Peptide Linker Research Articles

Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed.

The outcomes of unresectable gastric cancer (GC) are unfavorable even with chemotherapy; therefore, a new treatment modality is required. The combination of an oncolytic virus and photodynamic therapy can be one of the promising modalities to overcome this. Mammalian orthoreovirus (MRV) is an oncolytic virus that has been used in clinical trials for several cancers. In this study, we developed and evaluated a recombinant MRV strain type 3 Dearing (T3D) that expresses membrane-targeting KillerRed (KRmem), a phototoxic fluorescent protein that produces cytotoxic reactive oxygen species upon light irradiation. KRmem was fused in-frame to the 3' end of the σ2 viral gene in the S2 segment using a 2A peptide linker, enabling the expression of multiple proteins from a single transcript. RNA electrophoresis, Western blotting, and immunofluorescence analyses confirmed functional insertion of KRmem into the recombinant virus. The growth activity of the recombinant virus was comparable to that of the wild-type MRV in a cultured cell line. The recombinant virus infected two GC cell lines (MKN45P and MKN7), and a significant cytocidal effect was observed in MKN45P cells infected with the recombinant virus after light irradiation. Thus, recombinant MRV-expressing KRmem has the potential to serve as a novel treatment tool for GC.

A lipid/PLGA nanocomplex to reshape tumor immune microenvironment for colon cancer therapy.

Immune checkpoint blockade therapy provides a new strategy for tumor treatment; however, the insufficient infiltration of cytotoxic T cells and immunosuppression in tumor microenvironment lead to unsatisfied effects. Herein, we reported a lipid/PLGA nanocomplex (RDCM) co-loaded with the photosensitizer Ce6 and the indoleamine 2,3-dioxygenase (IDO) inhibitor 1MT to improve immunotherapy of colon cancer. Arginine-glycine-aspartic acid (RGD) as the targeting moiety was conjugated on 1,2-distearoyl-snglycero-3-phosphoethanolamine lipid via polyethylene glycol (PEG), and programmed cell death-ligand 1 (PD-L1) peptide inhibitor DPPA (sequence: CPLGVRGK-GGG-d(NYSKPTDRQYHF)) was immobilized on the terminal group of PEG via matrix metalloproteinase 2 sensitive peptide linker. The Ce6 and 1MT were encapsulated in PLGA nanoparticles. The drug loaded nanoparticles were composited with RGD and DPPA modified lipid and lecithin to form lipid/PLGA nanocomplexes. When the nanocomplexes were delivered to tumor, DPPA was released by the cleavage of a matrix metalloproteinase 2-sensitive peptide linker for PD-L1 binding. RGD facilitated the cellular internalization of nanocomplexes via avβ3 integrin. Strong immunogenic cell death was induced by 1O2 generated from Ce6 irradiation under 660 nm laser. 1MT inhibited the activity of IDO and reduced the inhibition of cytotoxic T cells caused by kynurenine accumulation in the tumor microenvironment. The RDCM facilitated the maturation of dendritic cells, inhibited the activity of IDO, and markedly recruited the proportion of tumor-infiltrating cytotoxic T cells in CT26 tumor-bearing mice, triggering a robust immunological memory effect, thus effectively preventing tumor metastasis. The results indicated that the RDCM with dual IDO and PD-L1 inhibition effects is a promising platform for targeted photoimmunotherapy of colon cancer.

Modular and automated synthesis of oligonucleotide-small molecule conjugates for cathepsin B mediated traceless release of payloads.

The reversible attachment of small molecules to oligonucleotides provides versatile tools for the development of improved oligonucleotide therapeutics. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement for traceless release of the payload in vivo. Herein, we describe a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB) for the automated synthesis of oligonucleotide-small molecule conjugates. Val-Ala(NB) was protected by a photolabile 2-nitrobenzyl group to improve the stability of the peptide linker during DNA synthesis. Intracellular cathepsin B digests the dipeptide efficiently, releasing the payload-phosphate which is converted to the free payload by endogenous phosphatase enzymes. With the advantages of modular synthesis and stimuli-responsive drug release, we believe Val-Ala(NB) will be a potentially valuable cleavable linker for use in oligonucleotide-drug conjugates.

