Role of Human -defensins
in HIV Infection
A. Weinberg1*, M.E. Quiones-Mateu2,
M.M. Lederman3
1Department of Biological Sciences, School of Dental Medicine, Case
Western Reserve University (CWRU), 10900 Euclid Ave., Cleveland, OH
44106, USA; 2Lerner Research Institute, Cleveland Clinic Foundation,
Center for AIDS Research, CWRU; and 3 University Hospitals of
Cleveland, OH; *corresponding author, aaron.weinberg@case.edu.
Adv Dent Res 19:42-48, April, 2006
M
echanisms of resistance to HIV-1 infection in the
human oral cavity are incompletely understood.
While salivary components have been implicated in
protection, there is growing evidence that human
beta-defensins (hBDs), originating in oral epithelial cells, may
be playing an important role in the prevention of HIV
infection. New antiviral, chemotactic, and immunosurveillance
properties are being attributed to hBDs, which are small
cationic antimicrobial innate response molecules expressed in
mucosal epithelium. Inducible hBDs are always expressed in
normal oral epithelium, a property not shared by other
mucosal barriers. Data reviewed in this paper demonstrate
that: (1) HIV-1 X4 and R5 phenotypes induce hBD-2 and -3
mRNA in normal human oral epithelial cells; (2) hBD-2 and -3
inhibit HIV-1 infection by both viral strains, with greater
activity against X4 viruses; and (3) this inhibition is due to a
direct interaction with virions and through modulation of the
CXCR4 co-receptor. These properties may be exploited as
strategies for mucosal protection against HIV-1 transmission.
Introduction
Epidemiological evidence suggests that exogenous infection
with HIV-1 through the oral cavity is quite rare, yet the
mechanisms for this protection are not understood. Moreover,
while persons chronically infected with HIV may be infected
with viruses that utilize CCR5 or CXCR4 co-receptors for
cellular entry, in acute infection, CCR5-tropic viruses almost
always predominate. The mechanisms underlying this
restriction are also not completely understood. While adaptive
cellular immune responses are critical regulators of HIV
replication once infection has occurred, and there is some
evidence that persons at high risk for HIV infection who
remain uninfected may have low-level cellular responses to
HIV-1, there is increasing interest in the importance of innate
immune factors emanating from mucosal tissues and fluids.
Recent findings in the field of innate immunity are
shedding new light on the importance of host defense
antimicrobial peptides. With the recent discoveries of human
-defensins (hBDs) in mucosal epithelium, a new line of
investigation is emerging to test the hypothesis that these
peptides, integral members of the innate host immune
response, function to protect the host against microbial
pathogenesis at the mucosal barrier. While their antibacterial
and antifungal properties are well-established (Weinberg et al.,
1998; Zasloff, 2002), new findings point to hBDs acting like
chemokines in 'cross-talking' with the adaptive immune
system, and possibly orchestrating immunosurveillance
through maturation of dendritic cells. They recruit immature
dendritic cells and T-cells by interacting with the chemokine
receptor CCR6 (Yang et al., 1999) and, in mice, have recently
been shown to activate immature dendritic cells through Toll-
42
like receptor 4 (Biragyn et al., 2002a). Since (1) the
constitutively expressed hBD-1 and inducible hBD-2 and hBD-
3 are present in normal human oral epithelium and cells
(NHOECs) (Krisanaprakornkit et al., 1998, 2000; Weinberg et al.,
1998; Dunsche et al., 2002), (2) they are important mediators of
innate mucosal defense against microbial infection (Weinberg
et al., 1998), (3) these peptides may be involved in
immunomodulation of the adaptive immune system (Biragyn
et al., 2002a,b), and (4) -defensins can impair adenoviral
infections (Gropp et al., 1999), we explored the possible role of
these molecules in defense against HIV infection. This review
describes novel information related to the antiretroviral
activity of mucosal beta-defensins.
Background
HIV-1 and the oral cavity
At the outset of the AIDS epidemic, there was concern that
HIV could be transmitted from the oral secretions of HIV
carriers during kissing, dental treatment, biting, or through
aerosolization. It became clear, however, that oral transmission
of HIV is actually rare (Rogers et al., 1990; Gooch et al., 1993;
Moore et al., 1993). Interestingly, the frequency with which
infectious HIV can be found in the saliva of HIV-1-infected
patients is low, approximately 1%, although infectious virus
can generally be isolated from the blood of untreated infected
persons (Barr et al., 1992; Moore et al., 1993; Coppenhaver et al.,
1994). This finding is not due to a lack of virus in the oral
cavity; HIV-1 RNA, proviral DNA, and infected cells are
detected in oropharyngeal tissues and salivary secretions of
infected persons (Goto et al., 1991; Baron et al., 1999). The
clinical importance of this dissociation is suggested by the
difficulty with which HIV is transmitted through the oral
mucosa (Herz et al., 2002). The question, therefore, is, why?
This is particularly important, since > 90% of HIV cases
worldwide have been transmitted via mucosal surfaces,
primarily across genital mucosal surfaces and across lower
gastrointestinal tract mucosa (Smith et al., 1999).
Several explanations have been suggested to explain the
paucity of oral HIV-1 infection: (i) a thick multilayered mucosal
surface (first line of defense against microbial invasion), (ii)
low salivary HIV-1 titers (Ho et al., 1985), and (iii) endogenous
antiviral factors present in oral secretions (Fultz, 1986; Shugars,
1999). During the last 15 years, multiple studies have been
conducted to identify the source(s) and identity of HIV-
inhibitory activity in the saliva of healthy and infected
individuals (Fultz, 1986; McNeely et al., 1995; Wahl et al., 1997;
Baqui et al., 1999; Becquart et al., 1999; Shugars, 1999; Pillay et
al., 2001). Many endogenous inhibitors of HIV-1 in saliva have
been proposed (e.g., amylase, lactoferrin, proline-rich peptides,
salivary mucins, thrombospondin; and secretory leukocyte
protease inhibitor [SLPI], reviewed in Shugars and Wahl, 1998).
Key Words
Human beta-defensin, epithelial cell, innate immunity, HIV.
