Serum-based ELISA (enzyme-linked immunosorbent assay) has been widely used to detect anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. However, to date, no study has investigated patient urine as a biological sample to detect SARS-CoV-2 virus-specific antibodies. An in-house urine-based ELISA was developed using recombinant SARS-CoV-2 nucleocapsid protein. The presence of SARS-CoV-2 antibodies in urine was established, with 94% sensitivity and 100% specificity for the detection of anti–SARS-CoV-2 antibodies with the urine-based ELISA and 88% sensitivity and 100% specificity with a paired serum-based ELISA. The urine-based ELISA that detects anti–SARS-CoV-2 antibodies is a noninvasive method with potential application as a facile COVID-19 immunodiagnostic platform, which can be used to report the extent of exposure at the population level and/or to assess the risk of infection at the individual level.

Currently, there is no licensed vaccine to protect against human visceral leishmaniasis (VL), a potentially fatal disease caused by infection with Leishmania parasites. In the current study, a recombinant chimeric protein ChimT was developed based on T-cell epitopes identified from the immunogenic Leishmania amastigote proteins LiHyp1, LiHyV, LiHyC and LiHyG. ChimT was associated with the adjuvants saponin (Sap) or monophosphoryl lipid A (MPLA) and used to immunize mice, and their immunogenicity and protective efficacy were evaluated. Both ChimT/Sap and ChimT/MPLA induced the development of a specific Th1-type immune response, with significantly high levels of IFN-γ, IL-2, IL-12, TNF-α and GM-CSF cytokines produced by CD4⁺ and CD8⁺ T cell subtypes (p < 0.05), with correspondingly low production of anti-leishmanial IL-4 and IL-10 cytokines. Significantly increased (p < 0.05) levels of nitrite, a proxy for nitric oxide, and IFN-γ expression (p < 0.05) were detected in stimulated spleen cell cultures from immunized and infected mice, as was significant production of parasite-specific IgG2a isotype antibodies. Significant reductions in the parasite load in the internal organs of the immunized and infected mice (p < 0.05) were quantified with a limiting dilution technique and quantitative PCR and correlated with the immunological findings. ChimT/MPLA showed marginally superior immunogenicity than ChimT/Sap, and although this was not statistically significant (p > 0.05), ChimT/MPLA was preferred since ChimT/Sap induced transient edema in the inoculation site. ChimT also induced high IFN-γ and low IL-10 levels from human PBMCs isolated from healthy individuals and from VL-treated patients. In conclusion, the experimental T-cell multi-epitope amastigote stage Leishmania vaccine administered with adjuvants appears to be a promising vaccine candidate to protect against VL.

Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.

In a proteomics approach conducted with Leishmania amazonensis, parasite proteins showed either an increase or a decrease in their expression content during extensive in vitro cultivation, and were related to the survival and the infectivity of the parasites, respectively. In the current study, a computational screening was performed to predict virulence factors among these molecules. Three proteins were selected, one of which presented no homology to human proteins. This candidate, namely small myristoylated protein-3 (SMP-3), was cloned, and its recombinant version (rSMP-3) was used to stimulate peripheral blood mononuclear cells (PBMCs) from healthy subjects living in an endemic area of leishmaniasis and from visceral leishmaniasis patients. Results showed high interferon-γ (IFN-γ) production and low levels of interleukin 10 (IL-10) in the cell supernatants. An in vivo experiment was then conducted on BALB/c mice, which were immunized with rSMP-3/saponin and later challenged with Leishmania infantum promastigotes. The rSMP-3/saponin combination induced high production of protein-specific IFN-γ, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the spleen cells of the immunized mice. This pattern was associated with protection, which was characterized by a significant reduction in the parasite load in distinct organs of the animals. Altogether, these results have revealed that this new virulence factor is immunogenic in both mice and humans, and have proven its protective efficacy against visceral leishmaniasis in a murine model.

The development of a vaccine for the prevention of visceral leishmaniasis (VL) still represents a significant unmet medical need. A human vaccine can be found if one takes into consideration that many people living in endemic areas of disease are infected but do not develop active VL, including those subjects with subclinical or asymptomatic infection. In this study, a phage display was used to select phage-exposed peptides that were specific to immunoglobulin G (IgG) antibodies from asymptomatic and symptomatic VL patients, separating them from non-infected subjects. Phage clones presenting valid peptide sequences were selected and used as stimuli of peripheral blood mononuclear cells (PBMCs) obtained from both patients’ groups and controls. Those with higher interferon-gamma (IFN-γ)/interleukin (IL)-10 ratios were further selected for vaccination tests. Among 17 evaluated clones, two were selected, B1 and D11, and used to immunize BALB/c mice in an attempt to further validate their in vivo protective efficacy against Leishmania infantum infection. Both clones induced partial protection against the parasite challenge, which was evidenced by the reduction of parasitism in the evaluated organs, a process mediated by a specific T helper (Th)1 immune response. To the best of our knowledge, this study is the first to use a rational strategy based on in vitro stimulation of human PBMCs with selected phage-displayed clones to obtain new immunogens against VL.

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the β-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.