Previous studies have demonstrated that
IL-2 and IL-15 can increase the function of NK cells isolated from
HIV-infected individuals [107,108]. This suggests that the cytokines
can allow the NK cells to overcome the exhaustion they exhibit
when utilized directly ex vivo. Alternatively, treatment of NK cells
with cytokines may allow NK cells to garner further education,
which would increase their functional potential. Indeed, it has
recently been demonstrated that one of the mechanisms behind
the increased NK cell functionality upon treatment with IL-2 or IL15 is increased expression of inhibitory KIR, which bind to self HLA-I
[109].
Several possibilities for Ab deficits could also explain the inability of patients to spontaneous clear reactivated infected cells. As
previously mentioned, the amount of ADCC-competent Abs is an
important indicator of the ability to elicit NK cell-mediated anti-HIV
ADCC responses [37]. The titers of anti-HIV Abs tend to be reduced
in chronically infected individuals after suppression of virus with
cART [110]. Our lab has recently replicated this observation and
demonstrated that after prolonged suppression of viral replication with cART anti-HIV ADCC Abs are reduced (unpublished data).
Another possible Ab-related problem for clearing reactivated latent
virus is that the reactivated virus may exhibit escape mutations to
the available Abs. Indeed, the ability of HIV to escape ADCC pressure
has previously been demonstrated by our research group [111].
Overcoming deficits in the anti-HIV Ab response also represents
a major potential hurdle for clearing reactivated latently infected
cells. The problem of lower Ab titers in individuals that have
been successfully treated with cART may be easily overcome
with a booster immunization against autologous virus prior to
reactivation. The problem of viral escape mutants to circulating
ADCC Abs, however, represents a more nefarious setback. Several
lines of research suggest Ab responses to HIV may be subject to a
phenomenon termed repertoire freeze, where Abs most well suited
to targeting earlier viral variants for destruction are sufficiently
cross-reactive with contemporary autologous viruses to prevent
the induction of more efficient novel Ab responses [112,113]. As
such, attempts to immunize individuals in a manner that would
create novel Ab specificities may fail due to the ability of the
currently available Abs and B-cells to recognize the new antigen,
be boosted, and mop-up antigen. One possibility to overcome this
potential problem would be to eliminate the presently available
anti-viral humoral immune response, and memory B-cells. Indeed,
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it has previously been shown in macaques that idiotypic-based
suppression of anti-viral Abs in the context of an active infection
allows for the development of novel Ab specificities [114,115]. This
mechanism of regulating the humoral immune response to HIV
may also be relevant for increasing the presence of useful ADCC
Abs in chronically infected individuals prior to viral reactivation.
The potential to reactivate and eliminate the latent viral
reservoirs of HIV-infected individuals represents an exciting and
promising avenue of research into curing HIV infection. This area
of research is still very much in its infancy, and many questions
remain about the best strategies and potential pitfalls. Although
we focus on utilizing ADCC to eliminate reactivated virally infected
cells, it should be noted that other cytolytic immune responses,
such as those mediated by cytotoxic CD4+ or CD8+ T-lymphocytes,
could also provide a mechanism to eliminate reactivated infected
cells. The observation that individuals with reactivated virus do
not spontaneously clear infected cells, however, suggests that
immunological roadblocks are in existence for all of these cytolytic
responses [101]. Indeed, HIV-specific CD8+ T-cells require preactivation to mediate robust cytolysis of reactivated latently
infected cells [116]. As such, the roadblocks to immune-mediated
clearance of reactivated latently infected cells need to be elucidated and immune modulating strategies designed to increase the
chances of an effective functional cure against HIV. A successful protocol for eliminating reactivated latently infected cells will likely
utilize more than one method of immune-mediated clearance.
8. Potential integration of factors for vaccination against
HIV
The search for an effective prophylactic HIV vaccine has been the
holy grail of HIV research since the identification of the viral agent
as the cause of AIDS in 1984 [117–120]. Traditional attempts to
design prophylactic HIV vaccines have focused on eliciting neutralizing Abs or CD8+ cytotoxic T-lymphocyte (CTL) responses [121].
The recent success of the RV144 trial, however, has suggested that
vaccine-induced ADCC Abs may provide a pathway to protection
from HIV infection [15,16]. Nevertheless, as alluded to in the previous sections of this review, there are several hurdles to utilizing
ADCC Abs as a prophylactic vaccine.
Perhaps the foremost problem facing an HIV vaccine that utilizes
Abs is that these proteins wane with duration from final immunization [22,23]. Diminishing Ab titers are associated with decreased
vaccine efficacy overtime, suggesting it is necessary to add components to the vaccine to maintain high Ab titers. Vaccination of
non-human primates with live-attenuated SIV has proven effective
at sustaining sufficient Ab levels to provide long-term protection
against challenge with pathogenic viral variants [36]. However, as
live-attenuated viruses have the potential to revert to pathogenic
forms [122], the utilization of live-attenuated viruses for the vaccination of humans is not up for consideration. As such, future
investigations need to evaluate alternative methods to sustain high
titers of anti-viral ADCC Abs. Such methods could include prolonged
immunization regimens, unique adjuvants and/or the implementation of long-lived vaccine vectors that lack pathogenic potential.
