Daniel W. Kulp

Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Promiscuous Glycan Site Recognition by Antibodies to the High-Mannose Patch of gp120 Broadens Neutralization of HIV

HIV broadly neutralizing monoclonal antibodies targeting the high-mannose patch of Env can use alternate glycan sites for neutralization. Neutralizing Antibodies with a Sweet Tooth Sugar can be quite tempting—as anyone who’s seen a kid rip into birthday cake can attest. Yet, antibodies can also have a sweet tooth, targeting glycan modifications on the surface of proteins. Indeed, some antibodies that neutralize multiple HIV strains—broadly neutralizing monoclonal antibodies (bnmAbs)—target a high-mannose patch on the HIV protein Env. Although this high-mannose patch is centered around the glycan at position 332 (N332), it has remained unclear if the N332 glycan is absolutely required for neutralization and, if not, why not. Sok et al. found that these mannose patch–targeting antibodies can bind alternate glycans in the absence of N332, which helps to explain their ability to neutralize many strains of HIV. Specifically, some bnmAbs can bind to the N334 site when that replaces the N332 site and some can form more interactions with other glycans, particularly complex-type glycans on variable loops, if the N332 sugar is absent. These data also suggest that mannose patch–targeting bnmAbs can work in combination to neutralize a wider range of different strains than single bnmAbs. The promiscuity of glycan binding by these sugar-loving antibodies is important to consider for both vaccine and therapeutic antibody development. Broadly neutralizing monoclonal antibodies (bnmAbs) that target the high-mannose patch centered around the glycan at position 332 on HIV Env are promising vaccine leads and therapeutic candidates because they effectively protect against mucosal SHIV challenge and strongly suppress SHIV viremia in established infection in macaque models. However, these antibodies demonstrate varying degrees of dependency on the N332 glycan site, and the origins of their neutralization breadth are not always obvious. By measuring neutralization on an extended range of glycan site viral variants, we found that some bnmAbs can use alternate N-linked glycans in the absence of the N332 glycan site and therefore neutralize a substantial number of viruses lacking the site. Furthermore, many of the antibodies can neutralize viruses in which the N332 glycan site is shifted to the 334 position. Finally, we found that a combination of three antibody families that target the high-mannose patch can lead to 99% neutralization coverage of a large panel of viruses containing the N332/N334 glycan site and up to 66% coverage for viruses that lack the N332/N334 glycan site. The results indicate that a diverse response against the high-mannose patch may provide near-equivalent coverage as a combination of bnmAbs targeting multiple epitopes. Additionally, the ability of some bnmAbs to use other N-linked glycan sites can help counter neutralization escape mediated by shifting of glycosylation sites. Overall, this work highlights the importance of promiscuous glycan binding properties in bnmAbs to the high-mannose patch for optimal antiviral activity in either protective or therapeutic modalities.

Salt bridges: Geometrically specific, designable interactions

Salt bridges occur frequently in proteins, providing conformational specificity and contributing to molecular recognition and catalysis. We present a comprehensive analysis of these interactions in protein structures by surveying a large database of protein structures. Salt bridges between Asp or Glu and His, Arg, or Lys display extremely well‐defined geometric preferences. Several previously observed preferences are confirmed, and others that were previously unrecognized are discovered. Salt bridges are explored for their preferences for different separations in sequence and in space, geometric preferences within proteins and at protein–protein interfaces, co‐operativity in networked salt bridges, inclusion within metal‐binding sites, preference for acidic electrons, apparent conformational side chain entropy reduction on formation, and degree of burial. Salt bridges occur far more frequently between residues at close than distant sequence separations, but, at close distances, there remain strong preferences for salt bridges at specific separations. Specific types of complex salt bridges, involving three or more members, are also discovered. As we observe a strong relationship between the propensity to form a salt bridge and the placement of salt‐bridging residues in protein sequences, we discuss the role that salt bridges might play in kinetically influencing protein folding and thermodynamically stabilizing the native conformation. We also develop a quantitative method to select appropriate crystal structure resolution and B‐factor cutoffs. Detailed knowledge of these geometric and sequence dependences should aid de novo design and prediction algorithms. Proteins 2011.

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

Baby steps toward bNAbs Some HIV-infected individuals develop heavily mutated, broadly neutralizing antibodies (bNAbs) that target HIV. Scientists aim to design vaccines that would elicit such antibodies. Jardine et al. report an important step toward this goal: They engineered an immunogen that could engage B cells from HIV-uninfected individuals that express the germline versions of the immunoglobulin genes harbored by a particular class of bNAbs. The frequencies of these B cells, their affinities for the immunogen, and structural analysis suggest that the immunogen is a promising candidate. Further shaping of the B cell response with subsequent immunogens may eventually elicit bNAbs in people. Science, this issue p. 1458 People that have not been infected with HIV can harbor HIV-1 broadly neutralizing antibody B cell precursors. Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Summary Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Summary Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.

Design of a switchable eliminase

The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

De novo proteins provide a unique opportunity for investigating the structure-function relationships of metalloproteins in a minimal, well-defined, and controlled scaffold. Herein, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the due ferri family. Originally created to catalyze O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyze the selective N-hydroxylation of arylamines by remodeling the substrate access cavity and introducing a critical third His ligand to the metal binding cavity. Additional second-and third-shell modifications were required to stabilize the His ligand in the core of the protein. These changes resulted in at least a 106 –fold increase in the relative rates of the two reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

Consensus motif for integrin transmembrane helix association

Interactions between transmembrane (TM) helices play an important role in the regulation of diverse biological functions. For example, the TM helices of integrins are believed to interact heteromerically in the resting state; disruption of this interaction results in integrin activation and cellular adhesion. However, it has been difficult to demonstrate the specificity and affinity of the interaction between integrin TM helices and to relate them to the activation process. To examine integrin TM helix associations, we developed a bacterial reporter system and used it to define the sequence motif required for helix–helix interactions in the β1 and β3 integrin subfamilies. The helices interact in a novel three-dimensional motif, the “reciprocating large-small motif” that is also observed in the crystal structures of unrelated proteins. Modest but specific stabilization of helix associations is realized via packing of complementary small and large groups on neighboring helices. Mutations destabilizing this motif activate native, full-length integrins. Thus, this highly conserved dissociable motif plays a vital and widespread role as an on-off switch that can integrate with other control elements during integrin activation.