Chapter 3 – Targeted disruption of pi-pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity
This work was conducted in collaboration with Steven R. King, Maureen Legendre,
Michael D. Swanson, Auroni Gupta, Sandra Claes, Jennifer L. Meagher, Arnaud Boonen,
Lihong Zhang, Birte Klaveram, Zoe Raglow, Craig Day, Alexander N. Freiberg, Mark Prichard,
Jeanne A. Stuckey, Dominique Schols, and David M. Markovitz. E.M.C.-D., A.B., Z.R., D.S., and D.M.M. contributed to manuscript preparation. E.M.C.-D., S.R.K., A.G., S.C., and A.B. characterized the mitogenic and antiviral activity of WT and F84T Malaysian banana lectin
(Malay BanLec). J.L.M. solved the crystal structures of WT and F84T Malay BanLec. M.D.S. cloned Malay BanLec and made intellectual contributions. E.M.C.-D. and A.G. created the F84T mutation. M.L. prepared WT and H84T BanLec, and WT and F84T Malay BanLec. L.Z. and
B.K. performed the anti-Ebola and Lassa fever virus assays. C.D., A.N.F., and M.P. supervised
F84T Malay BanLec antiviral activity testing; J.A.S. supervised crystallography studies; D.S. supervised mitogenicity, HIV binding, and anti-HIV activity testing; and D.M.M. supervised the overall project. The manuscript is in preparation for submission to the Journal of Biological
Chemistry at the time of this writing.
3.1 Abstract
Lectins have been regarded as potential antiviral agents as they can selectively bind glycans on viral surface glycoproteins. However, clinical development of lectins has been stalled
97 by their mitogenicity, which is the ability to stimulate proliferation, especially of immune cells.
We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin,
BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84
(H84T). The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, and showed that it is both mitogenic to peripheral blood mononuclear cells and potently antiviral. We therefore engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted based on structural considerations, F84T Malay BanLec (F84T) was less mitogenic than Malay BanLec. However, F84T maintained strong antiviral activity and inhibited replication of HIV, influenza, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.
3.2 Introduction
Viral infections are a major cause of morbidity and mortality and suboptimal therapeutic options exist for many diseases of viral etiology, underscoring the urgent need for new agents to treat most viral infections. Further, unlike the case with bacteria, few broad-spectrum antiviral agents exist. A group of proteins called lectins has emerged as promising potential antivirals.
Broadly defined, lectins encompass a category of proteins that bind carbohydrates, including
98 glycoproteins found on viral and cell surfaces. Lectins recognize and bind particular mono- or oligo-saccharides through their carbohydrate recognition domains in a highly specific manner.
The existence of lectins was first reported by Peter Hermann Stillmark in 1888, who described a hemagglutinin isolated from the castor tree, so named for its ability to agglutinate erythrocytes, and which he called ricin (97). Since that time, lectins have been described ubiquitously, including in bacteria, viruses, plants, and animals; they are also present endogenously in humans
(39, 98).
Lectins appear to play diverse roles in viral pathogenesis and the host immune response: they are capable of inhibiting viral attachment, entry, and fusion, and of activating both the innate and adaptive immune systems. For example, the transmembrane lectin Langerin, found on
Langerhans cells, has been shown to bind and degrade HIV-1 in Birbeck granules (99). The mammalian lectin mannose binding lectin (MBL) is capable of binding to viral surfaces and activating the complement cascade, thus facilitating viral elimination (41). Indeed, individuals with low levels of MBL may be more susceptible to infection with respiratory syncytial virus
(45). The soluble lectin surfactant protein D (SP-D) have been shown to inhibit influenza A virus activity by preventing viral attachment and entry, facilitating macrophage clearance, and activating neutrophil chemotaxis and the oxidative burst (42–44).
While lectins are promising as both prophylactic and therapeutic antiviral agents, their potential has been limited by their mitogenicity. The mitogenic effects of lectins were first reported in 1960 by Nowell, who described the ability of the plant lectin phytohemagglutinin
(PHA) to induce the growth and division of lymphocytes (100). Lectins exert their mitogenic activity through multiple mechanisms, including binding to T cell receptors (TCRs) and inducing second messenger-mediated stimulation and production of pro-proliferation cytokines (49, 101).
99 Indeed, this property of lectins has been utilized by the scientific community to study mechanisms of immune activation, immunodeficiencies, malignancies, and in other applications such as growing retroviruses. However, the mitogenicity of lectins presents a significant barrier to their more widespread clinical use as antivirals. For example, the prokaryote-derived lectin cyanovirin-N (CVN), which has anti-HIV activity, was shown to have marked stimulatory effects on treated cells, which could paradoxically increase susceptibility to HIV through activation of chemokines and T cells, upregulation of activation markers, and subsequent recruitment of susceptible immune cells to sites of infection (48). On a broader level, if lectins are to be used as antivirals, one of the most important potential routes of administration is the mucosal surface. Nonspecific, uncontrolled inflammation of the mucosa would likely increase viral transmission by facilitating breaks in the epithelium; thus, if the mitogenic and inflammatory effects of lectins cannot be mitigated, they could paradoxically increase the viral transmission they are intended to prevent.
Banana lectin (BanLec), a high-mannose-specific lectin isolated from the fruit of bananas, has been shown to have multivalent anti-viral activity through the inhibition of cellular attachment, viral entry, and endosomal fusion of viruses including HIV (60, 78), HCV (60), influenza (E. Covés-Datson and D. Markovitz, manuscript submitted), and Ebola (E. Covés-
Datson, J. White, and D. Markovitz, manuscript submitted). However, BanLec also has significant mitogenic activity, and has been shown to induce T cell proliferation and cytokine production (60, 102), as well as cause local injection site reactions, piloerection, and weight loss in mice (E. Covés-Datson and D. Markovitz, manuscript submitted), thus limiting its potential therapeutic use. In 2015, we demonstrated that through rational molecular engineering, it is possible to significantly decrease lectin mitogenicity while preserving antiviral activity. This
100 was, to our knowledge, the first example of the ability to separate these two distinct functions of a lectin through molecular engineering. The decrease in mitogenicity was achieved through a specific amino acid substitution in which a histidine at position 84 is changed to a threonine
(H84T), which, through disruption of pi-pi stacking, appears to diminish the capacity of H84T
BanLec (H84T) to interact with multiple glycan molecules, which is required for mitogenicity.
However, importantly, H84T retains the potent antiviral activity of wild-type (WT) BanLec (60).
Although disruption of pi-pi stacking in H84T is associated with its decreased mitogenicity, it remained to be determined whether disruption of this interaction in another mitogenic antiviral lectin could reduce mitogenic activity without sacrificing antiviral potential.
In this work, we thus sought to understand whether we could recapitulate in another lectin the ability to separate two distinct lectin functions via targeted molecular engineering. That is, we asked whether disruption of pi-pi stacking in a structurally related but distinct lectin,
Malaysian banana lectin (Malay BanLec), would reduce mitogenicity of the engineered lectin without abolishing antiviral activity. We demonstrate that a single amino acid substitution, F84T, akin to the H84T mutation and predicted to remove the pi-pi stacking interaction, does indeed decrease mitogenicity while preserving antiviral activity. F84T Malay BanLec (F84T) still maintains some mitogenic activity, but it stimulates peripheral blood lymphocytes (PBLs) to proliferate to a lesser degree than does WT Malay BanLec. In addition, F84T treatment leads to the production of fewer cytokines and chemokines from peripheral blood mononuclear cells
(PBMCs) and may activate fewer CD4+ PBMCs than does WT Malay BanLec treatment.
Antiviral activity against HIV is maintained, and F84T has robust antiviral activity against human cytomegalovirus, and influenza, Ebola, varicella-zoster, and Lassa fever viruses. These results serve as a further proof-of-principle that pi-pi stacking is a determinant of mitogenicity
101 that may be disrupted to reduce mitogenic activity without removing antiviral activity. This discovery may signal new therapeutic potential for lectins, confirming that molecular engineering may allow for more widespread and targeted use of lectins through improvements in their clinical safety profiles.
3.3 Results
3.3.1 Malay BanLec is structurally similar to BanLec
Our previous work demonstrated that a single amino acid substitution (H84T) in an antiviral, mitogenic banana lectin (BanLec) led to a significant reduction in its mitogenicity (60).
Loss of the pi-pi stacking interaction between tyrosine 83 and histidine 84 in the ligand recognition loop of the lectin was associated with this decrease; this loop is located between the two carbohydrate binding sites (CBS) of the lectin and had been thought to perhaps refine binding specificity to complex glycans (57) and to control binding to multiple glycans, which promotes mitogenicity (60). We aimed to determine whether disruption of pi-pi stacking in a structurally related but distinct lectin could similarly decrease the mitogenic activity of another antiviral lectin. To do so, we first sought to identify another lectin predicted to have two CBS separated by a ligand recognition loop containing two aromatic amino acids capable of pi-pi stacking. A BLAST protein sequence alignment search revealed a candidate lectin from Musa acuminata ssp. malaccensis, which we term Malay banana lectin (Malay BanLec) (Figure 3.1).
