1. No category

Slamming the door on unwanted guests: why preemptive
strikes at the mucosa may be the best strategy against HIV
Susanna Trapp, Stuart G. Turville, and Melissa Robbiani1
Center for Biomedical Research, Population Council, New York, New York, USA
Key Words: transmission 䡠 microbicide 䡠 dendritic cells 䡠 macrophage
EARLY EVENTS AS HIV BREACHES THE
BODY SURFACES
The highest incidence of HIV transmission is for the receptive
partner during sexual intercourse. The World Health Organization estimates that 60% of HIV-positive people in Africa are
women infected via the vaginal route (http://www.unaids.org/
epi2005/doc/report_pdf.html). Therefore, there is an urgent
need to develop preventive treatments, which are suitable for
women and men in these poor regions of the world. In addition
to preventative or therapeutic vaccines, a promising way to
assist the body to strengthen its antiviral barrier is to develop
microbicides, which act potently and early.
The rectal and vaginal environments represent natural barriers for the incoming virus. The squamous epithelium (often
multi-layered) lining the vagina provides a formidable, primary
obstacle for the virus to cross. Once crossing primary barriers,
HIV predominantly replicates in memory CD4⫹ T cells of the
Lamina propria. Macrophages (M⌽) and dendritic cells (DCs)
are infected less frequently, but the quality of virus produced
and targeted delivery of virus by these cells may endow them
to be also key disseminators. Thus, understanding how the
virus deals with these environments is critical in designing any
intervention strategy. This review will focus on the strategies
HIV has evolved to overcome the mucosal barrier, and subsequently, the different microbicides, which have/are being developed to prevent infection [1].
THE IMPORTANCE OF PRIMARY BARRIERS
This was highlighted in macaque studies, which showed how
estrogen treatment protected animals from infection [2], and
progesterone treatment enhanced an animal’s susceptibility to
infection [2]. However, the possible modulation of leukocytes
by sex hormones within these tissues [1], which might also
impact infection, could contribute. In the female reproductive
tract, the virus must not only pass the epithelial layer but also
overcome acidic pH, cervically secreted mucous, hydrogen
peroxide, and potentially, other innate defenses secreted at the
mucosa (see review in ref. [3]). This barrage of mechanisms is
quite effective when observing transmission in vivo, as there
are infrequent populations that are infected at the cervicovaginal mucosa. Such observations have led to the hypothesis that
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Journal of Leukocyte Biology Volume 80, November 2006
small, founder populations of infected cells are key for initial
dissemination [4]. The idea of a transmission bottleneck is
supported further by the observations of early, replicating
variants in humans being monophyletic [5] and thus, suggestive that few virions reach the point of a productive infection.
Although one must note that the rectal mucosa consisting only
of a monolayered epithelium is more prone to lesions, which
might make it even more susceptible for HIV/SIV transmission. In terms of early defense mechanism, the rectal milieu
may contain hydrogen peroxide, but the pH is rather neutral.
This makes it much easier for the virus to survive and to cross
the barrier and reach the L. propria. The mechanism(s) that
allow the small proportion of virus to survive, clearly following
vaginal or rectal exposure, warrants further study.
DEFINING THE CELLULAR SUBSTRATES
Breaking down cellular partners of HIV as “cellular substrates” will make it easier to address the actions of microbicides later. Recently, observations of predominant L. propria
CD4⫹ T cell infection in vivo have led to the hypothesis that
permissive leukocyte abundance dictates viral transmission
[6]. Shortcomings with this hypothesis are the lack of consideration of less-abundant, permissive cells, in addition to cells
that bind and traffic virus, independent of their infection. In
addition, in vivo observations of transmission often involve
high levels of viral inoculum and therefore, may or may not be
representative of potentially lower inocula in seminal fluid.
Virus binding and transfer independent of
infection: potential passive substrates (Fig. 1A)
Epithelial cells (ECs) are the first cells encountering virus
and/or infected cells. ECs may not be productively infected by
HIV in vitro [4, 7], but ECs can endocytose and transfer virus
to CD4⫹ T cells [8]. Fibronectin, present in seminal plasma,
might also facilitate ␤-integrin interaction of virus with ECs [7,
9], in addition to viral capture by surface heparan sulfates [10].
