Design of soluble HIV-1 envelope trimers free of covalent gp120-gp41 bonds with prevalent native-like conformation.

Soluble HIV-1 envelope (Env) trimers may serve as effective vaccine immunogens. The widely utilized SOSIP trimers have been paramount for structural studies, but the disulfide bond they feature between gp120 and gp41 constrains intersubunit mobility and may alter antigenicity. Here, we report an alternative strategy to generate stabilized soluble Env trimers free of covalent gp120-gp41 bonds. Stabilization was achieved by introducing an intrasubunit disulfide bond between the inner and outer domains of gp120, defined as interdomain lock (IDL). Correctly folded IDL trimers displaying a native-like antigenic profile were produced for HIV-1 Envs of different clades. Importantly, the IDL design abrogated CD4 binding while not affecting recognition by potent neutralizing antibodies to the CD4-binding site. By cryoelectron microscopy, IDL trimers were shown to adopt a closed prefusion configuration, while single-molecule fluorescence resonance energy transfer documented a high prevalence of native-like conformation. Thus, IDL trimers may be promising candidates as vaccine immunogens.

HIV-1-envelope trimer transitions from prefusion-closed to CD4-bound-open conformations through an occluded-intermediate state.

HIV-1 infection is initiated by the interaction between the gp120 subunit in the envelope (Env) trimer and the cellular receptor CD4 on host cells. This interaction induces substantial structural rearrangement of the Env trimer. Currently, static structural information for prefusion-closed trimers, CD4-bound prefusion-open trimers, and various antibody-bound trimers is available. However, dynamic features between these static states (e.g., transition structures) are not well understood. Here, we investigate the full transition pathway of a site specifically glycosylated Env trimer between prefusion-closed and CD4-bound-open conformations by collective molecular dynamics and single-molecule Förster resonance energy transfer (smFRET). Our investigations reveal and confirm important features of the transition pathway, including movement of variable loops to generate a glycan hole at the trimer apex and formation or rearrangements of α-helices and ß-strands. Notably, by comparing the transition pathway to known Env-structures, we uncover evidence for a transition intermediate, with four antibodies, Ab1303, Ab1573, b12, and DH851.3, recognizing this intermediate. Each of these four antibodies induce population shifts of Env to occupy a newly observed smFRET state: the "occluded-intermediate" state. We propose this occluded-intermediate state to be both a prevalent state of Env and an on-path conformation between prefusion-closed and CD4-bound-open states, previously overlooked in smFRET analyses.

Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Ultrapotent Broadly Neutralizing Human-llama Bispecific Antibodies against HIV-1.

Broadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV-1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion-stabilized HIV-1 envelope (Env) trimer, BG505 DS-SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4-binding site (CD4bs) of vulnerability. Two of the vaccine-elicited CD4bs-targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a-hinge region and human IgG1-constant region (G36×3-IgG2a and R27×3-IgG2a), neutralized 96% of a multiclade 208-strain panel at geometric mean IC80s of 0.314 and 0.033 µg mL-1, respectively. Cryo-EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV-1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2-apex-targeting broadly neutralizing antibody, CAP256V2LS. The resultant human-llama bispecific antibody CAP256L-R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV-1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn-Fc mice similar to the parent CAP256V2LS. Vaccine-elicited llama nanobodies, when combined with V2-apex broadly neutralizing antibodies, may therefore be able to fulfill anti-HIV-1 therapeutic and prophylactic clinical goals.

Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains.

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

Cholesterol reduction by immunization with a PCSK9 mimic.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a plasma protein that controls cholesterol homeostasis. Here, we design a human PCSK9 mimic, named HIT01, with no consecutive 9-residue stretch in common with any human protein as a potential heart attack vaccine. Murine immunizations with HIT01 reduce low-density lipoprotein (LDL) and cholesterol levels by 40% and 30%, respectively. Immunization of cynomolgus macaques with HIT01-K21Q-R218E, a cleavage-resistant variant, elicits high-titer PCSK9-directed antibody responses and significantly reduces serum levels of cholesterol 2 weeks after each immunization. However, HIT01-K21Q-R218E immunizations also increase serum PCSK9 levels by up to 5-fold, likely due to PCSK9-binding antibodies altering the half-life of PCSK9. While vaccination with a PCSK9 mimic can induce antibodies that block interactions of PCSK9 with the LDL receptor, PCSK9-binding antibodies appear to alter homeostatic levels of PCSK9, thereby confounding its vaccine impact. Our results nevertheless suggest a mechanism for increasing the half-life of soluble regulatory factors by vaccination.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Boron-intercalation-triggered crystalline transition of Pd nanosheet assemblies for an enhanced oxygen reduction reaction.

The development of effective and stable cathode electrocatalysts is highly desired for fuel cells. Controlling the composition and morphology of Pd-based materials can provide a great opportunity to improve their oxygen reduction reaction (ORR) performance. Here, we report the synthesis of hexagonal close-packed (hcp) Pd2B nanosheet assemblies (Pd2B NAs) via the boronation reaction between as-synthesized Pd NAs and N,N-dimethylformamide. The hcp Pd2B NAs with uniform pore distribution can provide sufficient active sites for ORRs. The insertion of B atoms can induce the phase transition from face-centered cubic structure to hcp structure, as the most thermodynamically stable phase in the Pd-B alloy, which is beneficial for enhancing the ORR stability and toxicity resistance. Therefore, the hcp Pd2B NAs exhibit superior mass activity, specific activity and excellent stability for ORR. The present strategy of boron-intercalation-triggered crystalline transition of Pd-based nanomaterials is valuable for the design of metal-nonmetal catalysts with enhanced performance.

Extraordinary Titer and Broad Anti-SARS-CoV-2 Neutralization Induced by Stabilized RBD Nanoparticles from Strain BA.5.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 µg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of ß-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.