CCR5: From natural resistance to a new Anti-HIV strategy

Abstract

The C-C chemokine receptor type 5 (CCR5) is a key player in HIV infection due to its major involvement in the infection process. Investigations into the role of the CCR5 coreceptor first focused on its binding to the virus and the molecular mechanisms leading to the entry and spread of HIV. The identification of naturally occurring CCR5 mutations has allowed scientists to address the CCR5 molecule as a promising target to prevent or limit HIV infection in vivo. Naturally occurring CCR5-specific antibodies have been found in exposed but uninfected people, and in a subset of HIV seropositive people who show long-term control of the infection. This suggests that natural autoimmunity to the CCR5 coreceptor exists and may play a role in HIV control. Such natural immunity has prompted strategies aimed at achieving anti-HIV humoral responses through CCR5 targeting, which will be described here.

Full text

Viruses 2010, 2, 574-600; doi:10.3390/v2020574
viruses
ISSN 1999-4915
www.mdpi.com/journal/viruses
Review
CCR5: From Natural Resistance to a New Anti-HIV Strategy
Lucia Lopalco
Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute,
via Stamira D’Ancona, 20, 20127 Milan, Italy; E-Mail: lopalco.lucia@hsr.it; Tel.: +39-02-2643-7936;
Fax: +39-02-2643-5381
Received: 23 October 2009; in revised form: 22 December 2009 / Accepted: 4 February 2010 /
Published: 5 February 2010
Abstract: The C-C chemokine receptor type 5 (CCR5) is a key player in HIV infection due
to its major involvement in the infection process. Investigations into the role of the CCR5
coreceptor first focused on its binding to the virus and the molecular mechanisms leading to
the entry and spread of HIV. The identification of naturally occurring CCR5 mutations has
allowed scientists to address the CCR5 molecule as a promising target to prevent or limit
HIV infection in vivo. Naturally occurring CCR5-specific antibodies have been found in
exposed but uninfected people, and in a subset of HIV seropositive people who show long-
term control of the infection. This suggests that natural autoimmunity to the CCR5
coreceptor exists and may play a role in HIV control. Such natural immunity has prompted
strategies aimed at achieving anti-HIV humoral responses through CCR5 targeting, which
will be described here.
Keywords: CCR5; HIV; vaccine
1. Introduction
More than 40 million people, mostly women and children, are presently infected by the human
immunodeficiency virus (HIV); almost all horizontal and vertical transmissions of HIV infection are
due to HIV strains that use the CCR5 coreceptor expressed on mucosal surface [1,2].
CCR5 is undoubtedly the main HIV coreceptor, involved in virus entry and cell-to-cell spread:
Such R5-tropic viruses are nearly always involved in the initial infection, while HIV strains using the
CXCR4 coreceptor are observed only seldomly in the early infection [3]. Due to the natural history of
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HIV infection, CCR5 is a key target for the development of drugs and immunogens that are able to
elicit systemic and especially mucosal responses to protect exposed people from infection. Easy-to-
use, cheap, and long-lasting mucosal protection could significantly limit HIV spread, especially in sub-
Saharan Africa, Eastern Asia, and other areas where sexually transmitted diseases are heavy health and
social burdens [4].
The discovery of CCR5 genetic polymorphisms associated with HIV-resistance or disease control
encouraged the research and development of inhibitor-drugs and antibodies that are able to counteract
HIV at its major portal of entry; some of these products are presently undergoing evaluation in clinical
trials or have even been licensed for therapy [5,6]. Stemming from such CCR5 investigations, natural
anti-CCR5 immunity was observed in special populations dealing with HIV, i.e., HIV-exposed
seronegative people and long-term non-progressing seropositive individuals [7]. Strikingly, such
antibodies ─ found in serum, but most importantly also in mucosal secretions ─ were associated with a
protective role or with control of the disease [8,9].
These observations confirm that CCR5 is a promising target in the prevention or therapy of HIV,
and suggest that even innovative approaches, such as anti-CCR5 vaccination, can provide useful
scientific insight; and but most importantly, valuable weapons to fight HIV and other immune-based
diseases. This review will discuss the role of CCR5 in HIV infection and the current approaches to
target CCR5, with particular attention to the cases of natural immunity to the coreceptor and
immunization experiments aimed at reproducing it.
2. CCR5 functions
CCR5 belongs to a large family of chemokine receptors that are expressed on surface of
lymphocytes and other cell types, where they are involved in signaling and coordination of immune
responses [10]. Similarly to CXCR4, CCR5 is also an HIV coreceptor [11-14]. CCR5 and other
chemokine receptors belong to an even larger family of seven transmembrane proteins coupled to G
proteins, a very important family that includes many signaling receptors, such as rhodopsin and beta-
adrenergic receptors [10]. Seven transmembrane receptors are large molecules (Figure 1), however
their three-dimensional structures are still poorly elucidated from physico-chemical spectroscopic
methods, such as X-ray crystallography. Only the structure of rhodopsin, the two beta adrenergic
receptors, and the adenosine receptor have been recently characterized [1]. The approximate structures
of other receptors, such as CCR5, have been modeled based on similarities revealed by the structures
of these related proteins [1].
CCR5 is expressed on immature (Th0) and memory and primed Th1 cells, monocytes,
macrophages, and immature DC; on neurons, astrocytes, and microglia; on epithelium, endothelium,
vascular smooth muscle, and fibroblasts [2]. Its preferential ligands are the pro-inflammatory
cytokines CCL3 (MIP-1 alpha), CCL4 (MIP-1beta) and CCL5 (RANTES), involved in the initiation of
effector responses [3]. Other cytokines, such as CCL7 (MCP-3), CCL8 (MCP-2) and CCL13 (MCP-
4), are a competitive antagonist and two weak agonists, respectively. Since chemokine binding may
interfere with HIV docking, natural CCR5 ligands were evaluated as HIV competitors, with varying
results: CCL3, CCL4, CCL5 and CCL8 displayed inhibiting properties to HIV, CCL7 was shown not
to interfere, while CCL2 (MCP-1) even enhanced HIV infection in vitro [4]. CCL3L1 and CCL4L1

