GM-3 Lactone Mimetic Interacts with CD4 and HIV-1 Env Proteins

Jun 20, 2016 - In fact, a high concentration of GM-3 may engage CD4, thus reducing the ... Interaction among OVA-glycoprotein 4, CD4, and Env Proteins...
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GM‑3 Lactone Mimetic Interacts with CD4 and HIV‑1 Env Proteins, Hampering HIV‑1 Infection without Inducing a Histopathological Alteration Barbara Richichi,† Claudia Pastori,‡ Stefano Gherardi,† Assunta Venuti,‡ Antonella Cerreto,† Francesca Sanvito,§ Lucio Toma,∥ Lucia Lopalco,*,‡ and Cristina Nativi*,† †

Department of Chemistry, University of Florence, 50019 Sesto F.no (FI), Italy Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20127 Milan, Italy § Pathology Department, Mouse Histopathology Unit, San Raffaele Scientific Institute, 20100 Milan, Italy ∥ Department of Chemistry, University of Pavia, Pavia, Italy ‡

S Supporting Information *

ABSTRACT: Glycosphingolipids (GSLs) are involved in HIV-1 entry. GM3 ganglioside, a widespread GSL, affects HIV entry and infection in different ways, depending on the concentration, through its anchoring activity in lipid rafts. This explains why the induction of an altered GSLs metabolism was a tempting approach to reducing HIV-1 cell infection. This study assayed the biological properties of a synthetic GM-3 lactone mimetic, 1, aimed at blocking HIV-1 infection without inducing the adverse events expected by an altered metabolism of GLSs in vivo. The mimetic, conjugated to immunogenic protein ovalbumin and multivalently presented, was able to bind the CD4 molecule with high affinity and block its engagement with gp120, thus inhibiting virus entry. Elicited antimimetic antibodies were also able to block HIV-1 infection in vitro, with activity complementary to that observed for 1. These preliminary results show that the use of GSLs mimetics can be a novel promising mode to block HIV-1 infection and that 1 and other GSL mimetics deserve further attention. KEYWORDS: glycosides, mimetics, HIV infection, gangliosides

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lines, acquire GSLs11 and that the incorporation of GSLs into the viral envelope very likely contributes to block viral entry. At physiological concentration, GM-3 and a second GSL, the Gb-3, are able to interact with both CD4 and gp120, inducing the formation of trimolecular complex CD4-GM-3-gp120.8,10 In this multicomponent system, GM-3 seems to facilitate the migration of the CD4-gp120 complex to an appropriate coreceptor and the subsequent fusion between the viral and target membranes. It is therefore clear that, depending on the cell surface concentration, GM-3 affects HIV-1 entry and infection in different ways. In this context, the induction of an altered GSLs metabolism might be a tempting approach to circumvent cell susceptibility to HIV-1 infection. In fact, a high concentration of GM-3 may engage CD4, thus reducing the formation of the trivalent complex gp120-CD4-coreceptor.8,12 However, it is still unclear which adverse events might be evoked by an altered regulation of GSLs.13 As a matter of fact, GM-3 metabolites, generated in vivo upon the pathological overexpression of GM-3, have been

he entry of human immunodeficiency virus type 1 (HIV1) into cells is a multistep process mediated by the interaction of viral envelope glycoproteins gp120 and gp41 (HIV Env) with the CD4 and the chemokine receptors expressed on the target cells.1 HIV entry inhibitors that interact with each single step of the process have been developed; nonetheless, at present only two of them (maraviroc and enfuvirtide) have been approved for clinical use and four have entered clinical trials.2 Since 1996, it has been documented that glycosphingolipids (GSLs) expressed on a target cells’ membrane are involved in CD4-mediated HIV-1 entry.1,3,4 In particular, the GM-3 ganglioside, a widespread GSL involved in many important physiological roles5−7 (Figure 1) at a high level in the plasma membrane has definitely been demonstrated to act as a barrier, blocking HIV-1 Env-mediated fusion8 and preventing subsequent events necessary for infection. More recently, GM-3 was recognized as the host-derived GSL responsible for mediating the unique, Env-independent, HIV-1 mature dendritic cells’ (mDC) attachment and infection.9 GM-3 is also the most abundant ganglioside on T-lymphocytes,10 and it is known that HIV-1 particles, produced by infected T-cell © 2016 American Chemical Society

Received: April 7, 2016 Published: June 20, 2016 564

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Figure 1. Structures of the GM-3 ganglioside, the GM-3 lactone, mimetic 1, glycoside 2, KLH-glycoprotein 3, and OVA-glycoprotein 4.

