Binding vs. adhesion

To characterize the interaction between gp120 and α₄β₇ we employed two assays. We developed a novel surface-plasmon resonance (SPR) based assay that utilized dextran surfaces coated with recombinant envelope (env) proteins, V1/V2 scaffolds, or synthetic V2 cyclic peptides. The analyte that we reacted with these surfaces was a recombinant soluble α₄β₇ heterodimer in which the carboxy-terminal transmembrane and cytoplasmic tail domains of both chains were removed and replaced by short peptides that function as an “α₄ chain acid-β₇ chain base coiled-coil clasp” [46]. This acid-base clasp was joined by a disulfide bond that served to stabilize the heterodimer. In one iteration of this assay we employed short linear peptides derived from V2 as competitive inhibitors. The second assay we employed was a static adhesion assay based on the method developed by Peachman and colleagues, in which RPMI8866 cells, that express α₄β₇ on the cell surface, were allowed to adhere to the recombinant env proteins, V1/V2 scaffolds or synthetic V2 cyclic peptides (S1A Fig). The α₄β₇-expressing RPMI8866 cell line was derived from a human B cell lymphoma, and expresses α₄β₇, but no detectable CD4 or CCR5. Cells were grown in media containing retinoic acid, which increased both levels of expression, and clustering of α₄β₇ (S2B Fig). In some assays we included anti-integrin and anti-gp120 mAbs as adhesion inhibitors. The SPR assay allowed us to evaluate the kinetics of integrin-gp120 binding, while the cell-based assay measured adhesion between two multivalent surfaces.

The specific affinity of α₄β₇ for gp120 V2 is similar to its affinity for MAdCAM

Previous studies describing the interaction between gp120 and α₄β₇ have demonstrated, in a qualitative way, the specific interaction between these two proteins, without establishing an estimate of affinity [9–12]. We reasoned that a quantitative comparison of the binding kinetics of gp120 vs. MAdCAM to α₄β₇ would help determine if α₄β₇-gp120 interactions mimic α₄β₇-MAdCAM interactions. We carried out a kinetic analysis using the SPR assay noted above. Soluble α₄β₇ (analyte) was passed over surfaces coupled with either recombinant gp120 or a MAdCAM-Ig fusion protein (ligands). A recombinant A244 gp120 (subtype A/E produced by Global Solutions for Infectious Diseases (GSID)) was employed in these assays. We initially measured binding kinetics in the presence of a buffer containing 1mM MnCl₂ in order to uniformly stabilize α₄β₇ in an extended/activated conformation (discussed below). Under these conditions MAdCAM and A244 gp120 demonstrated comparable high affinities (K_D (nM)) of 0.597 and 7.140, respectively (Fig 1A and 1B). These affinities are generally comparable to gp120-soluble CD4 binding kinetics (e.g 22 nM K_D) [47]. When MnCl₂ was removed, affinity for both MAdCAM and gp120 fell below the detection limit of this assay (Fig 1C and 1D). This requirement for Mn⁺⁺ is consistent with our previous report that gp120, similar to MAdCAM, interacts with an extended conformation of α₄β₇ [9]. This observation suggests that gp120 is likely to engage α₄β₇ only on cells with an enhanced potential to traffic to the gut. We next replaced A244 gp120 with a synthetic cyclic 42 amino acid peptide fragment (cV2) derived from the V2 domain (AA 157–196 (HXB2 numbering)) of 92TH023 gp120 (subtype A/E), in which N and C termini were joined by a disulfide bond, and the C terminus was biotinylated to facilitate coupling to NeutrAvidin coated biosensor chips. The 92TH023 V2 sequence is nearly identical to that of A244 gp120 V2 (S1A Fig). The affinity of this peptide (cV2 92TH023) for α₄β₇ (K_D (nM) 1.150) was close to that of A244 gp120 (Fig 1E), demonstrating that a cV2 is sufficient to mediate the high-affinity interaction shown in Fig 1B. Moreover, it indicates that this high-affinity interaction does not require the glycans that decorate the V2 of GSID A244 gp120, or any bridging protein [13]. When we replaced α₄β₇ with α₄β₁ affinity for cV2 92TH023 was reduced by >8000 -fold ((K_D (nM) 9710) (Fig 1F), demonstrating binding specificity and the fact that V2, like MAdCAM, preferentially binds to α₄β₇.

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Fig 1. Specific affinity of α₄β₇ for HIV gp120, MAdCAM and SIV gp120.

A) Sensorgram of increasing concentrations (2-fold) of soluble α₄β₇ passed over surface-immobilized MAdCAM in the presence of 1 mM MnCl₂ for 120 sec, followed by a 120 sec washout/dissociation phase. Mass of bound α₄β₇ (y-axis) expressed as response units (RU). Affinity expressed as K_D (nM) is shown. B) Same as in panel A, with immobilized A244 gp120. C) Same as in panel A with immobilized MAdCAM in the absence of divalent cations. D) Same as in panel A with immobilized A244 gp120 in the absence of divalent cations. E) Same as in panel A with immobilized cV2 92TH023 peptide. F) Same as in panel A with immobilized 92TH023 cV2 peptide and soluble α₄β₁ as the analyte. G) Same as in panel A with immobilized cV2 C06980v0c22 peptide. H) Same as in panel A with immobilized BG505 gp120. I) Same as in panel A with immobilized SIVmac766 gp120. Additional binding parameters are listed in S1 Table. Each sensorgram is representative of three independent measurements of each analyte-ligand interaction.

https://doi.org/10.1371/journal.ppat.1007278.g001

We extended this analysis by measuring binding kinetics for a cV2 peptide derived from C06980v0c22 (subtype C) and a recombinant gp120 BG505 protein (subtype A) (Fig 1G and 1H). α₄β₇ bound to each with similar high affinities, consistent with the conserved nature of this interaction across HIV clades as we had originally reported [9]. The V2 domains of HIV and SIV diverge both in primary sequence, as well as in length and the typical number of disulfide bridges (1 vs 2), suggesting significant structural differences between them. Using a recombinant gp120 derived from SIVmac766 we obtained an α₄β₇ affinity (K_D (nM)) of 104 (Fig 1I). This is ~10-fold lower than that observed for HIV A244 gp120. It is possible that this reduction in affinity reflects differences in the primary amino acid sequences of human vs. rhesus macaque α₄β₇ (Figs 2A and S1A), such that SIV gp120s might exhibit a higher affinity for a rhesus macaque versus a human derived α₄β₇ protein. Detailed α₄β₇ binding parameters for each of the analytes appears in S1 Table.

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Fig 2. Inhibition of α₄β₇ -mediated cell adhesion to MAdCAM and a cyclic V2 peptide.

A) Ribbon diagram of a human α₄β₇ heterodimer headpiece (α₄: green, β₇: blue) (PDB ID: 3V4P). Yellow indicates sequence divergence in rhesus macaque. Ligand binding groove is highlighted by a red arrow, and the SDL is highlighted by a green box. B) Ribbon diagram of the two N-terminal IgSF domains of human MAdCAM (PDB ID: 1GSM). The MAdCAM CC′ loop of IgSF domain 1 is highlighted in the red box and the DE loop of IgSF domain 2 is highlighted in a green box. C) A representative result of RPMI8866 cells adhering to immobilized MAdCAM or a cV2 92TH023 peptide in the absence or presence of vedolizumab, an LDV mimetic ELN-475772, the anti-α₄ specific mAb 2B4, soluble MAdCAM-Ig, or control human IgG, as indicated. Adhesion was determined at OD_590nm and listed as fluorescence units (y-axis). Background fluorescence (BF) of RPMI8866 cell adhesion to blank wells is denoted by a dashed line. Adhesion to immobilized MAdCAM serves as a specificity control for α₄β₇ adhesion and human IgG is employed as a reagent control. Conditions are run in triplicate and error bars indicate standard error of the mean (SEM). D) Schematic of three conformations of the cell surface expression of the α₄β₇ heterodimer. The influence of divalent cations (Mg⁺⁺ and Mn⁺⁺) on the type of adhesion is listed below. E) Adhesion of RPMI8866 cells to either immobilized MAdCAM or cV2 92TH023 as in panel C. Average results from four or more independent experiments are shown. Y-axis indicates % inhibition relative to either MAdCAM or cV2 92TH023 in the absence of any inhibitor. Inclusion of inhibitors is indicated by a + sign below. Error bars indicate standard deviation (SD). F) Adhesion of RPMI8866 cells to immobilized MAdCAM or a cV2 92TH023 peptide in the buffers containing a low concentration of divalent cations, or high concentrations of MnCl₂ or MgCl₂. Adhesion was determined at OD_590nm and listed as fluorescence units (y-axis). Conditions are run in triplicate and error bars indicate standard error of the mean (SEM). Background fluorescence (BF) of RPMI8866 cells to blank wells is denoted by a dashed line. Two additional replicate experiments are shown in S3 Fig.

https://doi.org/10.1371/journal.ppat.1007278.g002

α₄β₇ antagonists developed to block MAdCAM binding also block gp120 V2 adhesion

Similarities between MAdCAM and HIV gp120 with respect to affinity, cation-dependence, and preference for binding α₄β₇ over α₄β₁ suggests that gp120 V2 mimics MAdCAM in the manner in which it engages α₄β₇. A number of α₄β₇ antagonists, developed to treat inflammatory bowel disease (IBD) act by occupying MAdCAM binding sites on α₄β₇. To the extent that gp120 uses these same sites, such antagonist should also interfere with gp120 adhesion. These antagonists include a class of small molecule mimetics that resemble the Leu-Asp binding motif present in the CC′ β strands of MAdCAM IgSF domain 1 [48]. They compete directly with MAdCAM by binding to key residues in the ligand binding groove formed by the interface between α₄ and β₇ (Fig 2A). Vedolizumab is a mAb antagonist of α₄β₇ with a different and unique mechanism of action (MOA). It binds exclusively to the specificity-determining loop (SDL) in β₇ (Fig 2A) [23]. The SDL mediates secondary interactions with a negatively charged DE loop in MAdCAM IgSF domain 2 (Fig 2B) [23, 49–52]. We evaluated the capacity of a small molecule LDV mimetic, ELN-475772, and vedolizumab to interfere with gp120-α₄β₇ interactions using the RPMI8866-based adhesion assay described above. As expected, both ELN-475772 and vedolizumab blocked adhesion of α₄β₇ to immobilized MAdCAM (Fig 2C and 2E). We then tested their ability to block α₄β₇-mediated adhesion to the cV2 92TH023. The anti-α₄ mAb 2B4, which inhibits most α₄-ligand interactions, along with human IgG were employed as specificity controls. Both ELN-475772 and vedolizumab inhibited α₄β₇ adhesion to V2 by >90% (Fig 2C and 2E). Because the inhibitory MOAs of ELN-475772 and vedolizumab involve direct interactions with two discreet MAdCAM binding sites on α₄β₇, these results suggest that soluble MAdCAM would compete with gp120 V2 in binding to α₄β₇. Of note, soluble MAdCAM-Ig blocked V2-α₄β₇ adhesion (Fig 2C and 2E). We conclude that, at least in a general way, gp120 V2 effectively mimics MAdCAM in the manner in which it engages α₄β₇.

α₄β₇ activity is regulated by its conformation. Intracellular signaling events modulate the ectodomain to reversibly transition between bent and extended conformations (Fig 2D). In addition, the headpiece, which mediates ligand interactions, can assume closed, intermediate, and open conformations. Transitions between these conformations allows α₄β₇ to mediate both rolling and firm adhesion [23]. Rolling adhesion is associated with lower affinity binding to MAdCAM, while firm adhesion is associated with a higher affinity interaction. Manipulation of Ca⁺⁺, Mg⁺⁺, and Mn⁺⁺ concentrations provides a way to manipulate the affinity of α₄β₇-MAdCAM interactions in vitro [38]. Strength of adhesion is highest in Mn⁺⁺, followed by Mg⁺⁺ > Mg⁺⁺/Ca⁺⁺, > Ca⁺⁺. We asked whether the pattern of V2 adhesion to α₄β₇ was similar to that mediated by MAdCAM in buffers containing Mn⁺⁺ vs Mg⁺⁺, vs low cations (Ca⁺⁺). Adhesion of both MAdCAM and cV2 92TH023 was strongest in the presence of MnCl₂ and reduced in the presence of MgCl₂ by ~5-fold and 2.5-fold respectively (Figs 2F and S3). In low-cation conditions MAdCAM adhesion appeared close to background levels; however, we were still able to detect residual cV2 92TH023 adhesion. Overall, V2 adhesion to α₄β₇ responded to divalent cations in a similar manner as did MAdCAM. This is particularly noteworthy insofar as the dynamic changes in affinity required to mediate both rolling and firm adhesion reflect the unique and highly specialized nature of α₄β₇-MAdCAM interactions [23]. The way in which both HIV and SIV gp120 mimic this highly specialized interaction argues that it provides them a selective advantage.

