ABSTRACT
Human cytomegalovirus (HCMV) encodes for multiple surface glycoproteins and glycoprotein complexes1, 2. One of these complexes, the HCMV Pentamer (gH, gL, UL128, UL130 and UL131), mediates tropism to both epithelial and endothelial cells by interacting with the cell surface receptor neuropilin 2 (NRP2)3, 4. Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here we describe the cryo-EM structure of NRP2 bound to the HCMV Pentamer. The high-affinity interaction between these proteins is calcium-dependent and differs from the canonical C-terminal arginine (CendR) binding that NRP2 typically utilizes5, 6. The interaction is primarily mediated by NRP2 domains a2 and b2, which interact with UL128 and UL131. We also determine the structures of four human-derived neutralizing antibodies in complex with the HCMV Pentamer to define susceptible epitopes. The two most potent antibodies recognize a novel epitope yet do not compete with NRP2 binding. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization, and serve as a guide for the development of HCMV treatments and vaccines.
MAIN TEXT
Human cytomegalovirus (HCMV) is a ubiquitous pathogen and congenital infection can cause debilitating and permanent birth defects7–9. Despite the severity of these infections and the prevalence of this pathogen, there are currently no FDA-approved vaccines and therapeutic options are limited10–12. HCMV is an enveloped, double-stranded DNA virus of the family Herpesviridae13. The surface of the viral membrane is decorated by several glycoprotein complexes that mediate viral entry and membrane fusion14–16. One of these complexes is the HCMV Trimer, composed of glycoproteins gH, gL, and gO2, 17. The HCMV Trimer mediates tropism for fibroblasts by binding platelet derived growth factor receptor alpha (PDGFRα)18, 19. The HCMV Trimer is also capable of mediating infection of a broader variety of cell types by interacting with transforming growth factor beta receptor 3 (TGFβR3)4, 20. The other critical tropism-determining complex is the HCMV Pentamer, which is composed of glycoproteins UL128, UL130, UL131, and the same gH and gL proteins that comprise the bulk of the HCMV Trimer1, 3. This elongated heteropentamer mediates tropism for endothelial and epithelial cells by binding to neuropilin 2 (NRP2) and triggering the viral fusion protein, gB, to facilitate viral entry into host cells4, 15, 21–23.
Neuropilins 1 and 2 are single-pass transmembrane proteins that are expressed on the surface of neuronal, epithelial, and endothelial cells24, 25. Under normal conditions, these proteins function as receptors and co-receptors that engage in numerous physiological processes, including angiogenesis and development of the nervous system26, 27. NRP2 is composed of two N-terminal CUB domains (a1 and a2), two F5/8 domains (b1 and b2), a MAM domain, a transmembrane domain, and a C-terminal PDZ domain that is thought to mediate cytoplasmic signaling in response to extracellular stimuli28, 29. Perhaps the most thoroughly characterized of these stimuli is vascular endothelial growth factor (VEGF)30. The crystal structure of these proteins in complex with one another has been determined, revealing that the b1 domain of NRP2 engages the C-terminal arginine of VEGF6, 31. Since this initial characterization, the NRP2 b1 domain has been shown to interact with other binding partners via the same mechanism32, prompting the moniker “CendR” to refer to this exposed C-terminal arginine motif5. Although it has been shown that soluble NRP2 is capable of inhibiting HCMV infection of epithelial cells4, the molecular determinants that mediate this interaction remain unclear, and several additional Pentamer receptors have been proposed33, 34. The most potently neutralizing HCMV-directed antibodies are elicited against the Pentamer, suggesting that it represents a susceptible target for the development of vaccines and immunotherapeutics35–37.