Scheme 1. Synthesis of OVA-glycoprotein 4 and of Negative Control Glc-glycoprotein 6

specific antibodies (Abs) were elicited.20 We therefore evaluated whether mimetic 1 was able to recognize CD4 and/or gp120, thus blocking HIV infection in vitro. Herein we also reported our further investigation of the possible histopathological alteration induced in vivo by immunization with 1. To gain this goal and by considering the known low immunogenicity of saccharidic antigens, KLH-glycoconjugate 3 was employed in vivo. Activated glycoderivative 2 was linked to ovalbumin (OVA) to form OVA-glycoprotein 4, which was employed to test Abs specificity (Figure 1). Synthesis of KLH-glycoprotein 3, OVA-glycoprotein 4, and Glc-glycoprotein 6. KLH-glycoprotein 3 was prepared following the protocol previously reported.20 OVA-glycoprotein 4 (Figure 1 and Scheme 1) was obtained by coupling commercially available OVA with activated carboxylic derivative 220 (phosphate buffer, room temperature, 20 h). The saccharidic antigen/OVA loading was estimated (trinitrobenzensulfonic, TNBS, test) to be 50%.21 Glc-glycoprotein 6 was used as a negative control and synthesized by reacting Glc-

recognized as tumor-associated carbohydrate antigens (TACAs). This event, which takes place in melanoma and other cancers, increases tumor survival, promotes cell adhesion and spread, and, most importantly, blocks host immunity.6,14,15 GM-3 and GSLs in general are characterized by extremely complex structures and are commonly obtained in small amounts from blood or brain tissues.16 In the last few years, the availability of fully synthetic or semisynthetic GSLs, obtained in important amounts, enantiomerically pure and free from possible biological contamination, paved the way to their application in clinical trials and therapeutic settings.17 In this framework, the stereoselective synthesis of 1, a structurally simpler and in vivo stable mimetic of the GM-3 lactone, a GM3 metabolite, has been reported (Figure 1).18 The GM-3 lactone is formed as consequence of an overexpression of GM-3 and has been recognized as a TACA.19 When thioether-bridged mimetic 1 was transformed to KLH-glycocojugate 3 by reacting derivative 2 with an immunogenic carrier such as the KLH protein (Figure 1), in vivo immune activation was observed and 565

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IIIB or gp120 Bal indicates that there is no association with HIV Env coreceptor usage. Similar ELISA assays were performed using antihuman CD4 monoclonal antibody (mAb) SIM4 or anti-HIV1 gp120 mAbs b12 and 2G12. Figure 2B shows the binding of SIM4 to CD4; as expected, SIM4 did not recognize gp120 (either Bal or IIIB), but a low binding level was observed with complex gp120-CD4 because SIM4 recognizes a region on CD4 that is involved in the HIV-1 binding site. Figure 2C shows the binding of b12 mAbs to gp120 (Bal and IIIB) alone or to gp120-CD4 complexes. As expected, b12, which is directed to the CD4 binding site (CD4bs) on gp120, showed a decrease in the binding to gp120 when the two proteins were preincubated with CD4. Any difference in the binding of gp120, either alone or preincubated with CD4, was observed when antibody nontargeting CD4bs on gp120 was used (Figure 2D). Negative control 6 (Scheme 1) did not bind either CD4 or gp120 proteins (data not shown). Immunogenicity and Safety of KLH-glycoprotein 3. To confirm the immunogenicity of mimetic 120 and evaluate the effect of the immunization, KLH-glycoprotein 3 was employed to immunize Balc/C mice. The immune responses were evaluated 1 week after II, III, and IV immunization. The reactivity on ELISA tests of the antisera to 3 was assayed (Figure S1, Supporting Information) using OVA-glycoprotein 4 for the coating (Results and Discussion). Overall, a progressive increase in the binding was evident after each immunization; in particular, after the III and IV immunizations, the specific antisera showed positive binding over 1:70.000 serum dilution. We performed histopathological analysis to evaluate the effect of immunization on the different tissues (Supporting Information). Necropsies were performed on all of the immunized animals. We established the histopathological effect by evaluating the inflammatory level and the presence of granulomatous reactions; in detail, we defined four different levels: 0, when we did not observe any alterations; 1, if we observed a low level of inflammation; 2, when moderate inflammation was shown; and 3, when severe inflammation with a granulomatous reaction was shown. No histopathological abnormalities were observed in the majority of tissues although a very low level of inflammation was observed in lymph nodes, the urogenital level, and the stomach, with maximum level 1 as shown in Table 1. Overall, all animals were in good health and no histopathology indicative of pathologic responses was observed in most organs and tissues. Cell Surface Recognition by Antisera Anti-KLHglycoprotein 3. To evaluate the binding of antisera to KLH-glycoprotein 3 on the cell surface, a flow cytometry assay was performed with the 3T3 cell line, either transfected or not for the CD4 protein, to verify whether the binding of 3 was due to CD4 or to glycosphingolipid. The assay was also performed with the M7 cell line, which has been used in the neutralization assay (see below). As shown in Figure 4, positive binding was

