Profiling microRNAs through development of the parasitic nematode Haemonchus identifies nematode-specific miRNAs that suppress larval development

Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host

Toolbox for Non-Intrusive Structural and Functional Analysis of Recombinant VLP Based Vaccines: A Case Study with Hepatitis B Vaccine

Background: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine. Methodology: The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM). Principal Findings: SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turnaround, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipidto-protei

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs).</p> <p>Results</p> <p>VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced.</p> <p>Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire</p> <p>HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres.</p> <p>Conclusions</p> <p>D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.</p

The majority of ruptured aneurysms are small with low rupture risk scores.

BACKGROUND: Understanding the rupture risk of unruptured intracranial aneurysms has important clinical implications given the morbidity and mortality associated with subarachnoid hemorrhage (SAH). The ISUIA, UCAS, and PHASES studies provide rupture risk calculations. OBJECTIVE: We apply the risk calculations to a series ruptured intracranial aneurysms to assess the rupture risk for each aneurysm (had they been discovered in the unruptured state). METHODS: This is a retrospective study of 246 patients with SAH from a ruptured saccular aneurysm. The ISUIA, UCAS, and PHASES calculators were applied to each patient/aneurysm to demonstrate a theoretical annual risk of rupture dichotomized by aneurysm location. RESULTS: The average diameter of the aneurysms was 5.5 ± 3.1 mm. Three quarters (75%) of the aneurysms measured48.8% were(Acomm) was the most common location of rupture (24.7%). Posterior communicating artery aneurysms (Pcomm) were the third most common at 16.2%. The average ISUIA 1-year rupture risk was 0.46 ± 0.008%. The average UCAS 1-year rupture risk was 0.93% ± 0.01. The annualPHASESrupture risk was0.32 ± 0.004%. The highest risk locations were the vertebral artery (up to 10.3% per year) and superior cerebellar artery (up to 2.7% per year). On average, Acomm aneurysms had 1 year risk no higher than 1.1% and Pcomm aneurysms no higher than 1.2% per year. CONCLUSION: We observed that in a small retrospective series of ruptured aneurysms, the majority wereaneurysms, had they been discovered in the unruptured state, is low

Characterization of virus-like particles in GARDASIL (R) by cryo transmission electron microscopy

Chinese National Science Fund [81273327]; 863 Major Project [2012AA02A408]Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL (R). CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant. In addition, we determined the three-dimensional structure of the VLPs, both alone and when interacting with neutralizing antibodies. Two modes of binding of two different neutralizing antibodies were apparent; for HPV type 11 saturated with H11.B2, 72 potential Fab binding sites were observed at the center of each capsomer, whereas for HPV 16 interacting with H16.V5, it appears that 60 pentamers (each neighboring 6 other pentamers) bind five Fabs per pentamer, for the total of 300 potential Fab binding sites per VLP

CryoTEM map of rHBsAg lipid-protein particle.

<p>(A) The resulting 3D map presented with roughly spherical morphology with “knuckle”-like protrusions projecting from a smooth surface. (B) Segmentation of the map revealed regions of high density, presumed to be protein, surrounded by regions of lesser density, presumed to be lipid. Map shown end-on for the 4-fold (top left), 2-fold (top right), and 3-fold (bottom left) views.</p

Non-intrusive biophysical and immunochemical methods evaluated here for VLP characterization compared with conventional techniques.

*<p>The solution competitive ELISA measures the accessible epitopes on VLPs adsorbed to particulate adjuvant. This method is used to probe the stability samples upon prolonged storage in a more faithful manner as to the intact and accessible epitopes. In addition, this method may also mimic the <i>in vivo</i> antigen presentation to some degree without the needs to dissolve the aluminum adjuvant, which is co-injected with antigen during immunization.</p

Arrangement of protein and lipid in the rHBsAg particle.

<p>(A) The arm (ar) and shoulder (sh) regions of each S-protein containing unit wrap tightly around the body (bd) of a neighboring unit to form a tight framework of protein around which lipids are interspersed. (B) The body (bd) organizes the outer lipid layer (oL), whereas the inner lipid (iL) layer surrounds the arm region of the S-protein containing densities that protrude into the inside of the VLP.</p

Structural features of the protein containing protrusion and surrounding lipid layer.

<p>(A) A single protein protrusion with associated lipid was segmented from the 3D map and (B) various structural features present in the lipid and protein densities were identified. The protein unit is composed of a body (bd), shoulder (sh), and arm (ar) region, and the lipid layer is composed of an outer layer (oL) and an inner layer (iL).</p