Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells.

Molecular vaccines comprising antigen peptides and inflammatory cues make up a class of therapeutics that promote immunity against cancer and pathogenic diseases but often exhibit limited efficacy. Here, we engineered an antigen peptide delivery system to enhance vaccine efficacy by targeting dendritic cells and mediating cytosolic delivery. The delivery system consists of the nontoxic anthrax protein, protective antigen (PA), and a single-chain variable fragment (scFv) that recognizes the XCR1 receptor on dendritic cells (DCs). Combining these proteins enabled selective delivery of the N-terminus of lethal factor (LFN) into XCR1-positive cross-presenting DCs. Incorporating immunogenic epitope sequences into LFN showed selective protein translocation in vitro and enhanced the priming of antigen-specific T cells in vivo. Administering DC-targeted constructs with tumor antigens (Trp1/gp100) into mice bearing aggressive B16-F10 melanomas improved mouse outcomes when compared to free antigen, including suppressed tumor growth up to 58% at 16 days post tumor induction (P < 0.0001) and increased survival (P = 0.03). These studies demonstrate that harnessing DC-targeting anthrax proteins for cytosolic antigen delivery significantly enhances the immunogenicity and antitumor efficacy of cancer vaccines.

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d-Phe-6, and the N-Terminus Enabled by S-Lipidation.

With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol-ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics.

Synthetic vaccines targeting Mincle through conjugation of trehalose dibehenate.

The covalent fusion of immunostimulatory adjuvants to immunogenic antigens is a promising strategy for the development of effective synthetic vaccines for infectious diseases. Herein, we describe the conjugation of a mycobacterial peptide antigen from the 6 kDa early secretory antigenic target (ESAT6) to a suitably functionalised trehalose dibehenate (TDB), a potent glycolipid adjuvant targeting macrophage inducible C-type lectin (Mincle).

Generation of oligonucleotide conjugates via one-pot diselenide-selenoester ligation-deselenization/alkylation.

A breadth of strategies are needed to efficiently modify oligonucleotides with peptides or lipids to capitalize on their therapeutic and diagnostic potential, including the modulation of in vivo chemical stability and for applications in cell-targeting and cell-permeability. The chemical linkages typically used in peptide oligonucleotide conjugates (POCs) have limitations in terms of stability and/or ease of synthesis. Herein, we report an efficient method for POC synthesis using a diselenide-selenoester ligation (DSL)-deselenization strategy that rapidly generates a stable amide linkage between the two biomolecules. This conjugation strategy is underpinned by a novel selenide phosphoramidite building block that can be incorporated into an oligonucleotide by solid-phase synthesis to generate diselenide dimer molecules. These can be rapidly ligated with peptide selenoesters and, following in situ deselenization, lead to the efficient generation of POCs. The diselenide within the oligonucleotide also serves as a flexible functionalisation handle that can be leveraged for fluorescent labelling, as well as for alkylation to generate micelles.

Chemical Synthesis and Semisynthesis of Lipidated Proteins.

Lipidation is a ubiquitous modification of peptides and proteins that can occur either co- or post-translationally. An array of different lipid classes can adorn proteins and has been shown to influence a number of crucial biological activities, including the regulation of signaling, cell-cell adhesion events, and the anchoring of proteins to lipid rafts and phospholipid membranes. Whereas nature employs a range of enzymes to install lipid modifications onto proteins, the use of these for the chemoenzymatic generation of lipidated proteins is often inefficient or impractical. An alternative is to harness the power of modern synthetic and semisynthetic technologies to access lipid-modified proteins in a pure and homogeneously modified form. This Review aims to highlight significant advances in the development of lipidation and ligation chemistry and their implementation in the synthesis and semisynthesis of homogeneous lipidated proteins that have enabled the influence of these modifications on protein structure and function to be uncovered.

Immunological Assessment of Lung Responses to Inhalational Lipoprotein Vaccines Against Bacterial Pathogens.

Lipopeptides or lipoproteins show potential as safe and effective subunit vaccines for protection against bacterial pathogens. Provided suitable adjuvants are selected, such as the TLR2-stimulating molecules Pam2Cys and Pam3Cys, these may be formulated as inhalational vaccines to optimize localized pulmonary immune responses. Here, we present methods to assess antigen-specific memory lymphocyte responses to novel vaccines, with a focus on immune responses in the lung tissue and bronchoalveolar space. We describe detection of T-cell responses via leukocyte restimulation, followed by intracellular cytokine staining and flow cytometry, enzyme-linked immunosorbent spot assay (ELISpot), and sustained leukocyte restimulation for detection of antigen-specific memory responses. We also detail assessment of antibody responses to vaccine antigens, via enzyme-linked immunosorbent assay (ELISA)-based detection. These methods are suitable for testing a wide range of pulmonary vaccines.

An in vivo selection-derived d-peptide for engineering erythrocyte-binding antigens that promote immune tolerance.