To investigate NRP2 and mAb binding, we initiated structural and biophysical studies. Based on previous crystallographic experiments that reported conserved calcium-coordinating loops in both the a1 and a2 domains of NRP238, 39, we measured the affinity of recombinantly expressed NRP2 a1a2b1b2 for the soluble HCMV Pentamer ectodomain in both the presence and absence of calcium. We found that in the presence of 2 mM EDTA, no association between NRP2 and Pentamer could be detected. However, when the same experiment was performed in the presence of 2 mM CaCl2, the affinity of the interaction was determined to be 2.2 nM (Supplementary Fig. 1a-b). It is possible that a failure to add additional calcium is what necessitated the use of chemical cross-linkers during previous efforts to observe this complex by negative-stain electron microscopy4. The addition of 2 mM CaCl2 enabled us to form a stable ∼230 kDa complex that was suitable for cryo-EM screening. The addition of 0.1% amphipol A8-35 helped to prevent aggregation and allowed for the determination of a 4.0 Å resolution cryo-EM structure of the HCMV Pentamer bound by human NRP2 (Fig. 1a, Supplementary Figs. 2 and 3). Performing focused refinement on the NRP2-bound UL proteins yielded a 3.65 Å reconstruction that aided in model building and refinement.
These reconstructions revealed an extensive binding interface, with contacts formed by NRP2 domains a1, a2 and b2 (Fig. 1). Notably, the calcium-coordinating loop of domain a2 (residues 251−258) forms a sizable portion of this binding interface, likely providing an explanation as to why high-affinity NRP2 binding could only be observed after the addition of 2 mM CaCl2 (Fig. 1a-b). Additional contacts are formed between the C-terminal beta strands of ULs 130 and 131 and a loop formed by residues 453-461 of the b2 domain of NRP2 (Fig. 1c). This mode of NRP2 binding differs from the canonical CendR motif binding that has been described previously for other NRP2-binding partners40, 41. The CendR binding mechanism involves the engagement of a C-terminal arginine residue by the b1 domain, whereas Pentamer is exclusively bound by the a1, a2, and b2 domains. Furthermore, none of the three UL proteins contain a positively charged C-terminal arginine that makes up the CendR motif. As expected, the majority of the binding interface from the Pentamer is composed of the tropism-determining UL proteins, particularly UL128 and UL1311, 3, which respectively contribute 437.5 Å2 and 208.4 Å2 of buried surface area to the interface. Whereas the NRP2 a2, b1, and b2 domains are clustered tightly together at the head of the Pentamer, the N-terminal a1 domain is tethered via a flexible linker that allows it to bind near the middle of the Pentamer, where the C-terminus of UL128 associates with gL. The local resolution for this portion of the reconstruction was relatively poor compared to the rest of the complex, suggesting either conformational flexibility in this region or a loose association of a1. To test the importance of the a1 domain, we expressed NRP2 with a 144-residue N-terminal truncation and observed that even in the absence of this flexibly tethered a1 domain, NRP2 a2b1b2 was capable of binding to the HCMV Pentamer with 7.9 nM affinity (Supplementary Fig. 1c), supporting our structural observations that the critical determinants of Pentamer binding are contained within NRP2 domains a2b1b2. Intriguingly, our cryo-EM data processing also revealed that a second, more poorly resolved copy of NRP2 could be observed binding near the C-terminal arginine of gL via the b1 domain (Supplementary Figs. 2 and 4). Although this second NRP2 appears to exhibit the canonical CendR binding, it could only be observed in ∼40% of particles. Furthermore, its binding to the gL protein rather than the tropism-determining UL proteins suggests that this second copy of NRP2 is likely an artifact of the high concentrations of NRP2 that were used to form a stable complex. Overall, the conformation of the receptor-bound Pentamer ectodomain does not drastically differ from that of the unbound Pentamer42 (Supplementary Fig. 5), suggesting that rather than undergoing substantial conformational rearrangements, this complex acts as a tether to connect HCMV virions to the surface of epithelial and endothelial cells until the viral fusogen gB fuses the viral and cellular membranes.