DC are located within and just underneath the epithelial
layers, thereby positioning them as one of the first leukocyte
substrates. To sample pathogens/antigens, Langerhans cells
(LCs) in the outer epithelia and DCs within the L. propria [11]
1
Correspondence: Center for Biomedical Research, Population Council,
1230 York Avenue, New York, NY 10021. E-mail: [email protected]
Received February 28, 2006; revised April 6, 2006; accepted April 30,
2006; doi: 10.1189/jlb.0206121.
0741-5400/06/0080-1076 © Society for Leukocyte Biology
fer captured virus to CD4⫹ T cells across the “virologic synapse” [13–15]. The relative efficiency in transferring captured
viruses depends on their maturation status: mature ⬎ immature DCs, which interact with HIV via a variety of receptors,
such as CD4, C-type lectin receptors (CLRs), and CCRs.
Which receptors are involved depends on the DC subset and its
state of maturation [16]. However, early DCs transfer to CD4⫹
T cells is multifaceted and potentially involves one or more
CLRs and other unknown pathways [16, 17].
M⌽ and CD4⫹ T cells are seen predominately as infectious
cell (active) substrates, although both cell types retain the
capacity to traffic virus independent of infection [18]. Much
like DCs, M⌽ capture and internalize HIV using the CLR
CD206 (mannose receptor) [19], and this may provide another
pathway through which virus is trafficked to more permissive
CD4⫹ T cells.
Virus infection of cellular substrates (active
substrates; Fig. 1B)
Fig. 1. Trafficking of HIV through infected substrates (A) and noninfected
substrates (B). (Schematic is a model of the cervicovaginal epithelium with
squamous and columnar epithelia. For the rectal mucosa, the latter epithelia is
only present.) (A) HIV predominately infects resting memory T cells (T) of the
L. propria, although to lower levels, DCs and M⌽ (M) have the potential to be
infected. Virus may amplify locally within the mucosal tissues, primarily in T
cells directly infected or after transfer of newly produced virus coming from the
DC or M⌽ to the T cells. Cells infected in the periphery also probably migrate
to the draining lymph nodes to further amplify virus distally. (B) Cell types
involved in trafficking of virus, independent of their infection, are highlighted.
In particular, epithelial cells (ECs) are capable of binding large quantities of
virus, but virus trancytosis to the L. propria is limited (although sufficient for
infection). As in (A), large quantities of virus may be transported to the L.
propria as a result of damaged epithelial integrity (physical trauma or ulcerative lesions caused by another pathogen). Once in the L. propria, cells may
bind and transfer the virus to neighboring cells. This is particularly likely in
the case of DCs, which also have the potential to traffic virus efficiently to the
draining lymph nodes. STD, Sexually transmitted disease.
may reach their dendrites to the epithelial surfaces. Considering how ECs and DC influence each other and how they
interact with HIV will be important to appreciate these early
events in HIV transmission. Immature and mature DC bind
and internalize virus efficiently in vitro [12] and rapidly trans-
Infection of memory CD4⫹ T cells primarily occurs in distinct
crypts of the L. propria in vivo, underscoring the major obstacle
that the epithelial barrier represents to the virus [4, 6]. The
abundance of such infected cell types is believed to ensure
viral dissemination [6]. Memory CD4⫹ T cells allow replication
of HIV/SIV, but how these cells first “receive” the virus or get
the signals to drive replication (prior to the detected, amplified
virus) likely comes from local APC (DCs and M⌽).
It is unclear what role M⌽ might play during early viral
transmission, yet there is evidence of low-level infection of DCs in
vivo. Spira et al. [20] were the first to implicate DCs in peripheral
tissues as the first targets of HIV. Later work found DCs (intraepithelial LCs) to be the first substrates of viral infection [21].
Unlike DCs carrying virus, infected, immature DCs are exquisitely potent at transferring newly produced virus, even when the
level of DCs infection is below the limit of in vitro detection [22].
