Env Trimer Research Articles

Antibody Recognition of CD4-Induced Open HIV-1 Env Trimers.

Human immunodeficiency virus type 1 (HIV-1) envelope (Env), a heterotrimer of gp120-gp41 subunits, mediates fusion of the viral and host cell membranes after interactions with the host receptor CD4 and a coreceptor. CD4 binding induces rearrangements in Env trimer, resulting in a CD4-induced (CD4i) open Env conformation. Structural studies of antibodies isolated from infected donors have defined antibody-Env interactions, with one class of antibodies specifically recognizing the CD4i open Env conformation. In this study, we characterized a group of monoclonal antibodies isolated from HIV-1 infected donors (V2i MAbs) that displayed characteristics of CD4i antibodies. Binding experiments demonstrated that the V2i MAbs preferentially recognize CD4-bound open Env trimers. Structural characterizations of V2i MAb-Env-CD4 trimer complexes using single-particle cryo-electron microscopy showed recognition by V2i MAbs using different angles of approach to the gp120 V1V2 domain and the β2/β3 strands on a CD4i open conformation Env with no direct interactions of the MAbs with CD4. We also characterized CG10, a CD4i antibody that was raised in mice immunized with a gp120-CD4 complex, bound to an Env trimer plus CD4. CG10 exhibited characteristics similar to those of the V2i antibodies, i.e., recognition of the open Env conformation, but showed direct contacts to both CD4 and gp120. Structural comparisons of these and previously characterized CD4i antibody interactions with Env provide a suggested mechanism for how these antibodies are elicited during HIV-1 infection. IMPORTANCE The RV144 HIV-1 clinical vaccination trial showed modest protection against viral infection. Antibody responses to the V1V2 region of HIV-1 Env gp120 were correlated inversely with the risk of infection, and data from three other clinical vaccine trials suggested a similar signal. In addition, antibodies targeting V1V2 have been correlated with protections from simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) infections in nonhuman primates. We structurally characterized V2i antibodies directed against V1V2 isolated from HIV-1 infected humans in complex with open Env trimers bound to the host receptor CD4. We also characterized a CD4i antibody that interacts with CD4 as well as the gp120 subunit of an open Env trimer. Our study suggests how V2i and CD4i antibodies were elicited during HIV-1 infection.

Fine-mapping the immunodominant antibody epitopes on consensus sequence-based HIV-1 envelope trimer vaccine candidates

The HIV-1 envelope glycoprotein (Env) trimer is the key target for vaccines aimed at inducing neutralizing antibodies (NAbs) against HIV-1. The clinical candidate immunogen ConM SOSIP.v7 is a stabilized native-like HIV-1 Env trimer based on an artificial consensus sequence of all HIV-1 isolates in group M. In preclinical studies ConM SOSIP.v7 trimers induced strong autologous NAb responses in non-human primates (NHPs). To fine-map these responses, we isolated monoclonal antibodies (mAbs) from six cynomolgus macaques that were immunized three times with ConM SOSIP.v7 protein and boosted twice with the closely related ConSOSL.UFO.664 immunogen. A total of 40 ConM and/or ConS-specific mAbs were isolated, of which 18 were retrieved after the three ConM SOSIP.v7 immunizations and 22 after the two immunizations with ConSOSL.UFO.664. 22 mAbs (55%) neutralized the ConM and/or ConS virus. Cross-neutralization of ConS virus by approximately one-third of the mAbs was seen prior to ConSOSL.UFO.664 immunization, albeit with modest potency. Neutralizing antibodies predominantly targeted the V1 and V2 regions of the immunogens, with an apparent extension towards the V3 region. Thus, the V1V2V3 region is immunodominant in the potent NAb response elicited by two consensus sequence native-like HIV-1 Env immunogens. Immunization with these soluble consensus Env proteins also elicited non-neutralizing mAbs targeting the trimer base. These results inform the use and improvement of consensus-based trimer immunogens in combinatorial vaccine strategies.

Complementary antibody lineages achieve neutralization breadth in an HIV-1 infected elite neutralizer.

