Inhibition of Infection and Transmission of HIV-1 and Lack of Significant

J Antimicrob Chemother 2013; 68: 2026–2037 doi:10.1093/jac/dkt152 Advance Access publication 2 May 2013

Inhibition of infection and transmission of HIV-1 and lack of signiﬁcant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents

Mariya I. Petrova1,2†, Leen Mathys3†, Sarah Lebeer1,2, Sam Noppen3, Els J. M. Van Damme4, Haruo Tanaka5, Yasuhiro Igarashi6, Mario Vaneechoutte7, Jos Vanderleyden1 and Jan Balzarini3* Downloaded from https://academic.oup.com/jac/article/68/9/2026/782524 by guest on 27 September 2021

1Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, bus 2460, B-3001 Leuven, Belgium; 2Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium; 3Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium; 4Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, B-9000 Ghent, Belgium; 5Faculty of Pharmacy and College of Science and Engineering, Iwaki Meisei University, Iwaki, Fukushima 970-8551, Japan; 6Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan; 7Laboratory of Bacteriology Research, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium

*Corresponding author. Tel: +32-16-337367; Fax: +32-16-337340; E-mail: [email protected] †These authors contributed equally to this work.

Received 12 November 2012; returned 4 February 2013; revised 13 March 2013; accepted 25 March 2013

Objectives: A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. Methods: Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. Results: The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV- 1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. Conclusions: The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.

Keywords: adhesion, HIV/AIDS, antimicrobial agents, antiretroviral therapy, bacterial bioﬁlms, lactic acid bacteria, microbicides

Introduction will result in the exposure of previously hidden immunogenic epi- Carbohydrate-binding agents (CBAs) comprise a broad and struc- topes to the immune system.1 A variety of CBAs derived from turally diverse functional class of agents, which may become the several organisms other than mammals have been described to first chemotherapeutics with a dual mechanism of antiviral be endowed with anti-HIV activity (for an overview, see François action: first, through direct antiviral activity by binding the and Balzarini2). Such CBAs include plant, invertebrate and prokary- glycans of the virus envelope and blocking virus entry, and otic lectins. The CBAs may qualify as potential microbicidal agents second, through indirect antiviral action by exerting pressure on since several among them have the intrinsic potential not only to the virus to select for deletions in the envelope glycan shield that inhibit virus infection and interaction between (gp120-expressing)

2026 Lack of toxicity of anti-HIV CBAs against vaginal lactobacilli JAC virus-infected cells and uninfected cells, but also to prevent the Test compounds used in this study capture of virus particles by dendritic cell-specific intercellular ad- The CBAs used in the study are shown in Table 1 together with their carbo- hesion molecule-3 grabbing non-integrin (DC-SIGN)-expressing hydrate specificities.11 – 16 The plant lectins derived from Galanthus nivalis + cells and subsequent transmission to uninfected CD4 T lympho- (GNA), Hippeastrum hybrid (HHA) and Urtica dioica agglutinin (UDA) were 1,2 cytes. However, when introducing these molecules as microbi- kindly provided by E. J. M. Van Damme (Ghent, Belgium).17,18 Actinohivin cides in complex hosts for prevention and/or therapy of viral (AH) prepared from a cultured broth of an actinomycete, Longispora infections, the CBAs will also come into contact with the normal albida K97-0003T was provided by H. Tanaka (Fukushima, Japan).19 Pradi- microbiota, which are important for the health of the host and micin A (PRM-A)20 and pradimicin S (PRM-S)21,22 were obtained from Profes- which should preferentially not be affected by the CBAs. Lactoba- sor T. Oki and Professor Y. Igarashi (Toyama, Japan). The monoclonal cilli are important beneficial members of the human vaginal and antibody 2G12 was purchased from Polymun Scientific (Vienna, Austria). gastrointestinal microbiota.3 It has been previously reported that lactobacilli have various carbohydrate-containing macromole- Downloaded from https://academic.oup.com/jac/article/68/9/2026/782524 by guest on 27 September 2021 4 –6 Viruses cules on their surface that might be possible targets for CBAs. The aim of this study was to evaluate and confirm the potential HIV-1NL4.3 was producedbythe simultaneoustransfection of HEK 293Tcells of several distinct CBAs to inhibit both infection and transmission of with a DNA construct encoding recombinant gp160 and XbaI-digested pNL4.3_DEnv_eGFP, which led to homologous recombination, thereby HIV in cell culture, and to investigate whether CBAs with promising 23 antiviral potential affect the growth, survival and adhesion cap- inserting the gp160 gene in the pNL4.3_eGFP backbone. As a result, acity of a broad variety of relevant (human) Lactobacillus strains. infected cells express enhanced green fluorescent protein (eGFP). The construct pNL4.3_DEnv_eGFP was kindly provided by Dr M. E. Quinõnes-Mateu This study is therefore focusing on vaginal Lactobacillus species, (Lerner Research Institute, Cleveland, OH, USA).24 The produced wild-type since the vagina is the most important port of entry for HIV and virus was used to infect U87.CD4.CXCR4.CCR5, leading to an increased lactobacilli create a natural protection barrier in the human viral yield. vagina.6 –8 HIV-1IIIB was provided by R. C. Gallo and M. Popovic (Institute of Human Virology, University of Maryland, Baltimore, MD, at that time at the NIH, Materials and methods Bethesda, MD). Cell lines Bacteria Human CD4+ T lymphocytic C8166 and SupT1 cells were obtained from The bacterial strains, their origin and reference/source are shown in the ATCC (Manassas, VA, USA). HIV-1 -persistently infected Hut-78 cells IIIB Table 2.25 – 35 (Hut-78/HIV-1) were obtained after exposure of Hut-78 cells to wild-type