It is possible that similar low levels of DCs and M⌽ infection
occur in vivo and largely go undetected until the virus is amplified
significantly within the memory T cell pool. Moreover, the viruses
produced in M⌽ [23] or DCs [24] have different characteristics to
those replicating in T cells. Carrying different host molecules
within the viral membrane (DC-derived), more complex glycosylation patterns (M⌽-derived), and/or higher levels of envelope
(M⌽-derived) may also provide further problems for microbicide
development.
Extrapolation of in vitro and the limitations of in
vivo observations
Although there are many mechanisms that have been highlighted in vitro, care must be taken for their relevance and
thus, final extrapolation to the in vivo setting. For instance, the
mechanism of “trans” enhancement (the ability for a cell to
uptake virus and transfer it to a permissive cellular recipient at
an efficiency greater than the initial viral inoculum) has been
demonstrated by many in various, different cellular backgrounds in vitro, especially DCs. Yet the question still remains
as to whether this would be active in vivo, and currently, there
is little evidence from animal and/or epidemiological studies of
human transmission that it does. With respect to the logistiTrapp et al. Slamming the door on HIV
1077
cally difficult in vivo observations in animals and humans,
such studies are an important “snap shot” of transmission, but
sampling still remains a chronic problem. For example, the
infectious crypts observed recently in macaque models highlight the importance of primary barriers and the presence of
bottlenecks, but what of the events prior to this, and what is the
contribution of cells of low abundance (e.g., DC), which may be
lost within the thousands of tissue sections? Thus, one must
carefully consider the potential of many different pathways in
transmission, as current in vitro models may not be representative of key mechanisms, and in vivo sampling also has the
potential to miss them.
Pathogenic modulation of cellular substrates
The presence of other sexually transmitted pathogens (e.g.,
Neisseria gonorrhea, Chlamydia trachomatis, HSV-2), especially those causing ulcerative lesions, dramatically increases
the chances of acquiring or transmitting HIV [25, 26]. The
damaged epithelial barriers and subsequent infiltration of activated leukocytes into the lesions might allow HIV to access
target cells directly (Fig. 1) [27]. The recent evidence that
pathogens such as HSV-2 can paralyze DC function also suggests that dampened immune function mediated by HSV-2
might further enhance the establishment of HIV infection
[28 –30].
HIV may also directly modulate target populations (particularly DCs and T cells) [31] to support its own replication. Nef,
one accessory protein of HIV/SIV, might enhance viral replication [32, 33] via several specific mechanisms, including
modulation of DC-T cell contact, signaling cascades (e.g.,
p21-activated kinase) [34, 35], induction of chemokine and
cytokine secretion [36 –38], and/or via other unknown direct or
indirect mechanisms. Given the multifaceted nature of Nef and
its potential to drive viral replication, inhibition of this key
accessory protein may represent an unrecognized approach in
microbicide development.
External influences on substrates
Some pathogens affect HIV transmission/infection, but they
might also impact the development of AIDS. ECs and DC are
able to defend against incoming pathogens by activating innate
(and ultimately adaptive) immune functions; e.g., the interaction of TLRs and their pathogen-specific ligands [39] trigger
specific (immune) responses [40 – 42]. These signaling cascades lead to the expression of various factors involved in
immune activation [43] and might influence HIV spread.
Studies in the macaque demonstrated that topical application of a TLR9 ligand slightly increased vaginal infection [44].
In contrast, ex vivo studies about lymphoid tissues from infected macaques observed a dampening of virus replication in
response to TLR3 stimulation using the synthetic dsRNA analog polyinosinic:polycytidylic acid {poly(I:C)} [45]. TLR3 ligation on ECs elicits potent chemokine and cytokine responses
as well as the release of bactericidal and virucidal agents at
times when adaptive immunity is down-regulated by sex hormones to meet the constraints of procreation [1]. These studies
propose a considerable role of the mucosal innate immune
system with respect to HIV infection/transmission. We recently
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Journal of Leukocyte Biology Volume 80, November 2006
found that poly(I:C) treatment of HIV-loaded, immature DC
impedes DC-driven infection in vitro (S. Trapp and M. Robbiani, unpublished observations). Signaling through specific TLR
molecules might represent another strategy to limit sexual
transmission of HIV.