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses.

Host Molecule Incorporation into HIV Virions, Potential Influences in HIV Pathogenesis.

During the last phase of HIV viral production, nascent HIV virions acquire a fraction of the cellular lipid membrane to create the external lipid envelope, a process by which cellular proteins present on the surface of the infected cell can be incorporated along with Env trimers. Interestingly, several studies indicated that these incorporated host molecules could conserve their biological activity and consequently contribute to HIV pathogenesis either by enhancing the infectivity of HIV virions, their tissue tropism or by affecting immune cell functions. The following review will describe the main approaches used to characterize membrane bound host molecule incorporation into HIV virions, the proposed mechanisms involved, and the role of a non-exhaustive list of incorporated molecules.

Cryo-EM structures of prefusion SIV envelope trimer.

Simian immunodeficiency viruses (SIVs) are lentiviruses that naturally infect non-human primates of African origin and seeded cross-species transmissions of HIV-1 and HIV-2. Here we report prefusion stabilization and cryo-EM structures of soluble envelope (Env) trimers from rhesus macaque SIV (SIVmac) in complex with neutralizing antibodies. These structures provide residue-level definition for SIV-specific disulfide-bonded variable loops (V1 and V2), which we used to delineate variable-loop coverage of the Env trimer. The defined variable loops enabled us to investigate assembled Env-glycan shields throughout SIV, which we found to comprise both N- and O-linked glycans, the latter emanating from V1 inserts, which bound the O-link-specific lectin jacalin. We also investigated in situ SIVmac-Env trimers on virions, determining cryo-electron tomography structures at subnanometer resolutions for an antibody-bound complex and a ligand-free state. Collectively, these structures define the prefusion-closed structure of the SIV-Env trimer and delineate variable-loop and glycan-shielding mechanisms of immune evasion conserved throughout SIV evolution.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVCEnv) covalently complexed to two-domain CD4S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits

Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycoprotein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4S60C) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformationally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogenicity of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVCEnv) covalently complexed to 2dCD4S60C. Methods: Recombinant BG505 SOSIP.664 and FVCEnv SOSIP.664 were expressed in mammalian cells, purified, covalently coupled to 2dCD4S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C was investigated in New Zealand white rabbits and compared to unliganded FVCEnv and 2dCD4S60C. Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C elicited a potent, HIV-specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to-neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4S60C and FVCEnvSOSIP.664-2dCD4S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4S60C. These findings justify further investment into research exploring modified open, CD4-bound Env conformations as novel vaccine immunogens.

Stabilized HIV-1 envelope immunization induces neutralizing antibodies to the CD4bs and protects macaques against mucosal infection.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4+ T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine-induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus

SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated intravenous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

A naturally arising broad and potent CD4-binding site antibody with low somatic mutation.

The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs not only exhibit extraordinary breadth and potency but also rank among the most highly somatically mutated bNAbs. Here, we describe a VRC01-class antibody isolated from a viremic controller, BG24, that is much less mutated than most relatives of its class while achieving comparable breadth and potency. A 3.8-Å x-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach.

Viral and Host Factors Regulating HIV-1 Envelope Protein Trafficking and Particle Incorporation.

The HIV-1 envelope glycoprotein (Env) is an essential structural component of the virus, serving as the receptor-binding protein and principal neutralizing determinant. Env trimers are incorporated into developing particles at the plasma membrane of infected cells. Incorporation of HIV-1 Env into particles in T cells and macrophages is regulated by the long Env cytoplasmic tail (CT) and the matrix region of Gag. The CT incorporates motifs that interact with cellular factors involved in endosomal trafficking. Env follows an unusual pathway to arrive at the site of particle assembly, first traversing the secretory pathway to the plasma membrane (PM), then undergoing endocytosis, followed by directed sorting to the site of particle assembly on the PM. Many aspects of Env trafficking remain to be defined, including the sequential events that occur following endocytosis, leading to productive recycling and particle incorporation. This review focuses on the host factors and pathways involved in Env trafficking, and discusses leading models of Env incorporation into particles.

