JAC Advance Access originally published online on February 12, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):818-826; doi:10.1093/jac/dkn042
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Original research |
CD4 mimetic miniproteins: potent anti-HIV compounds with promising activity as microbicides
1 Virology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium 2 CEA, iBiTecS, Service d’Ingénierie Moléculaire des Protéines, Gif sur Yvette F-91191, France 3 Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
* Correspondence address. Virology Unit, Institute of Tropical Medicine, 155 Nationalestraat, B-2000 Antwerp, Belgium. Tel: +32-3-247-62-28; Fax: +32-3-247-63-33; E-mail: gvanham{at}itg.be
Received 19 October 2007; returned 3 December 2007; revised 28 December 2007; accepted 16 January 2008
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Abstract |
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Objectives: The antiviral activity of CD4 miniproteins was evaluated as potential HIV microbicides, using relevant in vitro models.
Methods: Compounds were tested in a single-cycle HIV-1 pseudovirus assay and against replication competent HIV-1 in co-cultures of monocyte-derived dendritic cells (MO-DC) and CD4+ T cells. Cytotoxic activity was evaluated in an MTT assay.
Results: Monomeric miniproteins (M47 and M48) showed 50% effective concentration (EC50) values of 79–105 nM against a subtype B, CCR5 co-receptor-using Ba-L pseudovirus. Higher activity was found for the dimeric miniproteins M48D30, M48D50 and M48D100 (EC50 between 15 and 30 nM), in contrast to the tetrameric miniproteins M48T30, M48T50 and M48T100 (EC50 between 107 and 377 nM). The hetero-bivalent miniprotein M48-Hep and miniproteins that targeted the Phe-43 cavity on gp120 (M48-U1, M48-U2 and M48-U3) were highly active, with EC50 values as low as 2 nM for M48-U1. All miniproteins showed high activity against CCR5 or CXCR4 co-receptor-using subtype B and CRF-01_A/E pseudoviruses. Many early M48-based compounds were much less active against subtype C pseudoviruses, whereas M48-U compounds that targeted the Phe-43 cavity were very active against all pseudoviruses, including subtype C. In MO-DC/CD4+ T cell co-cultures with replication-competent HIV-1 Ba-L, EC50 values ranged between 13 and 1719 nM depending on the miniprotein, with M48-U1, M48-U2 and M48-U3 again being the most potent. Importantly, the latter compounds completely prevented viral replication by treating the cultures from 2 h before until 24 h after infection, at non-toxic concentrations of 66–6564 nM.
Conclusions: These novel CD4 miniproteins might constitute a promising class of HIV microbicides.
Keywords: vaginal microbicides , in vitro activity , sexual transmission
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Introduction |
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Various drugs are currently being investigated both in vitro and in vivo for potential use as HIV microbicides to prevent sexual HIV transmission. These compounds include polyanionic entry inhibitors, chemokine receptor blockers and fusion inhibitors,1–5 surface-active agents (surfactants),6 vaginal pH buffers7 and nucleotide reverse transcriptase inhibitors as well as non-nucleoside reverse transcriptase inhibitors (NNRTIs).8–11 The latter group of compounds is especially interesting, since they combine an extremely high antiviral activity with a relative low toxicity.12 The former groups of compounds are much less active on a molar basis and their capacity to completely block infection without showing toxicity is questionable. Therefore, to stop HIV at the binding and fusion stages, a search for new and better compounds is needed.
In this regard, various CD4 mimetic miniproteins have been rationally designed using structural information on a CD4–gp120–17b antibody complex.13 These compounds are endowed with an optimal interaction with gp120 and bind to viral particles and diverse HIV-1 envelopes with CD4-like affinity. Moreover, they proved to inhibit infection of both immortalized and primary T cells.14 In general, CD4 miniproteins exhibit functional characteristics of native human CD4 while their small size and constrained structure are advantageous for gaining easier access to the CD4-binding site on the viral gp120. In addition, these peptides are stable in strong denaturing conditions, including acidic pH, and they are relatively resistant to degradation by proteases, which favours the development of these compounds as anti-HIV drugs in general and as vaginal microbicides in particular.
