Mechanism of dengue virus broad cross-neutralization by a monoclonal antibody

Serafin et al. Subsequently, Wahala et al. In contrast, genotype I and genotype II were neutralized at very low antibody concentrations - 0.

The DENV-3 clones recapitulate genotypic variation at those residues. Purified 8A1 was tested for ability to neutralize parent and isogenic clones Figure 2C and Table 1.

For mAb 8A1, as with mAb 1H9, a distinct pattern of neutralization was observed; genotype IV requires a high concentration of antibody for neutralization, genotype III requires intermediate concentration of antibody and finally genotype I and II are neutralized by low concentrations of antibody Figure 2C.

Moreover, Wahala et al. These two sites are located in adjacent loops of the lateral ridge, with position at the apex of the N-terminal linker and position in the FG loop. We hypothesized that variations at one or both sites , are responsible for the sensitivity difference of mAb 8A1 and our reverse genetics system provided us a chance to test this hypothesis and further study the mechanism of this differential neutralization at molecular level. We found that both mutations were required to increase sensitivity to neutralization Figure 3.

Single mutations led to partial increases in neutralization sensitivity but only the double mutant was as sensitive to neutralization as the DENV-3 genotype I and II sensitive strains. This suggests that the two amino acids at and both confer sensitivity to mAb 8A1, with double mutations at these two positions conferring the greatest effect.

To further explore the mechanism of neutralization sensitivity by the individual and combined effect of and site mutations, we studied the binding affinity difference of mutant viruses to mAb 8A1. After optimization, equal amount of virus was captured and 8A1 concentration at 0. Introducing NK alone increased the OD value only slightly, indicating that NK mutation failed to increase binding affinity. This is consistent with previous observation that mutation at this site did not cause change of 8A1 binding of EDIII recombinant protein Wahala, Donaldson et al.

We next employed surface plasmon resonance SPR to better understand how specific residues affect binding affinity and kinetics of 8A1 to the EDIII protein from each genotype.

The association rate clearly cannot explain the half-life difference or neutralization sensitivity variation. The dissociation rate data are consistent with the neutralization data and half-life data as slower dissociation results in the longer half-life of binding, and subsequently a higher 8A1 occupancy of the virus at any given moment, which ultimately leads to neutralization Pierson, Xu et al.

Expanding our kinetic data characterizing 8A1 binding to EDIII, we sought to determine the role of specific residues involved in this interaction. Our neutralization experiments suggested that sites and both contribute to neutralization sensitivity.

Mutation of position from a threonine to a leucine resulted in a half-life of In contrast, mutation of position from an asparagine to a lysine resulted in a decrease of only association rate and a half-life comparable to parental genotype III EDIII These results suggest that the dissociation rate is determined by position alone as T leads to short half-life 12—16 seconds while L leads to long half-life 50—68 seconds.

How natural strain variation within each DENV serotype influences virus-antibody interactions has important implications for rational vaccine design and virus evolution Wong, Abd-Jamil et al. We and others have recently reported genotype dependent variable neutralization within serotypes using both mAbs and human DENV-3 sera Wahala, Donaldson et al. Despite the accumulated evidence, the detailed mechanism of genotype dependent variable neutralization remains unknown.

To better understand this mechanism, we employed a DENV-3 reverse genetic clone system to capture the amino acid variation in E glycoprotein of all the four genotypes into the otherwise isogenic background of genotype III. The resulting isogenic viruses recapitulated the previously reported sensitivity differences of the DENV-3 genotypes to these mAbs Wahala, Donaldson et al. Using these isogenic viruses, we mapped the mutations critical to mAb 8A1 neutralization to two sites - and - on the lateral ridge region of EDIII.

We found that both mutations are needed to alter neutralization by mAb 8A1. Study of antibody-antigen affinity of whole virus suggested the distinct neutralization sensitivity is mainly attributed to binding affinity differences between different residues at these two positions. Using SPR and recombinant EDIII protein, we further found that binding differences are determined mainly by the dissociation rate and mutation at site alone affects the dissociation rate.

Our finding that only two residues are responsible for differential neutralization between DENV-3 genotypes I, II, III and IV sheds light on our understanding of how neutralization is modulated by viral genetic variations.