Weakly neutralizing HIV V2 mAbs elicited by vaccination and infection inhibit gp120-α₄β₇ adhesion

The conformation of the V2 domain of gp120 is dynamic, and consequently it was deleted from the recombinant proteins used to generate the initial high-resolution gp120-mAb cocrystals [53, 54]. Instead, V2 structures were obtained by grafting V1/V2 fragments onto scaffolds derived from unrelated proteins. These scaffolds stabilized V1/V2 in a way that, in complex with conformation-dependent V2 mAbs, allowed for the derivation of high resolution structures. The first V2 structure was obtained by presenting V1/V2 on a scaffold termed 1FD6 in complex with the broadly neutralizing, glycan dependent monoclonal antibody PG9 [43]. In this context, V2 adopted a Greek key β sheet structure (Fig 2A). Another study in which the same 1FD6-V1/V2 protein was complexed with mAb 830A provided additional detail and revealed V2 in a related β- barrel conformation [42], which is consistent with what has been observed in pre-fusion env trimers [40, 41]. However, when a linear V2 peptide is left unconstrained it can adopt α helical structure [55]. This is the case for a crystal structure of mAb CH58 in complex with a linear V2 peptide derived from HIV isolate 92TH023 gp120 [56]. CH58 does not exhibit broad potent neutralizing activity and we refer to here as weakly- neutralizing. It recognizes a helix structure (Fig 3A) and is noteworthy insofar as it was generated from an uninfected immunized individual who participated in the RV144 vaccine trial. It recognizes an epitope that maps within a short region of V2 (AA 168–181), that includes two residues, K¹⁶⁹ and I¹⁸¹, identified by sieve analysis as sites of vaccine elicited immune pressure in the RV144 trial (Fig 3B). Additional V2-specific mAbs that recognize helical structures in this same region have subsequently been described. We report here the cocrystal structure of a V2 peptide with one such mAb, Mk16C2, that was generated from a gp120 immunized rabbit (Fig 3A and S2 Table). It binds to the same helical structure as CH58 but approaches V2 from the opposite side. Of note, helix -preferring V2 antibodies are not limited to vaccine elicited immune responses. mAb CAP228-16H, which was derived from an HIV-infected subject, recognizes a V2 helix structure that is strikingly similar to that recognized by CH58 [57]. Thus, mAbs reacting with the region of V2 from AA 153–194 recognize at least two distinct types of epitopes: those like PG9 and 830A that recognize a constrained β- sheet, and those like CH58 that recognize a less constrained helical conformation.

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Fig 3. The structures of V2 mAbs in complex with peptides, and V2 mAb inhibition of α₄β₇ adhesion.

A) Crystal structure of three V2 domain mAbs, CH58 (PDB ID: 4HPO), Mk16C2 (PDB ID: 6CEZ), and 830A (PDB ID: 4YWG) in complex with V2 peptides, and a superimposition of CH58 and Mk16C2 binding to opposite sides of the same helical region of a V2 peptide. Only the Fv regions are shown with the heavy chain, light chain, and the V2 epitope colored cyan, green, and magenta, respectively. B) Amino acid sequence of the V2 domain of 92TH023. Sieve residues identified in the RV144 vaccine study are highlighted in red, along with schematics of the epitopes of CH58, Mk16C2 and 830A aligned below (with the corresponding HIV isolate listed). Residues in contact with each mAb are highlighted in magenta and the size of each amino acid is proportional to the contact surface area. C) Adhesion of RPMI8866 cells to cV2 92TH023 in the absence or presence of V2 mAbs: CH58, CAP228-16H, 830A, and Mk16C2. The mAb VRC01 is included as a nonspecific mAb control, and the anti-α₄ mAb 2B4 is employed as a positive control. Adhesion was determined at OD_590nm and listed as % inhibition in three or more independent experiments relative to cV2 92TH023 in the absence of any inhibitor (y-axis). Error bars indicate SD.

https://doi.org/10.1371/journal.ppat.1007278.g003

In an ELISA assay mAbs 830A, CH58, CAP228-16H and Mk16C2 bind to cV2 92TH023 (S1B Fig), indicating that this cyclic peptide is sufficiently long and flexible to present the epitopes recognized by each of these mAbs. Although the core epitopes of CH58 and 830A differ, the C-terminal end of the CH58 epitope overlaps the 830A epitope (Fig 3B), which is consistent with our observation that CH58 can compete with 830A (S1C Fig). We evaluated the ability of each of these mAbs to inhibit α₄β₇ interactions with V2 using the adhesion assay described above. mAbs 2B4 and VRC01 were employed as specificity controls. CH58 and CAP228-16H inhibited α₄β₇ adhesion to V2 by >90% (Fig 3C). Mk16C2 inhibited adhesion less effectively. Of note, 830A failed to inhibit adhesion in a significant way. Thus, while a V2 mAb (CH58) that recognizes a helical structure interfered with α₄β₇ -mediated adhesion, mAb 830A, that shows preference for the β strand, failed to show detectable interference.

α₄β₇ adhesion is mediated by an unconstrained form of gp120 V2

Rao and colleagues have recently reported that adhesion of recombinant gp120 to α₄β₇ requires the partial enzymatic removal of glycans [58]. This is consistent with our finding that removal of several potential N-glycosylation sites (PNGs) within V2 can enhance gp120 binding to α₄β₇ [16]. In agreement with these reports we find that GSID A244 gp120 required limited deglycosylation with PNGase, under nonreducing conditions, in order to mediate α₄β₇ adhesion (Fig 4A). This adhesion, similar to the adhesion of α₄β₇ to cV2 92TH023, was efficiently inhibited by ELN-475772, CH58, and CAP228-16H (>90%) (Figs 4A and S4). Again, Mk16C2 was less effective (~47% reduction). We also evaluated α₄β₇ adhesion to a BG505 SOSIP gp120/41 trimer and monomeric SIVmac766 gp120. Unlike the two monomeric gp120s, the SOSIP stabilized protein failed to mediate adhesion after removal of glycans (Figs 4B and S5). Inability to adhere to α₄β₇ is not a consequence of the primary sequence of BG505 V2 since a BG505 cyclic V2 (cV2 BG505) was able to mediate α₄β₇ adhesion (Figs 4C and S6). These finding are in agreement with the observations of Rao and colleagues. Of note, in an SPR -based assay, CH58 binds to the cV2 BG505 but not to the BG505 SOSIP (Fig 4D). Given that CH58, which recognizes a helix, efficiently blocks V2 adhesion to α₄β₇, we hypothesized that the failure of the BG505 SOSIP trimer to engage α₄β₇ reflects an underlying conformational constraint on V2 mediated by the SOSIP stabilization strategy in which this constraint precludes the formation of a structure required for α₄β₇ reactivity.

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Fig 4. Adhesion of α₄β₇ to recombinant gp120 and the V1/V2 scaffolds.

