Mare Cudic

Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2.

Matix protein 2 (M2) is a transmembrane protein of influenza type A virus. It contains a 23 aa long ectodomain (M2e) that is highly conserved amongst human influenza type A viruses. M2e-specific antibodies have been shown to restrict virus growth in vitro and in vivo and thus have the potential of providing cross-reactive resistance to influenza type A virus infection. We attempted to induce M2e-specific protection with synthetic multiple antigen peptide (MAP) constructs that contained covalently linked M2e- and Th-determinant peptides. Mice, vaccinated twice by the intranasal (i.n.) route with adjuvanted M2e-MAPs exhibited significant resistance to virus replication in all sites of the respiratory tract. Compared to mice primed by two consecutive heterosubtypic infections, resistance was of similar strength in nasal and tracheal tissue but lower in pulmonary tissue. Importantly, the protection in M2e-MAP- and infection-immunized mice appeared to be mediated by distinct immune mechanisms. This suggests that stronger protection may be achievable by combining both protective activities.

Complex Carbohydrates Are Not Removed During Processing of Glycoproteins by Dendritic Cells: Processing of Tumor Antigen MUC1 Glycopeptides for Presentation to Major Histocompatibility Complex Class II-restricted T Cells

In contrast to protein antigens, processing of glycoproteins by dendritic cells (DCs) for presentation to T cells has not been well studied. We developed mouse T cell hybridomas to study processing and presentation of the tumor antigen MUC1 as a model glycoprotein. MUC1 is expressed on the surface as well as secreted by human adenocarcinomas. Circulating soluble MUC1 is available for uptake, processing, and presentation by DCs in vivo and better understanding of how that process functions in the case of glycosylated antigens may shed light on antitumor immune responses that could be initiated against this glycoprotein. We show that DCs endocytose MUC1 glycopeptides, transport them to acidic compartments, process them into smaller peptides, and present them on major histocompatability complex (MHC) class II molecules without removing the carbohydrates. Glycopeptides that are presented on DCs are recognized by T cells. This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

Development of novel antibacterial peptides that kill resistant isolates

The rapid emergence of bacterial strains that are resistant to current antibiotics requires the development of novel types of antimicrobial compounds. Proline-rich cationic antibacterial peptides such as pyrrhocoricin kill responsive bacteria by binding to the 70 kDa heat shock protein DnaK and inhibiting protein folding. We designed and synthesized multiply protected dimeric analogs of pyrrhocoricin and optimized the in vitro antibacterial efficacy assays for peptide antibiotics. Pyrrhocoricin and the designed dimers killed beta-lactam, tetracycline- or aminoglycoside-resistant strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the submicromolar or low micromolar concentration range. One of the peptides also killed Pseudomonas aeruginosa. The designed dimers showed improved stability in mammalian sera compared to the native analog. In a murine H. influenzae lung infection model, a single dose of a dimeric pyrrhocoricin analog reduced the bacteria in the bronchoalveolar lavage when delivered intranasally. The solid-phase synthesis was optimized for large-scale laboratory preparations.

Extracellular proteases as targets for drug development

Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV) and cysteine proteases (cathepsin B) are discussed herein.

In vitro and in vivo activity of an antibacterial peptide analog against uropathogens

The alarming rate of bacterial resistance induction highlights the clinical need for antimicrobial agents that act by novel modes of action. Based on the activity profile, the general tissue distribution and renal clearance of peptide-based drugs, we hypothesized that our newly developed pyrrhocoricin derivative would be able to fight resistant uropathogens in vitro and in vivo. Indeed, the Pip-pyrr-MeArg dimer killed all 11 urinary tract infection-related Escherichia coli and Klebsiella pneumoniae strains we studied in the sub-low micromolar concentration range. Almost all control antibiotics, including the currently leading trimethoprim-sulfametoxazole combination for urinary tract infection, remained without considerable activity against two or more of these bacterial strains. In a mouse ascending urinary tract infection model with E. coli CFT073 as pathogen, two doses of intravenous, subcutaneous or oral treatment with the Pip-pyrr-MeArg derivative reduced the bacterial counts in the kidneys, bladder and urine to varying levels. Statistically significant elimination or reduction of bacteria compared to untreated animals was observed at dual intravenous or subcutaneous doses of 0.4 or 10mg/kg, respectively. Serial passage of the same E. coli strain in the presence of sublethal doses of the designed peptide failed to generate resistant mutants. The Pip-pyrr-MeArg dimer showed no toxicity to COS-7 cells to the highest 500microM concentration studied.