Presented at the Fifth World Workshop on Oral Health and Disease
in AIDS, Phuket, Thailand, July 6-9, 2004, sponsored by Prince of
Songkla University, Thailand, the International Association for Dental
Research, the World Health Organization, the NIDCR/National
Institutes of Health, USA, and the University of California-San
Francisco Oral AIDS Center.
Moreover, these agents are found in seminal fluid and vaginal
secretions, routinely harvested from sites that are very
susceptible to infection (Shugars, 1999). Importantly, there is a
paucity of information regarding the contributions of innate
immune factors emanating from the oral mucosal epithelium
itself.
Transmission of HIV-1
A significant genetic bottleneck is apparent during transmission
by any route of HIV-1 infection. The selective factors imposing
the bottleneck are diverse and likely include: (i) host factors
such as innate immune response, (ii) density of target cells
and/or their co-receptors at the site of infection, (iii) number of
transmitted virions, and (iv) the structure of transmitted viral
quasi-species (swarms of mutants). Environmental differences
(pH, target cells, mucosal composition) at the site of exposure
may affect the efficiency of transmission of the infecting isolates
(Overbaugh et al., 1999; Blauvelt et al., 2000). Although there
may be an element of chance in the expansion of a particular
HIV-1 clone, phenotypic selection does occur in nearly every
HIV-1 infectioni.e., selection of R5 isolates occurs in nearly
every HIV-1 infection. The heterogeneous HIV-1 envelope
glycoproteins have been identified as responsible for two major
viral bio-phenotypes: (i) macrophage-tropic non-syncytium-
inducing/CCR5-tropic (NSI/R5) HIV-1 isolates, and (ii) T-cell-
line tropic isolates, forming cell syncytia during active
replication in tumor T-cell lines, which utilize the CXCR4 co-
receptor for entry (SI/X4) (Alkhatib et al., 1996; Deng et al., 1996;
Dragic et al., 1996; Feng et al., 1996). SI/X4 HIV-1 isolates often
dominate the quasi-species late in disease, and yet the NSI/R5
variant is typically transmitted to a newly infected person
regardless of the route of transmission (reviewed in Fenyo et al.,
2000). Preferential transmission of NSI/R5 over SI/X4 HIV-1
isolates is contradictory to increased replication of SI/X4 HIV-
1 over NSI/R5 isolates in culture (Tersmette et al., 1988;
Bjorndal et al., 1997). Thus, selectivity of transmission is likely
not simply a reflection of replicative fitness, but may also be
observed in the transmission of different NSI/R5 HIV-1
isolates in the human population (Blackard et al., 2001).
Although in vivo findings suggest that NSI/R5 HIV-1 isolates
may out-compete the SI/X4 variants at the site of primary
infection, one report suggests that the NSI/R5 isolates
predominate only after a temporary expansion of SI/X4 HIV-1
isolates is quenched by an activated immune response
(Cornelissen et al., 1995). However, this observation is difficult
to reconcile with the finding that humans who are
homozygous for a 32-base-pair deletion in the CCR5 open-
reading frame, and who lack CCR5 on any cell surface, are
typically resistant to HIV-1 infection (Dean et al., 1996; O'Brien
and Moore, 2000). To date, the universal factors (i.e., those
found in almost every human host) involved in the selection
of NSI/R5 HIV-1 isolates during transmission and
asymptomatic disease are not well-defined. Langerhans cells
(LC) are found embedded in mucosa (i.e., vaginal and oral
mucosa) and may be the first cell targets for primary
heterosexual transmission (Soto-Ramirez et al., 1996; Blauvelt
et al., 2000). Thus, LC may play a role in NSI/R5 HIV-1
selection, since CCR5 is better expressed in situ and in the
absence of external stimuli (Blauvelt et al., 2000). For example,
a recent report describes increased replication of an NSI/R5
(HIV-1Bal) over an SI/X4 (HIV-1III-B) isolate in LC embedded
in skin-derived explants, even though the opposite is true in
PBMC cultures or other permissive cell lines (Soto-Ramirez et
al., 1996; Blauvelt et al., 2000). However, this is not altogether
clear, since another report indicates that CXCR4 is functionally
expressed on the surfaces of freshly isolated and unstimulated
LC (Tchou et al., 2001).
Defensin peptides and their antiviral activity
The human defensin antimicrobial peptide family, its
Adv Dent Res 19:42-48, April, 2006 Role of Human -defensins in HIV Infection
molecular characteristics, and purported modes of activity are
reviewed in Weinberg et al. (1998) and Zasloff (2002). The
defensin peptides are a superfamily of peptide antibiotics with
a characteristic -sheet structure stabilized by two or three
intramolecular disulfide bonds. They are strongly cationic by
virtue of their numerous arginine and lysine residues. Their
amphipathic and cationic characteristics are important for
binding to anionic microbial surfaces, such as LPS, through
displacement of divalent, lipopolysaccharide (LPS)-associated
cell-surface cations, followed by membrane insertion through
the 'self-promoted uptake' pathway (Hancock, 1997) and
generation of stable pores (for review, see Weinberg et al.,
1998).
There is a paucity of information regarding defensin
antiviral effects. What has been published suggests that
defensins can inactivate a host of different viruses. Epithelial-
cell-derived beta-defensins have been shown to inhibit
adenoviral infections (Gropp et al., 1999). Alpha-defensins,
found in azurophilic granules of human neutrophils, inhibit
adenoviral infection in vitro (Bastian and Schafer, 2001),
inactivate cytomegalovirus, vesicular stomatitis virus,
influenza virus, and herpes simplex virus 1 and 2, but not two
non-enveloped viruses, echovirus type 11 and reovirus type 3
(Daher et al., 1986). Alpha-defensins have also been shown to
inhibit HIV replication in vitro (Nakashima et al., 1993; Zhang et
al., 2002). Theta-defensins, originally isolated from the rhesus
monkey, Macacca mulatta, and not expressed in humans (Tang
et al., 1999), were recently shown to prevent infection by T- and
M-tropic strains of HIV-1 (Cole et al., 2002). The mode(s) of
viral inactivation in all of the cited studies were not
determined, and therefore leave a definite void in our
understanding of this potentially important biological activity.