Malay BanLec was predicted to have the desired structural features, including two CBS ligand binding loops characterized by the GXXXD sequence (56). Between the two CBS, as in wild- type (WT) BanLec, Malay BanLec has a tyrosine at position 83 (Y83), but instead of a histidine
102 at position 84 as in WT BanLec, Malay BanLec has a phenylalanine (F84). We would expect
Y83 and F84 in Malay BanLec to interact via pi-pi stacking. Thus, although Malay BanLec is quite a different lectin than BanLec with only 63% amino acid identity (differing at 52 of 141 amino acids), it is predicted to have important structural features in common with BanLec that make it a suitable candidate to engineer in a similar fashion with a view to reduce mitogenicity.
3.3.2 Malay BanLec has both mitogenic and antiviral activity
First, we assessed whether Malay BanLec was mitogenic and antiviral as we expected from its predicted structural similarity with BanLec. We cloned the codon-optimized sequence of
Malay BanLec containing a 6x His-tag with the sequence LEHHHHHH into the pET9a expression vector suitable for protein expression in E. coli. To assess the mitogenicity of Malay
BanLec, we incubated peripheral blood lymphocytes (PBLs) isolated from a healthy donor with a range of concentrations of WT, H84T, or D133G (non-carbohydrate-binding (60)) BanLec or with Malay BanLec for three days and then monitored incorporation of bromodeoxyuridine
(BrdU) over 18 h by ELISA (Figure 3.2A). As expected, treatment with D133G, which is neither antiviral nor mitogenic, resulted in minimal incorporation of BrdU. H84T treatment, which we have previously shown to be non-mitogenic, also resulted in minimal BrdU incorporation. In contrast, consistent with its known mitogenicity, WT BanLec caused PBLs to incorporate BrdU in a dose-dependent fashion, with increasing amounts of lectin resulting in increased incorporation of BrdU. Malay BanLec treatment resulted in greater BrdU incorporation than did
WT BanLec treatment at concentrations up to 10 nM, but less incorporation above that concentration, which may be due to cell death triggered by excessive mitogenic stimulation.
103 These results indicate that Malay BanLec is indeed mitogenic, and highly so as it is even more stimulatory than WT BanLec. To assess whether Malay BanLec has antiviral activity, we pretreated TZM-bl cells, which express a luciferase reporter for HIV infection, with varying concentrations of WT, H84T, or D133G BanLec, or Malay BanLec, and exposed them to HIV for 2 days (Figure 3.2B). Cells treated with D133G at even the highest concentration showed robust luciferase activity, indicating that D133G did not inhibit HIV infection. In contrast, treatment with all other lectins led to a dose-dependent decrease in luciferase activity with increasing concentration of lectin. H84T exhibited the least potent inhibition of viral infection, whereas Malay BanLec exhibited the most. Therefore, Malay BanLec, like WT and H84T
BanLec, does possess antiviral activity and perhaps has more potent activity against certain strains of HIV than does either WT or H84T BanLec. It should be noted that relative potency in the TZM-bl assay does not necessarily correlate with assays in primary cells (60). (Also see below and Table 3.3 in this chapter.)
Our previous work showed that it is not merely a matter of mutating the aromatic amino acid at position 84 in BanLec that allows for successful uncoupling of mitogenicity and antiviral activity, but also that the substituted amino acid is a threonine (60). Therefore, we next set out to generate a similar mutation in Malay BanLec as was done with WT BanLec. We designed primers to introduce the F84T mutation by changing the F84 codon to a threonine codon with the fewest number of nucleotide changes possible. We successfully generated the F84T mutation as confirmed by sequence analysis and were able to produce a recombinant protein in E. coli of the predicted size as assessed by Western blotting.
104
3.3.3 F84T Malay BanLec is less mitogenic than its WT counterpart
To compare the mitogenicity of WT versus F84T Malay BanLec, we exposed PBLs to
WT or F84T Malay BanLec for three days and then measured incorporation of BrdU over 18 h by ELISA (Figure 3.3A). For comparison, PBLs were exposed to WT or H84T BanLec in parallel. BanLec is a known potent T cell mitogen (58). In two different PBL donors, as expected, H84T-treated PBLs incorporated less BrdU than those that were treated with WT
BanLec. F84T-treated cells also incorporated less BrdU than WT Malay BanLec-treated cells, indicating that F84T is less mitogenic than WT Malay BanLec. It is evident, however, that F84T, though less mitogenic than its parent lectin, remained mitogenic and caused PBLs to proliferate more than either H84T or WT BanLec. We must note that for unclear reasons the removal of endotoxin from Malay BanLec and especially F84T is difficult and hence incomplete, which might contribute to mitogenicity as endotoxin is a B cell mitogen. Previous work with BanLec and H84T BanLec, originally performed when we did not routinely remove endotoxin from the preparations (60), suggests that this assay is not sensitive to certain levels of endotoxin, since
H84T BanLec preparations from which endotoxin has and has not been removed are both not mitogenic to PBLs. However, the levels of endotoxin in the preparations of F84T are much higher. That the preparation of F84T contained markedly more endotoxin than that of WT Malay
BanLec (when endotoxin removal was attempted, >10 versus 2.07 endotoxin units/mg, respectively) and was still less stimulatory to PBLs indicates that the F84T mutation did, as expected, decrease mitogenicity of the lectin and moreover suggests that the decrease in mitogenic activity in F84T may perhaps be underestimated.
105 Given that F84T treatment induced fewer PBLs to proliferate than did WT Malay BanLec treatment, we reasoned that CD4+ cells would become less activated in response to F84T as compared to WT Malay BanLec treatment. We therefore treated peripheral blood mononuclear cells (PBMCs) from three donors with WT or F84T Malay BanLec for 72 h and examined expression of activation markers (CD25 and CD69) on CD4+ PBMCs by flow cytometry
(Figures 3.3B and 3C). PBMCs were also treated with griffithsin (GRFT), a non-mitogenic antiviral lectin (103, 104), as a negative control and PHA, which is highly mitogenic, as a positive control. CD25 is the alpha chain of the IL-2 receptor and considered a mid-stage marker of T cell activation; CD69 is considered an early stage marker of T cell activation. As expected,
PHA induced high expression of both CD25 (Figure 3.3B) and CD69 (Figure 3.3C), whereas
GRFT did not. Although the differences were not statistically significant, F84T-treated CD4+
PBMCs tended to express less CD25 (Figure 3.3B) and less CD69 (Figure 3.3C) than those treated with WT Malay BanLec, suggesting that they were less activated and supporting the finding that F84T induces less proliferation of PBLs than does WT Malay BanLec. There appeared to be a dose effect trend, with higher concentrations of either lectin inducing higher expression of activation markers, more so with CD69 than CD25. Variation in human donor
PBMC responses likely accounts for the lack of statistically significant data, as examination of the individual responses reveals clear differences between the F84T- and WT Malay BanLec- treated cells (Table 3.1).
In order to further refine our understanding of the mitogenic differences between WT and
F84T Malay BanLec, we next treated PBMCs from multiple healthy donors with WT or F84T
Malay BanLec, or with GRFT for 72 h (Figures 3.3D-F). Supernatants from all lectin-treated
PBMCs were collected and analyzed for the expression of a panel of cytokines and chemokines
106 by Bio-Plex assay. Cytokine and chemokine signals secreted by lymphocytes in response to mitogenic stimuli cause them to proliferate (49). GRFT stimulated only minimal expression of cytokines and chemokines. Consistent with the two assays above, F84T-treated PBMCs induced the expression of cytokines and chemokines, but overall, greater cytokine and chemokine expression was seen in WT Malay BanLec-treated cells. F84T and WT Malay BanLec stimulated similar percentages of PBMCs to secrete IL-1b, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-15, eotaxin, basic FGF, GM-CSF, IFN-g, MCP-1, MIP-1a, MIP-1b at similar levels. F84T treatment induced somewhat higher expression of IL-7, IL-8, IL-9, PDGF-BB from a greater percentage of
PBMCs as compared to WT treatment. However, a greater percentage of WT-treated PBMCs expressed higher levels of IL-1RA, IL-2, IL-13, IL-17A, G-CSF, IP-10, RANTES, TNF-a, and
VEGF. IL-2 is an important stimulus for progression of T cells to S phase (49), so it is notable that F84T stimulates less expression of this cytokine from PBMCs than does WT. Additionally,
IL-1RA, IL-17, TNF-a, and VEGF are all key inflammatory cytokines that have been targeted clinically. Taken together, these studies indicate that F84T induces less proliferation of PBLs, less expression of key cytokines/chemokines from PBMCs, and perhaps less activation of CD4+
PBMCs than WT Malay BanLec, indicating that F84T is less mitogenic than WT Malay BanLec.
