O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Testing a Recombinant Form of Tetanus Toxoid as a Carrier Protein for Glycoconjugate Vaccines.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

Characterization of Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-Antigens in Human Sera.

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18-/- syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella.

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials.

Guiding Chemically Synthesized Peptide Drug Lead Optimization by Derisking Mast Cell Degranulation-Related Toxicities of a NaV1.7 Peptide Inhibitor.

Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

Optimization of linear and cyclic peptide inhibitors of KEAP1-NRF2 protein-protein interaction.

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.

Discovery, characterization and potential roles of a novel NF-YAx splice variant in human neuroblastoma.

BACKGROUND: Identification of novel cancer-associated splice variants is of potential diagnostic, prognostic and therapeutic importance. NF-Y transcription factor is comprised of NF-YA, NF-YB and NF-YC subunits, binds inverted CCAAT-boxes in ≈70% of gene promoters, regulates > 1000 cancer-associated genes and proteins involved in proliferation, staminality, differentiation, apoptosis, metabolism and is subject to component alternative splicing. RT-PCR evaluation of alternative NF-YA splicing in primary human neuroblastomas (NBs), led to discovery of a novel NF-YAx splice variant, also expressed during mouse embryo development and induced by doxorubicin in NB cells. Here, we report the discovery and characterisation of NF-YAx and discus its potential roles in NB. METHODS: NF-YAx cDNA was RT-PCR-cloned from a stage 3 NB (provided by the Italian Association of Haematology and Paediatric Oncology, Genova, IT), sequenced and expressed as a protein using standard methods and compared to known fully-spliced NF-YAl and exon B-skipped NF-YAs isoforms in: EMSAs for capacity to form NF-Y complexes; by co-transfection, co-immunoprecipitation and Western blotting for capacity to bind Sp1; by IF for localisation; in AO/EtBr cell-death and colony formation assays for relative cytotoxicity, and by siRNA knockdown, use of inhibitors and Western blotting for potential mechanisms of action. Stable SH-SY5Y transfectants of all three NF-YA isoforms were also propagated and compared by RT-PCR and Western blotting for differences in cell-death and stem cell (SC)-associated gene expression, in cell-death assays for sensitivity to doxorubicin and in in vitro proliferation, substrate-independent growth and in vivo tumour xenograft assays for differences in growth and tumourigenic capacity. RESULTS: NF-YAx was characterized as a novel variant with NF-YA exons B, D and partial F skipping, detected in 20% of NF-YA positive NBs, was the exclusive isoform in a stage 3 NB, expressed in mouse stage E11.5-14 embryos and induced by doxorubicin in SH-SY5Y NB cells. The NF-YAx protein exhibited nuclear localisation, competed with other isoforms in CCAAT box-binding NF-Y complexes but, in contrast to other isoforms, did not bind Sp1. NF-YAx expression in neural-related progenitor and NB cells repressed Bmi1 expression, induced KIF1Bß expression and promoted KIF1Bß-dependent necroptosis but in NB cells also selected tumourigenic, doxorubicin-resistant, CSC-like sub-populations, resistant to NF-YAx cytotoxicity. CONCLUSIONS: The discovery of NF-YAx in NBs, its expression in mouse embryos and induction by doxorubicin in NB cells, unveils a novel NF-YA splice mechanism and variant, regulated by and involved in development, genotoxic-stress and NB. NF-YAx substitution of other isoforms in NF-Y complexes and loss of capacity to bind Sp1, characterises this novel isoform as a functional modifier of NF-Y and its promotion of KIF1Bß-dependent neural-lineage progenitor and NB cell necroptosis, association with doxorubicin-induced necroptosis and expression in mouse embryos coinciding with KIF1Bß-dependent sympathetic neuroblast-culling, confirm a cytotoxic function and potential role in suppressing NB initiation. On the other hand, the in vitro selection of CSC-like NB subpopulations resistant to NF-YAx cytotoxicity not only helps to explain high-level exclusive NF-YAx expression in a stage 3 NB but also supports a role for NF-YAx in disease progression and identifies a potential doxorubicin-inducible mechanism for post-therapeutic relapse.