Fig 1.
Characterization of F4, F6, F7, F9, and R10 mAbs.
(A) SPR assays for characterizing binding affinities between Fab of F4, F6, F7, F9, R10, and HAV particles. The binding affinity is depicted in terms of KD (equilibrium dissociation constant, KD = kd/ka), which are listed in the figure. (B) Competition studies among the 4 different anti-HAV NAbs. The CM5 chip (BIAcore, GE Healthcare) fixed with HAV particles was first saturated with indicated R10. The capacity of an additional binding was monitored by measuring further shifts after injecting the second antibody in the presence of the first one. The pink dotted vertical line represents the second Nab’s loading time. CV60 (gray curve) was used as a non-HAV binding control. (C) Neutralization of HAV by F4, F6, F7, F9, and R10. F4 (blue curve), F6 (red curve), F7 (green curve), F9 (purple curve), and R10 (cyan curve) were used to block HAV infection at different concentrations by detecting de novo synthesized viral capsids. Data showing the levels of inhibition of virus are represented as the percentage of HAV antigen relative to antigen in the control wells. The values represent means of results from triplicate wells with SDs. The underlying data of panels B and C can be found in S1 Data.; Fab, fragment of antigen binding; HAV, hepatitis A virus; mAb, monoclonal antibody; NAb, neutralizing monoclonal antibody; SPR, surface plasmon resonance.
Fig 2.
Cryo-EM structures of F4-Fab-HAV complex, F6-Fab-HAV complex, F7-Fab-HAV complex, and F9-Fab-HAV complex.
(A) The maps of F4, F6, F7, and F9 Fab in complex with full particles are colored based on the radial distance (Å) of the capsid and Fab from the particle center. Only the front half of each type of particle is shown. Triangles indicate an icosahedral asymmetric unit. (B) The typical NAb’s Fab (F7) binds to the viral surface along the pentamer interface between the 2-fold and 3-fold axes. (C) The quality of Fab-HAV complex density map (gray) is illustrated by fit of backbone and side chains for 3 separate structures of F7-Fab-HAV complex. cryo-EM, cryo-electron microscopy; Fab, fragment of antigen binding; HAV, hepatitis A virus; Nab, neutralizing monoclonal antibody.
Table 1.
Cryo-EM data collection and atomic models refinement statistics.
Fig 3.
Typical interaction between HAV with F6 Fab.
(A) The F6 footprints on the HAV surface. The figure shows a 2D projection of the HAV surface produced using RIVEM [25]. Residues of VP1, VP2, and VP3 are outlined in pale blue, green, and purple, respectively; residues involved in binding to F6 are shown in brighter colors corresponding to the protein chain they belong to. The footprints of F6 heavy and light chains are indicated by magenta and green lines, respectively. Five-, 3-, and 2-fold icosahedral symmetry axes are marked as for 1 icosahedral asymmetric unit. (B) The front and back view of the interaction between F6 Fab and HAV capsid proteins. F6 Fab binds to the BC loop, EF loop, C terminal of a VP3, and BC loop from another VP2. HAV capsid proteins and the Fab molecule are shown in cartoon format, with VP1, VP2, VP3, light chain, and heavy chain colored in blue, green, red, cyan, and magenta, respectively. And below, the cartoon representation of the interacting residues on capsid proteins, the left is the front view, and the right is the back view. The residues in VP2 and VP3 involved in the interactions with F6 Fab are shown as sticks. (C) Interaction between F6 Fab and HAV capsid proteins. Some residues involved in the formation of hydrogen bonds are shown as sticks and labeled. VP2, VP3, light chain, and heavy chain are colored in green, red, cyan, and magenta, respectively. Three CDRs of the heavy chain and epitope on VP2 and VP3 are highlighted by black outlines CDR, complementary determining region; Fab, fragment of antigen binding; HAV, hepatitis A virus.
Fig 4.
Analysis of the conservation and structure activity correlates of F4, F6, F7, F9, and R10 Fab.
(A) The conservation analysis of epitopes recognized by F4, F6, F7, F9, and R10. The adjacent VP2 and VP3 of capsids are shown in surface representation with epitope residues colored according to conservation values. (B) ESPript [28] representation of sequence alignment of the variable regions of light chains and heavy chains of F4, F6, F7, F9, and R10. Residues in light chain and heavy chain that interact with HAV capsid proteins are outlined in cyan and purple, respectively. (C) Superposition of partially variable regions of light chains and heavy chains of F4, F6, F7, F9, and R10 are colored in blue, red, green, purple, and cyan. The interacted epitopes of HAV capsid are colored in gray. (D) The correlation between neutralization activity and interface area. (E) The correlation between neutralization activity (log) and binding energy. The underlying data of panels D and E can be found in S1 Data. CDR, complimentary determining region; Fab, fragment of antigen binding; HAV, hepatitus A virus.
Fig 5.
The single, conserved antigenic site on the surface of HAV.
(A) Multiple-sequence alignment analysis of the 6 genotypes of human HAV capsids. The alignment results are displayed with the program ESPript [28]. The epitopes that interacted with the 5 NAbs are marked by blue diamonds. (B) Residue conservation mapped onto the HAV capsid (VP2 and VP3) using Consurf [29] (residues are displayed as spheres in variable sizes; conserved residues are shown smaller, and variable residues are shown as bigger), based on the alignment of all the virus sequences. Residues involved in interactions with heavy and light chains are shown as magenta and cyan spheres, respectively. The boundary of epitope is indicated with a black dotted circle. HAV, hepatitis A virus; NAb, neutralizing monoclonal antibody.
Fig 6.
In silico–designed anti-HAV drug candidates.
(A) The overall structure of golvatinib and the binding pocket as well as the interaction details between golvatinib and HAV capsid. The binding pocket is shown as a secondary structure representation and is colored in gray; the key residues are shown as a ball and stick representation. (B) The assays of cytotoxic and inhibitory effects of screened compound on 2BS cells. 2BS cells were cultured in the absence or presence of various concentrations of compounds and incubated at 34°C for 7 d. Viable cell number was determined by LDH release assay using the CCK-8 kit (Sangon Biotech, Shanghai). Data are presented as mean ± SD of 3 independent experiments. Random errors within reasonable error range (<5%) exist. Various concentrations of inhibitors were preincubated with HAV for 1 hour at room temperature before infection of 2BS cells. The inhibitory abilities of screened chemicals were evaluated by determining HAV antigen content using indirect ELISA after 7 d of infection. Values are mean ± SD. Experiments were repeated in triplicate. The underlying data of panel B can be found in S1 Data. CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay; HAV, hepatitis A virus; LDH, lactate dehydrogenase.