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are variant chemokines encoded by genes with varying copy numbers. These chemokines inhibit the
binding of CCR5 to HIV through receptor down-regulation, in an inverse relationship; their gene copy
numbers and hence the expression levels influence HIV progression [5].
Figure 1. Structure of the CCR5 coreceptor; the HIV binding domains (N-terminal and
ECL2 domain) and the ECL1 domain are indicated.
CCR5 is not only a chemokine receptor that is able to induce cell chemotaxis towards chemokine
gradients, but it also takes part in immune synapses where it behaves as a costimulatory molecule [6].
More specifically, CCR5 is involved in the orchestration of cellular immunity, which is a
CCL5/RANTES-mediated cascade that is independent of the chemotactic response [7]. CCL5 was
shown to induce the expression of activation markers at the surface of primary T cells in vitro; in vivo,
it increased the proliferative response to antigens in CD4+ T cells and subsequent cytokine
secretion [6]. CCR5 was also shown to sustain recruitment of naive CD8+ T cells to antigen-
presenting dendritic cells [8]. CCR5 chemokine ligands can enhance effector responses by
potentiating APC and T cell activities in response to antigen-induced stimulation [9].
The mechanism of CCR5 signaling first requires the ligand binding to the extracellular domains of
the receptor, followed by receptor dimerization and phosphorylation [10]. Intracellular signal
transduction, mediated by GDP release, requires binding and hydrolysis of a new GTP molecule [1].
The activated G protein then dissociates from the cytoplasmic domain of CCR5 and activates a
second-messenger cascade, sustained by phospholipase C kinase, inositol-triphosphate (IP3-kinase)
and mitogen-activated (MAP) kinases or other tyrosine kinases [11]. The mechanism of CCR5

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signaling and regulation is complex, and most aspects are still not well understood. Experiments aimed
at correlating CCR5 structure and function by using monoclonal antibody panels suggests that CCR5
internalization may occur via phosphorylation and binding to arrestin before internalization in clathrin-
coated vesicles, as observed upon chemokine stimulation [12]. However, CCR5 internalization may
also involve a different pathway, dependent on cholesterol-rich membrane caveolae [13]. CCR5 was
identified in membrane raft microdomains and its subcellular localization was supposed to contribute
to chemotaxis as well as to HIV entry [14]. However, constitutive CCR5 turnover also occurs in the
absence of ligand, with a half-life of six to nine hours [15]. Thr in-and-out flux of CCR5 molecules is
highly regulated as surface receptor density is inversely correlated with CCL5/RANTES expression
levels [16].
CCR5 could be anchored to plasma membrane lipid rafts through the palmitoylated cysteine
residues located in its C-terminal domain [17]. CCR5 molecules placed in the rafts closely cluster
with CD4 on the cell surface, therefore providing a convenient “docking site” for HIV [18]. CD4 and
CCR5 were not only found physically associated as HIV coreceptors [19,20], but CD4 was also
shown to associate with CCR5 molecules from the endoplasmic reticulum, thus promoting their
exposure at the cell surface [21].
Wild-type CCR5 is able to polymerize, not only with itself [22], but also with its truncated delta-32
form [23], with other chemokine receptors, such as CCR2 [24], and even with other GPCR, such as
the opioid receptor [25]. The biological significance that homo- and hetero-dimerization has on CCR5
conformation, binding, and signaling are presently unknown.
3. CCR5 deletion and its consequences
CCR5 expression levels may vary in individuals without affecting immune function [26].
Depending on the number of exposed receptors, low and high “CCR5 expression” individuals have
been described [27]. This variation in CCR5 expression levels between individuals reflect genetic
factors as well as environmental stimuli, as reported in a comparative study that observed higher levels
of CCR5 expression and immune activation in European and African subjects residing in Africa ─
possibly due to parasitic infections ─ than in a cohort of the same ethnic groups residing in
Europe [28]. Genetic patterns that prevent CCR5 expression have been described in HIV-exposed
uninfected people who display natural resistance to HIV infection [29,30]; also, the enhanced
expression of chemokines has been reported to play a role in natural resistance to HIV [31]. Reduced
or abolished expression of the CCR5 receptor has been found in Caucasians and in other ethnic groups
worldwide; the delta-32 mutation - the first to be described - causes a deletion in the receptor sequence
that prevents exposure of the truncated receptor on the cell surface [4]. Consequently, homozygous
delta-32 individuals are substantially ─ but not completely ─ resistant to HIV infection, but do not
show any pathologic phenotype [32,33,34]. In some cases, infection of delta-32 homozygous
individuals was associated with dual tropic R5-X4 or to X4-tropic viral strains [35,36,37]. As
confirmation of delta-32 resistance to HIV, a recent clinical observation showed long-term control of
infection without antiretroviral therapy in an HIV-positive patient who had the CCR5+ genotype and
underwent CCR5-/- stem cells transplantation to treat acute myeloid lymphoma [38]. CCR5+
macrophages were identified in a patient biopsy from intestinal mucosa, taken several weeks after

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transplantation, showing that the circulating, but not the resident CCR5+ cells, had been replenished
by the transplant. However, viral RNA was undetectable despite the persistence of HIV-permissive
cells, and the patient did not experience a rebound in viral load in the absence of antiretroviral therapy.
Another important finding of the study was the absence of a virus shift in favor of X4 strains, whose
presence was unnoticed by current diagnostics practices, but was, however, detected by ultra-deep
sequencing [38]. Heterozygous CCR5-delta-32 alleles have been found to be more prevalent in long-
term non-progressing population than in progressing cohorts, therefore confirming that CCR5 load and
functions may play a more complex role than that of coreceptor in the pathogenesis of HIV
infection [39,40]. Some hypotheses have been drawn to explain the evolution and the selective
advantage conferred by CCR5 delta-32 allele in humans, such as an increased resistance to plague or
smallpox, but none is presently conclusive [41].
Other CCR5 mutations have been subsequently described; interestingly, high levels of circulating
beta-chemokines (e.g., CCL5/RANTES) also affects HIV binding to CCR5 molecules [26]. Both
events converge on the CCR5 receptor, but different mechanisms may be involved in each of these. In
fact, homozygous CCR5 mutation may prevent wild-type CCR5 from being exposed on the cell
surface; circulating chemokines can compete with HIV for binding, mask the viral binding site, or
subtract the whole receptor from the cell surface by inducing its internalization [4]. A large cohort
study, involving over 2000 HIV-positive and healthy people, compared the two major parameters of
clinical status in HIV infection, i.e., viral load and CD4+ T cell counts, and two parameters
representing immune activation and inflammation, i.e., CCR5 expression and gene copy number of the
CCL3-L1 molecule, a natural cytokine acting as the most powerful CCR5 inhibitor in vivo. The study
population was stratified according to CCR5 expression level (high vs. low) and CCL3L1 genotype
(high copy number vs. low copy number), and genetic profiling defined individuals with high-,
moderate-, or low-risk of HIV-progression. However, CCR5 expression and these clinical parameters
were not strongly associated, suggesting that CD4+ T cell depletion is not only due to the rates of HIV
infection and replication (and therefore to CCR5 expression), but also to other immune mechanisms,
such as cell-mediated immunity (CMI). The exact mechanism(s) leading to CMI impairment is
presently undetermined, but it is expected to exert more subtle effects than the mere down-regulation
of the CCR5 receptor or the competition with HIV binding [42]. CCR5 knockout mice (ccr5-/-) were
found to develop normally, and also showed a more robust T-cell response to a number of antigens
than wild-type (WT) mice [43]. When ccr5-/- mice were experimentally infected with West Nile Virus
(WNV), all of them succumbed to the infection, while the majority of WT mice survived. CCR5-
deficient mice failed to control virus replication in the CNS, due to reduced recruitment of infiltrating
CD4+ and CD8+ T cells, NK cells, and macrophages, suggesting a disequilibrium between pathogen
immunity and deleterious effects of inflammation [44]. In the experimental infection with HSV-2,
ccr5 -/- mice also showed higher brain titers than WT control animals, but were able to clear the
infection [45]. Similar results were observed in cohort studies on patients infected by WNV, which
showed an increased risk of symptomatic infection in homozygous carriers of the delta-32
mutation [46]. Other flavivirus infections, such as the tick-borne encephalitis (TBE), an endemic
infection in Europe and Asia, were found to be associated with delta-32 alleles [41].
CCR5 also takes part in the response to bacteria and bacterial products, such as lipopolysaccharide
(LPS) and heat-shock proteins. Macrophages from CCR5-deficient mice challenged with LPS show an