-defensins and the oral cavity
The recent discoveries that -defensins originate in
mammalian mucosal epithelium, including human (Diamond
et al., 1991; Schonwetter et al., 1995; Zhao et al., 1996; Harder et
al., 1997, 2001; McCray and Bentley, 1997; Boe et al., 1999;
O'Neil et al., 1999; Haynes et al., 2000; Garcia et al., 2001), has
led to the hypothesis that these antimicrobial peptides function
to protect the host against microbial pathogenesis at these
critical confrontational sites. We have extended this hypothesis
to encompass the oral epithelium as well (Krisanaprakornkit et
al., 1998, 2000; Weinberg et al., 1998; Dale et al., 2001). This
tissue, and cells derived from it, constitutively express hBD-1
and can be induced to express hBD-2 and -3.
The first evidence of -defensins in a mammalian oral
cavity was described by Schonwetter et al. (1995). The study
identified a -defensin in the upper surface of the bovine
tongue, which was markedly increased in the epithelium
surrounding naturally occurring tongue lesions, areas of both
acute and chronic inflammation. This agent was shown to be
an effective antibacterial and antifungal agent. Since then, we
and others have described the presence of -defensins in the
human oral cavity (Weinberg et al., 1998; Bonass et al., 1999;
Mathews et al., 1999; Sahasrabudhe et al., 2000; Dale et al., 2001;
Dunsche et al., 2002). In gingival tissue, mRNA for both hBD-1
and -2 was localized in suprabasal stratified epithelium, and
the peptides were detected in upper epithelial layers,
consistent with the formation of the stratified epithelial barrier
(Dale et al., 2001). hBD-1 and -2 were not detected in the
junctional epithelium (JE) that serves as the attachment to the
tooth surface. In contrast, -defensins and LL-37the only
other cationic antimicrobial peptide known to be expressed in
most mucosal epithelium, and belonging to the cathelicidin
family of antimicrobial peptides (reviewed in Weinberg et al.,
1998)are detected only in the polymorphonuclear
neutrophils (PMNs) that migrate through the JE (Dale et al.,
2001), a localization that persists during inflammation, when
the JE and surrounding tissue are highly infiltrated with
43
 system. Moreover, the fact that hBDs are
also expressed in less-differentiated
basally oriented epithelial cells indicates
that either differentiation is not essential
for hBD expression, as previously
suggested (Dale et al., 2001), or that hBD-
expressing cells, other than epithelial cells,
are infiltrating the oral mucosae. With
recent findings that hBD-1 and hBD-2 are
also expressed systemically, in human
monocytes, macrophages, and dendritic
cells (Duits et al., 2002), it is tempting to
speculate that they are the source, in part,
for hBD expression in the basal lamina.

Human Beta-defensins and HIV-1

As described above, the mechanisms
underlying mucosal transmission of HIV-1
are incompletely understood, as are the
mechanisms of resistance to HIV-1 infection
in the oral cavity. Infectious virus is rarely
isolated from saliva in HIV-1 infection, and
while salivary inhibitors of HIV-1i.e.,
mucins, amylase, SLPI, proline-rich
Fig. 1 - Immunohistochemical demonstration of human -defensin 1 (hBD-1) and hBD-2 in normal peptides, etc.have been identified, these
gingival epithelium. Gingival samples of periodontally healthy subjects were processed with polyclonal molecules are also found abundantly in
antibodies to hBD-1 (a,b) and hBD-2 (c,d) according to a standard immunohistochemical protocol. The vaginal and seminal secretions, sites where
tissue show intense expression of hBD-2 (c,d) and less intense expression of hBD-1 (a,b) in the granular, infection is more common. Finally, there is
spinous, and basal cell layers of gingival epithelia. The encircled areas in (a) and (c) are amplified in (b)
and (d), respectively, and show positive hBD expression in basal layers. Scale bar: 100 m. (Legend and
diminished infectivity of HIV-1 within oral
Fig. appear with permission from Dr. L.J. Jin [Lu et al., 2004]). tissue when compared with other
susceptible mucosae. For these reasons, we
proposed to test the hypothesis that
mucosal hBDs have anti-HIV-1 activity. Our
PMNs. Therefore, the undifferentiated JE contains exogenously findings are detailed in Quiones-Mateu et al. (2003) and are
expressed -defensins and LL-37, and the stratified epithelium reviewed herein.
contains endogenously expressed -defensins. Calprotectin, an
anionic epithelial cell and PMN-associated antimicrobial HIV-1 induces hBD-2 and -3 but not hBD-1 mRNA
peptide, is constitutively expressed in normal oral in normal human oral epithelial cells (NHOECs)
keratinocytes (Ross and Herzberg, 2001). These findings show NHOEC monolayers were challenged with HIV-1 strains
that defensins and other microbicidal peptides are localized in representing both viral bio-phenotypes, i.e., X4 and R5. Both
specific sites in the gingiva, are synthesized in different cell phenotypes were found to induce hBD-2 and -3 mRNA up to
types, and are likely to serve different roles in various regions 78-fold above baseline (see Quiones-Mateu et al., 2003). In
of the periodontium. addition, while HIV-1 can infect epithelial cells from other
A notable difference between oral and most other epithelia mucosal surfaces (Yahi et al., 1992; Han et al., 2000; Fotopoulos
is the expression of hBD-2 and -3. These defensins are et al., 2002), we could not detect infection of NHOECs by
expressed only in the presence of infection or inflammation in HIV-1 by viral reverse-transcriptase activity in culture
most tissues, including skin, trachea, and gut epithelium supernatant and after real-time PCR analysis to detect
(O'Neil et al., 1999). However, they are expressed in normal proviral DNA. These results differ from those of Liu et al.
uninflamed gingival tissue (Dale et al., 2001; Dunsche et al., (2003), who recently reported low-level replication of the
2002). Present evidence suggests that this baseline level of virus after high-dose viral challenge of NHOECs. The
hBD-2 and -3 could be due to the exposure of the tissue to discrepancy between their findings and ours may be due to
specific commensal bacteria. Our ongoing work in this area has differences in experimental conditions. We omitted polybrene
identified a novel strategy of Fusobacterium nucleatum, whereby from our infection assays, since this detergent is not
this ubiquitous Gram-negative organism of the human oral physiologic. They also infected cells using a viral inoculum
cavity stimulates hBD-2 and -3 expression in normal human eight times higher than ours. Finally, Liu et al. (2003) found
oral epithelial cells (NHOECs)a result that confers protection HIV-1 co-receptor expression on two-week-cultured
to the cells from P. gingivalis invasion (Weinberg et al., NHOECs, while our flow cytometric analyses did not reveal
unpublished observations; manuscript in review). CD4, CCR5, CXCR4, or galactosylceramide expression on
Most recently, Lu et al. (2004) demonstrated the three- to four-day-cultured NHOECs.