It still does, however, possess rather significant mitogenic properties, perhaps due to a high level of endotoxin that we have been unable to remove to this point.
3.3.4 F84T Malay BanLec retains robust antiviral activity
Before assessing whether F84T maintained antiviral activity against HIV, we first sought to assess whether F84T could bind HIV, as certain mutations (like D133G) in lectins abolish
107 carbohydrate-binding activity (60), which in turn disrupts antiviral activity. HIV is highly N- glycosylated and BanLec, as a mannose-binding lectin, binds to HIV and inhibits attachment of the virus to host cells (78). With its structural similarity to BanLec, Malay BanLec is predicted to also bind to HIV, which is supported by its inhibition of the virus (Figure 3.2B). To assess binding of WT versus F84T to HIV, we conducted surface plasmon resonance analysis to determine the binding times to, and binding affinities for, the gp140 trimer, which is made up of uncleaved ectodomains of the gp160 HIV envelope glycoprotein trimer (Figure 3.4). The results show that F84T and WT Malay BanLec, as well as GRFT, bind with high affinity to the gp140 trimer. This was expected as these lectins bind to high-mannose structures that are highly expressed on the trimer. Even after optimization of the assay, the WT and F84T Malay BanLec showed linear association curves, a hallmark of mass transport limitation, and the evaluation software also indicated that mass transport limitation was present. Mass transport limitation occurs when local concentration gradients of the analyte (lectin) develop after the analyte binds to and dissociates from the ligand (gp140 trimer) and affect the observed binding kinetics of the analyte to the ligand. The mass transport coefficient was therefore taken into account in the fitting model. It is clear that WT and F84T Malay BanLec bind 4 and 8 times slower, respectively, to the gp140 trimer than does GRFT and dissociate 5 and 3 times slower, respectively, than does GRFT. Even though their kinetic properties differ, WT Malay BanLec and GRFT obtain quite similar binding affinities (KD) and F84T Malay BanLec has a slightly higher KD (Table 3.2).
Having confirmed that F84T retains the ability to bind to the HIV glycoprotein, we next assessed whether it maintained anti-HIV activity. To do so, we pretreated TZM-bl cells with
F84T or WT Malay BanLec, or with H84T or WT BanLec, exposed them to HIV for 2 days, and
108 measured luciferase activity as a readout for HIV infection. As shown in Figure 3.5, WT BanLec had more potent antiviral activity than did H84T BanLec, and WT Malay BanLec exhibited the most potent anti-HIV activity. F84T robustly reduced luciferase activity, indicating that it retained the ability to inhibit HIV infection. The potency of F84T in this assay was similar to that of WT BanLec. From this and the above study, it is clear that F84T binds to the HIV glycoprotein and maintains anti-HIV activity.
To further characterize the antiviral activity of F84T, we tested its ability to inhibit various strains of HIV as compared to that of WT Malay BanLec via two approaches (Table 3.3).
Inhibitors of the HIV co-receptors CXCR4 and CCR5, AMD3100 and maraviroc, respectively, were included as controls. First, CD4+ MT-4 cells, which are CXCR4-positive but CCR5- negative, were pre-treated with a range of concentrations of lectin (F84T or WT Malay BanLec) or AMD3100 and then incubated with three different X4 strains of HIV-1 and HIV-2 for 5 days.
CPE was measured to calculate 50% inhibitory concentration (IC50) values. Secondly, PBMCs were pre-treated as above with lectin, AMD3100, or maraviroc before exposure to CXCR4-,
CCR5-, or dual-tropic HIV-1 strains for 10 days. Supernatants containing virus were collected and viral production measured by HIV-1 p24 capsid antigen ELISA to calculate IC50 values.
F84T inhibited all strains of HIV-1 (with all tropisms) and HIV-2 tested in these assays in a low nanomolar to low micromolar range. F84T was found to have a lower IC50 value than WT Malay
BanLec against two of three strains tested in MT-4 cells and a higher IC50 value than WT Malay
BanLec against two of the three strains tested in PBMCs, but overall it appears that the antiviral activity of F84T against HIV is not only preserved but also not diminished.
As H84T is known to have broad-spectrum antiviral activity, we next sought to characterize whether F84T could inhibit additional viruses that are associated with human
109 disease and are known to express mannose-containing N-glycans on their surfaces (Table 3.4).
F84T had moderate to high activity against influenza A, varicella-zoster, human cytomegalovirus, Ebola, and Lassa fever viruses, indicating that like H84T it has broad-spectrum antiviral activity.
3.3.5 The F84T mutation disrupts pi-pi stacking without significant structural changes
Given that the antiviral activity of F84T was maintained, we predicted that the F84T mutation did not significantly alter the structure of Malay BanLec beyond disruption of pi-pi stacking, as was the case with H84T versus WT BanLec. In order to compare F84T and WT
Malay BanLec, we solved their crystal structures (Table 3.5). First, we aligned the structure of
WT Malay BanLec to that of WT BanLec (Figure 3.6A). The structures are remarkably similar, with a root-mean-square deviation (RMSD) between all atoms of only 0.517 Å. Like BanLec,
Malay BanLec has a b-prism-I fold characteristic of the jacalin-related lectin family. Both Malay
BanLec and BanLec have a pi-pi stacking interaction between amino acid 84 (F and H, respectively) and Y83 within the ligand recognition group between CBS I and II. However, the aromatic rings involved in pi-pi stacking are arranged in a planar (sandwich) conformation in
BanLec, whereas they arranged in an edge-on conformation in Malay BanLec (Figure 3.6B). The
F84T mutation in Malay BanLec disrupts pi-pi stacking without significant changes to the overall structure of the monomer (Figure 3.6C) or to the dimer (Figure 3.6D). F84T and Malay
BanLec have an almost identical structure, with an RMSD of 0.424 Å between all atoms.
110 3.4 Discussion
Lectins have immense potential as antiviral agents as they are highly specific for their target glycans, but clinical development of promising broad-spectrum antiviral lectins has been halted by the inability to separate mitogenic from antiviral activity in these molecules (48). The ability to fine-tune lectin function by separating distinct activities of lectins, such as mitogenicity and inhibition of viral replication, would be of great usefulness in designing therapeutic lectins with reduced mitogenicity (and perhaps increased efficacy), and in understanding the basic functions of lectins. We showed in our previous work with BanLec (60), in the first apparent example of its kind, that the separation of mitogenic and antiviral activity is possible via targeted molecular engineering. A single amino acid substitution (H84T) that disrupts pi-pi stacking in the ligand recognition loop of the molecule is associated with the decrease in mitogenicity. Here we again demonstrate that disruption of pi-pi stacking in the ligand recognition loop of another lectin, Malay BanLec, is able to separate mitogenic and antiviral activity to an extent. It is quite exciting that molecular engineering aimed to eliminate the same structural element (pi-pi stacking) with the same substituted amino acid (threonine 84) resulted in separation of mitogenic and antiviral activities in two lectins, BanLec and Malay BanLec, that are structurally related but only ~60% similar at the amino acid level. This is to our knowledge only the second instance of separation of two distinct lectin functions, and suggests that, while pi-pi stacking is found at active sites in only a select number of lectins, molecular fine-tuning of lectins according to structural principles is feasible and can be predicted.
F84T itself clearly still retains a high degree of mitogenic activity and is thus not a likely candidate for clinical development. However, we provide proof-of-concept that pi-pi stacking
111 within the ligand recognition loop is a determinant of mitogenicity that can be disrupted to reduce mitogenic activity without abolishing antiviral activity. Given the high mitogenic activity retained by F84T, it is likely that other elements confer mitogenicity in this lectin, though incomplete removal of endotoxin from F84T might also account for some mitogenicity. There are perhaps other determinants of mitogenicity to discover by comparing a still mitogenic lectin
(F84T) to a non-mitogenic lectin (H84T), as Malay BanLec and BanLec differ at ~40% of their amino acid sequences. The H84T mutation is thought to disrupt the multivalent interactions required for mitogenic stimulation by decreasing binding of glycans to CBS II (60); the mitogenicity of lectins depends on the ability to cross-link receptors, such as the T cell receptor, on immune cells (49). It is therefore possible that the F84T mutation does not fully interfere with the ability of the lectin to participate in multivalent interactions such as those that would occur when cross-linking immune cell surface receptors, and so it retains some mitogenicity. Of particular interest to investigate would be the specific amino acid residues that differ within the carbohydrate binding sites and ligand recognition loops between F84T and H84T. Comparing antiviral lectins that are naturally mitogenic (such as Malay BanLec and BanLec) and those that are non-mitogenic (such as GRFT) could also help elucidate determinants of mitogenicity that could be removed without destroying antiviral activity.