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impaired function; this finding was associated with a reduced efficiency in clearance of Listeria
infection and with a protective effect against LPS-induced endotoxemia [43]. The possible association
of CCR5 deficiency with other diseases, such as hepatitis C, and with autoimmune disorders, such as
multiple sclerosis, has not been proven [47]. However, CCR5 deficiency was shown to play a
protective role in rheumatoid arthritis [48], supporting the use of CCR5 antagonists in clinical
treatment of autoimmune, inflammation-based disorders. In this case, CCR5 blockage may inhibit T
cell migration, a key pathway in the inflammatory process causing pain, tissue damage, and
disability [49]. Acute rejection is characterized by cell recruitment into clinical allografts via CCR5-
mediated cytokine signaling; for instance, immunosuppressed patients receiving renal transplants who
are homozygous carriers of the CCR5 delta-32 allele rarely exhibit late graft loss. The use of
cyclosporine A in association with a CCR5 inhibitor reduces leukocyte recruitment to grafts and
prolongs their survival in a cynomolgus model of monkey cardiac allograft model [50].
4. CCR5 role in HIV infection
HIV entry engages the viral env glycoprotein complex, the CD4 antigen, and a chemokine receptor,
nearly always CCR5 ─ sometimes CXCR4, especially in later stages of disease ─ both located on the
surface of the host cell. The virus envelope consists of two proteins, gp120 and gp41, which mediate
virus attachment on the host cell, binding, and fusion with the target cell membrane. The external
gp120 and the transmembrane gp41 subunits are generated by proteolytic cleavage of a larger
precursor, gp160, and are not covalently associated; three env complexes form trimeric spikes on the
virus particle. Although the three-dimensional structure of the env-receptor complex has not been fully
elucidated by spectrometric analysis, biochemical, genetic, and immunological investigations have
provided information about the order of event and the protein domains taking part in it [51].
Binding of gp120 to CD4 generates a conformational change in the env complex and exposes ─ or
induces ─ the CCR5 binding site, whose major domains are the bridging sheet and the variable V3
loop. Env domains interacting with the N-terminus and the second extracellular loop of CCR5 cause a
conformational change in the coreceptor, which activates the coreceptor signaling. Conversely, CCR5
binding triggers further conformational changes, leading to the extension of the gp41 fusogenic
domain and to refolding of the gp41 trimer in a six-helix bundle, bringing lipid bilayers into close
contact and eventually leading to fusion [4]. Comparative studies employing monoclonal antibody
panels, chimeric molecules, viral pseudotypes or site-directed mutagenesis, have helped to understand
the key determinants of binding. HIV binding has been shown to involve the N-terminus and the
second extracellular loop of the CCR5 molecule, while natural CCR5 ligands, such as CCL4/MIP-
1beta or CCL5/RANTES, bind to overlapping regions on the receptor, different for each ligand, and
compete for binding with the virus. Some monoclonal antibodies were also found to promote receptor
signaling and internalization, mediated by a conformational change requiring CCR5
oligomerization [52]. However, HIV binding may occur with wild-type and even with C-truncated
CCR5 receptors, which are unable to be internalized or to transduce signaling to G proteins, therefore
showing that this event is not required for efficient cell infection [53-55]. Direct crystallographic
approaches, as well as indirect biochemical or immunological studies, have led the way in the design

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and synthesis of drugs targeting CCR5, such as Maraviroc, which was approved for clinical use in
2007 [56].
4.1. CCR5 vs. CXCR4
Dendritic cells (DC) are natural sentinel cells that sample incoming pathogens or their antigens at
the mucosal epithelia, transport them to regional lymph nodes, and there present them to T and B cells
in order to initiate adaptive immune responses [57]. DC express CCR5, but not the CXCR4 receptor,
and therefore are exposed to infection by R5 virus strains. Such strains preferentially penetrate
mucosal barriers, leading to lymph nodes drainage, by using DC as Trojan horses [58]. Indeed, when
infected DC prime and activate CD4+ T cells within lymph nodes, the virus is placed in a perfect
environment that favors its rapid and efficient amplification, and R5 viruses dominate the scene
because of the expression of CCR5 on activated CD4+ T cells [59].
Extremely high levels of replication of SIV can occur in naturally infected monkeys without the
onset of immunodeficiency [60]. In HIV-infected humans, immune decline is associated with viral
impairment of the regenerative capacity of the defense system [61]. X4 viral strains can cause local
damage to the thymus by targeting developing T cells there. R5 viruses are less cytopathic than X4
strains in cultured thymocytes; they also replicate in the thymus in vivo, but without damaging the
developing T cells; In fact, CCR5 expression is lower than CXCR4 expression during T cell
development [62]. For instance, children carrying X4 viruses show a greater impairment of thymic
function and CD4+ T cells than those infected with R5 viruses [63]. Immune activation due to HIV
infection causes CCR5 up-regulation in CD4+ cells [64]. Overall, the relative expression levels of
CCR5 and CXCR4 in PBMC do not influence the rate of evolution of X4 variants; the availability of
CXCR4+ cells does not increase the evolution rate of X4 viruses; moreover, CXCR4 usage is not an
escape mechanism adopted to overcome propagation limits due to a lower count of CCR5+ target
cells [65]. The lack of a fast progression to AIDS due to expansion of X4 viruses in most individuals
could be due to virological and to immunological reasons. On one hand, R5 viruses are favored by the
prompt and abundant availability of CCR5+ cells in mucosae, in professional APC, and in lymph
nodes. On the other hand, the emergence of X4 strains in late stages of infection might reflect the
progressive accumulation of immune damage [66].
4.2. CCR5 in mucosal HIV transmission
Clinical observations confirm that mucosal transmission of HIV is nearly exclusively due to CCR5-
dependent HIV strains [67]. Dual-tropic, R5X4 viruses, or the rarer CXCR4-dependent viruses, are
observed in late phases of the infection, and are usually associated with a faster progression to AIDS
and to a marked decline of immune response [66].
The prevalence of R5-tropic HIV can be due to HIV biology as well as to host features; both factors
determine the natural history of infection. The mucosal environment is the place where virus-host
contact takes place and is where immunity should provide the maximal defense. The majority of HIV
infections, both horizontal and vertical transmissions, occur via genital mucosa, via sexual intercourse
or child delivery, even if HIV shows a low rate of infection through the genital route [68,69]. Mucosal

