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The Journal of Immunology, 2006, 176: 5627-5636.
Copyright © 2006 by The American Association of Immunologists

Lactobacilli-Expressed Single-Chain Variable Fragment (scFv) Specific for Intercellular Adhesion Molecule 1 (ICAM-1) Blocks Cell-Associated HIV-1 Transmission across a Cervical Epithelial Monolayer1

Caren J. Chancey2,*, Kristen V. Khanna2,*, Jos F. M. L. Seegers{dagger}, Guang Wen Zhang*, James Hildreth{ddagger},§, Abigail Langan* and Richard B. Markham3,*

* Johns Hopkins Bloomberg School of Public Health, Department of Molecular Microbiology and Immunology, Baltimore, MD 21205; {dagger} Erasmus University, Rotterdam, The Netherlands; {ddagger} Johns Hopkins School of Medicine, Department of Pharmacology and Molecular Science, Baltimore, MD 21205; and § Meharry Medical College, Nashville, TN 37208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The vaginal and cervical epithelia provide an initial barrier to sexually acquired HIV-1 infection in women. To study the interactions between HIV-1-infected cells or cell-free HIV-1 and the reproductive epithelium, the transmission of HIV-1 by infected cells or cell-free virus across human cervical epithelial cells was examined using a Transwell culture system. Cell-associated HIV-1 was transmitted more efficiently than cell-free virus, and monocyte-associated virus was transmitted most efficiently. Abs to ICAM-1 added to the apical side of the epithelium blocked cell-mediated transepithelial HIV-1 transmission in vitro. When used in a previously described model of vaginal HIV-1 transmission in human PBL-SCID mice, anti-murine ICAM-1 Abs (0.4 µg/10 µl) also blocked vaginal transmission of cell-associated HIV-1 in vivo. To evaluate a candidate delivery system for the use of this Ab as an anti-HIV-1 microbicide, anti-ICAM single-chain variable fragment Abs secreted by transformed lactobacilli were evaluated for their protective efficacy in the Transwell model. Like the intact Ab and Fab derived from it, the single-chain variable fragment at a concentration of 6.7 µg/100 µl was able to reduce HIV-1 transmission by 70 ± 5%. These data support the potential efficacy of an anti-ICAM Ab delivered by lactobacilli for use as an anti-HIV-1 microbicide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human immunodeficiency virus type 1 infections are acquired most often through sexual contact, with the majority of the sexual transmission of HIV-1 worldwide occurring as a result of heterosexual contact ( 1, 2). Women of childbearing age are at the greatest risk for HIV-1 infection ( 3, 4), resulting in a corresponding increase in HIV-1 infection of women, newborns, and infants worldwide. Studies of the mechanisms of sexual transmission of HIV-1 to women that also reveal targets for HIV-1 prevention in women are critical for limiting the global AIDS pandemic.

Despite the prominence of sexual transmission in the continued spread of HIV-1, the cellular interactions involved in this mode of transmission are poorly understood. For example, it is not known whether cell-free virus, cell-associated virus, or both are essential for HIV-1 transmission in humans. Animal models of related retroviral infections have been used to address the relative importance of cell-associated and cell-free HIV-1 transmission. Cell-associated virus is transmitted in the human PBL (HuPBL)4-SCID model of vaginal transmission ( 5), whereas both cell-free and cell-associated virus are infectious when administered vaginally in the HIV/chimpanzee ( 6) and feline immunodeficiency virus/cat models ( 7, 8). In the SIV/macaque model, cell-free SIV was transmitted vaginally, whereas infected mononuclear cells were incapable of transmitting SIV ( 9). The current studies use a Transwell culture system of human cervical epithelial cells ( 10) to examine the relative efficiency of cell-associated and cell-free virus HIV-1 transmission and to identify appropriately targeted interventions to block that transmission.

The majority of anti-HIV-1 microbicides tested to date have targeted transmission by cell-free virus. However, the microbicides currently being tested present several drawbacks, including reduced effectiveness against some CCR5-using isolates, disruption of the normal flora of the genitourinary tract, toxicity for the genital epithelium, carcinogenic potential, and undemonstrated efficacy against the cell-associated virus (reviewed in Ref. 11). The use of Abs as microbicides, such as the vaginally applied anti-gp120 Abs used to protect against the transmission of simian-human immunodeficiency in macaques, may avoid the toxicity problems associated with many chemical compounds ( 12).

One difficulty in designing chemically or Ab-based microbicides targeting cell-free transmission of HIV-1 is the high degree of variability of viral surface epitopes. This difficulty can be avoided by targeting the host cell protein epitopes involved in cell-associated and/or cell-free virus transmission. However, despite newer methods of Ab production, the cost for such a microbicide necessarily involving the passive administration of an Ab is likely to be prohibitive. A potentially feasible delivery system for an Ab involves the application of engineered lactobacilli for sustained in situ production of a short surface-bound or secreted heterologous protein known as the single-chain variable fragment (scFv). These scFvs can fold to resemble the variable region of Abs targeted toward specific HIV-related targets.

In these studies we have identified Ab to ICAM-1 as a candidate for disruption of cell-associated HIV-1 transmission across the cervical epithelium. We have also successfully engineered Lactobacillus casei to express scFv directed against human ICAM-1 and used the purified scFv to reduce the transmission of HIV-1 p24 and the migration of PBMCs from infected cultures across an epithelial barrier.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
HIV-1 preparation

HIV-1Ba-L (Advanced Biotechnologies), a CCR5-using variant of HIV-1, was purchased in 1-ml aliquots (1 x 106 50% tissue culture infectious doses (TCID50)/ml) and stored in liquid nitrogen.