It should be noted that precisely how lectins induce mitogenicity is not completely understood. Cross-linking of T cell receptors (TCR) by lectins is proposed to mediate mitogenicity, at least for certain lectins (49, 60), but how the lectin and the TCRs interact is not known. It is presumed that they interact via binding to glycans on TCRs, as TCRs are glycosylated. Part of the difficulty in elucidating where and how lectins might bind to TCRs is that a full understanding of how lectins bind to glycans beyond mono- and di-saccharides has not
112 yet been attained. While it is clear that the amino acid residues within the CBS and ligand recognition loops are important in determining binding specificity to different glycans (105), we have not yet achieved the ability to precisely predict to which complex glycans a lectin with known specificity will or will not bind. For example, though BanLec is known to have high- mannose-binding activity, it does not inhibit all viruses that express high-mannose N-glycans
(e.g., HSV-1, RSV), which may mean that it cannot efficiently bind to these viruses (unpublished data). The ability to decrease mitogenic activity in naturally mitogenic lectins is likely to help illuminate which specific interactions with cells lead to mitogenicity.
Effective therapies are lacking for most viral illnesses, and most of the existing antiviral agents are capable of inhibiting only one or a few viruses, i.e., they are narrow-spectrum. This is distinct from the case of treating bacterial illnesses, where broad-spectrum antibiotics are often available. In sharp contrast to many of the currently available antiviral therapies, lectins often have broad-spectrum antiviral activity, which would be beneficial for both targeted and empiric treatment of viral illnesses. Both H84T and F84T have broad-spectrum antiviral activity across a range of viruses from different families, including HIV, Ebola virus, influenza virus, and varicella-zoster virus, as they bind and presumably bind, respectively, to high-mannose type N- glycans that are on the surface of many viruses. Other lectins with broad-spectrum antiviral activity like CVN could potentially be made less mitogenic via molecular engineering.
In this work, we demonstrate that Malay BanLec and BanLec are structurally highly similar, though they share only ~60% amino acid identity. Disruption of the pi-pi stacking interaction is achieved via the F84T mutation in Malay BanLec without overall structural changes to the lectin. As hypothesized, taking down this “mitogenic wall” (60) leads to an overall decrease in mitogenicity. Importantly, F84T retains the ability to bind to HIV and retains
113 antiviral activity against multiple strains of HIV-1 and HIV-2 with different tropisms. It is also broad-spectrum, being quite active against human cytomegalovirus, and varicella-zoster, influenza, Ebola, and Lassa fever viruses. In contradistinction to most healthy human cells, a number of malignancies display high-mannose or other unusual carbohydrates on their surfaces
(20, 106, 107), and thus molecularly engineered lectins also hold promise as anti-cancer agents.
Lectins are highly specific for their targets, having evolved for millions of years, and have shown great antiviral potential that has been limited by their mitogenicity. If we could harness a greater understanding of the mitogenic and effector elements of lectins, we might be able to unlock their vast antiviral and anti-cancer potential to treat an array of human illnesses.
3.5 Experimental Procedures
3.5.1 Lectin production
Recombinant His-tagged D133G BanLec, WT Malay BanLec, and F84T Malay BanLec were produced in and harvested from E. coli as previously described (60). Non-His-tagged
H84T, which is used throughout this study, was produced similarly, except that Sephadex G-75 columns were used in place of Ni-NTA agarose columns for purification and elution was performed with 0.2 M methyl-a-D-glucopyranoside. Endotoxin testing for all lectins was performed with the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher
Scientific). Endotoxin removal was then performed by adding 1 M glucose and passing pooled eluates containing protein through Mustang E filters (Pall). Endotoxin removal was performed to yield lectin with < 0.1 endotoxin units/mg of protein for lectins besides F84T and WT Malay
BanLec; removal of endotoxin from these lectins was less efficient. After removal of endotoxin, glucose was removed and lectin concentrated in water using the Vivaspin 20 centrifugal unit
114 with 3K MWCO. Finally, endotoxin and protein concentrations were measured via LAL and
BCA assays, respectively.
3.5.2 F84T mutation of Malay BanLec
Site-directed mutagenesis to introduce the F84T mutation into Malay BanLec was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer’s instructions. Sense and antisense primers were designed using the
QuikChange Primer Design software: sense 5'-catggagggtcacgtggttgattatactggcctgaccatc-3' and antisense 5'-gatggtcaggccagtataatcaaccacgtgaccctccatg-3'. Two nucleotide changes were introduced via these primers to change the F84 codon (TTT) to a threonine codon (ACT).
Following the PCR reaction, plasmids were transformed into BL21-CodonPlus Competent Cells and colonies screened by Sanger sequencing for successful mutagenesis.
3.5.3 Assessment of anti-HIV activity by TZM-bl cell infection
As previously described (60), 100 µl of a 1 x 105 cells/ml suspension of TZM-bl cells
(HELA cells engineered to express CD4, CXCR4, and CCR5 and containing the HIV long terminal repeat upstream of a luciferase gene) were added to each well of a white 96-well plate.
Cells were pre-treated with lectins (or PBS as a control) at 2X the final concentration for 30 min before the addition of 100 µl of the HIV strain BaL. Two days post-infection, medium was removed and 100 µl of ONE-Glo Luciferase reagent (Promega) added to determine luciferase expression.
115 3.5.4 Assessment of mitogenic activity by BrdU incorporation in PBLs
As previously described (60), PBLs were isolated and resuspended in RPMI medium containing 10% fetal bovine serum at a concentration of 2 x 106 cells/mL. 50 µL of cell suspension were added per well of a white 96-well plate followed by 50 µL of medium containing lectins at various concentrations or PBS. The cells were incubated at 37°C for 3 d prior to an 18 h addition of BrdU. Proliferation was measured by BrdU incorporation, which was detected via a chemiluminescence-based ELISA (Cell Proliferation ELISA (chemiluminescent);
Roche) as per the manufacturer’s instructions.
3.5.5 Evaluation of cellular activation markers
PBMCs treated for 3 d with the indicated concentrations of lectins were analyzed by flow cytometry following dual fluorescent staining with anti-mouse antibodies purchased from BD
Pharmingen (San Diego, CA). Briefly, cell cultures were transferred from plates to 5 ml round bottom tubes and washed with PBS containing 5% inactivated FBS (washing solution). After 10 min blocking with purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block), cells were incubated in the dark with FITC-conjugated anti-CD4 mAb in combination with PE-conjugated anti-CD25 or anti-CD69 mAb for 30 min on ice. Finally, PBMCs were washed and analyzed with a FACSCelesta flow cytometer (BD, San Jose, CA), counting 10,000 events per sample.
Data were acquired and analyzed using FlowJo from BD. PHA and GRFT were used as positive and negative controls, respectively.
116 3.5.6 Bio-Plex cytokine/chemokine assay of human PBMC supernatants
PBMCs from multiple blood donors were cultured for 72 h in the presence of 10 µg/ml
WT or F84T Malay BanLec, 10 µg/ml GRFT, or 2 µg/ml PHA. In the culture supernatants, the concentrations of IL-1α, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-
15, IL-17, eotaxin, FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB,
RANTES, TNF-α, and VEGF were determined by the Bio-Plex 200 system (Bio-Rad) and Bio-
Plex Human Cytokine 27-plex assay according to the manufacturer's instructions. Data were generated with the Bio-Plex Manager 4.1 software.
3.5.7 Surface plasmon resonance (SPR) analysis
SPR technology was used to determine the binding of WT Malay BanLec, F84T Malay
BanLec, and GRFT to immobilized gp140 on a Biacore T200 instrument (GE Healthcare,
Uppsala, Sweden). BG505 SOSIP.664 gp140 trimers (108) (kindly provided by Dr. R.W.
Sanders) were immobilized on an NTA sensor chip using a capture coupling method. First, 0.5 mM Ni2+ was injected for 60 seconds to activate the NTA surface, then EDC/NHS 1:1 was injected for 60 seconds to activate the carboxyl groups. Following this, the gp140 trimers were captured and covalently bonded onto the surface by injecting them for 120 seconds with a concentration of 0.25 µg/ml diluted in HBS-P+ (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20; pH 7.4) immobilization buffer. Afterwards, the surface was deactivated by a 60- second injection of 1.0 M ethanolamine-HCl pH 8.5 and regenerated with 350 mM EDTA to remove any remaining bound ligand. All steps of the immobilization were performed with an injection speed of 10 µl/min. Interaction studies were performed at 25°C in HBS-EP+ (10 mM
HEPES, 150 mM NaCl, 30 mM EDTA, 0.05% surfactant P20; pH 7.4). The lectins were serially
117 diluted, covering a concentration range between 0.63 and 10 nM by using 2-fold dilution steps for the WT and F84T Malay BanLec and between 0.12 and 10 nM using 3-fold dilution steps for
GRFT. Samples were injected, using single cycle kinetics, for 2 minutes at a flow rate of 30 µl/min and the dissociation was followed for 10 minutes after the final injection. The sensor chip surface was regenerated with a 20 second injection of 50 mM NaOH followed by a
30 second injection of 10 mM glycine-HCl pH 1.5. For the WT and F84T Malay BanLec, this regeneration step was performed twice as they were difficult to regenerate. Several buffer blanks were used for double referencing. Apparent binding kinetics (KD, ka, kd) were derived after fitting the experimental data to the 1:1 binding model in the Biacore T200 Evaluation Software 3.1.