A) Adhesion of RPMI8866 cells to immobilized A244 gp120 in the absence or presence of HIV V2-specific mAbs: CH58, CAP228-16H, and Mk16C2. The LDV mimetic ELN-475772 provides a specificity control for adhesion to α₄β₇. Adhesion was determined by OD_590nm and listed as fluorescence units (y-axis). Conditions are run in triplicate and error bars indicate SEM. Background fluorescence (BF) of RPMI8866 cells is denoted by a dashed line. Deglycosylation (DG) of A244 gp120 as indicated. Results from three or more independent experiments are shown in S4 Fig. B) Comparison of α₄β₇ adhesion to glycosylated vs. DG forms of BG505 SOSIP trimer, A244 gp120, and SIVmac766 gp120 as described in panel A. Conditions are run in triplicate and error bars indicate SEM. BF is denoted by a dashed line. DG as indicated. Results from three independent experiments are shown in S5 Fig. C) Comparison of adhesion of RPMI8866 cells to BG505 SOSIP vs. cV2 BG505 peptide as described for panel A. The anti-α₄ mAb 2B4 is employed as a positive control and the gp120 mAb VRC01 is employed as a negative control. Error bars indicate SEM. This result is representative of three or more independent experiments as shown in S6 Fig. D) SPR analysis showing increasing concentrations of mAb CH58 (2-fold increases from 1.56 nM-25 nM) binding to BG505 SOSIP and cV2 BG505 coated surfaces. The parameters for the binding kinetics for cV2 BG505 are listed. E) Comparison of adhesion of RPMI8866 cells to V1/V2 92TH023-1FD6 vs. V1/V2 92TH023-tag scaffolds using increasing concentrations of each scaffold as indicated (x-axis). Adhesion was determined as describe for panel A. Conditions are run in triplicate and error bars indicate SEM. Results are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1007278.g004

To address whether the β- barrel conformation of V2 is incompatible with α₄β₇ -reactivity, we inserted the V1/V2 sequences of 92TH023 into two scaffolds. The first scaffold, termed 1FD6, has been previously shown to constrain V1/V2 in a way that increases its propensity to form a β- barrel [42]. The second scaffold, termed tag, consists of V1/V2 that is untethered at the C-terminus, allowing it to adopt an unconstrained, CH58 -reactive, helical conformation [42] [PMID: 27707920]. We found that the deglycosylated 92TH023 V1/V2 tag scaffold showed ~5x greater adhesion than did deglycosylated 92TH023 V1/V2 1FD6, which mediated only minor levels of α₄β₇ adhesion (Fig 4E). We conclude that α₄β₇ -reactivity requires a degree of V1/V2 flexibility that is not present in the recombinant BG505 SOSIP trimer. Our results suggest that this is due to constraints placed on V2 by other sequences encoded in the closed trimer. However, we cannot rule out interference by PNGase resistant glycans.

α₄β₇ -coated nanoparticles capture HIV virions derived from primary CD4⁺ T cells

The data presented above suggests that α₄β₇ recognizes a structure distinct from the closed spike on virions that is the target of many well characterized neutralizing mAbs. However, it is well established that env appearing on virions is conformationally heterogeneous [59]. We asked whether, among these various env conformations was one that is α₄β₇ -reactive. To address this question, we employed magnetic nanoparticles (MNPs) coated with either α₄β₇ or V2 mAbs CH58, PG9, and 830A to capture 92TH023 virions derived from primary CD4⁺ T cells (Fig 5). mAb 2G12 was employed as a positive control. After extensive washing, virion capture was measured by a Luminex -based p24 detection assay [60]. In three independent experiments CH58, PG9, and 2G12 each captured ~5X greater amounts of virus than non-specific IgG. Capture by mAb 830A was less efficient (~3X over IgG) (Fig 5A). The capacity of mAb CH58, which recognizes a helical structure and inhibits V2 adhesion to α₄β₇ is able to capture virions suggesting that these virions present an α₄β₇ -reactive form. To test this directly we incubated virions with α₄β₇- MNPs in the absence or presence of increasing amounts of the α₄β₇ inhibitor ELN-475772. In three independent experiments α₄β₇- MNPs captured virus, and this capture was inhibited by ELN-475772 in a dose -dependent manner (Fig 5B). Thus, 92TH023 virions derived from primary CD4⁺ T cells present gp120 in an α₄β₇ -reactive form.

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Fig 5. Virion capture by V2 mAbs and α₄β₇ coated nanoparticles.

A) Capture of 92TH023 by MNPs coated with gp120 mAbs including 2G12 (positive control) and V2 mAbs PG9, CH58 and 830A. A non-specific Ig was employed as a negative control. Virion capture was determined by detection of MNP-bound p24 (Y-axis). Results from three independent experiments are shown. B) 92TH023 virion capture by α₄β₇-MNPs in the absence or presence of increasing concentrations of ELN-475772. Results from three independent experiments are shown. Detection of MNP -captured virus as in panel A. Error bars indicate SEM. Significance measure by ANOVA followed by the Tukey multiple comparison test (***p <0.001 and ****p <0.0001).

https://doi.org/10.1371/journal.ppat.1007278.g005

A 15- amino acid linear V2 -derived peptide is sufficient to mediate both HIV and SIV gp120 binding to α₄β₇

Liao and colleagues demonstrated that a 15 AA peptide corresponding to residues 168–181 of V2 adopts a helical structure when complexed with mAb CH58 (Fig 3A) [56]. Using the SPR assay described above we asked whether a similar 15 AA peptide was sufficient to bind to α₄β₇. Soluble α₄β₇ was passed over a cV2 92TH023 coated surface in the absence or presence of 8 overlapping linear 15 amino acid peptides. These peptides corresponded to sequences in an HIV-1 subtype B consensus V2 domain (Fig 6A). Peptide H43 (Q¹⁷⁰KEYALFYKLDVVPI¹⁸⁴), which closely aligns with the peptide employed by Liao and colleagues, inhibited α₄β₇ -binding by >90% (Fig 6B). This peptide includes both the canonical L¹⁷⁹D¹⁸⁰ α₄β₇ binding site and a Q¹⁷⁰K¹⁷¹E¹⁷² that aligns with the QRV cryptic α₄β₇ epitope identified by Cardozo and colleagues [12]. We repeated this analysis but substituted cV2 92TH023 with A244 gp120 and obtained a similar result (Fig 6C). We then competed mAbs CH58 and CAP228-16H with these same peptides (Fig 6D and 6E). Peptides H42 (R¹⁶⁶DKVQKEYALFYKLD¹⁸⁰) and H43 each partially inhibited mAb CH58 binding and strongly inhibited (>90%) CAP228-16H binding. Taken together these results suggest that inhibition by peptide H43 involves direct binding to α₄β₇. To rule out allosteric inhibition (i.e. peptide H43 binding directly to, and altering the conformation of V2), we carried out a similar peptide inhibition assay with immobilized MAdCAM and determined that H43 inhibited α₄β₇ binding to MAdCAM by >90% (Fig 6F). This result is best explained by direct competition between the Leu¹⁷⁹-Asp¹⁸⁰ in peptide H43 and the critical Leu⁴¹-Asp⁴² encoded within MAdCAM IgSF domain 1 (Fig 6G). However, we believe it is very likely that other residues in H43 also engage α₄β₇. We conclude that an epitope contained within a linear peptide corresponding to residues 170–181 of V2 binds directly to α₄β₇. This same region of V2 overlaps the epitopes recognized by CH58 and CAP228-16H.

