Characterization of human papillomavirus type 16 pseudovirus containing histones

Lymphoproliferative responses following four immunizations with HPV16 PsVs from fraction I, II, or III. The mice were immunized four times with 50 ng of PsVs per dose at 2-week intervals. Mouse splenocytes were obtained 5 days after the fourth immunization. Mouse splenocytes were labeled with carboxyfluorescein succinimidyl ester (CFSE), stimulated with purified HPV16 L1 VLPs, and cultured for 4 days. The splenocytes were stained with allophycocyanin (APC)-conjugated anti-CD4 antibody (eBioscience, USA) and examined with a FACSCalibur flow cytometer (BD Bioscience, USA). To count CD4+ cells, the cells were gated according to forward and side scatter, and the upper-left segment of each graph was counted on FITC and APC scatter plots. Panel A shows the flow cytometry results for three individual mice. The value in panel B represents the mean ± SEM (n = 3). (DOCX 171 kb

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

The Choice of Resin-Bound Ligand Affects the Structure and Immunogenicity of Column-Purified Human Papillomavirus Type 16 Virus-Like Particles

Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity o

Analysis of the structural characteristics of hHPV16 and cHPV16 VLP.

<p>To analyze the structural integrity of the two VLP types, the HPV16 VLPs were fractionated on Optiprep density gradients (A). Eight fractions were collected in experiment A (0.5 ml each). The results of TEM analysis are presented in panel B. Magnification is 41,000× (bars 50 nm).</p

Camvir-1, H16.V5 and H16.E70 reactivity towards scHPV16 VLPs and schHPV16 VLPs.

<p>The amounts of L1 proteins contained in scHPV16 VLP and schHPV16 VLP were confirmed by SDS-PAGE and Western blotting prior to performing ELISAs (A). In panel A, loading amount indicates protein amount loaded for SDS-PAGE and Western blot. L1 amount indicates L1 protein amount contained in the loading sample. The L1 protein amount was confirmed by L1 band intensities on SDS-PAGE and Western blot. M indicates the molecular weight marker. The SDS-PAGE and western blot are representatives of duplicate assays. The Camvir-1, H16.V5 and H16.E70 reactivity towards schHPV16 VLPs and scHPV16 VLPs were determined by direct ELISA and are presented in B, C and D, respectively. The ODs of hHPV16 VLPs after reaction with 1 µg/ml of Camvir-1, 0.25 µg/ml of H16.V5 and 0.25 µg/ml of H16.E70 were set at 100% in B, C and D, respectively. The ELISA values are the means ± SD of two independent assays.</p

Immunization Protocol-1 Anti-HPV16 L1 IgG titer and neutralization activity resulting.

<p>Mice were immunized according to immunization protocol-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035893#pone-0035893-t002" target="_blank">Table 2</a>). The mice were immunized four times with 100 µl of PBS, 8 ng of hHPV16 VLP or 8 ng of cHPV16 VLP at two-week intervals, in the absence of adjuvant. Ten days after the 3^rd and 4^th immunization, the mice sera were obtained and analyzed. Panels A and B present the anti-HPV16 L1 IgG titer and neutralization activity after the 3^rd immunization, respectively. Panels C and D present the anti-HPV16 L1 IgG titer and neutralization activity after the 4^th immunization, respectively. The horizontal bars are median values of the IgG titer and neutralization activity (PBS, n = 6; hHPV16 VLP, n = 15; cHPV16 VLP, n = 15).</p

Camvir-1, H16.V5 and H16.E70 reactivity towards hHPV16 VLPs and cHPV16 VLPs.

<p>The HPV16 L1 protein residues recognized by H16.V5, H16.E70 and Camvir-1 are displayed graphically in (A). BC, CD, DE, EF, FG and HI indicate the loop structures in HPV16 L1. The numbers refer to the amino acid residues as counted from the N-terminus, the black boxes indicate loops covering the solvent-exposed face of the capsid, and the white box (CD) indicates an internal loop. The gray box (EF) indicates a loop partly located on the outside of the capsid. The hHPV16 VLP and cHPV16 VLP concentrations were confirmed by SDS-PAGE prior to running ELISAs (B). The protein concentration of each VLP preparation was determined by Bradford protein assay, and 500 to 62 ng of proteins were loaded for SDS-PAGE analysis. M indicates the molecular weight marker. Camvir-1, H16.V5 and H16.E70 reactivity towards hHPV16 VLPs and cHPV16 VLPs was determined by direct ELISA. The ELISA results are presented in C, D and E, respectively. The ODs of the hHPV16 VLPs after reaction with 1 µg/ml of Camvir-1, 0.25 µg/ml of H16.V5 and 0.25 µg/ml of H16.E70 were set at 100% in C, D and E, respectively. The ELISA values are the means ± SD of two independent assays.</p

Particle size distributions of hHPV16 VLP and cHPV16 VLP.

<p>The HPV16 VLP populations along with their associated hydrodynamic diameters were analyzed by DLS as described in the Materials and Methods section. Each HPV16 VLP was prepared in 25 mM MOPS containing 75 mM NaCl pH 7.0 and adjusted to 40 µg/ml. Panel A and B are representatives of duplicate measurements of the hHPV16 VLP and cHPV16 VLP populations, respectively.</p

Mouse immunization protocols used in this study.

<p>Mouse immunization protocols used in this study.</p

Procedures used to purify the HPV16 VLPs used in this study.

<p>All the HPV16 VLP preparations were finally dialyzed against 0.325 M NaCl in phosphate buffer pH 7.2.</p>a<p>The scHPV16 VLP was further separated by heparin chromatography.</p