Generation and biochemical characterization of anti-EV-D68 neutralizing MAbs.
It has been previously shown that recombinant VLPs of EV-D68 could induce high levels of broadly neutralizing antibodies capable of conferring protection against lethal infection in mice26, 27. In this study, we adopted the hybridoma technology to generate EV-D68-specific neutralizing MAbs from one mouse immunized with recombinant VLP of EV-D68 strain US/MO/14-18950 (hereinafter referred to as 18950, clade B). The resulting hybridoma cells were screened for their ability to neutralize EV-D68 clinical strain US/MO/14-18947 (hereinafter referred to as 18947; clade B). Note that strain 18947 was used in the initial screens because the immunogen strain (18950) was not available to us. 2 stable clones (2H12 and 8F12) were found to possess neutralizing activity (Fig. 1a). Isotyping analysis showed that 2H12 was IgG2a antibody while clone 8F12 was of IgG2b isotype (Fig. 1a). We determined the coding sequences of the two anti-EV-D68 MAbs. The sequence of the heavy chain variable region (VH) of antibody 2H12 was 79% identical to that of 8F12, while the two MAbs shared 57% sequence identity in light-chain variable region (VL) (Supplementary Fig. 1). Next, neutralization potency of the purified MAbs was initially assessed by standard neutralization assay with the 18947 strain. For MAbs 2H12 and 8F12, their neutralization concentrations (the lowest antibody concentration that could fully inhibit EV-D68-induced cytopathic effect [CPE]) against strain 18947 were determined to be 1.95 and 0.06 µg/mL, respectively (Fig. 1a). In quantitative neutralization assays, both anti-EV-D68 MAbs exhibited inhibitory effects in an antibody dose-dependent manner (Fig. 1b) with IC50s being 0.412 and 0.004 µg/mL for 2H12 and 8F12, respectively; in contrast, the two irrelevant MAbs, 1C11 (IgG2a isotype control) and 1F4 (IgG2b isotype control) did not show any neutralization effect regardless of the antibody dose.
The MAbs were tested by ELISA for their ability to recognize different antigens, including EV-D68 VLP, enterovirus 71 (EV71) VLP and coxsackievirus A16 (CVA16) VLP. Anti-EV-D68 MAbs 2H12 and 8F12, but not the isotype control antibodies 1C11 and 1F4, were found to react with EV-D68 VLP (Fig. 1c). It is worth pointing out that MAb 2H12 showed stronger binding to EV-D68 VLP than MAb 8F12 (Fig. 1c). The two anti-EV-D68 MAbs did not exhibit any reactivity in EV71 VLP- or CVA16 VLP-binding ELISA assays which were validated using anti-EV71 MAb D5 and anti-CVA16 MAb 9B5, respectively (Supplementary Fig. 2). These results indicated that MAbs 2H12 and 8F12 were indeed EV-D68-specific antibodies.
Compared with MAb 2H12, MAb 8F12 had relatively lower binding activity towards EV-D68 VLP (Fig. 1c) but exhibited stronger neutralizing activities on EV-D68 strain 18947 (Fig. 1b). This seeming contradiction prompted us to determine the binding affinity of the MAbs to the 18947 virion by BLI. The virion-binding affinity of MAb 8F12 was found to be significantly higher than that of 2H12 (Fig. 1a, 1d and 1e), in agreement with the neutralization potency of the MAbs (Fig. 1b). Hence, for the anti-EV-D68 MAbs, their virion-binding affinity, rather than the affinity towards the VLP immunogen, positively correlated with their neutralization ability.
Cross-neutralization capacities of the MAbs
The MAbs were further evaluated for their ability to cross-neutralize two other EV-D68 strains, including the prototype strain Fermon and a clinical strain US/KY/14-18953 (hereinafter referred to as 18953; clade D). MAb 2H12 effectively neutralized strain 18953 with a neutralization concentration of 0.12 µg/mL but had no neutralization effects on Fermon even at 500 µg/mL, the highest concentration tested (Fig. 1a). Conversely, MAb 8F12 showed very weak neutralization against strain 18953 (neutralization concentration: 125 µg/mL), but it could potently neutralize the Fermon strain (neutralization concentration: 0.49 µg/mL). As expected, the two isotype control antibodies, 1C11 and 1F4, did not display any neutralization effect on the two EV-D68 strains (Fig. 1a).
