Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.

Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.

Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Maturation and Diversity of the VRC01-Antibody Lineage over 15 Years of Chronic HIV-1 Infection.

HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

A Neutralizing Antibody Recognizing Primarily N-Linked Glycan Targets the Silent Face of the HIV Envelope.

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.

Structural basis of malaria RIFIN binding by LILRB1-containing antibodies.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to ß2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7.

The COVID-19 pandemic has had widespread effects across the globe, and its causative agent, SARS-CoV-2, continues to spread. Effective interventions need to be developed to end this pandemic. Single and combination therapies with monoclonal antibodies have received emergency use authorization1-3, and more treatments are under development4-7. Furthermore, multiple vaccine constructs have shown promise8, including two that have an approximately 95% protective efficacy against COVID-199,10. However, these interventions were directed against the initial SARS-CoV-2 virus that emerged in 2019. The recent detection of SARS-CoV-2 variants B.1.1.7 in the UK11 and B.1.351 in South Africa12 is of concern because of their purported ease of transmission and extensive mutations in the spike protein. Here we show that B.1.1.7 is refractory to neutralization by most monoclonal antibodies against the N-terminal domain of the spike protein and is relatively resistant to a few monoclonal antibodies against the receptor-binding domain. It is not more resistant to plasma from individuals who have recovered from COVID-19 or sera from individuals who have been vaccinated against SARS-CoV-2. The B.1.351 variant is not only refractory to neutralization by most monoclonal antibodies against the N-terminal domain but also by multiple individual monoclonal antibodies against the receptor-binding motif of the receptor-binding domain, which is mostly due to a mutation causing an E484K substitution. Moreover, compared to wild-type SARS-CoV-2, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4-fold) and sera from individuals who have been vaccinated (10.3-12.4-fold). B.1.351 and emergent variants13,14 with similar mutations in the spike protein present new challenges for monoclonal antibody therapies and threaten the protective efficacy of current vaccines.

Structure-based design of a single-chain triple-disulfide-stabilized fusion-glycoprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus.

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens.

Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth.

Detailed studies of the broadly neutralizing antibodies (bNAbs) that underlie the best available examples of the humoral immune response to HIV are providing important information for the development of therapies and prophylaxis for HIV-1 infection. Here, we report a CD4-binding site (CD4bs) antibody, named N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain. In addition, structural analysis revealed that the orientation of N6 permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNAb can achieve potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis.

Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Pneumatic Structural Deformation to Enhance Resonance Behavior for Broadband and Adaptive Radar Stealth.

Ideal radar absorbing materials (RAMs) require instantaneous, programmable, and spontaneous adaptability to cope with a complex electromagnetic (EM) environment across the full working frequency. Despite various material systems and adaptive mechanisms having been demonstrated, it remains a formidable challenge to integrate these benefits simultaneously. Here, we present a pneumatic matrix that couples morphable MXene/elastomer conductors with dielectric spacers, which leverages controllable airflow to reconfigure the spatial structure between a flat sheet and a hemispherical crown while maintaining resistance stability via wrinkle folding and unfolding. The interdimensional reconfigurations drastically induce multiple resonance behavior, enabling the matrix remarkable frequency tunability (144.5%), ultrawide bandwidth (15 GHz), weak angular dependence (45° incidence), ultrafast responsiveness (∼30 ms), and excellent reproducibility (1000 cycles). With multichannel fluidic and conceptual automated control systems, the final pneumatic device demonstrates a multiplexed, programmable, and autonomous transformable mode that builds a promising platform for smart radar cloaking.

Diverse Murine Vaccinations Reveal Distinct Antibody Classes to Target Fusion Peptide and Variation in Peptide Length to Improve HIV Neutralization.

While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.

Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses.

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody.

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti-HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

The influence of daily life events on learning crafting and the intervening roles of academic emotions and regulatory focus in high school students.

BACKGROUND: The adaptation of students to academic challenges in high school is crucial for academic performance. This study proposes the concept of "learning crafting," a previously under-researched area, and investigates its associated variables. METHODS: Using a diary method, we studied 187 Chinese high school students (64% female; Mage = 15.57) over a 9-day period. We examined the effect of daily life events on learning crafting, and considered academic emotions as mediators and regulatory focus as moderators. RESULTS: Hierarchical linear modeling revealed that daily positive events were positively correlated with learning crafting at both within-person and between-person levels. Positive academic emotions served as mediators of this relationship. Furthermore, promotion focus had a positive moderating effect on the relationship between daily positive events and positive academic emotions. Conversely, daily negative events were only negatively correlated with learning crafting at the between-person level, and no additional significant relationships were identified. CONCLUSION: This study elucidated the effect of daily life events on learning crafting, its mediating mechanisms, and conditional factors. These results not only contribute to crafting theory, but also provide theoretical underpinnings for future interventions targeting high school students' learning crafting.