We report here on the antiviral and cytotoxic in vitro activity of several novel mono- and multimeric CD4 mimetic miniproteins, a hetero-bivalent CD4 miniprotein containing a heparin moiety, and a set of miniproteins that specifically target the Phe-43 cavity on gp120. As a first screening, the antiviral activity of all CD4 miniproteins was evaluated in a single-cycle pseudovirus assay, using CD4+CCR5+ and CD4+CXCR4+ GHOST cells as target cells. Additionally, since mucosal dendritic cells and CD4+ T cells represent the primary target cells involved in sexual HIV transmission,15–17 we used our previously described in vitro model of monocyte-derived dendritic cells (MO-DC) and autologous CD4+ T cells8,9 to evaluate the antiviral activity of the different CD4 miniproteins and their potency to prevent productive infection of MO-DC/CD4+ T cell co-cultures with replication competent HIV-1 Ba-L, as a possible predictor of in vivo microbicide activity.
Moreover, we analysed the cytotoxicity of the compounds in MO-DC/CD4+ T cell co-cultures and in cultures of ME-180 endocervical cells. These latter cells represent the primary epithelial cells, lining the (endo)cervix, which is most probably the anatomical region of the female lower genital tract most susceptible to HIV infection.18,19
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CD4 miniproteins and reference compounds
The CD4-binding surface of the HIV-1 gp120 glycoprotein was transplanted onto a scorpion-toxin scaffold.14 The resulting miniprotein, CD4M33 (M33), contains 27 amino acids and shows CD4-like binding affinity to HIV-1 gp120 (Figure 1). Combinatorial chemistry was used to further optimize the gp120-binding affinity of the miniprotein. To this end, peptide libraries were generated by solid-phase synthesis to explore the side chain/main chain contribution in order to better interact with the β15 strand, being one of the hot spots of the gp120 site. Afterwards, peptide libraries were purified by HPLC and characterized by mass spectrometry and circular dichroism. Those peptide libraries were screened for binding affinity against HIV-1 LAI and HIV-1 SF162 gp120 and two new miniproteins, M47 and M48, were selected (Figure 1). Both miniproteins bind gp120 with a 2- to 10-fold increased affinity with reference to M33. Based on the optimized monomeric M48 miniprotein, dimeric (D) and tetrameric (T) M48 miniproteins were also generated (Figure 2), with different polyethylene glycol spacer lengths of 2 x 30, 2 x 50 or 2 x 100 Å within each group (respectively, M48D30, M48D50 and M48D100, and M48T30, M48T50 and M48T100). Those lengths were used to screen different spatial organization of the native trimeric form of envelopes. Indeed, applying cryoelectron microscopy tomography, several 3D models of the SIV envelope spike alone20,21 or in contact with T cells or neutralizing proteins were described.22,23 Starting from those various models, we estimated that the distance between two CD4-binding sites should be at least 60 Å.
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Additionally, a hetero-bivalent CD4 miniprotein, M48-Hep, was created containing the M48 miniprotein covalently linked to heparin. The most recent CD4 miniproteins under evaluation in this paper include compounds that specifically target the Phe-43 cavity on the CD4-binding site of HIV gp120. To better interact with that cavity, derivatization was introduced in the para-position of phenylalanine 23 of M48. Previous data showed that a biphenyl moiety (M33) in that cavity allowed increasing affinities, mainly for the subtype-B envelope. Nevertheless, the 3D crystallographic complex of the gp120 core in interaction with that peptide and the stabilizing CD4i Fab 17b highlighted that the additional phenyl ring, due to its rigidity, induced structural alterations in gp120.24 Thus, much more flexible groups such as cyclohexylmethoxy, cyclopentylethoxy and phenylethoxy were added in that position leading to three peptides named, respectively, M48-U1, M48-U2 and M48-U3 (Figure 3).
