Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004-2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Yersinia pestis Antigen F1 but Not LcrV Induced Humoral and Cellular Immune Responses in Humans Immunized with Live Plague Vaccine-Comparison of Immunoinformatic and Immunological Approaches.

The recent progress in immunoinformatics provided the basis for an accelerated development of target-specific peptide vaccines as an alternative to the traditional vaccine concept. However, there is still limited information on whether the in silico predicted immunoreactive epitopes correspond to those obtained from the actual experiments. Here, humoral and cellular immune responses to two major Yersinia pestis protective antigens, F1 and LcrV, were studied in human donors immunized with the live plague vaccine (LPV) based on the attenuated Y. pestis strain EV line NIIEG. The F1 antigen provided modest specific cellular (mixed T helper 1 (Th1)/Th2 type) and humoral immune responses in vaccinees irrespective of the amount of annual vaccinations and duration of the post-vaccination period. The probing of the F1 overlapping peptide library with the F1-positive sera revealed the presence of seven linear B cell epitopes, which were all also predicted by in silico assay. The immunoinformatics study evaluated their antigenicity, toxicity, and allergenic properties. The epitope TSQDGNNH was mostly recognized by the sera from recently vaccinated donors rather than antibodies from those immunized decades ago, suggesting the usefulness of this peptide for differentiation between recent and long-term vaccinations. The in silico analysis predicted nine linear LcrV-specific B-cell epitopes; however, weak antibody and cellular immune responses prevented their experimental evaluation, indicating that LcrV is a poor marker of successful vaccination. No specific Th17 immune response to either F1 or LcrV was detected, and there were no detectable serum levels of F1-specific immunoglobulin A (IgA) in vaccinees. Overall, the general approach validated in the LPV model could be valuable for the rational design of vaccines against other neglected and novel emerging infections with high pandemic potency.

New Promising Targets for Synthetic Omptin-Based Peptide Vaccine against Gram-Negative Pathogens.

Omptins represent a family of proteases commonly found in various Gram-negative pathogens. These proteins play an important role in host-pathogen interaction and have been recognized as key virulence factors, highlighting the possibility of developing an omptin-based broad-spectrum vaccine. The prototypical omptin, His-tagged recombinant Pla, was used as a model target antigen. In total, 46 linear and 24 conformational epitopes for the omptin family were predicted by the use of ElliPro service. Among these we selected highly conserved, antigenic, non-allergenic, and immunogenic B-cell epitopes. Five epitopes (2, 6, 8, 10, and 11 corresponding to Pla regions 52-60, 146-150, 231-234, 286-295, and 306-311, respectively) could be the first choice for the development of the new generation of target-peptide-based vaccine against plague. The partial residues of omptin epitopes 6, 8, and 10 (regions 136-145, 227-230, and 274-285) could be promising targets for the multi-pathogen vaccine against a group of enterobacterial infections. The comparative analysis and 3D modeling of amino acid sequences of several omptin family proteases, such as Pla (Yersinia pestis), PgtE (Salmonella enterica), SopA (Shigella flexneri), OmpT, and OmpP (Escherichia coli), confirmed their high cross-homology with respect to the identified epitope clusters and possible involvement of individual epitopes in host-pathogen interaction.

Humoral and cellular immune responses to Yersinia pestis Pla antigen in humans immunized with live plague vaccine.

BACKGROUND: To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination. CONCLUSIONS/SIGNIFICANCE: The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.