CHALLENGES OF MICROBICIDE
DEVELOPMENT IN LIGHT OF HIV
TRANSMISSION MODELS
Given the complexities faced by the virus at the mucosa, it is
surprising that the virus can establish an infectious niche.
Primary barriers play a key role in complicating HIV dissemination, and one approach in microbicide development is to
strengthen these existing mechanisms. A variety of strategies
are being explored and are summarized herein (Table 1). The
potential modes of action are considered in Figure 2.
Maintenance of the primary barriers
Mimicking the acidic, protective nature of lactobacilli is one
method by which BufferGel威 (ReProtect, Inc., Baltimore, MD)
and Acidform (TOPCAD, Chicago, IL) destroy incoming pathogens. In addition, thickening of the epithelial barrier through
estrogen pretreatment is another method that proved successful
in preventing SIV transmission [2].
Disruptive agents
Surfactants
Many pathogens are dependent on their membrane integrity to
maintain infectivity, and thus, viral membrane disruption represents one strategy to kill pathogens. The first surfactant
unsuccessfully tested in clinical trials was N-9, which might
have even promoted viral transmission by destroying physiological barriers [46]. As an alternative substance, C31G has
broader activity (HSV-2 and HIV) and is in clinical trials.
C31G has been effective in postcoital contraceptive activity
[47] and has promising safety profiles.
Specific permeabilization of the virions—2-hydroxy-propyl-␤cyclodextrin (␤-CD)
HIV buds selectively through areas in the plasma membrane
called lipid rafts. Therefore, interrupting lipid rafts by using
␤-CD seems to be a promising tool for microbicide approaches.
␤-CD is effective in vitro [48] and in vivo at blocking cellassociated HIV transmission in the murine severe combined
immunodeficiency disease (SCID) human (SCIDhu) model [49].
The advantage of this method over the broader-acting surfactants may indeed be that it specifically mediates viral lysis
while maintaining mucosal epithelial integrity.
Broad-acting chemical barriers
Several sulfated, sulfonated polymers and poly-anions, including polystyrene sulfonate, napthelene sulfonate (Pro 2000),
cellulose sulfate, CAP, and carrageenans derived from Red
Seaweed (Carraguard) have broad, antipathogen activity [HSV,
human papilloma virus, N. gonorrhea, C. trachomatis]. The
action of sulfated polymers toward ⫻4 isolates blocks the HIV
http://www.jleukbio.org
TABLE 1.
Candidate microbicide action
Broad:
pH regulators
Hormonal regulation
Sulfated/sufonated polymer, polyanions
Surfactants
Cholesterol depletion
HIV-specific:
Envelope/CD4/CCR5
i) Carbohydrates (gp120 and gp41)
ii) Fusion domain (gp41)
iii) HIV gp120 CD4-binding site
iv) CCR5 inhibitors
Reverse transcriptase
Combination:
Broad ⫹ specific (absorbtion and RT)
Specific (fusion & CD4-binding site)
Specific (fusion & CCR5)
Specific (CD4-binding site & CCR5)
Specific (Fusion, CD4-binding site & CCR5)
Candidate Microbicides for Blocking HIV Transmissions
Animal studiesa
Candidate compounds
—Acidform gel
—BufferGel
—Lime juice
—Protected Lactobacilli with
benzalkonium chloride (BZK)
—Estrogen
—Pro 2000 (napthelene sulphonate)
—Cellulose sulfate
—Carraguard (carrageenan)
—Polystyrene sulfate
—CAP
—SPL7013 (vivagel)
—N-9
—Savvy/C13G
—␤ cyclodextrin
—Cyanovirin
—(Plant lectins)
—Mannan
—C52L
—Escherichia coli-secreting C52L
—Enfuvirtide (T20)
—BMS-378806, b12 mAb
—PSC-RANTES, CMPD167
NRTI:
—Tenofovir/PMPA
NNRTI:
—TMC-120
—UC781
—PC-815 (MIV-150⫹Carraguard)
—MIV-150 & Carraguard (PC-815)
—C52L ⫹ BMS-378806
—C52L ⫹ CMPD167
—BMS-378806 ⫹ CMPD167
—C52L ⫹ BMS-378806 ⫹ CMPD167
—Macaque
—Macaque
—Macaque
—Macaque
In trialsb
Therapyc
(I)
(II/III)
(I)
—
—
—
(II)
—
(III)
(II)
(III)
—
—
—
—
—
—
—
—
—
—
—
(I)
(I)
—Macaque
—Murine
(III)
—
—Macaque
—
—
—
—
—
⫹
—
—
—Macaque
—Macaque
—Macaque
—Macaque
—Murine
(II/IIb)
⫹
—Macaque
(I)
(I)
(I*)
—
—
—
—Macaque
—Macaque
—Macaque
—Macaque
—Macaque
(I*)
—
—
—
—
—
—
—
—
—
a
