Robert A. Cordfunke

Posttranslational Modification of HLA-DQ Binding Islet Autoantigens in Type 1 Diabetes

Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis–restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-γ, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.

A Novel Secreted Metalloprotease (CD2830) from Clostridium difficile Cleaves Specific Proline Sequences in LPXTG Cell Surface Proteins

Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90β, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro–Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.

Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145.

The scarcity of current antibiotic-based strategies to prevent biomaterial-associated infections (BAI) and their risk of resistance development prompted us to develop a novel antimicrobial implant-coating to prevent Staphylococcus aureus-induced BAI. We incorporated the antimicrobial peptide OP-145 into a Polymer-Lipid Encapsulation MatriX (PLEX)-coating to obtain high peptide levels for prolonged periods at the implant-tissue interphase. We first confirmed that OP-145 was highly effective in killing S. aureus and inhibiting biofilm formation in vitro. OP-145 injected along S. aureus-inoculated implants in mice significantly reduced the number of culture-positive implants. OP-145 was released from the PLEX coating in a controlled zero-order kinetic rate after an initial 55%-burst release and displayed bactericidal activity in vitro. In a rabbit intramedullary nail-related infection model, 67% of rabbits with PLEX-OP-145-coated nails had culture-negative nails after 28days compared to 29% of rabbits with uncoated nails. In rabbits with PLEX-OP-145-coated nails, bone and soft tissue samples were culture-negative in 67% and 80%, respectively, whereas all bone samples and 71% of the soft tissue samples of rabbits with uncoated nails were infected. Together, PLEX-OP-145 coatings, of which both compounds have already been found safe in man, can prevent implant colonization and S. aureus-induced BAIs.

Phospholipid-driven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems.

OP-145, a synthetic antimicrobial peptide developed from a screen of the human cathelicidin LL-37, displays strong antibacterial activities and is--at considerably higher concentrations--lytic to human cells. To obtain more insight into its actions, we investigated the interactions between OP-145 and liposomes composed of phosphatidylglycerol (PG) and phosphatidylcholine (PC), resembling bacterial and mammalian membranes, respectively. Circular dichroism analyses of OP-145 demonstrated a predominant α-helical conformation in the presence of both membrane mimics, indicating that the different membrane-perturbation mechanisms are not due to different secondary structures. Membrane thinning and formation of quasi-interdigitated lipid-peptide structures was observed in PG bilayers, while OP-145 led to disintegration of PC liposomes into disk-like micelles and bilayer sheets. Although OP-145 was capable of binding lipoteichoic acid and peptidoglycan, the presence of these bacterial cell wall components did not retain OP-145 and hence did not interfere with the activity of the peptide toward PG membranes. Furthermore, physiological Ca++ concentrations did neither influence the membrane activity of OP-145 in model systems nor the killing of Staphylococcus aureus. However, addition of OP-145 at physiological Ca++-concentrations to PG membranes, but not PC membranes, resulted in the formation of elongated enrolled structures similar to cochleate-like structures. In summary, phospholipid-driven differences in incorporation of OP-145 into the lipid bilayers govern the membrane activity of the peptide on bacterial and mammalian membrane mimics.

The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms

Infections refractory to conventional antibiotics may be targeted by the antimicrobial peptide SAAP-148. New way to keep bacteria at bay Antibiotic resistance is a major threat to public health. To develop a new type of weapon in the arms race against bacteria, de Breij et al. generated a panel of synthetic peptides based on the human antimicrobial peptide LL-37. The lead candidate from this panel, SAAP-148, can kill dangerous antibiotic-resistant pathogens in many contexts, including on ex vivo human skin and in biofilms. Long-term exposure to SAAP-148 did not induce bacterial resistance. Topical application of SAAP-148 could one day be used in hospitals to help patients combat bacteria resistant to traditional antibiotics. Development of novel antimicrobial agents is a top priority in the fight against multidrug-resistant (MDR) and persistent bacteria. We developed a panel of synthetic antimicrobial and antibiofilm peptides (SAAPs) with enhanced antimicrobial activities compared to the parent peptide, human antimicrobial peptide LL-37. Our lead peptide SAAP-148 was more efficient in killing bacteria under physiological conditions in vitro than many known preclinical- and clinical-phase antimicrobial peptides. SAAP-148 killed MDR pathogens without inducing resistance, prevented biofilm formation, and eliminated established biofilms and persister cells. A single 4-hour treatment with hypromellose ointment containing SAAP-148 completely eradicated acute and established, biofilm-associated infections with methicillin-resistant Staphylococcus aureus and MDR Acinetobacter baumannii from wounded ex vivo human skin and murine skin in vivo. Together, these data demonstrate that SAAP-148 is a promising drug candidate in the battle against antibiotic-resistant bacteria that pose a great threat to human health.

