A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein.

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin.

Allogeneic B cell immunomodulatory therapy in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease. Immune system dysregulation plays an essential role in ALS onset and progression. Our preclinical studies have shown that the administration of exogenous allogeneic B cells improves outcomes in murine models of skin and brain injury through a process termed pligodraxis, in which B cells adopt an immunoregulatory and neuroprotective phenotype in an injured environment.

Targeting metabolic pathways to counter cancer immunotherapy resistance.

Immunotherapies have revolutionized the treatment of certain cancers, but challenges remain in overcoming immunotherapy resistance. Research shows that metabolic modulation of the tumor microenvironment can enhance antitumor immunity. Here, we discuss recent preclinical and clinical evidence for the efficacy of combining metabolic modifiers with immunotherapies.

Immunomodulatory drugs: a promising clinical ally for cancer immunotherapy.

While immunomodulatory imide drugs (IMiDs) have been authorised for treatment of haematological cancers for over two decades, the appreciation of their ability to stimulate antitumour T cell and natural killer (NK) cell responses is relatively recent. Clinical trial data increasingly show that targeted immunotherapies, such as antibodies, T cells, and vaccines, improve outcomes when delivered in combination with the IMiD derivatives lenalidomide or pomalidomide. Here, we review these clinical data to highlight the relevance of IMiDs in combinatorial immunotherapy for both haematological and solid tumours.

Cytometry profiling of recall responses to in previously naturally exposed individuals reveals long-term changes in both adaptive and innate immune cellular compartments.

Introduction: Q fever, caused by the intracellular bacterium , is considered an occupational and biodefense hazard and can result in debilitating long-term complications. While natural infection and vaccination induce humoral and cellular immune responses, the exact nature of cellular immune responses to is incompletely understood. The current study seeks to investigate more deeply the nature of long-term cellular recall responses in naturally exposed individuals by both cytokine release assessment and cytometry profiling.

Combinatorial islet protective therapeutic approaches in β-cell transplantation: Rationally designed solutions using a target product profile.

While progress has been made in the development of islet cell transplantation (ICT) as a viable alternative to the use of exogenous insulin therapy in the treatment of type 1 diabetes, it has not yet achieved its full potential in clinical studies. Ideally, ICT would enable lifelong maintenance of euglycemia without the need for exogenous insulin, blood glucose monitoring or systemic immune suppression. To achieve such an optimal result, therapeutic approaches should simultaneously promote long-term islet viability, functionality, and localized immune protection.

Q fever vaccine development: Challenges and progress in balancing safety and efficacy.

The existing human vaccine against Q fever, a zoonotic disease of biothreat concern, is approved only in Australia. In this issue of , Gregory and colleagues describe a new vaccine candidate that overcomes specific concerns hindering wider acceptance of the commercial vaccine..

B cells support the repair of injured tissues by adopting MyD88-dependent regulatory functions and phenotype.

Exogenously applied mature naïve B220 /CD19 /IgM /IgD B cells are strongly protective in the context of tissue injury. However, the mechanisms by which B cells detect tissue injury and aid repair remain elusive. Here, we show in distinct models of skin and brain injury that MyD88-dependent toll-like receptor (TLR) signaling through TLR2/6 and TLR4 is essential for the protective benefit of B cells in vivo, while B cell-specific deletion of MyD88 abrogated this effect.

Whole Blood Interferon γ Release Is a More Sensitive Marker of Prior Exposure to Than Are Antibody Responses.

For the zoonotic disease Q fever, serological analysis plays a dominant role in the diagnosis of infection and in pre-screening for past exposure prior to vaccination. A number of studies suggest that assessment of -specific T-cell IFNγ responses may be a more sensitive tool to assess past exposure. In this study, we assessed the performance of a whole blood IFNγ release assay in comparison to serological detection in an area of high Q fever incidence in 2014, up to seven years after initial exposure during the Dutch Q fever outbreak 2007-2010.

Coxiella burnetii Epitope-Specific T-Cell Responses in Patients with Chronic Q Fever.

Infection with , the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic infection (convalescents) to promiscuous HLA class II epitopes, providing the basis for a novel T-cell targeted subunit vaccine.