Current and published macaque and murine models using microbicides. b Microbicides currently in or approved* for clinical trials (for more details, see
Alliance for Microbicide Development: www.microbicide.org). c Current HIV microbicides, which are routinely available for therapeutic use. CAP, Cellulose
acetate 1,2-benzenedicarboxylate; N-9, nonoxynol-9; PSC-RANTES, L-Thia-Pro2, L-␣-cyclohexylen-gly3-NNY-RANTES; PMPA, 9-[2-(phosphonomethoxy)propyl]adenine; NRTI, nucleoside RT inhibitor; NNRTI, non-NRTI.
envelope and CXCR4 interaction [50], although the broad
activity of these compounds may be a function of pathogenic
absorption to the ECs and/or other target cells through molecules such as heparan sulfate [10] (Fig. 2D).
In vivo, the levels of compound currently used are far in
excess of what is needed for blocking in in vitro studies. Thus,
the gel-forming nature of large, charged polymers may also
provide a primary barrier similar to mucosal secretions and
may afford additional protection through lubrication and protection of the upper epithelial lining during sex (Fig. 2A).
Macaque studies have demonstrated the potential for several of
these compounds in limiting vaginal SIV infection (Table 1)
[51, 52] (Carraguard; David Phillips, Population Council, New
York, personal communication, and S. G. Turville and M.
Robbiani, unpublished observations). The added benefit of this
class of compounds is that they may serve as carriers for other
antiretroviral drugs, as recently seen with the NNRTI MIV-150
formulation with Carraguard [53]. The additional activities
against other sexually transmitted pathogens are also a major
appeal for this sort of approach (e.g., Carraguard activity
against multiple microorganisms [54 –56]).
HIV-specific microbicides
HIV glycan
The mechanism of HIV attachment, fusion, and entry has been
studied extensively, and specific compounds are now available,
which act on several of these pathways. In terms of attachment,
several compounds are targeted toward the glycosylation sites
of the HIV envelope. CNV has the ability to inactivate virions
potently and irreversibly by binding to high mannose envelope
surface glycans and is effective in vitro and in vivo [57, 58].
Given DCs and M⌽ also bind HIV through mannose residues
on HIV, there is likely to be an added benefit of competing
ligands such as CNV. DCs are capable of internalizing virus in
the presence of CNV, although virions are rendered inactive by
previous exposure to these compounds [22].
Trapp et al. Slamming the door on HIV
1079
cient to induce significant protection in macaques [60]. As with
CNV, CD4-IgG2 (similar in action to BMS-378806) does not
block virus capture by DCs but does block the subsequent
spread to T cells [61]. Thus, the CD4-binding site class may
afford significant protection by acting directly on incoming
virions (Fig. 2B). Even uptake of viruses bound by these agents
(by ECs, DCs, or M⌽) and subsequent transfer to CD4⫹ T cells
would not thwart the blocking activity, as it is highly probable
that the CD4-dependent infection of the recipient T cells would
have been inactivated.
Fusion inhibitors
Linear peptides derived from the membrane region of the gp41
are effective inhibitors of a broad range of HIV isolates as a
result of the conserved nature of the hydrophobic groove to
which these peptides bind [62]. T-20 (enfuvirtide) is currently
in clinical use as a salvage, antiretroviral therapy [63]. In
macaque studies, C52L only protected 50% of animals when
used at high levels, although in combinations known to be
synergistic in vitro, C52L with BMS-378806 or the CCR5
inhibitor CMPD167 (or both) were more effective in macaque
challenges [60]. It is difficult to establish whether this was
synergistic in vivo. Given the high levels of the fusion inhibitors that are needed for topical application, others have investigated the potential of using recombinant, colonizing bacteria to secrete active fusion peptides [64].