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Recombinant soluble human CD4 (sCD4) was used as a reference for compounds interacting with the CD4-binding site of gp120 and was obtained through the NIH AIDS Research and Reference Reagent programme, Division of AIDS, NIAID, NIH, from Dr Norbert Schuelke. Heparin (Sigma-Aldrich, Bornem, Belgium) was used as a control for the hetero-bivalent CD4 miniprotein M48-Hep.
HIV isolates and HIV-derived pseudoviruses
For single-cycle infection experiments, HIV pseudoviruses representative of different HIV-1 subtypes were produced by co-transfection of 2 x 105 HEK293 T cells using the calcium phosphate method (ProFection mammalian transfection system; Promega, Leiden, The Netherlands). Briefly, we used 1 µg of an HIV-1 env expressing vector (pcDNA4/TO-Env), under control of a CMV promotor and 400 ng of the defective HIV-1 genomic vector (pNL4-3.Luc R–E–), under control of the LTR promoter. pNL4-3.Luc R–E– contains the whole HIV genome, including a packaging signal, but has a frameshift in the env and vpr genes rendering it non-infectious. Furthermore, a luciferase reporter gene replaces the nef gene. After 24 h, the fetal bovine serum (FBS) (10%)-DMEM medium was replaced with medium containing 1 mM sodium butyrate (Sigma-Aldrich) and the cells were further incubated for an additional 24 h. Afterwards, the supernatant with the pseudovirus was harvested, filtered (Millex 0.45 µm filters; Millipore NV, Brussels, Belgium) and after adding FBS (10%) stored at –80°C. Pseudovirus constructs of different HIV-1 subtypes were created, including: pseudoviruses derived from the subtype B, CCR5 co-receptor-using strains Ba-L and SF162; the subtype B, CXCR4 co-receptor-using strains MN and IIIb; the subtype C, CCR5 co-receptor-using strains VI829, VI1358 and MJ4; and the CRF01_AE, CXCR4 co-receptor-using strain CA10-3.
For experiments in co-cultures of MO-DC and autologous CD4+ T cells, we used the replication-competent CCR5 co-receptor-using, non-syncytium-inducing, subtype B HIV-1 strain Ba-L, of which the infectious titre was determined on PHA/IL-2 stimulated peripheral blood mononuclear cells (PBMCs) according to the method of Reed and Muench25 and modified by Peden and Martin.26
HIV single-cycle pseudovirus assay
Fifty microlitres of HIV pseudovirus was pre-incubated with 50 µL of a dilution series of compound. After 15 min, 100 µL of GHOST target cells (1.2 x 105 cells/mL) expressing CD4 and either of the HIV co-receptors CCR5 or CXCR4 were added to the 96-well plates containing the pre-treated pseudovirus. Cells were cultured at 37°C and 5% CO2. The GHOST cells were lysed 48 h post-infection by addition of the luciferin substrate (SteadyLite HTS, Perkin Elmer Life Sciences, Zaventem, Belgium). The resulting light signal, expressed as relative lights units, was quantified in a luminometer (TopCountTM, Canberra-Packard, Zellik, Belgium). The percentage the compound-mediated inhibition of viral infection, when compared with untreated controls, was plotted against the concentration of the compound, and the linear regression analysis was performed to calculate the 50% effective concentration (EC50).
