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Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display. Ebola virus glycoprotein (GP) is a major target for vaccine design. Here, the authors identify mutations to improve GP stability and yield, design two multilayered nanoparticle carriers, and demonstrate good immunogenicity of the modified GP on nanoparticles in mice and rabbits.

Efficacy evaluation through human trials is crucial for advancing a vaccine candidate to clinics. Next-generation sequencing (NGS) can be used to quantify B cell repertoire response and trace antibody lineages during vaccination. Here, we demonstrate this application with a case study of Hecolin®, the licensed vaccine for hepatitis E virus (HEV). Four subjects are administered the vaccine following a standard three-dose schedule. Vaccine-induced antibodies exhibit a high degree of clonal diversity, recognize five conformational antigenic sites of the genotype 1 HEV p239 antigen, and cross-react with other genotypes. Unbiased repertoire sequencing is performed for seven time points over six months of vaccination, with maturation pathways characterize for a set of vaccine-induced antibodies. In addition to dynamic repertoire profiles, NGS analysis reveals differential patterns of HEV-specific antibody lineages and highlights the necessity of the long vaccine boost. Together, our study presents a quantitative strategy for vaccine evaluation in small-scale human studies., The authors provide a comprehensive characterization of the human antibody response to a licensed hepatitis E virus (HEV) vaccine, Hecolin, in four individuals over the course of six months post vaccination. They demonstrate diverse patterns of antibody response underlying the vaccine protection.

Salt stress threatens plant growth, development and crop yields, and has become a critical global environmental issue. Increasing evidence has suggested that the epigenetic mechanism such as DNA methylation can mediate plant response to salt stress through transcriptional regulation and transposable element (TE) silencing. However, studies exploring genome-wide methylation dynamics under salt stress remain limited, in particular, for studies on multiple genotypes. Here, we adopted four natural accessions of the model species

The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and repertoire sequencing to identify neutralizing antibodies (NAbs) from immunized animals. A panel of mouse NAbs was isolated from mice immunized with a 60-meric I3-01 NP presenting 20 stabilized trimers. Three mouse NAbs potently neutralized BG505.T332N by recognizing a glycan epitope centered in the C3/V4 region on BG505 Env, as revealed by electron microscopy (EM), X-ray crystallography, and epitope mapping. A set of rabbit NAbs was isolated from rabbits immunized with a soluble trimer and a 24-meric ferritin NP presenting 8 trimers. Neutralization assays against BG505.T332N variants confirmed that potent rabbit NAbs targeted previously described glycan holes on BG505 Env and accounted for a significant portion of the autologous NAb response in both the trimer and ferritin NP groups. Last, we examined NAb responses that were induced by non-BG505 Env immunogens. We determined a 3.4-Å-resolution crystal structure for the clade C transmitted/founder (T/F) Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend in gp41. This clade C Env, in a soluble trimer form and in a multivalent form with 8 trimers attached to ferritin NP, and the gp41-stabilized clade A Q482-d12 Env trimer elicited distinct NAb responses in rabbits, with notable differences in neutralization breadth. Although eliciting a broad NAb response remains a major challenge, our study provides valuable information on an HIV-1 vaccine design strategy that combines gp41 stabilization and NP display. IMPORTANCE Self-assembling protein nanoparticles (NPs) presenting BG505 envelope (Env) trimers can elicit tier 2 HIV-1-neutralizing antibody (NAb) responses more effectively than soluble trimers. In the present study, monoclonal NAbs were isolated from previously immunized mice and rabbits for structural and functional analyses, which revealed that potent mouse NAbs recognize the C3/V4 region and small NP-elicited rabbit NAbs primarily target known glycan holes on BG505 Env. This study validates the gp41 stabilization strategy for HIV-1 Env vaccine design and highlights the challenge in eliciting a broad NAb response.

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.