Previous efforts to characterize the humoral immune response to asymptomatic HCMV infection yielded an extensive panel of neutralizing antibodies directed against gB, the HCMV Trimer, and the HCMV Pentamer35, 43, 44. To learn more about the mechanisms of neutralization of high-affinity, Pentamer-directed antibodies, we determined cryo-EM structures of four naturally elicited human antibodies in complex with the Pentamer (Fig. 2, Supplementary Figs. 3, 6, 7, and 8). The flexibility and elongated shape of the Pentamer necessitated focused refinements of the Fabs along with the domains making up their respective epitopes. Model building was facilitated by high-resolution crystal structures of unbound Fabs (1-103: 1.9 Å, 1-32: 2.1 Å, 2-18: 2.8 Å, 2-25: 2.5 Å), which were then used as reference restraints and lightly refined as a part of the complex (Supplementary Tables 1 and 2). Three of these antibodies (1-103, 2-18, and 2-25) bound solely to the UL proteins at the head of the Pentamer, whereas the fourth (1-32) bound to gL, near the junction between the UL proteins and the conserved gH/gL scaffold (Fig. 2a). The Fab 1-103 epitope is solely composed of residues from the membrane-distal tip of UL128, sometimes referred to as Site 1 of immunogenic region (IR) 14, 35. The epitopes of Fabs 2-18 and 2-25 overlap substantially, with both Fabs binding to the junction between ULs 128 and 131. This junction where UL128 meets UL131 does not fit into one of the preexisting antigenic sites, but rather overlaps with both Site 2 and Site 5 of IR14, 35. The Fab 1-32 epitope spans the interface between gH and gL, slightly below Site 4/6 in IR24, 35 (Fig. 2b). This epitope is consistent with previous observations35 that 1-32 is the only one of the four antibodies evaluated that was capable of binding to both the fully assembled HCMV Pentamer and disulfide-linked homodimers of the gH/gL heterodimer. Despite the ability to recognize the gH/gL heterodimer that serves as the scaffold for assembly of both the HCMV Pentamer and Trimer, 1-32 was only capable of neutralizing HCMV infection in epithelial cells35.
By analyzing the structures of these immunocomplexes in conjunction with the structure of Pentamer bound by NRP2, it becomes possible to delineate more clearly the molecular basis for neutralization (Fig. 3). The CDR H3 and CDR L1 of Fab 1-103 compete with NRP2 by binding to the same portion of Pentamer that is engaged by the calcium-coordinating loop (residues 251 to 258) of NRP2 domain a2. By binding to the junction between gL and the UL head of Pentamer, Fab 1-32 occupies the same space as several of the loops of the a1 domain of NRP2 (residues 45−48; 72−77; 106−110) via its CDR L1 (Fig. 3b). Consistent with the relatively poor density for domain a1, we found that when bound by 1-32, Pentamer was still capable of interacting with NRP2, albeit with diminished affinity (133 nM) (Supplementary Fig. 9), further supporting our findings that the a1 domain is not strictly required for binding to Pentamer. This observation is also consistent with the relatively weak neutralization capacity of 1-32 compared to 1-103, which competes for the a2b1b2 interface.
Intriguingly, the two most potently neutralizing mAbs that we examined, 2-18 and 2-25 (Fig. 4a), do not appear to compete with NRP2 a1a2b1b2 based on our structural analysis. Furthermore, although it is possible that 2-18 and 2-25 compete with the C-terminal MAM domain, this seems unlikely based on the position of the C-terminus of b245. These mAbs are both directed against an epitope at the junction between ULs 128 and 131, and although this epitope is directly adjacent to the NRP2 binding interface, the binding angles of 2-18 and 2-25 result in these two Fabs being directed away from the β-sheet-rich face of the Pentamer that is responsible for engaging NRP2. A biolayer interferometry-based competition assay confirmed our structural observation that Fabs 2-18 and 2-25 do not disrupt the interaction between Pentamer and NRP2 a1a2b1b2 (Fig. 4b). These two Fabs are still capable of neutralizing HCMV in vitro even when administered up to 30 minutes after allowing viruses to adhere to human epithelial cells, although we observed a significant decrease in the neutralization potency of 2-25 Fab, as compared to 2-25 IgG (Figs. 4a, 4c). This broad and potent neutralization of multiple HCMV strains (Fig. 4d), which occurs without disrupting the interaction between NRP2 a1a2b1b2 and the Pentamer, suggests that 2-18 and 2-25 neutralize via a distinct mechanism from 1-103 and 1-32 (Supplementary Fig. 10). Whether 2-18 and 2-25 prevent the association of an unidentified secondary receptor or prevent some conformational change that is required to trigger gB-induced membrane fusion remains unclear and requires additional investigation.
