Biology subjects

Bardelli, M.

Publications and source records attributed to Bardelli, M..

6 recordsLinked to original sources

bioRxiv2025

Evaluation of Klebsiella pneumoniae antigen MrkA reveals expression-limited therapeutic potential

Klebsiella pneumoniae (KP) poses a major global health concern, particularly as hospital-acquired strains become increasingly antibiotic resistant. MrkA, the major type 3 fimbrial (T3F) subunit, is a recognised prospective vaccine and monoclonal antibody (mAb) target. We assessed T3F structural (mrkABCDF) and regulatory (mrkHIJ) gene prevalence and allelic variation across 1649 KP genomes, using 100 diverse isolates for detailed phenotypic characterisation. Despite high conservation MrkA expression itself is variable with genotype failing to predict phenotype. We constructed isogenic KP T3F mutants with absent and supraphysiological MrkA expression to investigate functional consequences and therapeutic responses during infection. In a severe pneumonia model blood dissemination inversely correlated with T3F expression. In vivo antigen profiling revealed only [~]20% of wild-type lung bacteria express MrkA, dropping to [~]5% in blood, with low MrkA expression defining bacteraemic KP irrespective of infection route. Critically, anti-MrkA mAb efficacy was contingent on antigen abundance, with therapeutic benefit restricted to supraphysiological MrkA expression. These findings reveal substantial in vivo MrkA expression variability with critical onward implications for vaccine and mAb development.

microbiology↗

bioRxiv2024

Evaluation of Precision of the Plasmodium knowlesi Growth Inhibition Assay for Plasmodium vivax Duffy-Binding Protein-based Malaria Vaccine Development

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted evaluating four different anti-PvDBPII human monoclonal antibodies (mAbs) at different concentrations, and three GIAs were conducted testing eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95%CI) for %GIA or GIA50 in repeat assays was calculated in this investigation. These results will support the development of future blood-stage malaria vaccines, specifically second generation PvDBPII-based formulations.

immunology↗

bioRxiv2023

Human monoclonal antibodies reveal subdominant gonococcal and meningococcal cross-protective antigens

Gonococcus (Gc), a bacterium resistant to most antibiotics causing more than 80 million cases of gonorrhea annually, is a WHO high priority pathogen. Recently, vaccine development prospects were boosted by reports that licensed meningococcus serogroup B (MenB) vaccines provided partial protection against Gc infection. To determine antigens responsible for cross-protection, memory B cells from 4CMenB vaccinated volunteers were single-cell sorted to identify antibodies that kill Gc in a bactericidal assay. Nine different antibodies, all deriving from the IGHV4-34 germline carrying unusually long HCDR3s, recognized the PorB protein, four recognized the lipooligosaccharide (LOS), and four unknown antigens. One of the PorB antibodies, tested in vivo, provided protection from Gc infection. The identification of PorB and LOS as key antigens of gonococcal and meningococcal immunity provides a mechanistic explanation of the cross-protection observed in the clinic and shows that isolating human monoclonal antibodies from vaccinees can be instrumental for bacterial antigen discovery.

immunology↗

bioRxiv2023

Naturally acquired and Vaccine-induced Human Monoclonal Antibodies to Plasmodium vivax Duffy Binding Protein Inhibit Invasion of Plasmodium knowlesi (PvDBPOR) Transgenic Parasites

The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to Plasmodium vivax (Pv) invasion of reticulocytes. Pv expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and their protein-protein interaction is central to vivax blood stage malaria. Here we compared the functional activity of humAbs derived from naturally exposed and vaccinated individuals for the first time using easily cultured P. knowlesi (Pk) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. Using this model, we evaluated the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 g/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 g/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10-100 g/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 g/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. This PkPvDBPOR model system enables efficient assessment of NA and VI humAbs individually and in combination. IMPORTANCEGiven the importance of Duffy blood group antigen and P. vivax Duffy binding protein (PvDBP) interaction leading to blood stage vivax malaria, development of vaccines or therapeutic human monoclonal antibodies (humAbs) targeting PvDBP are key strategies for treating and controlling Pv. The P. knowlesi-based PkPvDBPOR transgenic model system enables efficient assessment of NA and VI humAbs individually and in combination. As such, this model could prioritize specific humAb combinations ahead of clinical trials of these reagents.

microbiology↗

bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗

bioRxiv2018

A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity

Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy.\n\nAuthor summaryAntibody immunotherapy is considered a viable strategy against prion disease. We previously showed that antibodies against the so-called globular domain of Prion Protein (PrP) can cause PrP dependent neurotoxicity; this does not happen for antibodies against the flexible tail of PrP, which therefore ought to be preferred for therapy.\n\nHere we show that simultaneous targeting of both globular domain and flexible tail by a bispecific, combination of a toxic and a non-toxic antibody, results in stronger protection against prion toxicity, even if the bispecific is administered when prion pathology is already conspicuous.\n\nWe hypothesize that neurotoxicity arises from binding to specific \"toxicity triggering sites\" in the globular domain. We designed our bispecific with two aims: i) occupying one such site and preventing prion or other factors from docking to it and ii) binding to the flexible tail to engage the region of PrP necessary for neurotoxicity.\n\nWe also show that neurotoxic antibodies cause the formation of soluble PrP oligomers that cause toxicity on PrP expressing cell lines; these are not formed in the presence of prion protective antibodies. We suggest that these soluble species might play a role in prion toxicity, similarly to what is generally agreed to happen in other neurodegenerative disorders.

molecular biology↗