Safety and immunogenicity of Px563L,a recombinant anthrax vaccine candidate,in a two-dose regimen for post-exposure prophylaxis in healthy adults

Px563L is a next-generation anthrax vaccine candidate consisting of a protein subunit, mutant recombinant protective antigen SNKE167-ΔFF-315-E308D (mrPA), and liposome-embedded monophosphoryl lipid A (MPLA) adjuvant. Px563L has the potential to deliver an improved safety and immunogenicity profile relative to the currently licensed vaccine, which is produced from filtered B. anthracis culture supernatants.We conducted a Phase 1, double–blind, placebo–controlled, dose–escalation study in 54 healthy subjects to evaluate Px563L at 3 dose levels of mrPA (10, 50, and 80 mcg). For each dose level, 18 subjects were randomized in an 8:8:2 ratio to Px563L (mrPA with adjuvant), RPA563 (mrPA only) or placebo (saline). Each subject received an intramuscular (IM) injection on Day 0 and Day 28. Primary safety and immunogenicity analysis was conducted after all subjects completed the Day 70 visit, a duration deemed clinically relevant for post-exposure prophylaxis. Long-term safety was assessed through Day 393.Vaccinations with Px563L at all dose levels were well-tolerated. There were no serious adverse events or adverse events (AE) leading to early withdrawal. In all treatment groups, most AEs were due to injection site reactions, and all AEs at the 10 and 50 mcg dose levels were mild. For the primary immunogenicity endpoint (protective toxin neutralizing antibody 50% neutralization factor [TNA NF₅₀]), titers started to increase significantly after the second administration of Px563L, from Day 35 through Day 70, with the geometric mean and lower bound of the 95% confidence interval exceeding 0.56, a threshold correlating with significant survival in animal models of anthrax exposure.In conclusion, Px563L, administered as two IM doses 28 days apart, was well-tolerated and elicited a protective antibody response starting at seven days after the second vaccination. These findings support the continued development of Px563L in a two-dose regimen for anthrax post-exposure prophylaxis. ClinicalTrials.gov identifier NCT02655549.     

Randomized,double-blind,placebo-controlled,safety and immunogenicity study of 4 formulations of Anthrax Vaccine Adsorbed plus CPG 7909 (AV7909) in healthy adult volunteers

A new anthrax vaccine that could accelerate the immune response and possibly reduce the number of injections needed for protection would be desirable in a post-exposure setting.     

9例注射用重组人组织型纤溶酶原激酶衍生物致死亡病例分析

目的 探讨注射用重组人组织型纤溶酶原激酶衍生物致死性不良反应的特点和影响因素,为临床安全合理用药提供参考。方法 检索2012～2018年山东省药品不良反应数据库,筛选涉及怀疑用药为重组人组织型纤溶酶原激酶衍生物的死亡病例,进行统计、分析。结果 重组人组织型纤溶酶原激酶衍生物致死性不良反应主要表现为过敏反应和出血,与患者年龄、原患疾病、既往病史和合并用药均有关。结论 临床上要加强对重组人组织型纤溶酶原激酶衍生物致死性不良反应的认识,权衡利弊,准确把握溶栓治疗的指征和禁忌。     

基因重组毕赤酵母表达瑞替普酶过程中的酶活性分析

目的 研究毕赤酵母诱导表达瑞替普酶过程中的关键酶活性.方法 以摇瓶培养为研究对象,在用甲醇诱导后,连续取样,破碎菌体制成无细胞悬液,检测乙醇氧化酶、甲醛脱氢酶、PDC、G-6-PD、ID、α-KGD和SD的活性.结果 乙醇氧化酶比活在0～6 h逐渐增加,在第6h达到最大44.5 U/mg蛋白.随后迅速下降.在第24～48 h有所回升,然后又逐渐降低.FAD比活在第0～48h逐渐增加.在第48h达到最大值6.72U/mg蛋白,随后逐渐降低.直至放瓶.G-6-PD比活在第2h～6h逐渐增加,在第6～24h逐渐降低,在第24～48h逐渐升高,48h后又逐渐降低直至放瓶.PDC比活在第0～6h逐渐降低,随后略有升高的趋势.ID、α-KGD、SD的活性变化有相似趋势.在前6 h酶活均迅速下降,在第6～24 h缓慢下降,随后ID活性继续缓慢下降,而α-KGD和SD活性在第2～48 h逐渐升高,在第48 h均达到最高值,然后又逐渐降低,直至放瓶.结论 根据酶活变化规律,可将整个诱导期分为4个阶段:第1阶段为诱导0～6 h,是甲醇适应期;第Ⅱ阶段为诱导6～24h,是快速生长期;第Ⅲ阶段为诱导24～48h,是产物积累期;第Ⅳ阶段为诱导48～72h,是代谢缓慢期.在甲醇适应期,甲醇完全氧化代谢流占主导地位.在快速生长期和产物积累期,代谢流逐渐向糖酵解途径和TCA循环途径迁移.     

