27Al and 31P NMR spectroscopy method development to quantify aluminum phosphate in adjuvanted vaccine formulations.

A novel qNMR method is described for the quantitative determination of total aluminum and phosphate in aluminum phosphate (AlPO4) adjuvanted vaccine samples using solution 27Al and 31P nuclear magnetic resonance (NMR) spectroscopy. External standard calibrations of AlPO4 solutions established excellent linearity in the range of 15-40 × 10-3 M and additional studies determined the level of detection for both nuclei. A commercialized combination vaccine product (Quadracel®), along with several individual adsorbed antigen components used in the vaccine were employed as model systems for method development. The developed method is also capable of quantitating the free phosphate (i.e. the fraction not bound to AlPO4 particles) in adjuvanted vaccines. This study is the first demonstration of a solution NMR method that is suitable for measuring total aluminum, and free and total phosphate concentrations in vaccine formulations consisting of antigen(s) adsorbed to aluminum adjuvant, in a single analytical workflow.

Tuberculosis vaccine candidate: Characterization of H4-IC31 formulation and H4 antigen conformation.

Tuberculosis (TB) is one of the leading causes of death worldwide, making the development of effective TB vaccines a global priority. A TB vaccine consisting of a recombinant fusion protein, H4, combined with a novel synthetic cationic adjuvant, IC31®, is currently being developed. The H4 fusion protein consists of two immunogenic mycobacterial antigens, Ag85 B and TB10.4, and the IC31® adjuvant is a mixture of KLK, a leucine-rich peptide (KLKL5KLK), and the oligodeoxynucleotide ODN1a, a TLR9 ligand. However, efficient and robust methods for assessing these formulated components are lacking. Here, we developed and optimized phase analysis light scattering (PALS), electrical sensing zone (ESZ), and Raman, FTIR, and CD spectroscopy methods to characterize the H4-IC31 vaccine formulation. PALS-measured conductivity and zeta potential values could differentiate between the similarly sized particles of IC31® adjuvant and the H4-IC31 vaccine candidate and could thereby serve as a control during vaccine formulation. In addition, zeta potential is indicative of the adjuvant to antigen ratio which is the key in the immunomodulatory response of the vaccine. ESZ was used as an orthogonal method to measure IC31® and H4-IC31 particle sizes. Raman, FTIR, and CD spectroscopy revealed structural changes in H4 protein and IC31® adjuvant, inducing an increase in both the ß-sheet and random coil content as a result of adsorption. Furthermore, nanoDSF showed changes in the tertiary structure of H4 protein as a result of adjuvantation to IC31®. Our findings demonstrate the applicability of biophysical methods to characterize vaccine components in the final H4-IC31 drug product without the requirement for desorption.

Molecular mechanisms initiating amyloid beta-fibril formation in Alzheimer's disease.

The deposition of aggregated amyloid beta-protein (Abeta) in the human brain is a major lesion in Alzheimer' disease (AD). The process of Abeta fibril formation is associated with a cascade of neuropathogenic events that induces brain neurodegeneration leading to the cognitive and behavioral decline characteristic of AD. Although a detailed knowledge of Abeta assembly is crucial for the development of new therapeutic approaches, our understanding of the molecular mechanisms underlying the initiation of Abeta fibril formation remains very incomplete. The genetic defects responsible for familial AD influence fibrillogenesis. In a majority of familial cases determined by amyloid precursor protein (APP) and presenilin (PS) mutations, a significant overproduction of Abeta and an increase in the Abeta42/Abeta40 ratio are observed. Recently, it was shown that the two main alloforms of Abeta have distinct biological activity and behaviour at the earliest stage of assembly. In vitro studies demonstrated that Abeta42 monomers, but not Abeta40, form initial and minimal structures (pentamer/hexamer units called paranuclei) that can oligomerize to larger forms. It is now apparent that Abeta oligomers and protofibrils are more neurotoxic than mature Abeta fibrils or amyloid plaques. The neurotoxicity of the prefibrillar aggregates appears to result from their ability to impair fundamental cellular processes by interacting with the cellular membrane, causing oxidative stress and increasing free Ca(2+) that eventually lead to apoptotic cell death.

A direct interaction between transforming growth factor (TGF)-betas and amyloid-beta protein affects fibrillogenesis in a TGF-beta receptor-independent manner.