Human PBMC isolation and HIV-1 infection

Human PBMCs were isolated by centrifugation of leukopheresed blood (Hemapheresis Center, Johns Hopkins Hospital, Baltimore, MD) on a Ficoll-Hypaque gradient (GE Healthcare). Total PBMCs were plated at 1 x 107/ml in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg of streptomycin, 10 ng/ml gentamicin, and 2 mM L-glutamine (hereafter referred to as cRMPI, media and all supplements were from Invitrogen Life Technologies). Cells were suspended in RPMI 1640 as described above with 20% heat-inactivated FCS (56°C for 45 min; Gemini Bio-Products) or 10% heat-inactivated A/B human serum (HS; Nabi Biopharmaceuticals) during the initial 12 h of culture. Monocytes were collected using a previously described method ( 13). Briefly, nonadherent PBMCs were removed, and adherent cells were detached from the flasks using cold HBSS (Invitrogen Life Technologies). More than 95% of the adherent cells were determined to be monocytes by flow cytometric analysis of the expression of the surface Ag CD14. Monocytes were washed and suspended at 5 x 106/ml with 1 x 103 TCID50 of HIV-1Ba-L in 5 ml of cRPMI with 10% FCS or cRPMI with 10% HS in 25-cm2 flasks (Corning Scientific Products) and cultured at 37°C with 5% CO2. The virus inoculum was removed after 24 h, cells were washed once with warm cRPMI, and fresh cRPMI with 10% FCS or cRPMI with 10% HS was added. Cultures were fed with fresh media on days 3 and 7 postinfection. Infected monocytes were removed from the flasks using cold HBSS or 0.02% EDTA (Sigma-Aldrich) and used on day 10 postinfection.

PBLs or PBMCs were suspended at 2 x 106/ml in cRPMI with 10% FCS supplemented with PHA (5 µg/ml; Sigma-Aldrich) for 48 h. Following infection with 1 x 103 TCID50 HIV-1Ba-L, PBLs were cultured identically to monocytes, except that the PBL culture medium contained 10 U/ml IL-2 (Roche).

The extent to which the monocytes and PBLs were infected with HIV-1 was determined by limiting dilution-PCR (LD-PCR), performed with the appropriate standards and controls as described previously ( 14) and using HIV-1 Gag-specific primers (forward primer, 5'-GCG AGA GCG TCA GTA TTA AGC GG- 3' (nucleotides 795–816 of HXB2); reverse primer, 5'-TCT GAT AAT GCT GAA AAC ATG GG- 3' (nucleotides 1296–1318 of HXB2). PCR products were visualized by electrophoresis on a 1% agarose gel.

All studies using donor cells were performed with approval from the Johns Hopkins Institutional Review Board.

Cell-free HIV-1

The cell-free virus was collected from HIV-1Ba-L-infected monocytes or HIVBa-L-infected PBLs in cRPMI with 10% FCS or cRPMI with 10% HS. Supernatants were cleared of cells by centrifugation and filtered through a 0.2-µm syringe filter (Millipore). The cell-free virus was quantified by HIV-1 p24 ELISA (sensitivity of 12.5–350 pg/ml; PerkinElmer) and by coculture with PBMC from a healthy, HIV-negative donor to determine TCID50 as previously described ( 15).

Human cervical epithelial cell Transwell cultures

The human, spontaneously transformed, cervical epithelial cell lines ME-180 and HT-3 (ATCC HTB-33 and HTB-32, respectively) were cultured in cRPMI with 10% FCS and routinely subcultured every 3 days with cell displacement by 0.05% trypsin-EDTA (Invitrogen Life Technologies). Cervical epithelial cells were plated at 2 x 105 cells in cRPMI with 10% FCS or 10% HS per 12-mm diameter Transwell insert with a pore size of 5 µm (Corning Scientific Products). Due to a change in manufacturing process, PCF Transwell inserts with a pore size of 3 µm (Millipore) were used for the experiments presented in Figs. 5, 6, and 9. Cervical epithelial cells in the Transwells were maintained at 37°C under 5% CO2 conditions. Media were changed every 2–3 days. The cells formed a polarized, complete monolayer on the Transwell inserts in 7 days, which was confirmed by monitoring the resistance across the epithelium using a Millicell electrical resistance system ohmmeter (Millipore) and/or by monitoring the permeability of cell monolayers to HRP (Sigma-Aldrich). The cervical epithelial monolayers on Transwell inserts were used between days 7 and 10 of culture.


Figure 5
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  		FIGURE 5. Anti-ICAM-1 antibodies (20 µg/ml) block the transmission of cell-associated HIV-1 across a monolayer of HT-3 cervical epithelial cells. HIV-1-infected PBMCs (1 x 106) were added with the designated treatment to the apical side of HT-3 monolayers grown on permeable Transwell supports and allowed to transmigrate for 24 h. Results are expressed as mean ± SD HIV-1 p24 concentration from the basal side supernatant fluid and are representative of three separate experiments. *, p < 0.05.