We present a comprehensive vaccine strategy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by combining antigen optimization and nanoparticle display. We first developed a receptor binding domain (RBD)-specific antibody column for purification and displayed the RBD on self-assembling protein nanoparticles (SApNPs) using the SpyTag/SpyCatcher system. We then identified the heptad repeat 2 (HR2) stalk as a major cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on three SApNPs with high yield, purity, and antigenicity. Compared to the RBD, the RBD-ferritin SApNP elicited a more potent murine neutralizing antibody (NAb) response on par with the spike. S2GΔHR2 elicited two-fold-higher NAb titers than the proline-capped spike (S2P), while S2GΔHR2 SApNPs derived from multilayered E2p and I3–01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3–01v9 SApNP also induced critically needed T-cell immunity, thereby providing a next-generation vaccine candidate to battle the COVID-19 pandemic., ONE-SENTENCE SUMMARY The receptor binding domain and stabilized SARS-CoV-2 spike were displayed on nanoparticles as vaccine antigens and elicited potent immune responses.

BNAbs of VH1-69 germline origin from a Chinese patient recognize the V3 glycan supersite via a disulfide-locked long HCDR3 loop., An oligomannose patch around the V3 base of HIV-1 envelope glycoprotein (Env) is recognized by multiple classes of broadly neutralizing antibodies (bNAbs). Here, we investigated the bNAb response to the V3 glycan supersite in an HIV-1–infected Chinese donor by Env-specific single B cell sorting, structural and functional studies, and longitudinal analysis of antibody and virus repertoires. Monoclonal antibodies 438-B11 and 438-D5 were isolated that potently neutralize HIV-1 with moderate breadth, are encoded by the VH1-69 germline gene, and have a disulfide-linked long HCDR3 loop. Crystal structures of Env-bound and unbound antibodies revealed heavy chain–mediated recognition of the glycan supersite with a unique angle of approach and a critical role of the intra-HCDR3 disulfide. The mechanism of viral escape was examined via single-genome amplification/sequencing and glycan mutations around the N332 supersite. Our findings further emphasize the V3 glycan supersite as a prominent target for Env-based vaccine design.

An HCV vaccine strategy displays optimized E2 cores on nanoparticles for eliciting a cross-neutralizing B cell response., Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are responsible for cell entry, with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell–based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved substantial yield and purity, as well as enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B cell patterns associated with nanoparticle-induced antibody responses, which target the conserved neutralizing epitopes on E2 and cross-neutralize HCV genotypes.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are critical for cell entry with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved high yield, high purity, and enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B-cell patterns associated with nanoparticle-induced antibody responses, which cross-neutralized HCV by targeting the conserved neutralizing epitopes on E2.One Sentence SummaryAn HCV vaccine strategy is presented that displays redesigned E2 cores on nanoparticles as vaccine candidates for eliciting a broadly neutralizing B-cell response.

Zika virus (ZIKV) specific neutralizing antibodies hold a great promise for antibody-based interventions and vaccine design against ZIKV infection. However, their development in infected patients remain unknown. Here, we report on the dynamic development of a potent and protective ZIKV-specific human antibody ZK2B10 initially isolated from a ZIKV convalescent individual using next-generation sequencing (NGS). The unbiased repertoire analysis showed dramatic changes in many families of heavy and light chain variable regions. However, lineage tracing of ZK2B10 revealed limited somatic hypermutation throughout the 12 months since the onset of symptom. In particular, NGS-derived germline-like somatic variants neutralized and protected mice from lethal challenge of ZIKV without detectable cross-reactivity with Dengue virus (DENV). Site-directed mutagenesis identified two residues within λ chain, N31 and S91 that are essential to the functional maturation. The dynamic features unveiled here will assist us to better understand the pathogenesis of ZIKV infection and inform rational design of vaccines.Author summaryRecently emerged ZIKV is associated with severe neurological complications such as Guillain–Barré syndrome in adults and congenital microcephaly in newborns. No ZIKV-specific therapeutics or vaccines are currently available. We and others have identified a number of neutralizing antibodies capable of protecting experimental animals from ZIKV infection. However, the development of these potent antibodies during ZIKV natural infection remains unknown. Here, we report on the longitudinal analysis of one such antibody ZK2B10 using next-generation sequencing (NGS), bioinformatics and functional analysis. We found that the ZK2B10 germline-like antibodies possess strong neutralizing activity in vitro and impressive protectivity against lethal ZIKV infection in vivo. These findings suggest that the potent and protective antibody response against ZIKV can be generated within relative short term with high germline identity which provide great hope and promise for successful vaccine development against ZIKV.

