Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.

Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk with altered NTD dynamics.

SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3. As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

Structural characterization of HIV-1 Env heterotrimers bound to one or two CD4 receptors reveals intermediate Env conformations.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120/gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops, and in fully-saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops. To investigate changes resulting from sub-stoichiometric CD4 binding, we solved 3.4Å and 3.9Å single-particle cryo-EM structures of soluble, native-like Envs bound to one or two CD4 molecules. Env trimer bound to one CD4 adopted the closed, prefusion Env state. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state that included gp120 outward rotation but maintained the prefusion, three-stranded gp120 bridging sheet and showed only partial V1V2 displacement and V3 exposure. We conclude that engagement of one CD4 molecule was insufficient to stimulate CD4-induced conformational changes, while binding two CD4 molecules led to Env opening in CD4-bound protomers only. Together, these results illuminate HIV-1 Env intermediate conformations and illustrate the structural plasticity of HIV-1 Env.

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 ß2/ß3 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.

HIV-1 CD4-binding site germline antibody-Env structures inform vaccine design.

BG24, a VRC01-class broadly neutralizing antibody (bNAb) against HIV-1 Env with relatively few somatic hypermutations (SHMs), represents a promising target for vaccine strategies to elicit CD4-binding site (CD4bs) bNAbs. To understand how SHMs correlate with BG24 neutralization of HIV-1, we report 4.1 Å and 3.4 Å single-particle cryo-EM structures of two inferred germline (iGL) BG24 precursors complexed with engineered Env-based immunogens lacking CD4bs N-glycans. Structures reveal critical Env contacts by BG24iGL and identify antibody light chain structural features that impede Env recognition. In addition, biochemical data and cryo-EM structures of BG24iGL variants bound to Envs with CD4bs glycans present provide insights into N-glycan accommodation, including structural modes of light chain adaptations in the presence of the N276gp120 glycan. Together, these findings reveal Env regions critical for germline antibody recognition and potential sites to alter in immunogen design.

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.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

How Antibodies Recognize Pathogenic Viruses: Structural Correlates of Antibody Neutralization of HIV-1, SARS-CoV-2, and Zika.

The H1N1 pandemic of 2009-2010, MERS epidemic of 2012, Ebola epidemics of 2013-2016 and 2018-2020, Zika epidemic of 2015-2016, and COVID-19 pandemic of 2019-2021, are recent examples in the long history of epidemics that demonstrate the enormous global impact of viral infection. The rapid development of safe and effective vaccines and therapeutics has proven vital to reducing morbidity and mortality from newly emerging viruses. Structural biology methods can be used to determine how antibodies elicited during infection or vaccination target viral proteins and identify viral epitopes that correlate with potent neutralization. Here we review how structural and molecular biology approaches have contributed to our understanding of antibody recognition of pathogenic viruses, specifically HIV-1, SARS-CoV-2, and Zika. Determining structural correlates of neutralization of viruses has guided the design of vaccines, monoclonal antibodies, and small molecule inhibitors in response to the global threat of viral epidemics.

Construction, characterization, and immunization of nanoparticles that display a diverse array of influenza HA trimers.

Current influenza vaccines do not elicit broadly protective immune responses against multiple strains. New strategies to focus the humoral immune response to conserved regions on influenza antigens are therefore required for recognition by broadly neutralizing antibodies. It has been suggested that B-cells with receptors that recognize conserved epitopes would be preferentially stimulated through avidity effects by mosaic particles presenting multiple forms of a variable antigen. We adapted SpyCatcher-based platforms, AP205 virus-like particles (VLPs) and mi3 nanoparticles (NPs), to covalently co-display SpyTagged hemagglutinin (HA) trimers from group 1 and group 2 influenza A strains. Here we show successful homotypic and heterotypic conjugation of up to 8 different HA trimers to both VLPs and NPs. We characterized the HA-VLPs and HA-NPs by cryo-electron tomography to derive the average number of conjugated HAs and their separation distances on particles, and compared immunizations of mosaic and homotypic particles in wild-type mice. Both types of HA particles elicited strong antibody responses, but the mosaic particles did not consistently elicit broader immune responses than mixtures of homotypic particles. We conclude that covalent attachment of HAs from currently-circulating influenza strains represents a viable alternative to current annual influenza vaccine strategies, but in the absence of further modifications, is unlikely to represent a method for making a universal influenza vaccine.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies.

The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1-5 show therapeutic promise and are being evaluated clincally6-8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to "up" RBDs, (2) ACE2-blocking hNAbs that bind both "up" and "down" RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize "up" and "down" RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only "up" RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent "down" RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.