Convenient synthesis of a head-to-tail cyclic peptide containing an expanded ring

Here we describe a rapid and efficient solid-phase synthesis of a 29-mer cyclic antibacterial peptide 1, currently under pharmaceutical development. The linear peptide was assembled by standard Fmoc chemistry on an Fmoc-Asp(resin)-ODmab carrier. Intramolecular on-resin head-to-tail cyclization was enabled after selective deprotection of the Asp α-carboxy protecting group with 2% hydrazine–DMF at room temperature.

The efficacy of the antibacterial peptide, pyrrhocoricin, is finely regulated by its amino acid residues and active domains

Pyrrhocoricin, a highly active antibacterial peptide isolated from insects, inhibits chaperone-assisted protein folding via binding to the 70 kDa heat shock protein DnaK with its amino terminal half. The C-terminus functions as an intracellular delivery module. In the current study, chimeras consisting of the putative functional units of pyrrhocoricin and a related peptide, drosocin, were made, and it was found that some mixed and matched sequences retained their ability to killEscherichia coli, Salmonella typhimurium andAgrobacterium tumefaciens. While pyrrhocoricin appeared to have a more universal pharmacophore, drosocin featured a more robust intracellular delivery unit. We also identified the minimal length of pyrrhocoricin that is needed to efficiently kill bacteria. While for activity againstS. typhimurium the peptide could not be shortened, againstE. coli it was sufficient to have a Vall-Ile16 amino-terminal fragment. Although Vall was not part of the Asp2-Pro 10 pharmacophore (it could be replaced with other residues), it could not be eliminated and apparently played an important role in defining the activity of the peptide. Indeed, when Val1 was replaced with lysine, not only the efficacy of pyrrhocoricin to kill the sensitive strains increased significantly, resulting in the most active antimicrobial peptide against some clinical strains ever made, but the modified peptide was also able to killPseudomonas aeruginosa, an originally unresponsive bacterium in the low μg ml−1 concentration range. However, this substitution likely influenced the interaction with bacterial membranes rather than that with the target protein, and therefore the dominant mode of action of the Lysl-pyrrhocoricin peptide may feature membrane disintegration instead of DnaK inhibition.

Conformation of glycopeptides.

The presence of carbohydrate side-chains in native glycoproteins alters a number of biochemical properties of the peptide backbone. One of the most frequently studied questions is the conformation-modifying effect of sugar incorporation into asparagine, serine and threonine residues. When N-glycosylation modifies the conformation, the resulting structures are more ordered than the peptide chain without sugar addition. For O-glycopeptides the final conformations can be either more ordered or less ordered. In any event, only the innermost carbohydrates make contact with the peptide backbone. Through-space structural changes are mostly found downstream of the O-glycosylation site. In the repeat unit of epithelial mucin-1 protein, clustering of the carbohydrates results in an easily observable stabilization of the poly-proline II helix.

Synthesis, Conformation and T-Helper Cell Stimulation of an O-Linked Glycopeptide Epitope Containing Extended Carbohydrate Side-Chains

To answer the question whether or not T cells to immunodominant protein fragments recognize glycosylated antigens, we synthesized a series of glycopeptides corresponding to peptide 31D, a major T-helper cell epitope of the rabies virus nucleoprotein. Thr4 of the epitope is known to allow mono- or disaccharide side-chain substitutions in either alpha- or beta-anomeric configuration without interfering with MHC-binding. To model naturally occurring glycoprotein fragments that carry extended sugar chains, we prepared Fmoc-Ser/Thr-OPfp building blocks containing alpha- and beta-linked linear tri- and heptasaccharides. Peptide 31D was synthesized with the complex carbohydrates attached to Thr4, and the T-helper cell activity of the glycopeptides was determined. Addition of alpha-linked carbohydrates, that mimic most of the natural O-linked glycoproteins, resulted in a major drop in the T-cell stimulatory ability in a sugar length-dependent manner. In contrast, the cytosolic glycoprotein mimicking beta-linked glycopeptides retained their T-cell stimulatory activity, with the trisaccharide-containing analogue being almost as potent as the unglycosylated peptide. When the peptides were preincubated with diluted human serum, all peptides lost their ability to stimulate the 9C5.D8-H hybridoma. These findings indicated that (i) in contrast to cytosolic glycosylation, incorporation of long O-linked carbohydrates into T-helper cell epitopes abrogates the antigenicity of these protein fragments, and (ii) glycosylation is not a viable alternative to improve the immunogenic properties of subunit peptide vaccines. Glycosylation with all four carbohydrate moieties similarly destroyed the inducible alpha-helical structure of peptide 31D as detected by CD, indicating that the differences in the T-cell activity were not due to different peptide conformations.

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential.

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs.