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.

NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.

The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

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.

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.

Vaccination induces maturation in a mouse model of diverse unmutated VRC01-class precursors to HIV-neutralizing antibodies with >50% breadth.

Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains. Here, using sequential immunization of immunoglobulin (Ig)-humanized mice expressing diverse unmutated VRC01-class antibody precursors, we elicited serum responses capable of neutralizing viruses bearing glycan276 and isolated multiple lineages of VRC01-class bnAbs, including two with >50% breadth on a 208-strain panel. Crystal structures of representative bnAbs revealed the same mode of recognition as known VRC01-class bnAbs. Structure-function studies further pinpointed key mutations and correlated their induction with specific immunizations. VRC01-class bnAbs can thus be matured by sequential immunization from unmutated ancestors to >50% breadth, and we delineate immunogens and regimens inducing key SHM.

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.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.

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.

A mouse model to distinguish NLRP6-mediated inflammasome-dependent and -independent functions.

The NOD-like receptor (NLR) family pyrin domain containing 6 (NLRP6) serves as a sensor for microbial dsRNA or lipoteichoic acid (LTA) in intestinal epithelial cells (IECs), and initiating multiple pathways including inflammasome pathway and type I interferon (IFN) pathway, or regulating nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. NLRP6 can exert its function in both inflammasome-dependent and inflammasome-independent manners. However, there is no tool to distinguish the contribution of individual NLRP6-mediated pathway to the physiology and pathology in vivo. Here, we validated that Arg39 and Trp50 residues in the pyrin domain (PYD) of murine NLRP6 are required for ASC recruitment and inflammasome activation, but are not important for the RNA binding and PYD-independent NLRP6 oligomerization. We further generated the Nlrp6R39E&W50E mutant mice, which showed reduced inflammasome activation in either steady state intestine or during viral infection. However, the type I IFN production in cells or intestine tissue from Nlrp6R39E&W50E mutant mice remain normal. Interestingly, NLRP6-mediated inflammasome activation or the IFN-I production seems to play distinct roles in the defense responses against different types of RNA viruses. Our work generated a useful tool to study the inflammasome-dependent role of NLRP6 in vivo, which might help to understand the complexity of multiple pathways mediated by NLRP6 in response to the complicated and dynamic environmental cues in the intestine.

Genome- and transcriptome-wide association studies of 386,000 Asian and European-ancestry women provide new insights into breast cancer genetics.

By combining data from 160,500 individuals with breast cancer and 226,196 controls of Asian and European ancestry, we conducted genome- and transcriptome-wide association studies of breast cancer. We identified 222 genetic risk loci and 137 genes that were associated with breast cancer risk at a p < 5.0 × 10-8 and a Bonferroni-corrected p < 4.6 × 10-6, respectively. Of them, 32 loci and 15 genes showed a significantly different association between ER-positive and ER-negative breast cancer after Bonferroni correction. Significant ancestral differences in risk variant allele frequencies and their association strengths with breast cancer risk were identified. Of the significant associations identified in this study, 17 loci and 14 genes are located 1Mb away from any of the previously reported breast cancer risk variants. Pathways analyses including 221 putative risk genes identified multiple signaling pathways that may play a significant role in the development of breast cancer. Our study provides a comprehensive understanding of and new biological insights into the genetics of this common malignancy.

BloodNet: An attention-based deep network for accurate, efficient, and costless bloodstain time since deposition inference.

