Methods for modulating host cell surface interactions with human cytomegalovirus

Abstract

Provided herein are methods for treating or preventing human cytomegalovirus (HCMV) infection, comprising modulating the interaction between the HCMV gHgLgO trimer and a plasma membrane-expressed host cell protein, and methods for identifying modulators of such an interaction.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 118,859, filed November 27, 2020, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on November 15, 2021 is titled 50474-247WO2_Sequence_Listing_11_15_2021_ST25 and is 26,180 bytes in size.

[0003] Provided herein are methods for treating or preventing human cytomegalovirus (HCMV) infection, comprising modulating the interaction between the HCMV gHgLgO trimer and a plasma membrane-expressed host cell protein, and methods for identifying modulators of such an interaction. [Background technology]

[0004] Human cytomegalovirus (HCMV) is a member of the Betaherpesvirinae subfamily of the Herpesviridae family, establishing a lifelong infection in over 70% of the human population. Following primary infection, HCMV becomes latent, and its reactivation causes severe morbidity and mortality in individuals who are immunosuppressed or undergoing organ or hematopoietic stem cell (HSC) transplantation. HCMV poses a particular threat during pregnancy due to its ability to cross the placental barrier and infect the fetus. HCMV infection affects 0.3% to 2.3% of newborns and is a leading viral cause of congenital birth defects, including brain damage, hearing loss, learning disabilities, heart disease, and mental retardation. For these reasons, HCMV has been identified as a high-priority disease target by the Institute of Medicine. Effective antiviral therapeutics or vaccines should target the early stages of the HCMV infection cycle, including viral entry into host cells. HCMV enters different cell lines using several envelope glycoprotein complexes, including two gHgL envelope glycoprotein complexes, gHgLgO (trimer) and gHgLpUL128-131A (pentamer), and glycoprotein B (gB). HCMV trimer or pentamer binding to cellular host receptors provides a trigger signal for the HCMV glycoprotein gB to catalyze membrane fusion between the virus and the infected cell through a yet-to-be-identified mechanism. This fusion allows HCMV to enter the cell, replicate, and establish its latency.

[0005] HCMV exhibits a broad cytotropy, including fibroblasts, monocytes, macrophages, neurons, epithelial cells, and endothelial cells, through interactions with structurally and functionally distinct receptor proteins. Recent evidence has suggested a key role for the trimeric complex in infection of all cell types. Trimer-mediated infection of fibroblasts is the most well-studied and involves the interaction of the trimer with PDGFRα, a member of the receptor tyrosine kinase 3 (RTK3) family. TGFβR3 has also been found to bind HCMV trimers with high affinity, representing an additional putative cellular receptor that may explain the broad cytotropy of HCMV.

[0006] Over the past few decades, significant efforts have been made to develop vaccine candidates against HCMV infection. However, results from recent clinical trials have shown that HCMV vaccines have only moderate efficacy in preventing viral infection. Therefore, the development of effective therapeutic agents against HCMV represents an important unmet medical need. Summary of the Invention

[0007] In one aspect, the disclosure features a modulator of the interaction between the gO subunit of the human cytomegalovirus (HCMV) gHgLgO trimer and PDGFRα, wherein the modulator binds to a glycosylation-free surface of the gO subunit, causing a decrease in binding of the gO subunit to PDGFRα.

[0008] In some embodiments, the modulator binds to (a) one or more of residues R230, R234, V235, K237, and Y238 of the gO subunit, (b) one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit, and (c) one or more of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit.

[0009] In another aspect, the disclosure features a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα, wherein the modulator binds to (a) one or more of residues R230, R234, V235, K237, and Y238 of the gO subunit, (b) one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit; and (c) one or more of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit, causing a decrease in binding of the gO subunit to PDGFRα.

[0010] In some embodiments, the modulator binds to all 23 of the gO subunit residues: R230, R234, V235, K237, Y238, N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, V123, R336, Y337, K344, D346, N348, E354, and N358.

[0011] In some embodiments, the modulator further binds to one or more of residues R47, Y84, and N85 of the gH subunit of HCMV.

[0012] In some embodiments, the modulator is a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an ASO or siRNA.

[0013] In some embodiments, the antigen-binding fragment is a bis-Fab, Fv, Fab, Fab'-SH, F(ab')2, diabody, linear antibody, scFv, scFab, VH domain, or VHH domain.

[0014] In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the bispecific or multispecific antibody binds to at least three distinct epitopes of the gO subunit. In some embodiments, the at least three distinct epitopes include: (a) a first epitope comprising one or more of residues R230, R234, V235, K237, and Y238 of the gO subunit; (b) a second epitope comprising one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit; and (c) a third epitope comprising one or more of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit.

[0015] In some embodiments, the modulator is a mimetic of PDGFRα.

[0016] In another aspect, the disclosure features a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα, wherein the modulator binds to the D1 (SEQ ID NO: 11), D2 (SEQ ID NO: 12), and D3 (SEQ ID NO: 13) domains of PDGFRα and causes a decrease in binding of the gO subunit to PDGFRα.

[0017] In some aspects, the modulator binds to (a) one or more of residues N103, Q106, T107, E108, and E109 of PDGFRα, (b) one or more of residues M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRα, and (c) one of residues N240, D244, Q246, T259, E263, and K265 of PDGFRα.

[0018] In another aspect, the disclosure features a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα, wherein the modulator binds to (a) one or more of residues N103, Q106, T107, E108, and E109 of PDGFRα, (b) one or more of residues M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRα; and (c) one or more of residues N240, D244, Q246, T259, E263, and K265 of PDGFRα, causing a decrease in binding of the gO subunit to PDGFRα.

[0019] In some embodiments, the modulator binds to all ten of the following residues of PDGFRα: T107, E108, E109, M133, L137, I139, Y206, L208, E263, and K265.

[0020] In some embodiments, the modulator further binds to one or more of residues E52, S78, and L80 of PDGFRα.

[0021] In some embodiments, the modulator is a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an ASO or siRNA.

[0022] In some embodiments, the antigen-binding fragment is a bis-Fab, Fv, Fab, Fab'-SH, F(ab')2, diabody, linear antibody, scFv, scFab, VH domain, or VHH domain.

[0023] In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the bispecific or multispecific antibody binds to at least three distinct epitopes of PDGFRα. In some embodiments, the at least three distinct epitopes include: (a) a first epitope comprising one or more of residues N103, Q106, T107, E108, and E109 of PDGFRα; (b) a second epitope comprising one or more of residues M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRα; and (c) a third epitope comprising one or more of residues N240, D244, Q246, T259, E263, and K265 of PDGFRα.

[0024] In some embodiments, the modulator is a mimetic of the gO subunit of the HCMV gHgLgO trimer.

[0025] In some embodiments, the modulator reduces binding of the gO subunit of the HCMV gHgLgO trimer to PDGFRα by at least 50%. In some embodiments, the modulator reduces binding of the gO subunit of the HCMV trimer to PDGFRα by at least 90%.

[0026] In some embodiments, the modulator reduces binding of the gO subunit of the HCMV gHgLgO trimer to TGFβR3 by at least 50%.

[0027] In some embodiments, the reduction in binding is measured by surface plasmon resonance, biolayer interferometry, or enzyme-linked immunosorbent assay (ELISA).

[0028] In some embodiments, the modulator has minimal binding to a region of PDGFRα that induces downstream signaling.

[0029] In some embodiments, the modulator does not bind to a region of PDGFRα that induces downstream signaling.

[0030] In some embodiments, the region of PDGFRα that induces downstream signaling is the binding site for PDGF.

[0031] In some embodiments, the modulator causes less than a 20% decrease in signaling by PDGFRα compared to signaling in the absence of the modulator.

[0032] In some embodiments, the modulator does not cause a decrease in signaling by PDGFRα compared to signaling in the absence of the modulator.

[0033] In some embodiments, the modulator causes a decrease in infection of cells by HCMV compared to infection in the absence of said modulator, hi some embodiments, infection is reduced by at least 40% as measured in a viral infection or entry assay using pseudotyped particles.

[0034] In some aspects, the modulator further comprises a pharmaceutically acceptable carrier.

[0035] In another aspect, the disclosure features a method of treating an HCMV infection in an individual, the method comprising administering to the individual an effective amount of a modulator provided herein, thereby treating the individual. In some aspects, the duration or severity of the HCMV infection is reduced by at least 40% compared to an individual not administered the modulator.

[0036] In another aspect, the disclosure features a method of preventing HCMV infection in an individual, the method comprising administering to the individual an effective amount of a modulator provided herein, thereby preventing HCMV infection in the individual.

[0037] In another aspect, the disclosure features a method of preventing a secondary HCMV infection in an individual, the method comprising administering to the individual an effective amount of a modulator provided herein, thereby preventing a secondary HCMV infection in the individual. In some aspects, the secondary infection is an HCMV infection of an uninfected tissue. In some aspects, the individual is immunocompromised, pregnant, or an infant. [Brief explanation of the drawings]

