Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies

Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti–CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions

Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 envelope glycoprotein trimers on neutralizing antibody induction

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb speciUcity using rational heterologous boosts

Enhancing Humoral Responses Against HIV Envelope Trimers via Nanoparticle Delivery with Stabilized Synthetic Liposomes

An HIV vaccine capable of eliciting durable neutralizing antibody responses continues to be an important unmet need. Multivalent nanoparticles displaying a high density of envelope trimers may be promising immunogen forms to elicit strong and durable humoral responses to HIV, but critical particle design criteria remain to be fully defined. To this end, we developed strategies to covalently anchor a stabilized gp140 trimer, BG505 MD39, on the surfaces of synthetic liposomes to study the effects of trimer density and vesicle stability on vaccine-elicited humoral responses in mice. CryoEM imaging revealed homogeneously distributed and oriented MD39 on the surface of liposomes irrespective of particle size, lipid composition, and conjugation strategy. Immunization with covalent MD39-coupled liposomes led to increased germinal center and antigen-specific T follicular helper cell responses and significantly higher avidity serum MD39-specific IgG responses compared to immunization with soluble MD39 trimers. A priming immunization with liposomal-MD39 was important for elicitation of high avidity antibody responses, regardless of whether booster immunizations were administered with either soluble or particulate trimers. The stability of trimer anchoring to liposomes was critical for these effects, as germinal center and output antibody responses were further increased by liposome compositions incorporating sphingomyelin that exhibited high in vitro stability in the presence of serum. Together these data highlight key liposome design features for optimizing humoral immunity to lipid nanoparticle immunogens.National Institute of Allergy and Infectious Diseases (U.S.) (Award UM1AI100663)National Institutes of Health (U.S.) (Award P01-AI104715)National Institutes of Health (U.S.) (Award P01-AI048240)National Cancer Institute (U.S.) (Grant P30-CA14051

Exploring Epitope-Focused Vaccine Development: Design of Epitope Scaffolds and Nanoparticle Presentation Platforms, and Computational Prediction of Conformational Epitopes

Thesis (Ph.D.)--University of Washington, 2013Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) evolved a number of defense strategies to evade protective mechanisms of the immune system. Classical vaccine approaches have failed to elicit a protective response for these targets. Epitope Scaffolding is a theoretically attractive immunogen design strategy, which isolates known protective epitopes from their environments while stabilizing relevant conformations, as defined by a neutralizing antibody. In this work, we extend previous epitope scaffolding methods to scaffold a known protective HCV epitope and introduce preliminary immunization results. Additionally, in light of several mixed, protein and glycan, epitopes described for HIV, we apply the scaffolding strategy to an anti-HIV broadly neutralizing antibody - PG9. Next, using a derivative of an HIV envelope glycoprotein, we describe the development of a novel multimerization platform and illustrate potential applications. Finally, a computational protocol was developed to identify antibody accessible epitopes on flexible, glycosylated proteins

Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies

The envelope spike of HIV-1 employs a 'glycan shield' to protect itself from antibody-mediated neutralization. Paradoxically, however, potent broadly neutralizing antibodies (bnAbs) that target this shield have been isolated. The unusually high glycan density on the gp120 subunit limits processing during biosynthesis, leaving a region of under-processed oligomannose-type structures, which is a primary target of these bnAbs. Here we investigate the contribution of individual glycosylation sites in the formation of this so-called intrinsic mannose patch. Deletion of individual sites has a limited effect on the overall size of the intrinsic mannose patch but leads to changes in the processing of neighbouring glycans. These structural changes are largely tolerated by a panel of glycan-dependent bnAbs targeting these regions, indicating a degree of plasticity in their recognition. These results support the intrinsic mannose patch as a stable target for vaccine design.</p

An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

<div><p>Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.</p></div

Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for Self-Assembly into Nanoparticles

Self-replicating RNAs derived from alphaviruses have high potential for vaccine applications. Utilizing lipid nanoparticle-formulated RNA replicons as a vaccine platform to deliver self-assembling protein immunogens, Melo et al. demonstrate robust anti-HIV Env antibody production and significantly improved antigen-specific B cell activation in vaccinated mice when compared with protein immunization.NIAID (Awards UM1AI100663, AI104715, EB025854 and AI048240)National Cancer Institute (Grant P30-CA14051

Schematic and expression of the cleavable Env_IL-21.

<p>Linear (A) and cartoon (B) representation of the Env_IL-21 and clEnv_IL-21 proteins. Cleaved proteins were created by introducing a stop codon in front of the isoleucine zipper (IZ) trimerization domain in the Env_IL-21. (C) SDS-PAGE analysis of chimeric uncleaved and cleaved Env_IL-21 constructs.</p

Immunoglobulin production by B cells stimulated with Env_wt, Env_IL-4 and Env_IL-21 molecules and controls.

<p>IgG, IgA and IgM levels secreted by the B cells from human PBMCs cultured with (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10. Data are representative of three independent experiments showing similar results. Immunoglobulin secretion by B cells from different donors cultured with Env_wt and Env_IL-21 molecules in (C) CD40L/IL-10 and (D) CD40L/IL-4/IL-10 milieu. Culture supernatant from mock transfected 293T cells was used as a negative control and the values were deducted from the test values. Data represent the fold change values compared to Env_wt from at least 12 donors and each donor sample was tested in duplicate. (E) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env_wt, Env_IL-21, Env_ChimIL-21/4 supernatants and controls in CD40L/IL-10 milieu. Data are representative of three experiments using B cells from three different donors. (F) The expression of CD38 cell surface marker treated with different Env_IL-21 constructs and controls in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six different donors.</p