For example, in a recent review one of us suggested studying the
potential of anti-idiotypic Abs, directed against a non-antigen binding site of HIV Abs, to mediate an interaction that mutually sustains
both anti-viral and anti-anti-viral B-cell populations [113].
A major concern regarding utilization of HIV vaccines that
exploit ADCC Abs is that the potential of NK cells to mediate
ADCC is prejudiced by the process of NK cell education, which
is determined by genetic factors. As reviewed above, the NK cell
populations that have the greatest functional potential upon
successful direct or Ab-dependent activation are those that have
been educated through the interaction of inhibitory receptors with
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their HLA-I ligands [25–28,54]. Indeed, NK cells educated through
the interaction of KIR3DL1 and HLA-Bw4 exhibit a functional
advantage when stimulated with autologous HIV ADCC target
cells [26]. It is currently thought that other education competent
receptor/ligand combinations (i.e., KIR2DL1/HLA-C2, KIR2DL2 or
KIR2DL3/HLA-C1, & NKG2A/HLA-E) do not produce NK cell populations with functional advantages for mediating anti-HIV ADCC.
These educated NK cell populations are largely inhibited during
HIV ADCC assays [74]. Because of this role for NK cell education in
determining the ADCC potential of NK cells, individuals lacking the
educating KIR3DL1/HLA-Bw4 combination may have less potential
to eliminate early infected autologous CD4+ cells shortly after
HIV exposure. The potential bias of ADCC Ab inducing vaccines
to work more efficiently in individuals carrying genetic combinations of educating receptor/ligand combinations represents a
difficult problem to overcome. For example, techniques to turn
on non-educated NK cells, which lack inhibitory receptors for self,
could be undesirable, as these cells may have increased potential
to mediate autoimmune responses.
Lastly, considerations of utilizing anti-HIV ADCC for vaccine
design need to take into consideration the mechanism of HIV transmission. It is frequently assumed that HIV is transmitted primarily
as a free virus within seminal and vaginal fluids. There is, however,
much evidence that HIV can be transmitted via infected allogeneic
lymphocytes present in semen or vaginal fluids [123–127]. Indeed,
animal models have revealed that the levels of infected lymphocytes required to obtain infection are more reflective of the amount
of HIV-infected cells present in bodily fluids than are the levels of
free virions required to obtain infection. If infected allogeneic lymphocytes are indeed involved in the transmission of HIV, they add
an extra layer of complexity to efforts to design vaccines utilizing ADCC. Due to interpersonal variability infected lymphocytes
from different individuals will express different HLA-I combinations, as well as different peptides bound within the grooves of
these molecules. These complexes of HLA-I and peptide will have
the potential to interact differently with different inhibitory KIR,
potentially rendering the effectiveness of an ADCC-utilizing vaccine
dependent upon a very complex interaction of the KIR repertoire
and educational status of the NK cells of the recipient of the infected
cells and the HLA-I repertoire of the infected cell donor. Although
this represents a major potential problem for vaccines utilizing
ADCC, it is notable that some educated NK cells are capable of overcoming inhibition to mediate ADCC in the presence of their ligands
[26]. The mechanism behind this phenomenon needs to be elucidated. If this is due to an antibody factor, such as the polyclonal
nature of the response, it may be possible to design vaccines that
can render this concern irrelevant.
9. Conclusions
Long abandoned as an uninteresting or irrelevant anti-viral
immune response, recent evidence has implicated ADCC as a potent
anti-viral immune response associated with slower progression
towards AIDS and potentially important for preventing infection
[15,17,18,21]. Indeed, many investigators are now studying the
prospect of harvesting the power of ADCC responses to design therapeutics and vaccines.
Attempts to design vaccines against HIV have long focused on
the induction of broad neutralizing Ab responses and/or robust
CTL [121]. The recent RV144 efficacy trial revealed protection
from infection despite not sufficiently eliciting either of these
immune responses [12,15]. Instead, the protection conferred by
the RV144 regimen has been linked to non-neutralizing binding IgG. Furthermore, in the absence of anti-viral IgA, which can
inhibit IgG-mediated ADCC, the ADCC potential of the anti-viral
IgG has been correlated with the observed protection [15,16]. As
highlighted in this review, many factors are known to influence
ADCC responses. A collective synthesis of the current knowledge
regarding these factors is required to assist the design of future vaccine constructs attempting to utilize anti-viral ADCC. Furthermore,
intense research efforts are needed to answer the major questions
being raised by the current state of knowledge. Central to the design
of a credible ADCC-utilizing HIV vaccine are techniques to sustain
vaccine-induced Abs, as well as a more robust understanding of the
mechanisms involved in the mucosal transmission of HIV.
Conflict of interest statement
MSP is a member of the Scientific Advisory Board for Network
Immunology Incorporated.
Acknowledgement
This work was supported by program grant no. 510448 from the
National Health and Medical Research Council (NHMRC).
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