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Fig 6. Inhibition of α₄β₇ binding of HIV V2 peptides to HIV gp120, cyclic V2 and MAdCAM.

A) Alignment of the V2 domains of HIV consensus B, and the eight HIV consensus B 15mer peptides (H40-H47) used in this study, and the regions of V2 recognized by mAbs CH58 and CAP228-16H. B-C) HIV 15mer peptide inhibition of α₄β₇ binding to cV2 92TH023 and A244 gp120 as measured by SPR. Percent of α₄β₇ binding relative to binding in the absence of peptide (defined as 100%) is shown (y-axis). D-E) HIV 15mer peptide inhibition of V2 mAbs, CH58 and CAP228-16H binding to cV2 92TH023. Percent binding relative to binding in the absence of peptide is shown (y-axis). F) HIV 15mer peptide inhibition of α₄β₇ binding to MAdCAM. Percent α₄β₇ binding relative to binding in the absence of peptide is shown (y-axis). Results are representative of three or more independent experiments. G) Alignment of α₄β₇ binding site in MAdCAM IgSF domain 1, and HIV peptide H43. Conserved LDV/LDT binding motifs are boxed in blue.

https://doi.org/10.1371/journal.ppat.1007278.g006

The α₄β₇ binding epitope of V2 is conserved in SIV as exemplified by the specific affinity of

SIVmac766 gp120 for human α₄β₇ (Fig 1I). To determine whether this region in SIV V2 is also involved in α₄β₇ -binding, we carried out a similar competition binding experiment as described above in Fig 6, but utilized SIVmac239 derived V2 overlapping peptides, and immobilized SIVmac766 gp120, along with SIVsmE660-CR51 gp120 (Fig 7A). Of the eight SIV peptides we employed only S46 showed strong inhibition (>90%) (Fig 7B). S46 is the SIVmac239 V2 domain peptide that corresponds to HIV peptide H43 (Fig 7C). Peptides H43 and S46 show limited sequence identity, but notably 5 residues: K^171/183, E¹⁷²/¹⁸⁴, Y¹⁷³/¹⁸⁵, Y^177/190, D^180/193, appear to be conserved (Fig 7C).

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Fig 7. Inhibition of α₄β₇ binding to SIV gp120 by V2 peptides.

A) Alignment of the V1V2 domains of SIVmac766 and SIVmac239, and eight overlapping 15 mer peptides of SIVmac239 (S40-S47). B) Inhibition of soluble α₄β₇ binding to SIVsmE660-CR51 and SIVmac766 gp120s by overlapping SIV 15mer peptide. Binding was determined by SPR analysis and binding RU at 200 seconds following the addition of α₄β₇ is listed (y-axis). Results are representative of three or more independent experiments C) Alignment of the V2 domains of HIV consensus B and SIVmac239, and peptides H43 and S46. The tri-peptide motifs (aliphatic-Asp-aliphatic residue) are highlighted in red and regions of sequence identity are boxed in blue.

https://doi.org/10.1371/journal.ppat.1007278.g007

We next asked whether SIV V2 mAbs could inhibit α₄β₇ adhesion to V2. Five V2 mAbs, ITS03, ITS09, ITS12.01, ITS41, and NCI09 were evaluated using the same strategy outlined above for HIV V2 mAbs (see Fig 3C above). We also included ITS13, a V1 mAb and VRC01 an HIV CD4 binding-site mAb as reagent controls. Mapping studies of these V2 mAbs have been described in detail elsewhere [61] and are summarized in Fig 8A. The epitope for mAb ITS12.01 spans SIV V2 residues 187–197 and falls within S46. It includes the key Asp that is conserved in HIV, SIV and MAdCAM IgSF domain 1. However, ITS12.01 did not block α₄β₇ adhesion to SIVmac766 gp120 (Fig 8B). Instead, ITS03 and NCI09, which recognize residues NH₂-terminal to the ITS12.01 epitope inhibited adhesion most efficiently (>90%). ITS09 and ITS41 also inhibited adhesion, but to a lesser extent, while NCI05, which maps to a region overlapping the ITS09 epitope failed to inhibit adhesion in a detectable way. Of note, the epitopes for both ITS03 and NCI09 do not include the canonical Leu-Asp binding site (Ala¹⁹²-Asp¹⁹³ in SIV) that is conserved in HIV V2 and MAdCAM (Fig 8A). Both of these mAbs do however overlap with the corresponding region of HIV V2, that includes the epitopes for CH58 and CAP228-16H, both of which inhibit α₄β₇ adhesion to HIV V2. In summary, a 15-amino acid linear peptide derived from SIV V2 (AA 183–197) inhibits SIV gp120 binding to α₄β₇, and SIV mAbs whose epitopes overlap this peptide also inhibit α₄β₇ adhesion.

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Fig 8. Inhibition of α₄β₇ adhesion by SIV V2 specific mAbs.

A) Alignment of the amino acid sequences of the V2 domains of HIV consensus B and SIVmac239, and the regions of the V2 containing epitopes recognized by V2 specific mAbs. B) Adhesion of RPMI8866 cells to DG SIVmac766 gp120 in the presence of SIV V2-specific mAbs: ITS03, ITS09.01, ITS12.01, ITS41, and NCI09. ITS13, a V1 mAb and VRC01, an HIV gp120 mAb are included as nonspecific mAb controls. Presence of each inhibitor indicated by a + sign below. Y-axis indicates % inhibition relative to adhesion in the absence of any inhibitor in three or more independent experiments. Error bars indicate SD. C) SPR analysis showing increasing concentrations of ITS03 mAb (2-fold increases from 1.56 nM-25 nM) binding to an HIV cV2 92TH023 coated surface. The parameters of binding kinetics are listed.

https://doi.org/10.1371/journal.ppat.1007278.g008

One potential explanation for the conservation of α₄β₇ -reactivity between HIV and SIV, despite the divergent V2 sequences represented by peptides H43 and S46 (< 50% sequence similarity) is that the secondary structures of these two peptides share common features [62]. To address this possibility, we screened SIV mAbs for cross-reactivity with HIV V2 and found that ITS03, one of the SIV mAbs that blocked α₄β₇ binding most effectively, reacted with relatively high-affinity to HIV cV2 92TH023 (K_D (nM)) 0.105) (Fig 8C). This cross-reactivity is consistent with shared secondary structure between the α₄β₇ -binding epitopes localized within the V2 regions of HIV and SIV gp120.