Monocytes were separated from the buffy coats of HIV seronegative blood donors by counter-flow elutriation and E-rosetting. They were subsequently differentiated to MO-DC using GM-CSF and IL-4, as described previously.27 Fifty microlitres of HIV-1 Ba-L [corresponding to a multiplicity of infection (moi of 10–3)] was pre-incubated with 50 µL of a dilution series of compound. After 30 min, 100 µL of MO-DC was added (4 x 105 cells/mL) and incubated for 2 h (37°C, 5% CO2). Afterwards, cells were washed three times and autologous CD4+ T cells were added, in the presence of compound. Co-cultures were incubated for 24 h, after which the cells were washed again (three times) to remove the compound. Cells were incubated for 14 days of primary culture, during which half of the culture medium was refreshed twice weekly, without compound. At day 14, cultures were washed and PHA/IL-2-activated PBMCs (2 x 105 cells/well) were added to stimulate any latent virus or viral growth below the detection limit. This secondary culture was maintained for 14 days. Culture supernatants of primary (day 14) and secondary (day 28) cultures were analysed for the presence of HIV p24 antigen by a modified in-house monoclonal p24 (HuMab-HIVp24) (Biomaric NV, Gent, Belgium) ELISA.28 The percentage compound-mediated inhibition of viral infection, when compared with untreated controls, was plotted against the concentration of the compound, and the linear regression analysis was performed to calculate the EC50 after primary culture. The minimal concentration of the compound (Cmin) that blocked the infection below the detection limit of the ELISA (20 pg HIV p24 Ag/mL) was determined in the primary culture supernatants and absence of infection at this concentration of compound was further confirmed by ELISA analysis of the secondary cultures supernatants. We previously showed that the ELISA result of the secondary cultures was fully predictive of the presence or absence of proviral DNA,8 implying that the absence of an ELISA signal after secondary culture points to the absence of integrated provirus. In this assay, we used the NNRTI TMC-120 (dapivirine) as a reference with known nanomolar activity.7,8
Cytotoxicity of the compounds was evaluated using an enhanced colorimetric MTT assay, WST-1, according to the manufacturer’s instructions (Roche, Vilvoorde, Belgium). As target cells, we used either co-cultures of MO-DC and allogeneic CD4+ T cells or cultures of ME-180 endocervical cells. Briefly, MO-DC/CD4+ T cell co-cultures or ME-180 cells were incubated for, respectively, 5 days or 24 h, in the presence of compound, after which WST-1 reagent was added. Cellular cytotoxicity was quantified 6 h later by analysis in ELISA. The percentage cell viability, when compared with untreated controls, was plotted against the compound concentration, and the linear regression analysis was performed to calculate the 50% cytotoxic concentration (CC50).
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CD4 mimetic miniproteins show potent antiviral activity against HIV-1 subtype B pseudovirus infection
Different classes of CD4 mimetic miniproteins were evaluated in a single-cycle HIV-1 Ba-L pseudovirus assay. The first group of molecules consisted of the monomeric CD4 miniproteins M33, M47 and M48 (Figure 1). Whereas M47 and M48 were similarly active against the subtype B pseudovirus Ba-L, 
10-fold lower activity was found for M33 (Table 1). This result seems to prove that the neutralization activity is directly correlated with the binding affinity of the CD4 miniprotein with the gp120 variant.
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Since the HIV-1 envelope glycoprotein gp120 is displayed as a multivalent (trimeric) complex on the surface of virions and infected T cells, dimeric and tetrameric CD4 miniproteins were developed based on the most active miniprotein M48, whereby the length of the carbon spacer varied from 2 x 30 over 2 x 50 to 2 x 100 Å (Figure 2). The dimeric CD4 miniproteins showed up to 5-fold higher activity against Ba-L pseudovirus compared with the monomeric compound M48, with a minimal impact of the carbon spacer length on the antiviral activity of the compound (Table 1). In contrast, up to 5-fold lower antiviral activity was found for the tetrameric compounds, in which the antiviral activity was positively correlated with the carbon spacer length of the molecule (Table 1).
Besides the mono- and multimeric CD4 miniproteins, we also evaluated a hetero-bivalent CD4 miniprotein, M48-Hep, in which M48 was covalently linked to a heparin moiety. This peptide simultaneously targets the CD4-binding site (through the M48 core) and the V3 loop of gp120 (through the heparin moiety), which is involved in co-receptor binding. Upon binding of a CD4 miniprotein to gp120, the same conformational changes are expected in gp120 as after interaction of CD4 with gp120. As a result, CD4i epitopes on gp120 that mediate co-receptor binding should be unmasked, which facilitates heparin binding and thus prevent interaction of gp120 with the cellular co-receptor. The compound showed high antiviral activity, but it was 5-fold lower compared with the activity of the heparin control (Table 1).
