Development of a Stable Virus-Like Particle Vaccine Formulation against Chikungunya Virus and Investigation of the Effects of Polyanions

Chikungunya virus (CHIKV) is an alphavirus that infects millions of people every year, especially in the developing world. The selective expression of recombinant CHIKV capsid and envelope proteins results in the formation of self-assembled virus-like particles (VLPs) that have been shown to protect nonhuman primates against infection from multiple strains of CHIKV. This study describes the characterization, excipient screening, and optimization of CHIKV VLP solution conditions toward the development of a stable parenteral formulation. The CHIKV VLPs were found to be poorly soluble at pH 6 and below. Circular dichroism, intrinsic fluorescence, and static and dynamic light scattering measurements were therefore performed at neutral pH, and results consistent with the formation of molten globule structures were observed at elevated temperatures. A library of generally recognized as safe excipients was screened for their ability to physically stabilize CHIKV VLPs using a high-throughput turbidity-based assay. Sugars, sugar alcohols, and polyanions were identified as potential stabilizers and the concentrations and combinations of select excipients were optimized. The effects of polyanions were further studied, and while all polyanions tested stabilized CHIKV VLPs against aggregation, the effects of polyanions on conformational stability varied.     

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N-linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10 °C-90 °C) and solution pH (3-8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle-to-larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two-step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.     

Physical Characterization and Formulation Development of a Recombinant Pneumolysoid Protein-Based Pneumococcal Vaccine

Streptococcus pneumoniae is a major cause of death in children worldwide. There are more than 90 known pneumococcus serotypes that vary by geographical location. Pneumolysin is a protein toxin produced by virtually all invasive strains of S. pneumoniae and is considered an important virulence factor. Pneumolysin is immunogenic and has the potential to be a new vaccine antigen offering broad serotype-independent coverage. To develop a stable vaccine formulation, the conformational stability of a recombinant pneumolysin mutant (pneumolysoid L460D) was characterized by various techniques. Three data visualization diagrams were constructed to summarize the biophysical data of the L460D pneumolysoid; the protein is most stable in solution at pH 6–7, and loses conformational integrity above 48°C. Excipient screening assays were performed and sugars such as trehalose and sucrose stabilized the pneumolysin mutant with respect to improving thermal transition temperatures and minimizing aggregation. In addition, the protein antigen showed efficient binding to aluminum hydroxide adjuvant. The conformational stability of the L460D pneumolysoid on the surface of alhydrogel adjuvant was little affected by adsorption, either with or without excipients. These studies provide important preformulation characterization information useful for the development of a stable pneumolysin mutant-based vaccine.     

龙血竭胶囊治疗软组织挫伤230例

1 临床资料软组织挫伤460(男300,女160)例,年龄5～72(平均38)岁,面部伤86例,躯干伤98例,四肢伤200例,躯干合并四肢伤76例,均为12 h内收治病例. 实验组(n=230)给于龙血竭胶囊(龙血竭胶囊是由植物防卫素和龙血竭皂甙组成)口服,3次/d,每次1.2 g;对照组(n=230)给于跌打丸口服,2次/d,每次1丸,疗程均为6 d. 实验组总有效率为96.5%,对照组为83.5%,两组间有显著差别(P<0.01). 实验组用药后3和6 d血糖值均较对照组有明显下降(P<0.01). 实验组与对照组用药后总抗氧化能力(TAO, kU/L)均升高(12.5±0.08→42.5±0.03, 12.6±0.05→32.9±0.04),但实验组升高明显(P<0.01).     

Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide

A prospective randomized study was conducted comparing two conditioning regimens for the treatment of patients with chronic myeloid leukemia in chronic phase by marrow transplantation from HLA identical siblings. Sixty-nine patients received 60 mg/kg of cyclophosphamide on each of 2 successive days followed by 6 fractions of total body irradiation each of 2.0 Gy (CY-TBI), and 73 patients received 16 mg/kg of busulfan delivered over 4 days followed by 60 mg/kg CY on each of 2 successive days (BU-CY). There was no significant difference between the CY-TBI and the BU-CY groups in the 3-year probabilities of survival (0.80 for both), relapse (0.13 for both), or event-free survival (CY-TBI, 0.68; BU-CY, 0.71) or in speed of engraftment or incidence of venocclusive disease of the liver. The 4-year probabilities of survival and event- free survival for patients transplanted within 1 year of diagnosis were 0.86 and 0.72, respectively, for each group. Significantly more patients in the CY-TBI group experienced major creatinine elevations. There was significantly more acute graft-versus-host disease in the CY- TBI group. Fever days, positive blood cultures, hospitalizations, and inpatient hospital days were significantly more common in the CY-TBI group than in the BU-CY group. In conclusion, the BU-CY regimen was better tolerated than, and associated with survival and relapse probabilities that compare favorably with, the CY-TBI regimen.     