expression of hBD-1 and -2 not just in the granular and
spinous regions of normal gingival epithelia, but also in the hBD-2 and -3 inhibit HIV-1 replication without being
basal cell layers (Fig. 1). This is significant, since beta- cytotoxic to immunocompetent cells
defensins have been shown to possess immunomodulatory Pre-incubation of HIV-1 (X4 and R5 strains) with respective
activitysuch as chemoattraction of immature dendritic cells hBDs (recombinant forms generated in the Weinberg
(iDCs), monocytes, and T-cells (Yang et al., 1999; Wu et al., laboratory; see Quiones-Mateu et al. [2003] for details) at
2003)and maturation of iDCs (Biragyn et al., 2002a). concentrations ranging from 5 g/mL to 40 g/mL, followed
Expression of inducible hBDs throughout the oral epithelial by exposure of GHOST X4/R5 cells (osteosarcoma cells co-
mucosa supports the notion that these agents are also transfected with the HIV-2 long-terminal repeat driving
involved in 'cross-talk' with cells of the adaptive immune expression of the green fluorescent protein {hGFP}),

44 Weinberg et al. Adv Dent Res 19:42-48, April, 2006

demonstrated that hBD-2 and hBD-3 were
preferentially able to inhibit X4 virus from
infecting the GHOST cells, but not hBD-1
(Fig. 2B). Moreover, reduced GFP
fluorescence coincided with reduced viral
reverse-transcriptase activity (Fig. 2C).
Moreover, conditions that mimic the oral
mucosal interface, low salt and no serum
(Mandel, 1972), were found to elicit the
best in vitro protection by the hBDs
(Quiones-Mateu et al., 2003). Interestingly,
the concentration range of the hBDs that
were used fell well within the reported
concentrations of hBD-2 in normal oral
epithelium (Sawaki et al., 2002). A
thaizolyl-blue-based colorimetric assay
(MTT method) (Pauwels et al., 1988)
revealed no cytotoxicity against any of the
immunocompetent cells that were used in
the study, even at the highest hBD
concentrations that demonstrated anti-
HIV-1 activity.
hBD-2 and -3 down-modulate CXCR4,
but not CCR5
Flow cytometric analyses of sham-treated
vs. hBD-treated immunocompetent cells
(PBMCs) revealed a significant selective Fig. 2 - Anti-HIV-1 activity of hBDs. HIV-1 strains (X4 HXB2 and R5 93US142) were incubated in 10 mM
phosphate buffer with increasing concentrations of hBDs and used to infect GHOST X4/R5 cells. (a) Qualitative
reduction of the CXCR4 receptor in cells determination of HIV-1 infection, measured by GFP fluorescence, in the absence (-C) and presence (+C) of
pre-treated with hBD-2 or hBD-3, but not virus pre-incubated in 10 mM PB. (b) Anti-HIV-1 activity of hBDs in GHOST X4/R5 cells examined by
hBD-1. Similar results were obtained with fluorescence microscopy. (c) Anti-HIV-1 activity of hBDs measured by reverse-transcriptase (RT) activity in cell-
CEM cells (T-cell lines) expressing CXCR4 free culture supernatant, relative to the positive control (i.e., HIV-1 infection in the absence of hBD).
and CCR5. Confocal microscopy results Reproduced with permission from Quiones-Mateu et al. (2003).
suggested that hBD-2 and -3 bind to cell-
surface CXCR4 and induce internalization
of the bound complex (Fig. 3). In the case of hBD-3, this down-
regulation was maintained for at least 24 hrs. Interestingly,
hBD modulation of CXCR4 occurred even in the presence of
high salt concentrations, suggesting that the hBD activity could
be occurring on cells deeper in the mucosal epithelium. Finally,
immunoelectron microscopy confirmed hBD localization to the
cell membrane (Fig. 4).
hBD-2 and -3 interact directly with HIV-1
After noticing that multiple washings of HIV-1 virions that had
been pre-exposed to hBD2 or -3 did not reverse the anti-
retroviral effects of the hBDs, we conducted immunoelectron
microscopy and saw direct localization of the beta-defensins to
the virion particles (Fig. 4). Electrostatic interactions with
virion-specific membrane structures, such as gp120, are
plausible. Polyanionic compounds exert their anti-HIV-1
activity by binding to the positively charged sites in the V3
loop of gp120 (Schols et al., 1990; Witvrouw et al., 1994). Like
other polycationic peptides which block infection with X4 HIV-
1 isolates (e.g., T22, T134, and ALX40-4C) (De Clercq, 2002), the
direct anti-retroviral effects of beta-defensins might be
predicted to be very different, perhaps interacting with other
viral surface domains. Theta-defensins have been shown to
inhibit HIV-1 replication in vitro by binding to envelope sugar
residues (Wang et al., 2003). Whether hBD2 and -3 exhibit
Fig. 3 - Confocal microscopy analysis of hBD-2 and -3 on CXCR4 and CCR5
lectin-like properties remains to be seen.
surface expression. CEM X4/R5 cells were stained with anti-CXCR4 PE, anti-CCR5
Significance PE, anti-hBD-2, and anti-hBD-3 monoclonal antibodies in the presence and
absence of 20 g/mL of hBD-2 and -3. Pre-incubation with hBD-2 (a2) or hBD-3
More than any other single development, the advances in (b2) specifically blocked the detection of CXCR4 but not CCR5 on the cell surface.