The most recently developed CD4 miniproteins are compounds that specifically interact with the Phe-43 cavity of the viral gp120: M48-U1, M48-U2 and M48-U3 (Figure 3). These compounds showed most potent activity against Ba-L pseudovirus infection, with EC50 values ranging from 2 to 67 nM (Table 1).
We next evaluated whether the different CD4 miniproteins displayed similar antiviral activity against other subtype B pseudoviruses that use either CCR5 or CXCR4 as co-receptor including the subtype B, CCR5-using isolate SF162 and the subtype B, CXCR4-using isolates MN and IIIb (Table 1). Overall, miniproteins were most active against MN, followed by IIIb, SF162 and actually least against Ba-L. The EC50 values of the miniproteins against MN or IIIb pseudovirus were significantly different compared with either Ba-L or SF162 pseudovirus and EC50 values against either Ba-L versus SF162 or MN versus IIIb were also significantly different (all P values <0.05, non-parametric Wilcoxon signed ranks test).
CD4 mimetic miniproteins show decreased antiviral activity against subtype C pseudovirus isolates but not against a CRF01_AE isolate
The development of HIV microbicides is especially important for the African and Asian continent, in which the HIV/AIDS pandemic is most devastating. Therefore, candidate HIV microbicides should be evaluated against viral isolates that predominantly occur in those specific areas. To this end, we evaluated the activity of the CD4 miniproteins against three subtype C, CCR5 co-receptor-using isolates from our pseudovirus collection (VI829, VI1358 and MJ4) and one CRF01_AE, CXCR4 co-receptor-using isolate (CA10-3).
Clearly, the antiviral activity of most CD4 miniproteins greatly decreased when these compounds were evaluated against the VI829 pseudovirus (Table 2). The antiviral activity of the monomeric CD4 miniprotein M47 was at least 10-fold decreased in comparison with results obtained against subtype B isolates. Moreover, EC50 values of monomeric CD4 miniproteins M33 and M48, and all dimeric and tetrameric CD4 miniproteins were above the highest concentration tested. In contrast, high antiviral activity was found for the heparin-moiety containing CD4 miniprotein, M48-Hep, which showed an EC50 value of 30 nM. Importantly, potent antiviral activity was also found for the CD4 miniproteins that targeted the Phe-43 cavity on gp120 (M48-U1, M48-U2 and M48-U3), with EC50 values ranging from 11 to 951 nM.
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In order to confirm these striking results, we next evaluated the CD4 miniproteins against two other subtype C, CCR5-using isolates, i.e. VI1358 and MJ4 (Table 2). In general, these results indicated the same tendency towards a decrease in EC50 values as was seen with the VI829 isolate. However, the decrease in antiviral activity was less pronounced for the dimeric CD4 miniproteins and the heparin-moiety containing CD4 miniprotein M48-Hep, especially when the MJ4 isolate was used. Statistical analysis showed that the antiviral activity of the miniproteins was significantly different among all pseudovirus isolates tested (P values <0.05, non-parametric Wilcoxon signed ranks test).
Besides these subtype C isolates, we also evaluated the activity of the compounds against CA10-3, a CXCR4 co-receptor-using CRF01_AE isolate (Table 2). In contrast to the results obtained with the subtype C isolates, potent antiviral activity was found for most monomeric and dimeric CD4 miniproteins, with EC50 values ranging from 24 to 158 nM. As found with other HIV-1 subtypes, the tetrameric CD4 miniproteins showed to be the least potent with EC50 values ranging from 230 nM for the compound with the widest carbon spacer (M48T100) to above 671 nM for the compound with the smallest carbon spacer (M48T30). Similar to experiments with pseudovirus constructs that represented other subtypes, M48-U1, M48-U2 and M48-U3 showed the highest antiviral activity against CA10.3, with EC50 values ranging from 0.4 to 29 nM.