Comparing methods for immobilizing HIV-1 SOSIPs in ELISAs that evaluate antibody binding

Enzyme-linked immunosorbent assays (ELISAs) are used to evaluate binding of broadly neutralizing antibodies (bNAbs) and polyclonal sera to native-like HIV-1 Env SOSIPs. Methods for immobilizing SOSIPs on plates differ, which can lead to variable or, in some cases, misleading results. Three methods used to immobilize SOSIPs were compared to determine how antigen immobilization methods affect Env conformation and ELISA results. HIV-1 SOSIPs were directly coated on polystyrene plates, captured by a monoclonal antibody against a C-terminal affinity tag, or randomly biotinylated and coated on a streptavidin plate. Binding of bNAbs with known epitopes were compared for each immobilization method. Binding of bNAbs targeting the V1V2, V3, CD4 binding site, and gp120/gp41 interface was comparable for all antigen immobilization methods. However, directly coated HIV-1 SOSIP ELISAs showed detectable binding of 17b, a CD4-induced antibody that binds a V3 epitope that is concealed on closed prefusion Env trimers in the absence of added CD4, whereas antibody-immobilized and randomly biotinylated Env-coated ELISAs did not show detectable binding of 17b in the absence of CD4. We conclude direct coating of HIV-1 SOSIPs on ELISA plates can result in exposure of CD4-induced antibody epitopes, suggesting disruption of Env structure and exposure of epitopes that are hidden in the closed, prefusion trimer.

Design and Functional Characterization of HIV-1 Envelope Protein-Coupled T Helper Liposomes.

Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes. First, we passively encapsulated T helper peptides into a well-characterized liposome formulation displaying a dense array of Env trimers on the surface. We confirmed the closed pre-fusion state of the coupled Env trimers by immunogold staining with conformation-specific antibodies. These peptide-loaded Env-liposome conjugates efficiently activated Env-specific B cells, which further induced proliferation of CD4+ T cells by presentation of liposome-derived peptides on MHC-II molecules. The peptide encapsulation process was then quantitatively improved by an electrostatically driven approach using an overall anionic lipid formulation. We demonstrated that peptides delivered by liposomes were presented by DCs in secondary lymphoid organs after intramuscular immunization of mice. UFO (uncleaved prefusion optimized) Env trimers were covalently coupled to peptide-loaded anionic liposomes by His-tag/NTA(Ni) interactions and EDC/Sulfo-NHS crosslinking. EM imaging revealed a moderately dense array of well-folded Env trimers on the liposomal surface. The conformation was verified by liposomal surface FACS. Furthermore, anionic Env-coupled T helper liposomes effectively induced Env-specific B cell activation and proliferation in a comparable range to T helper VLPs. Taken together, we demonstrated that T helper VLPs can be substituted with customizable and GMP-scalable liposomal nanoparticles as a perspective for future preclinical and clinical HIV vaccine applications. The functional nanoparticle characterization assays shown in this study can be applied to other systems of synthetic nanoparticles delivering antigens derived from various pathogens.

SERINC5 Restricts HIV-1 Infectivity by Promoting Conformational Changes and Accelerating Functional Inactivation of Env.

SERINC5 incorporates into HIV-1 particles and inhibits the ability of Env glycoprotein to mediate virus-cell fusion. SERINC5-resistance maps to Env, with primary isolates generally showing greater resistance than laboratory-adapted strains. Here, we examined a relationship between the inhibition of HIV-1 infectivity and the rate of Env inactivation using a panel of SERINC5-resistant and -sensitive HIV-1 Envs. SERINC5 incorporation into pseudoviruses resulted in a faster inactivation of sensitive compared to resistant Env strains. A correlation between fold reduction in infectivity and the rate of inactivation was also observed for multiple Env mutants known to stabilize and destabilize the closed Env structure. Unexpectedly, most mutations disfavoring the closed Env conformation rendered HIV-1 less sensitive to SERINC5. In contrast, functional inactivation of SERINC5-containing viruses was significantly accelerated in the presence of a CD4-mimetic compound, suggesting that CD4 binding sensitizes Env to SERINC5. Using a small molecule inhibitor that selectively targets the closed Env structure, we found that, surprisingly, SERINC5 increases the potency of this compound against a laboratory-adapted Env which prefers a partially open conformation, indicating that SERINC5 may stabilize the closed trimeric Env structure. Our results reveal a complex effect of SERINC5 on Env conformational dynamics that promotes Env inactivation and is likely responsible for the observed restriction phenotype.