3.5.8 Anti-HIV cellular assays
We adapted the protocol as described in detail previously (109). Briefly,
CD4+CXCR4+ MT-4 cells (1x106 cells/ml; 50 µl) were seeded in cell culture medium in a 96- well plate (Falcon, BD Biosciences, Erembodegem, Belgium) and pre-incubated with 5-fold dilutions of test compound (100 µl) at 37°C for 30 min, in duplicate. Virus (50 µl; HIV strains
NL4.3, HE, or ROD) was added according to cell culture infectious dose (CCID50), which was determined by titration of the stock. After an incubation period of 5 days, virus-induced cytopathic effect (CPE) was scored with light microscopy and IC50 was calculated using the spectrophotometric MTS/PES viability staining assay (Cell-Titer96 Aqueous One Solution
Proliferation Assay kit; Promega, Leiden, The Netherlands). Absorbance was measured using the
Versamax microplate reader and SoftMax Pro software (Molecular Devices, Sunnyvale, CA,
USA). To determine the potential cellular cytotoxicity of the compounds, the assays were also
118 performed without the addition of virus. The reference compounds AMD3100 or maraviroc were included as controls in all assays.
The HIV-1 p24 Ag ELISA-based antiviral assay was performed in PBMCs, as described previously (110). PHA-stimulated PBMCs (5x105 cells/ml; 200 µl) were seeded in 48-well plates
(Costar, Elscolab NC, Kruibeke, Belgium) and pre-incubated for 30 minutes with various concentrations of test-compound (250 µl) in the presence of 2 ng/ml interleukin 2 (IL-2) (Roche
Applied Science, Vilvoorde, Belgium). Next, virus (HIV-1 NL4.3, HE, or BaL) was added and 2 ng/ml IL-2 was supplemented again at day 3 and 6 after infection. After 10 days of infection, supernatant was collected and stored. Viral replication was determined by HIV-1 p24 Ag ELISA
(Perkin Elmer) according to the manufacturer’s instructions.
3.5.9 Neutral red assay for assessment of anti-influenza virus activity
MDCK cell culture monolayers in a 96-well microplate were treated with 0.1, 1.0, 10, or
100 µg/ml of F84T in MEM medium supplemented with 10 mg/mL EDTA and 1 IU/mL trypsin.
Three wells were infected with <100 CCID50 virus and two remained uninfected for toxicity
(CC50) measurements. Plates were incubated for 3 days at 37ºC with 5% CO2, then stained with
0.011% neutral red, a vital stain. The incorporated dye was eluted with 50% Sorensen’s citrate buffer/50% ethanol then read on a spectrophotometer at 540 nm. ODs were converted to percent cytopathic effect compared with untreated cell controls and normalized to virus controls. The
50% effective concentrations (EC50) and 50% cytotoxic concentration (CC50) were then calculated by regression analysis.
119 3.5.10 Anti-varicella-zoster virus (VZV) cellular assays
VZV plaque reduction assays were performed as described previously (111). Briefly, human foreskin fibroblast (HFF) cells were seeded into 6-well plates. After seeding (48 h), they were infected with virus at a concentration that would yield 25-30 plaques per well. After 1 h incubation, the drug was serially diluted 1:5 at 6 different concentrations and added to the wells.
The plates were incubated for 10 days, stained with a 1% neutral red solution (Sigma), and counted using a stereomicroscope. EC50 and CC50 values were calculated using the MacSynergy program.
3.5.11 Anti-CMV cellular assays
CMV titer reduction assays were performed as follows: 96-well microtiter plates were seeded 24 h prior to infection. Cells were then infected with a high MOI of either AD169 or
GDGrK17 virus for 1 h. The cells were washed and 7 concentrations of serially diluted drugs were added. These plates were incubated for 72 h. The plates were then freeze-thawed twice and spun to remove debris. Using a BioMek 4000 autodilutor, the supernatants were serially diluted into a 384-well plate containing HFF cells. After 14 days the plates were either stained with
CellTiter Glo (Promega) and read on a luminometer or the DNA extracted with Extracta (Quanta
Bio) and qPCR assays performed. EC90 values were calculated using an Excel program.
3.5.12 Cytotoxicity and plaque reduction assays for assessment of anti-Ebola and Lassa fever virus activity
The cytotoxicity assay (In vitro Toxicology Assay Kit, Neutral red based; Sigma) was performed in 96-well plates following the manufacturer’s instructions. Briefly, growth medium
120 was removed from confluent Vero E6 cell monolayers and replaced with fresh medium (total of
100 µl) containing F84T. Control wells contained medium with the positive control or medium devoid of lectin. Wells without cells and growth medium only served as blank. A total of up to five replicates were performed for each condition. Plates were incubated for 5 days (length of incubation time for Lassa virus plaque assay) and 10 days (length of incubation time for Ebola virus plaque assay), respectively, at 37ºC with 5% CO2. The plates were stained with 0.033%
o neutral red for approximately two hours at 37 C in a 5% CO2 incubator. The neutral red medium was removed by complete aspiration, and the cells rinsed 1x with PBS to remove residual dye.
The PBS was completely removed and the incorporated neutral red eluted with 1% acetic acid/50% ethanol for at least 30 minutes. The dye content in each well was quantified using a 96- well spectrophotometer at 540 nm wavelength and 690 nm wavelength (background reading).
The 50% cytotoxic (CC50, cell-inhibitory) concentrations were calculated by linear regression analysis.
For the plaque reduction assay, confluent or near-confluent Vero E6 cell culture monolayers in 12-well disposable cell culture plates were prepared. Cells were maintained in
DMEM supplemented with 10% FBS. For antiviral assays the same medium was used but with
FBS reduced to 5% or less and supplemented with 1% penicillin/streptomycin. F84T was prepared in 2x DMEM and mixed 1:1 with virus prior to infection of cells. The virus control and cell control were run in parallel and the assay performed in biological triplicate. The assay was initiated by first removing growth media from the 12-well plates of cells, and infecting cells with
0.01 MOI of virus or about 50 to 100 plaque forming units (pfu) in the presence of lectin. Cells were incubated for 60 min: 100 µl inoculum/well, at 37°C, 5% CO2 with constant gentle rocking.
For Lassa virus, virus inoculum was removed, cells washed and overlaid with 0.8% tragacanth
121 diluted 1:1 with 2x DMEM and supplemented with 5% FBS and 1% penicillin/streptomycin and supplemented with the corresponding lectin concentration. For Ebola virus, virus inoculum was removed, cells washed and overlaid with 0.5% methylcellulose diluted 1:1 with 2x DMEM and supplemented with 2% FBS and 1% penicillin/streptomycin and supplemented with the corresponding lectin concentration. Cells were incubated at 37°C with 5% CO2 for 5 days for
Lassa virus and for 10 days for Ebola virus, respectively. Plotting the log10 of the inhibitor concentration versus log10 of virus produced at each concentration allowed calculation of the
50% (one log10) effective concentration by linear regression.
3.5.13 Crystallization and Structure Determination of WT and F84T Malay BanLec
WT and F84T Malay BanLec were each concentrated to 5 mg/mL in buffer containing 10 mM HEPES, pH 7.5 and 150 mM NaCl. Crystals of WT Malay BanLec grew at 20°C in drops containing equal volumes of protein and precipitating solution (1.6 M ammonium sulfate, 2% polyethylene glycol 2K, and 0.1 M HEPES, pH 7.5). Prior to data collection, crystals were cryoprotected in well solution containing 20% ethylene glycol. Crystals of F84T Malay BanLec also grew at 20°C from drops containing equal volumes of protein and well solution (1.8 M ammonium sulfate, 5% polyethylene glycol 400, and 0.1 M MES, pH 6.5) and were cryoprotected in 1.8 M lithium sulfate, 5% polyethylene glycol 400, and 0.1 M MES, pH 6.5.
Data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G at the
Advance Photon Source at Argonne National Laboratory. Data were processed with HKL2000
(112) and the structures were solved by molecular replacement using Phaser (113) for WT and
MolRep (114) for F84T Malay BanLec with PDB 2BMY as a search model. The structures were iteratively refined with Buster (115) and fit with COOT (116). WT Malay BanLec crystallized in
122 the P43212 space group with 1 dimer in the asymmetric unit and diffracted to 1.5 Å resolution.