[0038] [Figure 1A] Figure 1A is a global cryo-electron microscopy (cryo-EM) map showing the human cytomegalovirus (HCMV) gHgLgO glycoprotein trimeric complex bound to neutralizing Fab 13H11 and Msl-109. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. Gray (left): Fab 13H11. Gray (right): Fab Msl-109. [Figure 1B] Figure 1B is a pair of ribbon diagrams showing the front (left) and back (right) views of the HCMV gHgLgO trimeric complex. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. [Figure 1C] Figure 1C is a pair of images showing the electrostatic surface of the HCMV gHgLgO trimeric complex (same as Figure 1B) in the range of -10 to +10 keV. Red: negatively charged. Blue: positively charged. [Figure 1D] FIG. 1D is a pair of panels showing the distribution of glycosylation sites (colored) on the HCMV gHgLgO trimeric complex (same panel as FIG. 1B). [Figure 2A]FIG. 2A shows the superposition (coloring) of the gHgL subunit of the HCMV trimeric complex onto the gHgL subunit of the HCMV pentameric complex (gray, PDB code: 5VOB). [Figure 2B] FIG. 2B shows the superposition of the HCMV trimeric gHgL glycosylation site (colored) onto the HCMV pentameric gHgL glycosylation site (gray, PDB code: 5VOB). [Figure 2C] Figure 2C is a pair of panels showing a front view of the distal region of the HCMV trimer (top panel) showing the N-terminus of gH, gL, and gO, and a close-up of the gL-gO interaction region (bottom panel) highlighting the gL loop between residues A131 and V151 and the disulfide bond between gL residue C144 and gO residue C343. Red: gO subunit. Pink (top panel): gL subunit. Blue: gH subunit. [Figure 2D] Figure 2D is a pair of images showing a frontal view of the distal region of the HCMV pentamer (top panel) showing the N-terminus of gH, gL, UL130, UL131, and UL128, and a close-up view of the gL-UL128 interaction region (bottom panel) highlighting the gL helix between residues A131 and V151 and the disulfide bond between gL residue C144 and UL128 residue C162. Pink (top panel): gL subunit. Blue: UL131. Green: UL128. Orange: UL130. [Figure 3A] Figure 3A shows a front view of the HCMV gHgLgO trimeric complex bound to the neutralizing Fabs 13H11 and Msl-109. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. Green and light green: 13H11. Orange and light orange: Msl-109. [Figure 3B]Figure 3B shows the variable Fab regions of 13H11 and Msl-109 bound to the C-terminal region of gH. Inset panels 1-3 show a close-up of the key interaction site between 13H11 and the HCMV trimeric gH subunit. Inset panel 4 shows a close-up of the key interaction region between Msl-109 and the HCMV trimeric gH subunit. The Fab contact region on gH is highlighted in pink. Green: 13H11. Orange: Msl-109. [Figure 3C] Figure 3C is a set of figures showing a close-up of the variable Fab region of 13H11 and the highlighted interaction surfaces for the heavy (dark green) and light (light green) chains of 13H11 on gH (left) and the variable Fab region of Msl-109 and the highlighted interaction surfaces for the heavy (dark orange) and light (light orange) chains of Msl-109 on gH (right). [Figure 4A] FIG. 4A shows the structure of the HCMV gO subunit with the domain organization indicated by color. [Figure 4B] Figure 4B is a schematic diagram showing the domain organization of the HCMV gO subunit with secondary structure elements indicated: Domains 1-5: N-terminal beta strands; Domains 6, 9-10, and 12: central alpha helices; and Domains 16-17: C-terminal alpha helices. [Figure 4C] Figure 4C shows a pair of diagrams showing the C-terminal domain of the HCMV gO subunit (left) and the short-chain cytokine fold of the FLT3 ligand (right; PDB code: 3QS7) for structural comparison. The helices shown in pink represent the regions of gO and FLT3 that fold into the cytokine domain. [Figure 4D] FIG. 4D shows the distribution of cysteine ​​residues (pink) and disulfide bonds within the HCMV gO subunit. [Figure 4E] Figure 4E shows a pair of images showing the electrostatic surface of the HCMV gO subunit in the range of -10 to +10 keV. Red: negatively charged. Blue: positively charged. [Figure 4F]Figure 4F is a pair of panels showing the results of a conservation analysis of the HCMV gO subunit based on sequences from 93 herpesviruses and 5 strains. Conservation: low to high (green to purple). [Figure 4G] FIG. 4G is a pair of panels showing the distribution of glycosylation sites (colored) on the HCMV gO subunit. [Figure 5A] Figure 5A is a graph showing the levels of HCMV trimer (strains: Merlin and VR1814) binding to the indicated human receptor proteins (normalized binding signal of HCMV trimer expressed as a percentage of the maximum signal) resulting from the cell surface receptor discovery platform. [Figure 5B] Figure 5B is a schematic diagram showing the domain organization of human PDGFRα: domains D1, D2, D3, D4, D5, the transmembrane (TM) domain, and the kinase domain. [Figure 5C] Figure 5C shows a front view of the HCMV gHgLgO trimeric complex bound to PDGFRα domains D1-D3. Green: PDGFRα. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. [Figure 5D] Figure 5D is a set of figures showing a diagram of the HCMV trimer distal region, including the N-termini of gO, gL, and gH, and PDGFα D1-D4. PDGFα D4 is shown in low opacity to show the orientation of the receptor relative to the host cell membrane. The bottom panel shows residue interactions in sites 1-4. Green: D1-D4 of PDGFα. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. [Figure 5E] FIG. 5E shows the HCMV trimer distal region in a magnified view as described in FIG. 5D, with the highlighted surface region (green) involved in the interaction with PDGFRα. [Figure 5F] Figure 5F shows the results of a conservation analysis of the N-terminus of HCMV gO-gL+gH based on the sequences of 5 strains of 93 herpesviruses for gO and gH and 5 strains of 59 herpesviruses for gL. Conservation range: low to high (green to purple). [Figure 5G]Figure 5G is a bar graph showing the binding levels (expressed as percent binding compared to wild-type (WT) PDGFRα) of HCMV gHgLgO trimers to PDGFRα-Fc proteins into which single glycosylation sites or combinations of charge mutations (E52R, L80R, E108R, E111R, L137E, I139E, L208R, M260E, L261R, E263R, K265E) listed in Table 2 had been introduced. [Figure 5H] FIG. 5H is a set of graphs showing biolayer interferometry (BLI) binding curves for the HCMV gHgLgO trimer and PDGFRα-Fc interactions listed in Table 2. [Figure 6A] Figure 6A is a graph showing the levels of HCMV trimer (strains: Merlin and VR1814) binding (normalized binding signal of HCMV trimer expressed as a percentage of the maximum signal) with the indicated human receptor proteins resulting from the cell surface receptor discovery platform. [Figure 6B] Figure 5B is a schematic diagram showing the domain organization of human TGFβR3: orphan domain 2 (OD2), orphan domain 1 (OD1), N-terminal zona pellucida domain (ZP-N), C-terminal zona pellucida domain (ZP-C), transmembrane domain (TM), and intracellular domain (ICD). [Figure 6C] Figure 6C shows a size-exclusion chromatogram (upper panel) showing the absorbance at 280 nm for the eluted gHgLgO-TGFβR3-13H11-Msl-109 complex, and a corresponding SDS-PAGE gel image (lower panel) showing the components of the gHgLgO-TGFβR3-13H11-Msl-109 complex in the indicated SEC fractions. The dotted lines on the chromatogram and horizontally on the SDS-PAGE gel image indicate equivalent SEC elution fractions. [Figure 6D] Figure 6D shows a front view of the HCMV gHgLgO trimeric complex bound to TGFβR3 OD2. Dark green: TGFβR3. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. [Figure 6E]FIG. 6E shows the HCMV trimer distal region in a magnified view showing the gO, gL, and gH N-termini with highlighted surface regions (dark green) involved in interactions with TGFβR3 (gray). [Figure 6F] Figure 6F shows the results of a conservation analysis of the N-terminus of HCMV gO-gL+gH based on the sequences of 5 strains of 93 herpesviruses for gO and gH and 5 strains of 59 herpesviruses for gL. Conservation range: low to high (green to purple). [Figure 6G] Figure 6G is a set of figures showing a magnified view of the HCMV trimer distal region, showing TGFβR3 OD1-OD2, gO, gL, and gH N-termini. TGFβR3 OD1 is shown in low opacity to show the orientation of the receptor relative to the host cell membrane. The inset panel shows a magnified view of the key interaction sites (sites 1-3) between the HCMV gHgLgO trimer and TGFβR3. Red: gO subunit. Pink: gL subunit. Blue: gH subunit. Green: TGFβR3 OD2. [Figure 6H] Figure 6H is a set of figures showing a structural comparison of the OD2 domain of TGFβR3 with endoglin (PDB code: 5I04). The right panel shows a close-up of the corresponding loop region between β6 and β7 of TGFβR3 α1 and endoglin. [Figure 7A] FIG. 7A is a pair of images showing binding of PDGFRα (light green) and TGFβR3 (dark green) to HCMV gHgLgO (gray) in front (left) and top (right) views. [Figure 7B] Figure 7B is a size-exclusion chromatogram (upper panel) showing the absorbance at 280 nm for the eluted gHgLgO-TGFβR3 and gHgLgO-PDGFRα-TGFβR3 complexes, and the corresponding SDS-PAGE gel image (lower panel) showing the components of gHgLgO-TGFβR3 and gHgLgO-PDGFRα-TGFβR3 complexes in the indicated SEC fractions. [Figure 7C]Figure 7C shows PDGFRα (green) bound to HMCV gHgLgO (red) in a structural comparison, and a model of PDGF bound to PDGFRα based on the PDGFB-PDGFRβ co-crystal structure (PDGFB not shown; PDB code: 3MJG). [Figure 7D] Figure 7D is a set of graphs showing BLI binding curves for HCMV gHgLgO trimers with either wild-type gO (trimeric WT) and / or mutant gO (trimeric MUT; gO with M84R, F111R, R117E, F136R, R212E, R230E, R234E, R336E, F342E, A351R, and N358R amino acid substitution mutations) contacted with PDGFRα-Fc. [Figure 7E] Figure 7E is a Western blot analysis showing the levels of the indicated PDGFRα cell signaling components in MRC-5 cells. PDGFRα phosphorylation (pY762, pY849) and downstream signaling activity were assessed after addition of the growth factor PDGF-AA using wild-type or mutant gO in the absence (-) or presence (+) of HMCV gHgLgO trimer (as in Figure 7D). [Figure 7F] FIG. 7F is a schematic diagram showing a working model of receptor binding by HCMV gHgLgO trimers and neutralization of trimer binding by antibodies. [Figure 8A] Figure 8A is a schematic diagram showing the purification and reconstitution process of HCMV gHgLgO using Fab 13H11 and Msl-109: histidine (HIS); streptavidin (STREP); size exclusion chromatography (SEC). [Figure 8B] Figure 8B shows a size-exclusion chromatogram (top panel) showing the absorbance at 280 nm for the eluted gHgLgO-13H11-Msl-109 complex, and a corresponding SDS-PAGE gel image (bottom panel) showing the components of the gHgLgO-13H11-Msl-109 complex in the indicated SEC fractions. The dotted lines on the chromatogram and horizontally on the SDS-PAGE gel image indicate equivalent SEC elution fractions. [Figure 8C]Figure 8C is a cryo-EM micrograph showing the gHgLgO-13H11-Msl-109 complex. Scale bar: 10 nm. [Figure 8D] Figure 8D is a set of cryo-EM micrographs showing representative 2D class averages of monomeric and dimeric gHgLgO-13H11-Msl-109. Scale bar: 10 nm. [Figure 8E] Figure 8E is a schematic diagram of the processing workflow for obtaining the ab-initio 3D reconstruction of gHgLgO-13H11-Msl-109. [Figure 8F] FIG. 8F is a schematic diagram showing the data acquisition and processing scheme for obtaining a high-resolution 3D reconstruction of gHgLgO-13H11-Msl-109. [Figure 8G] Figure 8G shows an isosurface rendering of the gHgLgO-13H11-Msl-109 3D map before convergence refinement with surface coloring according to the local resolution estimated by windowed Fourier shell correlation (FSC). Resolution range: 2.7 to >4.7 Å (blue-red). [Figure 8H] Figure 8H is a heatmap representation of the distribution of assigned particle orientations. The heatmap shows the number of particles aligned at a defined orientation in 3D space. [Figure 8I] Figure 8I is a graph showing the FSC between half data sets of the overall gHgLgO-13H11-Msl-109 3D reconstruction and the convergent refinement reconstruction (shown in Figure 8F). [Figure 9A] FIG. 9A is a series of ribbon diagrams showing a structural comparison of the HCMV trimeric and pentameric (PDB code: 5VOB) gH and gL subunits divided into domains DI, DII, DII, and DIV. [Figure 9B] FIG. 9B is a pair of diagrams showing the interaction interface of gO mapped onto gL (top) and the interaction interfaces of UL130 and UL128 on gL (bottom: based on PDB code: 5VOB). [Figure 9C]FIG. 9C is a pair of images showing glycosylation site distribution (colored molecules) on the HCMV pentameric complex (PDB code: 5VOB) with the putative receptor binding site highlighted. [Figure 10] Figure 10 shows a close-up of the variable Fab regions of 13H11 and Msl-109 bound to the DII-DIV region of gH, highlighting the Fab contact regions on gH previously identified by hydrogen exchange mass spectrometry. [Figure 11A] Figure 11A shows a size-exclusion chromatogram (upper panel) showing the absorbance at 280 nm for the eluted gHgLgO-PDGFRα-13H11-Msl-109 complex, and the corresponding SDS-PAGE gel image (lower panel) showing the components of the gHgLgO-PDGFRα-13H11-Msl-109 complex in the indicated SEC fractions. The dotted lines on the chromatogram and horizontally on the SDS-PAGE gel image indicate equivalent SEC elution fractions. [Figure 11B] FIG. 11B is a representative cryo-EM micrograph showing the gHgLgO-PDGFRα-13H11-Msl-109 complex. [Figure 11C] FIG. 11C is a set of cryo-EM micrographs showing representative 2D class averages of the gHgLgO-PDGFRα-13H11-Msl-109 complex. [Figure 11D] FIG. 11D is a schematic diagram showing the data collection and processing scheme for obtaining high-resolution 3D reconstruction of the gHgLgO-PDGFRα-13H11-Msl-109 complex. [Figure 11E] FIG. 11E shows an isosurface rendering of the gHgLgO-PDGFRα-13H11-Msl-109 3D map before convergence refinement with surface coloring according to local resolution estimated by windowed FSC. [Figure 11F] Figure 11F shows a heatmap representation of the distribution of assigned particle orientations. The heatmap shows the number of particles aligned at a defined orientation in 3D space. [Figure 11G]FIG. 11G is a graph showing the FSC between half data sets of the gHgLgO-PDGFRα-13H11-Msl-109 3D reconstruction and the converged refinement reconstruction (shown in FIG. 11D). [Figure 12A] Figure 12A shows a comparison of the structures of D1 to D3 of PDGFRα (trimeric binding) and PDGFRβ (PDGFβ not shown; PDB code: 3MJG), Kit (SCF not shown; PDB code: 2E9W), or FMS (M-CSF not shown; PDB code: 3EJJ). [Figure 12B] FIG. 12B is a series of ribbon diagrams showing a structural comparison of the individual D1, D2, and D3 domains of PDGFRα (trimer-bound) and PDGFRβ (PDGFB not shown; PDB code: 3MJG). [Figure 12C] FIG. 12C is a series of ribbon diagrams showing a structural comparison of D1-D3 PDGFRα (trimer-bound) and PDGFRβ (PDGFB not shown; PDB code: 3MJG) after alignment on D2. [Figure 12D] FIG. 12D is a stick diagram showing a structural comparison of gHgLgO-PDGFRα and gHgLgO (Fabs 13H11 and Msl-109 not shown). [Figure 12E] Figure 12E shows a sequence alignment of the D1-D3 regions to the PDGFRβ sequence based on the PDGFRα structure. The interaction sites with HCMV trimer gHgLgO (sites 1-4) and PDGFβ are highlighted in red boxes. [Figure 13A] Figure 13A is a representative cryo-EM micrograph showing the gHgLgO-TGFβR3-13H11-Msl-109 complex. Scale bar: 10 nm. [Figure 13B] Figure 13B is a set of representative cryo-EM micrographs showing 2D class averages of the gHgLgO-TGFβR3-13H11-Msl-109 complex. Scale bar: 10 nm. [Figure 13C]FIG. 13C is a schematic diagram showing the data collection and processing scheme for obtaining a high-resolution 3D reconstruction of the gHgLgO-TGFβR3-13H11-Msl-109 complex. [Figure 13D] Figure 13D shows an isosurface rendering of the gHgLgO-TGFβR3-13H11-Msl-109 3D map before convergence refinement with surface coloring according to the local resolution estimated by windowed FSC: Resolution range: 2.5 to >5 Å (blue to red). [Figure 13E] Figure 13E is a heatmap representation showing the distribution of assigned particle orientations. The heatmap shows the number of particles aligned at a defined orientation in 3D space. [Figure 13F] FIG. 13F is a graph showing the FSC between half data sets of the gHgLgO-TGFβR3-13H11-Msl-109 3D reconstruction and the converged refinement reconstruction (shown in FIG. 13E). [Figure 13G] FIG. 13G shows a structural comparison between gHgLgO-TGFβR3 (green) and gHgLgO (Fab 13H11 and Msl-109 not shown). [Figure 13H] Figure 13H is a sequence alignment diagram showing the TGFβR3 structure-based sequence alignment of the OD2 region to the endoglin OD2 sequence. The interaction sites with HCMV trimer gHgLgO (sites 1-3) are highlighted in red boxes. [Figure 14A] Figure 14A is a pair of size-exclusion chromatograms (left panel) showing absorbance at 280 nm for eluted PDGFRα and TGFβR3, and the corresponding SDS-PAGE gel image (right panel) showing PDGFRα and TGFβR3 in the indicated SEC fractions. [Figure 14B] Figure 14B is a pair of size-exclusion chromatograms (left panel) showing the absorbance at 280 nm for the eluted HCMV gHgLgO trimer and gHgLgO-PDGFRα complex, and the corresponding SDS-PAGE gel image (right panel) showing the components of the gHgLgO-PDGFRα complex in the indicated SEC fractions. [Figure 14C] Figure 14C is a pair of size-exclusion chromatograms (left panel) showing the absorbance at 280 nm for the eluted gHgLgO-TGFβ3 and gHgLgO-PDGFRα-TGFβ3 complexes, and the corresponding SDS-PAGE gel image (right panel) showing the components of the gHgLgO-TGFβ3 and gHgLgO-PDGFRα-TGFβR3 complexes. gHgLgO-TGFβR3 was preincubated with an equimolar amount of PDGFRα. DETAILED DESCRIPTION OF THE INVENTION

[0039] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which the present invention pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0040] As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. As used herein, a reference to "about" a value or parameter includes (and describes) the aspect directed to that value or parameter itself.