HIV-1IIIB for 3–4 weeks. Human B-lymphocytic Raji/DC-SIGN cells, expressing DC-SIGN, were constructed by Geijtenbeek et al.9 and kindly provided by Dr L. Burleigh (Institut Pasteur, Paris, France). These cell lines were Antiretrovirus assays grownin RPMI-1640 medium(Invitrogen,Merelbeke, Belgium), supplemen- C8166 cells were grown in fresh culture medium (200 mL) in 96-well plates ted with 10% fetal calf serum (FCS) (Sigma, Bornem, Belgium), 2 mM at a density of 30000 cells/well. The appropriate concentrations of the test L-glutamine and 2% gentamicin (Invitrogen). compounds were added in 5-fold serial dilutions ranging between 20 mg/

Human embryonic kidney cells (HEK 293T) were obtained from the mL and 0.006 mg/mL. For ﬂow cytometric analysis, wild-type HIV-1NL4.3 ATCC. U87.CD4.CCR5.CXCR4 cells were provided by Professor D. Schols was added at viral loads able to result in a 10% infection after 72 h. For (KU Leuven, Belgium) and their construction and characterization have scoring of the cytopathogenic effect (CPE), cells were infected with a viral 10 been described previously. Both cell lines were grown in Dulbecco’s load of 5-fold the CCID50, resulting in abundant giant cell formation after modiﬁed Eagle medium (DMEM) (Invitrogen), supplemented with 10% 72 h in the absence of the compounds.

FCS (Sigma), 75 mM NaHCO3 and 2% gentamicin (Invitrogen). For the After 72 h of incubation at 378C, the cells were either microscopically U87.CD4.CXCR4.CCR5 cells, 1% puromycin (Invitrogen) and 0.02% geneti- examined for giant cell formation (CPE) or fixed in 3% formaldehyde for cin (Invitrogen) were added. flowcytometric analysis. The 50% effective concentration (EC50) wascalcu- Human cervix carcinoma HeLa cells were obtained from the ATCC. The lated asthe concentration of an agent required to suppress viral infection by cells were cultured at 378C in a 5% CO2/95% air atmosphere in DMEM 50%. Mutant HIV-1IIIB strains that had previously been isolated under CBA supplemented with 10% FCS. For the adhesion experiments, HeLa cells escalating selection conditions36,37 were also administered to C8166 cell were used at confluence, i.e. 4 days after seeding. cultures in the presence of a variety of CBA concentrations to determine

Table 1. CBAs used in the study

Lectin Derived from Carbohydrate speciﬁcity

HHA Hippeastrum hybrid agglutinin (plant) a(1,3)-a(1,6)Man11 GNA Galanthus nivalis agglutinin (plant) a(1,3)Man12 AH Longisporum albida high mannose [i.e. a (1,2)Man] glycans13 14 UDA Urtica dioica agglutinin (plant) (GlcNAc)n 2G12 monoclonal antibody against HIV-1 gp120 glycans high-mannose glycans15 PRM-A Actinomadura hibisca high-mannose glycans16 PRM-S Actinomadura spinosa high-mannose glycans16

2027 Petrova et al.

Table 2. Bacterial strains used in the study

Strain Relevant genotype/description Reference/source

Gastrointestinal Lactobacillus strain L. rhamnosus GG (ATCC 53103) wild-type, faecal isolate (25)

Vaginal Lactobacillus strains L. rhamnosus GR-1 ATCC 55826 wild-type, female urethra isolate (26–28)ATCC Lactobacillus reuteri RC-14 ATCC 55845 wild-type, female urethra isolate (27,28)ATCC Lactobacillus crispatus NCIMB 4505 wild-type, human isolate (29) LMG/BCCM

Lactobacillus jensenii ATCC 25258 wild-type, human isolate (30) LMG /BCCM Downloaded from https://academic.oup.com/jac/article/68/9/2026/782524 by guest on 27 September 2021 Lactobacillus salivarius ATCC 11742 wild-type, human isolate (31) LMG /BCCM Lactobacillus gasseri ATCC 33323 wild-type, human isolate (24,30) LMG /BCCM Lactobacillus helveticus LG0301 wild-type, human isolate Mario Vaneechoutte Laboratory Bacteriology Research, Ghent University L. helveticus LG0373 wild-type, human isolate Mario Vaneechoutte Laboratory Bacteriology Research, Ghent University L. plantarum LAB128 wild-type, human isolate Mario Vaneechoutte Laboratory Bacteriology Research, Ghent University L. plantarum LAB129 wild-type, human isolate Mario Vaneechoutte Laboratory Bacteriology Research, Ghent University

Non-vaginal bacteria strains L. plantarum WCFS1 wild-type, human saliva isolate (32) L. lactis ATCC 11454 wild-type, producer of nisin, dietary isolate ATCC E. coli MG1655 wild-type (33) B. subtilis AG174 wild-type (34) L. rhamnosus ATCC 9595 wild-type, dairy isolate (35)ATCC L. rhamnosus ATCC 7469 wild-type, dairy isolate (35)ATCC L. rhamnosus ATCC 10863 wild-type, dairy isolate (35)ATCC Lactobacillus casei ATCC 393 wild-type, cheese isolate ATCC their phenotypic sensitivity to these CBAs. Five-fold serial dilutions of the Transmission of captured virus from Raji/DC-SIGN cells CBAs starting with the following highest concentrations were used: to C8166 cells 50 mg/mL for HHA, GNA, UDA, PRM-A and PRM-S, and 10 mg/mL for 2G12 6 and AH. The 10 Raji/DC-SIGN cells were exposed to wild-type HIV-1NL4.3 particles (25000 pg of p24) for 2 h at 378C in a volume of 1 mL, allowing efﬁcient capture of the virus particles by the Raji/DC-SIGN cells. After thoroughly Co-cultivation assays washing to eliminate unbound virus, the cells were resuspended in 1 mL After persistently washing HIV-1-infected Hut-78/HIV-1 cells to remove of fresh culture medium. Next, 200 mL of the virus-bound Raji/DC-SIGN free virions, 105 Hut-78/HIV-1 cells were added to 105 SupT1 cells in cells was brought into contact with 200 mL of the test compounds in 200 mL wells of a 96-well plate. The appropriate concentrations of the wells of 48-well plates, at appropriate compound concentrations (1, 6.25, test compounds were added at 5-fold serial dilutions ranging between 25 and 125 times the EC50 of the agents). After 30 min of incubation at 5 100 mg/mL and 0.006 mg/mL. After 20 h of incubation at 378C, the cells 378C, 2×10 C8166 cells were added to obtain a total volume of 1 mL. Fol- were microscopically examined for syncytia formation. The EC50 was calcu- lowing an incubation of 72 h, samples were harvested to quantify the lated as the concentration of an agent required to suppress syncytia forma- amount of HIV-1 p24 antigen with a p24 ELISA (PerkinElmer) as a tion by 50%. measure of the viral production resulting from transmission of virions from particle-captured Raji/DC-SIGN cells to C8166 cells.