Advancing one health vaccination: In silico design and evaluation of a multi-epitope subunit vaccine against Nipah virus for cross-species immunization using immunoinformatics and molecular modeling.

The resurgence of the Nipah virus (NiV) in 2023 has raised concerns for another potentially severe pandemic, given its history of high mortality from previous outbreaks. Unfortunately, no therapeutics and vaccines have been available for the virus. This study used immunoinformatics and molecular modeling to design and evaluate a multi-epitope subunit vaccine targeting NiV. The designed vaccine construct aims to stimulate immune responses in humans and two other intermediate animal hosts of the virus-swine and equine. Using several epitope prediction tools, ten peptides that induced B-lymphocyte responses, 17 peptides that induced cytotoxic T-lymphocyte (CTL) responses, and 12 peptides that induced helper T-lymphocyte (HTL) responses were mapped from nine NiV protein sequences. However, the CTL and HTL-inducing peptides were reduced to ten and eight, respectively, following molecular docking and dynamics. These screened peptides exhibited stability with 30 common major histocompatibility complex (MHC) receptors found in humans, swine, and equine. All peptides were linked using peptide linkers to form the multi-epitope construct and various adjuvants were tested to enhance its immunogenicity. The vaccine construct with resuscitation-promoting factor E (RpfE) adjuvant was selected as the final design based on its favorable physicochemical properties and superior immune response profile. Molecular docking was used to visualize the interaction of the vaccine to toll-like receptor 4 (TLR4), while molecular dynamics confirmed the structural stability of this interaction. Physicochemical property evaluation and computational simulations showed that the designed vaccine construct exhibited favorable properties and elicited higher antibody titers than the six multi-epitope NiV vaccine designs available in the literature. Further in vivo and in vitro experiments are necessary to validate the immunogenicity conferred by the designed vaccine construct and its epitope components. This study demonstrates the capability of computational methodologies in rational vaccine design and highlights the potential of cross-species vaccination strategies for mitigating potential NiV threats.

The Potential of Single-Chain Variable Fragment Antibody: Role in Future Therapeutic and Diagnostic Biologics.

The advancement of genetic engineering has revolutionized the field of immunology by allowing the utilization of intrinsic antibody structures. One of the biologics that are being produced by recombinant antibody technology is single-chain fragments variable (scFv). Genes of variable regions, the heavy and light chains that are genetically linked into a single transcript by a short flexible linker peptide, are used to generate this fragment from cellular and synthetic libraries. The specificity and affinity of these molecules are comparable to those of parental antibodies. Fusion with marker proteins and other potent molecules improves their stability, circulation half-life, activity, and efficient purification. Besides, this review comprises construction protocols, therapeutics, and diagnostic applications of scFv, as well as related challenges. Nonetheless, there are still issues with efficacy, stability, safety, intracellular administration, and production costs that need to be addressed.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx.

Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx

AbstractBivalency is a prevalent natural mechanism to enhance receptor avidity. Various two‐domain disulfide‐rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high‐resolution solution structure of DkTx, revealing residual linker order stabilised by linker‐domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre‐organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre‐configured two‐domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

The flexible linker and CotG were more effective for the spore surface display of keratinase KERQ7.

Feather, horn, hoof, and other keratin waste are protein-rich but limited by natural keratinase synthesis, activity, pH, and temperature stability. It is challenging to realize its large-scale application in industries. Bacillus subtilis spores are a safe, efficient, and highly resistant immobilized carrier, which can improve target proteins' resistance. In this research, KERQ7, the keratinase gene of Bacillus tequilensis strain Q7, was fused to the Bacillus subtilis genes coding for the coat proteins CotG and CotB, respectively, and displayed on the surface of B. subtilis spores. Compared with the free KERQ7, the immobilized KERQ7 showed a greater pH tolerance and heat resistance on the spore surface. The activity of CotG-KERQ7 is 1.25 times that of CotB-KERQ7, and CotG-KERQ7 is more stable. When the flexible linker peptide L3 was used to connect CotG and KERQ7, the activity was increased to 131.2 ± 3.4%, and the residual enzyme activity was still 62.5 ± 2.2% after being kept at 60 ℃ for 4h. These findings indicate that the flexible linker and CotG were more effective for the spore surface display of keratinase to improve stress resistance and promote its wide application in feed, tanning, washing, and other industries.