HIV/AIDS therapeutics have caused a decrease in both AIDS Detection of hBD-2 (c,e) or -3 (d,f) with live, fixed, or live cells post-perfusion.
incidence and death in the US and Europe. Despite these Following incubation with hBD-2 or -3, polyclonal antibodies against the hBDs
gratifying advances, the worldwide incidence of HIV failed to detect them on the surfaces of live cells (c2, d2). 1% paraformaldehyde
fixation, followed by hBD-2 or -3 incubation and antibody labeling,
infection is rising, and strategies to prevent the acquisition of demonstrated hBDs bound to the cell membrane (e1, f1). To visualize hBD-2 and
HIV infection are urgently needed (Little et al., 2002). The -3 internalization, we incubated live CEM cells with hBD-2 or -3, followed by cell
discovery, in the mid-1990s, of the major HIV-1 co-receptors permeabilization (FACS/PERM) and staining with anti-hBD antibodies (e2, f2).
(Cocchi et al., 1995) has led to rapid development of novel Reproduced with permission from Quiones-Mateu et al. (2003).

Adv Dent Res 19:42-48, April, 2006 Role of Human -defensins in HIV Infection 45

Fig. 4 - Immunoelectron microscopy analysis showing the interaction of hBD-2
and -3 with HIV-1 and with T-cell membrane. X4 HIV-1 HXB2 strain and MT4
cells (T-cell line) were incubated with hBD-2 or -3 (20 g/mL), 37C, 1 hr.
Polyclonal anti-hBD-2 or -3 antibodies were added, followed by the addition
of secondary IgG conjugated with 10-nm gold particles. Arrows indicate hBD-
2 and -3 localization to virions and the cell membrane. Reproduced with
permission from Quiones-Mateu et al. (2003).
antiretroviral strategies that target binding of HIV to these co-
receptors. Moreover, observations of intrinsic resistance to the
acquisition of HIV infection have demonstrated that failure of
CCR5 expression is associated with high-level resistance to
HIV infection (Dean et al., 1996; Liu et al., 1996). Nonetheless,
studies in several cohorts of high-risk seronegative
individuals have clearly demonstrated that the 32-base-pair
mutation in the CCR5 open-reading frame, which results in
the failure of surface expression of this chemokine receptor, is
found at best in only a minority of high-risk HIV-seronegative
individuals (Kokkotou et al., 1998; Kaul et al., 2000; Salkowitz
et al., 2001). Thus, it is likely that other mechanisms
determine risk for the acquisition of HIV infection in these
groups. We propose that studies of the oral cavity may
provide insights into intrinsic mechanisms of resistance to
HIV infection. New drugs in development target different
steps in the viral life cycle (Jacobson et al., 2000), gp 120-
binding to the chemokine receptors CCR5 or CXCR4 (Cocchi
et al., 1995), and the fusion of the viral and cellular
membranes (Kilby et al., 1998). The fact that the oral cavity is
naturally resistant to HIV-1 infection prompts us to ask how
and why? While salivary components have been identified
and are continuing to be studied as potential antiretroviral
agents, we believe that there is much to be learned, and
potentially tapped, from the oral mucosal epithelium. There
is new, exciting information emanating from leading
laboratories that defensin molecules may be a new frontier
for studying the host's defense against HIV-1 (Yang et al.,
1999; Biragyn et al., 2002a,b; Zhang et al., 2002). Our
preliminary studies suggest that an intriguing dynamic is
occurring between the human oral epithelial cell and HIV-1
virions, possibly leading to mucosal protection through -
defensin expression. We hypothesize that 'turning on'
inducible beta-defensins, locally, could result in less viral
transmission through the mucosal barrier. Moreover, other
epithelial-cell-derived antimicrobial agents may be induced
along with the hBDs, upon HIV-1 challenge, and could then
act collectively in protecting the oral cavity from HIV-1
infection. This may include such agents as SLPI, whose
mechanism for selectively inhibiting R5 virus was recently
elucidated (Ma et al., 2004). Results presented herein are
intended to shed light on the properties of the oral mucosa
that could contribute to its relative resistance to HIV-1, which
could then be translated into promoting similar protection in
more vulnerable mucosal barriers.
46 Weinberg et al.
Acknowledgments
Work at the CCF (M.E.Q-M) was supported by grant 5-K01-
HL67610-03. Work at CWRU was supported by CFAR A136219
and A1 51649 (M.M.L.). Work at the CWRU School of Dental
Medicine was supported by R01 DE12589, R01 DE13992, and
R01 DE015510 (A.W.).
References
Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy
PM, et al. (1996). CC CKR5: a RANTES, MIP-1alpha, MIP-1beta
receptor as a fusion cofactor for macrophage-tropic HIV-1. Science
272:1955-1958.
Baqui AA, Meiller TF, Falkler WA Jr (1999). Enhanced secretory
leukocyte protease inhibitor in human immunodeficiency virus
type 1-infected patients. Clin Diagn Lab Immunol 6:808-811.
Baron S, Poast J, Cloyd MW (1999). Why is HIV rarely transmitted by
oral secretions? Saliva can disrupt orally shed, infected leukocytes.
Arch Intern Med 159:303-310.
Barr CE, Miller LK, Lopez MR, Croxson TS, Schwartz SA, Denman H,
et al. (1992). Recovery of infectious HIV-1 from whole saliva. J Am
Dent Assoc 123:36-37, 39-48.
Bastian A, Schafer H (2001). Human alpha-defensin 1 (HNP-1) inhibits
adenoviral infection in vitro. Reg Pept 101:157-161.
Becquart P, Gresenguet G, Hocini H, Kazatchkine MD, Belec L (1999).
Secretory leukocyte protease inhibitor in colostrum and breast
milk is not a major determinant of the protection of early postnatal
transmission of HIV. AIDS 13:2599-2602.
Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A,
Chertov O, et al.(2002a). Toll-like receptor 4-dependent activation
of dendritic cells by beta-defensin 2. Science 298:1025-1029.
Biragyn A, Belyakov IM, Chow YH, Dimitrov DS, Berzofsky JA, Kwak
LW (2002b). DNA vaccines encoding human immunodeficiency
virus-1 glycoprotein 120 fusions with proinflammatory
chemoattractants induce systemic and mucosal immune responses.
Blood 100:1153-1159.
Bjorndal A, Deng H, Jansson M, Fiore JR, Colognesi C, Karlsson A, et
al. (1997). Coreceptor usage of primary human immunodeficiency
virus type 1 isolates varies according to biological phenotype. J
Virol 71:7478-7487.