The distinct cross-neutralization profiles of 2H12 and 8F12 propelled us to investigate whether they can be used in combination to create synergy. A two-MAb cocktail was thus formulated by mixing 2H12 and 8F12 at a ratio of 1:1. Results from neutralization assays showed that the two-MAb cocktail exhibited greater neutralization breadth than any of single MAbs (Fig. 1a). Specifically, for the antibody cocktail, neutralization concentrations against strains 18947, 18953 and Fermon were determined to be 0.12, 0.24, and 1.95 µg/mL, respectively (Fig. 1a). These data indicated that 2H12 and 8F12 MAbs can complement each other in neutralizing diverse EV-D68 clades/strains.
Prophylactic efficacy of the MAbs
The protective efficacy of anti-EV-D68 MAbs was evaluated in a previously established mouse model of EV-D68 infection 28. For prophylactic efficacy evaluation, five groups of newborn ICR mice (n = 12–14/group) were adminstered PBS, or a single dose (10 µg/g) of 2H12, 8F12, IgG2a isotype control (1C11), or IgG2b isotype control (1F4), respectively. One day later, all mice were infected with EV-D68 clade B strain 18947 and subsequently observed for clinical signs and mortality for a period of 14 days. As shown in Supplementary Fig. 3, mice in the PBS and control antibody groups started to show clinical signs at 3 days post-infection (dpi), and majority of them eventually died (69% and 77% mortality, respectively). In contrast, 92% and 100% of the mice in the 2H12 and 8F12 groups survived from lethal challenge, respectively. These results indicated that both 2H12 and 8F12 MAbs are effective in preventing lethal EV-D68 infection in mice.
Therapeutic efficacy of the MAbs
We then assessed the therapeutic efficacy of the MAbs in the mouse model. The first experiment was designed to evaluate therapeutic efficacies of the MAbs administered at 1 dpi. Groups of one-day-old ICR mice were infected with strain 18947 and one day later given single injections of PBS, 2H12 (10 µg/g), or 8F12 (10 µg/g), respectively. Survival and clinical score were then monitored on a daily basis. As shown in Fig. 2a, mice in the PBS group became sick at 3 dpi and a large proportion (86%) of them eventually died; in contrast, all of the MAb-treated mice survived, indicating that the MAbs were therapeutically efficacious. We should point out that the mean clinical scores in the 2H12 group were significantly higher than those in the 8F12 groups (Fig. 2a).
The second experiment was aimed to evaluate the MAbs for their efficacy in treating mice that had already developed disease. Besides individual MAbs, a two-MAb cocktail (formulated by mixing 2H12 and 8F12 at a ratio of 1:1) was also included as a treatment option in the experiments. Mice infected with strain 18947 started to show clinical signs at 3 dpi (Fig. 2a, right panel). Therefore, MAb treatment was given at 3 dpi and the treated mice were monitored for two weeks. As shown in Fig. 2b, the survival rates for the 2H12, 8F12, and two-MAb groups were 85%, 92%, and 92%, respectively, whereas only 26% of the mice in the PBS control group survived. These data showed that the anti-EV-D68 MAb treatment remained effective when given at a delayed time point during the course of infection. Again, clinical manifestations shown in the 2H12-treated mice were more severe than those observed in the 8F12 and the cocktail groups (Fig. 2b, right panel), suggesting that 2H12 is relatively less efficient than 8F12 in protecting against 18947 infection, in agreement with the lower in vitro neutralization efficiency of 2H12 against 18947 strain (Fig. 1b).
In a following experiment, we evaluated the anti-EV-D68 MAbs for their therapeutic breadth by using a clade D strain, 18953, as the challenge virus. Mice were administered single doses of MAb 2H12 (10 µg/g), MAb 8F12 (10 µg/g), the antibody cocktail (10 µg/g of 2H12 plus 10 µg/g of 8F12), or PBS three days after 18953 infection and subsequently monitored for a period of 14 days. The results were shown in Fig. 2c. In the control (PBS) group, the disease severity gradually increased and the final mortality rate reached 38%. The survival rate and mean clinical scores of the 8F12 group were not significantly different (P value = 0.30) from those of the control group, suggesting that MAb 8F12, at the dose used, is not therapeutically effective against 18953 infection. In contrast, all of the mice receiving MAb 2H12 eventually recovered (100% survival), indicating that 2H12 is more effective than 8F12 in treating 18953 infection. Moreover, treatment with the 2H12/8F12 cocktail also led to full protection. Taken together, the above results demonstrate that the 2H12/8F12 cocktail is superior to single MAbs in treating diverse EV-D68 infections at a delayed time point.