The time since deposition (TSD) of a bloodstain, i.e., the time of a bloodstain formation is an essential piece of biological evidence in crime scene investigation. The practical usage of some existing microscopic methods (e.g., spectroscopy or RNA analysis technology) is limited, as their performance strongly relies on high-end instrumentation and/or rigorous laboratory conditions. This paper presents a practically applicable deep learning-based method (i.e., BloodNet) for efficient, accurate, and costless TSD inference from a macroscopic view, i.e., by using easily accessible bloodstain photos. To this end, we established a benchmark database containing around 50,000 photos of bloodstains with varying TSDs. Capitalizing on such a large-scale database, BloodNet adopted attention mechanisms to learn from relatively high-resolution input images the localized fine-grained feature representations that were highly discriminative between different TSD periods. Also, the visual analysis of the learned deep networks based on the Smooth Grad-CAM tool demonstrated that our BloodNet can stably capture the unique local patterns of bloodstains with specific TSDs, suggesting the efficacy of the utilized attention mechanism in learning fine-grained representations for TSD inference. As a paired study for BloodNet, we further conducted a microscopic analysis using Raman spectroscopic data and a machine learning method based on Bayesian optimization. Although the experimental results show that such a new microscopic-level approach outperformed the state-of-the-art by a large margin, its inference accuracy is significantly lower than BloodNet, which further justifies the efficacy of deep learning techniques in the challenging task of bloodstain TSD inference. Our code is publically accessible via https://github.com/shenxiaochenn/BloodNet. Our datasets and pre-trained models can be freely accessed via https://figshare.com/articles/dataset/21291825.

Ubiquitin receptor PSMD4/Rpn10 is a novel therapeutic target in multiple myeloma.

PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.

Genome-wide association study identifies genetic risk loci for adiposity in a Taiwanese population.

Overweight and obese are risk factors for various diseases. In Taiwan, the combined prevalence of overweight and obesity has increased dramatically. Here, we conducted a genome-wide association study (GWAS) on four adiposity traits, including body-mass index (BMI), body fat percentage (BF%), waist circumference (WC), and waist-hip ratio (WHR), using the data for more than 21,000 subjects in Taiwan Biobank. Associations were evaluated between 6,546,460 single-nucleotide polymorphisms (SNPs) and adiposity traits, yielding 13 genome-wide significant (GWS) adiposity-associated trait-loci pairs. A known gene, FTO, as well as two BF%-associated loci (GNPDA2-GABRG1 [4p12] and RNU6-2-PIAS1 [15q23]) were identified as pleiotropic effects. Moreover, RALGAPA1 was found as a specific genetic predisposing factor to high BMI in a Taiwanese population. Compared to other populations, a slightly lower heritability of the four adiposity traits was found in our cohort. Surprisingly, we uncovered the importance of neural pathways that might influence BF%, WC and WHR in the Taiwanese (East Asian) population. Additionally, a moderate genetic correlation between the WHR and BMI (γg = 0.52; p = 2.37×10-9) was detected, suggesting different genetic determinants exist for abdominal adiposity and overall adiposity. In conclusion, the obesity-related genetic loci identified here provide new insights into the genetic underpinnings of adiposity in the Taiwanese population.

Deficiency of Cullin 3, a Protein Encoded by a Schizophrenia and Autism Risk Gene, Impairs Behaviors by Enhancing the Excitability of Ventral Tegmental Area (VTA) DA Neurons.

The dopaminergic neuromodulator system is fundamental to brain functions. Abnormal dopamine (DA) pathway is implicated in psychiatric disorders, including schizophrenia (SZ) and autism spectrum disorder (ASD). Mutations in Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex, have been associated with SZ and ASD. However, little is known about the function and mechanism of CUL3 in the DA system. Here, we show that CUL3 is critical for the function of DA neurons and DA-relevant behaviors in male mice. CUL3-deficient mice exhibited hyperactive locomotion, deficits in working memory and sensorimotor gating, and increased sensitivity to psychostimulants. In addition, enhanced DA signaling and elevated excitability of the VTA DA neurons were observed in CUL3-deficient animals. Behavioral impairments were attenuated by dopamine D2 receptor antagonist haloperidol and chemogenetic inhibition of DA neurons. Furthermore, we identified HCN2, a hyperpolarization-activated and cyclic nucleotide-gated channel, as a potential target of CUL3 in DA neurons. Our study indicates that CUL3 controls DA neuronal activity by maintaining ion channel homeostasis and provides insight into the role of CUL3 in the pathogenesis of psychiatric disorders.SIGNIFICANCE STATEMENT This study provides evidence that Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex that has been associated with autism spectrum disorder and schizophrenia, controls the excitability of dopamine (DA) neurons in mice. Its DA-specific heterozygous deficiency increased spontaneous locomotion, impaired working memory and sensorimotor gating, and elevated response to psychostimulants. We showed that CUL3 deficiency increased the excitability of VTA DA neurons, and inhibiting D2 receptor or DA neuronal activity attenuated behavioral deficits of CUL3-deficient mice. We found HCN2, a hyperpolarization-activated channel, as a target of CUL3 in DA neurons. Our findings reveal CUL3's role in DA neurons and offer insights into the pathogenic mechanisms of autism spectrum disorder and schizophrenia.