Collectively, these data provide a molecular basis of HCMV tropism for both epithelial and endothelial cells. Due to the importance of these cell types during natural infection, this represents a critical advance in our understanding of how HCMV engages host cells at one of the earliest stages of infection22, 46. Similarly, the structure of the HCMV Trimer was recently reported in complex with PDGFRα and TGFβR3, two host cell receptors that both mediate tropism of fibroblasts18, 20. In an effort to explain how this interaction might lead to triggering of gB and viral fusion, the authors speculate that receptor engagement of the Trimer may induce a rigid-body rotation relative to the viral membrane that causes the attachment complex to destabilize prefusion gB47. Our findings agree with their observation that receptor binding does not induce any conformation changes, lending credence to the hypothesis that a rigid-body rotation of the receptor-binding complex may act as trigger to induce membrane fusion. However, the existence of neutralizing mAbs that do not disrupt NRP2 binding suggests that additional fusion triggers, perhaps in the form of secondary receptors33, 34 may exist, necessitating further investigation.
In addition to detailing the molecular determinants that mediate HCMV infection, these findings expand our understanding of how antibody-mediated neutralization of HCMV can be achieved. Previous structural work has delineated a series of antigenic sites covering the surface of the Pentamer4, 35, 42, 48, but in the absence of high-resolution information regarding the NRP2 binding interface, the mechanisms of neutralization for antibodies targeting these sites were unknown. Our data suggest that it may be possible to neutralize HCMV via multiple, distinct mechanisms simultaneously by administering a cocktail of Pentamer-directed antibodies36, 49, 50. By elucidating which epitopes on the surface of the Pentamer are susceptible to antibody-mediated neutralization, these findings will also help to guide future structure-based vaccine design efforts.
AUTHOR CONTRIBUTIONS
D.W., X.Y., Z.A., and J.S.M. conceived of and designed experiments. D.W., X.Y., H.G.J., N.W., and A.K.M. produced and purified proteins. D.W., H.G.J., and A.K.M. performed crystallographic studies. D.W. performed BLI and SPR experiments. D.W. and J.S.M. collected and analyzed cryo-EM data. X.Y., Z.K., and L.L. established the neutralization assays and X.Y., H.S., D.C.F., and F.L. performed the assays. X.Y., Z.K., A.T., and D. Wang analyzed the neutralization data. D.K.J. and H.Z. prepared the HCMV AD169rev-GFP virus. Z.A., T-M.F., N.Z., and J.S.M. supervised experiments. D.W., X.Y., Z.A., and J.S.M. wrote the manuscript with input from all authors.
DECLARATIONS OF INTEREST
Z.A. and T-M.F. have filed a patent related to the antibody 2-18. D.C.F., F.L., A.T., and D. Wang are Merck & Co., Inc. employees. Other authors declare no competing interests.
METHODS
Protein production and purification
Plasmids encoding the heavy and light chains of 1-103, 1-32, 2-18, 2-25 and 8I21 IgG with an HRV3C protease cleavage site engineered into the hinge between the CH1 and CH2 domains of the heavy chain were co-transfected into FreeStyle 293F cells using polyethylenimine. To produce the soluble ectodomain of the HCMV Pentamer (strain Towne), plasmids encoding residues 24-718 of gH with a C-terminal 6x HisTag, residues 31-278 of gL, residues 21-171 of UL128, residues 26-214 of UL130 and residues 19-129 of UL131, all with artificial signal sequences were simultaneously co-transfected at an equimolar ratio.
Similarly, plasmids encoding an artificial signal peptide, residues 23-595 of human NRP2 and a C-terminal HRV3C cleavage site with either an 8x HisTag and a TwinStrepTag or a monomeric IgG1 Fc tag and an 8x HisTag were transfected into FreeStyle 293F cells, as described above. An N-terminal truncation of NRP2 that encompassed residues 145-595 with an artificial signal sequence and a C-terminal HRV3C cleavage site with a monomeric IgG1 Fc tag and an 8x HisTag (NRP2 a2b1b2) was transfected using the same conditions. NRP2 and NRP2 a2b1b2 were purified from cell supernatants using either StrepTactin resin (IBA) or Protein A resin before being run over a Superdex 200 Increase column using a buffer composed of 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3 and 2 mM CaCl2.