 

Figure 6
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  		FIGURE 6. Anti-ICAM Fab blocks transmission of HIV-1-infected PBMC (A) and p24 (B) across an HT-3 cell monolayer. HIV-1-infected PBMCs (1 x 106) were added with the designated treatment to the apical side of HT-3 monolayers grown on permeable Transwell supports and allowed to transmigrate for 24 h. All intact antibodies were used at a concentration of 100 µg/ml, and all Fabs were used at 67 µg/ml to equalize the available binding sites. Data are expressed as mean ± SD basilar HIV-1 p24 concentration or viable PBMCs counted. *, p < 0.05; **, p < 0.01.

 

Figure 9
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  		FIGURE 9. Anti-ICAM scFvs block transmission of HIV-1 p24 (A) and infected PBMCs (B) across an HT-3 cell monolayer. HIV-1-infected PBMCs (1 x 106) were added with the designated treatment to the apical side of HT-3 monolayers grown on permeable Transwell supports and allowed to transmigrate for 24 h. All intact antibodies were used at a concentration of 100 µg/ml, and all Fabs and scFvs were used at 67 µg/ml to equalize the available binding sites. Data are expressed as mean ± SD basilar HIV-1 p24 concentrations or viable PBMC control. *, p < 0.05; **, p < 0.01.

 
Transepithelial HIV-1 transmission

The transepithelial migration assay was performed as described by Bomsel ( 10) with several modifications. Briefly, 1 x 106 HIVBa-L-infected monocytes, 1 x 106 HIVBa-L-infected PBLs, or cell-free HIV-1 in the supernatant fluid from the monocyte or PBL cultures and dilutions thereof were added to the apical sides of epithelial monolayers. Time course analyses were performed, with collection of the apical and basal side media between 1 and 48 h after the addition of the infected cells to the inserts. Nearly maximal transmission across the monolayer was observed at 24 h, and this time point was chosen for all subsequent experiments. The viability of monocytes and PBLs was assessed by trypan blue exclusion (Sigma-Aldrich), both before their addition to the Transwells and after the 24-h transmission period, and it was always found to be >90%. The amount of HIV-1 p24 Ag in the apical and basal supernatant fluid was determined by HIV-1 p24 ELISA. For some experiments, transmitted inoculum cells were collected from the basal compartment, pelleted by centrifugation, and counted on a hemacytometer with trypan blue exclusion to assess viability.

In vitro transmission blocking studies

Mouse anti-human ICAM-1 blocking Ab (HA58 (IgG1) from BD Biosciences or MT-M5 (IgG1) provided by J.H.), anti-human E-cadherin Ab (67A4 (IgG1) from Beckman Coulter Immunotech), anti-E-cadherin Ab E4.6 (IgG1 blocking Ab, a generous gift from Dr. M. Brenner, Harvard Medical School, Boston, MA) ( 16), anti-human CD103 blocking Abs (2G5 (IgG2a) from Beckman Coulter Immunotech), or their respective isotype controls were added to 1 x 106 HIV-infected monocytes or PBMCs or to a cell-free virus immediately before their addition to the cervical epithelial Transwell cultures. For Fab studies, mouse anti-human ICAM-1 MT-M5 was digested and purified using a ficin-based ImmunoPure Fab/F(ab')2 digestion kit (Pierce). Apical and basal supernatant fluids were collected after a 24-h incubation at 37°C with 5% CO2. Transepithelial migration assays were performed as stated above.

HuPBL-SCID mouse model of vaginal transmission

The HuPBL-SCID mouse model was previously described ( 5). Briefly, female mice with SCID (C.B-17 SCID) ( 17) were obtained from our SCID mouse colony (established using C.B-17 SCID mice from The Jackson Laboratory). The mice were treated s.c. with 2.5 mg of progestin (Depo-Provera; Upjohn Pharmaceuticals) on the same day as the i.p. administration of 5 x 107 unstimulated, uninfected human PBMCs (HuPBMCs) in 1 ml of PBS. Seven days following progestin treatment and reconstitution of the SCID mice with HuPBMCs, the mice were anesthetized with isoflurane (IsoFlo; Abbott Laboratories) in a 30–50% O2 mix delivered by a Vaporstick anesthesia apparatus (SurgiVet) and intravaginally administered either varying combinations of anti-ICAM-1 Abs as designated or the appropriate isotype control Abs 5 min before receiving 1 x 106 HIV-1Ba-L-infected HuPBMCs suspended in PBS with 1% BSA (Sigma-Aldrich). Mice remained anesthetized for 5 min following intravaginal inoculation by pipette. Extreme care was taken to avoid trauma to vaginal tissues. Two weeks later the mice were euthanized, and peritoneal cells were recovered by lavage with cold PBS. The cells recovered by lavage (of both murine and human origin) were assayed by DNA-PCR for human beta-globin to confirm the success of the human cell engraftment in the mice. Mice without human cells, typically between 0 and 30% of an experimental group, were excluded from analyses.

To assess virus recovery from cells harvested from the peritoneal cavities of challenged mice, uninfected HuPBMCs were stimulated with PHA and maintained in IL-2-supplemented medium (1 x 106 per mouse) in preparation for coculture with the peritoneal cells recovered from the HuPBL-SCID mice. Positive mice were determined by HIV-1 p24 ELISA on supernatants from cocultured cells, which in our experience has been the most sensitive method for detecting infected mice. In all cases the cells were obtained from a donor other than that from which cells were obtained for the original transplant into the peritoneal cavities of the mice.