glycoderivative 5 with commercially available OVA (details in Supporting Information) (Scheme 1). Interaction among OVA-glycoprotein 4, CD4, and Env Proteins. To determine whether OVA-glycoprotein 4 was able to interact with either CD4 and/or gp120 as described for the GM-3 ganglioside, we evaluated the capability of 4 to bind CD4, gp120 IIIB (CXCR4 tropic), gp120 Bal (CCR5 tropic), and complex CD4-gp120. ELISA tests were performed on a solid phase to assay the different proteins (CD4 and gp120). As shown in Figure 2A, 4 efficiently binds to CD4 and, to a lesser

Figure 2. ELISA on CD4, gp120, and the CD4-gp120 complex. Two different gp120s were used, named Bal (CCR5 tropic) and IIIB (CXCR4 tropic). Solid phases were performed with recombinant CD4, gp120-Bal, gp120-Bal+CD4, gp120-IIIB, and gp120-IIIB+CD4. (A) O.D. of OVA-glycoprotein 4 binding subtracted from the negative control (6, Scheme 1). (B) Binding of a monoclonal antibody to CD4 (SIM4) in different solutions ranging from 1:25 to 1:675. (C and D) Binding of two different monoclonal antibodies to gp120, named b12 (directed to the CD4 binding site, CD4bs, panel C) and 2G12 (directed to the V3 loop on gp120, panel D). Both antibodies were tested at several concentrations ranging from 80 to 2.96 ng/mL. The means plus standard deviation (bar) of two independent experiments are shown.

extent, to both gp120s. Of note, a lower level of binding was observed in the case of complex gp120+CD4 compared with the binding to CD4. This suggested that very likely 4 recognizes a region on CD4 involved in the binding with HIV. In addition, the positive binding of 4 with either gp120

Table 1. Effect of Immunization with KLH-glycoprotein 3: Histopathology Scoresa spleen, thumus

KLH-glycoprot. 3 controlb a

liver

kidney, lung

gut, pancreas

pelvis

adipo

spleen

thymus

lymph

parench

adipo

adipo

spleen

thymus

lymph

adipo

parench/ lymph

brain, heart, urogen

adipo

lymph

stomach

0 0

0 0

0 0

0.5 0

0 0

0 0

0 0

0 0

0 0

0.5 0

0 0

0/0 0/0

1 0

0 0

0 0

0.5 0

0, normal; 1, mild inflammation. bNon immunized mice. 566

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Figure 3. Binding of antisera to KLH-glycoprotein 3 on the cell surface of cell lines. Representative binding of the pool of antisera to 3 was observed by indirect flow cytometry analysis. Panels A−C show the binding on 3T3 parental, 3T3-CD4 transfected, and M7 cells, respectively. The white histogram is for secondary antibody-FITC. Light gray is for preimmune sera; dark gray is for sera to 3. CD4 expression was also evaluated on the same cell lines (D−F). White histograms are for mouse control phycoerythrin (PE), and light gray is for mouse antihuman CD4-PE antibody.

within gp120 favors the subsequent CD4 docking in the raft domain, thus the appropriate coreceptor molecules can be recruited within the raft, facilitating the virus entry and spread between cells.3 Raft-associated GSLs, such as GM-3 and Gb3, have been found to be involved in HIV infection and entry through their interaction with Env proteins.23 The effect of these two GSLs has been ascribed to their common structural features and, more precisely, to the free hydroxyl groups on C-4 of the galactose residue (the terminal one in Gb3).24 Interestingly, it was shown that GM-3 depletion or overexpression in rafts hamper HIV entry;8 moreover, GM-3 and Gb3 were recognized as the main GSLs able to bind free HIV particles, preventing their docking to rafts and the binding to CD4.23 These data strongly suggest that GSLs may offer a further attractive way to limit HIV infection and spread, either through the modulation of their levels within rafts or by the administration of soluble GSL mimetics. However, because of their role in cell signaling, immune trafficking, and oncogenesis, an up or down regulation of GSLs might induce severe adverse events.6,13,14 Therefore, the generation of synthetic molecules mimicking natural GSLs, endowed with selective antiviral activity, could enhance the efficacy and safety in GSL therapeutic application.18 In this study, spiro tioketal 1, a mimetic of the GM-3 lactone, a GM-3 metabolite formed on melanoma cells, was linked to immunogenic proteins KLH and OVA, and the glycoproteins obtained, 3 and 4, respectively, were tested to verify the hypothesis that a mimetic of GM-3 may interfere with HIV entry through a competitive mechanism, either at the cell membrane or at the viral envelope. Mimetic 1, structurally simpler than the native GM-3 lactone, presents a 2-deoxy branched mannopyranose residue mimicking the sialic residue, linked to a galactal-fused 1,4-oxathiine ring through a spiranic carbon. The tridimensional shape of 1 preserves the folded shape characteristic of the GM-3 lactone and, very likely, of the GM-3 ganglioside when abnormally overexpressed and packed.19 It is noteworthy that, like GM-3 and Gb-3, 1 shows a free hydroxyl on C-4 of the galactal moiety

evident with the pool of antisera to 3 on the cell surface of each cell line (3T3 untransfected cells, see Figure 1A; 3T3-CD4 transfected cells, see Figure 1B; and M7 cells, see Figure 1C), suggesting that the binding is possibly due to glycosphingolipid and that the antisera could mimic the CD4 molecule. Preimmune sera did not bind the cell lines. Each cell line has been treated with a specific anti-CD4 monoclonal antibody to verify the expression of CD4 (Figure 3D−F). Reduction of HIV-1 Infectivity by OVA-glycoprotein 4. OVA-glycoprotein 4 also underwent a neutralization assay to examine the effect of the GM-3 lactone mimetic-OVA conjugate on the reduction of HIV-1 infectivity; the assay was performed on the M7 cell line as target cells, employing laboratory HIV strain SF162. As unrelated virus, VSV (vesicular stomatitis virus, strain SVA.MLV#922) was used. As shown in Figure 4A, HIV replication was inhibited by 4 but not by negative control 6. Similar curves were observed when 4 was incubated either with cells (M7 cell line) or virus (HIV-1SF162), confirming that 4 binds GSLs present on both CD4 molecules expressed on the cell membrane and gp120, which represents the main protein on the viral envelop (Figure 4A). No inhibition was observed with VSV (Figure 4B), thus confirming the specificity of HIV infectivity reduction. The specific inhibition of HIV-1 infectivity was obtained with antisera to 3 as well at 1:60 sera dilution (Figure 4C). No effect was shown when VSV was used (Figure 4C). As positive controls, a monoclonal antibody to GM-3 (M2590), a neutralizing monoclonal antibody to HIV-Env (2F5, recognizing the gp41 MPER domain), and SIM4 were used, as shown in Figure 4D. M2590 antibody was preincubated with either M7 cells or HIV-SF162; SIM4, with M7 cells; and 2F5, with HIVSF162.

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RESULTS AND DISCUSSION GSLs are involved in membrane trafficking, receptor signaling, and virus interaction via their presence in lipid rafts22 of cell membranes. In particular, GSL interaction with the V3 loop 567