Capture of free HIV-1NL4.3 particles by Raji/DC-SIGN cells Surface plasmon resonance (SPR) analysis Wild-type HIV-1NL4.3 (25000 pg of p24) was incubated with appropriate concentrations of a test compound (several concentrations tested: 1, SPR was used to evaluate competition between DC-SIGN and AH for binding

6.25, 25 and 125 times the EC50 according to the cell-free virus infection to recombinant HIV-1IIIB gp120. Therefore DC-SIGN was covalently immo- assays) for 30 min at 378C in a volume of 500 mL. Next, 106 Raji/DC-SIGN bilized on a CM5 sensor chip in 10 mM sodium acetate, pH 5.5, using stand- cells were added to obtain a total volume of 1 mL. After incubation for ard amine coupling chemistry, resulting in a chip density of 1830 RU. 2 h at 378C, the cells were washed thoroughly to eliminate unbound A reference flow cell was used as a control for non-specific binding and virions. The cells were then resuspended in 1 mL of fresh culture medium, refractive index changes. All interactions were performed at 258Cona after which the concentration of the viral antigen p24 was quantified by a Biacore T200 instrument (GE Healthcare, Uppsala, Sweden). The analytes, p24 ELISA (PerkinElmer, Boston, MA, USA), to determine the amount of 2 nM of gp120 alone or premixed with AH (at different concentrations), virus particles captured by the Raji/DC-SIGN cells. were diluted in HBS-P (10 mM HEPES, 150 mM NaCl and 0.05% surfactant

2028 Lack of toxicity of anti-HIV CBAs against vaginal lactobacilli JAC

P20, pH 7.4) supplemented with 10 mM CaCl2. Samples were injected for shaking for 3 and 24 h at 378Cor308C. Subsequently, bacterial dilution 2 min at a flow rate of 30 mL/min and followed by a dissociation phase of series were plated out to determine the capacity of the Lactobacillus 2 min. The CM5 sensor chip surface was regenerated with a single injection strains to survive in the presence of the different CBAs. The effect of the of 20 mM NaOH. CBAs on bacterial viability was calculated as a percentage by comparing the survival percentage of the bacterial strain without adding the CBAs and the survival percentage after incubation with the CBAs. Each of the bac- Flow cytometric analysis of eGFP-encoding HIV-1-infected terial strains and the CBAs were examined in three replicates and each ex- C8166 cell cultures periment was independently performed at least three times. Concanavalin A (ConA) at a concentration of 100 mg/mL was also included in this experi- The number of HIV-1NL4.3-infected C8166 cells was quantified by measur- ment as a control. ing the eGFP expression upon infection with eGFP-encoding HIV-1NL4.3. For this purpose, a FACS CantoII flow cytometer (Becton Dickinson, San