Consistent with previous research, our ELISA data suggests that binding affinity differences cause neutralization sensitivity variation Gromowski and Barrett ; Lisova, Hardy et al.

However, mutant TL restores fully binding affinity but it restores only partially the neutralization sensitivity. This suggests that site also contributes to 8A1 neutralization sensitivity via a mechanism other than binding affinity.

The evidence is that mutant NK increased neutralization sensitivity about 4 times, without increasing binding affinity Figure 4 or half-life of binding Table 1. We can find a similar pattern for mutation TL as introducing this mutation to genotype III increased sensitivity for about 10 times and introducing this mutation into mutant NK also increased sensitivity for about the same extent.

This pattern strongly argues that both sites contribute to 8A1 neutralization sensitivity through different and independent mechanisms. We speculate that NK could alter neutralization by affecting virus structural dynamics so that each 8A1 antibody binding may exert more constraints on virus post-attachment steps such as conformational change for fusion, as mAb 8A1 neutralizes virus by blocking post-attachment steps 2unpublished data.

It has been shown that flavivirus dynamic equilibrium breathing can regulate antibody neutralization Lok, Kostyuchenko et al. SPR has been proven a powerful tool in studying and viewing antigen-antibody interactions from a kinetic and structure perspective Bedouelle, Belkadi et al. The correlation between the virus binding affinity data Figure 4 and the half-life data Table 1 suggests that 8A1 binding affinity is determined by off-rate.

We further found that both residues and can affect association rate as mutation at either residue changed the association rate Table 1. However, only residue , not residue , affects dissociation rate, and also half-life Table 1. These data are consistent with previous mapping data of 8A1 showing that mutation of residue did not affect 8A1 binding Wahala, Donaldson et al.

The association rate of genotype II is aberrant and extremely low, consequently the resulted unexpectedly high KD value contradicts with both genotype II EC50 and 8A1 neutralization titer Table 1.

This anomaly could be either due to a instability of this particular construct or b non-specific interaction between the DIII and MBP fusion partner that occludes recognition of the 8A1 epitope. Understanding the mechanisms of DENV neutralization by antibodies is, therefore, crucial for the design of vaccines that simultaneously protect against all four viruses. Here, we report a comparative, high-resolution crystallographic analysis of an "A-strand" murine monoclonal antibody, Mab 4E11, in complex with its target domain of the envelope protein from the four DENVs.

Mab 4E11 is capable of neutralizing all four serotypes, and our study reveals the determinants of this cross-reactivity. The structures also highlight the mechanism by which A-strand Mabs disrupt the architecture of the mature virion, inducing premature fusion loop exposure and concomitant particle inactivation.

References Articles referenced by this article 51 Electrostatics of nanosystems: application to microtubules and the ribosome. Diversity and junction residues as hotspots of binding energy in an antibody neutralizing the dengue virus. The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity.

Direct phase determination by entropy maximization and likelihood ranking: status report and perspectives. Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody. The CCP4 suite: programs for protein crystallography.

Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells Crill J. Humoral immune responses of dengue fever patients using epitope-specific serotype-2 virus-like particle antigens.

Cross-reacting antibodies enhance dengue virus infection in humans. A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus. Show 10 more references 10 of Smart citations by scite. The number of the statements may be higher than the number of citations provided by EuropePMC if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.

Explore citation contexts and check if this article has been supported or disputed. Antibody affinity versus dengue morphology influences neutralization. Potential neutralizing antibodies discovered for novel corona virus using machine learning.

Data Data that cites the article This data has been provided by curated databases and other sources that have cited the article. Protein Structures 4. View structure. Similar Articles To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III ED3 of dengue 2 virus.

Monoclonal antibodies that bind to common epitopes on the dengue virus type 2 nonstructural-1 and envelope glycoproteins display weak neutralizing activity and differentiated responses to virulent strains: implications for pathogenesis and vaccines.

One of the 'holy grails' in dengue research has been to identify DENV epitopes targeted by durable strongly neutralizing serotype-cross-reactive antibodies that are minimally enhancing across serotypes. In this issue of Nature Immunology , the findings of Dejnirattisai et al.