HuPBMCs that were used for vaginal inoculation were isolated as described above and maintained in cRPMI. PBMCs were stimulated with PHA for 2 days; cells were then exposed to 300 TCID50 of HIV-1Ba-L in cRPMI with IL-2 (10 U/ml). Infected-cell cultures were maintained in cRPMI supplemented with IL-2 for 10 days before inoculation into the mice. All anti-mouse Abs and controls used in the HuPBL-SCID mouse experiments are commercially available (BD Biosciences/BD Pharmingen).

All studies using animals were performed with approval from the Johns Hopkins Institutional Review Board and the Animal Care and Use Committee.

Cloning and expression of scFv in Lactobacillus

Following RNA isolation and cDNA preparation, the variable H chain and variable L chain regions from the anti-ICAM-1 hybridoma MT-M5 were amplified via PCR and cloned into the vector pSCN112 as illustrated in Fig. 7. The resulting plasmid pSCN112hIcam-1 was then electroporated into L. casei 393 as follows: L. casei 393 was grown at 37°C overnight in Mann-Rogosa-Sharpe medium (Difco; BD Biosciences). The overnight culture was diluted 50-fold in fresh MRS and incubated at 37°C for another five hours (OD600 = 0.8). Cells were collected through centrifugation, washed three times with electroporation wash buffer (5 mM NaH2PO4, pH 7.4, 1 mM MgCl2), and washed once with electroporation buffer (0.3 M sucrose, 5 mM NaH2PO4 (pH 7.4), and 1 mM MgCl2). Cells were resuspended in 1/100 volume electroporation buffer. Transformation was conducted via electroporation of 50 µl of cells with 200 ng of plasmid DNA, and transformants were selected on Mann-Rogosa-Sharpe plates with chloramphenicol at 10 µg/ml. Transformant lactobacilli were tested for expression of scFv using both Western blot analysis and detection of E-tag, a FLAG sequence that was translationally fused to the scFv for detection and purification purposes. Transformed lactobacilli were grown in Mann-Rogosa-Sharpe medium at 37°, and secreted scFvs were purified using the E-tag HiTrap purification system (GE Healthcare).


Figure 7
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  		FIGURE 7. Cloning vector used for expression of the human ICAM-I single-chain antibody. Vector allows for direct cloning of PCR-amplified fragments of the variable domains of the H and L chains.

 
The ability of scFvs both in culture supernatant and purified form to bind Chinese hamster ovary (CHO) cells overexpressing full-length ICAM-1 was assessed by flow cytometry. Briefly, CHO cells, transfected to overexpress ICAM-1 using a previously described method ( 18), were washed and incubated on ice for 1 h in either a 10-fold dilution of culture supernatant or dilutions of purified scFv. Cells were then washed and incubated on ice for 1 h in the presence of mouse anti-E-tag IgG (GE Healthcare). Following another wash, the cells were incubated for a final hour on ice in the presence of FITC-labeled goat anti-mouse IgG Abs. After a final wash, the cells were analyzed by flow cytometry, and binding of the scFv was compared with the binding ability of an intact MT-M5 Ab.

Statistical analysis

Statistical analysis was performed using the Instat (GraphPad Software) and Intercooled Stata 8 (Stata) statistical packages. One-way ANOVA with Bonferroni correction was used to compare the differences between groups for the in vitro studies and a Fisher’s exact analysis for the in vivo studies. Values of p ≤ 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cell-associated HIV-1 crosses human cervical epithelial cells more efficiently than cell-free virus

To evaluate the extent to which each crossed the intact human cervical epithelial monolayer, HIV-1-infected monocytes, HIV-1-infected PBLs, and cell-free virus derived from monocyte or PBL cultures, each in cRPMI supplemented with 10% FCS, were placed into the apical chambers of Transwell cultures containing a confluent monolayer of ME-180 cells (Fig. 1A). Monocyte-associated virus was approximately five times more efficient at crossing the cervical epithelium than the PBL-associated virus, although the respective percentages of infected cells in the inocula were statistically similar. Cell-free viruses derived from either cell type did not cross the epithelial monolayer under these experimental conditions and were typically below the limit of detection of the HIV-1 p24 ELISA. The p24 Ag concentration in the lower chamber medium of Transwell inserts without ME-180 cells present was measured to confirm the extent to which the cervical epithelial monolayer and the membrane dividing the Transwell chamber provided a barrier limiting the transepithelial transmission of HIV-1. In contrast to the difference in levels of basal HIV-1 p24 Ag transmitted by HIV-infected monocytes or the HIV-infected PBLs shown in Fig. 1A, the amount of p24 Ag measured in the lower chamber of the Transwell cultures without cervical epithelial cells did not differ significantly (p > 0.05) between monocyte-associated HIV-1 and PBL-associated HIV-1 (Fig. 1), and p24 from a cell-free virus was readily detected. Thus, the nylon support mesh of the Transwell did not pose a barrier to the movement of cells or cell-free viruses. In the experiment shown, we noted that when using monocytes as the infected cell inoculum, more HIV-1 p24 was detected on the basal side of Transwells with cervical epithelial cells as compared with membrane alone. However, this effect has not been consistently observed.


Figure 1
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  		FIGURE 1. Monocyte-associated HIV-1Ba-L crosses a human cervical epithelial cell barrier more efficiently than PB L-associated HIV-1Ba-L or a cell-free virus. Data are expressed as mean ± SD of HIV-1 p24 Ag (pg/ml) in the basal side supernatant fluid from the Transwell culture, with (w/) and without (w/o) ME-180 cervical cells plated in cRPMI plus 10% FCS on the insert membranes, respectively. A representative experiment (n = 3) is shown (p < 0.05 for comparisons between all inoculum types with and without ME-180 cells, and p < 0.05 for comparisons between inoculum types with ME-180 cells).

