Monoclonal antibodies directed against the envelope glycoproteins of membrane viruses such as human immunodeficiency virus type 1 (HIV-1), influenza, and ebolavirus are essential reagents for deciphering mechanisms of viral entry, and identifying epitopes for immunotherapy or vaccine development. Recent work in antibody engineering has demonstrated that specific, high-affinity antibodies can be isolated from simple phage display libraries in which diversity at the antibody complementarity determining regions (CDRs) is encoded by designed, synthetically-derived oligonucleotides ('synthetic antibodies'). Therefore, the synthetic antibody approach circumvents many limitations of traditional antibody isolation methods thereby expanding the scope and specificity with which antigens may be targeted. The overall goal of this proposal is to develop enabling synthetic antibody technologies for applications in the study of viral membrane fusion.
In Aim 1, we propose to develop synthetic antibody libraries focused toward viral antigens based on the promiscuous germline segment VH1-69. Many viral antibodies borne from this progenitor exhibit similar modes of interaction with their viral antigens, suggesting that VH1-69 could serve as a scaffold for development of virus- specific synthetic antibody libraries. We will produce VH1-69-based synthetic antibody libraries and screen them against viral targets to evaluate this hypothesis.
In Aim 2, we propose to identify conformation-specific antibodies that target the membrane-proximal external region (MPER) of HIV-1 gp41. The MPER is the target of several broadly neutralizing antibodies (NAbs) and therefore the subject of intense investigation for vaccine development. We will identify synthetic antibodies against structurally-constrained MPER peptides, and then characterize neutralization potency of these antibodies to gain insight into the role of MPER epitope conformation on inhibition of membrane fusion.
In Aim 3, we propose to isolate synthetic antibodies against the putative fusion intermediates of ebolavirus GP1 and GP2. In comparison to HIV-1 and influenza, relatively few antibodies against GP1 and GP2 have been isolated which has greatly impeded mechanistic understanding of membrane fusion for this virus. Our approach will overcome limitations of other antibody isolation methods that have failed to produce GP1- or GP2-specific antibodies, and yield novel reagents for dissection of fusion intermediates and potential immunotherapeutics.
Monoclonal antibodies have been an important source of therapeutic, diagnostic, and research reagents. Recent technological advances in antibody isolation methods have greatly expanded capabilities for isolation of antibodies. We are using this new technology to identify novel antibodies against viral targets. This work will provide new tools to understand viral infection and how this process can be inhibited by therapeutic agents or vaccines.
|Malashkevich, Vladimir N; Higgins, Chelsea D; Almo, Steven C et al. (2015) A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations. Biopolymers 104:178-85|
|Frei, Julia C; Kielian, Margaret; Lai, Jonathan R (2015) Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display. Virology 485:371-82|
|Dai, Zhou; Tao, Yisong; Liu, Nina et al. (2015) Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments. Biochemistry 54:1589-99|
|Liu, Nina; Tao, Yisong; Brenowitz, Michael D et al. (2015) Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop. J Infect Dis 212 Suppl 2:S146-53|
|Koellhoffer, Jayne F; Dai, Zhou; Malashkevich, Vladimir N et al. (2014) Structural characterization of the glycoprotein GP2 core domain from the CAS virus, a novel arenavirus-like species. J Mol Biol 426:1452-68|
|Koellhoffer, Jayne F; Higgins, Chelsea D; Lai, Jonathan R (2014) Protein engineering strategies for the development of viral vaccines and immunotherapeutics. FEBS Lett 588:298-307|
|Chen, Gang; Koellhoffer, Jayne F; Zak, Samantha E et al. (2014) Synthetic antibodies with a human framework that protect mice from lethal Sudan ebolavirus challenge. ACS Chem Biol 9:2263-73|
|Higgins, Chelsea D; Malashkevich, Vladimir N; Almo, Steven C et al. (2014) Influence of a heptad repeat stutter on the pH-dependent conformational behavior of the central coiled-coil from influenza hemagglutinin HA2. Proteins 82:2220-8|
|Stewart, Alex; Harrison, Joseph S; Regula, Lauren K et al. (2013) Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment. BMC Biochem 14:9|
|Harrison, Joseph S; Higgins, Chelsea D; O'Meara, Matthew J et al. (2013) Role of electrostatic repulsion in controlling pH-dependent conformational changes of viral fusion proteins. Structure 21:1085-96|
Showing the most recent 10 out of 18 publications