Blackard JT, Renjifo B, Fawzi W, Hertzmark E, Msamanga G,
Mwakagile D, et al. (2001). HIV-1 LTR subtype and perinatal
transmission. Virology 287:261-265.
Blauvelt A, Glushakova S, Margolis LB (2000). HIV-infected human
Langerhans cells transmit infection to human lymphoid tissue ex
vivo. AIDS 14:647-651.
Boe R, Silvola J, Yang J, Moens U, McCray PB Jr, Stenfors LE, et al.
(1999). Human beta-defensin-1 mRNA is transcribed in tympanic
membrane and adjacent auditory canal epithelium. Infect Immun
67:4843-4846.
Bonass WA, High AS, Owen PJ, Devine DA (1999). Expression of beta-
defensin genes by human salivary glands. Oral Microbiol Immunol
14:371-374.
Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P
(1995). Identification of RANTES, MIP-1 alpha, and MIP-1 beta as
the major HIV-suppressive factors produced by CD8+ T cells [see
comments]. Science 270:1811-1815. Comment in: Science 274:1393-
1395.
Cole AM, Hong T, Boo LM, Nguyen T, Zhao C, Bristol G, et al. (2002).
Retrocyclin: a primate peptide that protects cells from infection by
T- and M-tropic strains of HIV-1. Proc Natl Acad Sci USA 99:1813-
1818.
Coppenhaver DH, Sriyuktasuth-Woo P, Baron S, Barr CE, Qureshi MN
(1994). Correlation of nonspecific antiviral activity with the ability
to isolate infectious HIV-1 from saliva. N Engl J Med 330:1314-1315.
Cornelissen M, Mulder-Kampinga G, Veenstra J, Zorgdrager F, Kuiken
C, Hartman S, et al. (1995). Syncytium-inducing (SI) phenotype
suppression at seroconversion after intramuscular inoculation of a
non-syncytium-inducing/SI phenotypically mixed human
immunodeficiency virus population. J Virol 69:1810-1818.
Adv Dent Res 19:42-48, April, 2006
Daher KA, Selsted ME, Lehrer RI (1986). Direct inactivation of viruses
by human granulocyte defensins. J Virol 60:1068-1074.
Dale BA, Kimball JR, Krisanaprakornkit S, Roberts F, Robinovitch M,
O'Neal R, et al. (2001). Localized antimicrobial peptide expression
in human gingiva. J Periodontal Res 36:285-294.
De Clercq E (2002). New developments in anti-HIV chemotherapy.
Biochim Biophys Acta 1587:258-275.
Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets
R, et al. (1996). Genetic restriction of HIV-1 infection and
progression to AIDS by a deletion allele of the CKR5 structural
gene. Hemophilia Growth and Development Study, Multicenter
AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San
Francisco City Cohort, ALIVE Study [see comments] [erratum
appears in Science 274:1069, 1996]. Science 273:1856-1862. Comment
in: Science 273:1979-1798.
Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al.
(1996). Identification of a major co-receptor for primary isolates of
HIV-1 [see comments]. Nature 381:661-666. Comment in: Nature
381:647-648.
Diamond G, Zasloff M, Eck H, Brasseur M, Maloy WL, Bevins CL
(1991). Tracheal antimicrobial peptide, a cysteine-rich peptide from
mammalian tracheal mucosa: peptide isolation and cloning of a
cDNA. Proc Natl Acad Sci USA 88:3952-3956.
Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA,
et al. (1996). HIV-1 entry into CD4+ cells is mediated by the
chemokine receptor CC-CKR-5 [see comments]. Nature 381:667-
673. Comment in: Nature 381:647-648.
Duits LA, Ravensbergen B, Rademaker M, Hiemstra PS, Nibbering PH
(2002). Expression of beta-defensin 1 and 2 mRNA by human mono-
cytes, macrophages and dendritic cells. Immunology 106:517-525.
Dunsche A, Acil Y, Dommisch H, Siebert R, Schroder JM, Jepsen S
(2002). The novel human beta-defensin-3 is widely expressed in
oral tissues. Eur J Oral Sci 110:121-124.
Feng Y, Broder CC, Kennedy PE, Berger EA (1996). HIV-1 entry
cofactor: functional cDNA cloning of a seven-transmembrane, G
protein-coupled receptor [see comments]. Science 272:872-877.
Comment in: Science 272:809-810.
Fenyo EM, Schuitemaker B, Asjo B, McKeating J, Sattentau Q, EC
Concerted Action HIV Variability (2000). The history of HIV-1
biological phenotypes past, present and future [review]. In:
Human Retroviruses and AIDS 1997. Korber B, Hahn B, Foley B,
Mellors JW, Leitner T, Myers G, et al., editors. Los Alamos, NM:
Theoretical Biology and Biophysics Group, Los Alamos National
Laboratory.
Fotopoulos G, Harari A, Michetti P, Trono D, Pantaleo G, Kraehenbuhl
JP (2002). Transepithelial transport of HIV-1 by M cells is receptor-
mediated. Proc Natl Acad Sci USA 99:9410-9414.
Fultz PN (1986). Components of saliva inactivate human
immunodeficiency virus [letter]. Lancet 2:1215.
Garcia JR, Krause A, Schulz S, Rodriguez-Jimenez FJ, Kluver E,
Adermann K, et al. (2001). Human beta-defensin 4: a novel
inducible peptide with a specific salt-sensitive spectrum of
antimicrobial activity. FASEB J 15:1819-1821.
Gooch B, Marianos D, Ciesielski C, Dumbaugh R, Lasch A, Jaffe H, et
al. (1993). Lack of evidence for patient-to-patient transmission of
HIV in a dental practice [see comments]. J Am Dent Assoc 124:38-
44. Comment in: J Am Dent Assoc 124:12, 14.
Goto Y, Yeh CK, Notkins AL, Prabhakar BS (1991). Detection of
proviral sequences in saliva of patients infected with human
immunodeficiency virus type 1. AIDS Res Hum Retroviruses 7:343-
347.
Gropp R, Frye M, Wagner TO, Bargon J (1999). Epithelial defensins
impair adenoviral infection: implication for adenovirus-mediated
gene therapy. Hum Gene Ther 10:957-964.
Han Y, Ventura CL, Black KP, Cummins JE Jr, Hall SD, Jackson S (2000).
Productive human immunodeficiency virus-1 infection of
epithelial cell lines of salivary gland origin. Oral Microbiol Immunol
15:82-88.
Hancock RE (1997). Peptide antibiotics. Lancet 349:418-422.
Harder J, Bartels J, Christophers E, Schroder JM (1997). A peptide
Adv Dent Res 19:42-48, April, 2006 Role of Human -defensins in HIV Infection
antibiotic from human skin [letter]. Nature 387:861.
Harder J, Bartels J, Christophers E, Schroder JM (2001). Isolation and
characterization of human beta-defensin-3, a novel human
inducible peptide antibiotic. J Biol Chem 276:5707-5713.
Haynes RJ, McElveen JE, Dua HS, Tighe PJ, Liversidge J (2000).
Expression of human beta-defensins in intraocular tissues. Invest
Ophthalmol Vis Sci 41:3026-3031.
Herz AM, Robertson MN, Lynch JB, Schmidt A, Rabin M, Sherbert C, et
al. (2002). Viral dynamics of early HIV infection in neonatal
macaques after oral exposure to HIV-2287: an animal model with
implications for maternal-neonatal HIV transmission. J Med
Primatol 31:29-39.
Ho DD, Byington RE, Schooley RT, Flynn T, Rota TR, Hirsch MS (1985).
Infrequency of isolation of HTLV-III virus from saliva in AIDS
[letter]. N Engl J Med 313:1606.
Jacobson JM, Lowy I, Fletcher CV, O'Neill TJ, Tran DN, Ketas TJ, et al.
(2000). Single-dose safety, pharmacology, and antiviral activity of
the human immunodeficiency virus (HIV) type 1 entry inhibitor
PRO 542 in HIV-infected adults. J Infect Dis 182:326-329.
Kaul R, Plummer FA, Kimani J, Dong T, Kiama P, Rostron T, et al.
(2000). HIV-1-specific mucosal CD8+ lymphocyte responses in the
cervix of HIV-1-resistant prostitutes in Nairobi. J Immunol
164:1602-1611.
Kilby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, Lee JY, et
al. (1998). Potent suppression of HIV-1 replication in humans by T-
20, a peptide inhibitor of gp41-mediated virus entry [see
comments]. Nat Med 4:1302-1307. Comment in: Nat Med 4:1232-
1233.
Kokkotou E, Philippon V, Gueye-Ndiaye A, Mboup S, Wang WK, Essex
M, et al. (1998). Role of the CCR5 delta 32 allele in resistance to
HIV-1 infection in west Africa. J Hum Virol 1:469-474.
Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA (1998).
Expression of the peptide antibiotic human beta-defensin 1 in
cultured gingival epithelial cells and gingival tissue. Infect Immun
66:4222-4228.
Krisanaprakornkit S, Kimball JR, Weinberg A, Darveau RP, Bainbridge
BW, Dale BA (2000). Inducible expression of human beta-defensin
2 by Fusobacterium nucleatum in oral epithelial cells: multiple
signaling pathways and role of commensal bacteria in innate
immunity and the epithelial barrier. Infect Immun 68:2907-2915.
Little SJ, Holte S, Routy JP, Daar ES, Markowitz M, Collier AC, et al.
(2002). Antiretroviral-drug resistance among patients recently
infected with HIV [see comments]. N Engl J Med 347:385-394.
Comment in: N Engl J Med 347: 438-439. Author reply in: N Engl J
Med 347:1889-1890.
Liu L, Heng H, Zhao C, Ganz T (1996). Human alpha- and beta-
defensins evolved from a common pre-mammalian ancestor
(abstract). J Invest Med 44:294A.
Liu X, Zha J, Chen H, Nishitani J, Camargo P, Cole SW, et al. (2003).
Human immunodeficiency virus type 1 infection and replication in
normal human oral keratinocytes. J Virol 77:3470-3476.
Lu Q, Jin L, Darveau RP, Samaranayake LP (2004). Expression of
human beta-defensins-1 and -2 peptides in unresolved chronic
periodontitis. J Periodontal Res 39:221-227.
Ma G, Greenwell-Wild T, Lei K, Jin W, Swisher J, Hardegen N, et al.
(2004). Secretory leukocyte protease inhibitor binds to annexin II, a
cofactor for macrophage HIV-1 infection. J Exp Med 200:1337-1346.
Mandel ID (1972). Saliva. St. Louis: C.V. Mosby Co.
Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S, Johnson GK, et
al. (1999). Production of beta-defensin antimicrobial peptides by
the oral mucosa and salivary glands. Infect Immun 67:2740-2745.
McCray PB Jr, Bentley L (1997). Human airway epithelia express a
beta-defensin. Am J Respir Cell Mol Biol 16:343-349.
McNeely TB, Dealy M, Dripps DJ, Orenstein JM, Eisenberg SP, Wahl
SM (1995). Secretory leukocyte protease inhibitor: a human saliva
protein exhibiting anti-human immunodeficiency virus 1 activity
in vitro. J Clin Invest 96:456-464.
Moore BE, Flaitz CM, Coppenhaver DH, Nichols M, Kalmaz GD,
Bessman JD, et al. (1993). HIV recovery from saliva before and after
dental treatment: inhibitors may have critical role in viral
47
 inactivation. J Am Dent Assoc 124:67-74.
Nakashima H, Yamamoto N, Masuda M, Fujii N (1993). Defensins
inhibit HIV replication in vitro [letter]. AIDS 7:1129.
O'Brien SJ, Moore JP (2000). The effect of genetic variation in
chemokines and their receptors on HIV transmission and
progression to AIDS. Immunolog Rev 177:99-111.
O'Neil DA, Porter EM, Elewaut D, Anderson GM, Eckmann L, Ganz T,
et al. (1999). Expression and regulation of the human beta-
defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol
163:6718-6724.
Overbaugh J, Kreiss J, Poss M, Lewis P, Mostad S, John G, et al. (1999).
