Biology subjects

Idris, A.

Publications and source records attributed to Idris, A..

11 recordsLinked to original sources

bioRxiv2025

Anti-Malaria Antibody Engineering Broadens Recognition Motifs and Reveals New Homotypic Interactions that Enhance Protective Breadth

The monoclonal antibody L9 mediates high-level protection against malaria in children for up to 6 months in Africa. L9 preferentially binds with high affinity to the NVDP minor repeat on the P. falciparum circumsporozoite protein (PfCSP). Here, we sought to improve the affinity of L9 to enhance protection against rare strains with two spatially separated minor repeats or a single minor repeat. Site saturation mutagenesis and yeast display-screening identified a panel of affinity-improved variants. In vivo challenge showed one variant, L9_yd19, to be modestly more potent against a chimeric transgenic Plasmodium encoding PfCSP with two widely spaced minor repeats from a Kenyan parasite strain, with no loss in potency against the benchmark 3D7 strain with its standard complement of minor repeats. L9_yd19 also had high affinity against NANP major repeats and was protective against transgenic Plasmodium with PfCSP containing only NANP major repeats (NANP12). Cryo-EM studies revealed L9_yd19 to recognize PfCSP with two distinct homotypic interfaces, which combined to yield two trimeric layers of antibodies comprising asymmetric trimers that dimerized in a head-to-head fashion. These data reveal a new antibody mechanism that utilizes interfaces involving dual homotypic symmetry elements, a 2-fold and an asymmetric 3-fold, for potentially improved malaria prevention. HIGHLIGHTSO_LIL9 is a highly protective antimalarial antibody that preferentially binds the NVDP minor repeat on Plasmodium falciparum circumsporozoite protein (PfCSP) and also binds with low affinity to the NANP major repeat; due to these targeting preferences, it has shown reduced protection against designed transgenic malaria strains with only a single NVDP motif (Fig. 1). C_LIO_LIUsing yeast display, a panel of L9 variants were generated based on higher affinity against the minor NVDP and major NANP motifs to determine if they could improve protection against strains with fewer minor repeat regions or only containing major repeats (Figs 1-4). C_LIO_LIOne L9 variant, L9_yd19 showed enhanced protection against chimeric transgenic CSP variants with a single minor repeat or two minor repeats in which the spacing was separated; L9_yd19 also showed protection against chimeric transgenic CSP variants containing only the NANP major repeat (Figs. 4-5). C_LIO_LICryo-EM analyses revealed L9_yd19 recognition of CSP to comprise two distinct homotypic interfaces: a side-to-side interface within asymmetric antibody trimer and a head-to-head interface between antibody trimers related by 2-fold symmetry that combined to yield a higher-order complex comprising two trimeric layers of antibodies (Figs. 6-7) C_LIO_LIStructure-function studies reveal a new antibody-based structural mechanism with dual homotypic interfaces mediating protection against varying numbers and spacing of minor repeats and major repeats. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/679367v1_fig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1978620org.highwire.dtl.DTLVardef@e6b84forg.highwire.dtl.DTLVardef@14e750eorg.highwire.dtl.DTLVardef@1e6ff51_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Improving affinity to target the CSP minor repeat enhances L9 antibody protective breadth. (A) The CSP minor repeat (NVDP) minor repeat is the principal binding target of the potent anti-malarial antibody L9. Genetic polymorphisms are present among P. falciparum strains in the number and spacing of NVDP motifs contained in the CSP repeat regions, and the loss of NPNV epitopes reduces the in vivo protective potency of the L9 antibody. (B) To enhance L9 affinity against the CSP antigen, multi-mutation libraries were generated from the L9 template and selectively screened against CSP peptide repeat motifs using yeast display. Libraries were first selected against Peptide22 (Pep22), which contains two minor and two major repeats, and subsequently selected against NTDS_5/3, a truncated CSP molecule that presents peptides in a more native-like conformation. Identified L9 variants were characterized for binding affinity in vitro and for protective potency in vivo against a panel of malaria antigens. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/679367v1_fig4.gif" ALT="Figure 4"> View larger version (59K): org.highwire.dtl.DTLVardef@844f00org.highwire.dtl.DTLVardef@5ebe9borg.highwire.dtl.DTLVardef@33d634org.highwire.dtl.DTLVardef@1342c8c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO In vitro and in vivo characterization reveals functional improvement for affinity-enhanced L9 variants against multiple transgenic strains, including the Kenya2-3 KI strain and a major repeat-only transgenic. (A) Biolayer interferometry (BLI) analysis against Peptide 22 (containing 2 NVDPs) and full-length CSP (fl_CSP) of P. falciparum 3D7. L9_WT affinity is marked with a vertical line on the legend bar. L9 variants marked with an asterisk (*) were selected for detailed in vivo characterization. (B) In the transgenic sporozoite intravenous (IV) challenge model, mice were administered IgG from L9 variants or control antibodies (-2 h), followed by IV challenge with P. berghei sporozoites (PbSPZ) encoding different PfCSP knock-in sequences (0 h), and subsequent luciferin-based evaluation (42 h). Each group comprised n=10 mice, with animals receiving the indicated antibody dose. (C) Transgenic PbSPZ challenge. Liver infection burden was quantified, and data are shown as mean {+/-} standard deviation; mean liver burden total flux for L9_WT at 12.5 {micro}g/mouse is indicated by a dashed line. Statistical significance between L9_WT and each L9 variant group at 12.5 {micro}g were determined using the ordinary one-way ANOVA test with a two-tailed P value calculation and a Dunnetts multiple test correction. Statistical significance was also analyzed between L9_yd19 and MAM01 using an unpaired t test with a two-tailed P value calculation; the two groups were significantly different when tested against 1 NVDP KI PbSPZ. See also Table S1 and Fig. S5. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/679367v1_fig5.gif" ALT="Figure 5"> View larger version (33K): org.highwire.dtl.DTLVardef@58bed2org.highwire.dtl.DTLVardef@580075org.highwire.dtl.DTLVardef@1864220org.highwire.dtl.DTLVardef@15f6582_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5.C_FLOATNO Affinity correlations reveal that improved protection breadth correlates with affinity to multiple malaria antigens. Correlation between binding affinity to PfCSP Kenya2-3 strain, NTDS_5/3 or PfCSP 3D7 strain (x-axis, linear scale) and normalized liver burden against various transgenic PbSPZs (y-axis, linear scale). Pearsons correlation coefficient (R) was calculated using the median normalized liver burden values, with corresponding two-tailed P-values and 95% confidence intervals. A linear regression was fit based on a Pearsons correlation analysis. See also Fig. S6. C_FIG O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig6.gif" ALT="Figure 6"> View larger version (46K): org.highwire.dtl.DTLVardef@14bf2org.highwire.dtl.DTLVardef@12123d9org.highwire.dtl.DTLVardef@b8b17aorg.highwire.dtl.DTLVardef@18c0195_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Structural analyses of L9_yd19 and PfCSP (3D7 or Kenya2-3) reveals a new interaction mode. (A) CryoEM structure of L9_yd19 in complex with PfCSP (3D7) at 3.4 [A] resolution. Heavy chain mutations are shown in red and light chain mutations are shown in blue. 27 residue stretch of PfCSP is ordered and the rest of the PfCSP is disordered. (B) CryoEM structure of L9_yd19 in complex with PfCSP (Kenya2-3) at 3.3 [A] resolution. 2D classes show both three L9_yd19 Fabs (orange arrow) and six L9_yd19 Fabs (red arrow) bound to PfCSP (Kenya2-3). 3D reconstruction for six L9_yd19 Fabs bound to PfCSP (Kenya2-3) is shown. C_FIG O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig7.gif" ALT="Figure 7"> View larger version (54K): org.highwire.dtl.DTLVardef@bbe52corg.highwire.dtl.DTLVardef@1c2c4b2org.highwire.dtl.DTLVardef@1ffc3a5org.highwire.dtl.DTLVardef@85ac9e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO L9_yd19 engineered mutations stabilize a new head-to-head homotypic interface allowing up to six Fabs to bind to PfCSP (Kenya2-3). (A) Structural comparison of L9_yd19 bound to PfCSP-3D7 and PfCSP-Kenya2-3. PfCSP is shown in red sticks with the N- and C-termini labeled. (B) L9_yd19 in complex with PfCSP-Kenya2-3. Fabs (3,4), (2,5), and (1,6) are related by 2-fold (C2) symmetry. Critical residues generating the head-to-head homotypic interface are shown in insets. C_FIG

immunology↗

bioRxiv2024

Encephalomyocarditis virus non-structural protein 2C degrades NDP52 autophagy protein to promote its own survival

Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen that causes encephalitis and myocarditis, primarily in pigs and in some mammals. Nuclear dot protein (NDP) 52 is an important autophagy adaptor protein that is known to target microbial pathogens, including viruses into autophagosomes to facilitate the selective autophagy process. Here, we wanted to investigate how an important zoonotic virus such as EMCV interacts with NDP52. We found that NDP52 negatively regulates the entry and replication phases of EMCV and interacts with EMCV VP1/VP2 proteins to mediate its autophagic degradation. EMCV counteracts this by exerting autophagy induction through its encoded 2C protein. The autophagy machinery then hijacks NDP52 and transports it to lysosomes for subsequent degradation via late endosomal molecules Rab7 and Rab9. Our study describes a novel mechanism by which EMCV escapes the hosts antiviral response to promote its survival by hijacking the autophagy pathway using the non-structural protein 2C of EMCV. ImportanceEMCV is an important zoonotic pathogen that can induce autophagy, but the regulatory role of autophagy receptors in EMCV infection has been rarely reported. Herein, we show that NDP52 is a novel host limiting factor for EMCV, which not only inhibits early EMCV endocytosis, but also restricts EMCV replication in vitro by controlling autophagic degradation of EMCV structural proteins VP1 and VP2. In order to counteract this, EMCV uses the N-terminal region of its non-structural protein 2C to interact with NDP52, and hijacks the autophagy machine to transport NDP52 to lysosomes for degradation, which weakens NDP52-mediated degradation of VP1 and VP2. Importantly, we reveal a novel mechanism by which EMCV evades the host antiviral response, contributing to a deeper understanding of the infectious mechanism of EMCV and providing new ideas for the design of antiviral drug targets.

microbiology↗

bioRxiv2023

De novo design of allosterically switchable protein assemblies

Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central role in the control of metabolism and cell signaling1-3. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such "action at a distance" modulation and to create synthetic proteins whose functions can be regulated by effectors4-7. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge8,9. Here, inspired by the classic Monod-Changeux-Wyman model of cooperativity10, we investigate the de novo design of allostery through rigid-body coupling of designed effector-switchable hinge modules11 to protein interfaces12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to effector binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry13, and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of effectors and can have ligand-binding cooperativity comparable to classic natural systems such as hemoglobin14. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized sidechain-sidechain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines, and cellular feedback control circuitry.

biochemistry↗

bioRxiv2023

ACE2-Coated Virus-Like Particles Effectively Block SARS-CoV-2 Infection

A large body of research accumulated over the past three years dedicated to our understanding and fighting COVID-19. Blocking the interaction between SARS-CoV-2 Spike and ACE2 receptor has been considered an effective strategy as anti-SARS-CoV-2 therapeutics. In this study, we developed ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus. These ACE2-VLPs exhibited high neutralization capacity even when applied at low doses, and displayed superior efficacy compared to extracellular vesicles carrying ACE2, in the in vitro pseudoviral assays. ACE2-VLPs were stable under different environmental temperatures, and they were effective in blocking all tested variants of concern in vitro. Finally, ACE2-VLPs displayed marked neutralization capacity against Omicron BA.1 in the Vero E6 cells. Based on their superior efficacy compared to extracellular vesicles, and their demonstrated success against live virus, ACE2-VLPs can be considered as vital candidates for treating SARS-CoV-2. This novel therapeutic approach of VLP coating with receptor particles can serve as proof-of-concept for designing effective neutralization strategies for other viral diseases in the future. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/558424v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@30ac94org.highwire.dtl.DTLVardef@1926217org.highwire.dtl.DTLVardef@165d9f0org.highwire.dtl.DTLVardef@1c737f9_HPS_FORMAT_FIGEXP M_FIG In our study, we demonstrate the prevention of SARS-CoV-2 infection through the use of Ace2-coated VLPs. C_FIG

