title: CryoEM visualization of an adenovirus capsid-incorporated HIV antigen.
abstract: Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and cellular immune responses against HIV remain a major challenge. In a novel approach to generate 2F5- and 4E10-like antibodies, we engineered an Ad vector with the HIV membrane proximal ectodomain region (MPER) epitope displayed on the hypervariable region 2 (HVR2) of the viral hexon capsid, instead of expressed as a transgene. The structure and flexibility of MPER epitopes, and the structural context of these epitopes within viral vectors, play important roles in the induced host immune responses. In this regard, understanding the critical factors for epitope presentation would facilitate optimization strategies for developing viral vaccine vectors. Therefore we undertook a cryoEM structural study of this Ad vector, which was previously shown to elicit MPER-specific humoral immune responses. A subnanometer resolution cryoEM structure was analyzed with guided molecular dynamics simulations. Due to the arrangement of hexons within the Ad capsid, there are twelve unique environments for the inserted peptide that lead to a variety of conformations for MPER, including individual α-helices, interacting α-helices, and partially extended forms. This finding is consistent with the known conformational flexibility of MPER. The presence of an extended form, or an induced extended form, is supported by interaction of this vector with the human HIV monoclonal antibody 2F5, which recognizes 14 extended amino acids within MPER. These results demonstrate that the Ad capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. In summary, this cryoEM structural study provided a means to visualize an epitope presented on an engineered viral vector and suggested modifications for the next generation of Ad vectors with capsid-incorporated HIV epitopes.
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Justin W
authors.initials: JW
authors.lastname: Flatt
authors.forename: Tara L
authors.initials: TL
authors.lastname: Fox
authors.forename: Natalia
authors.initials: N
authors.lastname: Makarova
authors.forename: Jerry L
authors.initials: JL
authors.lastname: Blackwell
authors.forename: Igor P
authors.initials: IP
authors.lastname: Dmitriev
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kashentseva
authors.forename: David T
authors.initials: DT
authors.lastname: Curiel
authors.forename: Phoebe L
authors.initials: PL
authors.lastname: Stewart
mesh_terms: D000256:Adenoviridae / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry* / Q000378:metabolism; D020285:Cryoelectron Microscopy; D000939:Epitopes / Q000737:chemistry; D005822:Genetic Vectors / Q000737:chemistry; D015488:HIV Antigens / Q000737:chemistry* / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary
chemical_list: D036022:Capsid Proteins; D000939:Epitopes; D015488:HIV Antigens; D015700:HIV Envelope Protein gp41; D010455:Peptides; C018315:hexon capsid protein, Adenovirus; C043322:penton protein, adenovirus