When displayed on erythrocytes, peptides and proteins can drive antigen-specific immune tolerance. Here, we investigated a straightforward approach based on erythrocyte binding to promote antigen-specific tolerance to both peptides and proteins. We first identified a robust erythrocyte-binding ligand. A pool of one million fully d-chiral peptides was injected into mice, blood cells were isolated, and ligands enriched on these cells were identified using nano-liquid chromatography-tandem mass spectrometry. One round of selection yielded a murine erythrocyte-binding ligand with an 80 nM apparent dissociation constant, Kd We modified an 83-kDa bacterial protein and a peptide antigen derived from ovalbumin (OVA) with the identified erythrocyte-binding ligand. An administration of the engineered bacterial protein led to decreased protein-specific antibodies in mice. Similarly, mice given the engineered OVA-derived peptide had decreased inflammatory anti-OVA CD8+ T cell responses. These findings suggest that our tolerance-induction strategy is applicable to both peptide and protein antigens and that our in vivo selection strategy can be used for de novo discovery of robust erythrocyte-binding ligands.

Discovery, Synthesis, and Optimization of Peptide-Based Antibiotics.

The rise of multidrug resistant bacteria has significantly compromised our supply of antibiotics and poses an alarming medical and economic threat to society. To combat this problem, it is imperative that new antibiotics and treatment modalities be developed, especially those toward which bacteria are less capable of developing resistance. Peptide natural products stand as promising candidates to meet this need as bacterial resistance is typically slow in response to their unique modes of action. They also have additional benefits including favorable modulation of host immune responses and often possess broad-spectrum activity against notoriously treatment resistant bacterial biofilms. Moreover, nature has provided a wealth of peptide-based natural products from a range of sources, including bacteria and fungi, which can be hijacked in order to combat more dangerous clinically relevant infections.This Account highlights recent advances in the total synthesis and development of a range of peptide-based natural product antibiotics and details the medicinal chemistry approaches used to optimize their activity.In the context of antibiotics with potential to treat Gram-positive bacterial infections, this Account covers the synthesis and optimization of the natural products daptomycin, glycocin F, and alamethicin. In particular, the reported synthesis of daptomycin highlights the utility of on-resin ozonolysis for accessing a key kynurenine residue from the canonical amino acid tryptophan. Furthermore, the investigation into glycocin F analogues uncovered a potent lead compound against Lactobacillus plantarum that bears a non-native thioacetal linkage to a N-acetyl-d-glucosamine (GlcNAc) sugar, which is otherwise O-linked in its native form.For mycobacterial infections, this Account covers the synthesis and optimization of teixobactin, callyaerin A, lassomycin, and trichoderin A. The synthesis of callyaerin A, in particular, highlighted the importance of a (Z)-2,3-diaminoacrylamide motif for antimicrobial activity against Mycobacterium tuberculosis, while the synthesis of trichoderin A highlighted the importance of (R)-stereoconfiguration in a key 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD) residue.Lastly, this Account covers lipopeptide antibiotics bearing activity toward Gram-negative bacterial infections, namely, battacin and paenipeptin C. In both cases, optimization of the N-terminal lipid tails led to the identification of analogues with potent activity toward Escherichia coli and Pseudomonas aeruginosa.

Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice.

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.

Mucosal Vaccination with a Self-Adjuvanted Lipopeptide Is Immunogenic and Protective against Mycobacterium tuberculosis.

Tuberculosis (TB) remains a staggering burden on global public health. Novel preventative tools are desperately needed to reach the targets of the WHO post-2015 End-TB Strategy. Peptide or protein-based subunit vaccines offer potential as safe and effective generators of protection, and enhancement of local pulmonary immunity may be achieved by mucosal delivery. We describe the synthesis of a novel subunit vaccine via native chemical ligation. Two immunogenic epitopes, ESAT61-20 and TB10.43-11 from Mycobacterium tuberculosis (Mtb), were covalently conjugated to the TLR2-ligand Pam2Cys to generate a self-adjuvanting lipopeptide vaccine. When administered mucosally to mice, the vaccine enhanced pulmonary immunogenicity, inducing strong Th17 responses in the lungs and multifunctional peripheral T-lymphocytes. Mucosal, but not peripheral vaccination, provided substantial protection against Mtb infection, emphasizing the importance of delivery route for optimal efficacy.

Synthesis of a Self-Adjuvanting MUC1 Vaccine via Diselenide-Selenoester Ligation-Deselenization.

Access to lipopeptide-based vaccines for immunological studies remains a significant challenge owing to the amphipathic nature of the molecules, which makes them difficult to synthesize and purify to homogeneity. Here, we describe the application of a new peptide ligation technology, the diselenide-selenoester ligation (DSL), to access self-adjuvanting glycolipopeptide vaccines. We show that rapid ligation of glyco- and lipopeptides is possible via DSL in mixed organic solvent-aqueous buffer and, when coupled with deselenization chemistry, affords rapid and efficient access to a vaccine candidate possessing a MUC1 glycopeptide epitope and the lipopeptide adjuvant Pam2Cys. This construct was shown to elicit MUC1-specific antibody and cytotoxic T lymphocyte responses in the absence of any other injected lipids or adjuvants. The inclusion of the helper T cell epitope PADRE both boosted the antibody response and resulted in elevated cytokine production.

Solid-phase synthesis of peptide selenoesters via a side-chain anchoring strategy.

Peptide selenoesters have recently emerged as key building blocks for the ligation-based assembly of large polypeptides and proteins. Herein, we report an efficient solid-phase method for the high yielding and epimerisation-free synthesis of peptide selenoesters using a side-chain immobilisation strategy.