Macaques treated with an anti-α₄β₇ mAb produce anti-V2 antibodies that block α₄β₇-adhesion

SIV infected macaques treated with a combination of ART and a recombinant rhesus anti α₄β₇ -mAb (Rh-anti-α₄β₇) were able to durably control viremia at relatively low levels following treatment interruption [37]. Although these animals failed to mount neutralizing antibody responses, we noted that following withdrawal of ART, 8/8 generated anti-V2 antibody responses, while only 3/7 control animals administered normal IgG and ART, generated similar responses. Mapping studies indicated that this response was strongly focused on the region of V2 corresponding to S43, the region of V2 that includes the NCI09 and ITS03 epitopes. These V2 responses persisted for at least 50 weeks, despite low plasma viremia [37]. Because NCI09 and ITS03 block adhesion to gp120, we asked whether these polyclonal antibody responses included antibodies that could also inhibit α₄β₇ adhesion to gp120. Serum IgG was purified from three ART + anti- α₄β₇ mAb -treated animals, RLn12, RDa15, and RId14, by protein G affinity chromatography, and along with normal rhesus macaque IgG was evaluated in an α₄β₇ adhesion assay. While normal rhesus IgG showed minimal inhibition of α₄β₇-adhesion to SIVmac766 gp120, the sera from all three ART + α₄β₇ treated macaques inhibited adhesion in a dose dependent manner (Fig 9A). We then asked whether these sera contained antibodies with specificities similar to the SIV V2 mAbs (described above in Fig 8) that inhibited α₄β₇ adhesion to gp120. Using an SPR -based assay, SIVmac766 gp120 coated surfaces were preincubated with SIV V2 mAbs ITS03, ITS09.01, ITS12.01 or NCI09. A surface preincubated with RLn12 serum served as a positive control. Surfaces were then reacted with RLn12 serum. As expected, pre-bound RLn12 serum inhibited RLn12 binding (~70% reduction) (Fig 9B). ITS03, ITS09.01 and NCI09 -mediated similar levels of inhibition. ITS12.01, which showed minimal inhibition of α₄β₇ adhesion (Fig 8B), failed to inhibit RLn12 binding (Fig 9B). We conclude that antibody responses generated in SIV infected macaques treated with ART and Rh-anti-α₄β₇ included V2 antibodies that target the α₄β₇ binding epitope of SIV V2.

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Fig 9. Inhibition of α₄β₇ adhesion by serum Ig from macaques treated with ART and Rh-anti-α₄β₇.

A) Adhesion of RPMI8866 cells to the DG form of SIVmac766 gp120 in the absence or presence of 10.0, 5.0, and 2.5 μg of serum Ig from a healthy macaque or three SIV infected macaques, RLn12, RDa15, RId14 treated with ART and anti-α₄β₇ mAb. Adhesion was determined at OD_590nm and listed as fluorescence units (y-axis). Each condition is run in triplicate. Error bars indicate SEM. These results are representative of three independent experiments. B) SPR analysis of serum Ig from SIV infected macaque RLn12 to SIVmac766 cV2 peptide in the absence or presence of pre-bound (blocking) SIV mAbs, ITS03, ITS09.01, ITS12.01 or NCI09, as indicated. Binding is expressed as percent binding RU at 200 seconds after injection of the analyte, relative to binding in the absence of Ig, defined as 100% (y-axis).

https://doi.org/10.1371/journal.ppat.1007278.g009

Ethics statement

Generation of mAb Mk16C2 was approved and carried out under animal use protocol A-1896 by the Institutional Animal Care Committee (IACUC) of the University of Massachusetts Medical School. The University of Massachusetts Medical School is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW). Assurance number: A-3306-01.

Cell line and reagents

RPMI8866 cells, a human B lymphoma cell line that constitutively expresses α₄β₇ was purchased from Sigma-Aldrich. Cells were maintained in RPMI-1640 (Lonza) containing 10% heat inactivated fetal bovine serum (Gibco), 2% penicillin/streptomycin/glutamine (Gemini Bio-Products) and 0.1% retinoic acid (RA). Cells were cultured for a minimum of 7 days prior to use in adhesion assays. RA was obtained from Sigma Chemical. CH58, human mAb isolated from RV144 vaccinated individuals, and VRC01 were provided by the NIAID AIDS reagent program. CAP228-16H mAb was generated in the laboratory of Dr. Lynn Morris (CAPRISA) [57]. The 830A mAb was provided by Dr. Susan Zolla-Pazner (Mt. Sinai Medical School) [42]. Rabbit Mk16C2 mAb (provided by Dr. Shan Lu, University of Massachusetts Medical School) was isolated from a rabbit that received a gp120-JRFL DNA prime- protein boost immunization, using V2-peptide specific single B cell sorting, and produced by transfection of cloned Ig genes in 293F cells at the University of Massachusetts Medical School [83]. The SIV mAbs ITS03, ITS09.01, ITS12.01, ITS13, and ITS41 were provided by Dr. Rosemarie Mason, NIAID VRC [61]. NCI09 was provided by Genoveffa Franchini and produced in the laboratory of Rosemarie Mason in a manner identical to mAbs mAbs ITS03, ITS09.01, ITS12.01, ITS13, and ITS41 [61]. Rhesus macaque serum from animals RLn12, RDa15, RId14 was provided by Dr. Aftab Ansari (Emory University) [37], was obtained after 72 weeks post-infection, and more than 40 weeks after the last infusion of anti-α₄β₇ mAb. Serum was purified by protein G column chromatography and dialyzed into HBS. Cyclic V2 peptides with > 90% purity and having an amino-terminal biotin, derived from 92TH023, BG505 and C06980v0c22 were supplied by JPT Peptide Technologies. Linear HIV and SIV 15 amino acid peptides were obtained from the NIAID AIDS Reagent Repository, or from Biopeptide Co. and supplied at >90% purity. Scaffolds V1/V2 92TH023-1FD6 and V1/V2 92TH023-Tag were constructed, expressed and purified as described elsewhere [42]. CHO cell derived A244 gp120 (Lot 26539–1) was provided by Global Solutions for Infectious Diseases (San Francisco, CA). Purification employed an anti-gD immunoaffinity resin, followed by both cation and anion exchange chromatography steps. Purity was estimated at 97.1%. CHO cell derived SIVmac766 gp120 was provided by Advanced Biotechnologies Laboratories. BG505 SOISP trimer was generously provided by Dr. Paolo Lusso, LIR/NIAID. Vedolizumab was obtained from the NIH Clinical Center Pharmacy Department. Human integrin α₄ mAb 2B4, MAdCAM-Ig, soluble α₄β₇ and α₄β₁ were obtained from R&D Systems. The LDV mimetic ELN-475772 was provided by ELAN Pharmaceuticals [84]. Conjugation of HRP to mAbs was carried out using a LYNX HRP conjugation kit obtained from Bio-Rad, using the manufacturer’s instructions.