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Figure 4. OVA-glycoprotein 4 and mouse antisera to KLH-glycoprotein 3 reduce HIV infectivity. Two viral strains (HIV-1-SF162 and negative control VSV) were used to infect the 5.25.EGFP.Luc.M7 cell line. Panel A shows neutralization curves obtained with OVA-glycoprotein 4 preincubated either on 5.25.EGFP.Luc.M7 or on the SF162 virus strain. Neg Ctrl 6 was preincubated either on M7 cells or SF162 and was used as a negative control. The values are expressed as the used concentration. (B) Neutralization curves obtained with OVA-glycoprotein 4 preincubated either on 5.25.EGFP.Luc.M7 or on the VSV virus strain. Neg Ctrl 6 was preincubated either on M7 cells or on VSV and was used as a negative control. The values are expressed as the used concentration. (C) Neutralization curves of a pool of mouse sera obtained after the last immunization diluted 1:60 to OVA-glycoprotein 3 preincubated either on 5.25.EGFP.Luc.M7 or on the two virus strains (HIV-1-SF162 and VSV). The values are expressed as the percentage of infectivity reduction subtracted from the values obtained with preimmune sera on each virus strain. (D) Neutralization curves obtained with several positive controls including 2F5 (monoclonal antibody to gp41) preincubated on SF162 and SIM4 (antibody to CD4 purified from the supernatant of a relative hybridoma) preincubated on the 5.25.EGFP.Luc.M7 cell line and a monoclonal antibody to GM3 (M2590 from Gentaur, Italy) preincubated either on HIV-1-SF162 or on the 5.25.EGFP.Luc.M7 cell line. Mean values of three independent experiments plus the standard deviation are shown for each concentration of reagent in all panels.

(see above).24 Mimetic 1 was conjugated with KLH to generate glycoprotein 3, a suitable immunogen for assaying its antigenic properties in vivo. Upon immunization, a high yield of specific antibodies was indeed obtained because of the II immunization (Figure S1, Supporting Information). To prevent a false positive of mouse antisera to the carrier, mice have been immunized with KLH-conjugated GM-3 mimetic 3 and the screening of antisera has been performed using a glycoprotein with a different carrier. Mimetic derivative 2 was thus conjugated with OVA, and OVA-glycoprotein 4 so obtained was employed to test the specificity of antisera to antigen mimetic 1.

OVA-glycoprotein 4 binds the CD4 molecule with high affinity, and, to a lesser extent, the CD4-gp120 complex. The binding of the mimetic was comparable to that shown by SIM4, a CD4-specific monoclonal antibody (Figure 2). The comparison between CD4 complexes hosting gp120-Bal and gp120-IIIB belonging to viruses proven to use different coreceptors (R5 and CXCR4, respectively) showed that the binding of the mimetic was not affected by the coreceptor type. This finding (i.e., independence from tropism of the virus strain) is of extreme interest in therapeutics for the wide applicability of a GSL-based drug to primary as well as to chronic infections. 568

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should be conveniently investigated because GSLs are involved in a large number of cell−cell interactions and signaling, within the immune system and in other cell types. The minor antiviral activity exerted by anti-3 antibodies is noteworthy and deserves further attention. Even though anti-glycoprotein 3 antibodies could prove useful in enhancing mimetic activity, their binding in vivo to endogenous GSLs might induce undesired side effects that need to be investigated. In conclusion, GM-3 lactone mimetic 1 and its glycoconjugates 3 and 4 could greatly help in the full comprehension of the potentials of GSLs in addressing HIV and deserve attention.

A key issue to be addressed concerning the efficacy and safety of mimetic 1 has been its antigenicity. Similarly to other bioinspired drugs, 1 was able to elicit an immune response in vivo.20 Antibodies to 1 might bind native GM-3 on cell membrane and also on free virus particles, thus inducing multiple effects, positive or detrimental, for the therapy. As expected,20 KLH-glycoconjugate 3 was highly immunogenic in mice, as a matter of fact, its antibody titer was high after the II immunization cycle and progressively increased in five boosts. Nonetheless, immunization did not induce severe histopathological alterations in mice; only mild signs of inflammation were noticed in brain, heart, and urogenital organs (Table 1), without any sign of tissue damage. These findings, albeit deserving further evaluation, sound encouraging for a possible future therapeutic development of this glycoconjugate. OVA-glycoconjugate 4 was assayed either on cells or on virus preparations to evaluate its ability to block infection in in vitro assays. Similar to the monoclonal antibodies used as a control (Figure 4D), 4 was able to block the HIV infection almost completely, showing even better results when tested on virus particles (Figure 4A). Interestingly, antibodies anti KLHglycoprotein 3 also showed moderate antiviral activity vs HIV (Figure 4C); this anti-HIV effect might provide a further contribution to the overall antiviral activity observed for 4 in a therapeutic scenario. Both 4 and antibody anti-KLHglycoprotein 3 provided stronger virus blocking when incubated with virus suspensions before the infection, therefore confirming that HIV particles carry GSLs and/or their ligands on the surface. The results of this preliminary study showed that GSL mimetics do offer a novel and promising way to block HIV infection, although further studies on the magnitude of neutralization should be performed.