Abstract C113: Discovery of a novel auristatin antibody drug conjugate drug linker with equal efficacy and reduced bone marrow toxicity compared to vedotin

Abstract There are four vedotin drug linker containing antibody drug conjugates currently approved for treating cancer patients, proving that this technology platform is an effective modality for the treatment of both hematologic and solid tumors. The primary mechanism of vedotin ADCs, direct cytotoxicity, is the result of intracellular release of monomethyl auristatin E (MMAE) after cleavage of the valine-citrulline (Val-Cit) dipeptide by lysosomal proteases. However, off-tumor delivery of MMAE can result in dose limiting bone marrow toxicity in preclinical models. Maintaining potency in cancer cells while reducing the bone marrow toxicity of vedotin ADCs could allow higher or more frequent clinical dosing and potentially improved response rates and patient outcomes. To decrease bone marrow toxicity of MMAE empowered ADCs, we developed a process to identify peptidic linker systems that provide selectivity for cancer cells over bone marrow cells. From this process we identified a tripeptide sequence (D-leucine-alanine-glutamate, dLAE) that is preferentially cleaved in cancer cells and used it to develop a drug linker that releases MMAE upon cleavage of this tripeptide. Here, we present in vitro and in vivo data comparing ADCs containing vedotin and dLAE tripeptide drug linkers. While ADCs with the dLAE drug linker had comparable in vitro cytotoxicity and antitumor activity to vedotin, the dLAE ADCs had a higher maximum tolerated dose in exploratory rat and cynomolgus macaque toxicity studies due to a reduction in bone marrow toxicity. Reduced bone marrow toxicity was correlated with decreased linker catabolism in bone marrow cells for conjugates bearing the dLAE tripeptide compared to Val-Cit, while similar catabolism was observed in cancer cells. Together, these preclinical data highlight that changing the peptide of the vedotin drug linker from Val-Cit to dLAE can yield ADCs with an improved preclinical therapeutic window. Given the preclinical characteristics of the dLAE drug linker, a clinical trial is being planned to evaluate an anti-CD30 ADC (SGN-35T). Citation Format: Noah A Bindman, Roma Yumul, Erica E McKinney, Scott Blackburn, Nicole Blesie, Xinqun Zhang, Brendan Drouhard, Sarah Anderson, Calvin Neace, David Ortiz, KC Crowder, Nancy Everds, Chris Carosino, Forgivemore Magunda, Django Sussman, Esther Trueblood, Nicole M. Okeley, Peter Senter. Discovery of a novel auristatin antibody drug conjugate drug linker with equal efficacy and reduced bone marrow toxicity compared to vedotin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C113.

Synthesis of Fluorescent Cyclic Peptides via Gold(I)-Catalyzed Macrocyclization.

Rapid and efficient cyclization methods that form structurally novel peptidic macrocycles are of high importance for medicinal chemistry. Herein, we report the first gold(I)-catalyzed macrocyclization of peptide-EBXs (ethynylbenziodoxolones) via C2-Trp C-H activation. This reaction was carried out in the presence of protecting group free peptide sequences and is enabled by a simple commercial gold catalyst (AuCl·Me2S). The method displayed a rapid reaction rate (within 10 min), wide functional group tolerance (27 unprotected peptides were cyclized), and up to 86% isolated yield. The obtained highly conjugated cyclic peptide linker, formed through C-H alkynylation, can be directly applied to live-cell imaging as a fluorescent probe without further attachment of fluorophores.