[0041] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an isolated peptide" means one or more isolated peptides.

[0042] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0043] The terms "patient," "subject," or "individual," used interchangeably herein, refer to a human patient.

[0044] An "intravenous" or "iv" dose, administration or formulation of a drug is administered through a vein, for example, by infusion.

[0045] A "subcutaneous" or "sc" dose, administration, or formulation of a drug is administered beneath the skin, for example, via a pre-filled syringe, auto-injector, or other device.

[0046] For purposes of this specification, "clinical status" refers to the health status of a patient. For example, whether the patient is getting better or worse. In one embodiment, the clinical status is based on an ordinal scale of clinical status. In one embodiment, the clinical status is not based on whether the patient has a fever.

[0047] An "effective amount" refers to an amount of an agent (e.g., a therapeutic agent) effective to provide a therapeutic / prophylactic benefit (e.g., as described herein) that does not outweigh any unwanted / undesirable side effects.

[0048] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations are sterile. In one embodiment, the formulation is for intravenous (iv) administration. In another embodiment, the formulation is for subcutaneous (sc) administration.

[0049] A "native sequence" protein, as used herein, refers to a protein comprising the amino acid sequence of a protein found in nature, including variants of a naturally occurring protein. As used herein, the term includes a protein isolated from its natural source or a protein that is recombinantly produced.

[0050] As used herein, the term "protein," unless otherwise specified, refers to any naturally occurring protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice, rats). The term encompasses "full-length," unprocessed proteins and all forms of proteins resulting from processing within a cell. The term also encompasses naturally occurring variants of proteins, such as splice variants or allelic variants, including amino acid substitution or deletion variants. The term also encompasses isolated regions or domains of proteins, such as the extracellular domain (ECD).

[0051] An "isolated" protein or peptide is one that is separated from a component of its natural environment. In some embodiments, the protein or peptide is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0052] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0053] As used herein, the terms "human cytomegalovirus (HCMV) trimer," "HCMV gHgLgO trimer," and "HCMV trimer" refer to a glycoprotein complex located on the outer surface of the viral envelope of human cytomegalovirus (HCMV) and composed of gH, gL, and gO glycoprotein subunits.

[0054] As used herein, the terms "human cytomegalovirus (HCMV) gO subunit," "gO subunit," and "gO" refer broadly to any native gO from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses full-length gO and isolated regions or domains of gO. The term also encompasses naturally occurring variants of gO, such as splice variants or allelic variants. An exemplary amino acid sequence of human gO is provided as SEQ ID NO: 1. Minor sequence variations, particularly conservative amino acid substitutions of gO that do not affect the function and / or activity of gO, are also contemplated by the present invention.

[0055] As used herein, the terms "human cytomegalovirus (HCMV) gH subunit," "gH subunit," and "gH" refer broadly to any native gH from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses full-length gH and isolated regions or domains of gH. The term also encompasses naturally occurring variants of gH, such as splice variants or allelic variants. The amino acid sequence of an exemplary human gH is provided as SEQ ID NO: 2. Minor sequence variations, particularly conservative amino acid substitutions of gH that do not affect the function and / or activity of gH, are also contemplated by the present invention.

[0056] As used herein, the terms "human cytomegalovirus (HCMV) gL subunit," "gL subunit," and "gL" refer broadly to any native gL from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses full-length gL and isolated regions or domains of gL. The term also encompasses naturally occurring variants of gL, such as splice variants or allelic variants. An exemplary amino acid sequence of a human gL is provided as SEQ ID NO: 3. Minor sequence variations, particularly conservative amino acid substitutions of gL that do not affect the function and / or activity of gL, are also contemplated by the present invention.

[0057] As used herein, a "modulator" is an agent that modulates (e.g., increases, decreases, activates, or inhibits) a given biological activity, e.g., an interaction or a downstream activity resulting from the interaction. A modulator or candidate modulator can be, for example, a small molecule, an antibody (e.g., a bispecific or multispecific antibody), an antigen-binding fragment (e.g., a bis-Fab, Fv, Fab, Fab'-SH, F(ab')2, diabody, linear antibody, scFv, ScFab, VH domain, or VHH domain), a peptide, a mimetic, an antisense oligonucleotide, or an inhibitory nucleic acid (e.g., an antisense oligonucleotide (ASO) or small interfering RNA (siRNA)).

[0058] By "increase" or "activate" is meant the ability to cause an overall increase, e.g., of 20% or more, 50% or more, or 75%, 85%, 90%, or 95% or more. In certain aspects, increasing or activating can refer to downstream activity of a protein-protein interaction.

[0059] "Decrease" or "inhibit" refers to the ability to cause an overall decrease, e.g., by 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more. In certain aspects, decrease or inhibit can refer to a downstream activity of a protein-protein interaction.

[0060] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, "binding affinity," unless otherwise indicated, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). In general, the affinity of a molecule X for its partner Y is determined by the dissociation constant (K D Affinity can be measured by methods common in the art, including those described herein.

[0061] As used herein, "complex" or "complex-type" refers to an association of two or more molecules that interact with each other through bonds and / or forces (e.g., van der Waals, hydrophobic, hydrophilic forces) that are not peptide bonds. In one aspect, the complex is a heteromultimer. As used herein, the term "protein complex" or "polypeptide complex" should be understood to include complexes having non-protein entities (e.g., including, but not limited to, chemical molecules such as toxins or detection agents) conjugated to proteins in the protein complex.

[0062] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transfected cells," "transformed cells," and "transformants," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. In some aspects, host cells are stably transformed with exogenous nucleic acid. In other aspects, host cells are transiently transformed with exogenous nucleic acid.

[0063] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0064] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., bis-Fab), so long as they exhibit the desired antigen-binding activity.

[0065] An "antigen-binding fragment" or "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, bis-Fab; Fv; Fab; Fab, Fab'-SH; F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, scFab); and multispecific antibodies formed from antibody fragments.

[0066] A "single domain antibody" refers to an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain aspects, a single domain antibody is a human single domain antibody (see, e.g., U.S. Pat. No. 6,248,516 B1). Examples of single domain antibodies include, but are not limited to, VHHs.

[0067] A "Fab" fragment is an antigen-binding fragment produced by papain digestion of an antibody and consists of an entire light chain, the variable region domain (VH) of the heavy chain, and the first constant domain (CH1) of one heavy chain. Papain digestion of an antibody produces two identical Fab fragments. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in that they contain additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues in the constant domains bear free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0068] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to its carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include antibody populations in which all Lys447 residues have been removed, antibody populations in which the Lys447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the Lys447 residue.

[0069] An "Fv" consists of a dimer of one heavy-chain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops from each H-chain and L-chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, although often with lower affinity than the entire binding site.

[0070] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0071] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Malmborg et al., J. Immunol. Methods 183:7-13, 1995.

[0072] The term "small molecule" refers to any molecule having a molecular weight of about 2000 daltons or less, for example, about 1000 daltons or less. In some embodiments, a small molecule is a small organic molecule.

[0073] As used herein, the term "mimetic" or "molecular mimetic" refers to a polypeptide that has sufficient similarity (e.g., secondary structure, tertiary structure) in conformation and / or binding capacity to a given polypeptide or to a portion of said polypeptide to bind to a binding partner. A mimetic may bind to a binding partner with equal, lesser, or greater affinity than the polypeptide it mimics. A molecular mimetic may or may not have appreciable amino acid sequence similarity to the polypeptide it mimics. Mimetics can be naturally occurring or engineered. In some embodiments, a mimetic is a mimetic of a member of a binding pair. In yet other embodiments, a mimetic is a mimetic of another protein that binds to a member of a binding pair. In some embodiments, a mimetic can perform all of the functions of the mimicked polypeptide. In other embodiments, a mimetic does not perform all of the functions of the mimicked polypeptide.

[0074] As used herein, the term "conditions that permit binding" of two or more proteins to one another refers to conditions (e.g., protein concentration, temperature, pH, salt concentration) under which two or more proteins would interact in the absence of a modulator or candidate modulator. Conditions that permit binding will vary for individual proteins and can differ between protein-protein interaction assays (e.g., surface plasmon resonance assays, biolayer interferometry, enzyme-linked immunosorbent assays (ELISAs), extracellular interaction assays, and cell surface interaction assays).

[0075] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0076] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or comprising a particular % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y In this case, X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​as used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0077] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence (e.g., prevention of HCMV infection or symptoms thereof), reduction or prevention of secondary infection in patients with an infection (e.g., reduction or prevention of secondary infection of nervous tissue, immune cells, lymphatic tissue, and / or pulmonary tissue), alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing of disease progression, improvement or amelioration of the disease state, and remission or improved prognosis.

[0078] The "pathology" of a disease or condition includes all phenomena that compromise the well-being of the patient.

[0079] "Amelioration," "ameliorating," "alleviation," "alleviating," or the like refers to both therapeutic treatment and prophylactic or preventative measures, where the purpose is to ameliorate, prevent, slow down, reduce, or inhibit a disease or condition, HCMV infection. Those in need of treatment include those already with the disease or condition as well as those prone to have the disease or condition, or those in need of disease or condition prevention.

[0080] II. Modulators of protein-protein interactions In some aspects, the disclosure features an isolated modulator of the interaction between PDGFRα or TGFβR3 and an HCMV gHgLgO trimer, wherein the modulator causes a decrease in binding of the HCMV gHgLgO trimer to PDGFRα or TGFβR3 compared to binding in the absence of the modulator.

[0081] A. Modulators of the interaction between PDGFRα and the HCMV gHgLgO trimer i. Modulators that bind to the HCMV gHgLgO trimer In some aspects, the disclosure features a modulator of the interaction between the gO subunit of the human cytomegalovirus (HCMV) gHgLgO trimer and PDGFRα, wherein the modulator binds to a glycosylation-free surface of the gO subunit, causing a decrease in binding of the gO subunit to PDGFRα.

[0082] In some aspects, the modulator is a modulator of (a) one of residues R230, R234, V235, K237, and Y238 of the gO subunit (e.g., one, two, three, four, or all five of R230, R234, V235, K237, and Y238), (b) one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit (e.g., N81, L82, M84, M86, , F109, F111, T114, Q115, R117, K121, and V123), and (c) one of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit (e.g., one, two, three, four, five, six, or all seven of R336, Y337, K344, D346, N348, E354, and N358).

[0083] In some aspects, the disclosure provides a modulator of the interaction between the gO subunit of an HCMV gHgLgO trimer and PDGFRα, comprising: (a) one of residues R230, R234, V235, K237, and Y238 of the gO subunit (e.g., one, two, three, four, or all five of R230, R234, V235, K237, and Y238); (b) one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit (e.g., N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123); and (c) one of residues R336, Y337, K344, D346, N348, E354 and N358 of the gO subunit (e.g., one, two, three, four, five, six or all seven of R336, Y337, K344, D346, N348, E354 and N358), and cause a decrease in binding of the gO subunit to PDGFRα.

[0084] In some embodiments, the modulator binds to all 23 of the gO subunit residues: R230, R234, V235, K237, Y238, N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, V123, R336, Y337, K344, D346, N348, E354, and N358.

[0085] In some embodiments, the modulator further binds to one or more of residues R47, Y84, and N85 (e.g., one, two, or all three of R47, Y84, and N85) of the gH subunit of HCMV. In some embodiments, the modulator further causes a decrease in binding of the gH subunit to PDGFRα.

[0086] In some embodiments, the modulator is a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid (e.g., an ASO or siRNA). Modulators are further described below.

[0087] In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the bispecific or multispecific antibody binds to at least three distinct epitopes of the gO subunit. In some embodiments, the at least three distinct epitopes include: (a) a first epitope comprising one or more of residues R230, R234, V235, K237, and Y238 of the gO subunit; (b) a second epitope comprising one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit; and (c) a third epitope comprising one or more of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit.

[0088] In some embodiments, the modulator is a mimetic of PDGFRα.

[0089] ii. Modulators that bind PDGFRα In some aspects, the disclosure features a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα, wherein the modulator binds to the D1, D2, and D3 domains of PDGFRα and causes a decrease in binding of the gO subunit to PDGFRα.