Deglycosylation of purified gp120 and gp140 protein

Prior to use purified gp120 and gp140 proteins utilized in the α₄β₇ adhesion assays were first treated with a deglycosylation protocol [58]. Purified proteins were treated with 500U of PNGase F (NEB) per 20 μg of protein under non-denaturing conditions (1X GlycoBuffer 2 (NEB), 5mM DTT, and water) at 37°C for 3 hours.

Surface plasmon resonance analysis of α₄β₇ or anti-V2 loop mAb binding to Hu-MAdCAM, gp120 or cyclic V2 peptides

Experiments were performed using a Biacore 3000 instrument (GE Life Sciences) using CM4 or CM5 sensor chips. The data were evaluated using BIAevaluation 4.1 software (GE Life Sciences). The chip surface was activated by injecting 35 μl of a 1:1 mixture of 0.05 M N-hydroxysuccinimide and 0.2 M N-ethyl-N-(dimethylaminopropyl) carbodiimide at 5 μl/min. NeutrAvidin, HIV gp120 or Hu-MAdCAM-Ig (R&D Systems) at concentrations of 5 μg/ml in 10mM NaOAc, pH 4.5, were immobilized to approximately 750 resonance units (RU). After the proteins were immobilized to the desired densities, unreacted sites on each surface were blocked with 35 μl of 1 M Tris-HCl (pH 8.0). Biotinylated cyclic V2 peptides (1 μg/ml in 20 mM Tris-HCl, pH 8.0) were bound to the NeutrAvidin surfaces to densities of approximately 250–300 RU. One surface was activated and blocked without ligand to act as a control surface for non-specific binding of the soluble ligand. Any binding was subsequently subtracted from the remaining surfaces. Running buffer was HBS (pH 7.4), 0.01 mM CaCl₂, either 1 mM MgCl₂ or MnCl₂, 0.005% Tween-20, 0.05% soluble carboxymethyl-dextran. Binding experiments were carried out at a flow rate of 25 μl/min at 25°C. After a 2 min injection, the surface was washed for an additional 2 min in running buffer to follow dissociation of the bound ligand from the surface. The surfaces were regenerated by multiple injections of 4.5 M MgCl₂ at a flow rate of 100 μl/min. Inhibition of α₄β₇ or anti-V2 loop antibodies by linear V2-loop peptides was carried out by pre-incubating the proteins with the peptides in running buffer at the indicated concentrations for 2 hours at room temperature prior to passing them over the prepared surfaces as described above.

Kinetic analysis of anti-V2 loop mAbs binding to cyclic V2 peptides

Antibodies were diluted to the indicated concentrations in running buffer prior to being sequentially passed over the surface bound cyclic peptides as described above. The resulting sensorgram series were analyzed using the BiaEvaluation 4.1 software (GE Life Sciences) and fitted using a 1:1 Langmuir binding model to determine the kinetic rate and affinity constants.

Crystallization, data collection, structure determination and refinement of Mk16C2 in complex with V2 peptide

The Fab fragment of rabbit mAb Mk16C2 (provided by Dr. Shan Lu, Univ. of Massachusetts Medical School) was prepared by papain digestion as described (PMID: 19913488 and 20622876). Briefly, the IgG molecule was mixed with papain (Worthington, Lakewood, NJ) at a 20:1 molar ratio in 100 mM Tris (pH 6.8) with 1 mM cysteine hydrochloride and 4 mM EDTA. The mixture was incubated for 1 hour at 37°C and the reaction was stopped by 10 mM iodoacetamide. The Fab fragment was separated from the Fc fragment and the undigested IgG by a protein A column and further purified by size exclusion chromatography. The Fab fragment was then concentrated to about 10 mg/ml for crystallization.

The 15mer V2_ConB peptide (RDKVQKEYALFYKLD) was dissolved in water and mixed with Fab of rabbit mAb Mk16C2 in excess at a 10:1 molar ratio. Crystallizations conditions were screened and optimized using the vapor diffusion hanging drop method. Well-diffracted crystals of Mk16C2 Fab/V2_ConB complex were obtained with a well solution of 23% polyethylene glycol 3350, 0.2 M LiCL, 0.1 M 2-ethanesulfonic acid (MES) pH 6.5, and soaked briefly in the crystallization solution with an additional 20% glycerol before being flash frozen in liquid nitrogen. X-ray diffraction data sets were collected at the synchrotron beamline 14–1 of Stanford Synchrotron Radiation Lightsource (SSRL) of Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory. All data sets were processed using the XDS (PMID: 20124692), and structures determined by molecular replacement using another rabbit mAb R56 Fab structure (PDB ID 4JO1) as the initial model. Cycles of refinement for each model were carried out in COOT (PMID: 15572765) and Phenix (PMID: 20124702). Final structural analyses were carried out using ICM and figures were generated using PyMOL (pymol.org) and ICM (www.molsoft.com). The antigen-antibody interactions described in Fig 3A are calculated by PDBePISA (EMBL-EBI). Coordinate and structure factor of the complex have been deposited in the Protein Data Bank under PDB ID 6CEZ.

