TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen‐associated molecular patterns. TLR2 is the receptor for a functional recognition ...of bacterial lipopeptides (LP) and is up‐regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1‐ or TLR6‐dependent manner. There are also di‐ and triacylated LP, which stimulate TLR1‐deficient cells and TLR6‐deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)2C‐(VPGVG)4VPGKG, fibroblast‐stimulating LP‐1, and Pam2C‐SK4. Dominant‐negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram‐positive and Gram‐negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.
Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication ...and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally‐acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope ‘E2EP3’. E2EP3 is located at the N‐terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3‐specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV‐specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non‐human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre‐clinical basis for the design of effective vaccine against arthralgia‐inducing CHIKV and other alphaviruses.
Abstract
T-cell immunity is central for control of COVID-19, particularly in patients incapable of mounting antibody responses. CoVac-1 is a peptide-based T-cell activator composed of SARS-CoV-2 ...epitopes with documented favorable safety profile and efficacy in terms of SARS-CoV-2-specific T-cell response. We here report a Phase I/II open-label trial (NCT04954469) in 54 patients with congenital or acquired B-cell deficiency receiving one subcutaneous CoVac-1 dose. Immunogenicity in terms of CoVac-1-induced T-cell responses and safety are the primary and secondary endpoints, respectively. No serious or grade 4 CoVac-1-related adverse events have been observed. Expected local granuloma formation has been observed in 94% of study subjects, whereas systemic reactogenicity has been mild or absent. SARS-CoV-2-specific T-cell responses have been induced in 86% of patients and are directed to multiple CoVac-1 peptides, not affected by any current Omicron variants and mediated by multifunctional T-helper 1 CD4
+
T cells. CoVac-1-induced T-cell responses have exceeded those directed to the spike protein after mRNA-based vaccination of B-cell deficient patients and immunocompetent COVID-19 convalescents with and without seroconversion. Overall, our data show that CoVac-1 induces broad and potent T-cell responses in patients with B-cell/antibody deficiency with a favorable safety profile, which warrants advancement to pivotal Phase III safety and efficacy evaluation. ClinicalTrials.gov identifier NCT04954469.
Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears ...promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 10.sup.2 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The polypeptide Pep19-2.5 (Aspidasept
) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) ...and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.
Efficient intracellular drug delivery in nanomedicine strongly depends on ways to induce cellular uptake. Conjugation of nanoparticles (NPs) with cell-penetrating peptides (CPPs) is a known means to ...induce uptake
via
endocytosis. Here, we functionalized NPs consisting of either poly(
d
,
l
-lactide-
co
-glycolide) (PLGA) or polyethene glycol (PEG)-PLGA block-copolymer with a lactoferrin-derived cell-penetrating peptide (hLF). To enhance the association between the peptide and the polymer NPs, we tested a range of acyl moieties for N-terminal acylation of the peptide as a means to promote noncovalent interactions. Acyl moieties differed in chain length and number of acyl chains. Peptide-functionalized NPs were characterized for nanoparticle size, overall net charge, storage stability, and intracellular uptake. Coating particles with a palmitoylated hLF resulted in minimal precipitation after storage at −20C and homogeneous particle size (<200 nm). Palmitoyl-hLF coated NPs showed enhanced delivery in different cells in comparison to NPs lacking functionalization. Moreover, in comparison to acetyl-hLF, palmitoyl-hLF was also suited for coating and enhancing the cellular uptake of PEG-PLGA NPs.
Noncovalent functionalization with acylated cell-penetrating peptides achieves an efficient cellular uptake of PLGA and PEG-PLGA nanoparticles.
Bacterial cell walls contain lipoproteins/peptides, which are strong modulators of the innate immune system. Triacylated lipopeptides are assumed to be recognized by TLR2/TLR1-, whereas diacylated ...lipopeptides use TLR2/TLR6 heteromers for signaling. Following our initial discovery of TLR6-independent diacylated lipopeptides, we could now characterize di- and triacylated lipopeptides (e.g. Pam2C-SK4, Pam3C-GNNDESNISFKEK), which have stimulatory activity in TLR1- and in TLR6-deficient mice. Furthermore, for the first time, we present triacylated lipopeptides with short length ester-bound fatty acids (like PamOct2C-SSNASK4), which induce no response in TLR1-deficient cells. No differences in the phosphorylation of MAP kinases by lipopeptide analogs having different TLR2-coreceptor usage were observed. Blocking experiments indicated that different TLR2 heteromers recognize their specific lipopeptide ligands independently from each other. In summary, a triacylation pattern is necessary but not sufficient to render a lipopeptide TLR1-dependent, and a diacylation pattern is necessary but not sufficient to render a lipopeptide TLR6-dependent. Contrary to the current model, distinct lipopeptides are recognized by TLR2 in a TLR1- and TLR6-independent manner.
Bacterial lipoproteins/peptides are composed of di‐O‐acylated‐S‐(2,3‐dihydroxypropyl)‐cysteinyl residues N‐terminally coupled to distinct polypeptides, which can be N‐acylated with a third fatty ...acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester‐bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide‐bound and another ester‐bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno‐stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.
Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce ...signaling in cells of the immune system through Toll‐like receptor 2 (TLR2)–TLR1 or TLR2–TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N‐palmitoyl‐S‐2,3‐bis(palmitoyloxy)‐(2RS)‐propyl‐(R)‐cysteine (Pam3)‐CSK4, signal through TLR2–TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage‐activating lipopeptide from Mycoplasma fermentans (MALP2) or S‐2,3‐bis(palmitoyloxy)‐(2RS)‐propyl‐(R)‐cysteine (Pam2)‐CGNNDESNISFKEK, induce signaling through TLR2–TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK‐SK4 (MALP2‐SK4) induced B lymphocyte proliferation and TNF‐α secretion in macrophages in a TLR6‐independent manner as determined with cells from TLR6‐deficient mice. Our results indicate that both the lipid and the N‐terminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand–receptor interaction on host cells.
Radioprotective 105 kDa (RP105, CD180) is a member of the Toll‐like receptor (TLR) family that interacts with TLR2 and facilitates recognition of mature lipoproteins expressed by Mycobacterium ...tuberculosis and Mycobacterium bovis BCG. In this study, we used synthetic lipopeptide analogs of the M. tuberculosis 19 kDa lipoprotein to define structural characteristics that promote RP105‐mediated host cell responses. A tripalmitoylated lipopeptide composed of the first 16 N‐terminal amino acids of the M. tuberculosis 19 kDa lipoprotein induced RP105‐dependent TNF and IL‐6 production by macrophages. Di‐ and tripalmitoylated variants of this lipopeptide elicited an equivalent RP105‐dependent response, indicating that while the lipid moiety is required for macrophage activation, it is not a determinant of RP105 dependency. Instead, substitution of two polar threonine residues at positions 7 and 8 with nonpolar alanine residues resulted in reduced RP105 dependency. These results strongly suggest that the amino acid composition of the M. tuberculosis 19 kDa lipoprotein, and likely other mycobacterial lipoproteins, is a key determinant of RP105 agonism.
PDF