[0090] In some aspects, the modulator is selected from the group consisting of: (a) one or more of residues N103, Q106, T107, E108, and E109 of PDGFRa (e.g., one, two, three, four, or all five of N103, Q106, T107, E108, and E109); (b) one or more of residues M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRa (e.g., M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRa); and (c) one, two, three, four, five, six, seven, or all eight of N240, D244, Q246, T259, E263, and K265 of PDGFRα (e.g., one, two, three, four, five, or all six of N240, D244, Q246, T259, E263, and K265).

[0091] In some aspects, the disclosure provides a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα, comprising: (a) one or more of residues N103, Q106, T107, E108, and E109 of PDGFRα (e.g., one, two, three, four, or all five of N103, Q106, T107, E108, and E109); (b) one or more of residues M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRα (e.g., M133, L137, I139, E141, I147, S145, Y206, and L208); and (c) one, two, three, four, five, six, seven, or all eight of residues N240, D244, Q246, T259, E263, and K265 of PDGFRα (e.g., one, two, three, four, five, or all six of N240, D244, Q246, T259, E263, and K265), causing a decrease in binding of the gO subunit to PDGFRα.

[0092] In some aspects, the modulator binds to all 19 of PDGFRα residues: N103, Q106, T107, E108, E109, M133, L137, I139, E141, I147, S145, Y206, L208, N240, D244, Q246, T259, E263, and K265.

[0093] In some embodiments, the modulator further binds to one or more of residues E52, S78, and L80 of PDGFRα, hi some embodiments, the modulator further causes a decrease in binding of the gH subunit to PDGFRα.

[0094] In some embodiments, the modulator is a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid (e.g., an ASO or siRNA). Modulators are further described below.

[0095] In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the bispecific or multispecific antibody binds to at least three distinct epitopes of PDGFRα. In some embodiments, the at least three distinct epitopes include: (a) a first epitope comprising one or more of residues N103, Q106, T107, E108, and E109 of PDGFRα; (b) a second epitope comprising one or more of residues M133, L137, I139, E141, I147, S145, Y206, and L208 of PDGFRα; and (c) a third epitope comprising one or more of residues N240, D244, Q246, T259, E263, and K265 of PDGFRα.

[0096] In some embodiments, the modulator is a mimetic of the gO subunit of the HCMV gHgLgO trimer.

[0097] iii. Reduced binding and / or infection In some embodiments, the modulator reduces binding of the gO subunit of the HCMV gHgLgO trimer to PDGFRα by at least 50%. In some embodiments, the reduction in binding is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% (i.e., binding is abolished) relative to binding in the absence of the modulator, e.g., the reduction is 5%-15%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 95%-100%. In some embodiments, the modulator reduces binding of the gO subunit of the HCMV trimer to PDGFRα by at least 90%, hi some embodiments, the reduction in binding is at least 50%, e.g., as measured by surface plasmon resonance, biolayer interferometry, or enzyme-linked immunosorbent assay (ELISA).

[0098] In some embodiments, the modulator reduces binding of the gO subunit of the HCMV gHgLgO trimer to TGFβR3 by at least 50%. In some embodiments, the reduction in binding is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% (i.e., binding is abolished) relative to binding in the absence of the modulator, e.g., the reduction is 5%-15%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 95%-100%. In some embodiments, the modulator reduces binding of the gO subunit of the HCMV trimer to TGFβR3 by at least 90%, hi some embodiments, the reduction in binding is at least 50%, e.g., as measured by surface plasmon resonance, biolayer interferometry, or enzyme-linked immunosorbent assay (ELISA).

[0099] In some embodiments, the modulator causes a decrease in infection of cells by HCMV compared to infection in the absence of said modulator, hi some embodiments, infection is reduced by at least 40% as measured in a viral infection or entry assay using pseudotyped particles. In some embodiments, the reduction is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% (i.e., no infection occurs), e.g., the reduction is between 5% and 15%, 15% and 25%, 25% and 35%, 35% and 45%, 45% and 55%, 55% and 65%, 65% and 75%, 75% and 85%, 85% and 95%, or 95% and 100%.

[0100] In some embodiments, the modulator minimally binds to or does not bind to the region of PDGFRα that induces downstream signaling. In some embodiments, the region of PDGFRα that induces downstream signaling is the binding site for PDGF. In some embodiments, the modulator does not sterically interfere with or minimally interferes with the binding of a PDGFRα ligand to the region of PDGFRα that induces downstream signaling, e.g., does not sterically interfere with or minimally interferes with the binding of PDGF to PDGFRα.

[0101] In some embodiments, a modulator causes less than a 20% decrease in signaling by PDGFRα compared to signaling in the absence of the modulator, hi some embodiments, a modulator causes less than a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% decrease in signaling by PDGFRα compared to signaling in the absence of the modulator (e.g., a 0%-5%, 5%-15%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, or 85-95% decrease in signaling by PDGFRα compared to signaling in the absence of the modulator). In some embodiments, the modulator does not cause a decrease in signaling by PDGFRα compared to signaling in the absence of the modulator.

[0102] In some embodiments, the modulator comprises a pharmaceutically acceptable carrier.

[0103] B. Modulators of the interaction between TGFβR3 and the HCMV gHgLgO trimer i. Modulators that bind to the HCMV gHgLgO trimer In some aspects, the disclosure features a modulator of the interaction between an HCMV gHgLgO trimer and TGFβR3 that binds to (a) one or more of residues Q115, L116, R117, and K118 (e.g., one, two, three, or all four of Q115, L116, R117, and K118) of the gO subunit of the HCMV gHgLgO trimer, (b) one or both of residues Y188 and P191 of the gO subunit of the HCMV gHgLgO trimer, and residue N97 of the gL subunit of the HCMV trimer; or (c) one or both of residues T92 and E94 of the gL subunit of the HCMV gHgLgO trimer, causing a decrease in binding of the HCMV gHgLgO trimer to TGFβR3.

[0104] In some embodiments, the modulator is a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid (e.g., an ASO or siRNA). In some embodiments, the antibody is a bispecific or multispecific antibody.

[0105] ii. Modulators that bind TGFβR3 In some aspects, the disclosure features a modulator of the interaction between HCMV gHgLgO trimer and TGFβR3 that binds to (a) one or more of residues V135, Q136, F137, and S143 of TGFβR3 (e.g., one, two, three, or all four of V135, Q136, F137, and S143), (b) one or more of residues R151, N152, and E167 of TGFβR3; and (c) one or both of residues W163 and K166 of TGFβR3, causing a decrease in binding of HCMV gHgLgO trimer to TGFβR3.

[0106] In some embodiments, the modulator is a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid (e.g., an ASO or siRNA). In some embodiments, the antibody is a bispecific or multispecific antibody.

[0107] iii. Reduced binding and / or infection In some embodiments, the modulator reduces binding of the gO subunit of the HCMV gHgLgO trimer to TGFβR3 by at least 50%. In some embodiments, the reduction in binding is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% (i.e., binding is abolished) relative to binding in the absence of the modulator, e.g., the reduction is 5%-15%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 95%-100%. In some embodiments, the modulator reduces binding of the gO subunit of the HCMV trimer to TGFβR3 by at least 90%, hi some embodiments, the reduction in binding is at least 50%, e.g., as measured by surface plasmon resonance, biolayer interferometry, or enzyme-linked immunosorbent assay (ELISA).

[0108] In some embodiments, the modulator reduces binding of the gO subunit of the HCMV gHgLgO trimer to PDGFRα by at least 50%. In some embodiments, the reduction in binding is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% (i.e., binding is abolished) relative to binding in the absence of the modulator, e.g., the reduction is 5%-15%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 95%-100%. In some embodiments, the modulator reduces binding of the gO subunit of the HCMV trimer to PDGFRα by at least 90%, hi some embodiments, the reduction in binding is at least 50%, e.g., as measured by surface plasmon resonance, biolayer interferometry, or enzyme-linked immunosorbent assay (ELISA).

[0109] In some embodiments, the modulator causes a decrease in infection of cells by HCMV compared to infection in the absence of said modulator, hi some embodiments, infection is reduced by at least 40% as measured in a viral infection or entry assay using pseudotyped particles. In some embodiments, the reduction is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% (i.e., no infection occurs), e.g., the reduction is between 5% and 15%, 15% and 25%, 25% and 35%, 35% and 45%, 45% and 55%, 55% and 65%, 65% and 75%, 75% and 85%, 85% and 95%, or 95% and 100%.

[0110] In some embodiments, the modulator comprises a pharmaceutically acceptable carrier.

[0111] C. Small molecules In some embodiments, the modulator or candidate modulator is a small molecule. A small molecule is a molecule other than a binding polypeptide or antibody as defined herein that can bind, preferably specifically, to PDGFRα (e.g., its D1 domain, D2 domain, and / or D3 domain), TGFβR3, or HCMV gHgLgO trimer (e.g., gO and / or gH). Binding small molecules can be identified and chemically synthesized using known methodologies (see, e.g., PCT Publication Nos. WO00 / 00823 and WO00 / 39585). The size of a binding small molecule is typically less than about 2000 daltons (e.g., less than about 2000, 1500, 750, 500, 250, or 200 daltons in size), where such small organic molecules capable of preferably specifically binding to the polypeptides described herein can be identified without undue experimentation using known techniques. In this regard, it is noted that techniques for screening small molecule libraries for molecules capable of binding to polypeptide targets are well known in the art (see, e.g., PCT Publication Nos. WO00 / 00823 and WO00 / 39585). The conjugated small molecule can be, for example, an aldehyde, ketone, oxime, hydrazone, semicarbazone, carbazide, primary amine, secondary amine, tertiary amine, N-substituted hydrazine, hydrazide, alcohol, ether, thiol, thioether, disulfide, carboxylic acid, ester, amide, urea, carbamate, carbonate, ketal, thioketal, acetal, thioacetal, aryl halide, aryl sulfonate, alkyl halide, alkyl sulfonate, aromatic compound, heterocyclic compound, aniline, alkene, alkyne, diol, amino alcohol, oxazolidine, oxazoline, thiazolidine, thiazoline, enamine, sulfonamide, epoxide, aziridine, isocyanate, sulfonyl chloride, diazo compound, acid chloride, and the like.

[0112] In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the small molecule (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is increased in the presence of the small molecule (e.g., by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% increase, e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, 95% to 100%, or more than 100% increase). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the small molecule (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction).

[0113] D. Antibodies and Antigen-Binding Fragments In some embodiments, the modulator or candidate modulator is an antibody or antigen-binding fragment thereof that binds to PDGFRα, TGFβR3, and / or HCMV gHgLgO trimer. In some embodiments, the antigen-binding fragment is a bis-Fab, Fv, Fab, Fab'-SH, F(ab')2, diabody, linear antibody, scFv, ScFab, VH domain, or VHH domain.

[0114] In some embodiments, the modulator is a multispecific antibody, e.g., a bispecific antibody. In some embodiments, the modulator is a bispecific or multispecific antibody that binds to multiple epitopes on the HCMV gHgLgO trimer, multiple epitopes on PDGFRα, or multiple epitopes on TGFβR3. In some embodiments, the modulator is a bispecific or multispecific antibody that binds to two or all three of the HCMV gHgLgO trimer, PDGFRα, and TGFβR3.

[0115] In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the antibody or antigen-binding fragment (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is increased in the presence of the antibody or antigen-binding fragment (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% increase, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, 95% to 100%, or greater than 100% increase). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the antibody or antigen-binding fragment (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction).

[0116] E. Peptides In some embodiments, the modulator or candidate modulator is a peptide that binds to PDGFRα, TGFβR3, and / or HCMV gHgLgO trimer. The peptide can be a naturally occurring peptide or an engineered peptide. In some embodiments, the peptide is a fragment of PDGFRα (e.g., its D1 domain, D2 domain, and / or D3 domain), TGFβR3, or HCMV gHgLgO trimer (e.g., gO and / or gH), or another protein that binds to PDGFRα, TGFβR3, and / or HCMV gHgLgO trimer. The peptide can bind to a binding partner with equal, less, or greater affinity than the full-length protein. In some embodiments, the peptide performs all of the functions of the full-length protein. In other embodiments, the peptide does not perform all of the functions of the full-length protein.

[0117] In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the peptide (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is increased in the presence of the peptide (e.g., by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% increase, e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, 95% to 100%, or more than 100% increase). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the peptide (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction).

[0118] F. Imitation In some embodiments, the modulator or candidate modulator is a mimetic, e.g., a molecular mimetic, that binds to PDGFRα, TGFβR3, or HCMV gHgLgO trimer (e.g., gO and / or gH). The mimetic can be a molecular mimetic of PDGFRα (e.g., its D1 domain, D2 domain, and / or D3 domain), TGFβR3, or HCMV gHgLgO trimer (e.g., gO and / or gH), or another protein (e.g., gO and / or gH) that binds to PDGFRα, TGFβR3, or HCMV gHgLgO trimer. In some embodiments, the mimetic can perform all of the functions of the mimicked polypeptide. In other embodiments, the mimetic does not perform all of the functions of the mimicked polypeptide.

[0119] In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the mimetic (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is increased in the presence of the mimetic (e.g., by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% increase, e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, 95% to 100%, or more than 100% increase). In some embodiments, binding of PDGFRα and / or TGFβR3 to the HCMV gHgLgO trimer is reduced in the presence of the mimetic (e.g., a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, e.g., a 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction).