Enhanced HIV SOSIP Envelope yields in plants through transient co-expression of peptidyl-prolyl isomerase B and calreticulin chaperones and ER targeting

High yield production of recombinant HIV SOSIP envelope (Env) trimers has proven elusive as numerous disulfide bonds, proteolytic cleavage and extensive glycosylation pose high demands on the host cell machinery and stress imposed by accumulation of misfolded proteins may ultimately lead to cellular toxicity. The present study utilized the Nicotiana benthamiana/p19 (N.b./p19) transient plant system to assess co-expression of two ER master regulators and 5 chaperones, crucial in the folding process, to enhance yields of three Env SOSIPs, single chain BG505 SOSIP.664 gp140, CH505TF.6R.SOSIP.664.v4.1 and CH848-10.17-DT9. Phenotypic changes in leaves induced by SOSIP expression were employed to rapidly identify chaperone-assisted improvement in health and expression. Up to 15-fold increases were obtained by co-infiltration of peptidylprolvl isomerase (PPI) and calreticulin (CRT) which were further enhanced by addition of the ER-retrieval KDEL tags to the SOSIP genes; levels depending on individual SOSIP type, day of harvest and chaperone gene dosage. Results are consistent with reducing SOSIP misfolding and cellular stress due to increased exposure to the plant host cell’s calnexin/calreticulin network and accelerating the rate-limiting cis–trans isomerization of Xaa-Pro peptide bonds respectively. Plant transient co-expression facilitates rapid identification of host cell factors and will be translatable to other complex glycoproteins and mammalian expression systems.

Commonly Elicited Antibodies against the Base of the HIV-1 Env Trimer Guide the Population-Level Evolution of a Structure-Regulating Region in gp41.

The antibody response against the HIV-1 envelope glycoproteins (Envs) guides evolution of this protein within each host. Whether antibodies with similar target specificities are elicited in different individuals and affect the population-level evolution of Env is poorly understood. To address this question, we analyzed properties of emerging variants in the gp41 fusion peptide-proximal region (FPPR) that exhibit distinct evolutionary patterns in HIV-1 clade B. For positions 534, 536, and 539 in the FPPR, alanine was the major emerging variant. However, 534A and 536A show a constant frequency in the population between 1979 and 2016, whereas 539A is gradually increasing. To understand the basis for these differences, we introduced alanine substitutions in the FPPR of primary HIV-1 strains and examined their functional and antigenic properties. Evolutionary patterns could not be explained by fusion competence or structural stability of the emerging variants. Instead, 534A and 536A exhibited modest but significant increases in sensitivity to antibodies against the membrane-proximal external region (MPER) and gp120-gp41 interface. These Envs were also more sensitive to poorly neutralizing sera from HIV-1-infected individuals than the clade ancestral form or 539A variant. Competition binding assays confirmed for all sera tested the presence of antibodies against the base of the Env trimer that compete with monoclonal antibodies targeting the MPER and gp120-gp41 interface. Our findings suggest that weakly neutralizing antibodies against the trimer base are commonly elicited; they do not exert catastrophic population size reduction effects on emerging variants but, instead, determine their set point frequencies in the population and historical patterns of change. IMPORTANCE Infection by HIV-1 elicits formation of antibodies that target the viral Env proteins and can inactivate the virus. The specific targets of these antibodies vary among infected individuals. It is unclear whether some target specificities are shared among the antibody responses of different individuals. We observed that antibodies against the base of the Env protein are commonly elicited during infection. The selective pressure applied by such antibodies is weak. As a result, they do not completely eliminate the sensitive forms of the virus from the population, but maintain their frequency at a low level that has not increased since the beginning of the AIDS pandemic. Interestingly, the changes in Env do not occur at the sites targeted by the antibodies, but at a distinct region of Env, the fusion peptide-proximal region, which regulates their exposure.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