bioengineering↗

bioRxiv2023

Design of stimulus-responsive two-state hinge proteins

Proteins that switch between two structural states as a function of environmental stimuli are widespread in nature. These proteins structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Engineering challenges ranging from biological computing devices to molecular motors require such two-state switches, but designing these is an unsolved problem as it requires sculpting an energy landscape with two low-energy but structurally distinct conformations that can be modulated by external inputs. Here we describe a general design approach for creating "hinge" proteins that populate one distinct state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, and double electron-electron resonance spectroscopy demonstrate that despite the significant structural differences, the two states are designed with atomic level accuracy. The kinetics and thermodynamics of effector binding can be finely tuned by modulating the free energy differences between the two states; when this difference becomes sufficiently small, we obtain bistable proteins that populate both states in the absence of effector, but collapse to a single state upon effector addition. Like the transistor, these switches now enable the design of a wide array of molecular information processing systems.

biophysics↗

bioRxiv2022

Intranasal delivery of lipid nanoparticle encapsulated SARS-CoV-2 and RSV-targeting siRNAs reduces lung infection

RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) in infected mouse lungs. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The studies presented here demonstrates an attractive alternate therapeutic delivery strategy for the treatment of both future and emerging respiratory viral diseases.

microbiology↗

bioRxiv2022

Visual detection of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.

Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity, called CluMPS (Clusters Magnified by Phase Separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context.

bioengineering↗

bioRxiv2022

Genomic Insights of Bruneian Malays

The Malays and their many sub-ethnic groups collectively make up one of the largest population groups in Southeast Asia. However, their genomes, especially those from Brunei, remain very much underrepresented and understudied. Here, we analysed the publicly available WGS and genotyping data of two and 39 Bruneian Malay individuals, respectively. NGS reads from the two individuals were first mapped against the GRCh38 human reference genome and their variants called. Of the total [~]5.28 million short nucleotide variants and indels identified, [~]217K of them were found to be novel; with some predicted to be deleterious and associated with risk factors of common non-communicable diseases in Brunei. Unmapped reads were next mapped against the recently reported novel Chinese and Japanese genomic contigs and de novo assembled. [~]227 Kbp genomic sequences missing in GRCh38 and a partial open reading frame encoding a potential novel small zinc finger protein were successfully discovered. Interestingly, although the Malays in Brunei, Singapore and Malaysia share >83% common variants, principal component and admixture analysis comparing the genetic structure of the local Malays against other Asian population groups suggested that they are genetically closer to some Filipino ethnic groups than the Malays in Malaysia and Singapore. Taken together, our work provides the first comprehensive insight into the genomes of the Bruneian Malay population.

genomics↗

bioRxiv2021

Complete protection by a single dose skin patch delivered SARS-CoV-2 spike vaccine

SARS-CoV-2 has infected over 160 million people and resulted in more than 3.3 million deaths, and we still face many challenges in the rollout of vaccines. Here, we use the high-density microarray patch to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show the vaccine, dry-coated on the patch is thermostable, and delivery of spike via HD-MAP induced greater cellular and antibody immune responses, with serum able to potently neutralize clinically relevant isolates including those from the B.1.1.7 and B.1.351 lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge, demonstrating that HD-MAP delivery of a SARS-CoV-2 vaccine is superior to traditional needle-and-syringe vaccination and has the potential to greatly impact the ongoing COVID-19 pandemic.

microbiology↗

bioRxiv2021

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

synthetic biology↗

bioRxiv2018

Development of novel lipidic particles for siRNA delivery that are highly effective after 12 months storage

Liposomes are versatile and well-proven as a means to deliver nucleic acids into cells. Most of the formulation procedures used are labour intensive and result in unstable end products. We have previously reported on the development of a simple, yet efficient, hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles. Here we show that the particles are stable up to 12 months after storage room temperature (RT), 4{degrees}C or - 20{degrees}C. While RT storage results in changes in particle size and polydispersity, gene silencing of all particles was similar to freshly prepared particles following storage for 3, 6, 9 or 12 months at all temperatures. This is the first report of such long-term stability in siRNA-loaded liposomes.

biochemistry↗