Postnatal Growth Deficits in Prenatal Ethanol-Exposed Mice: Characteristics and Critical Periods

We previously reported that offspring of C57BL/6 mice maintained on liquid diets containing 20% or 25% ethanol-derived calories throughout pregnancy had birth weights comparable with controls but had weight reductions that became manifest around 23 to 28 days postnatally. Since this pattern of weight reduction may represent an unrecognized condition for human ethanol exposure, we completed a number of experiments to more thoroughly characterize the altered growth of prenatal ethanol exposed C57BL/6 mice. The results of this study indicate that consumption of liquid diets containing either 17% or 25% ethanol-derived calories during pregnancy can reduce growth of male and female offspring. Although attenuated postnatal growth of prenatal ethanol-exposed rodents is not commonly reported, prospective studies in humans suggest that in addition to having lower birth weights, children prenatally exposed to ethanol are postnatally growth retarded. Mice exposed to the low ethanol doses used in the present study had normal birth weights; however, their growth was attenuated between 19 and 28 days of age (preadolescent growth spurt) resulting in a weight reduction for at least 35 days, and which according to our previous report could extend into adulthood. The latter stages of gestation appear to be more sensitive to the postnatal growth retarding effect of prenatal ethanol exposure than early gestation. Caloric deficiency and postnatal maternal factors were eliminated as possible mechanisms for the growth deficit.     

Sickle cell anemia patients have low erythropoietin levels for their degree of anemia

We have studied serum immunoreactive erythropoietin (SIE) levels in 28 patients with sickle cell anemia (SCA) without renal insufficiency and in 17 patients with nonhemoglobinopathy anemias of comparable severity using a sensitive radioimmunoassay procedure. An exponential relationship between SIE level and degree of anemia was noted in all patients. However, in nonhemoglobinopathy anemia, a sharp rise in the SIE level occurred as hemoglobin (Hb) levels fell below about 12 g/dL, whereas in sickle cell patients the increase was not marked until hemoglobin fell to about 9 g/dL. The response was more blunted in older SCA patients than in younger ones. A linear regression model relating SIE level to Hb level, presence/absence of SCA, and age explained 63% of the variation in SIE. We conclude that the serum erythropoietin levels in SCA increased at a lower hemoglobin concentration and are of a lower magnitude than that of the other anemias.     

High titer anti-HIV antibody reactivity associated with a paraprotein spike in a homosexual male with AIDS related complex

We observed a human immunodeficiency virus (HIV)-infected homosexual male with AIDS related complex (ARC) who had a serum globulin level of 80 g/L. Serum protein electrophoresis revealed a gamma globulin fraction of 40 g/L, of which 50% (20 g/L) was contained within a paraprotein spike, comprised predominantly of IgG kappa. This patient also had high titer anti-HIV antibodies in his serum, which were Western blot reactive at a final dilution of 1:500,000, and recognized gp120env, p66pol, p55gag, p53pol, p41gag, and p24gag. Because paraproteins in the past have been shown to be directed against specific antigens, we purified this patient's paraprotein using a modified high performance liquid chromatography (HPLC)-hydroxylapatite procedure and tested the purified paraprotein for anti-HIV antibody activity. The purified paraprotein retained anti-HIV antibody activity to a final dilution of 1:100,000, and recognized p66pol, p55gag, p53pol, p41gag, and p24gag. The recognition of both "gag" and "pol" gene products suggested that the purified paraprotein might not be monoclonal in origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified paraprotein contained at least two immunoglobulin light chain species (Mol wt 30 to 33 Kd). Affinity chromatography of the purified paraprotein using a p24- Sepharose 4B matrix separated the "gag" and "pol" antibody activities. Immunoglobulin gene rearrangement analysis of a bone marrow aspirate (which contained 15% plasma cells) failed to reveal a clonal population of immunoglobulin producing cells. We conclude that this patient's paraprotein accounted for most of the anti-HIV activity present in whole serum, and that this paraprotein was not monoclonal in origin.     

The 1st annual meeting of the American Society of Gene Therapy

The inaugural meeting of the American Society of Gene Therapy (ASGT) attracted over 1700 participants to the Pacific gateway city of Seattle, Washington for a multifaceted 4 day meeting organised into a series of symposia, workshops, poster sessions and educational opportunities representative of gene therapy's immense diversity. Presentations from the international assemblage of industrial and academic scientists covered a blend of data from cutting edge research to current clinical investigations across a spectrum of therapeutic targets. Unique educational sessions allowed participants to gain basic information regarding areas of gene therapy research in which they lacked familiarity, whereas several 'Meet the Investigator' sessions allowed participants to interact directly with experts in small group settings in order to obtain a more sophisticated perspective through informal dialogue. A large majority of the symposia and poster presentations showcased the development and current successes of viral-mediated gene therapy although non-viral approaches received their share of attention. Despite the fact that promising results were presented from ongoing clinical trials using viral-mediated gene therapy, much of the symposia addressed the accompanying problems of immunogenicity, low levels of gene expression and lack of control of gene expression that are presently limiting the clinical success of virally mediated gene transfer.     

Growth monitoring: testing the new guidelines

OBJECTIVE: To assess the impact of recent guidelines from the UK joint working party of child health surveillance recommending that all children be measured at age 5 and again between 7 and 9 years of age to determine how many normal school age children are likely to be referred for specialist assessment. METHODS: The longitudinal data of 486 children measured by school nurses in a community setting were examined and compared with measurements made in a research setting by a single, skilled observer. MAIN OUTCOME MEASURES: Number of children identified as having abnormal stature (< 0.4th or > 99.6th centile) and abnormal growth rate height standard deviation score (HSDS) change > 0.67). RESULTS: The community survey identified seven (1.4%) children as having abnormal stature (four short, three tall), 11 (2.3%) were identified as "slow growing", and nine (1.9%) increased their HSDS by more than 0.67. These results were comparable to data collected in ideal research conditions. CONCLUSIONS: Following the recommendations would not result in an excess number of inappropriate referrals. However, this study highlights several unresolved issues such as interobserver variability and time interval between measurements. A large scale prospective study should be considered to establish realistic and cost-effective criteria before implementation of a national screening programme.