[0120] G. Assays for Modulation of Protein-Protein Interactions In some embodiments, binding of PDGFRα or TGFβR3 and HCMV gHgLgO trimers in the presence or absence of a candidate modulator is assessed in an assay for protein-protein interaction. Modulation of an interaction may be identified as an increase in protein-protein interaction in the presence of a modulator compared to protein-protein interaction in the absence of the modulator, e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, 100%, or greater than 100% (e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, 95% to 100%, or greater than 100%) increase in protein-protein interaction. Alternatively, modulation can be identified as a decrease in protein-protein interaction in the presence of a modulator compared to protein-protein interaction in the absence of the modulator, e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 100% (e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100%) decrease in protein-protein interaction. The protein-protein interaction assay can be, for example, an SPR assay, a biolayer interferometry (BLI) assay, an enzyme-linked immunosorbent assay (ELISA), an extracellular interaction assay, or a cell surface interaction assay.

[0121] Exemplary methods for identifying modulators of protein-protein interactions, as well as agents capable of modulating such interactions, are described in PCT / US2020 / 025471, which is incorporated by reference in its entirety.

[0122] IV. Methods of Treating or Preventing HCMV Infection A. Methods of Treating Individuals with HCMV Infection In some aspects, the disclosure features a method of treating an HCMV infection in an individual, the method comprising administering to the individual an effective amount of a modulator described herein (e.g., a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα and / or a modulator of the interaction between the HCMV gHgLgO trimer and TGFβR3), thereby treating the individual. In some aspects, the individual is immunocompromised, pregnant, or an infant.

[0123] In some embodiments, the duration or severity of HCMV infection is reduced by at least 40% compared to an individual not administered the modulator, hi some embodiments, the duration or severity of HCMV infection is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or 100% (e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100%).

[0124] B. Methods for preventing HCMV infection or secondary infection In some aspects, the disclosure features a method for preventing HCMV infection in an individual, the method comprising administering to the individual an effective amount of a modulator described herein (e.g., a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα and / or a modulator of the interaction between the HCMV gHgLgO trimer and TGFβR3), thereby preventing HCMV infection in the individual.

[0125] In some embodiments, the modulator reduces the likelihood of HCMV infection in an individual compared to infection in the absence of the modulator. In some embodiments, the likelihood, extent, or severity of HCMV infection is reduced, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction), in patients treated according to the methods described above compared to untreated patients or compared to patients treated using a control method (e.g., SOC).

[0126] In some aspects, the disclosure features a method for preventing a secondary HCMV infection in an individual (e.g., an individual with an HCMV infection), the method comprising administering to the individual an effective amount of a modulator described herein (e.g., a modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα and / or a modulator of the interaction between the HCMV gHgLgO trimer and TGFβR3), thereby preventing a secondary HCMV infection in the individual. In some aspects, the secondary infection is an HCMV infection of an uninfected tissue.

[0127] In some embodiments, the modulator reduces the likelihood of a secondary HCMV infection in an individual compared to a secondary infection in the absence of the modulator. In some embodiments, the likelihood, extent, or severity of a secondary HCMV infection is reduced, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (e.g., 5% to 15%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 95% to 100% reduction), in patients treated according to the methods described above compared to untreated patients or compared to patients treated using a control method (e.g., SOC).

[0128] C. Combination Therapy In some embodiments of the above-described treatment and prevention methods, the method comprises administering to the individual at least one additional therapy (e.g., one, two, three, four, or more than four additional therapies). The modulator of the interaction between PDGFRα or TGFβR3 and the HCMV gHgLgO trimer can be administered to the individual prior to, concurrently with, or after the at least one additional therapy.

[0129] D. Method of Delivery The compositions utilized in the methods described herein (e.g., modulators of the interaction between PDGFRα or TGFβR3 and the HCMV gHgLgO trimer, e.g., small molecules, antibodies, antigen-binding fragments, peptides, mimetics, antisense oligonucleotides, or siRNA) can be administered in any suitable manner, including, but not limited to, intravenously, intramuscularly, subcutaneously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, transdermally, intravitreally (e.g., by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local perfusion directly bathing target cells, by catheter, by lavage, in a cream, or in a lipid composition. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the symptom, disease, or disorder being treated). In some embodiments, the modulator of protein-protein interactions is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various administration schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time points, a bolus dose, or a pulse infusion.

[0130] The modulators of protein-protein interactions described herein (and any additional therapeutic agents) may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The modulators need not be, but are optionally, formulated with and / or co-administered with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of modulator present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and by the same routes of administration as those described herein, or about 1-99% of the dosages described herein, or at any dosage and by any route empirically / clinically determined to be appropriate.

[0131] All patents, patent publications and references cited herein are hereby incorporated by reference in their entirety.

[0132] V. Working Examples Example 1. Structure of HCMV trimer gHgLgO Characterization of the HCMV gHgLgO trimer structure has proven difficult in the past due to its flexibility, extensibility, and numerous glycosylation sites, which may prevent the formation of crystals that diffract to high resolution (Ciferri et al., Proc Natl Acad Sci USA, 112:1767-1772, 2015). To determine the high-resolution structure of the HCMV trimer, also known as the gHgLgO complex, we recombinantly expressed the soluble region of the gHgLgO complex in Expi293 cells and purified the complex to high purity (Figure 8A). Consistent with previous reports, HCMV gH, gL, and gO are covalently linked by disulfide bonds and migrated as a single band by SDS-PAGE (Figure 8B) (Ciferri et al., Proc Natl Acad Sci USA, 112:1767-1772, 2015). To overcome limitations imposed by the size, shape, and flexibility of the gHgLgO complex, we focused on cryoEM and single-particle analysis after reconstituting gHgLgO with the fragment antigen-binding (Fab) regions of the gH-specific neutralizing monoclonal antibodies (mAbs) 13H11 and Msl-109 (Figures 8A-8I) (Macagno et al., J Virol, 84:1005-1013, 2010; Fouts et al., Proc Natl Acad Sci USA, 111:8209-8214, 2014). Binding of both Fabs increased the size, stability, and dimensions of the gHgLgO complex, facilitating high-resolution cryoEM investigations (Figures 8C and 8D). 2D class averages revealed a high degree of detail, including secondary structural features and particle alignment in different orientations (Figure 8D). Furthermore, a subpopulation of dimeric gHgLgO particles was observed in 2D classes and 3D ab initio volumes (Figures 8D and 8E). The dimeric gHgLgO particles oriented head-to-tail in opposing directions with only a small contact area at the interface. The head-to-tail orientation suggests positioning of the viral membrane at opposite ends of the complex and is therefore unlikely to be physiologically relevant. Using a mask around the monomeric gHgLgO complex, we determined a high-resolution structure of the gHgLgO-13H11-Msl-109 complex, extending to approximately 2.9 Å resolution (Figure 1A and Figures 8F-8I and Table 1).Dividing the complex into three subregions allowed us to further improve map quality across the entire gHgLgO-13H11-Msl-109 molecule using specific masks in convergent local refinement (Figure 8F). The combination of convergent 3D reconstructions allowed us to build the structure and assign sequences to gH, gL, and a large portion of the previously unknown gO subunit, as well as to the variable domains of both Fabs (Fv) (Figures 1A and 1B). [Table 1]

[0133] The overall structure of the HCMV trimeric gHgLgO adopts a boot-like structure with relative dimensions of 170 Å in length and 70 Å in width (Figure 1B). The three subunits interact in a linear order, with the C-terminus of gH oriented most proximal to the HCMV viral membrane and gO pointing toward the distal end of the molecule for receptor binding (Figures 1A and 1B). The gL subunit bridges the gH and gO subunits at the center of the complex (Figures 1A and 1B). Electrostatic surface charges are asymmetrically distributed across the gHgLgO complex, with a negative charge cluster in the proximal gH region and a positive charge cluster in the distal gO region (Figure 1C). Similarly, the 22 observed N-linked glycosylation sites are asymmetrically distributed along the gHgLgO complex, with five on gH, one on gL, and 16 on gO. Notably, there is an enrichment of glycosylated residues in the distal gO region, particularly along the backside of the entire complex (Figure 1D). In contrast, the anterior side of the trimeric complex appears to lack glycosylated residues in all three subunits (Figure 1D). This asymmetric distribution of surface charge and glycosylation has important implications for receptor interactions and potential interactions with the prefusion conformation of gB and can therefore be used to inform the design of antiviral strategies.

[0134] Protein expression and purification. The optimized coding DNAs of human herpesvirus 5 (HH) (1-716), gL, and gO (HCMV strain Merlin for gH and gL, and VR1814 for gO) were cloned into the pRK vector behind the CMV promoter. A C-terminal Myc-Avi-His tag was added to gH, and a C-terminal Twin-Strep tag was added to gO.

[0135] Expi293 cells in suspension were cultured in SMM 293T-I medium at 37°C under 5% CO2 until a cell density of 4 × 10 6 When the cell density reached 100 cells / ml, the cells were transfected with polyethylenimine (PEI) containing DNA at a ratio of 1:1:1 for gHgLgO expression. The transfected cells were cultured for 7 days, and the expression supernatant was then harvested.

[0136] The HCMV trimer gHgLgO was purified as follows: Expression supernatant corresponding to a 35 L expression volume was concentrated to a volume of 1–2 L by tangential flow filtration (TFF), loaded onto 20 mL of Ni Sepharose Excel (Cytiva) resin, washed with 13 column volumes (CV) of wash buffer (30 mM TRIS (pH 8.0), 250 mM NaCl, 5% glycerol, 20 mM imidazole), and eluted with 5 CV of elution buffer (30 mM TRIS (pH 8.0), 250 mM NaCl, 5% glycerol, 400 mM imidazole). The eluate was applied to 3 mL of Strep-Tactin XT high affinity resin (IBA) and allowed to bind for 2 h. The resin was washed with 10 CV Strep-wash buffer (25 mM HEPES (pH 7.5), 300 mM NaCl, 5% glycerol) and eluted from the beads in Strep-wash buffer supplemented with 50 mM biotin. The eluate was concentrated using an AMICON® Ultra centrifugal filter device (30 kDa molecular weight cutoff (MWCO)) and loaded onto a Superdex 200 10 / 300 or 10 / 60 column equilibrated with Trimer-SEC buffer (25 mM HEPES (pH 7.5), 300 mM NaCl, 5% glycerol).

[0137] The heavy and light chains of Fab Msl-109 were coexpressed under the phoA promoter in E. coli 34B8 cells in phosphate-limited medium (CRAP) at 30°C for 20 hours. The pellet from 1 L of expression was resuspended in 70 ml of lysis buffer (1x PBS, 25 mM EDTA) supplemented with Roche protease inhibitor tablets and lysed by sonication. The lysate was clarified by centrifugation at 25,000 x g for 1 hour and subsequently filtered through a 0.45 μm filter. The clarified lysate was loaded onto a 5 ml HiTrap Protein G HP (cytiva) column equilibrated in lysis buffer. The column was washed with 10-20 CV of lysis buffer and eluted with 0.58% (v / v) acetic acid. The pH of the eluate was immediately adjusted by adding SP-A buffer (20 mM MES, pH 5.5) and loaded onto a 5 ml HiTrap SP HP cation exchange chromatography column (Cytiva). Fab was eluted with a linear 20 CV gradient against SP-B buffer (20 mM MES, pH 5.5, 500 mM NaCl). The eluate was concentrated using an AMICON® Ultra Centrifugal Filter Device (10 kDa MWCO) and further purified on a Superdex 200 10 / 300 column equilibrated with Fab-S200 buffer (25 mM Tris, pH 7.5, 300 mM NaCl). The purified Fab was concentrated using an AMICON® Ultra Centrifugal Filter Device (10 kDa MWCO), frozen in liquid nitrogen, and stored at -80°C. The 13H11 antibody was purified as previously described (Ciferri et al., PLoS Pathog, 11:e1005230, 2015).

[0138] Reconstitution of HCMV gHgLgO trimers by human receptor proteins and neutralization of Fab The gHgLgO-13H11-Msl-109 complex was assembled by incubating 18.3 μM gHgLgO (300 μg) with 30 μM excess Fab 13H11 (150 μg) and 30 μM Msl-109 (150 μg) on ​​ice for 30 min. Excess Fab was removed by purification on a Superose 6 3.2 / 300 column equilibrated with SEC-reconst-1 buffer (25 mM HEPES, pH 7.5, 200 mM NaCl). For cryo-EM sample preparation, the main peak fraction of gHgLgO-13H11-Msl-109 was diluted to a concentration of 0.4 mg / ml with SEC-reconst-1 buffer.

[0139] Cryo-EM sample preparation and data acquisition The gHgLgO-13H11-Msl-109 complex was prepared as described below. A Holey carbon grid (C-Flat 45 nm R 1.2 / 1.3 300 mesh coated with Au / Pd 80 / 20; Protochips) was glow-discharged for 10 seconds using a Solarus™ plasma cleaner (Gatan). The complex was gently cross-linked with 0.025% EM-grade glutaraldehyde for 10 minutes at room temperature and quenched with 9 mM Tris, pH 7.5. 3 μl of sample (approximately 0.4 mg / ml here) was applied to the grid. The grid was blotted using a Vitrobot Mark IV (Thermo Fisher) with a 2.5-second blotting time at 100% humidity and quenched in liquid ethane cooled with liquid nitrogen.

[0140] Movie stacks were collected using Serial EM on a Titan Krios operating at 300 keV with a biological quantum energy filter equipped with a K2 Summit direct electron detector camera (Gatan) (Mastronarde et al., J Struct Biol. Oct;152(1):36-51, 2005). Images were recorded at 165,000x magnification using a 20 eV energy slit, corresponding to 0.824 Å per pixel. Each image stack contains approximately 50 eÅ. -2The image contained 50 frames recorded every 0.2 seconds for an accumulated dose of 1000 kJ and a total exposure time of 10 seconds. Images were recorded over a set defocus range of 0.5 to 1.5 μm.

