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Principal Investigator: ELLIS L REINHERZ
Organization: DANA-FARBER CANCER INST
Fiscal Year: 2024
Award: $940,588
Funding agency: National Institute of Allergy and Infectious Diseases
SUMMARY – PROJECT 2
There are only a handful of sites on the HIV-1 envelope protein (Env) that represent regions of
vulnerability for elicitation of broadly neutralizing antibodies (bnAbs). Four of these sites are associated with
the gp120 subunit while one of the sites resides in the membrane-proximal external region (MPER) of gp41.
We previously showed that the MPER is a conserved lipid-immersed, structural element consisting of two
helices separated by a hinge with tandem joints that facilitate viral hemifusion and fusion. MPER-specific
bnAbs manifest extraordinary HIV-1 strain and clade neutralization breadth, exerting anti-viral effect by
disturbing gp41 conformational change required for viral entry into host cells. The MPER is poorly
immunogenic, however, either as a component of the intact gp160 trimeric spike or in the context of an isolated
gp41 subunit immunogen. Our recent cryo-EM and molecular dynamics (MD) studies of membrane-embedded
Env trimers reveal that each MPER protomer radiates toward the 3-fold axis, generating an occluded
membrane-associated tripod structure beneath the upright Env spike. Notwithstanding, spontaneous spike
tilting exposes a 20-Å slot for an Fab arm to insert in a restricted vectorial space. By creating mRNA-based
chimeric vaccines, replacing the Env ectodomain with a trimeric host self-protein protomer linked to a viral
MPER-TM segment as detailed in Project 1, misguiding and sequence-variable viral structural elements are
replaced by a non-immunogenic structure. MPER immunogenicity should then no longer be subdominant. Aim
1 will express these MPER immunogens, incorporate them into nanodiscs and compare their structure and
dynamics, by themselves and in complex with Fabs of interest, with those of gp160 in nanodiscs. Aim 2 will
clone and characterize human-derived MPER-specific IgG1 and IgG3 bnAbs from patient biobanked samples.
Serial longitudinal serological and single memory B-cell analyses will allow for recombinant monoclonal
antibody (rmAb) cloning, thereby defining MPER epitope locales, somatic hypermutation (SHM), neutralization
breadth/potency, affinity, CDRH3 length and lipid binding/polyreactivity, with explicit comparison of IgG1 and
IgG3 isotypes and their aggregate molecular evolution. Aim 3 shall perform X-ray crystallographic studies
using Fabs generated in Aim 2 in complex with MPER epitopes, comparing the features of these bnAbs with
others defined previously in patients as well as those elicited through vaccination in Project 1. Structural and
MD studies of germline and mature IgG1 and IgG3 bnAbs shall reveal whether CH1-CL interdomain motions,
elbow angle flexibility, and/or antibody-to-framework distance are similar or different and impacted by hinge
length. We anticipate that the additional dexterity afforded by the long IgG3 hinge will allow for facile entry of
their Fab arms to approach the MPER, augmenting the ability of unmutated common ancestors (UCAs) and
their progeny with minimal mutation to bind to MPER-containing immunogens.
Terms: <3-D><3-Dimensional><3D><Address><Affinity><Antibodies><Antibody Response><Antigenic Determinants><Antigens><B blood cells><B cell><B cells><B-Cells><B-Lymphocytes><B-cell><Behavior><Binding><Binding Determinants><Cell Body><Cells><Characteristics><Chimera><Chimera organism><Clinical Treatment Moab><Cloning><Complex><Conserved Sequence><Cryo-electron Microscopy><Cryoelectron Microscopy><Data><Development><Elbow><Electron Cryomicroscopy><Elements><Envelope Protein><Epitopes><Family member><Frequencies><Genetic Alteration><Genetic Change><Genetic defect><Germ Lines><Glycans><Grain><Grant><HIV Envelope Glycoprotein gp120><HIV Envelope Protein gp120><HIV env Protein gp120><HIV-1><HIV-I><HIV1><HTLV-III gp120><Human><Human Immunodeficiency Virus Type 1><Human immunodeficiency virus 1><Ig Somatic Hypermutation><IgG1><IgG3><Image><Immunoglobulin Somatic Hypermutation><In Vitro><Joints><Kinetics><Length><Link><Lipid Binding><Lipids><Locales><Membrane><Memory B Cell><Memory B-Lymphocyte><Messenger RNA><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Evolution><Molecular Interaction><Molecular Stereochemistry><Monoclonal Antibodies><Motion><Mutation><Non-Polyadenylated RNA><Patients><Peptides><Peripheral><Polysaccharides><Production><Proteins><Protomer><RNA><RNA Gene Products><Recombinants><Ribonucleic Acid><Sampling><Serology><Single Crystal Diffraction><Site><Sorting><Specific qualifier value><Specified><Structure><Vaccination><Vaccine Design><Vaccines><Viral><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><arm><biobank><biorepository><chimeras><conformation><conformational><conformational state><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><developmental><dexterity><env Antigens><env Gene Products><env Polyproteins><env Protein><flexibility><flexible><genome mutation><gp120><gp120 ENV Glycoprotein><gp120(HIV)><gp160><imaging><immunogen><immunogenic><immunogenicity><interest><lipid bound><mAbs><mRNA><membrane structure><molecular dynamics><monoclonal Abs><nanodisk><neutralizing antibody><neutralizing vaccine><particle><reconstitute><reconstitution><screening><screenings><somatic hypermutation><three dimensional><vaccine strategy>