Jose, CA, USA) was used. The data were analysed by FACS Diva Software Downloaded from https://academic.oup.com/jac/article/68/9/2026/782524 by guest on 27 September 2021 (Becton Dickinson). Adhesion assays In order to investigate whether the CBAs affect the adhesion capacity of Growth conditions of the bacterial strains lactobacilli to cervical epithelium, HeLa cells were used. The in vitro adhe- 5 All Lactobacillus strains were routinely grown non-shaken in Man sion assayusing cervical HeLacells was performed as previously described, Rogosa Sharpe (MRS) medium (Difco, Becton Dickinson)31 at 378Cor308C with some modifications. Two different types of experiments were per- depending of the strain. Escherichia coli and Bacillus subtilis were grown in formed. The first set of adhesion experiments aimed to investigate the ad- Luria–Bertani (LB) medium (BioLab New England) with aeration at 378C. hesion blocking capacity of the different CBAs by pre-incubation of the HeLa Lactococcus lactis was grown in M17 medium (Becton Dickinson), cells with the CBAs. Monolayers of HeLa cells in 12-well plates were washed supplemented with 0.5% glucose. For some analyses, DMEM (Invitrogen) twice with pre-warmed PBS and subsequently incubated for 30 min with m supplemented with 2% or 0.2% glucose was used. different CBAs. ConA at a concentration of 100 g/mL was also used. To remove the unbound CBAs, the HeLa cell cultures were washed twice with pre-warmed PBS and subsequently incubated for 1 h with 1.5 mL of 107 Growth and survival assay of lactobacilli in the presence bacterial cells at 378C. Then the epithelial cell cultures were washed twice of CBAs with pre-warmed PBS. Subsequently 100 mL of trypsin-EDTA (Invitrogen) was added to each well and incubated for 10 min at 378C. Finally, 900 mL Two different assays were used to investigate whether the CBAs affect the of PBS was added and mixed, and serial dilutions were plated out. Plates growth and survival capacity of different Lactobacillus strains. were incubated at 378C for 72 h. The adhesion ratio (percentage) was cal- In the first set of experiments, the capacity of different Lactobacillus culated by comparing the number of adherent cells with the cell number of strains to grow in the presence of various CBAs was determined. Other the added bacterial suspension (107 cfu/mL). The effect of the CBAs on ad- bacterial species such as L. lactis, E. coli and B. subtilis were also included hesion was determined by comparing the adhesion percentage of the bac- as controls. Lactobacillus strains grown overnight to the stationary phase terial strain without adding the CBAs (positive control) and the adhesion [optical density at 600 nm (OD600) 1.8–2.0] were used as inocula for percentage after incubation with the CBAs. Each bacterial strain and CBA the assay. The assay was performed in 96-well microtitre plates. Sterile combination was evaluated in three replicate assays and each experiment wells were supplied with 200 mL of medium (MRS for all Lactobacillus was independently performed at least twice. strains, LB for E. coli and B. subtilis and M17 for L. lactis). Approximately In the second set of adhesion experiments, the CBAs were first incu- × 7 0.5 10 cfu of the bacterial strains (1/100 of an overnight culture) were bated with the vaginal Lactobacillus strains. Therefore the bacterial suspen- added. Five-fold dilution series of different concentrations of CBAs were sion at a concentration of 107 cfu/mLwas centrifuged at 2000 g for 10 min added (i.e. for HHA, GNA, AH and PRM-A, concentrations ranging from at 48C, washed twice with PBS and incubated for 30 min with the different 0.016 to 250 mg/mL were used; for UDA, concentrations ranging from CBAs. ConA at a concentration of 100 mg/mL was also used as a positive 0.04 to 125 mg/mL were used; and for PRM-S, concentrations ranging control. Subsequently the bacterial strains were incubated for 1 h with from 0.16 to 500 mg/mL were used). The plates were incubated without the confluent HeLa cell cultures. The effect of the CBAs on adhesion was shaking for 24 h at 378Cor308C, depending on the optimal temperature determined by comparing the adhesion number of the bacteria to the for growth of the individual bacterial strains. The OD600 was measured HeLa cell cultures with and without CBAs. Each bacterial strain and CBA after 24 h. As a negative control, each strain was grown in medium combination was evaluated in three replicate assays and each experiment without CBAs. As a positive control, the antimicrobial cationic peptide was independently performed at least twice. LL-37 was used.6 Alternatively, the activity of the CBAs was evaluated in chemically defined DMEM supplemented with 2% glucose. Each bacterial strain and each CBA was tested in duplicate. Each experiment was independently performed at least three times. Results In a second set of experiments, each CBAwas used at a concentration of 10 mg/mL, except for UDA and PRM-S, for which 25 mg/mL and 100 mg/mL Inhibition of HIV-1NL4.3 by CBAs in C8166 cell cultures were used, respectively. The chosen concentrations are much higher than The CBAs were exposed to human CD4+ T lymphocyte C8166 cul- their observed EC50 for HIV-1 inhibition (e.g. for HHA, 175 times, for GNA tures in the presence of cell-free HIV-1NL4.3 and analysis of their in- 243 times, for UDA and AH 140 times, for PRM-A 2.2 times and for PRM-S hibitory effect was based on either microscopic evaluation of giant 24 times the EC ). Lactobacillus strains grown to stationary phase (OD 50 600 cell formation (CPE) or on the flow cytometric quantification of 1.8–2.0, corresponding to approximately 107 cfu/mL cells) were used as inocula for the assays. The bacterial cultures were centrifuged for eGFP-expressing cells. This is possible because the eGFP gene is 10 min at 2000 g and washed once with PBS. After centrifugation, the inserted in the RNA genome of HIV-1NL4.3 and expressed in pro- cells were resuspended in 1 mL of DMEM, supplemented with 0.2% ductively HIV-1NL4.3-infected cells. Both the cytometric quantifica- glucose and containing the different CBAs. As a control, each bacterial tion assay and the microscopic CPE scoring assay gave very similar strain was resuspended in 1 mL of DMEM supplemented with 0.2% results. The resulting antiviral activities (EC50 values) of the com- glucose without adding CBAs. The tubes were then incubated without pounds are listed in Table 3.