[0141] Cryo-EM data processing Cryo-EM data were processed using a combination of RELION (Scheres, J Struct Biol., 180(3):519-30, 2012) and cisTEM (Grant et al., Elife, 7(7):e35383, 2018) software packages.

[0142] For the gHgLgO-13H11-Msl-109 complex, a total of 14,717 videos were corrected for frame motion using the MotionCor2 (Zheng et al., Nat Methods, 14(4):331-332, 2017) implementation in RELION, and contrast transfer function parameters were fitted using the 30-4.5 Å bandpass of the spectrum with CTFFIND-4 (Rohou and Grigorieff, J Struct Biol., 192(2):216-2, 2015). For the generation of the first ab-initio 3D reconstruction, images were filtered based on a detected fit resolution better than 4 Å. A total of 974,766 particles were sorted using the circular blob sorting tool within cisTEM. Particles were sorted through two rounds of cisTEM 2D classification to select the best-aligned particles, yielding 313,196 particles. These particles were subjected to ab initio generation in a cisTEM with three target volumes. The volume corresponding to a single HCMV trimer was used as a reference for cisTEM automatic and manual refinement by using a mask around the single (monomeric) gHgLgO-13H11-Msl-109 complex and applying a low-pass filter (LPF) outside the mask. This map was used as a 3D reference for high-resolution 3D refinement.

[0143] To generate a high-resolution 3D reconstruction of the gHgLgO-13H11-Msl-109 complex, the CTF-fit images were filtered based on a detected fit resolution better than 6 Å. Using a 30 Å low-pass filtered gHgLgO-13H11-Msl-109 complex reference structure, a total of 1,478,640 particles were collected by template matching with gautomatch (MRC Laboratory of Molecular Biology). Particles were sorted during the RELION 2D classification, and the selected 1,350,211 particles were imported into cisTEM for 3D refinement. The gHgLgO-13H11-Msl-109 3D reconstruction was obtained after automatic and manual refinement using a mask around the single (monomer) gHgLgO-13H11-Msl-109 complex, applying a low-pass filter (LPF) outside the mask (filter resolution 20 Å), and a score threshold of 0.25. This resulted in a gradual reduction in the outer weight from 0.5 to 0.15 with iterative manual refinement rounds. The 3D reconstruction converged to a map resolution of 2.9 Å (Fourier shell correlation (FSC) = 0.143, measured by cisTEM). To improve map quality, convergent refinement was obtained after dividing the map into three distinct regions using a mask, and manual refinement outside the mask was performed as described above with the following parameters: flattening from 8 Å resolution, -90 Å from the origin of reciprocal space. 2 The focused maps were sharpened in the cisTEM using a pre-cutoff B-factor of 1.0 and a figure of merit filter (Rosenthal and Henderson, J Mol Biol., 333(4):721-45, 2003). For model building and drawing, a composite map was generated from the three individual focused 3D maps using phenix combine_focused_maps.

[0144] Model construction and structural analysis The gH and gL subunits of the HCMV pentamer structure (Chandramouli et al., Sci Immunol, 2:eaan1457, 2017) were fitted as rigid bodies to the cryo-EM map. The gO subunit was built de novo into the high-resolution cryo-EM map. The resulting model was fitted as a rigid body to the cryo-EM map. After extensive rebuilding and manual adjustments, global structural differences between the initial model and the map were corrected using multiple rounds of real-space refinement using the phenix.real_space_refinement (Afonine et al., Acta Crystallogr D Struct Biol, 74(Pt 9):814-840, 2018) tool. The model was further manually refined in Coot (Emsley et al., Acta Crystallogr D Biol Crystallogr., 66(Pt 4):486-501, 2010) through iterative rounds of model building and real-space refinement in phenix. The model was validated using phenix.validation_cryoem (Afonine et al., Acta Crystallogr D Struct Biol., 74(Pt 9):814-840, 2018) incorporating MolProbity scoring (Williams et al., Protein Sci., 27(1):293-315, 2018). Figures were generated using PyMOL (The PyMOL Molecular Graphics System, v.2.07, Schrodinger, LLC) and UCSF ChimeraX (Goddard et al., Protein Sci., 27(1):14-25, 2018). 3D homology structure analysis was performed using the DALI server (Holm, Methods Mol Biol., 2112:29-42, 2020).Sequences were aligned using Clustal Omega (Sievers et al., Mol Syst Biol., 7:539, 2011) within JalView (Waterhouse et al., Bioinformatics, 25(9):1189-91, 2009), illustrated with ESPript 3.0 (Robert and Gouet, Nucleic Acids Res., 42(Web Server Issue):W320-4, 2014), and subsequently manually adjusted based on insights from the PDGFRα-gHgLgO-13H11-Msl-109 or TGFRβ3-gHgLgO-13H11-Msl-109 structural models.

[0145] Example 2. Structural basis for assembly of HCMV trimer- and pentamer-specific subunits The structural determinants that mediate trimer- and pentamer-specific assembly in HCMV remain unknown. Both trimers and pentamers share their gH and gL subunits, but the composition of the distal subunits gO and UL128-131, which mediate receptor recognition, differ (Ciferri et al., Proc Natl Acad Sci USA, 112:1767-1772, 2015). In trimers, the four gH domains (DI-IV) extend linearly away from the membrane-proximal face, and the N-terminal region of gH (DI) folds with gL near the membrane-distal region of the molecule (Figures 1B and 9). Structural comparison of the gHgL subunit between the crystal structures of the trimer and pentamer bound to the UL130-specific neutralizing Fab 8I21 (Chandramouli et al., Sci Immunol, 2:eaan1457, 2017) reveals nearly identical structures for gH and gL in both complexes (RMSD 0.7 Å / 582 Ca) (Figure 2A). Accordingly, most glycosylated residues on gH and gL are oriented in the same direction for the trimer and pentamer, except for residue N641, which is found in a less resolved irregular loop region of the trimer structure (Figure 2B).

[0146] Previously, it was established that gH and UL128 / UL130 / UL131A bind to the same site on gHgL through the formation of a disulfide bond with gL-Cys144 (Ciferri et al., Proc Natl Acad Sci USA, 112:1767-1772, 2015). However, the details of how trimer- and pentamer-specific proteins can form such stable interactions with the same gL interface remained unclear. Detailed structural comparison of individual gH domains and gL subunits between the trimer and pentamer confirms the expected high degree of structural similarity. Despite this overall similarity, we observed key differences in the distal end of the gL subunit, which interacts with either gH in the trimer or the UL128 and UL130 subunits in the pentamer (Figures 2C and 2D). gH binds as a capping crown around gL, covering approximately 2400 Å of its surface. 2 The interaction surface between gL and gO is approximately 1018 Å in the HCMV pentamer. 2 ) or UL130 (≒910Å 2 ) spans a wider area than the corresponding interface between either the trimer or the pentamer (Figure 9B). Comparison of the trimer and pentamer shows that the overall fold of gL is conserved, but there are differences in the configuration of residues centered around the critical Cys144 residue. Notably, in the trimer, this set of residues adopts a loop-like structure (Figure 2C), whereas in the pentamer, this region folds into a regular alpha helix to coordinate UL128 binding (Figure 2D). Thus, gL interacts with the gL C domain depending on the cytotropic and cytotropic decisions. 144 It appears to be an important adaptor protein that has evolved to recognize both gO and UL128 / UL130 proteins through a conformational switch centered on gO, allowing it to load trimeric or pentameric complexes onto the HCMV viral surface.

[0147] Example 3. Binding sites of HCMV trimer and pentamer neutralizing antibodies An important goal of HCMV research is to understand the structural basis and mechanisms of broadly neutralizing monoclonal antibodies (mAbs). Notably, mAbs isolated from healthy HCMV-seropositive donors that target conformational epitopes in HCMV pentamer and trimer have been previously reported (Macagno et al., J Virol, 84:1005-1013, 2010; Falk et al., J Infect Dis, 218:876-885, 2018; Nokta et al., Antiviral Res, 24:17-26, 1994). Among these, Msl-109 and 13H11 target gH in both HCMV trimer and pentamer complexes and are capable of broad HCMV neutralization (Nokta et al., Antiviral Res, 24:17-26, 1994). Here, a new structure of the HCMV trimeric gHgLgO-13H11-Msl-109 complex resolves to high resolution the Fv regions of both Fabs and their corresponding epitopes on gH (Fig. 1A, Fig. 3A, Fig. 8G).

[0148] 13H11 and Msl-109 bind to opposite faces of the gH kink C-terminal region. Using both its heavy and light chains, 13H11 recognizes a large footprint in the gH DII-DIII domain (Figures 3B and 3C). Specifically, the 13H11 heavy chain engages residues R223, D241, and D243 in the gH DII domain through polar interactions (Figure 3B, panel 1). The 13H11 light chain establishes polar contacts with residues R329, L218, and T387 in the gH-DII domain and residues S553, S556, H530, and E576 in the gH-DIII domain (Figure 3B, panels 2 and 3). In contrast to 13H11, Msl-109 utilizes its heavy chain to recognize the heel region of gH and recognizes a relatively small footprint in the DIII-DIV domain. The Msl-109 interaction involves polar contacts between its CDRs and residues W167, M168, P170, and D445 of the gH-DIII and DIV domains. Notably, the residues contacted by Msl-109 are identical to the escape mutations (W167C / R, P170S / H, D445N) isolated by growing HCMV VR1814 virus in epithelial or fibroblast cells under suboptimal Msl-109 antibody concentrations (Fouts et al., Proc Natl Acad Sci USA, 111:8209-8214, 2014). The newly established structure, including the Fab contact region on gH, significantly expands our knowledge of the 13H11 and Msl-109 epitopes previously characterized by mass spectrometry (Ciferri et al., PLoS Pathog, 11:e1005230, 2015) (Figure 10).

[0149] Example 4. Structure of gO reveals a novel fold The structure of gO is one of the most puzzling HCMV glycoproteins, as its amino acid sequence does not align well with previously published structures. In the cryo-EM structure described in Example 1, gO adopts a claw-like shape composed of one N-terminal domain and one C-terminal domain (Figure 4A). The N-terminal globular domain consists of five beta strands, and the C-terminal domain is primarily alpha helical. Notably, the central four alpha helices of the C-terminal domain share high structural similarity with the classical cytokine fold, the closest member of which is FLT3 (Figures 4A-4C).

[0150] The two domains of gO are Cys 167 -Cys 218 and Cys 149 -Cys 141 (Figure 4D) and Cys 343 (gO)-Cys 167The gO subunits, including the disulfide between gL and gO, align in one diagonal plane across the gO subunit. All cysteines in gO are conserved across all HCMV strains, suggesting that this configuration is important for the function of the HCMV trimer and may be crucial for receptor recognition. Comprehensive bioinformatic sequence analysis of the primary sequences of HCMV subunits showed that gO is one of the least conserved envelope glycoproteins, with a degree of conservation equal to 81% (Foglierini et al., Front Microbiol, 10:1005, 2019). Notably, mapping of gO conservation to newly established structures indicates that although the overall conservation of gO is lower than that of other HCMV proteins, there are, in fact, large surface patches on both domains of gO that are highly conserved (Figure 4E). Electrostatic surface analysis of gO identified a large region containing both the N- and C-terminal domains rich in positive charges (Figure 4F). This region overlaps with the conserved gO surface (Figure 4E-4F). Notably, glycosylation sites on the gO subunits were heterogeneously distributed and clustered on only one surface of the trimer, leaving one face of gO completely unmodified (Fig. 4G). This conserved, charged, glycosylation-free surface of gO was predicted to be the primary region involved in receptor binding.

[0151] Example 5. Trimers establish multiple contacts with PDGFRα PDGFRα has recently been identified as a receptor for HCMV trimers required for viral entry into fibroblasts (Martinez-Martin et al., Cell, 174:1158-1171 e19, 2018; Kabanova et al., Nat Microbiol, 1:16082, 2016; Wu et al., PLoS Pathog, 13:e1006281, 2017; Wu et al., Proc Natl Acad Sci USA, 115:E9889-E9898, 2018). However, the structural basis for PDGFRα recognition remains unclear. Notably, HCMV trimers bind with high affinity and selectivity to PDGFRα, but not to the closely related PDGFRβ or other members of class III receptor tyrosine kinases (RTKs) or related VEGF receptors (FLT1, KDR, and FLT4) (Figure 5A). These interaction data with selected human receptor proteins were from previously published Cell-Surface Receptor Discovery Platform results (Figure 5A) (Martinez-Martin et al., Cell, 174:1158-1171 e19, 2018). Members of the Class III RTK family include PDGFRα, PDGFRβ, KIT, FMS, and FLT3. The structures of these receptors consist of five extracellular Ig domain segments (D1-D5 domains), a short single-span transmembrane domain, and an intracellular kinase domain (Figure 5B). The HCMV trimer was reconstituted in complex with Fab 13H11 / Msl-109 and the PDGFRα D1-D5 extracellular domain, and its structure (Figure 11A) was determined using cryo-EM to an overall resolution of approximately 2.8 Å (Figures 5C and 11A-11G). The high resolution of the cryo-EM maps allowed us to assemble the amino acid sequences of most of the HCMV trimer-13H11-Msl109 complex and the PDGFRα D1-D3 domains (Figures S11E-S11F and S5C-S5D). The PDGFRα D4 and D5 domains were only weakly represented, likely due to the lack of direct contact with the HCMV trimer.Conceivably, PDGFRα receptor interaction could propagate conformational changes to the trimer that allow or prevent binding of other HCMV glycoproteins (e.g., prefusion gB). However, when comparing the structures before and after PDGFRα binding, nearly identical conformations of the trimer were observed (Fig. ​(Fig.12D),12D), suggesting that different mechanisms may be involved in activating the HCMV fusion machinery.

