Assessment of Preanalytical Cerebrospinal Fluid Handling and Storage Factors on Measurement of Aβ1-42, Aβ1-40, and pTau181 Using an Automated Chemiluminescent Platform.

Background: Standardizing cerebrospinal fluid (CSF) laboratory protocols will improve the reliability and availability of clinical biomarker testing required for prescription of novel Alzheimer disease (AD) therapies. This study evaluated several preanalytical handling and storage factors common to β-amyloid1-42 (Aβ1-42), β-amyloid1-40 (Aβ1-40), and phosphorylated tau (pTau181) concentrations including storage at different temperatures, extended cap contact, various mixing methods, and multiple freeze-thaw cycles.

Methods: Aβ1-42, Aβ1-40, and pTau181 concentrations were measured using LUMIPULSE G1200 automated assays.

A Quantile-Quantile Toolbox for Reference Intervals.

Background: Parametric statistical methods are generally better than nonparametric, but require that data follow a known, usually normal, distribution. One important application is finding reference limits and detection limits. Parametric analyses yield better estimates and measures of their uncertainty than nonparametric approaches, which rely solely on a few extreme values.

Engineered DNA-encoded monoclonal antibodies targeting Plasmodium falciparum circumsporozoite protein confer single dose protection in a murine malaria challenge model.

Novel approaches for malaria prophylaxis remain important. Synthetic DNA-encoded monoclonal antibodies (DMAbs) are a promising approach to generate rapid, direct in vivo host-generated mAbs with potential benefits in production simplicity and distribution coupled with genetic engineering. Here, we explore this approach in a malaria challenge model.

Strategic Variants of CSP Delivered as SynDNA Vaccines Demonstrate Heterogeneity of Immunogenicity and Protection from Infection in a Murine Model.

Malaria infects millions of people every year, and despite recent advances in controlling disease spread, such as vaccination, it remains a global health concern. The circumsporozoite protein (CSP) has long been acknowledged as a key target in antimalarial immunity. Leveraging the DNA vaccine platform against this formidable pathogen, the following five synthetic DNA vaccines encoding variations of CSP were designed and studied: 3D7, GPI1, ΔGPI, TM, and DD2.

The Alzheimer's Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid β and tau.

The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aβ42 and Aβ40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high diagnostic performance for AD, including the early phases of the disease. However, between-center differences in pre-analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry.

Effect of Patient-Specific Preanalytic Variables on CSF Aβ1-42 Concentrations Measured on an Automated Chemiluminescent Platform.

Background: Cerebrospinal fluid (CSF) biomarkers are increasingly used to confirm the accuracy of a clinical diagnosis of mild cognitive impairment or dementia due to Alzheimer disease (AD). Recent evidence suggests that fully automated assays reduce the impact of some preanalytical factors on the variability of these measures. This study evaluated the effect of several preanalytical variables common in clinical settings on the variability of CSF β-amyloid 1-42 (Aβ1-42) concentrations.

Protection against infection mediated by a synthetically engineered DNA vaccine.

Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease.

Concordance of Lumipulse cerebrospinal fluid t-tau/Aβ42 ratio with amyloid PET status.

Introduction: Cerebrospinal fluid (CSF) biomarkers can identify individuals with Alzheimer's disease (AD) pathology (eg, amyloid plaques, neurofibrillary tangles), but defined analyte cut-points using high-throughput automated assays are necessary for general clinical use.

Methods: CSF amyloid β42 peptide (Aβ42), t-tau, and t-tau/Aβ42 were quantified by the Lumipulse platform in two test cohorts (A/B: Eisai BAN2401-201/MISSION AD E2609-301/302, n = 138; C: Knight Alzheimer's Disease Research Center (ADRC), n = 198), and receiver operating characteristic (ROC) curve analyses defined cut-points corresponding best to amyloid determinations using positron emission tomography (PET) imaging. The best-performing cut-point was then validated as a predictor of amyloid status in an independent cohort (D: MISSION AD E2609-301/302, n = 240).

In Vivo Delivery of a DNA-Encoded Monoclonal Antibody Protects Non-human Primates against Zika Virus.

Zika virus (ZIKV) infection is endemic to several world regions, and many others are at high risk for seasonal outbreaks. Synthetic DNA-encoded monoclonal antibody (DMAb) is an approach that enables in vivo delivery of highly potent mAbs to control infections. We engineered DMAb-ZK190, encoding the mAb ZK190 neutralizing antibody, which targets the ZIKV E protein DIII domain.

Anti-OspA DNA-Encoded Monoclonal Antibody Prevents Transmission of Spirochetes in Tick Challenge Providing Sterilizing Immunity in Mice.

We recently developed anti-OspA human immunoglobulin G1 monoclonal antibodies (HuMAbs) that are effective in preventing Borrelia transmission from ticks in a murine model. Here, we investigated a novel approach of DNA-mediated gene transfer of HuMAbs that provide protection against Lyme disease. Plasmid DNA-encoded anti-OspA HuMAbs inoculated in mice achieved a serum antibody concentration of >6 μg/mL.

In Vivo Delivery of Synthetic Human DNA-Encoded Monoclonal Antibodies Protect against Ebolavirus Infection in a Mouse Model.

Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model.

Rapid response to an emerging infectious disease - Lessons learned from development of a synthetic DNA vaccine targeting Zika virus.

Vaccines are considered one of the greatest advances in modern medicine. The global burden of numerous infectious diseases has been significantly reduced, and in some cases, effectively eradicated through the deployment of specific vaccines. However, efforts to develop effective new vaccines against infectious pathogens such as influenza, Human immunodeficiency virus (HIV), dengue virus (DENV), chikungunya virus (CHIKV), Ebola virus, and Zika virus (ZIKV) have proven challenging.

Screening of a Haloferax volcanii Transposon Library Reveals Novel Motility and Adhesion Mutants.

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella-structures required for motility-share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain.

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation.

N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown.

Archaeal type IV pili and their involvement in biofilm formation.

Type IV pili are ancient proteinaceous structures present on the cell surface of species in nearly all bacterial and archaeal phyla. These filaments, which are required for a diverse array of important cellular processes, are assembled employing a conserved set of core components. While type IV pilins, the structural subunits of pili, share little sequence homology, their signal peptides are structurally conserved allowing for in silico prediction.

A conserved type IV pilin signal peptide H-domain is critical for the post-translational regulation of flagella-dependent motility.

In many bacteria and archaea, type IV pili facilitate surface adhesion, the initial step in biofilm formation. Haloferax volcanii has a specific set of adhesion pilins (PilA1-A6) that, although diverse, contain an absolutely conserved signal peptide hydrophobic (H) domain. Data presented here demonstrate that these pilins (PilA1-A6) also play an important role in regulating flagella-dependent motility, which allows cells to rapidly transition between planktonic and sessile states.

Haloferax volcanii cells lacking the flagellin FlgA2 are hypermotile.

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2.

Novel archaeal adhesion pilins with a conserved N terminus.

Type IV pili play important roles in a wide array of processes, including surface adhesion and twitching motility. Although archaeal genomes encode a diverse set of type IV pilus subunits, the functions for most remain unknown. We have now characterized six Haloferax volcanii pilins, PilA[1-6], each containing an identical 30-amino-acid N-terminal hydrophobic motif that is part of a larger highly conserved domain of unknown function (Duf1628).