Studies of human immunodeficiency virus type 1 mucosal viral
shedding and transmission in Kenya. J Infect Dis 179(Suppl
3):S401-S404.
Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P, et al.
(1988). Rapid and automated tetrazolium-based colorimetric assay
for the detection of anti-HIV compounds. J Virolog Meth 20:309-321.
Pillay K, Coutsoudis A, Agadzi-Naqvi AK, Kuhn L, Coovadia HM,
Janoff EN (2001). Secretory leukocyte protease inhibitor in vaginal
fluids and perinatal human immunodeficiency virus type 1
transmission. J Infect Dis 183:653-656.
Quiones-Mateu ME, Lederman MM, Feng Z, Chakraborty B, Weber J,
Rangel HR, et al. (2003). Human epithelial beta-defensins 2 and 3
inhibit HIV-1 replication. AIDS 17:F39-F48.
Rogers MF, White CR, Sanders R, Schable C, Ksell TE, Wasserman RL,
et al. (1990). Lack of transmission of human immunodeficiency
virus from infected children to their household contacts. Pediatrics
85:210-214.
Ross KF, Herzberg MC (2001). Calprotectin expression by gingival
epithelial cells. Infect Immun 69:3248-3254.
Sahasrabudhe KS, Kimball JR, Morton TH, Weinberg A, Dale BA
(2000). Expression of the antimicrobial peptide, human beta-
defensin 1, in duct cells of minor salivary glands and detection in
saliva. J Dent Res 79:1669-1674.
Salkowitz JR, Purvis SF, Meyerson H, Zimmerman P, O'Brien TR,
Aledort L, et al. (2001). Characterization of high-risk HIV-1
seronegative hemophiliacs. Clin Immunol 98:200-211.
Sawaki K, Mizukawa N, Yamaai T, Yoshimoto T, Nakano M, Sugahara
T (2002). High concentration of beta-defensin-2 in oral squamous
cell carcinoma. Anticancer Res 22:2103-2107.
Schols D, Pauwels R, Desmyter J, De Clercq E (1990). Dextran sulfate
and other polyanionic anti-HIV compounds specifically interact
with the viral gp120 glycoprotein expressed by T-cells persistently
infected with HIV-1. Virology 175:556-561.
Schonwetter BS, Stolzenberg ED, Zasloff MA (1995). Epithelial
antibiotics induced at sites of inflammation. Science 267:1645-1648.
Shugars DC (1999). Endogenous mucosal antiviral factors of the oral
cavity. J Infect Dis 179(Suppl 3):S431-S435.
Shugars DC, Wahl SM (1998). The role of the oral environment in HIV-
1 transmission. J Am Dent Assoc 129:851-858.
Smith PD, Li L, Meng G (1999). Mucosal events in the pathogenesis of
human immunodeficiency virus type 1 infection. J Infect Dis
48 Weinberg et al.
179(Suppl 3):S436-S440.
Soto-Ramirez LE, Renjifo B, McLane MF, Marlink R, O'Hara C,
Sutthent R, et al. (1996). HIV-1 Langerhans' cell tropism associated
with heterosexual transmission of HIV [see comments]. Science
271:1291-1293. Comment in: Science 278:786-788.
Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, Miller CJ, et al. (1999). A
cyclic antimicrobial peptide produced in primate leukocytes by the
ligation of two truncated alpha-defensins [see comments]. Science
286:498-502. Comment in: Science 286:420-421.
Tchou I, Misery L, Sabido O, Dezutter-Dambuyant C, Bourlet T, Moja P,
et al. (2001). Functional HIV CXCR4 coreceptor on human
epithelial Langerhans cells and infection by HIV strain X4. J Leukoc
Biol 70:313-321.
Tersmette M, de Goede RE, Al BJ, Winkel IN, Gruters RA, Cuypers HT,
et al. (1988). Differential syncytium-inducing capacity of human
immunodeficiency virus isolates: frequent detection of syncytium-
inducing isolates in patients with acquired immunodeficiency
syndrome (AIDS) and AIDS-related complex. J Virol 62:2026-2032.
Wahl SM, McNeely TB, Janoff EN, Shugars D, Worley P, Tucker C, et al.
(1997). Secretory leukocyte protease inhibitor (SLPI) in mucosal
fluids inhibits HIV-I. Oral Dis 3(Suppl 1):S64-S69.
Wang W, Cole AM, Hong T, Waring AJ, Lehrer RI (2003). Retrocyclin,
an antiretroviral theta-defensin, is a lectin. J Immunol 170:4708-
4716.
Weinberg A, Krisanaprakornkit S, Dale BA (1998). Epithelial
antimicrobial peptides: review and significance for oral
applications. Crit Rev Oral Biol Med 9:399-414.
Witvrouw M, Este JA, Quiones-Mateu ME, Reymen D, Andrei G,
Snoeck R, et al. (1994). Activity of a sulfated polysaccharide
extracted from the red seaweed Aghardhiella tenera against human
immunodeficiency virus and other enveloped viruses. Antivir
Chem Chemother 5:297-303.
Wu Z, Hoover DM, Yang D, Boulegue C, Santamaria F, Oppenheim JJ,
et al. (2003). Engineering disulfide bridges to dissect antimicrobial
and chemotactic activities of human beta-defensin 3. Proc Natl
Acad Sci USA 100:8880-8885.
Yahi N, Baghdiguian S, Moreau H, Fantini J (1992). Galactosyl
ceramide (or a closely related molecule) is the receptor for human
immunodeficiency virus type 1 on human colon epithelial HT29
cells. J Virol 66:4848-4854.
Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, et al.
(1999). Beta-defensins: linking innate and adaptive immunity
through dendritic and T cell CCR6 [see comments]. Science
286:525-528. Comment in: Science 286:420-421.
Zasloff M (2002). Antimicrobial peptides of multicellular organisms.
Nature 415:389-395.
Zhang L, Yu W, He T, Yu J, Caffrey RE, Dalmasso EA, et al. (2002).
Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1
activity of CD8 antiviral factor. Science 298:995-1000.
Zhao C, Wang I, Lehrer RI (1996). Widespread expression of beta-
defensin hBD-1 in human secretory glands and epithelial cells.
FEBS Lett 396:319-322.
Adv Dent Res 19:42-48, April, 2006