Mode of action of 2H12 and 8F12 antibodies
To elucidate the working mechanism of the anti-EV-D68 MAbs, we firstly determined at which stage the MAbs exert inhibitory function by performing time-of-addition assays. MAbs 2H12 and 8F12 were separately mixed with EV-D68 strain 18947 before virus binding to cells at 4 °C (pre-attachment), or added to virus-bound cells that had been incubated at 33 °C for various times, such as 0, 0.5, 1, 2, or 5 h (post-attachment). The samples were collected at 8 h post-infection and then subjected to RNA extraction and quantitative RT-PCR analysis. As shown in Fig. 3a, pre-treatment with 2H12 or 8F12 almost completely abolished viral infection, while the MAbs were partially effective when added right before virus-bound cells were transferred to 33 °C (0 h post-infection). In contrast, no inhibitory effect was seen when the MAbs were added at delayed time points (0.5–5 h post-infection) (Fig. 3a). These results indicate that both 2H12 and 8F12 exert inhibition primarily at the pre-attachment stage.
Next, we performed attachment-inhibition assays to evaluate the effect of MAb treatment on virus attachment. Pre-incubation of the 18953 virus with 2H12 MAb reduced the amount of virus adsorbed onto the cells in an antibody dose-dependent manner whereas the isotype control antibody 1C11 had no inhibitory effect regardless of the antibody dose (Fig. 3b). Similarly, 8F12 pretreatment dose-dependently inhibited the 18947 virus attachment onto cells whereas the corresponding isotype control antibody 1F4 did not show any inhibition (Fig. 3c). These results demonstrate that both 2H12 and 8F12 antibodies can efficiently block EV-D68 attachment onto target cells.
Sialic acid is a receptor for EV-D68 that promotes viral attachment onto host cells 21. It has been reported that EV-D68 can agglutinate red blood cells (RBC) through their sialic acid moiety 29. We therefore performed hemagglutination inhibition (HI) assay to determine whether MAb treatment could interfere with the interaction between EV-D68 virion and sialic acid receptor. As shown in Fig. 3d, RBC alone sank to the bottom of the plate and formed red dots in the center of the wells, while the addition of EV-D68 18947 virion alone caused complete hemagglutination of RBC as indicated by the formation of red sheets across the wells. Pre-incubation of EV-D68 with the isotype control MAbs did not show any inhibition on hemagglutination regardless of the antibody dose. In contrast, MAbs 2H12 or 8F12 was found to exert HI in an antibody dose-dependent manner. It is worth noting that MAb 8F12 showed greater HI activity than MAb 2H12, in line with the higher neutralization efficiency of 8F12 against the 18947 strain (Fig. 1b). These results suggest that both 2H12 and 8F12 antibodies can inhibit the binding of EV-D68 to sialic acid receptor.
Cryo-EM revealed structural basis of 8F12-mediated neutralization of EV-D68
To investigate the molecular basis of EV-D68 neutralization by the 8F12 MAb, we determined cryo-EM structure of EV-D68 mature virion in complex with the antigen-binding fragments (Fab) of 8F12 to a resolution of 2.89 Å (Fig. 4a, Supplementary Fig. 4). Inspection of the original micrographs of the EV-D68/8F12 Fab (abbreviated as EV-D68/8F12) complex indicated a high occupancy of the Fab on the capsid with a hedgehog-like appearance (distinct from the smooth spherical appearance of EV-D68 virion18, 28) (Supplementary Fig. 4a). We then built an atomic model for the EV-D68/8F12 complex, which matches the corresponding density map very well (Fig. 4b-c, Supplementary Fig. 5), illustrating the atomic resolution of the map.
The Fabs bind to the tips of the three-bladed propeller-like protrusion of EV-D68 and face each other across the icosahedral two-fold axis (Fig. 4a). The capsid structure in the EV-D68/8F12 complex is essentially identical to that of the unbound native EV-D68 virion 18, and the two-fold axis channel remains closed in the complex (Fig. 4c), indicating that 8F12 Fab binding does not induce obvious conformational changes of the EV-D68 virion. According to the structure, each protomer of EV-D68 engages with an 8F12 Fab (Fig. 4d). 8F12 Fab binds to the south rim of the canyon, and its light chain is tilted towards the five-fold axis of the virus, obscuring the canyon region (Fig. 4d, 4f), which is involved in receptor binding 21. 8F12-binding footprints overlap with the sialic acid binding site (Fig. 4f), thus explaining the experimental observation that MAb 8F12 could block virus attachment to the sialic acid receptor (Fig. 3).