Structural dynamics reveal isolate-specific differences at neutralization epitopes on HIV Env

SummaryThe envelope glycoprotein (Env) is the sole target for neutralizing antibodies against HIV and the most rapidly evolving, variable part of the virus. High-resolution structures of Env trimers captured in the pre-fusion, closed conformation have revealed a high degree of structural similarity across diverse isolates. Biophysical data, however, indicate that Env is highly dynamic, and the level of dynamics and conformational sampling is believed to vary dramatically between HIV isolates. Dynamic differences likely influence neutralization sensitivity, receptor activation, and overall trimer stability. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), we have mapped local dynamics across native-like Env SOSIP trimers from diverse isolates. We show that significant differences in epitope order are observed across most sites targeted by broadly neutralizing antibodies. We also observe isolate-dependent conformational switching that occurs over a broad range of timescales. Lastly, we report that hyper-stabilizing mutations that dampen dynamics in some isolates have little effect on others.

Data-driven determination of number of discrete conformations in single-particle cryo-EM

Background and objectiveOne of the strengths of single-particle cryo-EM compared to other structural determination techniques is its ability to image heterogeneous samples containing multiple molecular species, different oligomeric states or distinct conformations. This is achieved using routines for in-silico 3D classification that are now well established in the field and have successfully been used to characterize the structural heterogeneity of important biomolecules. These techniques, however, rely on expert-user knowledge and trial-and-error experimentation to determine the correct number of conformations, making it a labor intensive, subjective, and difficult to reproduce procedure. MethodsWe propose an approach to address the problem of automatically determining the number of discrete conformations present in heterogeneous single-particle cryo-EM datasets. We do this by systematically evaluating all possible partitions of the data and selecting the result that maximizes the average variance of similarities measured between particle images and the corresponding 3D reconstructions. ResultsUsing this strategy, we successfully analyzed datasets of heterogeneous protein complexes, including: 1) in-silico mixtures obtained by combining closely related antibody-bound HIV-1 Env trimers and other important membrane channels, and 2) naturally occurring mixtures from diverse and dynamic protein complexes representing varying degrees of structural heterogeneity and conformational plasticity. ConclusionsThe availability of unsupervised strategies for 3D classification combined with existing approaches for fully automatic pre-processing and 3D refinement, represents an important step towards converting single-particle cryo-EM into a high-throughput technique.

Covalent binding of human two-domain CD4 to an HIV-1 subtype C SOSIP.664 trimer modulates its structural dynamics

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) mediates host cell infection by binding to the cellular receptor CD4. Recombinant Env bound to CD4 has been explored for its potential as an HIV vaccine immunogen as receptor binding exposes otherwise shielded, conserved functional sites. Previous preclinical studies showed an interchain disulphide linkage facilitated between Env and 2dCD4S60C generates an immunogenic complex that elicits potent, broadly neutralizing antibodies (bNAbs) against clinically relevant HIV-1. This study investigated conformational dynamics of 2dCD4WT and 2dCD4S60C bound to an HIV–1C SOSIP.664 Env trimer using hydrogen-deuterium exchange mass spectrometry. The Env:2dCD4S60C complex maintains key contact residues required for MHCII and Env/gp120 binding and the residues encompassing Ibalizumab's epitope. Important residues remaining anchored, with an increased flexibility in surrounding regions, evidenced by the higher exchange seen in flanking residues compared to Env:2dCD4WT. While changes in Env:2dCD4S60C dynamics in domain 1 were moderate, domain 2 exhibited greater variation. Lack of stability-inducing H-bonds in these allosteric sites suggest the improved immunogenicity of Env:2dCD4S60C result from exposed CD4 residues providing diverse/novel antigenic targets for the development of potent, broadly neutralizing Ibalizumab-like antibodies.