Leishmaniases are complex vector-borne diseases caused by intracellular parasites of the genus Leishmania. The visceral form of the disease affects both humans and canids in tropical, subtropical, and Mediterranean regions. One health approach has suggested that controlling zoonotic visceral leishmaniasis (ZVL) could have an impact on the reduction of the human incidence of visceral leishmaniasis (VL). Despite the fact that a preventive vaccination could help with leishmaniasis elimination, effective vaccines that are able to elicit protective immune responses are currently lacking. In the present study, we designed a chimeric multi-epitope protein composed of multiple CD8⁺ and CD4⁺ T cell epitopes which were obtained from six highly immunogenic proteins previously identified by an immunoproteomics approach, and the N-termini of the heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis served as an adjuvant. A preclinical evaluation of the candidate vaccine in BALB/c mice showed that when it was given along with the adjuvant Addavax it was able to induce strong immune responses. Cellular responses were dominated by the presence of central and effector multifunctional CD4⁺ and CD8⁺ T memory cells. Importantly, the vaccination reduced the parasite burden in both short-term and long-term vaccinated mice challenged with Leishmania infantum. Protection was characterized by the continuing presence of IFN-γ⁺TNFα⁺-producing CD8⁺ and CD4⁺ T cells and increased NO levels. The depletion of CD8⁺ T cells in short-term vaccinated mice conferred a significant loss of protection in both target organs of the parasite, indicating a significant involvement of this population in the protection against L. infantum challenge. Thus, the overall data could be considered to be a proof-of-concept that the design of efficacious T cell vaccines with the help of reverse vaccinology approaches is possible.

T he CMY-type enzymes are the most frequent acquired class C ␤-lactamases (1). Spread of the respective genes is mediated mostly by IncI and IncA/C plasmids (2-4). bla CMY-carrying ColE1 replicons have also been reported (5). Here, we describe a CMY-4-encoding IncQ plasmid from Escherichia coli. E. coli EL231 was derived from a hospitalized patient in Athens in 2012. The isolate exhibited a "cephalosporinase" phenotype. PCR-sequencing and isoelectric focusing documented carriage of bla TEM-1 and bla CMY-4 and production of the respective enzymes. The isolate transferred its ␤-lactam resistance traits in mating-out assays (6). Transconjugants produced both TEM-1 and CMY-4 and harbored two plasmids of ϳ7 and ϳ80 kb, of which only the former (pQEL231) hybridized with a bla CMY-specific probe. Transformation of E. coli DH5␣ by purified pQEL231 resulted in CMY-4-producing, ␤-lactam-resistant clones (Table 1). Incompatibility typing by PCR-based replicon typing (7) classified the 80-kb plasmid as IncI, while pQEL231 was untypeable. pQEL231 was sequenced by a primer-walking approach (Fig. 1A; see also Table S1 in the supplemental material). Its size was 6,925 bp, comprising an IncQ-derived sequence (5,571 bp) and a bla CMY-4-containing acquired segment. pQEL231 possesses a backbone typical of the IncQ-1␣ subgroup, thus far including plasmids such as RSF1010, pSTU-288-2, and pCCK1900 (8-11) (Fig. 1B). The replication control region of pQEL231 was similar to that of RSF1010, differing only in the number of iterons controlling a single-strand displacement replication mechanism unique in IncQ plasmids (12, 13). This region included a 15-bp conserved sequence and two inverted repeats serving as single-strand initiation sites (ssiA/OriR and ssiB/OriL),

The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory.

The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.

The emerging SARS-CoV and SARS-CoV-2 belong to the family of “common cold” RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue—L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific—yet to be discovered—functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence.

Currently, the spread of multidrug-resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective β-lactamases is of vital importance.