2029 Petrova et al.

All CBAs markedly inhibited the HIV-1 infection and resulting Inhibitory effect of the CBAs on the ability of virus-induced cytopathogenesis. The mannose-specific CBAs, DC-SIGN-expressing cells to capture HIV-1 particles HHA, GNA and AH had comparable EC values (0.02–0.07 mg/ 50 Since the CBAs can efficiently block cell-free virus infection of T mL). The (GlcNAc) -specific UDA had a somewhat higher EC n 50 lymphocyte cell cultures, as well as the syncytium formation (0.17–0.22 mg/mL). The monoclonal high-mannose glycan- between persistently HIV-1-infected T lymphocytes and uninfect- recognizing antibody 2G12 showed an EC value of 0.65– 50 ed T lymphocytes, they may qualify as potential microbicide drug 0.97 mg/mL. The mannose-specific non-peptidic PRM-A and candidates. Therefore it was of interest to investigate their effect PRM-S antibiotics were endowed with the lowest antiviral activity, on HIV-1 capture by DC-SIGN-expressing cells since such cells are with EC s in the lower microgram/mL range. 50 thought to play an important role in HIV transmission. We made Based on microscopic examination of the cells, none of the use of human DC-SIGN-expressing Raji cells since this was previ- agents resulted in cytotoxicityat the highest tested concentrations ously shown to be a relevant cell model to investigate virus Downloaded from https://academic.oup.com/jac/article/68/9/2026/782524 by guest on 27 September 2021 (i.e. 50 mg/mL for PRM-A, PRM-S, 2G12; 20 mg/mL for HHA, UDA, AH; capture and transmission to uninfected CD4+ T lymphocytes.1,9 and 4 mg/mL for GNA). Raji/DC-SIGN cells were given the opportunity to capture HIV-1NL4.3 through DC-SIGN interaction with gp120 in the Prevention of syncytium formation between uninfected absence or presence of different concentrations of CBAs. The CD41 T lymphocytes and HIV-1-infected CD41 amount of captured virus on the Raji/DC-SIGN cells was quantified T lymphocytes by CBAs by HIV-1 antigen p24 ELISA and the results obtained in the presence of CBAs were then compared with the virus capture results The CBAs were evaluated for inhibition of syncytium formation in obtained in the absence of any compound (Figure 1). The CBAs co-cultures of persistently HIV-1-infected Hut-78/HIV-1IIIB cells dose-dependently prevented virus capture by the Raji/DC-SIGN and uninfected SupT1 cells. The control co-cultures show abun- cells. The mannose-specific HHA, GNA, AH and 2G12 CBAs were dant syncytium formation within 24–30 h after co-cultivation able to suppress virus capture by 50%–70% at the highest concen- due to the persistent expression of HIV-1 gp120 on the cell mem- trations tested (125 times EC50 for HHA, GNA and AH, and 25 times brane of the HUT-78/HIV-1 cells and the presence of CD4 on the un- EC50 for 2G12). The highest concentration of UDA (125 times its infected SupT1 cells. The EC50 values are shown in Table 4. All CBAs EC50) resulted in a decrease of capture efficiency by 90%. The efficiently prevented giant cell formation in the co-cultures. most remarkable observations were obtained for PRM-A and Whereas HHA, GNA, UDAwere 5- to 10-fold less effective compared PRM-S, which were able to substantially (85%–90%) inhibit with their ability to prevent cell-free virus infection in the virus capture at concentrations that were only 25-fold their EC50. virus-exposed C8166 cell cultures, the monoclonal antibody 2G12 was 3-fold less inhibitory and AH and the non-peptidic pradimicins kept an equal inhibitory potential in both the co-culture Inhibitory action of CBAs against the transmission syncytium formation (Table 4) and the virus-infection (Table 3) of Raji/DC-SIGN-captured HIV-1 particles to assays. uninfected C8166 cells Virus-bound Raji/DC-SIGN cells were co-cultured with C8166 T lym- Table 3. Anti-HIV activities of CBAs in C8166 cell cultures phocytes in the presence of various concentrations of CBAs. After a co-cultivation period of 72 h at 378C, samples were harvested and EC50 based on flow cytometric EC50 based on CPE microscopic analysed with a p24 ELISA assay to quantify the virions in the cell CBA analysis (mg/mL) scoring (mg/mL) cultures. The results obtained for transmission in the presence of CBAs were compared with the results obtained in the absence of HHA 0.057+0.023 0.046+0.015 the compounds (Figure 2). GNA 0.041+0.009 0.045+0.001 For HHA, GNA, AH and UDA, a decrease of 50% of transmission AH 0.070+0.015 0.017+0.004 efficiency was observed at the lowest tested concentration UDA 0.173+0.002 0.216+0.035 (1 times the EC50 as previously obtained in the cell-free virus 2G12 0.969+0.166 0.649+0.072 assay). PRM-A, PRM-S and 2G12 were even more potent in inhibiting PRM-A 4.4+0.4 3.9+0.7 virus transmission, since the lowest tested concentration (1 times PRM-S 4.1+0.6 4.5+0.0 the EC50) suppressed virus transmission at 75%–.95%. When higher concentrations of the CBAs were used, an almost complete Data are the means+SD of at least two to three independent experiments. annihilation of transmission could be achieved (Figure 2).

Table 4. Inhibition of giant cell formation in HUT-78/HIV-1 and SupT1 co-cultures by CBAs

EC50 (mg/mL)

HHA GNA AH UDA 2G12 PRM-A PRM-S

0.271+0.179 0.464+0.308 0.070+0.078 1.1+0.5 2.6+3.2 3.7+0.3 4.0+0.4

Data are the means+SD of at least two to three independent experiments.

2030 Lack of toxicity of anti-HIV CBAs against vaginal lactobacilli JAC

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Capture efﬁciency (percentage of control) of (percentage efﬁciency Capture 0

AH 1x HHA 1x GNA 1x AH 25x UDA 1x HHA 25x GNA 25x AH 125x UDA 25x 2G12 1x HHA 125x GNA 125x AH 6.25x UDA 125x 2G12 25xPRM-A 1x PRM-S 1x HHA 6.25x GNA 6.25x UDA 6.25x 2G12 6.25x PRM-A 25x PRM-S 25x PRM-A 6.25x PRM-S 6.25x No compound Compound concentration (fold EC50)

Figure 1. Virus capture by DC-SIGN-expressing cells in the presence of CBAs. DC-SIGN-expressing Raji cells were exposed to HIV-1 in the presence of different CBA concentrations. HIV-1 capture efﬁciency by Raji/DC-SIGN cells was measured by p24 antigen ELISA. The CBA concentrations were chosen to represent x-fold their EC50 values as reported in Table 1 for each CBA.

120

100

Virus transmission (percentage of control) (percentage Virus transmission 0

Figure 2. HIV-1NL4.3 transmission from virus-captured Raji/DC-SIGN cells to C8166 cells. Raji/DC-SIGN cell cultures containing captured HIV-1 particles through binding of HIV-1 gp120 with DC-SIGN were co-cultured with C8166 cells in the presence of CBAs. Virus production in the cell cultures was determined after 72 h by p24 antigen ELISA.

when the a1,2-mannose-speciﬁc AH was added to the gp120 so- Effect of CBAs on the interaction of gp120 with DC-SIGN lution prior to exposure to DC-SIGN, the binding amplitude of It is known that DC-SIGN preferentially recognizes a1,2-mannose gp120 to DC-SIGN was dose-dependently decreased from 22 RU oligomers present in high-mannose N-glycans.38 The interaction to 17.5 RU when 0.1 mM AH was present and to 10 RU when of gp120 with DC-SIGN was therefore examined by SPR technology. 0.5 mM AH was present. A higher concentration of AH did not DC-SIGN was covalently bound to the sensor chip (at 1830 RU). further decrease the gp120 binding to DC-SIGN. Thus it could be When 2 nM HIV-1 gp120 was exposed to DC-SIGN for 120 s, a concluded that AH decreased the binding of gp120 to DC-SIGN pre- binding amplitude of 22 RU was obtained. AH did not bind to sumably by competing for the same binding sites on gp120 as DC-SIGN under identical experimental conditions. However, DC-SIGN does.

