The humoral response after vaccination was evaluated in 1248 individuals who received different COVID-19 vaccine schedules. The study compared subjects primed with adenoviral ChAdOx1-S (ChAd) and boosted with BNT162b2 (BNT) mRNA vaccines (ChAd/BNT) to homologous dosing with BNT/BNT or ChAd/ChAd vaccines. Serum samples were collected at two, four and six months after vaccination, and anti-Spike IgG responses were determined. The heterologous vaccination induced a more robust immune response than the two homologous vaccinations. ChAd/BNT induced a stronger immune response than ChAd/ChAd at all time points, whereas the differences between ChAd/BNT and BNT/BNT decreased over time and were not significant at six months. Furthermore, the kinetic parameters associated with IgG decay were estimated by applying a first-order kinetics equation. ChAd/BNT vaccination was associated with the longest time of anti-S IgG negativization and with a slow decay of the titer over time. Finally, analyzing factors influencing the immune response by ANCOVA analysis, it was found that the vaccine schedule had a significant impact on both the IgG titer and kinetic parameters, and having a Body Mass Index (BMI) above the overweight threshold was associated with an impaired immune response. Overall, the heterologous ChAd/BNT vaccination may offer longer-lasting protection against SARS-CoV-2 than homologous vaccination strategies.

I-152 combines two pro-glutathione (GSH) molecules, namely N-acetyl-cysteine (NAC) and cysteamine (MEA), to improve their potency. The co-drug efficiently increases/replenishes GSH levels in vitro and in vivo; little is known about its mechanism of action. Here we demonstrate that I-152 not only supplies GSH precursors, but also activates the antioxidant kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (KEAP1/NRF2) pathway. The mechanism involves disulfide bond formation between KEAP1 cysteine residues, NRF2 stabilization and enhanced expression of the γ-glutamil cysteine ligase regulatory subunit. Accordingly, a significant increase in GSH levels, not reproduced by treatment with NAC or MEA alone, was found. Compared to its parent compounds, I-152 delivered NAC more efficiently within cells and displayed increased reactivity to KEAP1 compared to MEA. While at all the concentrations tested, I-152 activated the NRF2 pathway; high doses caused co-activation of activating transcription factor 4 (ATF4) and ATF4-dependent gene expression through a mechanism involving Atf4 transcriptional activation rather than preferential mRNA translation. In this case, GSH levels tended to decrease over time, and a reduction in cell proliferation/survival was observed, highlighting that there is a concentration threshold which determines the transition from advantageous to adverse effects. This body of evidence provides a molecular framework for the pro-GSH activity and dose-dependent effects of I-152 and shows how synergism and cross reactivity between different thiol species could be exploited to develop more potent drugs.

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10⁻⁴–1 × 10⁻⁶ ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

We report the evaluation of a small library of azole-bisindoles for their antileishmanial potential, in terms of efficacy on Leishmania infantum promastigotes and intracellular amastigotes. Nine compounds showed good activity on L. infantum MHOM/TN/80/IPT1 promastigotes with IC₅₀ values ranging from 4 to 10 μM. These active compounds were also tested on human (THP-1, HEPG2, HaCaT, and human primary fibroblasts) and canine (DH82) cell lines. URB1483 was selected as the best compound, with no quantifiable cytotoxicity in mammalian cells, to test the efficacy on intracellular amastigotes. URB1483 significantly reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. URB1483 emerges as a new anti-infective agent with remarkable antileishmanial activity and no cytotoxic effects on human and canine cells.

Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

We evaluated the post-vaccination humoral response of three real-world cohorts. Vaccinated subjects primed with ChAdOx1-S and boosted with BNT162b2 mRNA vaccine were compared to homologous dosing (BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S). Serum samples were collected two months after vaccination from a total of 1248 subjects. The results showed that the heterologous vaccine schedule induced a significantly higher humoral response followed by homologous BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S vaccines (p < 0.0001). Moreover, analyzing factors (i.e., vaccine schedule, sex, age, BMI, smoking, diabetes, cardiovascular diseases, respiratory tract diseases, COVID-19 diagnosis, vaccine side effects) influencing the IgG anti-S response, we found that only the type of vaccine affected the antibody titer (p < 0.0001). Only mild vaccine reactions resolved within few days (40% of subjects) and no severe side effects for either homologous groups or the heterologous group were reported. Our data support the use of heterologous vaccination as an effective and safe alternative to increase humoral immunity against COVID-19.