To define antibody-binding epitopes, we analyzed the interactions between 8F12 Fab and EV-D68 virion. This analysis suggested that the heavy chain (specifically only complementary determining region [CDR] 1) of 8F12 Fab binds to the EF loop of VP2, while the light chain (framework [FR] 1, CDR1, FR3, and CDR3) of 8F12 interacts with the VP1 BC and GH loops, VP2 EF loop, and VP3 C-terminus of EV-D68 (Fig. 4e, Supplementary Table 2). Note that VP1 BC loop resides at the north rim of the canyon, while VP1 GH loop and VP2 EF loop are situated at the south rim. The 8F12/EV-D68 interaction interface covers ~ 1130 Å2 of the surface area on each protomer, and the heavy and light chains of 8F12 contribute 30.4% and 69.6% of the interaction interface, respectively (Supplementary Table 4).
Cryo-EM revealed structural basis of 2H12-mediated neutralization of EV-D68 and 2H12-induced particle transformation
To reveal the structural basis of 2H12-mediated neutralization of EV-D68, we also performed cryo-EM study of the EV-D68 mature virion in complex with the 2H12 Fab. Surprisingly, the original cryo-EM micrographs of the EV-D68/2H12 Fab complex (abbreviated as EV-D68/2H12) showed a small proportion (~ 15%) of the 2H12 Fab-bound capsids were broken into pieces, leaking genomic RNAs into the external fluid surrounding the particles (Supplementary Fig. 6a). Note that the two immune complexes, EV-D68 complexed with 8F12 or 2H12, were prepared at the same time using the same batch of EV-D68 antigen. Thus, it is very likely that 2H12, but not 8F12, could to some extent destroy EV-D68 viral particles.
Interestingly, from one EV-D68/2H12 complex dataset we obtained three distinct conformational states, namely S1, S2, and S3, at resolutions of 3.09 Å, 3.60 Å, and 3.60 Å, respectively (Fig. 5a-c, Supplementary Fig. 6), which allowed us to build an atomic model for each of the three states and the models match the corresponding map very well (Fig. 5d-f, Supplementary Fig. 7). Among them, S1 occupies the highest population distribution (39.3%), while S2 and S3 occupy 20.6% and 29.9% of the population, respectively (Supplementary Fig. 6a). The overall conformation of the S1 state of EV-D68/2H12 is very similar to that of 8F12 Fab-bound EV-D68 virion (Fig. 4a and 5a), suggesting that 2H12 and 8F12 Fabs may target the same region of the EV-D68 capsid. The S2 and S3 states largely resemble S1 in their external appearance (Fig. 5b, c). However, detailed examination revealed differences in their capsid structural features (Fig. 5d-f). Specifically, our S2 and S3 maps exhibit open channels at the two-fold axis (Fig. 5e, f), a characteristic feature of expanded enteroviral particles; whereas it appears closed in S1 (Fig. 5d) and in the native EV-D68 virion structure (PDB: 6CSG) 18. Noteworthy, enterovirus genomes have been reported to be released through the two-fold axis channel 30, 31. Moreover, the capsids in both S2 and S3 are expanded relative to S1 and the native virion (the radii of the capsids in native virion, S1, S2, and S3 are 157 Å, 157 Å, 163 Å, and 162 Å, respectively) (Supplementary Fig. 8). Interestingly, compared with S1 and S2, S3 shows different internal genomic RNA organization and significantly weaker RNA genome density (Fig. 5g-i). Comparison of our maps with the available cryo-EM maps of the virion and A particle of the 18947 strain 18 indicated: (1) the viral particle in S1 adopts the native configuration and remains intact; (2) the viral particle in S2 is in the expanded 1 (E1) conformation; (3) the viral particle in S3 represents a previously undescribed uncoating intermediate state, which likely occurs in the transition from the 135S A-particle (with genome RNA) to the 80S emptied particle (without genomic RNA). Taken together, our structural analyses revealed that binding of 2H12 Fab can trigger EV-D68 virus uncoating.
