Simon Hoffenberg

Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120.

A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan–dependent epitope from its 3.1-Å crystal structure with gp120, CD4 and Fab 17b. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield and access the gp120 protein surface. EM reveals that PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. Combined structural studies of PGT 135, PGT 128 and 2G12 show that this Asn332-dependent antigenic region is highly accessible and much more extensive than initially appreciated, which allows for multiple binding modes and varied angles of approach; thereby it represents a supersite of vulnerability for antibody neutralization.

Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9

PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody–trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region.

Identification of an HIV-1 Clade A Envelope That Exhibits Broad Antigenicity and Neutralization Sensitivity and Elicits Antibodies Targeting Three Distinct Epitopes

ABSTRACT Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector.

Identification of a clade A HIV envelope immunogen from Protocol G that elicits neutralizing antibodies to tier 2 viruses

Background Broadly neutralizing antibodies PG9 and PG16 have been isolated from the B cells of one clade A-infected individual from IAVI Protocol G. PG16 is relatively trimer-specific whereas PG9 binds trimer preferentially, but can bind monomeric gp120 from several viral isolates. Both antibodies are potent neutralizers that recognize greater than 70% of tier 2 pseudovirues in the TZM-bl assay. We sought to begin immunogen design efforts based on sequences from the Protocol G donor, however all viruses isolated from the donor were resistant to neutralization by PG9 and PG16. We used a bioinformatics approach to infer the most recent common ancestor (MRCA) sequence for the viral envelope (Env) to identify closely related viruses sensitive to PG9/16.

Inverse dose-response to gp140 YU2 foldon trimer formulated with aluminum phosphate and ISCOMATRIX® adjuvants

Background Conventional vaccine approaches based on delivery of HIV-1 envelope (Env) proteins or peptides derived from Env sequences have failed to generate broadly neutralizing antibodies (bNAbs) to the virus. Even with large doses (200 ug) of adjuvanted gp120 proteins administered multiple times to human volunteers, the subsequent antibody response boosts only moderately with each succeeding vaccination, and titers drop precipitously thereafter. We hypothesized that the usual practice of administering a moderate to high antigen doses may be counter productive to the goal of eliciting durable, high-affinity antibody responses. Methods

Viral vector delivery of Env trimer immunogens

Methods We are using vesicular stomatitis virus (VSV) as a vector platform for delivery of Env immunogens as transmembrane glycoproteins. We have investigated a variety of vector designs and Env modifications to identify combinations that balance the practical requirement for vector genetic stability with factors influencing antibody responses including immunogen abundance, efficient post-translational processing, and presentation of antigenic determinants representative of a functional trimeric spike.

Optimizing expression of functional HIV envelopes in rVSV-ΔG vaccine vectors.

Methods Using a combination of nucleotide sequence optimization and protein domain swapping, we have generated a panel of novel gene inserts for VSV vectors that encode chimeric HIV-1 and VSV glycoprotein immunogens (EnvG). A stable VERO cell line engineered to express human CD4 and CCR5 was used to rescue rVSV vectors in which the G gene was replaced with coding sequence for several different EnvG proteins.

P12-14. Design of hydrophilic, helical peptides that mimic the 4E10 epitope of HIV-1 gp41

Background The broadly neutralizing monoclonal antibody 4E10 binds a linear, alpha-helical epitope in the membraneproximal external region (MPER) of HIV-1 gp41. The epitope, which lies immediately N-terminal of the single transmembrane segment of gp41, is partially inserted into the lipid bilayer. Efforts to design peptide immunogens containing the 4E10 epitope have been hampered by the hydrophobic nature of the sequence, and so far no neutralizing immune response could be elicited either with attached solubility tags, or with the peptide embedded in a lipophilic environment.