The crystal structure of insecticidal protein Txp40 from Xenorhabdus nematophila reveals a two-domain unique binary toxin with homology to the toxin-antitoxin (TA) system

Txp40 is a ubiquitous, conserved, and novel toxin from Xenorhabdus and Photorhabdus bacteria, toxic to a wide range of insect pests. However, the three-dimensional structure and toxicity mechanism for Txp40 or any of its sequence homologs are not yet known. Here, we are reporting the crystal structure of the insecticidal protein Txp40 from Xenorhabdus nematophila at 2.08 Å resolution. The Txp40 was structurally distinct from currently known insecticidal proteins. Txp40 consists of two structurally different domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), primarily joined by a 33-residue long linker peptide. Txp40 displayed proteolytic propensity. Txp40 gets proteolyzed, removing the linker peptide, which is essential for proper crystal packing. NTD adopts a novel fold composed of nine amphipathic helices and has no shared sequence or structural homology to any known proteins. CTD has structural homology with RNases of type II toxin-antitoxin (TA) complex belonging to the RelE/ParE toxin domain superfamily. NTD and CTD were individually toxic to Galleria mellonella larvae. However, maximal toxicity was observed when both domains were present. Our results suggested that the Txp40 acts as a two-domain binary toxin, which is unique and different from any known binary toxins and insecticidal proteins. Txp40 is also unique because it belongs to the prokaryotic RelE/ParE toxin family with a toxic effect on eukaryotic organisms, in contrast to other members of the same family. Broad insect specificity and unique binary toxin complex formation make Txp40 a viable candidate to overcome the development of resistance in insect pests.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Vaccinomics-aided next-generation novel multi-epitope-based vaccine engineering against multidrug resistant Shigella Sonnei: Immunoinformatics and chemoinformatics approaches.

Shigella sonnei is a gram-negative bacterium and is the primary cause of shigellosis in advanced countries. An exceptional rise in the prevalence of the disease has been reported in Asia, the Middle East, and Latin America. To date, no preventive vaccine is available against S. sonnei infections. This pathogen has shown resistances towards both first- and second-line antibiotics. Therefore, an effective broad spectrum vaccine development against shigellosis is indispensable. In the present study, vaccinomics-aided immunoinformatics strategies were pursued to identify potential vaccine candidates from the S. sonnei whole proteome data. Pathogen essential proteins that are non-homologous to human and human gut microbiome proteome set, are feasible candidates for this purpose. Three antigenic outer membrane proteins were prioritized to predict lead epitopes based on reverse vaccinology approach. Multi-epitope-based chimeric vaccines was designed using lead B- and T-cell epitopes combined with suitable linker and adjuvant peptide sequences to enhance immune responses against the designed vaccine. The SS-MEVC construct was prioritized based on multiple physicochemical, immunological properties, and immune-receptors docking scores. Immune simulation analysis predicted strong immunogenic response capability of the designed vaccine construct. The Molecular dynamic simulations analysis ensured stable molecular interactions of lead vaccine construct with the host receptors. In silico restriction and cloning analysis predicted feasible cloning capability of the SS-MEVC construct within the E. coli expression system. The proposed vaccine construct is predicted to be more safe, effective and capable of inducing robust immune responses against S. sonnei infections and may be worthy of examination via in vitro/in vivo assays.

Revealing Novel-Strain-Specific and Shared Epitopes of Infectious Bronchitis Virus Spike Glycoprotein Using Chemical Linkage of Peptides onto Scaffolds Precision Epitope Mapping.

The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.