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METHODS Synthesis of Glycoconjugate 4. To a solution of OVA (20 mg, 4.48 × 10−4 mmol) in 2.0 mL of buffer (0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.2), 220 (20 mg, 0.027 mmol) was added, and the mixture was stirred overnight at rt. The solution was dialyzed (dialysis tubing cutoff 3.5 kDa) and then lyophilized to give 19.0 mg of glycoconjugate 4 (Scheme 1). The antigen/protein ratio was evaluated by the determination of free lysine amino groups, before and after the conjugation, by titration with trinitrobenzensulfonic acid.25 Synthesis of Glycoconjugate 6. To a solution of Ova (20 mg, 4.48 × 10−4 mmol) in 2.0 mL of buffer (0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.2), glucose derivative 5 (16 mg, 0.030 mmol, SI) was added, and the mixture was stirred overnight at rt. The solution was dialyzed (dialysis tubing cutoff 3.5 kDa) and then lyophilized to give 20.0 mg of glycoconjugate 6. The glucose/protein ratio was evaluated by the determination of free lysine amino groups, before and after conjugation, by titration with trinitrobenzensulfonic acid.25 ELISA on CD4, gp120, and gp120+CD4 Complexes. Recombinant soluble CD4 and recombinant gp120 (either IIIB or Bal) were coated at 0.1 and 0.25 μg/well, respectively, in phosphate-buffered saline (PBS, 50 mM, pH 7.2) for 18 h at 4 °C on 96-well plates (Maxisorp, Corning). Subsequent steps were performed at 37 °C as previously reported.26 Wells were blocked with 0.5% BSA for 1 h, and SIM4 (mouse monoclonal antibody to human CD4), b12 (human monoclonal antibody directed to CD4 binding site), and 2G12 (human monoclonal antibody to V3 loop of gp120) were tested at different concentrations. SIM4 supernatant was diluted 1:25. Flow Cytometry Analysis. 3T3 parental, 3T3-CD4 transfected cells, and the 5.25.EGFP.Luc.M7 cell line (5 × 105) were fixed in 4% formaldehyde in PBS for 30 min at 4 °C and incubated with a pool of serum samples from five mice after the last immunization diluted 1:200 in PBS 3% FCS for 1 h at 4 °C and then washed with RPMI 3% FCS and incubated with FITC-conjugated goat antimouse antiserum for 30 min at 4 °C. Binding control was achieved using fixed 5 × 105 cells incubated with FITC-conjugated goat antimouse antibody for 30 min at 4 °C. As negative controls, either FITC-conjugated rabbit antimouse Ig or a pool of serum samples from five preimmune mice was used. A PE-conjugated mouse antihuman monoclonal antibody to CD4 (clone RPA-T4 from BD Biosciences, Italy) was used to verify the CD4 expression on each cell line. A PE-conjugated mouse isotype control (BD Biosciences) was used as a negative control. Ten thousand gated events were acquired using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA), and data analysis was performed with Cytomix RXP software. Live cells

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CONCLUSIONS HIV entry inhibitors still represent one of the most captivating and challenging targets in the search for new drugs to hamper cell infection. Recent acquisitions on HIV-1 entry demonstrated that GSL rafts participate in the fusion process stabilizing and conveying the viral particles to high-affinity receptors. In particular, GM-3 seems to be the host-derived GSL that mediates the Env independent interaction between HIV-1 and mDCs and that virion-associated GM-3 is the ligand necessary for trans-infection.9 Even though the up regulation or the administration of exogenous GM-3 could be a possible strategy for interfering with cell infection, the side effects triggered in vivo by the enrichment in GM-3 make this approach unreliable and unsuitable for reinforcing the current arsenal of antiviral drugs. In this article, we report on the synthesis of OVAglycoprotein 4 and on the ability of KLH-glycoprotein 3 and OVA-glycoprotein 4 presenting residues of a GM-3 lactone mimetic to interfere with HIV-1 infection. OVA-glycoprotein 4 was able to block the HIV infection almost completely. Different from other drug-targeting coreceptors, this GM-3 mimetic-containing glyconjugate is not affected by coreceptor usage; therefore, it cannot induce a virus switch because it is usually observed during the course of infection as a result of selective pressure caused by host immunity and when aggressive inhibitors are used. Histopathologic analysis has not revealed signs of severe toxicity due to KLH-glycoprotein 3 and to its antibodies; however, safety issues were not extensively addressed and 569