SUMMARYOvercoming envelope metastability is crucial to trimer-based HIV-1 vaccine design. Here, we present a coherent vaccine strategy by minimizing metastability. For ten strains across five clades, we demonstrate that gp41 ectodomain (gp41ECTO) is the main source of envelope metastability by replacing wild-type gp41ECTOwith BG505 gp41ECTOof the uncleaved prefusion-optimized (UFO) design. These gp41ECTO-swapped trimers can be produced in CHO cells with high yield and high purity. Crystal structure of a gp41ECTO-swapped trimer elucidates how a neutralization-resistant tier 3 virus evades antibody recognition of the V2 apex. UFO trimers of transmitted/founder (T/F) viruses and UFO trimers containing a consensus-based ancestral gp41ECTOsuggest an evolutionary root of the metastability. Gp41ECTO-stabilized trimers can be readily displayed on 24- and 60-meric nanoparticles, with incorporation of additional T cell help illustrated for a hyperstable 60-mer. In mice and rabbits, gp140 nanoparticles induced more effective tier 2 neutralizing antibody response than trimers with statistical significance.HIGHLIGHTSgp41 is the main source of HIV-1 envelope metastabilityBG505 gp41 of the UFO design stabilizes gp140 trimers of diverse subtypesgp41 stabilization facilitates gp140 nanoparticle assembly and improves productionNanoparticles elicit tier 2 neutralizing antibodies more effectively than trimers

Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches., IMPORTANCE Both epitope-focused and trimer-based strategies are currently being explored in HIV-1 vaccine development, which aims to elicit broadly neutralizing antibodies (bNAbs) targeting conserved epitopes on the viral envelope (Env). However, little is known about the differences in antibody response to these bNAb targets presented by foreign scaffolds and native Env. In this study, a systematic effort was undertaken to design multivalent epitope scaffolds and soluble gp140.681 trimers with a complete antigenic surface, and to comparatively analyze the antibody responses elicited by these antigens to the N332 supersite and MPER in a mouse model. This study will inform both epitope-focused and trimer-based vaccine design and will facilitate integration of the two vaccine strategies.

Aims. Electron capture in collisions of highly charged O6+ions with ground-state hydrogen atoms is a very important process in solar wind X-ray studies.Methods. In the present study, the full quantum-mechanical molecular-orbital close-coupling method is employed to study electron capture reactions in collisions of O6+ion with ground-state atomic hydrogen in the energy region from 10−4keV u−1to 5 keV u−1. The ab initio multi-reference single- and double-excitation configuration interaction (MRD-CI) method is used to calculate the potential and coupling data used in the QMOCC calculations.Results. Total and state-selective cross sections for the dominant and subdominant reaction channels are calculated and compared with the available experimental and theoretical data. The branching ratios for Lithium-like O5+excited ions are used to calculate the contribution of cascade radiative transitions fromn = 5 levels to the population of 4l states. From the calculated cross sections, reaction rate coefficients are obtained for temperatures between 1000 and 1 × 109K and compared with other calculations.

Aims. The radiative charge transfer and association processes in Li − + H collisions are studied in the 10 -10 −10 eV center-of-mass energy range. Methods. we carried out total and ν -resolved state-selective cross sections have been carried out by using the fully quantum, optical potential, and semiclassical methods. Results. In the energy region below ~ 0.8 eV, the radiative association process is the dominant decay channel, while radiative charge transfer dominates at higher energies. Rich resonance structures are observed in the cross sections of both processes in the 0.1−1.5 eV energy range; These structures are associated with the quasi-bound states below the top of the centrifugal barrier of the effective potential in the entrance channel for specific vibrational and angular momentum states. It is found that with the increase of collision energy, the resonances occur for higher angular momentum states and lower vibrational states. Besides the cross sections for the studied processes we also present their reaction rate coefficients in the 10 -6 –10 6 K temperature range.