Analyses of the interactions between EV-D68 virion and 2H12 in S1 state revealed that each 2H12 Fab binds to a single protomer (Fig. 5j); compared with 8F12 Fab, 2H12 Fab shows similar position and orientation on the virion surface (Fig. 4d, 5j). Specifically, 2H12 Fab contacts the south wall of the canyon, and its VL is tilted to the five-fold axis of EV-D68 virion, spatially covering the canyon and thus blocking the binding of the sialic acid receptor (Fig. 5j and 5 l). Furthermore, the 2H12 epitope includes residues from VP1 GH loop, VP2 EF loop, and VP3 C-terminus (Fig. 5k, Supplementary Table 3), among which five residues are also involved in interactions between EV-D68 virion and 8F12. To be specific, the EF loop of VP2 interacts with CDR1, CDR2, CDR3, and FR3 of 2H12-VH, as well as CDR3 of 2H12-VL ; the VP1 GH loop contacts with FR1 and CDR1 of 2H12-VL; the VP3 C-terminus interacts with CDR1 and FR3 of 2H12-VL (Fig. 5k). Putting together, EV-D68/2H12 interaction interface covers ~ 1153.7 Å2 of the surface area, and the 2H12 heavy chain and light chain contribute 44.1% and 55.9% of the interaction interface, respectively (Supplementary Table 4).
Structural comparison of EV-D68/2H12 and EV-D68/8F12 complexes
For the EV-D68/2H12 (state S1) and EV-D68/8F12 immune complexes, their interaction interfaces are in similar sizes, but the contributions of heavy chain and light chain to the contact area are very distinct, e.g., the interaction area of 8F12-VH and EV-D68 (343.4 Å2) is significantly smaller than that of 2H12-VH and EV-D68 (508.9 Å2) (Supplementary Table 4). Superposition of the structures of EV-D68/2H12 (S1) and EV-D68/8F12 showed that the two immune complexes have broadly similar overall structures, but there are several apparent local structural differences between them (Fig. 6a). In particular, despite 2H12-VL and 8F12-VL appear very similar, the structures of 2H12-VH and 8F12-VH are quite different (Fig. 6b-c) especially in the three CDR regions. CDR1–3 of 2H12-VH are closer to the viral surface than CDR of 8F12-VH (Fig. 6c), leading to a larger interaction area between 2H12-VH and the virus.
We also superimposed the models of EV-D68/2H12 (S1), EV-D68/8F12, and native EV-D68 virion (PDB: 6CSG) 18 to analyze the structural variations in capsid proteins. Overall, the capsid proteins in EV-D68/8F12 complex and in EV-D68/2H12 (S1) resemble closely those in native virion (Fig. 6d), however, several notable local structural variations were observed for EV-D68/2H12 (S1). Upon 2H12 binding, one can observe a large shift in residues 206 to 218 region within the VP1-GH loop of EV-D68 which lies in the floor and south wall of the canyon (Fig. 6d). This 2H12 binding-induced conformational change of the VP1 GH loop might further lead to greater changes in the overall structure of EV-D68 virion, e.g. the expansion of capsid structure and opening of the two-fold channel in states S2 and S3.
Humanized 2H12 and 8F12 antibodies are therapeutically efficacious
Having determined the efficacy and working mechanisms of 2H12 and 8F12 antibodies, we then asked whether these two murine MAbs could be humanized for future application in humans. Humanized MAbs were created by separately linking the variable domains of murine 2H12 and 8F12 to the constant domains of human IgG1 heavy chain and human kappa light chain (Fig. 7a). The engineered human-mouse chimeric antibodies were successfully produced in HEK 293F cells (Fig. 7b). Chimeric MAbs 2H12 (c2H12) and 8F12 (c8F12) reacted with anti-human IgG antibody but not anti-mouse IgG antibody in western blot assays (Fig. 7b), confirming their humanized nature. Both c2H12 and c8F12 had high affinities to the 18947 virion with KD values below 14 nM (Fig. 7c, 7d, 7f). The results from neutralization assays showed that both c2H12 and c8F12 potently neutralized the EV-D68 strain 18947 with IC50s being less than 180 ng/mL (Fig. 7e, 7f). Taken together, these data indicate that the humanized antibodies c2H12 and c8F12 retain high binding affinities and neutralizing activities.
The therapeutic potential of the humanized MAbs was also assessed in the EV-D68 infection mouse model. Groups of one-day-old ICR mice were inoculated with the 18947 strain and one day later given single injections of PBS, c2H12 (10 µg/g), or c8F12 (10 µg/g), respectively. Survival and clinical score were then monitored daily. As shown in Fig. 7g, h, all mice in the control (PBS) group died by 12 dpi; by contrast, 92.9% of the c2H12-treated mice and all of the c8F12-treated mice survived. These results indicate that both c2H12 and c8F12 retain excellent therapeutic efficacy.