To form the complex of Pentamer + 1-103 + 1-32 + 2-25, purified 1-103 IgG was immobilized to Protein A resin and this 1-103 resin was then used to capture Pentamer from co-transfected cell supernatants. The 1-103 + Pentamer complex was then eluted by incubation with HRV3C protease and purified over a Superose 6 Increase column in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3. This complex was then passed over a column containing 2-25 IgG immobilized to Protein A resin. Again, the complex was eluted by incubation with HRV3C protease and a molar excess of 1-32 Fab was added before a final round of purification over a Superose 6 Increase column using the same buffer.
To form the Pentamer + 2-18 + 8I21 complex, purified 2-18 IgG was immobilized to Protein A resin and used to capture Pentamer from co-transfected cell supernatants. The 2-18 + Pentamer complex was then eluted by incubation with HRV3C protease and mixed with a molar excess of 8I21 Fab before being run over a Superose 6 Increase column in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3.
To form the Pentamer + NRP2 complex, purified Pentamer was mixed with a threefold molar excess of 8x His/TwinStrep-tagged NRP2 in a buffer composed of 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3 and 2 mM CaCl2 and the two components were allowed to bind on ice for 1 hour. This mixture was then purified over a Superose 6 Increase column (Cytiva) using the same buffer.
X-ray crystallographic studies
Purified IgGs 1-103, 1-32, 2-18 and 2-25 were incubated with 10% (wt/wt) His-tagged HRV3C protease on ice for 2 hours before being passed over Protein A and NiNTA resin to removed cleaved Fc and excess protease. The remaining Fab was purified by SEC using a Superdex 200 Increase column in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3 (1-132 and 2-18) or 2 mM Tris pH 8.0, 50 mM NaCl and 0.02% NaN3 (1-103 and 2-25).
1-103 Fab was concentrated to 15.00 mg/mL and used to prepare sitting-drop crystallization trays. Diffraction-quality crystals grew in a mother liquor composed of 2.1 M sodium formate, 25% PEG 3350, 0.1 M sodium acetate pH 4.5 and 0.1 M calcium chloride. 1-103 Fab crystals were cryoprotected using mother liquor supplemented with 20% glycerol before being plunge frozen into liquid nitrogen.
1-32 Fab was concentrated to 11.00 mg/mL and used to prepare hanging-drop crystallization trays. Diffraction-quality crystals were grown in 2.0 M ammonium sulfate, 0.2 M sodium chloride and 5% isopropanol. 1-32 Fab crystals were cryoprotected using mother liquor supplemented with 20% glycerol before being plunge frozen into liquid nitrogen.
2-18 Fab was concentrated to 12.00 mg/mL and used to prepare sitting-drop crystallization trays. Small crystalline needles, grown in 0.1 M HEPES pH 7.5 and 45% PEG 400 were used to perform microseed matrix screening, ultimately yielding diffraction-quality crystals in a mother liquor composed of 0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate pH 5.6 and 30% PEG 4000. 2-18 Fab crystals were cryoprotected using mother liquor supplemented with 20% glycerol before being plunge frozen into liquid nitrogen.
2-25 Fab was concentrated to 15.4 mg/mL and used to prepare sitting-drop crystallization trays. Diffraction-quality crystals were grown in 30% PEG 4000, a mixture of 0.2 M divalent cations51 and 0.1 M BIS-TRIS pH 6.5. 2-25 Fab crystals were looped without cryoprotectant and directly plunge frozen into liquid nitrogen.
All diffraction data were collected at Argonne National Labs, Advanced Photon Source, SBC-19ID. Datasets were indexed in iMOSFLM52 and scaled in AIMLESS53. Molecular replacement solutions were determined using PhaserMR54 and models were iteratively built and refined using Coot55, PHENIX56 and ISOLDE57. Full crystallographic data collection and refinement statistics can be found in Supplementary Table 1. Crystallographic software packages were curated by SBGrid58.
Cryo-EM sample preparation and data collection
Purified HCMV Pentamer + 2-18 + 8I21 complex was diluted to a concentration of 0.25 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3 and 0.01% amphipol A8-35. 8I21 Fab was added after initial attempts to visualize the Pentamer + 2-18 complex were hampered by a lack of distinguishable features (data not shown). 3 μL of the ternary complex was deposited on a CF-1.2/1.3 grid that was glow discharged at 25 mA for 1 minute using an Emitech K100X (Quorum Technologies). Excess liquid was blotted away for 6 seconds in a Vitrobot Mark IV (FEI) operating at 4° C and 100% humidity before being plunge frozen into liquid ethane. Data were collected on a Titan Krios (FEI) operating at 300 kV, equipped with a K3 direct electron detector (Gatan). Movies were collected using SerialEM59 at 22,500x magnification, corresponding to a pixel size of 1.047 Å.