Promiscuous CD4 Epitope Clusters Associated With Human Recall Responses Are Candidates for a Novel T-Cell Targeted Multi-Epitope Q Fever Vaccine.

, the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine.

Dual blockade of CXCL12-CXCR4 and PD-1-PD-L1 pathways prolongs survival of ovarian tumor-bearing mice by prevention of immunosuppression in the tumor microenvironment.

Blockade of immune-checkpoint programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 can enhance effector T-cell responses. However, the lack of response in many patients to checkpoint-inhibitor therapies emphasizes the need for combination immunotherapies to pursue maximal antitumor efficacy. We have previously demonstrated that antagonism of C-X-C chemokine receptor type 4 (CXCR4) by plerixafor (AMD3100) can decrease regulatory T (T)-cell intratumoral infiltration.

Standardized guinea pig model for Q fever vaccine reactogenicity.

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls.

AMD3100 Augments the Efficacy of Mesothelin-Targeted, Immune-Activating VIC-008 in Mesothelioma by Modulating Intratumoral Immunosuppression.

AMD3100 (plerixafor), a CXCR4 antagonist, has been demonstrated to suppress tumor growth and modulate intratumoral T-cell trafficking. However, the effect of AMD3100 on immunomodulation remains elusive. Here, we explored immunomodulation and antitumor efficacy of AMD3100 in combination with a previously developed mesothelin-targeted, immune-activating fusion protein, VIC-008, in two syngeneic, orthotopic models of malignant mesothelioma in immunocompetent mice.

Application and utility of mass cytometry in vaccine development.

Mass cytometry enables highly multiplexed profiling of cellular immune responses in limited-volume samples, advancing prospects of a new era of systems immunology. The capabilities of mass cytometry offer expanded potential for deciphering immune responses to infectious diseases and to vaccines. Several studies have used mass cytometry to profile protective immune responses, both postinfection and postvaccination, although no vaccine-development program has yet systematically employed the technology from the outset to inform both candidate design and clinical evaluation.

Q-vaxcelerate: A distributed development approach for a new Coxiella burnetii vaccine.

Development of vaccines that are both safe and effective remains a costly and time-consuming challenge. To accelerate the pace of development and improve the efficacy and safety of candidate vaccines for both existing and emerging infectious agents, we have used a distributed development approach. This features the managed integration of individual expert groups having the requisite vaccine platforms, pre-clinical models, assays, skills and knowledge pertinent to a specific pathogen into a single, end-to-end development team capable of producing a new vaccine tailored to that particular agent.

Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions.

Chronic wounds affect 12-15% of patients with diabetes and are associated with a drastic decrease in their quality of life. Here, we demonstrate that purified mature naive B220 /CD19 /IgM /IgD B cells improve healing of acute and diabetic murine wounds after a single topical application. B cell treatment significantly accelerated acute wound closure by 2-3 days in wild-type mice and 5-6 days in obese diabetic mice.

The Caenorhabditis elegans NR4A nuclear receptor is required for spermatheca morphogenesis.

The gene nhr-6 encodes the Caenorhabditis elegans ortholog of the NR4A nuclear receptor. We determined the biological functions of NHR-6 through the isolation and characterization of a deletion allele of nhr-6, lg6001. We demonstrate that nhr-6 has an essential role in the development of the C.

Draft genome of the filarial nematode parasite Brugia malayi.

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units.

RNAi-based discovery and validation of new drug targets in filarial nematodes.

Human filarial nematodes cause river blindness and lymphatic filariasis, both of which are diseases that produce considerable morbidity. Control of these diseases relies on drug treatments that are ineffective against macrofilariae and are threatened by the development of resistance. New validated drug targets are required to allow development of new classes of antifilarial drugs.

From genes to drugs: target validation in Caenorhabditis elegans.

A central challenge in post-genomic drug discovery is selection of relevant therapeutic targets from a large pool of candidates, so that resources are invested productively. Key to meeting this challenge is demonstrating target function in disease-related pathways in vivo. The nematode Caenorhabditis elegans, a well-characterized experimental system for genetic analysis of biological regulatory pathways, provides a powerful means of assessing the impact of modulating target function on biological processes, thus facilitating selection of high-value targets.