CD4 mimetic miniproteins block replicative HIV-1 Ba-L infection of MO-DC/CD4+ T cell co-cultures
We next asked if the CD4 miniproteins maintained their antiviral activity in co-cultures of MO-DC and autologous CD4+ T cells, as representative target cells for sexual HIV transmission. For these experiments, the replication-competent HIV-1 strain Ba-L was used as a reference. The virus was shortly pre-incubated with a dilution range of compound before addition of the MO-DC target cells. The compound remained present during infection and during the first 24 h of the co-culture of the MO-DC with the autologous CD4+ T cells, but was washed away afterwards. Our first results, analysed after 7 days of primary culture, indicated EC50 values in the lower nanomolar range for most compounds tested (data not shown). Surprisingly, whereas the CD4 miniprotein with the heparin-moiety appeared to be one of the most potent compounds in the single-cycle pseudovirus assay, it showed the weakest activity in the MO-DC/CD4+ T cell co-culture assay after 7 days of culture (EC50: 1514 nM). In this regard, it should be noted that heparin, included as a reference, showed no inhibition up to a concentration of 6500 nM. The monomeric compounds M47 and M48 were most potent, with EC50 values <300 nM.
After 14 days of the ‘primary’ culture, all monomeric, dimeric and tetrameric compounds showed EC50 values ranging from 212 to 1719 nM, but the most recently developed CD4 miniproteins, targeting the gp120 Phe-43 cavity, confirmed their status as most potent miniproteins, with EC50 values ranging from 13 to 217 nM (Table 3).
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In order to detect any latent or subliminal infection of the MO-DC/CD4+ T cell co-cultures, we added PHA/IL-2-activated PBMCs at the end of the 14 day primary culture. After this ‘secondary’ culture, no viral infection was found in cultures that, during the primary culture, were shortly treated with
2500 nM of various dimeric CD4 miniproteins or 1300 nM of the tetrameric miniprotein M48T50. Most importantly, protection from replicative infection was also achieved with 66 nM M48-U1, 656 nM M48-U2 or 6564 nM M48-U3 (Table 3). As an illustration of the reproducibility of these results, the dose–response curves at the end of the secondary cultures are shown in two experiments (using MO-DC and CD4 T cells from two different donors), in which M48, its Phe-43-targeting derivatives and the reference NNRTI TMC-120 were used side-by-side (Figure 4).
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CD4 mimetic miniproteins show no toxicity towards MO-DC/CD4+ T cell co-cultures or towards ME-180 endocervical cells
Compounds under development as microbicides should combine a high antiviral activity and a low cytotoxicity. We therefore investigated if a random selection of CD4 miniproteins showed cytotoxic activity to co-cultures of MO-DC and allogeneic CD4+ T cells. CD4 miniproteins representative of each group of molecules (mono- and multimeric CD4 miniproteins, the hetero-bivalent miniprotein M48-Hep and the miniproteins that target the Phe-43 cavity on gp120) were analysed in parallel, including the heparin control. After a 5 day treatment, no cellular toxicity was found in comparison with untreated controls for any of the compounds at the highest concentration tested (up to 6800 nM). Figure 5 shows the results of a representative experiment, in which the toxicity of M48, its Phe-43-targeting derivatives and the reference NNRTI TMC-120 were used side-by-side. As can be seen, none of the miniproteins shows a tendency towards toxicity, whereas in fact TMC-120 was slightly toxic at the highest concentration used.
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Since a microbicide will be applied into the vaginal lumen, the cytotoxicity towards genital epithelial cells is also of importance. We used the ME-180 cell line as a model for the epithelial cells that line the cervix in vivo. Also in this set-up, none of the miniproteins or the reference compounds sCD4 and heparin showed any cellular toxicity (data not shown).
Clearly, to precisely calculate the selectivity index, even higher concentrations of miniproteins should be used, until some toxicity can be observed. However, for the most active Phe-43-targeting compounds, one can deduce from the EC50 values in Table 3 and the flat toxicity curve in Figure 5 that the selectivity index in the MO-DC/CD4 T system is >500 for M48-U1, >200 for M48-U2 and >30 for M48-U3.
