A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.

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A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man9 at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

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A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble SOSIP gp140 trimer in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 A resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1 and HR2 domains, and shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.

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Cryo-EM Structure of a Fully Glycosylated Soluble Cleaved HIV-1 Envelope Trimer

INTRODUCTION A major goal of HIV-1 vaccine development is to identify immunogens capable of inducing protective titers of broadly neutralizing antibodies (bNAbs) against circulating, neutralization-resistant (tier 2) viruses. The envelope glycoprotein (Env) trimer on the virus surface is the only bNAb target and accordingly serves as the basis for recombinant protein immunogens intended to induce bNAbs. We have engineered soluble, recombinant trimers based on the BG505 clade A and B41 clade B tier 2 viruses. These proteolytically cleaved and stabilized trimers, designated BG505 and B41 SOSIP.664 gp140, display multiple bNAb epitopes and have antigenic properties that mimic viral Env. High-resolution structures of the BG505 trimers reinforce this perspective. In contrast, other trimer designs (uncleaved gp140) that lack the SOSIP modifications and are based on eliminating the intersubunit proteolytic cleavage site adopt non-native configurations. RATIONALE To determine the NAb response elicited by native-like trimers, we immunized rabbits and macaques with BG505 SOSIP.664 gp140 proteins, and rabbits with their B41 counterparts. For comparison with the BG505 trimers, we also tested gp120 monomers and uncleaved gp140 proteins of the same genotype. We characterized the resulting antibody responses by quantifying their ability to neutralize the autologous tier 2 viruses and multiple heterologous tier 1 (neutralization-sensitive) and tier 2 viruses. We mapped the BG505 NAb responses using various techniques, including a large panel of Env-pseudotyped virus mutants. RESULTS BG505 SOSIP.664 trimers consistently induced potent NAbs against the autologous tier 2 virus in rabbits and similar but weaker responses in macaques. Cross-reactive NAbs against the more sensitive tier 1 viruses were also induced in all the immunized animals, but heterologous neutralization of tier 2 viruses was seen only weakly and sporadically. The rabbit responses to B41 SOSIP.664 trimers were qualitatively similar to those seen in the BG505 trimer recipients. The autologous tier 2 NAb titers to both SOSIP.664 trimers were greater than any such responses previously observed using various uncleaved gp140 proteins, including the BG505 version used in this study. The rabbit tier 2 and tier 1 NAb responses to the BG505 SOSIP.664 trimers were uncorrelated. Mapping studies showed the tier 2 NAbs recognized conformational epitopes that differed between animals. In some cases, these NAbs targeted components of the glycan shield previously associated with bNAb epitopes. In contrast, the tier 1 NAbs targeted linear epitopes in the gp120 V3 region. CONCLUSIONS Although an autologous tier 2 NAb response is not sufficient for vaccine protection against HIV-1, it may be a necessary step in various strategies intended to induce bNAbs. Thus, our findings indicate that native-like trimers represent a promising starting point for the further development of recombinant Env immunogens intended to broaden the NAb response. The high-resolution structures of SOSIP.664 trimers allow improvements to be rationally designed. Relevant strategies include the further stabilization of trimer-associated bNAb epitopes while reducing the antigenicity of V3 and other non-neutralizing epitopes that may be distractive; immunizing with longitudinal series of SOSIP.664 trimers from infected individuals who generate bNAbs; using trimer cocktails (e.g., from clades A, B, and C); and priming with trimer variants that trigger desirable germline responses before boosting with wild-type trimers.

https://science.sciencemag.org/content/sci/early/2015/06/17/science.aac4223.full.pdf

HIV-1 neutralizing antibodies induced by native-like envelope trimers

HIV employs multiple means to evade the humoral immune response, particularly the elicitation of and recognition by broadly neutralizing antibodies (bnAbs). Such antibodies can act antivirally against a wide spectrum of viruses by targeting relatively conserved regions on the surface HIV envelope trimer spike. Elicitation of and recognition by bnAbs are hindered by the arrangement of spikes on virions and the relatively difficult access to bnAb epitopes on spikes, including the proximity of variable regions and a high density of glycans. Yet, in a small proportion of HIV-infected individuals, potent bnAb responses do develop, and isolation of the corresponding monoclonal antibodies has been facilitated by identification of favorable donors with potent bnAb sera and by development of improved methods for human antibody generation. Molecular studies of recombinant Env trimers, alone and in interaction with bnAbs, are providing new insights that are fueling the development and testing of promising immunogens...

Broadly Neutralizing Antibodies to HIV and Their Role in Vaccine Design

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.

https://www.pnas.org/content/pnas/108/28/11440.full.pdf

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

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Yun Kenneth Kang

Papers

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies

Structural and immunogenicity studies of a cleaved, stabilized envelope trimer derived from subtype A HIV-1.