RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al.

Ligand accessibility to the HIV-1 Env co-receptor binding site can occur prior to CD4 engagement and is independent of viral tier category.

HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state.

Identification and characterization of a naturally occurring, efficiently cleaved, membrane-bound, clade A HIV-1 Env, suitable for immunogen design, with properties comparable to membrane-bound BG505.

Efficient cleavage of HIV-1 Env gp160 into its constituent subunits correlates with selective binding to neutralizing antibodies and are the closest mimetic of native, functional Envs. This was first demonstrated with the clade B Env, JRFL. The correlation between efficient cleavage and selective binding to neutralizing antibodies is the guiding principle for immunogen design for HIV vaccine.

Evolution of B cell analysis and Env trimer redesign.

HIV-1 and its surface envelope glycoproteins (Env), gp120 and gp41, have evolved immune evasion strategies that render the elicitation of effective antibody responses to the functional Env entry unit extremely difficult. HIV-1 establishes chronic infection and stimulates vigorous immune responses in the human host; forcing selection of viral variants that escape cellular and antibody (Ab)-mediated immune pressure, yet possess contemporary fitness. Successful survival of fit variants through the gauntlet of the human immune system make this virus and these glycoproteins a formidable challenge to target by vaccination, requiring a systematic approach to Env mimetic immunogen design and evaluation of elicited responses.

HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation.

The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs "knock out" trimers.

Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores.

Kinetic analysis of antibodies is one of the important study for characterization of antibodies and screening of ligands. In our recent study, we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as K, K, and K in a 96-well format.

Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays.

Analysis of the functional activity in polyclonal serum following immunization of a complex protein or glycoprotein immunogen is a very important but tedious process. Fine mapping of epitope specific antibodies is difficult when they are elicited at relatively low levels. In our recent study focused at developing an HIV-1 vaccine, we immunized rabbits with hyperglycosylated stable core immunogens, which were designed using high resolution structural information to elicit antibodies against the primary receptor-binding, CD4-binding site on HIV-1 gp120.

Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies.

Unlabelled: The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4(+) CCR5(+) host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies.

CD4-mimetic small molecules sensitize human immunodeficiency virus to vaccine-elicited antibodies.

Unlabelled: Approaches to prevent human immunodeficiency virus (HIV-1) transmission are urgently needed. Difficulties in eliciting antibodies that bind conserved epitopes exposed on the unliganded conformation of the HIV-1 envelope glycoprotein (Env) trimer represent barriers to vaccine development. During HIV-1 entry, binding of the gp120 Env to the initial receptor, CD4, triggers conformational changes in Env that result in the formation and exposure of the highly conserved gp120 site for interaction with the coreceptors, CCR5 and CXCR4.

Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity.

On the prereceptor-engaged HIV-1 envelope glycoprotein (Env) spike, epitope access by the membrane-proximal external region (MPER)-directed broadly neutralizing antibodies 2F5 and 4E10 remains unresolved. Data on binding to cell surface Env and entry data using primary isolates suggest inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and slow kinetics of shedding induced by 2F5 and 4E10 indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed in their antibody-bound state (or at least sampled) prior to receptor/coreceptor engagement or if receptor interactions both expose and form the MPER epitopes, presumably in the putative prefusion transitional intermediate.