CCR5-based inhibitors
Fig. 2. Microbicide strategies. (A) Chemical barriers (e.g., sulfated polymers)
and those microbicides that encourage primary barriers (e.g., estrogen, BufferGel, and Acidform) act by making the L. propria and/or permissive targets
unavailable. In the schematic, virus cannot penetrate the barrier created by the
microbicide. (B) Specific HIV inhibitors that attach to HIV envelope [e.g.,
BMS-378806, cyanovirin (CNV)] act by directly inactivating HIV (virions
inactivated, coated with microbicide). Virus may or may not penetrate the L.
propria, but in either case, the virus is not infectious. (C) HIV-specific
microbicides, which inhibit specific enzymatic activities (e.g., NNRTIs), can
directly inactivate cell-free virions (inactive virions marked internally with X).
In this case, contact with microbicide renders the virions inactive before or as
they enter the body (similar to that described in B). (D) CCR5 inhibitors (or
other HIV receptors involved in infection, e.g., CD4, CXCR4) block the
capacity for virus to infect permissive targets (represented in the figure by
“X”). These agents likely diffuse into the tissues to limit infection by virus,
which penetrates into the L. propria to access the most permissive cells. This
prevents the establishment of an infectious seeding site to allow for further
dissemination. act-T, Activated CD4⫹ T cells of the L. propria.
HIV envelope-CD4 interactions
The first in vivo study to look at disrupting the CD4-HIV
envelope interaction at the mucosa used the neutralizing antibody b12 [59]. In a similar manner, BMS-378806 was suffi1080
Journal of Leukocyte Biology Volume 80, November 2006
Individuals carrying the homozygous CCR5-⌬32 mutation do
not appear to show any clinical abnormalities and are significantly resistant to HIV acquisition [65]. Thus, inhibitors toward CCR5 have been pursued for therapeutic and microbicidal applications. CMPD167 (a small CCR5-specific molecule)
is capable of preventing transmission in vitro and in vivo [60].
In addition, analogs of the natural CCR5 ligand RANTES,
PSC-RANTES, also demonstrated dose-dependent inhibition
of infection in macaques [66]. Being able to block CCR5dependent infection following topical application further emphasizes that the early events during HIV transmission are
dependent on local infection. This seeds sites that ultimately
amplify virus to reach a critical threshold and eventually spill
over into the draining lymphatics to colonize the rest of the
body [4] (Figs. 1 and 2D).
RTIs
As CCR5 inhibitor observations support the importance of
infection in peripheral tissue prior to further viral dissemination, then other well-characterized inhibitors of viral replication may also be effective. NRTIs and NNRTIs are currently
being tested as topical microbicides in Phases I and II clinical
trials, the NRTI Tenofovir, and the NNRTIs TMC-120 and
UC-781 (MIV-150 in the form of PC-185 is scheduled for
Phase I clinical trials; Table 1). To date, the only published
animal studies have been conducted in the murine SCIDhu
model. Naturally, there are concerns that the use of RTIs may
lead to the development of resistant strains or that they might
prove less effective as a result of existing RTI resistance. As
TMC-120, UC-781, and MIV-150 are not used in any current
therapy regimes, and as resistance is difficult to achieve in
http://www.jleukbio.org
vitro, this might not become reality. In addition, the ability for
NNRTIs to cross virus membranes and irreversibly inactivate
virions makes them attractive candidates, as they act directly
on cell-free virions (Fig. 2C).