Purified HCMV Pentamer + 1-103 + 1-32 + 2-25 complex was diluted to a concentration of 0.2 mg/mL in 2 mM Tris pH 8.0, 400 mM NaCl, 0.02% NaN3 and 0.01% amphipol A8-35. 3 μL of protein was deposited on a CF-1.2/1.3 grid that was plasma cleaned at 25 mA for 30 seconds using a Solarus plasma cleaner (Gatan). Excess liquid was blotted away for 6 seconds in a Vitrobot Mark IV (FEI) operating at 4° C and 100% humidity before being plunge frozen into liquid ethane. Data were collected on a Titan Krios (FEI) operating at 300 kV, equipped with a K2 direct electron detector (Gatan). Movies were collected using Leginon60 at 22,500x magnification, corresponding to a pixel size of 1.075 Å.
Purified HCMV Pentamer + NRP2 complex was diluted to a concentration of 0.4 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl, 2 mM CaCl2, 0.02% NaN3 and 0.01% amphipol A8-35. 3 μL of protein was deposited on an UltrAuFoil 1.2/1.3 grid that was plasma cleaned at 25 mA for 2 minutes using a Solarus plasma cleaner (Gatan). Excess liquid was blotted away for 3 seconds in a Vitrobot Mark IV (FEI) operating at 4° C and 100% humidity before being plunge frozen into liquid ethane. Data were collected on a Titan Krios (FEI) operating at 300 kV, equipped with a K3 direct electron detector (Gatan). Movies were collected using SerialEM59 at 22,500x magnification, corresponding to a pixel size of 1.073 Å.
Cryo-EM data processing and model building
Motion correction, CTF-estimation and non-templated particle picking using BoxNet were performed in Warp61. Extracted particles were imported into cryoSPARC62 for 2D classification, ab initio 3D reconstruction calculation, 3D classification and non-uniform refinement63. Based on the flexibility of the interface between the gH/gL and UL proteins, particle subtraction and focused refinement were also performed in cryoSPARC. Final reconstructions were sharpened with DeepEMhancer64. A full description of the cryo-EM data processing workflows can be found in Supplementary Figs. 2, 6 and 7. Crystal structures were docked into cryo-EM density maps using Chimera65 before being refined in Coot55, PHENIX56 and ISOLDE57. A detailed description of the cryo-EM data processing workflow can be found in Supplementary Figs. 2, 6 and 7. Full cryo-EM data collection and refinement statistics can be found in Supplementary Table 2.
Surface plasmon resonance (SPR)
Purified His-tagged Pentamer was immobilized to a single flow cell of a NiNTA sensor in a Biacore X100 (GE Healthcare) to a level of ∼800 response units (RUs) using HBS-P+ buffer adjusted to a pH of 8.0. Two-fold serial dilutions of Fabs 1-103, 1-32, 2-18 and 2-25 were injected over both flow cells to measure binding kinetics. The sensor was doubly regenerated using 350 mM EDTA and 100 mM NaOH in between cycles. Data were double reference-subtracted and fit to a 1:1 binding model using Biacore Evaluation Software (GE Healthcare).
Biolayer Interferometry (BLI)
Purified monoFc-tagged NRP2 or NRP2 a2b1b2 was immobilized to anti-human capture (AHC) tips (ForteBio) in a buffer composed of 10 mM HEPES pH 8.0, 150 mM NaCl, 0.05% Tween 20, 1 mg/mL BSA and 2 mM CaCl2. Sensors were then dipped into wells containing purified HCMV Pentamer, ranging in concentration from 50 nM to 3.125 nM. Data were reference subtracted and processed using Octet Data Analysis software v10.0 (ForteBio) with a 1:1 binding model. To evaluate the impact of calcium on Pentamer binding, the same experiment was performed using monoFc-tagged NRP2 in a buffer composed of 10 mM HEPES pH 8.0, 150 mM NaCl, 0.05% Tween 20, 1 mg/mL BSA and 2 mM EDTA.