α₄β₇ adhesion to MAdCAM-1, HIV gp120, HIV cyclic V2 peptide and SIV gp120

The binding of α₄β₇ expressed by the RPMI8866 cell line to MAdCAM, HIV A244 gp120, HIV cyclic V2 peptides, and SIVmac766 gp120 gp120 in the absence or presence of vedolizumab or 2B4 or ELN-475772 to MAdCAM, HIV A244 gp120 were analyzed by an adhesion assay (adapted from KK Peachman et al., [11] (S2A Fig). This assay was modified by culturing RPMI8866 cells in media containing 1μM RA for at least 7 days prior to use in adhesion assays. Inclusion of RA increases adhesion to cV2 peptides, gp120 and MAdCAM (S2C Fig). Briefly, triplicate wells of a 96-well flat-bottom polypropylene plate (Greiner Bio-One) were coated overnight at 4°C with 100 μl of 2 μg/ml of MAdCAM-1 (R & D Systems) or 100 μl of 2 μg/ml NeutrAvidin or 100 μl of 0.5–2.0 μg of deglycosylated SIV and HIV gp120 diluted in 50mM bicarbonate buffer, pH 9.6. The NeutrAvidin-coated plates were then incubated with biotinylated cyclic V2 peptides (5 μg/ml in bicarbonate buffer) for 1 hour at 37°C. The solution from the plates was discarded and the plates were then blocked with blocking buffer (25mM Tris, 2.7mM potassium chloride, 150 mM sodium chloride, 0.5% BSA, 4mM manganese chloride, pH 7.2) for 1 hour at 37°C. The solution was discarded and plates were manually washed 4 times with blocking buffer. After blocking and washing the plate, RPMI8866 cells in a volume of 50 μl/well were pre-incubated for 40 min at 37°C with sample buffer in the absence or presence of 10 μg/ml of vedolizumab (α₄β₇ mAb) or 2B4 (α₄ mAb) or ELN-475772 (α₄β₇/α₄β₁ dual inhibitor). Plates were then incubated with 50 μl/well of 2x10⁵ RPMI8866 cells at 37°C (5% CO₂) for 1 hour, washed 5 times with PBS followed by the addition of 100 μl of RPMI-1640 containing 1% FBS, 1% pen/strep/glutamine, 25mM HEPES with 10 μl/well of AlamarBlue dye. Fluorescence (excitation 560 nm and emission 590 nm) was measured immediately after the addition of the AlamarBlue dye for 8 hours.

Capture of 92TH023 virions by MNPs coated with gp120 mAbs or α₄β₇

92TH023 virions were captured with 15 nm magnetic nanoparticles (MNPs) coupled to α₄β₇ or 2G12, CH58, PG9, and 830A mAbs as previously described [85]. Briefly, carboxyl-terminated iron oxide nanoparticles (Ocean Nanotech, San Diego) via two step carbodiimide reaction were coated with 1mg of anti-gp120 mAbs or recombinant soluble α₄β₇ according to manufacturer’s protocol. Virus preparations were derived from primary CD4⁺ T cells infected with an infectious molecular clone derived from 92TH023, using standard conditions. To capture virions, MNPs coated with mAbs (3.9 x10¹²) in 60μl were incubated with viral preparation (33 ng/ml based on p24 content) for 1hour at 37°C. Captured virions were separated on MACS magnetic columns attached to an OctoMacs magnet (Miltenyi Biotech) washed 4 times with 600 μl wash buffer (0.5% bovine serum albumin, 2mM EDTA in PBS), eluted in 200 μl PBS and analyzed on Luminex X200 for p24 content using a dynamic immunofluorescent cytometric bead assay [60].

In experiments with α₄β₇-MNPs, virions were incubated in the absence or presence of increasing amounts (1.25nM, 12.5nM, 125nM) of the α₄β₇ inhibitor ELN-475772. Incubation and washing were performed in complete medium with 1mM MnCl₂.

Inhibition of α₄β₇ adhesion to HIV gp120, HIV cyclic V2 peptides and SIV gp120 by V2 monoclonal antibodies and polyclonal sera

Triplicate wells of a 96-well flat-bottom polypropylene plate (Greiner Bio-One) were coated with biotinylated cyclic V2 peptides, deglycosylated SIV and HIV gp120 as described above. After blocking and washing, plates were incubated with 20–100 μg/ml of the designated anti-V2 mAbs or 2.5–10.0 μg of protein G purified IgG from sera drawn from SIV infected rhesus macaques (RLn12, RDa15, RId14 (provided by Dr. Aftab Ansari, Emory University School of Medicine)) in sample buffer (25 mM Tris, 2.7 mM KCl, 150 mM NaCl, 4 mM manganese chloride, 1% fetal bovine sera, pH 7.2) for 1 hour at 37°C. RPMI8866 cells were pre-incubated for 40 min at 37°C with sample buffer. Plates were then incubated with 50 μl/well of 2x10⁵ cells at 37°C (5% CO₂) for 1 hour, washed 5 times with PBS followed by the addition of 100 μl of RPMI-1640 containing 1% FBS, 1% pen/strep/glutamine, 25mM HEPES with 10 μl/well of AlamarBlue dye (Invitrogen). Following the addition of AlamarBlue dye (excitation 560 nm and emission 590 nm) fluorescence was measured for a period of 8 hours.

α₄β₇ adhesion to constrained and unconstrained V1/V2 92TH023 scaffolds

92TH023 V1/V2 was cloned into both 1FD6 (constrained) and tag (unconstrained) scaffolds. Plates were then coated with 0.5, 1.0, and 2.0 μg of either V1/V2 92TH023 1FD6 or V1/V2 92TH023 tag scaffolds followed by addition of RPMI8866 cells. The plates were washed and 100 μl of media and 10 μl of AlamarBlue dye was added to each well as described above. Fluorescence (excitation 560 nm and emission 590 nm) was measured immediately after the addition of the AlamarBlue dye.

Binding of cyclic V2 peptide to V2 monoclonal antibodies by ELISA

Briefly, Corning Costar 96-Well plates were coated with NeutrAvidin at 4°C overnight. Wells were washed six times with wash buffer (water, 1 mM MnCl₂, and 1X plate wash buffer) using Microplate Washer ELx50, (Bio Tek Instruments), and then blocked with blocking buffer (HBS, 5% bovine serum albumin) for 1 hour at room temperature. Plates were coated with 1 μg/ml biotinylated cyclic V2 peptides in binding buffer (HBS, 1 mM MnCl₂) for 1 hour at room temperature. The plates were washed six times with wash buffer and HRP-conjugated anti-V2 mAb was added to wells for 1 hour at room temperature. After washing, 100 μl/well of substrate was added for 10 min to develop color at room temperature in the dark. Plates were read at OD₄₅₀ nm using an EnSpire Multimode Plate Reader (PerkinElmer).

CH58 mAb and 830A mAb competition ELISA

10, 20, 50, 80 and 160 ng of the CH58 mAbs in binding buffer (HBS, 1 mM MnCl₂) were incubated with 50 ng of HRP-conjugated 830A mAb and then added to a plate coated with 100 ng of cV2 92TH023. Substrate was added using conditions specified by the manufacturer. Plates were read at OD₄₅₀ nm using an EnSpire Multimode Plate Reader, PerkinElmer.

Immunofluorescence staining

RPMI8866 cell line cultured in the presence or absence of RA were seeded at 1×10⁵/well in Poly-d-lysine coated glass bottom dishes (MatTek) with cover glasses and incubated overnight at 37°C. The cells were fixed with 2% paraformaldehyde (PFA), blocked with 1% BSA, and stained with an anti-β₇ PE mAb (BD Biosciences) or an IgG2a-PE isotype control mAb (R & D Systems). Stained RPMI8866 cells were microscopically analyzed using a Leica SP8 confocal microscope (Leica Microsystem, Inc.) and images were processed with Leica LAS AF software (Leica Microsystem, Inc.) and Imaris software v.9.0.1 64x (Bitplane AG).