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immunity to infectious agents begins with the physical protection conferred by intact epithelial
barriers, then by innate and adaptive immunity (Figure 2).
Figure 2. The mucosal scenario of HIV infection. Reproduced from Klasse et al., 2008 [69].
Pluristratified epithelia protect oral, vaginal, and anal accesses, while monostratified mucosa lines
the gut and the endocervix. The presence of mucus, as in the gut and in the cervix, is another physical
barrier, which can entrap pathogens and prevent infection by sexually-transmitted or food-borne
pathogens. Under the epithelial barrier, genital and intestinal stromal tissues are densely populated
with dendritic cells, macrophages, and T cells, which play a role in immune surveillance. The majority
of these cell types express CD4 and CCR5 molecules, and therefore offer a large and convenient
population of target cells to HIV [66]. In monostratified barriers, such as the columnar cervical
epithelium and the gut mucosa, HIV particles may diffuse by transcytosis due to their monostratified
structure - these layers offer poor resistance to virus penetration. Conversely, the vaginal mucosa
offers a stronger barrier to pathogens, due to the pluristratified structure and to the relatively restricted
surface. Microabrasions due to sexual intercourse and concomitant sexually-transmitted infections may
weaken or break the vaginal epithelium, facilitating HIV direct diffusion to submucosal tissues [69].
The gut surface, which is considerably larger (up to 400 square meters, i.e., the surface of a tennis
court), is monostratified and is therefore less resistant than the vaginal mucosa to microtrauma caused
by sexual intercourse. Moreover, submucosal GALT (gut-associated lymphoid tissues) hosts up to
90% of CD4+ and CD8 lymphocytes, making it a more important immune organ than even the

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blood [69,70]. CCR5 is expressed on intestinal epithelial cells, therefore allowing preferential
transmission of R5 viruses via the rectal route. CXCR4 is not expressed on intestinal epithelia, and
high levels of SDF-1, CXCR4-blocking ligand, are secreted in the intestinal lumen, thereby hampering
the transmission of X4-tropic HIV strains [66].
Mucosal surfaces are characterized by various molecules, including innate mucosal receptors, lipid
raft microdomains, and HIV coreceptors [71]. Notably, both CCR5 and CXCR4 receptors are
expressed on the genital mucosa. CXCR4 receptors are usually rare, due to down-regulation, and
mediated by high local SDF-1 expression [72]. The R5 virus is preferentially transmitted upon its
interaction with immune cells residing in the submucosal tissues, such as DC, Langerhans cells, and
macrophages; X4 viruses have multiple disadvantages in infecting target cells in deeper mucosal
layers. DC may transport HIV to regional lymph nodes where the virus can encounter CD4+ T cells,
other targets susceptible to infection by R5 HIV. In the human gut, organized mucosal lymphoid
follicles are aggregated to form Peyer’s patches, which are committed to sense microbes and antigens
in the lumen and provide a prompt immune response.
The gut epithelium contains specialized sensitive cells, the M cells, which form intraepithelial
pockets where submucosal lymphocytes can migrate; M cells capture and deliver samples of
intraluminal material by vesicular transport to underlying DCs [73]. Vaginal mucosa lacks organized
lymphoid follicles, like those found in Peyer’s pathches and M cells; therefore, DC themselves migrate
between epithelial cells, interrupt tight junctions, and obtain samples of foreign material directly from
the luminal compartment [74,75]. DC-SIGN, a C-type lectin expressed on submucosal DC in the
genital tract, may act as a Trojan horse that facilitates the induction of primary immune responses [59]
and at the same time, carries HIV particles to lymph nodes, where naïve T cells will be activated [76].
Further HIV dissemination will proceed from the mucosa-associated lymphatic tissues to other target
organs, such as spleen, brain, liver, and lungs, via infected macrophages or T-cells; infected cells can
also return to mucosal tissues, through infected mucosal secretions and semen [73,77].
Similarly to infection, the immune response also begins in and spreads from lymph nodes, in the
form of plasma cells that secrete neutralizing antibodies, T helper cells that produce cytokines, and
cytotoxic lymphocytes. Increased vascular permeability, subsequent to inflammatory stimuli driven by
the infection event, facilitates both the local recruitment of macrophages, NK, and T cells, as well as
the drainage of IgG molecules in situ [71]. Different antibodies isotypes, such as IgG, IgM and IgA,
take part in several effector pathways that may protect the host from mucosal infection and clear the
virus [78]. Soluble antibodies can compete with HIV for attachment to epithelial cells, participate in
opsonization, activate complement-mediated cell lysis, induce antibody-dependent cell cytotoxicity,
and mediate transcytosis inhibition [68,77].
5. Natural history of individuals carrying anti-CCR5 antibodies
Different types of HIV-blocking antibodies to CCR5 have been isolated from HIV-infected and
from HIV-exposed (ESN) subjects.
Antibodies to the HIV binding domain, i.e., the second external loop of the CCR5 molecule, appear
in response to HIV infection and block HIV entry through binding competition [79].