The potential problems with monomicrobicides:
the concern of resistance
Current microbicide formulations, especially those in clinical
trials, all consist of one active, antiviral component. Most of
these antivirals/microbicides are not being used in therapy to
date (with the exception of Tenofovir), although many are being
developed in parallel with future therapeutics (e.g., CCR5based compounds and fusion inhibitors). The coadministration
of compounds in therapeutics and as microbicides within a
population does raise concerns of an increasing number of
resistant strains being transmitted over time. Resistance to
related compounds, which are currently in antiretroviral therapy, may also impact on those currently being used as microbicides. For example, resistance to the NNRTI Efavirenz (currently in therapy) has a significant impact on the potency of the
NNRTI UC781 as a microbicide [67]. Although NNRTIs are
used as an example herein, the same may apply to any singlebased microbicide, where there is potential for resistance. As
highlighted above, the dual use of compounds in therapeutics/
microbicide strategies may indeed accelerate the existence of
circulating, resistant strains in a given population, but there
still may be the potential for resistance to microbicides, which
are designated solely for microbicide use.
Combinations
Given the concerns of resistant viral isolates arising from
single, compound-based microbicides, the support for multiple
combination strategies is growing. Indeed, when synergistic
combinations are found, the level of compound theoretically
needed is reduced, and the benefit of this is clear in terms of
mucosal bioavailability and cost of production. Currently, the
only combination microbicide scheduled for clinical trials is
PC-815 (carrageenan and MIV-150 combination, Table 1). We
recently confirmed that PC-815 could protect animals against
vaginal simian human immunodeficiency virus-RT challenge
(S. G. Turville and M. Robbiani, unpublished). Using other
products in combination, macaque studies provide encouraging
evidence that synergistic combinations found in vitro do provide good protection in vivo [60], although the licensing of new
microbicides, in addition to potential competition between
microbicide developers/manufacturers, may influence and potentially restrict future combinations and therefore, may not be
equivalent to the therapeutic situation of combination antiretroviral therapy. The latter situation can be resolved with collaboration or release of licenses for open microbicide use, and
precedent for such has recently taken place [68] and thus, may
encourage others to do so.
CONCLUSION
HIV transmission in vivo is multifactorial, and it remains
unclear as to which cells are first targeted by HIV to establish
infection and facilitate dissemination. It is likely an orches-
trated series of events involving multiple cell types, which
eventually result in the robust infection of memory CD4⫹ T
cells. The virus will not likely have to infect all of the cells it
encounters (ECs, DCs, and M⌽) but will exploit traffic through
these cells to be ferried rapidly to the more permissive targets
(low-level infection of DCs/M⌽ and vigorous replication in
CD4⫹ T cells). Thus, how to address these complex pathways
in microbicide design becomes somewhat daunting. Microbicides, which are in clinical trials, have taken a broad approach
to block several incoming pathogens. One common feature of
the broad inhibitors is to strengthen existing primary barriers
(e.g., pH regulation, estrogen treatment, additional absorption
barrier with sulfated polymers). Inhibitors specifically inactivating the incoming viruses (e.g., CNV, BMS-378806,
NNRTIs) also hold promise. Agents working at the level of the
cell are also yielding surprising and effective results in animal
microbicide studies (e.g., CCR5 inhibitors). However, the future of microbicides may lie in combinations, which impair the
virus at multiple levels. For instance, a combination, which
encourages the strength of the vaginal barrier in addition to
specific HIV targeting, may not only provide two lines of
defense against HIV but also may hinder the acquisition of
other sexually transmitted pathogens associated with increasing HIV infection. Whether a microbicide is in combination
with another or in isolation, knowing that putative formulations
function in the face of (or even protect against) other pathogens
is critical for their success.
ACKNOWLEDGMENTS
The laboratory of M. R. is supported by National Institutes of
Health Grants R01 AI040877, DE015512, DE016526, U19
AI065413, R21 AI060405, and DE016534. Support is also
provided by the Elizabeth Glaser Pediatric AIDS Foundation,
and M. R. is an Elizabeth Glaser Scientist. Additional support
has been provided by the Tulane National Primate Research
Center Base Grant RR00164. This publication was also made
possible through support provided by the Office of Population
and Reproductive Health, Bureau for Global Health, U.S.
Agency for International Development, under the terms of
Award No. HRN-A-00-99-00010. The opinions expressed
herein are those of the author(s) and do not necessarily reflect
the views of the U.S. Agency for International Development.
S. G. T. is supported by a C.J. Martin fellowship from the
National Health and Medical Research Council of Australia.
The authors thank Evan Read for his help with the original
version of the graphics. S. T. and S. G. T. contributed equally.
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