硫氧还蛋白-(His)6融合的瑞替普酶的表达、纯化和活性检测

目的:构建硫氧还蛋白-(His)6-瑞替普酶融合表达载体,提高瑞替普酶在大肠杆菌中的表达量,简化rPA的分离纯化.方法:将rPA基因克隆于原核表达载体pET32a硫氧还蛋白-(组氨酸)6标签下游,在大肠杆菌BL21(DE3)中用乳糖进行诱导表达.采用一步稀释法对融合蛋白体外复性后,使用Ni2 亲和层析柱,对包涵体复性液进行初步纯化.采用体外溶圈法对复性后和纯化后的融合蛋白进行生物活性的测定.结果:得到分子量约为56 kDa的融合蛋白,表达量达到总蛋白的30%以上.比本实验室构建的非融合表达载体表达量提高了约50%.经过一步Ni2 亲和层析,融合蛋白纯度达到80%以上.体外溶圈法实验表明融合蛋白复性后和纯化后具有溶栓活性.结论:与非融合表达相比,融合表达的表达量明显提高.即使N端额外融合一段融合多肽,rPA仍然具有生物学活性.     

Temperature-mediated recombinant anthrax protective antigen aggregate development: Implications for toxin formation and immunogenicity

Anthrax vaccines containing recombinant PA (rPA) as the only antigen face a stability issue: rPA forms aggregates in solution after exposure to temperatures ⩾40 °C, thus losing its ability to form lethal toxin (LeTx) with Lethal Factor. To study rPA aggregation’s impact on immune response, we subjected rPA to several time and temperature combinations. rPA treated at 50 °C for 30 min formed high mass aggregates when analyzed by gel electrophoresis and failed to form LeTx as measured by a macrophage lysis assay (MLA). Aggregated rPA-formed LeTx was about 30 times less active than LeTx containing native rPA. Mice immunized with heat-treated rPA combined with Al(OH)₃ developed antibody titers about 49 times lower than mice immunized with native rPA, as measured by a Toxicity Neutralization Assay (TNA). Enzyme Linked Immunosorbent Assay (ELISA) of the same immune sera showed anti-rPA titers only 2–7 times lower than titers elicited by native rPA. Thus, rPA’s ability to form LeTx correlates with its production of neutralizing antibodies, and aggregation significantly impairs the protein’s antibody response. However, while these findings suggest MLA has some value as an in-process quality test for rPA in new anthrax vaccines, they also confirm the superiority of TNA for use in vaccine potency.     

CT scan findings in chronic thromboembolic pulmonary hypertension: predictors of hemodynamic improvement after pulmonary thromboendarterectomy

STUDY OBJECTIVES: The aim was to correlate CT scan findings with hemodynamic measurements in patients who had undergone pulmonary thromboendarterectomy (PTE) and to evaluate whether CT scan findings can help to predict surgical outcome.Patients and method: Sixty patients who underwent PTE and preoperative helical CT scanning were included. Preoperative and postoperative hemodynamics were correlated with preoperative CT imaging features. RESULTS: The diameter of the main pulmonary artery (PA) and the ratio of the PA and the diameter of the ascending aorta correlated with preoperative mean pulmonary artery pressure (PAP) [r = 0.42; p < 0.001; and r = 0.48; p < 0.0001, respectively]. There was a significant correlation of subpleural densities with preoperative pulmonary vascular resistance (PVR) [r = 0.44; p < 0.001] and of the number of abnormal perfused lobes with preoperative PAP (r = 0.66; p < 0.0001) and PVR (r = 0.76; p < 0.0001). Postoperative PVR correlated negatively with the presence and extent of central thrombi (r = -0.36; p = 0.007) and dilated bronchial arteries (p = 0.03) seen on preoperative CT scans. Sixty percent of patients (3 of 5 patients) without visible central thromboembolic material on CT scans had an inadequate hemodynamic improvement in contrast to 4% of patients (2 of 51 patients) with central thrombi (p = 0.003). Preoperative PVR (r = 0.31; p = 0.018) and the extent of abnormal lung perfusion (r = 0.37; p = 0.007) and of subpleural densities (r = 0.32; p = 0.017) were positively correlated with postoperative PVR. CONCLUSIONS: In patients with thromboembolic pulmonary hypertension, CT scan findings can help to predict hemodynamic improvement after PTE. The absence of central thrombi is a significant risk factor for inadequate hemodynamic improvement.     

