Dose response curve is given in Figure S3, Supplementary Material.

**Figure 4.** Hepatitis C virus (HCV) inhibition by LUMS1: (**a**) Anti-HCV activity in Huh-7.5 cells. Cells were pretreated with increasing concentrations of LUMS1 for 2 h followed by infection with HCVcc (JFH1) for 72 h in the presence of proteins. (**b**) HCV infectivity and total cell number were assessed by determining the number of GFP-positive cells (green) and nuclei (red), respectively, for 3 days in the presence of LUMS1). Images were acquired by confocal microscopy. (**c**) HCV subgenomic replicon cells were treated with LUMS1 and sofosbuvir. (**d**) HCVpp was mixed with different concentrations of LUMS1 and subsequently added to Huh-7.5 cells. After 72 h incubation, cells were lysed and percent infection was measured through luciferase activity (BrightGlo, Promega, USA) for each dilution of inhibitor with respect to control containing no inhibitor.

### *3.4. LUMS1 Does Not Stimulate Cellular Activation Markers and Shows Negligible Cytotoxic E*ff*ect*

In the context of evaluating immunogenic effects of a protein in vitro, activation of Th and B cells is considered an important marker of immunogenicity as these cells are involved in inducing monoclonal antibody-based immunogenicity [26,27]. In this regard the effect of LUMS1 and in parallel of MVN, was analyzed on the expression of CD4, CD25, and CD20 activation markers through flow cytometry (FACS) using freshly isolated PBMCs from healthy individuals. CVN was used as a positive control for the expression of CD4 and CD25 as its effect on these cellular activation markers have already been reported [28]. Both LUMS1 and MVN did not increase the population of CD4<sup>+</sup> and CD25<sup>+</sup> cells (Th cells), while CVN in this case showed significantly high activation even at a concentration of 50 nM (Figure 5).

**Figure 5.** Effect of LUMS1 on the activation of Th cells: (**a**) Flow cytometry analysis of PBMCs to determine the population of CD4<sup>+</sup> and CD25<sup>+</sup> cells in freshly isolated PBMCs in response to treatment with LUMS1, MVN, cyanovirin-N (CVN). After treating PBMCs (106 cells/mL) with varying concentrations of LUMS1, MVN, and CVN for 72 h at 37 ◦C and 5% CO2, cells were washed with PBS and incubated with APC-conjugated anti-CD4 and PE-conjugated anti-CD25 antibodies for 30 min at 4 ◦C. Finally, cells were washed with PBS (2% FBS), fixed with 1% formaldehyde, and analyzed by FACS, using CellQuest software for data acquisition. Data were statistically analyzed using GraphPad Prism software. (**a**) Left panel, representative dot plots of forward scatter (FSC) and side scatter (SSC) indicating the subpopulation of cells in PBMCs; right panel, dot plots showing the relative population of cells with CD4 and CD25 activation markers. (**b**) Plot showing the percent population of CD4<sup>+</sup> and CD25<sup>+</sup> cells after treating with LUMS1, MVN, and CVN separately. The data represent the mean of three independent experiments and one-way ANOVA was used to compare different groups. \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001.

However, LUMS1 showed more a pronounced difference with MVN on the activation of CD20<sup>+</sup> cells. Treatment of LUMS1 at a concentration as high as 4 μM did not significantly increase the population of CD20<sup>+</sup> cells, while the effect of MVN in this regard was significantly high even at 2 μM concentration (Figure 6a). In addition to evaluating the cytotoxicity of LUMS1 against Huh7.5 cells (Figure 4a,c) we also determined the effect of LUMS1 on the viability of TZM-bl cells, PBMCs, and HepG2 cells using MTT assay. LUMS1 did not show a cytotoxic effect on Huh7.5 cells, HepG2 cells, and PBMCs at a concentration as high as 10 μM whereas its CC50 value against TZM-bl cells was calculated as 4.9 ± 0.166 μM (Figure 6b and Table 2).

**Figure 6.** Evaluation of B cells activation by LUMS1 and its cytotoxicity: (**a**) graph presenting the flow cytometry analysis to determine the population of CD20<sup>+</sup> cells in freshly isolated PBMCs. PBMCs were isolated from freshly collected blood using density gradient centrifugation and washed with PBS. After treating PBMCs (10<sup>6</sup> cells/mL) with varying concentrations of LUMS1 and MVN for 72 h at 37 ◦C and 5% CO2, cells were washed with PBS and incubated with percp cy5.5-conjugated anti-CD20 antibodies for 30 min at 4 ◦C. Finally, cells were washed with PBS (2% FBS), fixed with 1% formaldehyde and analyzed by FACS, using CellQuest software for data acquisition. Data were statistically analyzed using GraphPad Prism software. Analyses were performed in triplicate and one-way ANOVA with multiple comparisons was used to compare different groups. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001. (**b**) The plot showing concentration dependent cytotoxic effect of LUMS1 on PBMCs, HepG2, and TZM-bl cells.

**Table 2.** Overview of the cytotoxicity of LUMS1 against different cell lines.


#### **4. Discussion**

In this study, we engineered a lectin, LUMS1, by modifying MVN to incorporate two carbohydrate-binding sites and reduce chemical heterogeneity, a major factor in potential immunogenicity of a protein. The NMR analysis of the carbohydrate binding of LUMS1 suggested that it exhibited two carbohydrate-binding sites, and it has the same carbohydrate specificity as MVN—both recognize the α(1-2)mannobiose glycan as the minimum epitope.