2031 Petrova et al.

Table 5. Fold resistance of CBAs against mutant HIV-1 strains in C8166 In another set of experiments, the survival of the bacterial cell cultures strains was investigated by brieﬂy exposing the bacteria to CBAs. After 3 h of incubation of the bacterial cells with different CBAs in

Fold resistance (EC50 mutant HIV-1/EC50 wild-type HIV-1) DMEM supplemented with 0.2% glucose, no significant difference in the survival capacity of the variety of vaginal and non-vaginal CBA HIV-1/HHA-500a HIV-1/PRM-S-100b (shown in Table 1) Lactobacillus strains could be observed compared with the survival of the bacteria without adding the HHA .877 .877 lectins (not shown). In addition, the survival of E. coli, B. subtilis GNA .1219 .1219 and L. lactis was also not significantly affected (results not AH .143 .143 shown). Similar results were also obtained for the bacterial UDA 191 289 strains after 24 h of CBA exposure (Figure 3). In addition, the 2G12 .10 .10 effect of 100 mg/mL ConA was evaluated since it has been Downloaded from https://academic.oup.com/jac/article/68/9/2026/782524 by guest on 27 September 2021 PRM-A .11 .11 previously shown to recognize mannose/glucose residues at the PRM-S ≥12 12 cell surface of L. rhamnosus GG and other lactobacilli.4 However, no significant difference in the survival capacity of the different aMutant HIV-1/HHA-500 strain36 containing the following N-glycosylation Lactobacillus strains after incubation with ConA was observed site mutations in gp120: 230NKM232, 234NGI236, 276NFI278, (data not shown). 295NCI297, 332NIN334, 339YNT341 and 392DST394/392NSI394. bMutant HIV-1/PRM-S-100 strain37 containing the following N-glycosylation site mutations in gp120: 88NVI90, 230NKM232, 289NQF291, 295NCI297, Effect of CBAs on the adhesion capacity of vaginal 339NNI341 and 392DST394. Lactobacillus strains An important characteristic of vaginal Lactobacillus strains is their capacity to adhere to the urogenital epithelium. Therefore CBAs Inhibitory action of CBAs against HIV-1 strains selected in with activity against HIV—or new microbicides in general— the presence of high concentrations of HHA and PRM-S should not affect the adhesion capacity of the normal vaginal microbiota. In our experiments, adhesion to cervical epithelial Previously, HIV-1 strains have been exposed to escalating CBA IIIB HeLa cell cultures was therefore assessed. concentrations over time and several virus isolates have been geno- In a first set of experiments, the CBAs were briefly (30 min) typically characterized.37,38 We used an HHA- and a PRM-S-exposed exposed to the HeLa cell cultures, after which the bacteria were virus strain for phenotypical analysis against CBAs in virus-infected added. Figure 4 shows that the CBAs did not specifically affect C8166 cell cultures. The mutant virus strains contained a series of the adhesion capacity of the vaginal bacterial strains and L. rham- N-glycosylation site deletions in their gp120 envelope glycoprotein nosus GG after pre-incubation of the HeLa cells with the CBAs. (Table 5). The different CBAs invariably lost marked inhibitory In a second set of experiments, the CBAs were briefly (30 min) potency against the two mutant virus strains, which thus displayed exposed to the bacterial cells before the bacteria were added to a high degree of cross-resistance to the CBAs. the HeLa cell cultures. Again, no significant difference in the adhesion capacity of the vaginal (Figure 5) bacteria was observed after Effect of CBAs on the growth and survival capacity of (pre-) incubation of the bacterial strains with the different CBAs, vaginal Lactobacillus strains except for Lactobacillus plantarum LAB129. A significant decrease in the adhesion capacity of L. plantarum LAB129 was observed All bacterial strains showed the ability to grow (24 h assay) in the after incubation with CBAs recognizing a(1,2)Man and GlcNAc. presence of the different CBAs added to their growth medium (MRS for Lactobacillus strains, LB for E. coli and Bacillus subtilis and M17 for L. lactis). In these assays, the CBA concentrations Discussion (125–500 mg/mL) by far exceeded their 50% antivirally effective concentrations. However, MRS medium is a nutrient-rich culture In the last decade, the effect of CBAs against viral pathogens, and medium containing many components such as mannan- more precisely HIV, has been widely studied and reported.2 In add- containing yeast extract, which could have interacted with the ition, several studies indicated that well-defined CBAs such as the CBAs and inactivated them. Therefore MRS was replaced by a non-peptidic antibiotics PRM-S and PRM-A showed activity chemically defined medium, i.e. DMEM, which was solely supple- against a wide variety of fungi and yeasts in vitro21,39 and were mented with 2% glucose to provide the necessary carbohydrate demonstrated to have in vivo efficacy against systemic Candida source for the Lactobacillus strains. Again, growth of the vaginal albicans infections in mice.21 However, the effect of anti-HIV and and non-vaginally isolated strains (shown in Table 1) in DMEM antifungal CBAs against bacterial species that are part of the with 2% glucose in the presence of different CBAs was not signifi- gastrointestinal and urogenital microbiota is poorly known. In cantly affected (results not shown). Cathelicidin LL-37 was used this study, we therefore not only investigated the effect of a selec- as a positive control because it had been previously shown to tion of CBAs on the infection/transmission of HIV-1, but also on the affect the growth of Lactobacillus rhamnosus GG and other lacto- integrity and functionality of a broad variety of Lactobacillus strains bacilli.6 As expected, LL-37 at a concentration of 3 mg/mL affected with a focus on vaginal isolates. the growth of the tested strains, as exemplified by a significant There are at least four events in HIV infection and transmission (30%–60%) reduction in optical density measurements (data where the use of a microbicide drug may be advisable. Seminal not shown). fluid of virus-infected individuals contains both cell-free virus

