Andrew T. McGuire

Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors

Building Better Vaccines In the past few years, several highly potent, broadly neutralizing antibodies (bNAbs) specific for the gp120 envelope protein of HIV-1 have been discovered. The goal of this work is to use this information to inform the design of vaccines that are able to induce such antibodies (see the Perspective by Crowe). However, because of extensive somatic hypermutation, the epitope bound by these antibodies often does not bind to the germline sequence. Jardine et al. (p. 711, published online 28 March; see the cover) used computational analysis and in vitro screening to design an immunogen that could bind to VRC01-class bNAbs and to their germline precursors. Georgiev et al. (p. 751) took advantage of the fact that only four sites on the HIV viral envelope protein seem to bind bNAbs, and sera that contain particular bNAbs show characteristic patterns of neutralization. An algorithm was developed that could successfully delineate the neutralization specificity of antibodies present in polyclonal sera from HIV-infected patients. Structural knowledge of broadly neutralizing antibodies against HIV-1 guides the design of an immunogen to elicit them. Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1–infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens.

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

Eliminating key glycosylation sites on HIV envelope (Env) restores binding of the germline versions of known broadly neutralizing anti-Env antibodies.

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.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

Diverse recombinant HIV-1 Envs fail to activate B cells expressing the germline B cell receptors of the broadly neutralizing anti-HIV-1 antibodies PG9 and 447-52D

ABSTRACT Broadly neutralizing antibodies (bNAbs) against HIV-1 are generated during HIV-1-infection but have not yet been elicited by immunization with recombinant forms of the viral envelope glycoprotein (Env; the target of anti-HIV-1 neutralizing antibodies). A particular type of bNAb targets the CD4-binding site (CD4-BS) region of Env. These antibodies are derived from a limited number of VH/VL genes and can bind to and neutralize diverse HIV-1 strains. Recent reports have demonstrated the limited potential of Env to activate B cells expressing the germline B cell receptor (BCR) forms of anti-CD4-BS bNAbs. A potential reason for the lack of elicitation of anti-CD4-BS bNAbs by Env immunogens is the absence of stimulation of naive B cells expressing the germline BCRs of such antibodies. Several bNAbs have been isolated from HIV-1-infected subjects that target other structurally conserved regions of Env. How frequently Env immunogens stimulate the germline BCRs that give rise to bNAbs that target Env regions other than the CD4-BS is not well understood. Here, we investigated the interactions between diverse Envs and the BCRs of known bNAbs targeting not only the CD4-BS but also conserved elements of the second and third variable Env regions. Our results indicate that Env is generally ineffective in engaging germline BCRs of bNAbs irrespective of their epitope target. Potentially, this is the result of viral evolutionary mechanisms adopted to escape broadly neutralizing antibody responses. Our results also suggest that a single Env capable of activating germline BCRs that target distinct Env epitopes will be very difficult to identify or to design. IMPORTANCE Broadly neutralizing antibodies against HIV-1 are thought to be an important component of the immune responses that a successful vaccine should elicit. Broadly neutralizing antibodies are generated by a subset of those infected by HIV-1, but so far, they have not been generated by immunization with recombinant Envelope (Env, the target of anti-HIV-1 neutralizing antibodies). Here, we provide evidence that the inability of Env to elicit the production of broadly neutralizing antibodies is due to the inability of diverse Envs to engage the germline B cell receptor forms of known broadly neutralizing antibodies.

Structural basis for germline antibody recognition of HIV-1 immunogens.

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design. DOI: http://dx.doi.org/10.7554/eLife.13783.001

Germline-targeting immunogens

In 2009, Dimitrovs group reported that the inferred germline (iGL) forms of several HIV‐1 broadly neutralizing antibodies (bNAbs) did not display measurable binding to a recombinant gp140 Env protein (derived from the dual‐tropic 89.6 virus), which was efficiently recognized by the mature (somatically mutated) antibodies. At that time, a small number of bNAbs were available, but in the following years, the implementation of high‐throughput B‐cell isolation and sequencing assays and of screening methodologies facilitated the isolation of greater numbers of bNAbs from infected subjects. Using these newest bNAbs, and a wide range of diverse recombinant Envs, we and others confirmed the observations made by Dimitrovs group. The results from these studies created a paradigm shift in our collective thinking as to why recombinant Envs are ineffective in eliciting bNAbs and has led to the “germline‐targeting” immunization approach. Here we discuss this approach in detail: what has been done so far, the advantages and limitations of the current germline‐targeting immunogens and of the animal models used to test them, and we conclude with a few thoughts about future directions in this area of research.

Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.