To evaluate competition between Fabs and NRP2, monoFc-tagged NRP2 was immobilized to AHC tips in a buffer composed of 10 mM HEPES pH 8.0, 150 mM NaCl, 0.05% Tween 20, 1 mg/mL BSA and 2 mM CaCl2. Sensors were then dipped into wells containing a mixture of purified HCMV Pentamer at a concentration of 50 nM and 100 nM Fab. Data were reference subtracted using Octet Data Analysis software v10.0.
To measure the binding kinetics of NRP2 to Pentamer in the presence of mAb 1-32, 1-32 IgG was immobilized to AHC sensors using a buffer composed of 10 mM HEPES pH 8.0, 150 mM NaCl, 0.05% Tween 20, 1 mg/mL BSA and 2 mM CaCl2. Tips with immobilized 1-32 were then dipped into wells containing 100 nM Pentamer. The 1-32-captured Pentamer was then dipped into wells containing untagged NRP2, ranging in concentration from 400 nM to 25 nM. Data were reference subtracted and processed using Octet Data Analysis software v10.0 with a 1:1 binding model.
HCMV neutralization assay
All of the Pentamer-specific antibodies used for the purposes of neutralization and inhibition assays were produced as described previously43. A dengue virus specific human IgG1 antibody66 was used as isotype control. Fabs for neutralization and inhibition assays were generated by digesting IgG with papain (Sigma, P4762) and purifying as described previously67. A standard neutralization assay with the Towne-ts15-rR, AD169rev, and 12 clinical isolates as shown in Supplementary Fig. 10 were performed in ARPE-19 cells using an immunostaining method68. Neutralization assays in Fig. 4 were performed in ARPE-19 cells using AD169rev-GFP strain and virus infection was examined through GFP expression as described previously69. For the standard neutralization assay, 50 µL/well of AD169rev-GFP, generating about 100 GFP-positive cells was incubated with 50 µL/well of serial 2-fold diluted IgG or Fab (at indicated concentrations) at 37 °C for 30 min and then added to confluent ARPE-19 cells grown in a 96-well plate. Mock-infected cells and cells infected with virus-only served as controls. For the post-attachment assay, ARPE-19 cells grown in a 96-well plate were pre-cooled at 4 °C for 10 min. 50 µL/well of AD169rev-GFP was allowed to attach to cells for 1 h at 4 °C. After removing unattached virus through a single wash using cold media, the indicated IgG, diluted at concentrations of ∼200 times of corresponding IC50 was added after culturing AD169rev-GFP-attached cells for different lengths of time (0 min, 30 min, and 60 min) in a 37 °C incubator. The antibody-containing media was replaced with fresh media without antibody 2 h later. Mock-infected cells and cells infected with virus but not treated with antibodies served as controls. For all above assays, triplicate wells were determined for each condition and viral infection was examined at 48 h post-infection. A C.T.L. Immunospot analyzer was used to capture whole-well images of GFP expression and quantification of GFP-positive cells. The percentage of viral inhibition by the antibody and the IC50 of each antibody was calculated by non-linear fit of virus inhibition % vs. concentration (ng/mL) using GraphPad Prism® 5 software.
ACKNOWLEDGEMENTS
We thank Emilie Shipman and Dr. John Ludes-Meyers for their assistance with cell transfection and protein production; Drs. Aguang Dai and Sasha Dickinson at the Sauer Structural Biology Laboratory for their assistance with microscope alignment and data collection; the 19-ID beamline staff at the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory; and Dr. Georgina Salazar for assistance with manuscript preparation. This study was funded in part by grants from Merck & Co., Inc. Kenilworth, NJ, USA, the Texas Emerging Technology Fund, and the Welch Foundation Grant No. AU-0042-20030616. The Sauer Structural Biology Laboratory is supported by the University of Texas College of Natural Sciences and by award RR160023 from the Cancer Prevention and Research Institute of Texas (CPRIT). Argonne National Laboratory is operated by UChicago Argonne, LLC, for the U.S. Department of Energy (DOE), Office of Biological and Environmental Research under Contract DE-AC02-06CH11357.