Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

Development and validation of an expedited 10g protein counter (EP-10) for dietary protein intake quantification

Precise protein quantification is essential in clinical dietetics, particularly in the management of renal, burn and malnourished patients. The EP-10 was developed to expedite the estimation of dietary protein for nutritional assessment and recommendation. The main objective of this study was to compare the validity and efficacy of the EP-10 with the American Dietetic Association’s “Exchange List for Meal Planning” (ADA-7g) in quantifying dietary protein intake, against computerised nutrient analysis (CNA). Protein intake of 197 food records kept by healthy adult subjects in Singapore was determined thrice using three different methods – (1) EP-10, (2) ADA-7g and (3) CNA using SERVE program (Version 4.0). Assessments using the EP-10 and ADA-7g were performed by two assessors in a blind crossover manner while a third assessor performed the CNA. All assessors were blind to each other’s results. Time taken to assess a subsample (n=165) using the EP-10 and ADA-7g was also recorded. Mean difference in protein intake quantification when compared to the CNA was statistically non-significant for the EP-10 (1.4 ± 16.3 g, P = .239) and statistically significant for the ADA-7g (-2.2 ± 15.6 g, P = .046). Both the EP-10 and ADA-7g had clinically acceptable agreement with the CNA as determined via Bland-Altman plots, although it was found that EP-10 had a tendency to overestimate with protein intakes above 150 g. The EP-10 required significantly less time for protein intake quantification than the ADA-7g (mean time of 65 ± 36 seconds vs. 111 ± 40 seconds, P < .001). The EP-10 and ADA-7g are valid clinical tools for protein intake quantification in an Asian context, with EP-10 being more time efficient. However, a dietician’s discretion is needed when the EP-10 is used on protein intakes above 150g.

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and β-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane.

Cellular settings mediating Src Substrate switching between Focal Adhesion Kinase Tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) Tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Polycaprolactone-based scaffold plus recombinant human bone morphogenetic protein (rhBMP-2) in an ovine model of anterior spinal fusion

Synthetic scaffolds combined with growth factors have the potential to replace allograft or autograft as a graft material for spinal interbody fusion. Such tissue engineering approaches may be useful in Adolescent Idiopathic Scoliosis (AIS) surgery, however there are no studies to date examining the use of such biodegradable implants in combination with biologics in a thoracic spine model. This in vivo study examines the use of biodegradable polycaprolactone (PCL) based scaffolds with rhBMP-2 as a bone graft substitute in a sheep thoracic fusion model, where an anterior approach is used to simulate minimally invasive surgical deformity correction in the setting of AIS.

The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Hepatitis C virus (HCV ) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site(IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly.

Using expedited 10g protein counter (EP-10) for meal planning

Precise protein quantification and recommendation is essential in clinical dietetics, particularly in the management of individuals with chronic kidney disease, malnutrition, burns, wounds, pressure ulcers, and those in active sports. The Expedited 10g Protein Counter (EP-10) was developed to simplify the quantification of dietary protein for assessment and recommendation of protein intake.1 Instead of using separate protein exchanges for different food groups to quantify the dietary protein intake of an individual, every exchange in the EP-10 accounts for an exchange each of 3g non-protein-rich food and 7g protein-rich food (Table 1). The EP-10 was recently validated and published in the Journal of Renal Nutrition recently.1 This study demonstrated that using the EP-10 for dietary protein intake quantification had clinically acceptable validity and reliability when compared with the conventional 7g protein exchange while requiring less time.2 In clinical practice, the use of efficient, accurate and practical methods to facilitate assessment and treatment plans is important. The EP-10 can be easily implemented in the nutrition assessment and recommendation for a patient in the clinical setting. This patient education tool was adapted from materials printed in the Journal of Renal Nutrition.1 The tool may be used as presented or adapted to assist patients to achieve their recommended daily protein intake.

Nutritional intervention incorporating expedited 10g protein counter (EP-10) to improve the albumin and transferrin of chronic hemodialysis patients

Objective: The expedited 10g protein counter (EP-10) is a quick and valid clinical tool for dietary protein quantification. This study aims to assess the clinical effectiveness of the EP-10 in improving serum albumin and transferrin in chronic hemodialysis patients. Methods: Forty-five patients with low serum albumin (< 38 g /L) were enrolled in this study. Parameters measured included dry weight, height, dietary intake, and levels of serum albumin, transferrin, potassium, phosphate and kinetic modeling (Kt/v). The nutritional intervention incorporated the EP-10 in two ways (1)lto quantify protein intake of patients and (2)ito educate patients to meet their protein requirements. Mean values of the nutritional parameters before and after intervention were compared using paired t-test. Results: Three months after nutritional intervention, mean albumin levels increased significantly from 32.2+4.8g/L to 37.0+3.2g/L (p<0.001). Thirty-eight (84%) patients showed an increase in albumin levels while two (4%) maintained their levels. Of the thirty-six (80%) patients with low transferrin levels (<200 mg/dL), 28 (78%) had an increase and two maintained their levels post-intervention. Mean transferrin levels increased significantly from 169.4+39.9mg/dL to 180.9+38.1mg/dL (p< 0.05). Conclusion: Nutritional intervention incorporating the EP-10 method is able to make significant improvements to albumin and transferrin levels of chronic hemodialysis patients.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: Comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means. © 2007 SGM.

Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum

Background We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine. Results Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and - to a lesser degree - the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants - a significant increase over previous efforts. Immunization of New Zealand white rabbits with ∼50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro. Conclusion We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants. © 2007 Kohl et al; licensee BioMed Central Ltd.