 
To address whether cell-free HIV-1 may be inhibited from crossing an epithelial monolayer by FCS as suggested by Kage and colleagues ( 19), studies were performed using HIV-infected monocytes or cell-free viruses cultured in cRPMI containing 10% HS as inocula. In these experiments, monocyte-associated HIV-1 cultured in HS crossed the epithelium more efficiently than cell-free virus in HS (Fig. 2). In contrast to the lack of transepithelial transmission measured in FCS, cell-free virus cultured in HS were able to cross the intact epithelium, although at a significantly lower level than cell-associated virus (p < 0.05) (Fig. 2). The concentration of HIV-1 p24 Ag in apical supernatant fluid from HIV-infected monocytes and HIV-infected PBLs cultured in either FCS or HS did not differ significantly over the course of infection, indicating that the type of cell or serum did not affect the extent to which virus was shed from infected cells (data not shown).


Figure 2
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  		FIGURE 2. Cell-free virus crosses the cervical epithelium under human serum culture conditions, although less efficiently than the cell-associated virus. Results are expressed as the mean ± SD concentration of HIV-1 p24 antigen (pg/ml) from the basal supernatant fluid of Transwell cultures (p < 0.05 for comparisons between each inoculum in cRPMI plus 10% FCS with the corresponding inoculum in cRPMI plus 10% HS, and p < 0.005 for comparisons between monocyte HS and cell-free HS). A representative experiment of donors (n = 3) distinct from those in Fig. 1 is shown. The cell-free virus HIV-1Ba-L inoculum was routinely 1–3 x 104 TCID50/ml and up to 1.7 x 106 TCID50/ml.

 
The HIV-1 measured by p24 concentration in the basilar supernatant was found to be infectious when cocultured with uninfected monocytes or PHA-stimulated PBLs, as indicated by an increase in the amount of HIV-1 p24 Ag in the cultures over time (data not shown).

Monocyte-associated HIV-1 crosses the cervical epithelium in a time- and dose-dependent manner

To analyze the kinetics of transepithelial transmission, 1 x 106 HIV-infected monocytes in cRPMI with 10% HS were added to the apical side of the human cervical epithelial Transwell cultures. Infected monocytes that have migrated and/or transmitted HIV-1 transepithelially were detected as early as 4 h (Fig. 3A) with increasing basal HIV-1 p24 levels until 48 h, when a plateau was reached. Similarly, 1 x 106 HIV-infected monocytes or 5-fold dilutions thereof were added to the apical side of the epithelium, and the basal supernatant fluid was sampled for the presence of HIV-1 p24 Ag to determine the minimal cell inoculum required to detect virus on the basal side of the epithelium. Detectable HIV-1 p24 Ag from the basal side supernatants was obtained in some experiments with as few as 1000 monocytes applied to the apical side of the Transwell (Fig. 3B), and p24 Ag was consistently detected in the basal medium after applying 5000 HIV-1-infected monocytes. Between 1 and 5% of this population of cells are infected with HIV-1 as determined by LD-PCR (data not shown), indicating that as few as 50 infected cells consistently transmitted HIV-1.


Figure 3
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  		FIGURE 3. HIV-1Ba-L-infected monocytes transmit virus across a cervical epithelial monolayer in a time-dependent (A) and dose-dependent (B) manner. Symbols used in B represent the following: +, basilar HIV-1 p24 between 100 and 1,000 pg/ml; ++, basilar HIV-1 p24 between 1001 and 5,000 pg/ml; +++, basilar HIV-1 p24 between 5,001 and 30,000 pg/ml; and –, basilar HIV-1 p24 below the limit of detection (12.5 pg/ml). Experiments were performed with HIV-1Ba-L-infected monocytes in cRPMI with 10% HS. Results are expressed as the mean ± SD concentration of HIV-1 p24 antigen (pg/ml) in the basal supernatant. A representative experiment for the time course (n = 4) and inoculum dose (n = 2) is shown.

 
Anti-ICAM-1 Abs block monocyte-associated HIV-1 transmission

To examine the role of adhesion molecules on HIV-1-infected monocytes and epithelial cells in transepithelial migration and HIV-1 transmission, Abs against several adhesion molecules involved in the interactions between epithelial cells and T cells or monocytes were added to the Transwell cultures. Blocking Abs (1–20 µg) to specified adhesion molecules were added at the same time as HIV-1-infected monocytes and remained in the cultures for the duration of the assay.

ICAM-1 is expressed on HIV-1-infected monocytes ( 1, 4) and epithelial cells ( 1) and plays a significant role in LFA-1 mediated transendothelial migration of T lymphocytes and monocytes ( 20, 21, 22, 23). Anti-ICAM-1 Abs added to the inoculum cells immediately before their addition to the epithelial monolayer reduced by 90% (range of 70–90% in multiple experiments) the transmission of monocyte-associated HIV-1, and no blocking was observed in the relevant isotype controls (Fig. 4A). Adding the Ab to the epithelial cells just before the addition of inoculum cells yielded the same result (data not shown).