2032 Lack of toxicity of anti-HIV CBAs against vaginal lactobacilli JAC

(a) (b) 160 HHA-10 µg/mL GNA-10 µg/mL 140 AH-10µg/mL UDA-25µg/mL 140 120 120 100 100 80 80 60 60 40 40 Percentage survival Percentage Percentage survival Percentage 20 20

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LGG LGG LGR-1 LGR-1

L. reuteri RC-14 L. reuteri RC-14 L. crispatus 4505 L. jensenii 25258L. gasseriL. plantarum33323L. plantarum 128 129 L. crispatus 4505 L. jensenii 25258L. gasseriL. plantarum33323L. plantarum 128 129 L. salivarius 11742 L. helveticusL. helveticus 0301 0373 L. salivarius 11742 L. helveticusL. helveticus 0301 0373

Percentage survival Percentage 20 0

LGG LGR-1

L. reuteri RC-14 L. crispatus 4505 L. jensenii 25258L. gasseriL. plantarum33323L. plantarum 128 129 L. salivarius 11742 L. helveticusL. helveticus 0301 0373

Figure 3. Survival of L. rhamnosus GG and different vaginal Lactobacillus strains after incubation with CBAs in DMEM with 0.2% glucose for 24 h. (a) CBAs (HHA and GNA) with specificity for a(1,3)Man ora(1,3)/a(1,6)/Man. (b) CBA (AH) with specificity for high-mannose glycans and CBA (UDA) with specificity for

(GlcNAc)n. (c) Non-peptidic CBAs (PRM-S and PRM-A) with specificity for high-mannose glycans, especially a(1,2)Man oligomers. particles and virus-infected cells that express gp120 on their mem- intravaginal cream or gel formulation. In fact, several of the CBAs brane and are a source of cell-associated hidden virus particles.40 It included in this study have previously been demonstrated to effi- is therefore important that a microbicide drug candidate not only ciently inhibit—with the exception of monoclonal antibody prevents infection of cells by cell-free virus particles but also 2G12—infection of peripheral blood mononuclear cells by a wide close contact of virus-infected cells with uninfected cells. In variety of HIV-1 clade strains [i.e. clade A (UG273), clade B (US2), addition, it is well known that dendritic cells, Langerhans cells clade C (ETH2220), clade D (UG270), clade A/E (ID12), clade F and monocyte/macrophages are among the first cells that en- (BZ163), clade G (BCF-DIOUM) and group O (DCF06)].37,41 – 43 counter the virus upon HIVexposure and transmission.40 Therefore However, it is of crucial importance that vaginally administered microbicide drug candidates should also be investigated for their microbicide drugs do not significantly affect the vaginal micro- interaction with the DC-SIGN-driven virus capture and transmis- biota. The intravaginal ecosystem predominantly consists of a sion event. CBAs represent the only agents that seem to be able variety of Lactobacillus strains that create a defensive shield to efficiently and concomitantly interact with these four different against invading pathogens, including viruses.44 – 47The lactobacilli events, and thus they may qualify as potential microbicide lead not only produce H2O2, but also lactic acid, thereby establishing an agents for further (pre-) clinical investigations.1 It is interesting acidic environment unfavourable for bacterial, fungal and viral to observe that CBAs with predominant a1,2-, a1,3- and pathogens. The vaginal microbiota might sometimes be domi- a1,6-mannose and GlcNAc glycan specificities are able to interact nated by some non-lactobacilli species, although the isolated with these four important processes of virus infection, capture and strains are often associated with bacterial vaginosis and detected transmission (1,2 and data in this study). Depending on the nature in rather rare cases. Their role in the healthy vaginal ecosystem is of the CBA, their antiviral activity ranged in the nanomolar to the under discussion. Therefore the most important species in the lower micromolar range, i.e. at concentrations that can be easily vaginal ecosystem are lactobacilli, and any therapeutic interven- obtained in the clinical setting when locally administered as an tion that negatively affects their physiological function may

2033 Petrova et al.

(a) (b) 140 HHA-10µg/mL GNA-10 µg/mL 140 AH-10 µg/mL UDA-25 µg/mL 120 120 100 100 80 80 60 60 40 40 Percentage adhesion Percentage 20 adhesion Percentage 20

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LGG LGG LGR-1 LGR-1

Percentage adhesion Percentage 20 0

LGG LGR-1

L. reuteri RC-14 L. crispatus 4505 L. jensenii 25258L. gasseriL. 33323plantarumL. plantarum 128 129 L. salivarius 11742 L. helveticusL. helveticus 0301 0373

