Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.

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A multivalent vaccine is much needed to achieve protection against predominant Shigella serotypes. Recently, we demonstrated the clinical applicability and immunogenic potential of tri-acylated S. flexneri 2a lipopolysaccharide (Ac3-S-LPS). Using a similar approach, we designed a pentavalent LPS candidate vaccine against S. flexneri 1b, 2a, 3a, 6, and Y (PLVF). In this study, we performed molecular and antigenic characterization of the vaccine candidate and its preclinical evaluation. There were no signs of acute toxicity after subcutaneous administration of PLVF in rabbits at a proposed human dose of 125 μg. No pyrogenic reactions and adverse effects associated with chronic toxicity after repeated administration of PLVF were revealed either. The immunization of mice with PLVF led to ≥16-fold increase in S. flexneri 1b-, 2a-, 3a-, 6-, and Y-specific antibodies. In a serum bactericidal antibody (SBA) assay, we registered 54%, 66%, 35%, 60%, and 60% killing of S. flexneri 1b, 2a, 3a, 6, and Y, respectively. In the guinea pig keratoconjunctivitis model, the efficacy was 50% to 75% against challenge with all five S. flexneri serotypes. These studies demonstrate that PLVF is safe, immunogenic over a wide range of doses, and provides protection against challenge with homologous S. flexneri strains, thus confirming the validity of pentavalent design of the combined vaccine.

Резюме. С помощью ингибиторного анализа исследованы механизмы действия двух мурамилпептидов, входящих в состав препарата «Полимурамил»: 1) N-ацетил-D-глюкозаминил-(β1→4)-Nацетил-D-мурамоил-L-аланил-D-изоглютаминил-мезо-диаминопимелиновой кислоты (ГМтри); 2) димерного мурамилпептида (диГМтетра), в котором два мономера [N-ацетил-D-глюкозаминил-(β1→4)-N-ацетил-D-мурамоил-L-аланил-D-изоглютаминил-мезо-диаминопимелоил-D-аланин] соединены путем амидной связи между карбоксильной группой терминального D-аланина одного мономера и ω-аминогруппой мезо-диаминопимелиновой кислоты другого мономера. Продукция фактора некроза опухолей макрофагами (МФ) человека, стимулированными in vitro ГМтри и диГМтетра, подавлялась веществами SB203580 (ингибитор киназы RIP2), генистеином (ингибитор протеинтирозинкиназ) и BAY 11-7082 (ингибитор IκB-киназы). Ответ на диГМтетра, кроме того, подавлялся дайнасором (ингибитор клатрин-зависимого эндоцитоза) и коктейлем ингибиторов протеаз широкого спектра действия. Таким образом, активирующее влияние компонентов «Полимурамила» на МФ обеспечиваются минимум тремя биологическими процессами: 1) клатрин-зависимым эндоцитозом; 2) пептидазным процессингом диГМтетра; 3) активацией сигнальной цепочки «RIP2-IκB-киназа-фактор транскрипции NF-κB».

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