In Silico Prediction of Plasmodium falciparum PfRipr Epitopes as Vaccine Candidates

Abstract Background The global malarial death toll in 2020 was ~627,000, 77% of which occurred in children <5 years of age. Plasmodium falciparum is the most lethal parasite of its genus but has evaded vaccine development due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise due to their ability to target dominant and/or conserved regions in highly antigenically variable pathogens, including malaria. The recently characterized P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. (Figure 1) We sought to analyze P. falciparum sequences from highly endemic areas using bioinformatic approaches that identify candidate PfRipr epitope-based bloodstage vaccine antigens for inclusion in a multiantigen vaccine. Figure 1: Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. (https://doi.org/10.1038/s41564-022-01261-2 , https://creativecommons.org/licenses/by/4.0/) Methods Using two P. falciparum sequence datasets from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes for T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan and NetMHCIIpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. (Figure 2) Putative epitopes were filtered using binding thresholds, allele coverage, conservation, antigenicity, and allergenicity. (Figure 3) Figure 2: Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction. Figure 3: Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope). Results The Burkina Faso PfRipr dataset predicted 1729 unique T-cell and 51 unique B-cell epitopes, and 28 T-cell and 4 B-cell epitopes remained post-filtration. Epitope prediction using the Ugandan dataset predicted 576 unique T-cell and 53 unique B-cell epitopes, and 57 T-cell and 7 B-cell epitopes remained post-filtration. Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. (Tables 1-2, Figure 4) These epitopes were used to design a multi-epitope-based vaccine construct with a Flagellin adjuvant and peptide linkers to separate functional regions for immune processing. (Figure 5) Figure 4: 3D model of PfRipr with labeled B-cell epitopes. Predicted tertiary structure adapted from AlphaFold database. (https://doi.org/10.1038/s41586-021-03819-2, https://doi.org/10.1093/nar/gkab1061, https://creativecommons.org/licenses/by/4.0/) Figure 5: Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Conclusions Immunoinformatic tools employed in sequence identified candidate PfRipr epitopes used to design a bloodstage vaccine construct against P. falciparum. To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to a larger database of diverse P. falciparum sequences collected from other malaria endemic regions to develop vaccines effective against circulating strains.

Putative novel outer membrane antigens multi-epitope DNA vaccine candidates identified by Immunoinformatic approaches to control Acinetobacter baumannii

Multi-epitope polypeptide vaccines, a fusion protein, often have a string-of-beads system composed of various specific peptide epitopes, potential adjuvants, and linkers. When choosing the sequence of various segments and linkers, many alternatives are available. These variables can influence the vaccine’s effectiveness through their effects on physicochemical properties and polypeptide tertiary structure.The most conserved antigens were discovered using BLASTn. To forecast the proteins’ subcellular distribution, PSORTb 3.0.2 was used. Vaxign was used for the preliminary screening and antigenicity assessment. Protein solubility was also predicted using the ccSOL omics. Using PRED-TMBB, it was anticipated that the protein would localize across membranes. The IEDB and BepiPred-2.0 databases were used to predict the immunogenicity of B cell epitopes. A multi-epitope construct was developed and analyzed to evaluate. Twenty epitopes from A. baumannii’s outer membrane protein (omp) were included in the vaccination. TLR4 agonist explosibility was investigated. The physicochemical characteristics, secondary and tertiary structures, and B-cell epitopes of vaccine constructs were assessed. Additionally, docking and MD experiments were used to examine the relationship between TLR4 and its agonist.Thirteen antigens were discovered, and eight of the 13 chosen proteins were predicted to be surface proteins. The 34 kDa outer membrane protein, Omp38, Omp W, CarO, putative porin, OmpA, were chosen as having the right antigenicity (≥0.5). FhuE and CdiA were eliminated from further study because of their low antigenicity. The vaccine design was developed by combining the most effective 10 B-cell and 10 MHC-I/MHCII combined coverage epitopes. The molecular formula of the vaccine was determined to be C1718H2615N507O630S17. The vaccine form has a molecular weight of 40,996.70 Da and 47 negatively charged residues (Asp + Glu), whereas 28 positively charged residues (Arg + Lys). The estimated half-life was 7.2 hours (mammalian reticulocytes, in vitro), > 20 hours (yeast, in vivo) and > 10 hours (Escherichia coli, in vivo) for the vaccine. The multi-epitope vaccine insertion is carried via the expression vector pcDNA3.1 (+).The multi-epitope vaccine may stimulate humoral and cellular immune responses, according to our findings, and it may be a candidate for an A. baumannii vaccine.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

