Prediction and characterization of antibody-protein interactions in Dengue virus
Research area: Biologia Strutturale
Group leaders: Luca Varani
Status: In progress
Individuals that survive a viral infection have antibodies (Abs) capable of detecting and neutralizing subsequent attacks by the same virus. These Abs bind antigens (Ags), often viral proteins, through specific atomic interactions between the Ab and the region of the Ag that it recognizes (called epitope). If we understand the structural rules governing Ab-Ag interactions to a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines (since most vaccines generate an antibody response) or optimize the antibodies themselves for passive immunization strategies. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition.
We recently proposed an experimentally validated computational approach for the rapid and systematic characterization of Ab-Ag complexes. Schematically, we isolate Abs from the blood of human donors infected with a given virus; produce and purify human monoclonal antibodies (in collaboration with A. Lanzavecchia, IRB); characterize their immunological and biophysical properties; determine their epitope through NMR epitope mapping (Figure 1) and use the NMR results to drive and validate computational docking simulations of their complex with the desired antigen. Finally, the structural analysis of the complexes is the starting point for the design of antibody mutations aimed at modifying their properties in a predictable manner, with the goal of validating our results and engineer new antibodies with improved properties.
Figure 1: The epitope of several antibodies against Dengue was mapped with solution NMR spectroscopy. Epitopes are shown in red over the surface representation of the so-called DIII, a part of the protein forming the surface of Dengue virus and the site recognized by most neutralizing antibodies against Dengue. Each line indicates a different antibody; the vertical columns correspond to the four main Dengue serotypes.
Dengue Virus: a case study
Dengue Virus (DENV) is a flavivirus responsible for 100 million annual human cases, including 500,000 hospitalizations and 20,000 deaths with an economic burden rivaling that of malaria. Although DENV has been mainly restricted to the tropical region, both its epidemic activity and its geographic expansion are increasing as travel, urbanization and climate changes create favorable conditions for vector and virus dissemination. An estimated 2.5 billion people are at risk of infection.
No cure or vaccine for DENV is currently available. The effort to find one has been hampered by the presence of four different dengue serotypes (DENV1–4) and by a poorly understood process almost unique in human medicine: antibody-dependent enhancement (ADE). Abs raised against a previous Dengue infection facilitate subsequent infection by a different serotype and lead to dengue hemorrhagic fever, an often lethal form of the disease. This feature complicates the task of finding a vaccine, since a vaccine that would not protect equally against all four serotypes would actually contribute to the emergence of dengue hemorrhagic fever.
At the structural level, the most interesting region to study is the so-called Domain III of Dengue E protein (DIII), which forms the surface of the virus. DIII is the main target of neutralizing antibodies against DENV and it is relatively small, making it ideal for NMR and computational studies.
Our aim is to compare several antibodies bound to DIII of the four Dengue serotypes, searching for correlations between immunological and structural trends and exploiting them to further our understanding of antibody-antigen interaction and ADE, as well as a basis for drug design and improved vaccine strategies. In a simplistic example, should we find that all Abs effective against DENV4 have a positive charge in a particular three-dimensional position, we would try to introduce such a charge in Abs lacking it, thus improving their characteristics. Conversely, should all effective Abs against a certain serotype recognize a particular epitope, then it is conceivable to prepare an antigen sharing the best epitopes of each serotype as a possible vaccination agent.
This work was done in collaboration with Antonio Lanzavecchia and Federica Sallusto, IRB.
Rational antibody engineering to better neutralize Dengue
We determined the binding site of monoclonal antibody DV32.6 on DIII of all Dengue serotypes with NMR epitope mapping and used this information to guide and validate computational docking simulations yielding the three-dimensional structure of the antibody/antigen complexes. Visual analysis of such structures allowed us to design antibodies with altered binding sites that could: A) avoid binding to all serotypes. B) Bind only to one or two serotypes; eliminating unwanted cross-reactivity is a useful endeavor for therapeutic antibodies or when designing bio-recognition elements. C) Neutralize Dengue virus serotype 1 40 times more effectively than the original antibody or neutralize all serotypes between 10 and 17 fold better than the original molecule.
Overall, 18 out of 22 point mutations that we designed had the effect predicted by the computational models. The work proves that experimentally validated computational docking is an accurate, rapid and powerful tool for the characterization and rational engineering of antibodies (Figure 2).
Figure 2: We designed two antibody mutants with the intent of improving its neutralization properties. H-S52D (gray) neutralizes DenV1 40 times more efficiently than the wild-type (black) and L-N27E (violet) is better than the wild-type in all serotypes, albeit to a lesser extent. Viral neutralization assays are shown; the amount of infected cells (y axis) decreases at increasing antibody concentration (x axis). In comparison to the wild-type, a lower concentration of mutants is required to neutralize the same amount of virus.
Structural Characterization of a potent neutralizer of Dengue Virus
DV87.1 is probably the most potent Dengue antibody described so far in the literature. It binds the surface protein of Dengue virus with nanomolar affinity and has an EC50 of 0.004µg/ml.
We used solution NMR spectroscopy to define its epitope on Dengue serotype 2 (figure 3) and then used this information to validate computational simulations aimed at obtaining the three-dimensional structure of the antibody in complex with its antigen. The antibody binds to an epitope not before described for other Dengue antibodies. Its position in the structure suggests that DV87.1 may neutralize Dengue Virus by blocking the conformational changes required for membrane fusion and infection of the host cells. Experiments are undergoing to obtain direct evidence on this hypothesis.
Figure 3: NMR spectrum of the Dengue antigen (DIII of DenV2) by itself (blue) and in complex with antibody DV87.1 (red). In this type of experiment, each peak corresponds to an individual antigen residue. Differences in the position of the peaks between free and bound spectrum allow us to determine which residues are affected by antibody binding and are, thus, at the interface (epitope). Some of the residues whose signal differs between free and bound form are indicated in yellow.