重组rPA基因在毕赤酵母细胞中的胞内表达研究

目的构建含rPA全长基因的重组酵母胞内表达质粒pPIC9K rPA ,并在毕赤酵母中进行表达。方法用限制性内切酶BamHI和NotI将含有rPA全长基因的质粒pJZ1 6双酶切后 ,克隆至酵母表达载体pPIC9K中 ,构建酵母重组表达质粒。rPA基因经DNA序列测定准确无误。通过电穿孔法转染毕赤酵母菌GS1 1 5 ,PCR鉴定阳性酵母转化子 ,将rPA基因整合入酵母基因组DNA ,在毕赤酵母中实现了胞内非融合表达。表达产物进行SDS PAGE、Western blots以及溶纤维蛋白活性检测。结果表达产物以胞内包涵体的形式存在 ,未糖基化。SDS PAGE结果显示表达蛋白质的相对分子量约为 39kD。Western blots检测表明表达蛋白质能与单克隆抗体发生特异性反应。包涵体经 8mol L脲溶液溶解后 ,有明显的溶纤维蛋白活性。结论在毕赤酵母中成功地实现了rPA重组蛋白的胞内表达     

Disparate adjuvant properties among three formulations of “alum”

Aluminum adjuvants, commonly referred to as “alum,” are the most widespread immunostimulants in human vaccines. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to form antigen depots and induce inflammatory signals that, in turn, promote antibody production. It was recently noted that Imject^® alum, a commercial aluminum-containing adjuvant commonly used in animal studies, is not the physicochemical equivalent of aluminum adjuvant present in human vaccines. This difference raises concerns about the use of Imject^® alum in animal research as a model for approved aluminum adjuvants. Here, we compared the capacity of Imject^® alum, Alhydrogel^®, and a traditional alum-antigen precipitate to induce humoral responses in mice to the hapten-carrier antigen, NP-CGG [(4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin]. The magnitude of humoral responses elicited by Alhydrogel^® and precipitated alum was significantly greater than that induced by Imject^® alum. The strength of the humoral responses elicited by different alum formulations was correlated with the quantity of pro-inflammatory cytokines induced and the numbers of inflammatory cells at the site of immunization. Moreover, Imject^® exhibited a severely reduced capacity to adsorb protein antigens compared to Alhydrogel^® and precipitated alum. These findings reveal substantial differences in the immunostimulatory properties of distinct alum preparations, an important point of consideration for the evaluation of novel adjuvants, the assessment of new alum-based vaccines, and in mechanistic studies of adjuvanticity.     

rPA溶栓治疗急性肺血栓栓塞的观察与护理

目的观察应用r—PA溶栓治疗急性肺动脉血栓栓塞的疗效,总结护理经验。方法对52例经CT证实为急性肺动脉血栓栓塞的患者给予r—PA18mg加NS10ml静脉注射进行溶栓治疗,注射时间大于2min,观察溶栓前及溶栓4h后患者的临床症状、血氧饱和度、螺旋CT肺动脉成像及动脉血氧分压的变化。结果应用r—PA对52例急性肺动脉血栓栓塞患者溶栓后,84．61％的患者临床症状改善,氧饱和度上升,动脉氧分压升高,肺动脉压下降,与溶栓前比较差异显著（P〈0．01）。结论r—PA静脉溶栓治疗急性肺动脉血栓栓塞有较好的临床疗效与安全性,严密观察病情及时采取相关护理措施是促使病情恢复的保障。