Figure 4
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  		FIGURE 4. Anti-ICAM-1 antibodies block the transmission of monocyte-associated HIV-1. Culturing HIV-1-infected monocytes with anti-ICAM-1 antibodies (20 µg/ml; HA58 (IgG1)) inhibits the transepithelial transmission of cell-associated HIV-1 across a ME-180 monolayer. Anti-E-cadherin (20 µg/ml; 67A4 (IgG1) and E.4.6 (IgG1)) ( 11 ), anti-CD103 (20 µg/ml; 2G5 (IgG2a)), murine IgG1 (mIgG1; 20 µg/ml), and murine IgG2a (mIgG2a; 20 µg/ml) antibodies added with HIV-1-infected monocytes to the Transwell cultures have no effect on the transepithelial transmission of cell-associated HIV-1. Results are expressed as mean ± SD HIV-1 p24 concentrations from the basal side supernatant fluid and are representative of three separate experiments. *, p < 0.05.

 
To determine whether the inhibition of transepithelial transmission was specific to the ICAM-1 adhesion molecule, Abs against another relevant adhesion-receptor pair, E-cadherin and CD103, were tested for their ability to inhibit HIV-1 transmission. E-cadherin, expressed on epithelial cells ( 24, 25), and its ligand CD103 ( 26), expressed on monocytes ( 27) and a subset of PBLs ( 27, 28, 29), have been shown to play a role in the adhesion of T lymphocytes to the epithelium of the gastrointestinal mucosa ( 26, 30). Anti-E-cadherin Abs, anti-CD103 Abs, and their respective isotype controls did not block the transepithelial transmission of cell-associated HIV-1 (Fig. 4, A and B). It was determined that the incubation of HIV-1-infected monocytes with the Abs used in these experiments did not alter the amount of virus released from the infected cells, as measured in the apical supernatant fluid after the 24-h incubation period (data not shown).

To ensure that the blocking of cell-associated HIV-1 transmission by the Ab to ICAM-1 was not specific to the ME-180 cervical epithelial cell line, the assay was repeated using another cervical epithelial cell line, HT-3. The HT-3 line was selected based on its ability to form cadherin-based cell-cell junctions at a level comparable to that of ME-180 ( 31). As with the ME-180 cells, Ab to ICAM-1 significantly blocked transmission of cell-associated HIV-1 across HT-3 cell monolayers when compared with untreated or isotype-control treated monolayers (Fig. 5). Because these cell lines performed equivalently in these assays, they were used interchangeably for later experiments.

Anti-ICAM-1 Abs inhibit transepithelial transmission of cell-associated HIV-1 in vivo

To determine the extent to which ICAM-1 is involved in the process of vaginal HIV-1 transmission in vivo, we administered anti-ICAM-1 Abs to HuPBL-SCID mice exposed vaginally to HIV-1-infected PBMCs as described ( 5). Briefly, anti-ICAM-1 or appropriate isotype control Abs (0.4 µg in 10 µl of PBS with 1% BSA) were administered vaginally followed 5 min later by 1 x 106 human PBMCs, of which between 1 and 5% were infected with HIV-1Ba-L as determined by LD-PCR. When anti-human-ICAM-1, which would bind only the human cell inoculum, was administered before introduction of the HIV-infected PBMCs, no protection was observed (Table I, Experiment A). When a combination of anti-human and anti-mouse-ICAM-1 Abs were used, the mice that were exposed to the anti- ICAM-1 Abs were protected from HIV-1 infection (p < 0.01), whereas the mice exposed to a mixture of the appropriate isotype control Abs or PBS with 1% BSA were not protected from vaginal transmission (Table I, Experiment B). When anti-mouse-ICAM-1 alone, which binds only to the murine reproductive epithelium, was administered, the mice treated with the anti-ICAM-1 were again protected from HIV-1 transmission (p < 0.01) (Table I, Experiment C). Therefore, binding of ICAM-1 on the reproductive epithelium, but not the infected cell inoculum, is required to block transmission in this system.


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  		Table I. Anti-mouse ICAM-1 blocks transmission of cell-associated HIV-1 in vivo

 
Fabs of Abs to ICAM-1 block transmission of cell-associated HIV-1 with an efficacy equivalent to that of the intact Ab

To determine whether an Ab fragment similar in size and structure to lactobacillus-secreted scFv could block cell-associated transmission of HIV-1, we fragmented mouse IgG1 against ICAM-1 into Fab and Fc using a ficin-based digestion kit. The recovered Fabs were capable of blocking transmission of HIV-1 p24 to the basal compartment at levels equivalent to those of intact Ab when normalized for available binding sites (p < 0.05; Fig. 6A). In addition, anti-ICAM Fab also significantly reduced the number of cells crossing the cervical epithelial monolayer and Transwell membrane when compared with both untreated cells and corresponding isotype controls (p < 0.01; Fig. 6B). These results suggested that Lactobacillus-secreted anti-ICAM scFvs, which are similar in structure to Fab, would be capable of blocking cell-associated HIV-1.

Lactobacillus-produced anti-ICAM scFvs block transmission of cell-associated HIV-1 in vitro

The variable regions of the H and L chains of the anti-ICAM-1 mouse IgG1 hybridoma MT-M5 were cloned into the vector pSCN112 as described in Materials and Methods (Fig. 7). Anti-ICAM scFvs secreted by the transfected lactobacilli in both culture supernatant and purified forms were capable of binding CHO cells overexpressing human ICAM-1, even at a 1/10 dilution of the crude bacterial culture supernatant (Fig. 8).


