Published online before print August 14, 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Center for Biomedical Research, Population Council, New York, New York, USA
1 Correspondence: Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021. E-mail: mrobbiani{at}popcouncil.org
Key Words: transmission • microbicide • dendritic cells • macrophage
 TOP EARLY EVENTS AS HIV... THE IMPORTANCE OF PRIMARY...DEFINING THE CELLULAR SUBSTRATESCHALLENGES OF MICROBICIDE...CONCLUSIONREFERENCES |
|---|
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
].
|
TOPEARLY EVENTS AS HIV...THE IMPORTANCE OF PRIMARY...DEFINING THE CELLULAR SUBSTRATESCHALLENGES OF MICROBICIDE...CONCLUSIONREFERENCES |
|---|
|
TOPEARLY EVENTS AS HIV...THE IMPORTANCE OF PRIMARY...DEFINING THE CELLULAR SUBSTRATESCHALLENGES OF MICROBICIDE...CONCLUSIONREFERENCES |
|---|
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 ß-integrin interaction of virus with ECs [7
, 9
], in addition to viral capture by surface heparan sulfates [10
].
 View larger version (28K): [in a new window] |
Figure 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.
|
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 )
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 logistically 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
29
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 37 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
41
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 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.
|
TOPEARLY EVENTS AS HIV...THE IMPORTANCE OF PRIMARY...DEFINING THE CELLULAR SUBSTRATESCHALLENGES OF MICROBICIDE...CONCLUSIONREFERENCES |
|---|
|
View this table: [in a new window] |
Table 1. Candidate Microbicides for Blocking HIV Transmissions
|
 View larger version (38K): [in a new window] |
Figure 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.
|
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-ß-cyclodextrin (ß-CD)
HIV buds selectively through areas in the plasma membrane called lipid rafts. Therefore, interrupting lipid rafts by using ß-CD seems to be a promising tool for microbicide approaches. ß-CD is effective in vitro [48
] and in vivo at blocking cell-associated 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 x4 isolates blocks the HIV 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 55 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
].
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 sufficient 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
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 CCR5-dependent 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 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., CCR5-based 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 single-based 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.
|
TOPEARLY EVENTS AS HIV...THE IMPORTANCE OF PRIMARY...DEFINING THE CELLULAR SUBSTRATESCHALLENGES OF MICROBICIDE...CONCLUSIONREFERENCES |
|---|
) 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.
Received February 28, 2006; revised April 6, 2006; accepted April 30, 2006.
|
TOPEARLY EVENTS AS HIV...THE IMPORTANCE OF PRIMARY...DEFINING THE CELLULAR SUBSTRATESCHALLENGES OF MICROBICIDE...CONCLUSIONREFERENCES |
|---|
Nef is manifest in cultures of immature dendritic cells and T cells J. Virol. 74,2406-2413
-helical peptides: implications for the viral fusion mechanism Proc. Natl. Acad. Sci. USA 94,13426-13430This article has been cited by other articles:
 |
|
 |
|
  L. G. Perez, M. R. Costa, C. A. Todd, B. F. Haynes, and D. C. Montefiori Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41 J. Virol., August 1, 2009; 83(15): 7397 - 7410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Trapp, N. R. Derby, R. Singer, A. Shaw, V. G. Williams, S. G. Turville, J. W. Bess Jr., J. D. Lifson, and M. Robbiani Double-Stranded RNA Analog Poly(I:C) Inhibits Human Immunodeficiency Virus Amplification in Dendritic Cells via Type I Interferon-Mediated Activation of APOBEC3G J. Virol., January 15, 2009; 83(2): 884 - 895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Frank, H. Stossel, A. Gettie, S. G. Turville, J. W. Bess Jr., J. D. Lifson, I. Sivin, N. Romani, and M. Robbiani A Fusion Inhibitor Prevents Spread of Immunodeficiency Viruses, but Not Activation of Virus-Specific T Cells, by Dendritic Cells J. Virol., June 1, 2008; 82(11): 5329 - 5339. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