F84T Malay BanLec crystallized in space group P212121 with 16 dimers in the asymmetric unit and diffracted to 1.8 Å resolution. See Supplementary Table 3.1 for data collection and refinement statistics.
123
Figure 3.1 Malay BanLec is structurally similar to BanLec. Amino acid sequence alignment of BanLec and Malay BanLec (middle rows) from amino acids 1 (top left) to 142 (bottom right). The 6x His-tag (LEHHHHHH) is indicated at the end of the Malay BanLec sequence. The amino acids predicted to undergo pi-pi stacking (“pi-pi”) and the predicted ligand binding loops of the carbohydrate binding sites (“ligand binding”) in Malay BanLec are outlined in black and red, respectively, aligned with those known sites in BanLec. Sites of amino acid identity (“consensus”) are indicated in the top row in the colored boxes; “x” indicates that identity is not shared at a particular site. The bottom row (“sequence logo”) depicts the sequence of both lectins, with amino acids from both sequences represented if there is not identity at a particular site.
124
Figure 3.2 Malay BanLec has both mitogenic and antiviral activity. (A) BrdU incorporation into peripheral blood lymphocytes (PBLs). PBLs from a healthy human donor were treated with a range of concentrations of WT, H84T, or D133G BanLec, or Malay BanLec for 3 d and then BrdU incorporation over 18 h was measured by chemiluminescent ELISA. RLU = relative light units. (B) TZM-bl cells were pre-treated with the same lectins as in A and exposed to HIV BaL for 2 d. To assess levels of infection, luciferase activity was measured. Data in A and B are representative of one independent experiment each. Error bars represent the SEM of triplicate values. Work in A and B performed by S.R.K.
125
Figure 3.3 F84T Malay BanLec is less mitogenic than is WT Malay BanLec. (A) BrdU incorporation into PBLs. PBLs from healthy human donors were treated with a range of concentrations of WT or F84T Malay BanLec, or WT or H84T BanLec for 3 d and then BrdU incorporation over 18 h was measured by chemiluminescent ELISA. Data are representative of two independent experiments using cells from two donors, with data from one donor shown. Error bars represent the SEM of triplicate values. (B and C) Activation markers in lectin-treated peripheral blood mononuclear cells (PBMCs). PBMCs from three healthy human donors were treated with a range of concentrations of WT Malay BanLec (WT) or F84T Malay BanLec (F84T), griffithsin (GRFT), or PHA and the percentage of CD4+ CD25+ (B) or CD4+ CD69+ (C) cells assessed by flow cytometry. GRFT, known to be virtually non-mitogenic, serves as the negative control, whereas PHA serves as the positive control. Bars represent the average values from the three donors and error bars the SEM. Black asterisks indicate statistically significant differences between the means in the GRFT group and the corresponding means in the WT group. Blue asterisks indicate statistically significant differences between the mean in the PHA group and the mean in the control group. *P < 0.05, **P < 0.01, ****P < 0.0001. (D to F) Cytokine/chemokine secretion in lectin-treated PBMCs. PBMCs from multiple healthy donors
126 were treated with GRFT (D), WT (E), or F84T Malay BanLec (F) and supernatants collected for analysis of expression of various cytokines and chemokines by BioPlex assay. Results are expressed as the percentage of PBMC donors whose stimulated PBMCs express the indicated fold increase values of cytokine/chemokine concentrations with respect to untreated PBMCs. Work in B-D performed by S.C.
127
Figure 3.4 F84T Malay BanLec binds to HIV gp140 as determined by surface plasmon resonance. Single cycle kinetics of WT Malay BanLec (A), F84T Malay BanLec (B) and GRFT (C) binding to gp140. The concentrations of the injected lectins ranged from 0.63 to 10 nM for WT and F84T Malay BanLec and from 0.12 to 10 nM for GRFT. The red lines indicate the actual response measured during the experiment, the black lines indicate the 1:1 Langmuir model fitting. WT and F84T Malay BanLec bind 4 and 8 times slower, respectively, to the gp140 trimer than does GRFT and dissociate 5 and 3 times slower, respectively, than does GRFT. The binding affinities, -10 KD, of WT Malay BanLec, F84T, and GRFT are 1.22 ± 0.62, 3.68 ± 0.41, 1.37 ± 0.35 (10 ) (M), respectively. The graphs depict one of the three replicate experiments performed. Work in A-C performed by A.B.
128
Figure 3.5 F84T retains antiviral activity against HIV. TZM-bl cells were pre-treated with WT or F84T Malay BanLec, or WT or H84T BanLec and exposed to HIV BaL for 2 d. To assess levels of infection, luciferase activity was measured. Data are representative of two independent experiments. Error bars represent the SEM.
129
Figure 3.6 The F84T mutation interrupts pi-pi stacking between Y83 and F84 without altering the overall structure of Malay BanLec. (A and B) Crystal structures comparing WT BanLec (cyan) to WT Malay BanLec (navy). H84 in BanLec and F84 in Malay BanLec are indicated. Y83 exists in both structures. In (A) the alignment of the monomers is depicted, and in (B) a close-up of the ligand binding loops showing the pi-pi stacking interactions between H84 and Y83 and F84 and Y83 in WT BanLec and Malay BanLec, respectively. (C and D) Crystal structures comparing WT Malay BanLec (navy) to F84T Malay BanLec (yellow). In (C) two views of the alignment of the monomers are depicted. F84 in WT Malay BanLec and T84 in F84T Malay BanLec are indicated, along with the Y83 in common. CBS I and II are also shown, with the ligand binding loop containing the residues of interest in between them. NT = N-terminus. CT = C-terminus. In (D) the alignment of the dimers is depicted. Work in A-D performed by J.L.M.
130 Table 3.1 Expression of cellular activation markers by F84T- versus WT-treated CD4+ PBMCs. Work performed by S.C.
Donor 1 Donor 2 Donor 3 CD4+ CD4+ CD4+ CD4+ CD4+ CD4+ CD25+ CD69+ CD25+ CD69+ CD25+ CD69+
Control 2.69 0.60 2.14 0.67 2.69 0.78
0.4 8.02 5.10 7.50 6.42 9.68 11.70 µg/ml 2 WT 9.28 7.96 8.09 9.74 11.00 17.20 µg/ml 10 10.40 7.73 8.18 15.60 10.50 16.70 µg/ml
0.4 5.31 3.63 5.73 4.75 7.12 8.26 µg/ml 2 F84T 5.27 3.75 5.83 6.06 8.13 10.80 µg/ml 10 5.87 3.83 6.45 6.40 8.29 11.40 µg/ml
0.4 3.44 0.36 2.34 0.78 2.51 0.77 µg/ml 2 GRFT 2.94 0.32 2.41 0.72 2.94 0.62 µg/ml 10 3.18 0.46 2.54 1.41 2.73 0.68 µg/ml
2 PHA 17.40 23.80 11.70 19.50 12.80 22.30 µg/ml
131 Table 3.2 Average and standard deviation of kinetics obtained from three replicate SPR experiments measuring the binding response between BG505 SOSIP.664 gp140 trimers and lectins (WT Malay BanLec, F84T Malay BanLec, and GRFT). Work performed by A.B. ka, association rate constant; kd, dissociation rate constant; KD, binding affinity.
gp140 chip 6 -4 -10 Sample ka (10 )(1/M.s) kd (10 )(1/s) KD (10 )(M) WT 1.33 ± 0.36 1.40 ± 0.25 1.22 ± 0.62 F84T 0.72 ± 0.03 2.66 ± 0.29 3.68 ± 0.41 GRFT 5.56 ± 0.80 7.40 ± 1.09 1.37 ± 0.35
132 Table 3.3 Inhibition of multiple strains of HIV by F84T versus WT Malay BanLec. Work performed by S.C.
IC50 (ng/ml)
HIV-1 HIV-1 HIV-2 CC50 (µg/ml) MT-4 NL4.3 HE ROD
WT 24.3 18.5 23.6 1.7 F84T 34.6 6.3 18.5 >20 AMD3100 15.3 11.8 7.4 >10
IC50 (ng/ml)
HIV-1 HIV-1 HIV-1 CC50 (µg/ml) PBMC NL4.3 (X4) HE (dual) BaL (R5)
WT 8.3 36.5 143.8 >1 F84T 29.6 66.5 122.8 >1 AMD3100 17.1 >100 >100 >1 Maraviroc >100 >100 4.9 >1