[0152] When bound to the trimer, PDGFRα D1-D3 adopts a twisted conformation similar to previously determined structures of the D1-D3 domains of PDGFRβ and other class III RTKs (Figure 12A). Structural comparison of the individual D1-D3 domains between PDGFRα and PDGFRβ (determined in complex with PDGF) confirms the high similarity between the two receptors (RMSD of 0.7-0.8 Å, Figure 12B). However, comparison of the PDGFRα and PDGFRβ D1-D3 domains aligned along the D2 domain revealed a relative rotation of approximately 105° of the D3 domain between these two structures, with little change in the position of D1 (Figure 12C).

[0153] The PDGFRα D1-D3 domains established extensive interactions at four major conserved surfaces spanning the N-termini of gO and gH (sites 1-4; Figures 5D-5F and Table 3). Specifically, the first major interaction surface (site 1) included the N-terminus of gH between strands D1-b and D1-c, and between strands D1-d and D1-e, and the loop region of PDGFRα D1 (Figures 5D and 12E). In site 1, PDGFRα E52 formed salt bridges with gH R47 and PDGFRα S78 and L80 contact residues N85 and Y84, respectively (Figures 5D and 12E). Site 2 had electrostatic properties, with two acidic side chains in the extended loop between PDGFRα D1-f and D1-g (E108 and E109) ​​bound to the basic groove between the N- and C-terminal domains of gO (Figures 5D and 12E). At site 3, hydrophobic residues (M133, L137, I139, L208, and Y206) of the PDGFRα D2 domain were oriented toward the hydrophobic groove in the N-terminal region of gO (Figure 5D). Site 4 involved E263 and K265 on strand D of the PDGFRα D3 domain, which established charged and polar interactions with R336, Y337, and N358 on gO (Figure 5D, site 4). Notably, subtle but unique side chain differences at each of these four major interaction sites, taken together, may rationalize the high specificity of the trimer, which binds to PDGFRα but not PDGFRβ (Figure 12E). PDGFRα bound with low nanomolar affinity to the trimer (Table 2 and Figures 5G–5H), consistent with the four large contact sites observed in the structure. Interestingly, the introduction of mutations designed to add bulky N-linked glycans to each individual site in gH or gO did not significantly reduce binding of PDGFRα to the trimer (Table 2 and Figures 5G-5H). However, introducing a combination of N-glycans or charge mutations at all four sites almost completely abolished the interaction between PDGFRα and the trimer (Figure 5G). Thus, the extensive interaction interface between the trimer and PDGFRα consisted of numerous highly conserved residues that promoted high-affinity and highly selective binding of the trimer to the surface of fibroblasts via PDGFRα binding.The residues involved in binding of the HCMV gHgLgO trimer to PDGFRα are shown in Table 3. [Table 2] Single-site point mutations (site 1: E52N, E54S; site 2: E108N, N110S; site 3: E208N, S210S; site 4: E263N, K265S). D , dissociation constant; WT, wild type. [Table 3]

[0154] Protein expression and purification Optimized coding DNA for human PDGFRα (1–528) was cloned into the pRK vector behind a CMV promoter. A C-terminal human IgG1 (Fc) tag was added to the PDGFRα construct. Expi293 cells in suspension were cultured in SMM 293T-I medium at 37°C under 5% CO2 until a cell density of 4 × 10 6 When the cell density reached 100 cells / ml, the cells were transfected with polyethylenimine (PEI) containing DNA at a ratio of 1:1:1 for gHgLgO expression. The transfected cells were cultured for 7 days, and the expression supernatant was then harvested.

[0155] PDGFRα (1–524) (DDDDK) (Sino Biological) with five amino acids at the C-terminus was used for cryo-EM sample preparation and in vitro competition experiments. The lyophilized powder was resuspended in ddH2O, concentrated using an AMICON® Ultra centrifugal filter device (30 kDa MWCO), purified on a Superose 6 3.2 / 300 column equilibrated with PDGFRα-SEC buffer (25 mM HEPES, pH 7.5, 250 mM NaCl), and then reconstituted with HCMV trimer gHgLgO and neutralizing Fab.

[0156] Reconstitution of HCMV gHgLgO trimer with PDGFRα and neutralizing Fab The PDGFRα-gHgLgO-13H11-Msl-109 complex was assembled by incubating 5 μM (83.3 μg) gHgLgO with excess PDGFRα (6 μM, 33.3 μg) and Fabs (13H11 and Msl-109) at 18 μM (50 μg) each on ice for at least 30 min. Excess Fab was removed by purification on a Superose 6 3.2 / 300 column equilibrated with SEC-reconst-2 buffer (25 mM HEPES, pH 7.5, 300 mM NaCl). The main peak fractions of gHgLgO-13H11-Msl-109 were combined and concentrated to 0.5 mg / ml for cryo-EM sample preparation.

[0157] Biolayer Interferometry The interaction between PDGFRα protein and CMV trimers was analyzed by biolayer interferometry using the Octet Red system. Recombinant PDGFRα protein was captured on an anti-human Fc-coated sensor (Forte Pall) and tested for binding to CMV trimers as soluble analytes, assayed in PBS. Data were acquired using Forte Pall software version 9.0. To compare the relative binding of WT trimers to PDGFRα WT and mutant proteins, trimers were assayed at concentrations of 50 nM or 100 nM, and binding units at the end of association were plotted. For estimation of binding kinetics, low levels of PDGFRα protein were captured on the sensor. Data were acquired using the Octet Red instrument, followed by calculation of kinetic parameters using Biaevaluation software version 4.1 (GE Healthcare).

[0158] Cryo-EM sample preparation and data acquisition The PDGFRα-gHgLgO-13H11-Msl-109 complex was prepared as described below. A Holey carbon grid (C-Flat 45 nm R 1.2 / 1.3 300 mesh coated with Au / Pd 80 / 20; Protochips) was glow-discharged for 10 seconds using a Solarus plasma cleaner (Gatan). The complex was gently crosslinked with 0.025% EM-grade glutaraldehyde for 10 minutes at room temperature and quenched with 9 mM Tris (pH 7.5). 3 μl of sample (approximately 0.4 mg / ml here) was applied to the grid. The grid was blotted using a Vitrobot Mark IV (Thermofisher) with a 2.5-second blotting time at 100% humidity and quenched in liquid ethane cooled with liquid nitrogen.

[0159] Cryo-EM data processing The PDGFRα-gHgLgO-13H11-Msl-109 complex was processed similarly to that described in Example 1 for the gHgLgO-13H11-Msl-109 complex. A total of 34,829 videos were corrected for frame motion using the MotionCor2 (Zheng et al. Nat Methods, 14(4):331-332, 2017) implementation, and contrast transfer function parameters were fitted using the 30-4.5 Å band of the spectrum with CTFFIND-4 (Rohou and Grigorieff, J Struct Biol, 192(2):216-2, 2015). The CTF-fitted images were filtered based on a detected fit resolution better than 8 Å. A total of 4,151,085 particles were collected by template matching using gautomatch (MRC Laboratory of Molecular Biology) using a 30 Å low-pass filtered gHgLgO-13H11-Msl-109 complex reference structure. Particles were sorted during the RELION 2D classification, and 3,560,620 selected particles were imported into cisTEM for 3D refinement. The PDGFRα-gHgLgO-13H11-Msl-109 3D reconstruction was obtained after automatic and manual refinement using a mask, by applying a low-pass filter (LPF) outside the mask (filter resolution 20 Å) and a score threshold of 0.25. This gradually reduced the outside weight from 0.5 to 0.15 through iterative manual refinement rounds. The 3D reconstruction converged to a map resolution of 2.8 Å (Fourier shell correlation (FSC) = 0.143, measured by cisTEM). To improve map quality, a mask was used to divide the map into three distinct regions before obtaining convergent refinement, and manual refinement outside the mask was obtained using a low-pass filter (LPF) as described above. The convergent maps were sharpened in a cisTEM and combined using phenix as described above in Example 1.

[0160] Model construction and structural analysis The structure of PDGFRβ (PDB: 3MJG) was used as a template for modeling PDGFRα D1 to D3. Model construction and structural analysis were carried out in the same manner as in Example 1. Example 6. TGFβR3 binds to the interface between gH, gL, and gO

[0161] The trimer is required for HCMV tropism to all cell types, including endothelial and epithelial cells (Zhou et al., J Virol, 89:8999-9009, 2015; Wille et al., mBio, 4:e00332-13, 2013; Ryckman et al., J Virol, 82:60-70, 2008). This requirement suggests that the trimer may directly contribute to HCMV host tropism by directly interacting with multiple receptors. Accordingly, TGFβR3 was recently identified as a high-affinity binder for the trimer and putative HCMV receptor (Martinez-Martin et al., Cell, 174:1158-1171 e19, 2018). The TGFβR3 glycoprotein is a member of the TGF-β signaling pathway receptor superfamily and plays an essential role in mediating cell proliferation, apoptosis, differentiation, and cell migration in most human tissues (Zhang et al., Cold Spring Harb Perspect Biol, 9:a022145, 2017). The extracellular domain of TGFβR3 consists of two N-terminal membrane-distal orphan domains (OD2 and OD1) and a membrane-proximal zona pellucida (ZP) domain (Kim et al., Structure, 27:1427-1442 e4, 2019). Each OD consists of two β-sandwich domains, while the ZP domain adopts a classical immunoglobulin-like fold (Figure 6B) (Lin et al., Proc Natl Acad Sci USA, 108:5232-5236, 2011). Despite the homology between TGFβR3 and TGFβR1, TGFβR2, or endoglin, no binding between these additional proteins and the HCMV trimer was observed (Figure 6A) (Martinez-Martin et al., Cell, 174:1158-1171 e19, 2018), which was previously published in the results of the Cell-Surface Receptor Discovery Platform. [Table 4]

[0162] To gain direct structural insight into TGFβR3 recognition by the trimer, we reconstituted a stoichiometric complex of the HCMV trimer and TGFβR3 containing the OD and ZP domains with Fabs 13H11 and Msl-109 and used cryo-EM to determine the structure to an overall resolution of approximately 2.6 Å (Figures 6C-6G and 13A-13F). Because the trimer associates with the Ig-like D1-D3 domains of PDGFRα, binding of TGFβR3 was predicted to occur via that Ig-like domain. Unexpectedly, the newly revealed structure showed that TGFβR3 exclusively utilized the OD2 domain to bind to conserved residues on gO and gL at three major sites (Figures 6D-6G and Table 4), whereas the density of the TGFβR3 OD1 domain appeared weak, apparently due to the lack of direct contact with the trimer. TGFβR3 OD1 did not specifically contact the HCMV trimer, was poorly resolved in the cryo-EM map, and was not modeled in the structure. Notably, TGFβR3 binding did not induce any major structural rearrangements on the trimer, similar to those observed for PDGFRα (Figure 13G).

[0163] The human TGFβR3 OD2 domain is composed of 10 β-strands and two α-helices, one between β6 and β7 (α1) and the other between β7 and β8 (α2) (Fig. S13H). The regions surrounding both helices formed important contacts with the HCMV trimer. Specifically, TGFβR3 utilizes a loop structure surrounding the α1 region to contact the gO side chain K and the gO N-terminal domain, respectively. 118 and R 117 In contrast to S 143 Carbonyl and Q 136 Hydrogen bonds with the side chain (site 1, Figures 6G and 13H). TGFβR3 F 137 and gO L 116 Hydrophobic contacts between the TGFβR3 R and the TGFβR3 R in chain β7b further supported the interaction at site 1 (Fig. 6G, site 1). 151 is gO Y 188 and gLN, which forms π-stacking interactions with 97At site 3, the TGFβR3 W of α2 binds to the α2 domain (Fig. 6G, site 2). 163 and K. 166 The carbonyls of the gL side chain E 94 and T 92 formed hydrogen bonds with (Fig. 6G, site 3).

[0164] The OD2 domains of TGFβR3 and endoglin were very similar in structure (Figure 6H). Detailed mapping, particularly of the key interaction regions in sites 1 and 2, revealed that the endoglin OD2 domain lacked a loop structure in α1 and no β-strand secondary structure in β7b (Figures 13H and 6H). Overall, the numerous interacting residues with contrasting properties rationalized the strong interaction between TGFβR3 and the HCMV trimer, and the key differences in sites 1 and 2 relative to endoglin provided an explanation for the high specificity and selectivity of the TGFβR3 trimer interaction.

[0165] Protein expression and purification The optimized coding DNA for human TGFβR3 (1–787) was cloned into the pRK vector behind the CMV promoter. A C-terminal FLAG tag was added to the TGFβR3 construct. Expi293 cells in suspension were cultured in SMM 293T-I medium at 37°C under 5% CO2 until a cell density of 4 × 10 6 When the cell density reached 100 cells / ml, the cells were transfected with polyethylenimine (PEI) containing DNA at a ratio of 1:1:1 for gHgLgO expression. The transfected cells were cultured for 7 days, and the expression supernatant was then harvested.