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Anti-CCR5 antibodies recognizing the first external loop of the protein do not interfere with HIV
binding directly, but rather induce coreceptor down-regulation, thus abolishing virus
infectivity [80,81]. The generation of anti-CCR5 antibodies to the first external loop, observed in
healthy subjects not previously exposed to HIV, could be probably explained by autoimmune
phenomena triggered by membrane perturbations unrelated to HIV stimuli, such as the activity of
exogenous or endogenous viruses or local inflammation [81,82].
During HIV infection, allo-immune responses to polymorphic surface molecules, such as HLA,
may be generated in response to antigens entrapped in HIV particles during budding, or may be
elicited via molecular mimicry between gp120, gp41 and host antigens. Allo-immunization can elicit
chemokine and CD8 effector cells to HIV in humans [83] and was also shown to be effective to
protect monkeys from experimental challenge with infectious SIV [84]. The binding of HIV antigens
may induce alterations in host antigens, which are subsequently presented to the immune system in the
form of cryptic or uncommon self-epitopes, thereby generating anti-self responses. Responses leading
to the generation of anti-self antibodies can also involve idiotype-anti-idiotype networking via an
homology/mimicry interplay between HIV and host antigens. This mechanism may lead to the
generation of cross-reactive antibodies and to the development of immune complexes, which may
entrap viral particles and host antigens and potentially give rise to other uncommon antibody
specificities [85].
Antibodies to the first extracellular loop (ECL1 domain) of CCR5 have been only detected in HIV-
exposed but uninfected subjects (ESN) and in long-term non-progressing HIV-positive subjects
(LTNP), supporting the hypothesis that these antibodies could be involved in HIV protection or in
infection control. One clinical study searched for such anti-CCR5 antibodies in 497 subjects, including
85 LTNPs, 70 progressors, 135 HIV+ patients receiving highly active antiretroviral therapy (HAART),
and 207 HIV-negative donors [86]. Anti-CCR5 antibodies were isolated in 23% of the LTNP but not
in the other subpopulations studied (P<.001; Figure 4). Anti-CCR5 Abs were shown to recognize a
conformational epitope within the first external loop, and to induce a stable and long-lasting
downregulation of CCR5 from the surface of T lymphocytes, thereby inhibiting HIV entry. Receptor
internalization was shown to be specifically inhibited by sucrose, but not by filypin or nystatin,
nocodazole or cytochalasin D, therefore supporting a specific role for clathrin-coated pits and
excluding the caveolae compartments [86]. In addition, CD4+ lymphocytes from the LTNP subset
displaying anti-CCR5 Abs were found to be resistant to in vitro infection with R5-tropic HIV-1 strains.
The level of anti-CCR5 antibodies appeared to be correlated with levels of HIV exposure, being lower
in seronegative ESN subjects and higher in seropositive LTNP individuals (0.1% vs. 8% of the total
antibodies, respectively).
Interestingly, the loss of anti-CCR5 antibodies was observed in the course of clinical follow-up, and
this event was significantly associated with clinical progression toward disease in 9 out of 20 LTNP
enrolled in the study, some of who experienced a statistically significant increase in viremia and
required the resumption of therapy, thus becoming progressors. Strikingly, subjects who retained anti-
CCR5 Abs maintained a stable LTNP status without any treatment. According to the finding, the loss
of anti-CCR5 Abs was associated with disease progression; toward this observation was strongly
supported by the development of AIDS despite antiretroviral therapy in some subjects [86].

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Figure 3. Anti-CCR5 antibodies to the first extracellular loop, isolated in various cohorts
of HIV-exposed or HIV-infected, Long-Term non progressing people. Modified from
Pastori et al., 2006 [86].
The persistence of very low, undetectable levels of HIV replication may provide a continuous
antigen boost that does not result in a strong generalized immune activation, similar to what is
observed in the course of natural latent viral infections (e.g., herpes viruses) or in food-delivered
antigens and/or vaccines, which may establish tolerance but also retain their antigenic
potential [70,87]. In the lucky subset of ESN and LTNP individuals able to control HIV, physiological
and immunological conditions might have established a positive feedback loop that maintains
undetectable levels of virus replication and a suitable antigen presentation on one hand; and on the
other hand, long-lasting responses that are able to block HIV through its major coreceptor provide a
key mechanism for fighting HIV replication [85]. Another key point in the study is the observation that
the viral phenotype in LTNPs carrying anti-CCR5 antibodies did not shift in the presence of such
antibodies, thus confirming that the selective pressure of CCR5 inhibitors does not induce a change of
viral phenotype per se, as already reported in a monkey model [88]. In addition, anti-CCR5 antibodies
were not found to induce any apparent alterations in immune function, as demonstrated by the
continued health of subjects who retained anti-CCR5 antibodies; both these findings provide
arguments against theoretical concerns about CCR5 targeting with specific antibodies.
6. Strategies for CCR5 targeting
Due to its features and its natural history, CCR5 is a key target in HIV therapy and prevention.
CCR5-fostered therapeutic approaches to block HIV infection to date, including small molecule