133 Table 3.4 Inhibition of multiple viruses by F84T Malay BanLec. Work performed by L.Z., B.K., C.D., A.N.F., and M.P.
Virus Strain Cell line EC50 CC50 Influenza A California/07/2009 (H1N1)pdm09 MDCK 0.21 µg/mla 24 µg/mla Ellen HFF 0.021 µMa 0.4 µMa Varicella-zoster 15-6-001 (resistant isolate) HFF 0.016 µMa 0.4 µMa Human AD169 HFF <0.05 µMb 2.93 µMb cytomegalovirus GDGrK17 (resistant isolate) HFF 0.08 µMc 7.21 µMb Ebola Zaire Vero <0.1 µg/mld 4.9 µg/mla Lassa fever Josiah Vero 0.3 µg/mld 4.95 µg/mla Assay used to determination the 50% effective concentration (EC50) or 50% cytotoxicity concentration a b c d (CC50): neutral red, CellTiter-Glo, qPCR for viral DNA, crystal violent plaque reduction
134 Table 3.5 Crystallography Data Collection and Refinement Statistics. Work performed by J.L.M.
Data Collection WT Malay BanLec F84T Malay BanLec PDBID 6OPR 6OQ0 Space Group P43212 P212121 Unit Cell (Å) 52.526, 52.526, 79.247, 157.871, 221.307 204.101 Wavelength (Å) 0.9786 0.9786 Resolution (Å)1 1.51 (1.51 – 1.54) 1.80 (1.80 – 1.83) Rsym2 0.060 (0.414) 0.084 (0.662) 3 20 (5) 10 (2) Completeness (%)4 99.5 (98.4) 99.8 (99.5) Redundancy 14.0 (11.7) 7.1 (6.9)
Refinement Resolution (Å) 1.51 1.80 R-Factor 5 18.4 20.2 Rfree 6 20.2 23.4 Protein atoms 2117 16861 Water Molecules 363 2240 Unique Reflections 49784 229852 R.m.s.d.7 Bonds 0.008 0.008 Angles 0.99 1.07 MolProbity Score8 0.92 0.93 Clash Score8 0.70 0.76 1Statistics for highest resolution bin of reflections in parentheses. 2 Rsym =ShSj | Ihj-
135 Chapter 4 – Discussion
4.1 Determinants of mitogenicity in antiviral lectins
The stark contrast in mitogenicity between H84T (Musa acuminata) and F84T (Musa acuminata ssp. malaccensis), with F84T being much more mitogenic than H84T and more mitogenic than even WT BanLec, presents the interesting opportunity to investigate other defining characteristics of mitogenicity in antiviral lectins beyond the pi-pi stacking interaction in the ligand recognition loop. Mitogenicity, at least in the case of BanLec, is easily decreased if carbohydrate binding activity is disrupted (60), but we were the first group to separate mitogenicity from a lectin that retained antiviral activity. Other determinants of mitogenicity that if disrupted would not compromise antiviral activity remain poorly understood. Our previous work demonstrates that H84T, though safe in animals and non-mitogenic towards peripheral blood lymphocytes at concentrations up to 1 µM, is minimally mitogenic and retains a minor mitogenicity peak as indicated by NMR spectroscopy (60). Pi-pi stacking is clearly a determinant of mitogenicity as mutating both WT BanLec and WT Malay BanLec to disrupt this interaction resulted in a decrease in mitogenicity in the engineered lectins (Chapter 3). However, BanLec and Malay BanLec are only 63% similar on the amino acid level, meaning that differences in the non-identical regions of the lectins could account for the marked differences in mitogenicity between the mutant lectins. Other potential determinants of mitogenicity, which may be located outside of the ligand recognition loop, might also be relevant to defining mitogenic
136 characteristics in lectins that are mitogenic but have only one carbohydrate binding site unlike
BanLec and Malay BanLec. Studying the structural differences between mitogenic and non- mitogenic, or less mitogenic, lectins might also be useful in this investigation. Examples of non- mitogenic antiviral lectins include galanthus nivalis agglutinin, hippeastrum hybrid agglutinin
(117), and griffithsin (GRFT) (103, 104).
Using the knowledge generated above, other lectins with demonstrated antiviral activity could be modified in order to decrease and/or eliminate their mitogenicity, opening up the possibility of using other antiviral lectins in a clinical setting. For example, one of the best studied lectins of bacterial origin, cyanovirin-N (CV-N), has been found by many groups to have anti-HIV activity, but it is also known to be highly stimulatory towards peripheral blood mononuclear cells. CV-N is also reported to have activity against HCV, influenza virus, ebolavirus, and HSV-1 (39). Decreasing the mitogenicity of this lectin might make more feasible possible clinical use of this broad-spectrum antiviral lectin. Individual lectins have high specificity for the glycans to which they bind, but as a group, lectins have broad reactivities. It is possible that one could find a lectin to bind to most or all pathogenic enveloped viruses, and it would be a matter of rational engineering to reduce mitogenicity to unlock this broad potential of lectins to target viruses. A broad-spectrum cocktail of antiviral lectins could be created for empiric treatment of particular categories of viral illness, such as upper respiratory infections, which can be caused by an array of viruses. Some combinations of lectins have been shown to have synergistic activity against infections, and so using a panel of lectins for empiric treatment of disease could also impart a therapeutic benefit.
Beyond the structural determinants of mitogenicity, the precise mechanism by which some lectins, and in particular BanLec, are mitogenic is not well understood. It is thought that
137 lectin-induced mitogenic stimulation of lymphocytes is due to crosslinking of cellular receptors on these cells (e.g., the T cell receptor) by the lectin, triggering downstream signaling events that lead to proliferation following a rise in the intracellular calcium ion concentration (49). Binding to cellular receptors may occur through recognition of glycans on the receptor. The valence of a lectin sometimes predicts its mitogenic activity, as in the case of the lima bean lectin, which is non-mitogenic in its divalent, tetrameric form yet mitogenic in its tetravalent, octameric form
(118); a higher valence predicts a greater ability to bind to and crosslink cell surface receptors.
Emerging work suggests that the mitogenic activity of WT BanLec depends on MHC Class II
(HLA-DR) and cytokine input from antigen-presenting cells, in particular, dendritic cells (DCs)
(M. Yu, A. Smed Sörensen, and D. Markovitz, unpublished data). It is not clear, however, exactly how WT BanLec might interact with DCs and T cells in order to mediate the increase in
T cell proliferation. Immunoprecipitation experiments could be performed in order to assess with which cellular receptors BanLec interacts.
4.2 Glycan binding specificity of H84T
Although it has been well documented by our group and others that BanLec and H84T
BanLec recognize and interact with glycans containing mannose and high-mannose residues
(57–60), understanding the glycan specificity of H84T is not that simple. There are viruses to which H84T was predicted to have activity based on the presence of high-mannose N-glycans in the viral envelopes and did not, for example herpes simplex virus and respiratory syncytial virus
(unpublished data). Lectins are quite specific and the internal linkages between monosaccharides in a given glycan are important determinants of whether a lectin will bind that glycan, as well as
138 the orientation of the non-reducing or reducing ends of the sugars as they sit in the carbohydrate binding site. BanLec is known to bind internal a-1,3-mannosyl/glucosyl and a-1,4- mannosyl/glucosyl units at non-reducing ends, terminal b-1,3-glucosyl units at reducing ends, and terminal a-D-mannosyl/glucosyl units at non-reducing ends (56, 105).
Part of the difficulty in predicting to which glycans and viruses H84T will bind also lies in the difficulty of predicting the exact nature of the N-glycans that occur on viral glycoproteins.
Sequons do not predict the identify of N-glycans deposited on a glycoprotein (16), and so glycan profiling can only be performed experimentally. It is known which viruses contain high-mannose type N-glycans on their surfaces based on approaches that allow a broad sampling of glycans without reference to where on the glycoprotein the glycans occur, but it is almost certain that
H84T binds only to a specific arrangement of mannoses on a glycan or only to glycans within a certain context relative to other glycans on the cell. Determining what precise types of glycans
H84T (and other mannose-binding lectins) can and cannot bind beyond a simple binary of high- mannose or not high-mannose type has not yet been attempted. H84T might also bind to certain types of hybrid glycans depending on their precise makeup of mannose residues. A new technology (119) has facilitated high-throughput analysis of site-by-site glycan determination, which could be leveraged to determine in further detail the carbohydrate structures to which
H84T binds and does not bind. In this technology, trypsin-digested whole cell lysates are enriched for glycopeptides using hydrophilic interaction liquid chromatography and glycopeptides are subsequently analyzed by tandem mass spectrometry. Analysis of the fine specificity of H84T would be useful not only for predicting the activity of H84T against a given virus, but also more broadly for gaining a deeper understanding of the sugar code, which dictates interactions between lectins and glycans.
139 It would be quite interesting to attempt to alter the binding specificity of H84T by changing the sequence of either the ligand recognition loop (residues 83-88) (60), which is thought to fine-tune binding to different oligosaccharides (57), or to the ligand binding domain itself (within the parameters of the GXXXD motif). Steric hindrance determines which ends of sugars can bind to H84T, and so mutating these might change the specificity of H84T and accommodate binding to new glycans. Binding to other types of glycans might widen the antiviral potential of the molecule, allowing H84T derivatives to inhibit a broader array of viruses than H84T alone, which is already quite broad-spectrum.