[0166] Human TGFβR3-Flag was purified from 10 L of expression supernatant. The supernatant was incubated with 10 ml of M2 agarose-Flag resin (Sigma) and incubated at 4°C for 20 hours. The resin was washed with 10 CV of FLAG wash buffer (30 mM HEPES, pH 7.5, 300 mM NaCl, 5% glycerol) and eluted with FLAG wash buffer supplemented with 0.2 mg / ml FLAG peptide. The eluate was concentrated using an AMICON® Ultra Centrifugal filter device (30 kDa MWCO) and loaded onto a Superdex 200 10 / 60 column equilibrated with TGFβR-SEC-1 buffer (30 mM HEPES, pH 7.5, 300 mM NaCl, 5% glycerol).

[0167] TGFβR3 (1-781) with a C-terminal HIS tag (Sino Biological) was used for cryo-EM sample preparation. The lyophilized powder was resuspended in ddH2O, concentrated using an AMICON® Ultra Centrifugal filter device (30 kDa MWCO), purified on a Superdex 200 3.2 / 300 column equilibrated with TGFβR-SEC-2 buffer (25 mM HEPES, pH 7.5, 200 mM NaCl), and then assembled with HCMV trimer gHgLgO and neutralizing Fab. Reconstitution of HCMV gHgLgO trimers with human TGF.BETA.R3 and neutralizing Fab

[0168] A 7.6 μM (85.5 μg) TGFβR3-gHgLgO-13H11-Msl-109 complex was assembled by incubation on ice for at least 30 min with 9.2 μM (54 μg) excess TGFβR3, 22.6 μM (78 μg) Fab 13H11, and 61 μM (210 μg) Msl-109. Excess Fab was removed by purification on a Superose 6 3.2 / 300 column equilibrated with SEC-reconst-2 buffer (25 mM HEPES, pH 7.5, 300 mM NaCl). The main peak fractions of gHgLgO-13H11-Msl-109 were combined and concentrated to 0.5 mg / ml for cryo-EM sample preparation.

[0169] Cryo-EM sample preparation and data acquisition The TGFβR3-gHgLgO-13H11-Msl-109 complex was prepared as follows: Holly carbon grids (Ultrafoil 25 nM Au® 1.2 / 1.3 300 mesh; Quantifoil) were glow discharged for 10 seconds using a Solarus plasma cleaner (Gatan). 3 μl of sample was applied to the grid, blotted on one side with a Leica EM GP (Leica) using a blotting time of 3.5 seconds at 100% humidity, and cooled in liquid ethane cooled with liquid nitrogen.

[0170] The TGFβR3-gHgLgO-13H11-Msl-109 complex was processed similarly to the gHgLgO-13H11-Msl-109 complex described above in Example 1. A total of 19,993 videos were corrected for frame motion using the MotionCor2 implementation in RELION (Zheng et al. Nat Methods, 14(4):331-332, 2017), and contrast transfer function parameters were fitted using the 30-4.5 Å bandpass of the spectrum with CTFFIND-4 (Rohou and Grigorieff, J Struct Biol, 192(2):216-2, 2015). A total of 2,780,519 particles were collected by template matching with gAutomach using a 30 Å low-pass filtered gHgLgO-13H11-Msl-109 complex reference structure. Particles were sorted during the RELION 2D classification, and the selected 2,780,519 particles were imported into cisTEM for 3D refinement. The TGFβR3-gHgLgO-13H11-Msl-109 3D reconstruction was obtained by automatic and manual refinement using a mask, followed by applying a low-pass filter (LPF) outside the mask (filter resolution 20 Å) and a score threshold of 0.25. This gradually reduced the outer weight from 0.5 to 0.15 through iterative rounds of manual refinement. The 3D reconstruction converged to a map resolution of 2.6 Å (Fourier shell correlation (FSC) = 0.143, measured in cisTEM). To improve map quality, convergent refinement was obtained after dividing the map into two separate regions using a mask, followed by manual refinement outside the mask using a low-pass filter (LPF) as described above. The converged maps were sharpened in cisTEM and combined using phenix as described above. Local resolution was determined in cisTEM using an in-house reimplementation of the Brockles algorithm (Cardone et al 2013).

[0171] Model construction and structural analysis The structure of zebrafish TGFβR3 (PDB: 6MZN) was used as a template to model human TGFβR3 OD2. Model construction and structural analysis were performed as in Example 1.

[0172] Example 7. PDGFRα and TGFβR3 compete for HCMV trimer binding The HCMV trimer could bind with high affinity to two completely different domain structures present on diverse receptors: the Ig-like D1-D3 domain of PDGFRα and the OD2 domain of TGFβR3 (Figures 5 and 6). Although both receptors interacted in the membrane-distal region of the trimer, PDGFRα and TGFβR3 bound to the trimer through different interaction surfaces (Figures 5E and 6E). Despite this, superposition of the trimer-PDGFRα and trimer-TGFβR3 complex structures suggested that these receptors share partially overlapping binding sites at the interface between gH, gL, and gO and therefore cannot simultaneously bind the trimer (Figure 7A). Furthermore, PDGFRα N 179 The upper N-linked glycan chain was found to point toward the TGFβR3 binding site, which may further restrict co-receptor binding (FIG. 7A).

[0173] To test the hypothesis that PDGFRα and TGFβR3 binding to the trimer is mutually exclusive, we performed a competition experiment by incubating HCMV trimers bound to TGFβR3 with equimolar amounts of PDGFRα (Figure 7B and Figure 14). The results showed that PDGFRα could completely displace bound TGFβR3 (Figure 7B), consistent with the reported higher affinity between HCMV trimers and PDGFRα compared to TGFβR3 (Martinez-Martin et al., Cell, 174:1158-1171 e19, 2018). Together, these structural and biophysical data suggest that PDGFRα and TGFβR3 likely mediate HCMV tropism by acting as independent receptors rather than as coreceptors.

[0174] Binding competition experiments of PDGFRα and TGFβR3 to HCMV trimer gHgLgO HCMV trimers gHgLgO, PDGFRα, and TGFβR3 alone or in combination with gHgLgO + PDGFRα, gHgLgO + TGFβR3, or gHgLgO + PDGFRα + TGFβR3 were co-incubated at a concentration of 3 μM in SEC competition buffer (25 mM HEPES (pH 7.5), 300 mM NaCl) for at least 60 min on ice and loaded onto a Superose 6 3.2 / 300 column equilibrated in SEC competition buffer.

[0175] Example 8. HCMV trimers compete with the growth factor PDGF for binding to PDGFRα In Examples 4–6, cryo-EM structures of the trimer, trimer-PDGFRα, and trimer-TGFβR3 revealed functionally important, highly conserved surfaces on the trimer involved in receptor binding and potential targets for potent neutralizing antibodies (Figures 4, 5, and 6). Therefore, we investigated whether the trimeric interaction with PDGFRα could interfere with important cell signaling pathways. In the case of PDGFRα, binding of the PDGF growth factor dimerizes the receptor and activates the intracellular kinase domain, inducing a signaling cascade (Shim et al., Proc Natl Acad Sci USA, 107:11307–11312, 2010). Structural superposition of the trimer-PDGFRα complex with a homology model of the dimeric (signaling-active) PDGFRα-PDGF complex revealed multiple steric clashes between gO and PDGF at the PDGFRα D2 and D3 interaction interfaces (Figures 7C and 12E). Considering the strong interaction of the trimer with PDGFRα (Table 2) at low nanomolar affinity (Kabanova et al., Nat Microbiol, 1:16082, 2016) and the moderate binding affinity of PDGF-AA for PDGFRα, characterized by three-digit nanomolar affinity (Mamer et al., Sci Rep, 7:16439, 2017), we tested the hypothesis that the trimer could compete with PDGF for binding to PDGFRα, thereby preventing the induction of the signaling cascade. To test this hypothesis, HCMV trimers were first purified with charge mutations of gO (trimers). mut ; sites 2–4: M84R, F111R, R117E, F136R, R212E, R230E, R234E, R336E, F342E, A351R, N358R), an approximately 10,000-fold decrease in in vitro binding to PDGFRα was observed (KD 三量体-WT :2.25×10 -9 + / -1.1M vs KD 三量体-mut :4.25×10 -5 + / - 0.5M) (Figure 7D). Next, autophosphorylated PDGFRα residue Y 762 and Y 849 and phosphorylated AKT as a downstream substrate, thereby detecting PDGF-AA and trimers. WT or trimermut PDGF-AA alone induced strong activation signals in PDGFRα and AKT, but the trimeric form WT Titration of PDGF-AA strongly reduced the PDGF-AA-induced activity of PDGFRα (Figure 7E). In contrast, the PDGFRα binding-deficient trimer Mut The addition of PDGF-AA did not reduce the activity of PDGFRα in the presence of PDGF-AA (Figure 7E). Thus, HCMV trimers directly compete with PDGF-AA for binding to PDGFRα and disrupt PDGFRα signaling, an important consideration for the design of effective and safe trimer-based antiviral strategies.

[0176] PDGFRα activation and signaling The fibroblast cell line MRC-5 was used to study receptor phosphorylation and downstream signaling. MRC-5 cells were grown in RPMI medium supplemented with 10% FBS, glutamine, and antibiotics. Cells were cultured at 37°C and 5% CO2. Cells were seeded into M6-well plates, grown to approximately 75% confluence, and starved overnight before stimulation. On the day of the assay, cells were stimulated with PDGF-AA (3.7 nM concentration), CMV trimer, or PDGF-AA:CMV trimer at increasing molar ratios. Stimulation was performed for 10 minutes at 37°C in serum-free medium. After treatment, cells were washed with cold PBS and lysed (lysis buffer: 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% (v / v) NP40, supplemented with protease (Roche) and phosphatase inhibitors (Sigma)). Samples were diluted in loading buffer (Thermo Fisher Scientific) using denaturing conditions and analyzed by Western blotting using a LI-COR® instrument.

[0177] Antibodies and Recombinant Proteins All primary antibodies used in these examples were purchased from Cell Signaling Technology®. The secondary antibody used for detection (IRDYES®) was obtained from LI-COR® Biosciences. All antibodies were used at the dilution recommended by the manufacturer, and incubation was overnight (primary antibodies) or for 1 hour at room temperature (LI-COR® antibodies).

[0178] Human PDGF-AA used for cell stimulation was purchased from STEMCELL™ Technologies. All other recombinant proteins were produced in-house.

[0179] conclusion These examples present the structure of the HCMV trimer, revealing unprecedented insights into the structure of the trimeric complex, binding of broadly neutralizing antibodies, the mechanism of trimer-mediated HCMV receptor interaction, and the consequences for cell signaling pathways. These results have important implications for the design of trimer-based vaccines and antiviral therapeutics.

[0180] Importantly, these examples directly demonstrate the potential for the development of novel broadly neutralizing antibodies against the glycan-free surface of gO. Furthermore, blocking trimer interactions with PDGFRα and TGFβR3 would also provide a new strategy for targeting HCMV entry. Notably, the trimer forms extensive contacts across multiple interaction sites with PDGFRα and TGFβR3, and attempts to disrupt binding at a single site failed to completely abolish PDGFRα binding (Figure 5). Instead, multiple interaction sites in gO were demonstrated and needed to be simultaneously targeted to block the interaction between HCMV trimers and PDGFRα (Figure 5G). Therefore, broadly neutralizing antibodies with a sufficiently large interaction footprint against gO, including multispecific (e.g., bispecific) antibodies, could be used to displace the interaction of both the PDGFRα and TGFβR3 receptor proteins. Alternatively, the D1-D3 domains of PDGFRα could be utilized to block trimer binding to endogenous host receptors for the development of antiviral therapeutics.

Claims

1. A modulator of the interaction between the gO subunit of the human cytomegalovirus (HCMV) gHgLgO trimer and PDGFRα, comprising: binds to the glycosylated-free surface of the gO subunit, causing a decrease in binding of the gO subunit to PDGFRα; A modulator that is a small molecule, peptide, mimetic, or inhibitory nucleic acid.

2. The modulator (a) one or more of residues R230, R234, V235, K237, and Y238 of the gO subunit; (b) one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit; and (c) one or more of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit. The modulator of claim 1 , which binds to 3. A modulator of the interaction between the gO subunit of the HCMV gHgLgO trimer and PDGFRα, comprising: (a) one or more of residues R230, R234, V235, K237, and Y238 of the gO subunit; (b) one or more of residues N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, and V123 of the gO subunit; and (c) one or more of residues R336, Y337, K344, D346, N348, E354, and N358 of the gO subunit. and causing a decrease in binding of the gO subunit to PDGFRα; a small molecule, an antibody or antigen-binding fragment thereof, a peptide, a mimetic, or an inhibitory nucleic acid; Modulator.

4. A modulator described in claim 2 or 3, which binds to all 23 residues of the gO subunit: R230, R234, V235, K237, Y238, N81, L82, M84, M86, F109, F111, T114, Q115, R117, K121, V123, R336, Y337, K344, D346, N348, E354 and N358.

5. A modulator described in claim 1 or 3, wherein the inhibitory nucleic acid is ASO or siRNA.

6. A modulator according to any one of claims 1 to 5 for use in treating HCMV infection in an individual, comprising: A modulator, wherein the duration or severity of HCMV infection is reduced by at least 40% compared to an individual not receiving said modulator.

7. A modulator described in any one of claims 1 to 5 for the prevention of HCMV infection in an individual.

8. A modulator according to any one of claims 1 to 5 for use in the prophylaxis against secondary HCMV infection in an individual, comprising: The modulator, wherein said secondary infection is an HCMV infection of an uninfected tissue.

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