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New HIV prevention strategies such as the development of potent HIV microbicides are urgently needed to stop the HIV epidemic. Therefore, it will be of great interest to identify antiviral compounds that specifically target the very early step of infection, i.e. binding of the viral gp120 envelope protein with the cellular CD4 receptor. Efficient targeting of gp140 presents a major challenge because of its inherent genetic diversity, explaining the poor potency of gp120 inhibitors against diverse clinical isolates. However, as the CD4-binding site on gp120 is one of the better conserved antigenic epitopes,29,30 compounds that target this site might show enhanced clinical efficacy. In the present in vitro study, we evaluated various novel CD4 mimetic miniproteins, which were optimized from previously described miniproteins by combinatorial approach14,30,31 and by taking advantage of structural information on the gp120–CD4 complex.32,33
As a first screening, we evaluated the miniproteins in a single-cycle pseudovirus assay, in which different viral subtypes were represented. The newly synthesized monomeric compounds M47 and M48 showed potent antiviral activity against the subtype B, CCR5 co-receptor-using HIV-1 pseudoviruses Ba-L and SF162, with EC50 values in the lower nanomolar range.
As the HIV-1 envelope is displayed as a trimeric complex, multimerization of the CD4 miniproteins was expected to enhance antiviral activity. This was shown previously for a synthetic bivalent and a trivalent CD4M9 miniprotein where, depending on the viral isolate, a 1–2 log increased potency was found compared with the monomeric CD4M9.34,35 This tendency for increased antiviral activity was also found for the dimeric analogues of the novel monomeric M48 miniprotein we evaluated, independent of the length of the carbon spacer that linked the molecules. Although the increase in antiviral activity of the dimeric M48 compounds was limited, it was still much higher compared with the previously evaluated dimeric CD4M9 compounds. In contrast, all tetrameric M48 analogues, we evaluated, showed clearly decreased antiviral activity in comparison with the monomeric and dimeric compounds, whereby the antiviral activity was positively correlated with the carbon spacer length. These results could be compared with the unexpected observation that the trivalent CD4M9 described by Li et al.35 did not show enhancement of neutralization activity.
Binding of cellular CD4 to the viral gp120 has been reported to induce conformational rearrangements in the envelope complex, unmasking CD4i epitopes on gp120 that facilitates recognition of the CCR5 co-receptor and consequent viral entry into the cells.36 As shown previously, CD4 mimicking molecules with surrogate structural elements for the Phe-43/Arg-59 components of CD4 are sufficient to elicit a gp120 conformational isomerization similar to that induced by CD4 itself.14,37 Thus, we designed a hetero-bivalent construct based on the M48 CD4 mimic covalently linked to heparin that could simultaneously target the CD4-binding site and the V3 loop of gp120 involved in co-receptor binding. Although the M48-Hep compound showed an 8-fold increased activity against Ba-L pseudovirus compared with the M48 miniprotein, it seemed that this beneficial additive effect is solely attributed to heparin, as the latter alone showed equally high activity as such. Remarkably, in the MO-DC/T4 co-cultures with replication-competent Ba-L, antiviral activity was strongly decreased for both M48-Hep and the heparin control, with EC50 values above the highest concentration tested.
M48-UX miniproteins were designed to interact more deeply with the Phe-43 cavity of gp120. This interest was highlighted with the gain of affinity obtained with the addition of the phenyl residue in the para-position of Phe23 of M33.14 However, analyses of the crystallographic structure have shown some limitations, in which this second ring was found to bring too much rigidity into the structure. More structural flexibility was introduced in designing M48-U1. As we had hoped, the latter compound indeed presented the highest antiviral activity with a big tolerance through all the isolates tested. As recently shown by Xie et al.,38 this enhancement in neutralization activity showed that the ‘Phe-43 cavity’ could be a potential target and that the binding contacts inside the cavity were preferentially dominated by van der Waals type of interactions.