4.3 H84T as a broad-spectrum antiviral and anti-cancer agent
H84T has activity against a number of viruses with high-mannose N-glycans on their envelopes, including influenza (in vitro and in mice, as presented in Chapter 2) (60), HIV (in vitro and in humanized mice) (60, 78), Ebola (in vitro and in mice) (E. Covés-Datson, J. White, and D. Markovitz, manuscript submitted), HCV (in vitro) (60), cytomegalovirus (in vitro), varicella zoster virus (in vitro), and measles (in vitro), among other viruses (unpublished data).
In the case of influenza virus, H84T efficaciously inhibits all strains tested, including oseltamivir-resistant and -sensitive strains; diverse subtypes of influenza A, even pandemic and avian; and influenza B. Not all influenza virus inhibitors have efficacy against both influenza A and B, and so it is notable that H84T does. It is possible that current anti-viral therapy for the viruses against which H84T has activity would synergize with H84T and/or other antiviral lectins. Indeed, against at least some influenza virus strains, H84T and oseltamivir drug combinations exhibit minor but significant synergy. In addition, the anti-HIV agents tenofovir,
140 maraviroc, and enfuvirtide are each known to synergize with GRFT against certain clades of
HIV (120).
Certain cancer cells, in addition to viruses, can express high-mannose N-glycans on glycoproteins at much higher levels than would be present on most normal human cells. These cancers include non-small lung cancer (NSCLC), hepatocellular carcinoma, and breast cancer, among other malignancies (20, 106, 107). NSCLC is an appropriate potential target for H84T as many NSCLCs express abnormally glycosylated epidermal growth factor receptor (EGFR) with enrichment for high-mannose N-glycans, and as the large majority of NSCLCs are “addicted” to
EGFR, requiring its input for survival. Indeed, the survival of lung cancer cell lines both with and without mutations in EGFR is markedly decreased by H84T but not by the carbohydrate- binding mutant D133G (A. Pal, A. Rehemtulla, and D. Markovitz, unpublished data). Moreover,
H84T administered IP significantly reduces tumor volume in a cell line xenograft model of flank tumors (A. Pal, M. Legendre, A. Rehemtulla, and D. Markovitz, unpublished data). Preliminary data suggest that H84T and EGFR colocalize in the endosomal/lysosomal compartment, which has an interesting parallel in the colocalization of H84T and influenza virus in the endosome/lysosome. Further, it appears that the H84T-EGFR interaction might lead to the destruction of the latter in the lysosome (A. Pal, A. Rehemtulla, and D. Markovitz, unpublished data).
4.4 Diverse mechanisms of action of H84T
The mechanisms of action of H84T against different viruses, where elucidated, differ to a surprising degree. Historically, the field of antiviral lectins posited that lectins would interfere
141 mostly at the viral life cycle step of attachment, because the lectins were hypothesized to bind to viral glycoproteins and interfere with binding of the virus to the cellular receptor (39). WT
BanLec does work in this fashion against HIV, interfering with attachment of the virus by binding to the glycoprotein gp120 (78). It also to a lesser extent inhibits HIV fusion with the plasma membrane. In the case of influenza virus, the major inhibitory effect of H84T is at the level of fusion and the minor effect at the level of attachment (Chapter 2). It was not expected that the majority of the anti-influenza effect would be via fusion inhibition. The mechanism of action of H84T against Ebola virus is more perplexing still, with its antiviral effect being partially due to entry inhibition and partially due to downstream steps other than assembly/budding, perhaps at the level of genome transcription/replication. How a lectin would interfere with genome transcription/replication is not well understood as there are not reported to be glycoproteins involved in this step of the Ebola virus life cycle.
Separately, it would be interesting to know if the mechanism of action of F84T, a similar lectin but with potentially different binding properties based on different ligand recognition and binding loops, is similar to H84T and if it might inhibit viruses not disrupted by H84T. So far,
H84T and F84T seem to have similar antiviral activity profiles, being active and inactive against the same viruses, but the mechanisms of action might still differ depending on the exact glycan binding specificities of the two molecules.
4.5 Emergence of resistance to H84T in influenza virus
We provide strong evidence that the antiviral activity of H84T is mediated by binding to
HA via high-mannose N-glycans and inhibiting attachment to a minor extent and fusion to a
142 major extent (Chapter 2). We would predict that emergence of resistance to H84T among influenza virus strains would be due to a mutation or mutations that would alter the N-glycan profile of HA, perhaps by disruption of sequons and removal of particular N-glycans. Generating a viral escape mutant by growing virus in increasing concentrations of H84T would allow us to analyze mutations in HA and perhaps other viral proteins (e.g., NA) that confer resistance to
H84T. Any mutations or changes in N-glycosylation would potentially suggest that the parent sites were important for binding to H84T. N-glycosylation, though variable in the head group of
HA, is required in the stem of HA for proper folding and transport of the protein (24, 25). One could thus hypothesize that it might be difficult for viruses to become resistant to H84T because, as H84T likely interacts with both the head and the stem of HA, there might be a fitness cost to decreasing N-glycan expression.
4.6 Considerations for clinical development of H84T
4.6.1 Efficient production of H84T
Our group currently produces H84T in an E. coli protein expression system, which necessitates endotoxin removal, especially for administration of H84T to animals. Producing the quantity of lectin required on a research scale is relatively efficient via this method. However, if
H84T were to be used in a clinical setting, large-scale affordable production would potentially require a different method. Growing E. coli cultures on an industrial scale is relatively low-cost, but the added step of endotoxin removal, which is not trivial, could ideally be avoided. Tobacco plants (121) and rice seeds (122) have been considered for large-scale endotoxin-free production
143 of therapeutic proteins, but the plants traditionally must be homogenized to harvest protein, which means they have a limited capacity. However, newer methods allow for secretion of proteins from the leaves of the tobacco plant without homogenizing them (123); these strategies would allow the plants to be used more than once for production, increasing yield and decreasing cost. We have begun to explore use of this method for production of H84T.
4.6.2 Delivery of H84T
There are multiple potential routes of H84T administration that would be clinically useful: intravenous (IV), subcutaneous (SC), and aerosol. Both IP (akin to IV) and SC administration of H84T have been shown to be efficacious in a murine model of influenza virus disease. IV administration would be most appropriate in hospitalized patients, whereas for outpatients, SC administration would be more convenient. The long half-life of H84T increases the likelihood that intermittent SC administration, or even a single dose, would be a feasible route in patients. In preliminary studies, aerosolization of H84T has appeared efficacious against influenza, but methods for aerosolization require additional optimization (S. Evans, unpublished data). Aerosol would allow for delivery of H84T directly to the lungs, which could be advantageous for viruses such as influenza that target the lungs and for limiting systemic exposure.
144 4.6.3 Binding of H84T to normal tissue
Although in general the N-glycans with which H84T interacts, especially high-mannose type glycans, are thought to be minimally expressed on most normal human cells (except embryonic and macrophage cells), it will be important to clarify whether some binding of H84T to normal cells does occur and what its effect might be. That H84T becomes internalized into cells suggests that it interacts to some degree with the cell surface. The murine model has shown
H84T to be very safe and quite well tolerated in mice, which suggests that if binding of H84T to normal cells does occur, it is perhaps not harmful. This question should be addressed with both mouse and human tissue.
4.6.4 Antibody response to H84T
Many or perhaps almost all humans have pre-existing titers of IgG4 antibodies against banana lectin (75), presumably due to exposure to bananas by ingestion. These antibodies also bind to H84T. Whether these antibodies would limit therapeutic efficacy of H84T in humans, though they do not appear to do so in mice, remains an important question. In the future, we would like to begin to address this issue by administering H84T to humanized mice in an influenza challenge experiment to assess whether the anti-influenza activity of H84T is blocked by pre-existing anti-BanLec antibodies. In addition, it will be important to further assess whether repeated exposure to systemic H84T leads to an immune response that could later limit therapeutic efficacy (50).
145 4.7 Summary
We have identified H84T BanLec, a broad-spectrum antiviral lectin with robust activity against all strains of influenza virus tested both in vitro and in vivo, as a new anti-influenza therapy with potential clinical usefulness via multiple routes of administration. H84T binds to
HA and enters into the endosomal-lysosomal system of cells, therein inhibiting the fusion activity of influenza, with a minor inhibitory effect on attachment. To test whether disruption of the pi-pi stacking interaction between two aromatic amino acid residues reduces the mitogenicity of antiviral lectins, we modified a different lectin using an approach similar to that we had employed with H84T. We found that F84T, though still mitogenic, has significantly reduced mitogenicity as compared to its parent lectin, which serves as a proof-of-concept that mitogenicity can be disrupted by targeted removal of pi-pi stacking. These insights highlight the promise of reducing mitogenicity by rational engineering to unlock the therapeutic potential of antiviral lectins.
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