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inhibitors, chemically modified ligands, and anti-CCR5 antibodies, have shown their antiviral
properties in cell-based tests and in in vivo trials [56,89,90]. These approaches, shown in Figure 3, can
be defined as “extracellular”; other approaches, still the subject of preclinical research, target CCR5
expression from an “intracellular” point of view; for example, taking advantage of drugs such as
rapamycin [91] or statins [92], which prevent CCR5 surface expression. Interfering mRNAs
(siRNAs) [93] and ribozymes [94] have also been shown to interfere with CCR5 expression. Other
methods being developed, such as “intrabodies” (single-chain, intracellular antibodies) [95] or
“intrakines” (intracellular chemokines) [96], also aim at trapping CCR5 within cells, thus preventing
its surface expression and/or its recycling [2].
6.1. Small molecule inhibitors
The binding of HIV to CCR5 occurs after the CD4-gp120 interaction, and usually triggers a
conformational change in the HIV envelope, which allows gp41 activity and promotes virus-cell
fusion. Small molecule antagonists of CCR5 that bind to CCR5 within a pouch created by the seven
membrane-spanning hydrophobic helices, stabilize the receptor conformation and prevent HIV binding
to the extracellular domains, namely to the N-terminus and ECL-2, thereby blocking gp41-mediated
fusion and subsequent viral replication [89]. CCR5 antagonists do not act on CXCR4, so their use is
not recommended in patients hosting X4 or R5/X4 dual tropic viruses. Usually X4 and dual-tropic
virus isolates appear later in the course of infection, and their appearance is usually associated with a
faster decrease in CD4+ T cells count and the progression towards symptomatic disease.
Two joint clinical trials (MOTIVATE one and two) have demonstrated the efficacy and safety of
maraviroc, which is currently approved for use in treatment-experienced patients; other drugs are
currently under preclinical or clinical development. After 48 weeks, patients receiving maraviroc
showed at least a 1.5 log reduction in viral RNA copies/mL - significantly higher than the control - and
more than 40% of patients in all groups of treatment showed virus replication levels of <50 copies/mL.
Moreover, treated patients also displayed a significantly greater increase in CD4+ cell count than the
controls. Conversely, a consistent percentage of treatment failure was observed, mostly due to the
emergence of dual-tropic or X4 viral strains that were not blocked by antiviral therapies associated
with the study drug [56].
HIV is notorious for its ability to overcome immune defenses ─ and antiretroviral therapy ─
through Darwinian selection, i.e., by random gene mutations and selection of drug-resistant viral
strains. At the molecular level, resistance to small inhibitors involves point mutations in some env
domains, such as the variable V3 loop and C2-V5 mutations; V3 mutations by themselves were found
necessary but not sufficient for resistance [97,98]. Changes in the V3 region can strengthen the env
interaction with the N-terminal domain of CCR5, while the conformation of the first and second
extracellular loops, regions, altered by most CCR5 inhibitors, do not affect env binding and viral
entry [99]. Indeed, CCR5 blockage could offer the virus the obvious alternative to use CXCR4
molecules to bypass the drug effect. A switch towards a X4 phenotype has been shown to occur in
vitro and in vivo, however, X4 viruses appear to be selected in control as well as in cell cultures treated
with inhibitor molecules, suggesting that the viral genetic drift may occur independently of drug-
induced selection, e.g., as an adaptation to infect PBMC cells [89]. Probably, MOTIVATE patients

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already hosted X4 virus strains at the time of enrollment; according to other in vitro assays, the R5
viruses tended to retain their phenotype under inhibitor selection, probably because the original R5
viruses were less sensitive to a selective environment than the newly arisen ones [89].
Figure 4. Current approaches to extracellular CCR5 blocking.
Two major mechanisms have been described to explain HIV resistance, the competitive and the
non-competitive (allosteric) mechanisms. Competitive resistance appears as a shift in the drug IC50;
higher drug doses still achieve a 100% inhibition. Allosteric inhibition, conversely, does not change
IC50 values, but reduces the maximal viral inhibition below 100%, which is insensitive to further drug
addition. In competitive resistance, viruses most efficiently use CCR5 molecules that are still free from
drugs, while in allosteric resistance, new variant viruses become able to use drug-bound coreceptor as
well as free-CCR5 molecules [89].
Various studies and clinical observations support the idea that the R5 to X4 switch is a complex
event, which is not directly associated with the use of CCR5 inhibitors or other anti-retroviral drugs.
The coreceptor switch was observed in approximately 50% of HIV-infected patients carrying sub-type
B viruses, but progression to AIDS also occurred in patients hosting uniquely the R5 virus with
various functional changes in multiple env domains [100,101]. More strikingly, HIV entry efficiency
via CCR5 was found to improve in patients that remained R5-positive only, whereas it declined in
viruses isolated from patients carrying R5X4 viruses [102]. This apparently paradoxical finding can
be explained by results of other studies, which depicted dual-tropic R5X4 viruses as an heterogeneous

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population, in which “dual-R” (CCR5-preferering) or “dual-X” (CXCR4-preferring) viruses can be
distinguished [103].
Small CCR5 inhibitors do not always induce cross-resistance to other antiviral drugs, either acting
via CCR5, such as antibodies or modified chemokines, and to drugs endowed with other mechanisms
of action, such as protease inhibitors or RT-inhibitors [90]. In some cases, the acquisition of resistance
does not appear to compromise viral “fitness”, i.e., its replicative ability; in other cases, once the
selective agent is no longer administered, the virus phenotype reverts to sensitivity, showing that that
in-vitro resistance can carry a fitness cost [97]. However, env glycoproteins are under continual
selection pressure of neutralizing antibodies (NAbs) in vivo [104]; when small inhibitors are
administered, HIV undergoes a double selective pressure, which may impose functional constraints on
virus variability. For example, a virus variant able to evade an inhibitor can maintain sensitivity to a
Nab. According to a study evaluating mutant HIV strains resistant to small inhibitors, viruses resistant
to vicriviroc or AD101 also showed cross-resistance to other small inhibitors, such as aplaviroc or
maraviroc. However, they retained sensitivity to other antiretroviral drugs, such as reverse
transcriptase inhibitors, protease inhibitors and fusion inhibitors, such as enfuvirtide, which are
endowed with mechanisms of action independent of CCR5 [105]. Strikingly, such escape mutants
were sensitive to the chemokine ligand PSC-RANTES, to neutralizing mAbs, such as the humanized
antibody PRO140, and to sera from HIV-infected people [105]. These findings show that env
mutations conferring resistance to CCR5 inhibitors do not necessarily affect the efficacy and safety of
other antiviral strategies, particularly the activity of humoral immunity ─ chemokines and antibodies ─
either natural, elicited or administered.
6.2. Monoclonal antibodies to CCR5
Humanized monoclonal antibodies recognizing the CCR5 extracellular N-terminal and/or second
extracellular loop domains have been developed and were able to compete with gp120 binding [2].
When assayed in vitro at nanomolar concentrations, PRO140 was able to block HIV strains belonging
to different clades, both in primary macrophages and in PBMC [106]. PRO140 and another mAb,
HGS004, have been tested in HIV-infected subjects [107,108]. PRO140 was able to inhibit HIV
without blocking CCR5 response to chemokines, whereas HGS004 prevented both viral infection and
chemokine signaling. Notably, antibodies and small molecule antagonists do not share the same
mechanism and site of action; therefore, their activity may be synergic or contrasting, and no cross-
resistance has been observed [2].
6.3. Engineered chemokines
Natural chemokines have been found to prevent HIV binding to its coreceptors, due to steric
hindrance or competition for binding sites or to receptor internalization. Conditions which induce
sustained production of beta chemokines (CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES)
have been associated with a lower risk of HIV transmission; similarly, natural mutations that enhance
production of SDF-1, and therefore increase competition for the CXCR4 coreceptor, were shown to
play a protective role in HIV infection [72,83]. Due to the short half-lives (<10 min) of natural
chemokines, various N-terminally modified chemokines have been synthesized and tested for the