Our results indicate that the evaluated CD4 miniproteins show high antiviral activity against subtype B pseudoviruses that use either CCR5 or CXCR4 as cellular co-receptor for entry into the target cells. They were similarly active against a CRF01_AE pseudovirus, but their potency was strongly decreased when evaluated against subtype C pseudoviruses. In order to explain this differential activity of the miniproteins, sequence analysis was done. This revealed a difference at amino acid position 369 (according to the HXB2 sequence), in which the subtype C isolates we evaluated are characterized by a leucine compared with a proline in the subtype B isolates and the CRF01_AE isolate. Since the residue Asp-368 is a crucial point for the gp120–CD4 interaction, a mutation at position +1 could perturb the orientation of the aspartate residue involved in interaction with Arg-59 of CD4. Moreover, proline may be very important to give the orientation of the β15-sheet, which contributes to perform an antiparallel β-sheet alignment with both the CD4 strand C'' but also with a β-sheet of the CD4 mimetic miniproteins. Further experiments are planned in which we aim to induce the reverse mutation of Leu to Pro in subtype C isolates in order to find out if good antiviral activity of the compounds can be regained.
In general, assays, using pseudoviruses, provide sensitive and rapid screening tools for the evaluation of the antiviral activity of compounds, which interfere with HIV entry. However, as our screening assay is based on the use of replication-deficient (single-cycle) pseudoviruses, it is important to evaluate also the compounds in assays based on replication-competent HIV isolates. Therefore, we used MO-DC/CD4+ T cell co-cultures as representative target cells in sexual HIV transmission that are susceptible to infection with the replication-competent HIV-1 reference strain Ba-L. As expected, the antiviral activity of the CD4 miniproteins decreased when replication-competent virus was used in comparison with single-cycle pseudovirus. Nevertheless, a 24 h treatment of the cells with one of the dimeric CD4 miniproteins could still completely block infection of the MO-DC/CD4+ T cell co-cultures. Importantly, as low as 66 nM M48-U1, a miniprotein that targets the Phe-43 cavity, sufficed to completely prevent replicative HIV infection. In this regard, it was reported previously that 143 nM PRO542, a tetrameric fusion protein between CD4 and IgG2 under development for occupational or prenatal exposure to HIV-1,39 potently inhibited localized mucosal infection and dissemination pathways in a cervical explant model.40 However, whereas both the CD4-IgG2 protein and sCD4 might elicit an anti-CD4 immune response, the limited epitope sharing of CD4 mimetic miniproteins with human endogenous CD4 could be a distinctive advantage.14
We were unable to show any cellular toxicity of the various miniproteins in MO-DC/CD4 T cell co-cultures and against the ME-180 endocervical cell line at the maximum concentrations used in the microbicide assays. Although still higher concentrations should be used to allow precise calculation of selectivity indices, we can safely state that at least the most active M48 derivates pass this first filter of in vitro activity/toxicity ratio.
In summary, our results show that CD4 mimetic miniproteins potently inhibit subtype B pseudoviruses and a CRF01_AE pseudovirus, with EC50 values often <100 nM. Remarkably, the antiviral activity of the first generation of compounds was greatly reduced when evaluated against subtype C pseudovirus isolates, possibly as a consequence of the occurrence of a leucine at position 369 instead of a proline. Most importantly, potent antiviral activity remained for the most recently developed CD4 miniproteins that specifically targeted the Phe-43 cavity on gp120. Additionally, infection of primary target cells with replication-competent HIV could be blocked at non-toxic concentrations using different CD4 miniproteins. As we are now focusing on the development of a next generation of CD4 miniproteins with an even enhanced and broader range of antiviral activity, these compounds might represent a new class of promising HIV microbicides.
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This work was supported by grants from the European Microbicide Project (EMPRO—6th framework programme) and from the International Partnership on Microbicides (IPM).
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Transparency declarations |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
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None to declare.
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Acknowledgements |
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We thank the Antwerp Red Cross Blood Transfusion Center for providing buffy coats.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
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