Figure 4. Adhesion capacityof vaginal lactobacilli after treatment of HeLa cells with CBAs. The results are expressed as the percentage of the bacterial cells adhering to HeLa cells versus without CBAs. (a) CBAs (HHA and GNA) with specificity for a(1,3)Man or a(1,3)/a(1,6)/Man. (b) CBA (AH) with specificity for a(1,2)Man and CBA (UDA) with specificity for GlcNAc. (c) Non-peptidic CBAs (PRM-S and PRM-A) with specificity for a(1,2)Man. compromise the integrity of the protective intravaginal barrier. It is hazardous interaction with a variety of commensal bacteria, but important that none of the CBAs in this study measurably inter- it would be advisable that any potential clinical CBA candidate be fered with the three important physiological functions of the lacto- evaluated against as broad a variety of vaginal commensals as bacilli that are of crucial importance to maintaining the possible. homeostasis of the vaginal environment: growth, viability and ad- The CBAs are assumed to bind to the carbohydrate content of hesion ability. The lack of interaction with these bacteria was the HIV envelope to exert their inhibitory activity against HIV. In observed at CBA concentrations that far exceeded their ability to fact, HIV-1 strains exposed to increasing CBA concentrations inhibit virus infection, DC-SIGN-mediated capture and virus trans- have previously been shown to select for mutant virus strains mission. Although the lactobacilli represent the most important that contain a variety of N-glycosylation site mutations (glycan commensal microorganisms in the vaginal environment, it might deletions) in HIV-1 gp120. This has been demonstrated for be necessary to ensure that candidate microbicide CBAs do not ad- HHA,36 GNA,36 UDA,42 2G12,50 AH43 and the pradimicins A and versely interfere with other bacteria as well. It has been reported S.37,51 Not unexpectedly, mutant HIV-1 strains containing dele- that pradimicins do not interact with the growth of other bacterial tions in their N-glycosylation motifs in gp120 show phenotypic re- species such as intestinal B. subtilis and E. coli.37 It has also been sistance to CBAs. The degree of this drug resistance highly depends show that the potent anti-HIV prokaryotic lectin cyanovirin does on thenumberof N-glycandeletionsin HIV-1gp120, thelocationof not inhibit the Gram-positive commensal of the oral cavity Strepto- the deleted glycan in gp120 and the nature of the particular coccus gordonii48 and that AH does not affect the growth of the CBA.36,37,42,43,50,51 When the CBAs were evaluated for their inhibi- Gram-negative E. coli.49 In this study we were also able to show tory activity in C8166 cell cultures exposed to mutant HIV-1 strains that a variety of CBAs did not affect the growth of E. coli, B. subtilis that contained seven (HIV-1/HHA-500) or six (HIV-1/PRM-S-100) and L. lactis. Thus some CBAs may be safe in terms of their non- N-glycosylation site deletions in their gp120, they showed poor, if

2034 Lack of toxicity of anti-HIV CBAs against vaginal lactobacilli JAC

(b) (a) HHA-10 µg/mL GNA-10 µg/mL AH-10 µg/mL UDA-25 µg/mL 140 140 120 120 100 100 80 80 60 60 40 40 Percentage adhesion Percentage Percentage adhesion Percentage 20 20

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LGG LGG LGR-1 LGR-1

L. reuteri RC-14 L. reuteri RC-14 L. gasseri 33323 L. crispatus 4505 L. jensenii 25258L. gasseriL. plantarum33323L. plantarum 128 129 L. crispatus 4505 L. jensenii 25258 L. plantarumL. plantarum 128 129 L. salivarius 11742 L. helveticusL. helveticus 0301 0373 L. salivarius 11742 L. helveticusL. helveticus 0301 0373

Percentage adhesion Percentage 20

LGG LGR-1

L. reuteri RC-14 L. crispatus 4505 L. jensenii 25258L. gasseriL. plantarum33323L. plantarum 128 129 L. salivarius 11742 L. helveticusL. helveticus 0301 0373

Figure 5. Adhesion capacity of vaginal lactobacilli after treatment of the bacterial cells with CBAs. The results are expressed as the percentage of CBA-exposed bacterial cells adhering to HeLa cells versus CBA-non-exposed cells. (a) CBAs (HHA and GNA) with specificity for a(1,3)Man or a(1,3)/ a(1,6)/Man. (b) CBA (AH) with specificity for high-mannose glycans and CBA (UDA) with specificity for (GlcNAc)n. (c) Non-peptidic CBAs (PRM-S and PRM-A) with specificity for high-mannose glycans, especially a(1,2)Man oligomers. any, inhibitory effect (Table 5). These findings strongly point again In conclusion, our results show that various CBAs are active to a specific interaction of CBAs with N-glycans of gp120 to produce against HIV in terms of prevention of viral infection as shown by their antiviral activity. a pronounced inhibition of infection of CD4+ T lymphocytes bycell- It is interesting to note that CBAs are able to block fourdifferent free virus particles and efficient prevention of syncytia formation aspects of HIV-1 infection and transmission. Inhibition of cell-free between persistently virus-infected cells and uninfected CD4+ T virus infection and syncytium formation between persistently cells. They also proved active against viral transmission as shown virus-infected cells and uninfected cells can be explained by the by efficient prevention of DC-SIGN-directed virus capture and sub- binding of CBAs to the gp120 of the virus particle and the sequent transmission to uninfected CD4+ T lymphocytes. Import- virus-infected cells, and its subsequent prevention of conform- antly, these CBAs do not affect the growth and survival capacity of ational changes of CBA-bound gp120 necessary to further the a broad varietyof differentLactobacillusstrains. Moreover,the eval- virus entry/fusion process.1 The inhibitory effect of CBAs on virus uated CBAs also do not markedly affect the adhesion capacity of capture by DC-SIGN-expressing cells can be explained by the these bacterial strains. Our results add further impetus to the po- binding of CBAs to the glycans of the HIV-1 envelope particles, tential of CBAs to act as new microbicidal agents without pro- preventing the C-type lectin DC-SIGN to efficiently interact with nounced toxicity against the vaginal microbiota. the gp120 glycans. This was demonstrated by our SPR-based experiments that showed a dose-dependent lesser binding of gp120 to DC-SIGN in the presence of the a1,2-mannose Acknowledgements oligomer-specific AH. Thus DC-SIGN has to compete with CBAs We are grateful to Tine L. A. Verhoeven and Leen Ingels for excellent for HIV binding and subsequent transmission to uninfected technical assistance. We thank Christiane Callebaut for dedicated + CD4 T lymphocytes. editorial assistance.

2035 Petrova et al.

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