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ability to prolong or shorten CCR5 internalization [2]. AOP-RANTES (aminooxypentane-RANTES)
inhibits different HIV strains infecting PBMC through the induction of gamma-IFN and the parallel
reduction of IL-10 expression [109]. Its mechanism of action relies on coreceptor internalization and a
long-lasting inhibition of CCR5 recirculation [110]. PSC-RANTES ([N-nonanoyl, des-Ser1[L-
thioproline2, L-cyclohexylglycine3]) is a strong CCR5 agonist and a highly potent inhibitor of HIV
entry in vitro; it confers full protection from R5-mediated infection in an animal model of vaginal
transmission, due to long-lasting receptor internalization. However, CCR5 binding by CCL5/RANTES
derivatives is also associated with mucosal inflammation, a phenomenon which would enhance HIV
infection [111]. Pharmaceutical research and development is presently striving to identify new
derivatives that can be produced in inexpensive expression systems by the fermentative industry
without requiring post-synthetic chemical modifications. Moreover, ideal CCL5/RANTES derivatives
should also separate HIV-inhibition activity from CCR5 signaling, in order to prevent in vivo
potentially harmful pro-inflammatory activity [111,112]. It should also be remembered that
engineered chemokines, similarly to small molecule inhibitors, were shown to exert selective pressure
on CCR5-tropic viruses, with consequent development of R5 inhibitor-resistant HIV strains and
finally, leading the way to dual-tropic or X4-tropic virus shift [113].
6.4. Anti-CCR5 vaccination
Anti-CCR5 antibodies recognizing the first external loop of the protein do not interfere with HIV
binding directly, but induce coreceptor down-regulation, thus eliminating virus infectivity [81,114].
Such rare antibodies raise questions about their genesis, which may be natural (via genetic
mechanisms) rather than elicited by some still undetectable HIV-unrelated or very low level antigenic
stimulation. However, despite some still open questions, some vaccination experiments have
successfully elicited anti-CCR5 auto-antibodies and have investigated both in vitro and in vivo the
immune protection they confer.
Immunization experiments and in vitro studies of elicited antibodies were performed by
Chain et al. [115], who immunized rabbits with chimeric peptides encoding a very short fragment of
the N-terminal sequence of CCR5 (Met1-Ser7 or Asp2-Ser7), and a T-specific peptide from Tetanus
toxoid. T-specific CCR5 epitopes were not included in the immunogen, in order to prevent the
development of host autoimmune responses. The immunization generated a strong antibody response.
Binding experiments to N-terminal and full-length CCR5 suggest that only a small percentage of the
antibodies elicited by immunization were able to bind CCR5; nevertheless, anti-CCR5-specific
antibodies blocked HIV infection in macrophages in vitro. In a subsequent study, Devito et al. [116]
èerformed a long-term immunization with intranasal DNA prime followed by a peptide booster
immunization. Delivered antigens were from gp120 V3 loop, gp41 (MPER peptides containing the
ELDKWAS epitope) and CCR5-ECL domain (aa.168-185). The vaccination schedule elicited specific
IgG and IgA in sera and in mucosal secretions (intestinal, vaginal and lung) in immunized mice. More
interestingly, long-term IgG and IgA responses were still observed 12 months after boosting - both in
serum and in mucosal secretions. HIV-1-neutralizing antibodies were still detected in serum 12 months
after boosting. According to this study, intranasal DNA prime followed by one peptide/L3 adjuvant
booster immunization, ─ but not vice versa ─ induced long-lasting neutralizing antibodies and B

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memory cells to poorly immunogenic, conformational epitopes. Barassi et al. [81] generated chimeric
immunogens containing a CCR5 peptide from the first excellular domain (Tyr89-Trp102) in the
context of the capsid protein of flock house virus, a conformation-constrained expression
system [117]. When administered to mice by systemic or mucosal route, the immunogens elicited anti-
CCR5 IgG and IgA both in sera and in vaginal fluids. Similarly to HIV-exposed seronegative
individuals, mice producing anti-CCR5 autoantibodies expressed significantly reduced levels of CCR5
on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies showed that
murine IgG and IgA (i) specifically bound human and mouse CD4+ lymphocytes and the CCR5-
transfected U87 cell line; (ii) down-regulated CCR5 expression of CD4+ cells from both humans and
untreated mice, (iii) inhibited CCL4/MIP-1beta chemotaxis of CD4+ CCR5+ lymphocytes, and (iv)
blocked HIV R5 strains. Finally, Pastori et al. [118] performed a peptide-scanning assay on a panel of
synthetic peptides spanning the CCR5-ECL1 region; the resulting peptides were assayed with a pool of
natural anti-CCR5 antibodies and used to immunize mice and chickens. Further structural
characterization of the peptides was provided by NMR spectroscopy and molecular dynamics
simulations. Amino acid substitutions in positions 95 and 96 (Ala95-Ala96) increased antibody-
peptide binding compared to the wild-type peptide (Asp95-Phe96). The Ala95-96 peptide was able to
induce antibodies, both in mice and chickens, which displayed biological activity at very low
concentrations. Strikingly, chicken antibodies to the Ala95-96 peptide specifically recognized human
CCR5 molecules, down-regulated receptors from lymphocytes, inhibited CCR5-dependent chemotaxis,
and prevented infection by several R5 viruses, displaying IC50 values lower than 3 ng/ml. NMR
spectroscopy and molecular dynamics simulations confirmed the high level of flexibility of the
isolated epitopes and suggested that A95-A96 substitutions led to a slightly higher tendency to
generate helical conformations combined with a lower steric hindrance of the side chains in the
peptides. The different structural behavior of the mutagenized loop may account for a better molecular
structural organization, allowing the induction of the fittest antibodies. Optimized antibodies
recognized and bound native CCR5 with higher affinity and displayed enhanced biological activity.
Other in vivo studies coupled immunization studies with in vivo challenges to vaccinated animals to
evaluate whether a break in B-tolerance had been achieved and the extent of immune protection
conferred by the tested immunogens. Chackerian et al. [119] used the N-terminal domain of pigtailed
macaque CCR5 fused to streptavidin, which when conjugated at high densities to bovine
papillomavirus major capsid protein L1 virus-like particles induces high-titer anti-CCR5 IgG that
blocks infection by CCR5-tropic simian-human immunodeficiency virus (SHIV) in vitro. No decline
in the number of CCR5-expressing T cells was detected in immunized animals. In SHIV-challenged
macaques, viral loads and time to control of viremia were significantly decreased (relative to controls),
indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.
Bogers et al. [120] assayed a vaccine consisting of three extracellular peptides of CCR5, an
N-terminal HIV gp120 fragment generated in transgenic plants, and recombinant simian
immunodeficiency virus p27. They were linked to the microbial heat shock protein HSP70 used as a
carrier, and the vaccine was administered by mucosal and systemic routes. Vaginal challenge with
SHIV infected all macaques, but showed a significant variation in viral loads between the animals, and
the virus was cleared in five of nine immunized animals. Misumi et al. [121] adopted synthetic cyclic
peptides from the second external loop (Arg168 to Thr177) to induce anti-CCR5 antibodies in

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cynomolgus macaques. The immunization with cDDR5-conjugated multiple-Ag peptide
(cDDR5-MAP) induced long lasting anti-cDDR5 antibodies reacting with both human and macaque
CCR5 molecules, which were able to suppress infection by the R5 HIV-1 laboratory isolate (HIV
JRFL), R5 HIV-1 primary isolates (clade A:HIV 93RW004 and clade C:HIV MJ4), and a pathogenic
simian/HIV (SHIV SF162P3) bulk isolate in vitro. After SHIV challenge, cynomolgus macaques
showed an attenuated acute infection and a lower viral load than unvaccinated control animals.
According to in vitro and in vivo findings, immunization does elicit antibodies endowed with
neutralizing properties, showing that B-tolerance can be effectively broken; despite none of the
immunogens assayed in vivo being able to confer full protection from the virus challenge; infection of
vaccinated subjects was milder than in the controls and virus control was achieved in most subjects.
Finally, in vitro studies also demonstrate that conformational changes in the CCR5 protein, together
with host factors, have the potential to modulate protein immunogenicity in vivo and could also play a
role in the natural resistance to HIV infection.
7. Conclusion and perspectives for a vaccination intervention
CCR5 is a key player in HIV entry and many attempts to prevent its role in infection have been
developed and assayed. The clinical use of small CCR5 inhibitors has proven the feasibility and the
efficacy of CCR5 targeting, but it has also raised concerns about the safety of this approach:
R5-resistant HIV strains have been isolated in cell cultures and in patients receiving maraviroc and
other CCR5 inhibitors [56,89,105]. The use of humanized monoclonal antibodies has been shown to
be effective, safe, and long-lasting in HIV-infected patients, suggesting that passive immunization may
also offer therapeutic advantages [107,108]. The use of engineered chemokines induces receptor
down-regulation, therefore preventing the binding of CCR5 to HIV. However, despite its effectiveness,
this approach might be associated with adverse inflammatory events in vivo [111]. An HIV vaccine
remains the most expected goal to be accomplished in HIV research, proving its value both in
therapeutic intervention and in prevention [122]. Vaccination may offer long-lasting protection with
few administrations, an alternative in many geographical and social contexts where other forms of
prevention for sexually-transmitted diseases could be impractical or rejected [69].
Anti-CCR5 vaccination is an innovative anti-HIV strategy, which could provide effective protection
or safe containment of the spread of the virus. Indeed, the feasibility of anti-CCR5 vaccination has
been already demonstrated by two groups of naturally occurring CDC5-deficient people. Individuals
deprived of CCR5 receptor by genetic deletion [30,123,124] and those carrying naturally occurring
anti-CCR5 antibodies that down-regulate the receptor in vivo [82,86,114], were found to be healthy
and very resistant to HIV-infection. Very importantly, such natural anti-CCR5 antibodies were
observed in sera and in mucosal fluids from individuals who remained uninfected despite repeated,
unprotected, sexual exposure to HIV, and in infected individuals with long-term, asymptomatic
infection. The finding that both ESN and LTNP subpopulations exert a high and durable control on the
virus supports the hypothesis that natural anti-CCR5 antibodies could be associated with protection.
This concept is further strengthened by the good health and immune status shown by the LTNP cohort,
suggesting that long-lasting CCR5 down-regulation is not harmful; conversely, the loss of anti-CCR5
responses experienced by some patients in cohort follow-up was associated with a decline in virus

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control [86]. These findings are noteworthy, because genetic CCR5 deletion is associated with an
increased susceptibility to some viral and bacterial pathogens [41]. Moreover, anti-self immunity was
one of the mechanisms evoked to explain the generation of natural anti-CCR5 antibodies [85] and a
possible adverse event associated with anti-CCR5 vaccination [2]. Conversely, CCR5 targeting could
offer therapeutic advantages just in some autoimmune-based diseases, such as rheumatoid
arthritis [47], or in transplantation therapy - all situations where chemokine signaling and cell
recruitment are immune mechanisms sustaining tissue damage [50]. Another key finding from the
follow-up of the LTNP cohort was the lack of a R5-to-X4 shift, a fact supporting the safety of
antibody-mediated coreceptor targeting [86]. This is a key point to be considered due to the concerns
raised by the therapeutic use of small CCR5 inhibitors, which are prone to developi of in vitro and in
vivo drug resistance and might favor the selection of dual-tropic or X4-tropic virus
strains [88,89,125]. Indeed, immunization experiments performed in animals have shown that anti-
CCR5 antibodies can be obtained in vivo, provided that suitable vector systems are used, either to
break B-tolerance to the self CCR5 antigen and to constrain the ECL1 peptide (i.e., the target domain
of natural anti-CCR5 antibodies) in a conformation similar to the naturally occurring, immunogenic
one [81,118]. Moreover, anti-CCR5 antibodies elicited by mucosal route were shown to be long-
lasting and promptly re-boosted upon immunization, in sera and most importantly in mucosal fluids,
demonstrating the feasibility of local immunity at major portals of HIV entry [81].
Taken together, all of the findings we have reviewed here support the significance of interventions
aimed at targeting the CCR5 molecule as a principal HIV coreceptor. Among all the strategies now
available or under development, naturally occurring anti-CCR5 antibodies show the therapeutic
potential to provide durable, effective, and safe systemic, and especially, local immunity to HIV. From
follow-up studies and immunization experiments, antibody-mediated CCR5 targeting has been shown
to be not only feasible but also well tolerated. Together with other immune-modulating strategies, this
unconventional approach could open unprecedented scenarios not only in HIV vaccinology, but
possibly also in the treatment and prevention of other disorders where harmful pro-inflammatory
responses can develop.
Acknowledgements
The Author wishes to thank Silvia Russo for her editorial help. The study was supported by
Grant n° 201433 from European Commission/Seventh Framework Programme (URL: www.ngin.eu),
Grant n° 1 U19 AI062150 from NIH and Grants GCE n° 53030 and n° PP1008144 from Bill and
Melinda Gates Foundation.
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