Effectiveness of slow-release systems in CD40 agonistic antibody immunotherapy of cancer

Slow-release delivery has great potential for specifically targeting immune-modulating agents into the tumor-draining area. In prior work we showed that local treatment of slowly delivered anti-CD40 antibody induced robust anti-tumor CD8+ T cell responses without systemic toxicity. We now report on the comparison of two slow-release delivery systems for their use in antibody-based immunotherapy of cancer. Anti-CD40 agonistic antibody delivered locally in mineral oil Montanide ISA 51 or in dextran-based microparticles activated tumor-specific T cell activation. Both slow-release formulations significantly decreased systemic side-effects compared to systemic administration of anti-CD40 antibody. However, dextran-based microparticles caused serious local inflammation associated with unwanted rapid outgrowth of tumors instead of the tumor clearance observed with delivery in Montanide. We therefore conclude that Montanide ISA 51 is to be preferred as a slow-release agent for CD40 agonist immunotherapy of cancer.

Development of a nose cream containing the synthetic antimicrobial peptide P60.4Ac for eradication of methicillin-resistant Staphylococcus aureus carriage.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing problem, and current treatment options are suboptimal. Nasal carriage of MRSA is a major risk factor for infection, but nasal eradication strategies are increasingly considered to be insufficiently effective. In this study, a water-in-oil cream formulation was developed for nasal application with an antimicrobial peptide, P60.4Ac, aimed at the eradication of MRSA carriage. Quality control of the cream included the measurement of the content and release of the peptide by a validated high-performance liquid chromatography method. Stability of the peptide in the formulation was investigated including the evaluation of the effect of stress conditions. Preliminary shelf-life study of the drug formulation demonstrated that the peptide is stable in the formulation at least for 5 months. Microbial-killing assays with MRSA LUH14616 as a target demonstrated the dose-dependent antimicrobial activity of the peptide formulation.

Antimicrobial peptide P60.4Ac-containing creams and gel for eradication of methicillin-resistant Staphylococcus aureus from cultured skin and airway epithelial surfaces

ABSTRACT We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and BEMs.

Abrogation of Immunogenic Properties of Gliadin Peptides through Transamidation by Microbial Transglutaminase Is Acyl-Acceptor Dependent

Wheat gluten confers superior baking quality to wheat based products but elicits a pro-inflammatory immune response in patients with celiac disease. Transamidation of gluten by microbial transglutaminase (mTG) and tissue transglutaminase (tTG) reduces the immunogenicity of gluten; however, little information is available on the minimal modification sufficient to eliminate gliadin immunogenicity nor has the effectiveness of transamidation been studied with T-cell clones from patients. Here we demonstrate that mTG can efficiently couple three different acyl-acceptor molecules, l-lysine, glycine ethyl ester, and hydroxylamine, to gliadin peptides and protein. While all three acyl-acceptor molecules were cross-linked to the same Q-residues, not all modifications were equally effective in silencing T-cell reactivity. Finally, we observed that tTG can partially reverse the mTG-catalyzed transamidation by its isopeptidase activity. These results set the stage to determine the impact of these modifications on the baking quality of gluten proteins and in vivo immunogenicity of such food products.

Linking T cell epitopes to a common linear B cell epitope: A targeting and adjuvant strategy to improve T cell responses

HighlightsHumoral responses against tetanus toxoid aid de novo generation of cellular immune responses.A unique B cell epitope from tetanus toxin has been identified through a peptide library screen.A defined peptide conjugate including the B cell epitope can be used to improve T cell responses. Abstract Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti‐cancer immunity. However, a viable approach to use antibody‐complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof‐of‐concept we investigated if anti‐tetanus IgG could induce potent cross‐presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA‐specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide–peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high‐titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti‐MTTE antibodies with MTTE‐containing conjugates are able to induce DC and T cell activation using model antigens.