Figure 8
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  		FIGURE 8. Anti-ICAM scFvs produced by lactobacilli specifically bind ICAM-expressing cells. CHO cells overexpressing ICAM-1 were incubated with intact anti-ICAM MT-M5 (blue), negative (neg) control antibody (red), a 1/10 dilution of lactobacillus culture supernatant (A), or dilutions of purified scFv from a 20 µg/ml stock (green) (B–D). scFv binding was detected using a mouse anti-E tag followed by FITC-labeled goat anti-mouse.

 
The purified anti-ICAM scFvs were assayed for their ability to block transmission in vitro using the cervical epithelial Transwell model discussed above. Anti-ICAM scFvs significantly blocked both transepithelial cell migration and transmission of HIV-1 p24 at levels equivalent to those of anti-ICAM Fabs and intact Abs when compared with untreated cells and to isotype control Abs (Fig. 9).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These studies demonstrate the potential feasibility of using Lactobacillus-expressed anti-ICAM-1 to block sexual transmission of cell-associated HIV-1. In the experimental systems analyzed, cell-associated HIV-1 is more efficiently transmitted across a cervical epithelial cell monolayer than cell-free virus. Specifically, monocyte-associated virus is transmitted more efficiently than PBL-associated virus. Moreover, transepithelial transmission of cell-associated HIV-1 may be blocked by anti-ICAM-1 Abs, their corresponding Fabs, and by anti-ICAM scFvs secreted by engineered lactobacilli.

A previously published Transwell culture method ( 10) was modified to examine transepithelial transmission. Alterations included the measurement of basilar HIV-1 p24 Ag as an indicator of the transmitted virus and the use of cervical epithelial cells rather than endometrial cells. We found that both monocytes and PBLs were able to migrate through the epithelial monolayer and were found in the basal chamber of the Transwell. These in vitro data are consistent with our findings in vivo ( 5), which show that cells introduced vaginally into HuPBL-SCID mice migrate across the intact reproductive epithelium and can be found in regional lymph nodes within 4 h. The data are also consistent with in vitro findings from Phillips et al. demonstrating that monocytes can cross cervical epithelial tissue by migrating between cells ( 32).

The observation that monocytes were more efficient at transmitting HIV-1 across the cervical epithelium is highly relevant for the in vivo situation. The theory that HIV-infected cells and, specifically, monocytes, transmit infection was proposed in the late 1980s ( 33, 34). The mononuclear cells in semen are largely of the monocyte lineage ( 35), with an average of four million immune cells in a human ejaculate. These cells are infected with HIV-1 in vivo ( 36), although the percentage of cells infected with HIV-1 within the ejaculate varies by donor. It is notable that we observed measurable HIV-1 (expressed as p24 Ag) that crossed an intact epithelial monolayer when as few as 10–50 infected monocytes were present in the inoculum.

Although these observations support the hypothesis that cell-mediated HIV-1 transmission is significantly more efficient than cell-free HIV-1 transmission in vitro and in our huPBL-SCID mouse model, they do not exclude the possibility that cell-free virus, which is also found in seminal fluid, may also be able to transmit HIV-1 infection. Indeed, in transmission models that have been developed in nonhuman primates, it has been reported that cell-free virus is preferentially transmitted ( 37). However, viral challenge in this model was conducted at the acidic pH of the macaque vagina ( 38), as opposed to the more neutral conditions that would exist in the clinical setting in the presence of alkaline seminal fluid. At acidic pH cell migration is greatly reduced ( 39), which may account for the poor cell-associated transmission observed in the macaque studies. The mouse vagina maintains a neutral pH as does the feline vagina, which has been shown to support transmission of cell-associated feline immunodeficiency virus ( 7).

The failure to observe cell-free HIV-1 transmission in our in vitro and in vivo model systems may be attributable to the absence of human dendritic cells. Some studies in the macaque model have claimed a role for these cells in the vaginal transmission process ( 40), whereas others have not found evidence for such a role ( 41).

Collins and colleagues ( 42) reported that in an in vitro organ culture system transmission of cell-associated virus occurred over a much longer time period than that observed for the cell-free virus. Cell-associated transmission, therefore, may occur under conditions that are suboptimal for cell-free virus survival and transmission.

The observation that monocyte-associated HIV-1 crosses the epithelium more efficiently is consistent with studies that have found that the viruses transmitted are macrophage-tropic and use the CCR5 coreceptor for entry into CD4+ target cells ( 43, 44, 45). HIV-1Ba-L, an R5 virus that infects unactivated monocytes and PHA-activated but not unstimulated PBLs, was used in these experiments. It is a relevant strain for use in these studies, which aim to model in vitro the sexual transmission of HIV-1. The mechanism leading to the restricted transmission of R5 variants is not well understood, but these studies suggest that one factor contributing to this restriction is the preferential movement of a monocyte-associated virus across an epithelial barrier.

Thorough characterization of vaginal and cervical epithelial cells, as well as HIV-1-infected mononuclear cells, has identified several relevant adhesion molecules, including ICAM-1 ( 46, 47), that may facilitate the binding of infected cells to the epithelium ( 47, 48). ICAM-1 is highly expressed by monocytes ( 49) and epithelial cells ( 46) and, to a lesser extent, by activated lymphocytes including HIV-1 infected CD4+ T cells ( 47), and it plays a significant role in transendothelial migration of T lymphocytes and monocytes ( 20, 21, 22, 23). In examining the mechanism by which anti-ICAM-1 Abs may block transepithelial transmission of HIV-1, our results show that in the in vivo HuPBL-SCID mouse model, the binding of the Ab to ICAM-1 on the murine reproductive epithelium is critical for blocking cell-associated transmission. This does not exclude the possibility that, in the clinical setting, anti-ICAM-1 Abs may bind ICAM-1-bearing inoculum cells; however, binding of human ICAM-1 on the inoculum cells was insufficient for blocking the transmission of cell-associated HIV-1 in this in vivo model. Our studies do not address the issue of whether the Ab to ICAM-1 specifically disrupts either adhesion or transmigration of infected cells or both. Previous studies using Abs to explore this particular issue using endothelial cells have yielded conflicting results ( 20, 21, 50, 51, 52).

It is of particular note that the interference with transmission observed in the in vitro Transwell assay correlated with a high level of protection in the in vivo Hu-PBL-SCID mouse system. An advantage of this in vivo system is that it recapitulates the host immune environment at the time of infection. Previous studies from this laboratory have shown that the low level of expression of cellular activation markers in cells recently transplanted into SCID mice are similar to that observed clinically and differs markedly from that in the in vitro tissue culture setting ( 53). This difference has a profound effect on the selection of which viruses are transmitted and favors the transmission of CCR5-using viruses, as is observed clinically.

The ability of anti-ICAM-1 to block cell-associated HIV-1 transmission supports its use as an anti-HIV-1 microbicide. The majority of anti-HIV-1 microbicides currently in development target cell-free virus transmission and viral epitopes, although host cell epitopes including CD4 and CCR5 have been targeted with some success ( 54, 55). Anti-ICAM-1 has the advantage of targeting a conserved host epitope that is not subject to the variability of viral epitopes. In addition to its ability to block cell-associated transmission as demonstrated in these studies, it has also been shown to reduce the infectivity of ICAM-1-bearing cell-free virus ( 56, 57).

Our results suggest that vaginal colonization by anti-ICAM-1 scFv-expressing lactobacilli merits further development for use as an anti-HIV-1 microbicide. Lactobacillus-produced scFvs have been shown to be effective in a variety of settings; purified soluble two-domain CD4 molecules expressed by engineered lactobacilli have reduced HIV-1 infectivity in an in vitro model, and scFvs secreted by engineered lactobacilli in situ have reduced dental caries caused by Streptococcus mutans in vivo ( 54, 58). An advantage of this system for microbicidal applications is that lactobacilli are Gram-positive commensal bacteria commonly found as part of the normal microflora of the female genitourinary tract. The presence of certain strains of lactobacilli may be advantageous in itself, because the absence of peroxide-producing lactobacilli at the cervicovaginal mucosa is positively associated with bacterial vaginosis ( 59), a risk factor for HIV-1 transmission ( 60, 61). Compared with both passive administration of Ab and traditional microbicides, scFv-producing lactobacilli offer high efficacy with minimal toxicity and, dependent on their persistence in the genital tract, would not require application within the immediate precoital time frame. In addition, because lyophilized lactobacilli would not require refrigeration, are inexpensive to produce, and are transparent to users, they offer an opportunity to create a female-controlled microbicide feasible for use in cultural settings in which condom use is frequently rejected by the male partners of at-risk women.

The observations showing that monocytes transmit HIV-1 most efficiently across a cervical epithelial monolayer and that ICAM-1 can be successfully targeted to reduce transepithelial transmission of HIV-1 provide a basis for understanding the mechanisms involved in the sexual transmission of HIV-1 to women. Achieving a greater understanding of the process of HIV-1 transmission will facilitate the identification of target molecules for interrupting HIV-1 transmission, which, in turn, could inspire novel immunologically based strategies to prevent the sexual transmission of HIV-1.


   Acknowledgments
 
The authors greatly appreciate the technical assistance of David H. Ford and the secretarial and bibliographic assistance of Roberta Prevost. We thank Dr. Michael Brenner, Harvard Medical School, Boston MA, for the generous gift of anti-E-cadherin and anti-CD103 Abs. We thank Drs. Thomas Moench, Kevin Whaley, Xiao-Fang Yu, and Larry Zeitlin for thoughtful comments on the manuscript and the experiments included herein.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
R. B. Markham and K. V. Khanna are inventors of a U.S. patent for use of the anti-ICAM-1 Ab as an HIV-1 microbicide.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 R.B.M. was supported by National Institutes of Health Grants AI-55424, AI-45967, and AI-60615, and K.V.K. was supported by National Institutes of Health Training Grants AI-07417 and DA-05972. Results of these studies were presented in part at the 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998, and at the Conference on Retroviruses and Opportunistic Infections, February 2003 and February 2005. Back

2 These authors contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Richard B. Markham, Johns Hopkins Bloomberg School of Public Health, Department of Molecular Microbiology and Immunology, 615 North Wolfe Street, Baltimore, MD 21205. E-mail address: rmarkham{at}jhsph.edu Back

4 Abbreviations used in this paper: HuPBL, human PBL; CHO, Chinese hamster ovary; cRPMI, RPMI 1640 with supplements; HuPBMC, human PBMC; HS, human serum (A/B); LD-PCR, limiting dilution PCR; scFv, single-chain variable fragment; TCID50, 50% tissue culture infectious dose. Back

Received for publication October 20, 2005. Accepted for publication February 13, 2006.


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 Introduction
 Materials and Methods
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