Transforming growth factor-beta (TGF-beta) receptor-mediated signaling has been proposed to mediate both the beneficial and deleterious roles for this cytokine in amyloid-beta protein (Abeta) function. In order to assess receptor dependence of these events, we used PC12 cell cultures, which are devoid of TGF-beta receptors. Surprisingly, TGF-beta potentiated the neurotoxic effects of the 40-residue Abeta peptide, Abeta-(1-40), in this model suggesting that there may be a direct, receptor-independent interaction between TGF-beta and Abeta-(1-40). Surface plasmon resonance confirmed that TGF-beta binds with high affinity directly to Abeta-(1-40) and electron microscopy revealed that TGF-beta enhances Abeta-(1-40) oligomerization. Immunohistochemical examination of mouse brain revealed that hippocampal CA1 and dentate gyrus, two regions classically associated with Abeta-mediated pathology, lack TGF-beta Type I receptor immunoreactivity, thus indicating that TGF-beta receptor-mediated signaling would not be favored in these regions. Our observations not only provide for a unique, receptor-independent mechanism of action for TGF-beta, but also help to reconcile the literature interpreting the role of TGF-beta in Abeta function. These data support a critical etiological role for this mechanism in neuropathological amyloidoses.

Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways.

Amyloid beta-protein (Abeta) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of Abeta in AD is new evidence that oligomeric forms of Abeta are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Abeta, Abeta42, with the disease. Detailed knowledge of the structure and assembly dynamics of Abeta thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Abeta oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Abeta40 and Abeta42. Carefully prepared aggregate-free Abeta40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Abeta42 preferentially formed pentamerhexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Abeta40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Abeta42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Abeta42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by amyloid deposition in the cerebral neuropil and vasculature. These amyloid deposits comprise predominantly fragments and full-length (40 or 42 residue) forms of the amyloid beta-protein (Abeta) organized into fibrillar assemblies. Compelling evidence indicates that factors that increase overall Abeta production or the ratio of longer to shorter forms, or which facilitate deposition or inhibit elimination of amyloid deposits, cause AD or are risk factors for the disease. In vitro studies have demonstrated that fibrillar Abeta has potent neurotoxic effects on cultured neurons. In vivo experiments in non-human primates have demonstrated that Abeta fibrils directly cause pathologic changes, including tau hyperphosphorylation. In concert with histologic studies revealing a lack of tissue injury in areas of the neuropil in which non-fibrillar deposits were found, these data suggested that fibril assembly was a prerequisite for Abeta-mediated neurotoxicity in vivo. Recently, however, both in vitro and in vivo studies have revealed that soluble, oligomeric forms of Abeta also have potent neurotoxic activities, and in fact, may be the proximate effectors of the neuronal injury and death occurring in AD. A paradigm shift is thus emerging that necessitates the reevaluation of the relative importance of polymeric (fibrillar) vs. oligomeric assemblies in the pathobiology of AD. In addition to AD, an increasing number of neurodegenerative disorders, including Parkinson's disease, familial British dementia, familial amyloid polyneuropathy, amyotrophic lateral sclerosis, and prion diseases, are associated with abnormal protein assembly processes. The archetypal features of the assembly-dependent neuropathogenetic effects of Abeta may thus be of relevance not only to AD but to these other disorders as well.

Structure determination of micelle-like intermediates in amyloid beta -protein fibril assembly by using small angle neutron scattering.

Increasing evidence supports the hypothesis that amyloid beta-protein (Abeta) assembly is a key pathogenic feature of Alzheimer's disease. Thus, understanding the assembly process offers opportunities for the development of strategies for treating this devastating disease. In prior studies, Abeta was found to form micelle-like aggregates under acidic conditions. These structures exhibited an average observed hydrodynamic radius of 7 nm. They were found to be in rapid equilibrium with Abeta monomers or low molecular weight oligomers, and were centers of fibril nucleation. Here the technique of small angle neutron scattering has been used to determine the structure of these Abeta micelles. The data reveal that the micellar assemblies comprise 30-50 Abeta monomers and have elongated geometries. The best fit of the data to a uniform spherocylinder yields a radius approximately 2.4 nm and cylinder length approximately 11 nm. These structure parameters remain constant over more than a decade in concentration range. The concentration independence of the length of the cylindrical aggregate indicates the presence of an internal nonrepetitive structure that spans the entire length of the Abeta assembly.