Immunoinformatics-based multi-epitope containing fused polypeptide vaccine design against visceral leishmaniasis with high immunogenicity and TLR binding

Visceral leishmaniasis (VL) is the most lethal among all leishmaniasis diseases and remains categorized as a neglected tropical disease (NTD). This study aimed to develop a peptide-based multi-epitope vaccine construct against VL using immunoinformatics methodologies. To achieve this, four distinct proteins were screened to identify peptides consisting of 9–15 amino acids with high binding affinity to toll-like receptors (TLRs), strong antigenicity, low allergenicity, and minimal toxicity. The resulting multi-epitope vaccine construct was fused in a tandem arrangement with appropriate linker peptides and exhibited superior properties related to cytotoxic T lymphocytes (CTLs), helper T lymphocytes (HTLs), and B-cell epitopes. Subsequently, a three-dimensional (3D) model of the vaccine construct was generated, refined, and validated for structural stability and immune response capabilities. Molecular docking and simulations confirmed the vaccine construct's stability and binding affinities with TLRs, with TLR4 displaying the highest binding affinity, followed by TLR2 and TLR3. Additionally, simulations predicted robust cellular and humoral antibody-mediated immune responses elicited by the designed vaccine construct. Notably, this vaccine construct includes proteins from various pathways of Leishmania donovani (LD), which have not been previously utilized in VL vaccine design. Thus, this study opens new avenues for the development of vaccines against diverse protozoan diseases.

High-Affinity Host-Guest Recognition for Efficient Assembly and Enzymatic Responsiveness of DNA Nanostructures.

The combination of multiple orthogonal interactions enables hierarchical complexity in self-assembled nanoscale materials. Here, efficient supramolecular polymerization of DNA origami nanostructures is demonstrated using a multivalent display of small molecule host-guest interactions. Modification of DNA strands with cucurbit[7]uril (CB[7]) and its adamantane guest, yielding a supramolecularcomplex with an affinity of order 1010 m-1 , directs hierarchical assembly of origami monomers into 1D nanofibers. This affinity regime enables efficient polymerization; a lower-affinity β-cyclodextrin-adamantane complex does not promote extended structures at a similar valency. Finally, the utility of the high-affinity CB[7]-adamantane interactions is exploited to enable responsive enzymatic actuation of origami nanofibers assembled using peptide linkers. This work demonstrates the power of high-affinity CB[7]-guest recognition as an orthogonal axis to drive self-assembly in DNA nanotechnology.

A bifunctional anti-PCSK9 scFv/Exendin-4 fusion protein exhibits enhanced lipid-lowering effects via targeting multiple signaling pathways in HFD-fed mice

Fusion protein which encompasses more than one functional component, has become one of the most important representatives of macromolecular drugs for disease treatment since that monotherapy itself might not be effective enough to eradicate the disease. In this study, we sought to construct a bifunctional antibody fusion protein by fusing anti-PCSK9 scFv with Exendin-4 for simultaneously lowering both LDL-C and TG. Firstly, three Ex4-anti-PCSK9 scFv fusion proteins were constructed by genetically connecting the C-terminal of Exendin-4 to the N-terminal of anti-PCSK9 scFv through various flexible linker peptides (G4S)n (n = 2, 3, 4). After soluble expression in E. coli, the most potent Ex4-(G4S)4-anti-PCSK9 scFv fusion protein was selected based on in vitro activity assays. Then, we investigated the in vivo therapeutic effects of Ex4-(G4S)4-anti-PCSK9 scFv on the serum lipid profile and bodyweight changes as well as underlying molecular mechanism in HFD-fed C57BL/6 mice. The data showed that Ex4-(G4S)4-anti-PCSK9 scFv exhibits enhanced effects of lowering both LDL-C and TG in serum, reducing food intake and body weight via blocking PCSK9/LDLR, activating AMPK/SREBP-1 pathways, and up-regulating sirt6. Conclusively, Ex4-(G4S)4-anti-PCSK9 has the potential to serve as a promising therapeutic agent for effectively treating dyslipidemia with high levels of both LDL-C and TG.