Single-cell transcriptomic analysis in clear cell renal cell carcinoma: Deciphering the role of APP within the tumour microenvironment.

Clear cell renal cell carcinoma (ccRCC) represents a significant challenge in oncology, primarily due to its resistance to conventional therapies. Understanding the tumour microenvironment (TME) is crucial for developing new treatment strategies. This study focuses on the role of amyloid precursor protein (APP) in tumour-associated macrophages (TAMs) within the ccRCC TME, exploring its potential as a prognostic biomarker. Basing TAM-related genes, the prognostic model was important to constructed. Employing advanced single-cell transcriptomic analysis, this research dissects the TME of ccRCC at an unprecedented cellular resolution. By isolating and examining the gene expression profiles of individual cells, particularly focusing on TAMs, the study investigates the expression levels of APP and their association with the clinical outcomes of ccRCC patients. The analysis reveals a significant correlation between the expression of APP in TAMs and patient prognosis in ccRCC. Patients with higher APP expression in TAMs showed differing clinical outcomes compared to those with lower expression. This finding suggests that APP could serve as a novel prognostic biomarker for ccRCC, providing insights into the disease progression and potential therapeutic targets. This study underscores the importance of single-cell transcriptomics in understanding the complex dynamics of the TME in ccRCC. The correlation between APP expression in TAMs and patient prognosis highlights APP as a potential prognostic biomarker. However, further research is needed to validate these findings and explore the regulatory mechanisms and therapeutic implications of APP in ccRCC.

Interaction between p21-activated kinase 4 and ß-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

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.

DLRAPom: a hybrid pipeline of Optimized XGBoost-guided integrative multiomics analysis for identifying targetable disease-related lncRNA-miRNA-mRNA regulatory axes.

The lack of a reliable and easy-to-operate screening pipeline for disease-related noncoding RNA regulatory axis is a problem that needs to be solved urgently. To address this, we designed a hybrid pipeline, disease-related lncRNA-miRNA-mRNA regulatory axis prediction from multiomics (DLRAPom), to identify risk biomarkers and disease-related lncRNA-miRNA-mRNA regulatory axes by adding a novel machine learning model on the basis of conventional analysis and combining experimental validation. The pipeline consists of four parts, including selecting hub biomarkers by conventional bioinformatics analysis, discovering the most essential protein-coding biomarkers by a novel machine learning model, extracting the key lncRNA-miRNA-mRNA axis and validating experimentally. Our study is the first one to propose a new pipeline predicting the interactions between lncRNA and miRNA and mRNA by combining WGCNA and XGBoost. Compared with the methods reported previously, we developed an Optimized XGBoost model to reduce the degree of overfitting in multiomics data, thereby improving the generalization ability of the overall model for the integrated analysis of multiomics data. With applications to gestational diabetes mellitus (GDM), we predicted nine risk protein-coding biomarkers and some potential lncRNA-miRNA-mRNA regulatory axes, which all correlated with GDM. In those regulatory axes, the MALAT1/hsa-miR-144-3p/IRS1 axis was predicted to be the key axis and was identified as being associated with GDM for the first time. In short, as a flexible pipeline, DLRAPom can contribute to molecular pathogenesis research of diseases, effectively predicting potential disease-related noncoding RNA regulatory networks and providing promising candidates for functional research on disease pathogenesis.