MVN has been reported to inhibit HIV-1 entry with EC50 values ranging from 2 to 12 nM against HIV-1, and we reproduced the reported EC50 against HXB2 strain of HIV-1. LUMS1, however, inhibited the same strain with ≈4.5-fold lower potency (EC50 37 nM). LUMS1 contains two carbohydrate-binding sites and is expected to exhibit higher avidity as compared to MVN by engaging more than one glycan or glycan branches at the surface of the virus. Lower potency of LUMS1 against HIV-1 as compared to MVN could be attributed to the different possible mechanism by which these lectins attain high avidity of interactions with the viral envelope; multivalent recognition in the case of LUMS1 and the bind-and-hop mechanism through single site interactions in the case of MVN [19], although these mechanisms remain to be experimentally validated. Binding studies of these lectins with the stabilized HIV-1 gp120 trimer, the form of the envelope protein exists on the viral spike, through isothermal titration calorimetry (ITC) or fluorescence resonance energy transfer (FRET) measuring microevents of binding could illustrate the detailed mechanism [29,30]. Moreover, multivalent recognition can be identified by solving the structure of the complex of lectin and gp120-trimer through X-ray or cryo electron microscopy. While comparing the carbohydrate binding of these lectins, the cross-peaks of carbohydrate-binding site amino acids of MVN in 1H-15N correlation NMR spectra experienced chemical shift changes on the addition of ligand suggesting slow exchange on the NMR time scale [19]. On the other hand, in the case of LUMS1, chemical shift perturbation in most of the cross-peaks were in the form of line broadening suggesting intermediate exchange on the NMR time scale. Slow exchange on the NMR time scale is related to higher binding affinity (lower KD values) as compared to intermediate exchange [31]. The two lectins, therefore, demonstrate the difference in binding to carbohydrate in terms of affinity. The apparent lower carbohydrate-binding affinity of LUMS1 as compared to MVN indicates that its carbohydrate-binding sites may not be structurally optimal, which could be understood only after the structure of the complex of LUMS1 and carbohydrate is available. However, one of the significant aspects of LUMS1 was found to be its ability to potently inhibit HCV infection with an EC50 of 45 and 142.1 nM as determined in HCVcc and HCVpp assays, respectively. HCV inhibition by different oligomeric forms of MVN has been reported but only qualitatively [32]. By testing LUMS1 in HCVpp and replicon system, in addition to HCVcc assay, we clearly demonstrated that exclusively HCV E1/E2-mediated viral entry was inhibited. Many HIV-1 entry inhibitor lectins have been reported also to inhibit the entry of HCV [33–35], which could be attributed to likely similar glycan density on the surface of both viruses [8,36]. This apparent similarity potentiates the development of universal therapy against both viruses. Moreover, LUMS1 demonstrated its specificity for HIV-1 and HCV, as it did not inhibit an amphotropic virus VSV.

The major obstacle in the advancement towards clinical application of anti-viral lectins is their potential cytotoxicity and immunogenicity [17,27]. In cell viability assays using four different types of cells, LUMS1 demonstrated negligible cytotoxic effects with CC50 > 10 μM against Huh7.5 cells, HepG2 cells and PBMCs, and 4.9 ± 0.166 μM against the TZM-bl cells with a selectivity index (SI) value of 108, calculated by the ratio of the smallest CC50 value (4.9 μM) and the EC50 value (45.3 nM, against HCVcc) indicating its promising safety profile. As foreign peptides are prone to induce immunogenicity in patients, we tested LUMS1 for its effect on the activation of B and Th cells in vitro and observed that LUMS1 demonstrated no significant increase in the expression of activation markers for these cells at a concentration as high as 4 μM. LUMS1 demonstrated significantly lesser effect in inducing the CD20 activation marker however its effect on the induction of CD4 and CD25 activation markers was comparable to MVN but slightly less. The detailed safety profile of LUMS1, however, remains to be investigated in animal models in the follow-up study. Taken together, LUMS1 represents an attractive potential therapeutic candidate against HIV-1 and HCV, as it potently inhibits both of these viruses, demonstrates lack of cytotoxicity and negligible activation of B and Th cells. With the emerging trend of protein drugs, further optimization of LUMS1 to enhance its carbohydrate-binding affinity leading to increase anti-viral potency, and its detailed investigation in vitro and in vivo are the further aspects to be considered.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/12/2/199/s1, Detailed methods about protein expression; Figure S1, Image containing overlaid structures of MVN and LUMS1; Figure S2, Effect of LUMS1 on VSV infection; and Figure S3, Anti-HCV activity of MVN.

**Author Contributions:** M.S., A.Q., J.Y., I.A., H.Z. and S.M. performed experiments. All authors contributed to the data analysis and interpretation, and M.P.W. and S.S.-u.-H. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Higher Education Commission of Pakistan through NRPU grant (20-4437/NRPU/R&D/14), and the National Research Foundation of Korea (MSIT 2017M3A9G6068246 and NRF-2014R1A2A1A11052535) and the Gyeonggi Provincial Government.

**Acknowledgments:** We are thankful to Carole Bewley at NIDDK, National Institutes of Health, USA, for providing plasmids containing genes for MVN and CVN. We are also thankful to Punjab AIDS Control Program, Pakistan for providing a monthly stipend to Munazza Shahid.

**Conflicts of Interest:** The authors declare no conflict of interest.