Dejnirattisai, W. Pierson, T. Tsai, W. Smith, S. Cherrier, M. EMBO J. PLoS Negl. Article Google Scholar. Beltramello, M. Cell Host Microbe 8 , — Science , — USA , — Teoh EP. Fibriansah, G. EMBO Mol. For each isolate, a negative value depicts favorable contribution to energetics when bound to mAb , whereas a positive value depicts favorable contribution to energetics when bound to mAb 4E This is in agreement with previous experimental observations that were made for the interactions of mAb 4E11 with DENV Additionally, an interaction made by with polymorphic residue Fig.

Mutations at Glu L57 and Trp L60 also lead to substantial increases in affinity for 33 , Glu H55 has a very large contribution in improving the interactions of with the four serotypes. Indeed, computational Ala mutation analysis Fig. It also has a large network score Fig. In addition to the contacts observed in the crystal structure, MD simulations suggest that Glu H55 in forms a stable interaction with the strictly conserved charged Lys in the DENV serotypes.

The charge complementarity is further maintained in this region as the adjacent conserved acidic residue, Glu, forms a stable interaction with Lys L31 in the CDR-L1 paratope. In addition, Glu H55 recognizes and stabilizes residue , which is a polymorphic residue on the DENV epitope, with significantly higher binding energy. In contrast to the observations made in the crystal structure wherein Lys H31 is observed to form a H-bond with Lys , MD simulations for all four DENV serotypes bound to indicate that Asp H31 and Glu H55 stabilize this polymorphic site in concert.

The Ala55Glu mutation increases charge complementarity across the paratope-epitope in this region, while the extended side chain of Glu endows broader specificity with increased contributing surface area.

S5 and Table 3. The second major contributor towards the binding efficacy of identified from our work is the Asn57Glu CDR-L2 mutation, which specifically elicits favorable response due to charge complementarity for DENV2 and 4. These two paratope residues, conjointly with Asp H32 and Lys L31 , engage in stable H-bond interactions with the A-strand , , B-strand and G strand residues Table S2 forming strong interactions at the periphery of the interface.

This bond is not observed in other DENV serotypes due to conformational variability. In general, is endowed with cross-neutralizing and enhancing activity for DENV serotypes, however significant differences in the binding efficacies were observed within DENV4 strains see Table 2 of ref. From our MD simulations, subtle sequence variations affect the local conformation of the functional epitope, altering shape complementarity and hence antibody binding.

However, even though the binding constants are significantly different, the adjacent regions around the mutations largely preserve charge complementarity and the paratope-epitope interface. In conclusion, a combination of physico-chemical and structural changes brought about by engineering six mutations in 4E11 result in the generation of mAb , which potently inhibits all four DENV serotypes. Combining MD simulations and residue-network analyses can therefore be a powerful complement to crystallographic and binding data and illuminate detailed interactions that underpin the differential binding of antibodies to homologous viral antigens.

To identify the interactions and hot spots of binding energies between or 4E11 with various dengue serotypes, we built their respective homology models. Table S2 shows the list of PDB structures used to model the virus isolates as well as the mAbs and the complex structures. Point mutations in various virus serotypes were incorporated in the models and missing regions, if any, were modeled. The scFv and 4E11 antibody structures were extracted from the crystal structures 4UDZ and 3UZQ, followed by modeling the complexes with the respective serotypes.

The modeled complexes were subjected to molecular dynamics simulations for further refinement. In all the complexes, hydrogen atoms were added using the Xleap module of Amber12 39 to prepare the systems for MD simulations. The Sander module was used for minimization of the complexes with steps of steepest decent algorithm, followed by steps of conjugate gradient algorithm. Initially, the antibody atoms, solvent water molecules and counter ions were relaxed, by keeping the virus residues restrained.

This was followed by unrestrained energy minimization to remove any steric clashes. Analyses of the simulation trajectories were performed using the ptraj module in Amber.

The effective binding energies were decomposed into contributions of individual residues using MMGBSA energy decomposition scheme. To investigate the impact of mutations in the antibodies on serotype recognition, selected hot-spot residues from the antibodies in each complex were computationally mutated to alanine.

The solvent accessible surface areas were calculated for every 50 th frame in the equilibrated trajectory. Using the average values obtained above, BSA values were then calculated as the sum of the solvent accessible surface area of individual molecules minus the solvent accessible surface area of their complex. Calculations were performed using Naccess V2. The proteins were then overexpressed by the addition of 0. For scFv WT, the titrations consisted of a single initial injection of 0.

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