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*E-mail: cristina.nativi@unifi.it. Mail: via della Lastruccia, 3-13, Sesto F.no (FI) 50019, Italy.

initially gated by forward and side scatter were analyzed for FL1 expression. At least 10 000 events were counted. Histopathological Analysis. Necropsies were performed on all immunized animals 1 week after the last immunization, corresponding to day 34 after the first immunization. The skin, lymph nodes (iliac and cervical), spleen, stomach, gut, liver, kidney, lung, heart, female reproductive system, urinary bladder, and bone marrow were harvested, immediately fixed in 4% buffered formalin, and embedded in paraffin. Paraffin sections (3 μm) were stained with hematoxylin and eosin for histopathological examination. HIV Neutralization. The assay is performed in 5.25.EGFP.Luc.M7 cells and requires multiple rounds of virus replication to achieve adequate levels of reporter gene expression to measure virus neutralization. 5.25.EGFP.Luc.M7 is a genetically engineered clone of CEMx174 that expresses multiple entry receptors (CD4, CXCR4, CCR5, GPR15/Bob). The cells also possess tat-responsive reporter genes for luciferase (Luc) and green fluorescent protein (GFP). A total of 7.5 × 104 5.25.EGFP.Luc.M7 cells/well in complete RPMI medium were plated in 96-well plates and DEAE-dextran (10 μg/mL) was added to the cells to enhance virus infection, and then 5000 TCID50 of SF162 or VSV (vesicular stomatitis virus used as a negative control) viral isolates was added to each well. Several concentrations of OVA-glycoprotein 4 and relative negative control 6 ranging from 225 to 0.926 mg/mL were used. A pool of antisera to KLH-glycoprotein 3 after the the last immunization and a pool of preimmune sera were also tested at several dilutions ranging from 1:60 to 1:4860. All reagents were preincubated for 1 h with virus or cells and then added to wells containing cells or virus, respectively. Different positive controls have been used, including several concentrations of 2F5 (monoclonal antibody to HIV-env-gp41) and SIM4 (monoclonal antibody to CD4) preincubated with virus or cells, respectively, and a monoclonal antibody to GM-3 (M2590; Gentaur, Brussels, Belgium) was preincubated with either cells or virus and was tested at concentrations ranging from 33.3 to 0.13 μg/mL. The cells were cultured for 2 days, diluted 1:2 in complete RPMI medium and DEAE-dextran (10 μg/mL), and cultured for another 3 days. Infection was monitored by evaluating the luciferase activity. Titers were calculated as ID50, the sample dilution at which relative luminescence units (RLUs) were reduced 50% compared to virus control wells (wells without inhibitor) after the subtraction of background RLUs in control cell wells (wells without virus infection). The reaction was read with the use of a Top Count apparatus (Packard, Meriden, CT).

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Author Contributions

B.R. and C.P. contributed equally to this work. L.T., L.L., and C.N. conceived and designed the experiments. B.R., C.P., S.G., A.V., A.C., and F.S. performed the experiments. B.R., C.P., L.L., and C.N. analyzed the data. L.L. and C.N. wrote the article. All authors reviewed and approved the article. Funding

This work was supported by Ente Cassa di Risparmio di Firenze (ECR), Regione Toscana (grant to A.C.), AIRC (IG 2012, for the synthesis of 2), and MiUR (PRIN 2010). Notes

Experiments on live animals received the approval of the ethical committee and were performed in accordance with all national and local guidelines and regulations. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mrs. Arianna Vino and Mrs. Martina Rocchi for technical help with histopathological analysis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.6b00056. Materials, immunization protocol, flow and histopathological analysis, ELISA on OVA-glycoprotein 4, and synthesis and NMR spectra of compounds 2 and 5 (PDF)

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REFERENCES

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DOI: 10.1021/acsinfecdis.6b00056 ACS Infect. Dis. 2016, 2, 564−571

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DOI: 10.1021/acsinfecdis.6b00056 ACS Infect. Dis. 2016, 2, 564−571