CD4+ T cells against human papillomavirus-18 E7 in patients with high-grade cervical lesions associate with the absence of the virus in the cervix

Cervical neoplastic lesions are associated with infection by high‐risk human papilloma‐viruses (HPV). The two genotypes most frequently found in the lesions are HPV‐16 and HPV‐18 with a prevalence of 50–60% and 15–18%, respectively. The E6 and E7 viral oncoproteins are involved in the transformation process and represent foreign antigens for the host. We previously reported that anti‐HPV‐18 E6 CD4⁺ T cells are present in patients with high‐grade HPV‐18‐expressing cervical lesions but also in 50% of the total consecutive patients tested, independently of the HPV type carried. These results indicated that HPV‐18 E6 is immunogenic and suggested that all responsive patients, irrespective of the HPV expressed, had encountered HPV‐18 and cleared the infection. Here, we investigated anti‐HPV‐18 E7 CD4⁺ T‐cell immunity in a cohort of 23 HPV‐18 E6‐responsive patients. We found that, although E7‐specific CD4⁺ T cells were present in all women, a robust T helper type (Th1)/Th2 type response against E7 was associated with HPV‐18‐negative status, suggesting that indeed these patients might have cleared the virus. In agreement with this hypothesis, we found strong anti‐E7 CD4⁺ T‐cell immunity in 20% of 24 healthy donors without evidence of disease. In contrast, a robust Th1/Th2 type response against E6 but not E7 correlated with a lack of disease relapse and/or infection recurrence but did not discriminate between HPV‐18‐positive and HPV‐18‐negative patients. Collectively, our data suggest different roles for anti‐HPV‐18 E6 and E7 CD4⁺ T cells in anti‐viral and anti‐tumour immunity.     

Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1

We have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 109 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment.     

重组人骨形态发生蛋白2联合自体骨行椎间植骨融合治疗胸腰椎结核

文题释义：重组人骨形态发生蛋白2：是一种利用基因重组技术在体外克隆出来的蛋白,属于骨形态发生蛋白家族中活性最强的成员,是骨形成中重要的调控因子。它能够将体内未分化的间充质干细胞定向诱导分化为骨细胞,形成新生骨和软骨,促进骨组织修复。 椎间植骨融合术：是临床上重建和保持脊柱稳定性的手术方式,它不但能够恢复脊柱的稳定性,避免因脊柱不稳带来的疼痛,而且能维持椎体和椎间孔的高度,降低神经根压迫的风险,能够起保护神经功能和保证远期疗效作用。 背景：重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2, rhBMP-2)联合自体骨行椎间植骨融合术治疗腰椎滑脱、椎管狭窄、椎间盘突出等脊柱退行性变疾病的疗效和安全性已经得到认可,但对于治疗脊柱结核等脊柱感染性疾病的疗效和安全性少有临床研究。 目的：评价rhBMP-2联合自体骨行椎间植骨融合术对脊柱结核的临床疗效及其安全性。 方法：回顾性分析2010年11月至2018年5月广州中医药大学第一附属医院收治的胸腰椎结核患者的临床资料,所有患者均采用后路经椎弓根螺钉内固定+植骨融合术,根据植骨时是否使用rhBMP-2将其分为2组,试验组33例采用1 mg rhBMP-2联合自体骨植入大小适宜的支撑体中,对照组35例单纯用自体骨植入支撑体。术后随访1年以上,并对术前、术后疼痛目测类比评分、脊髓损伤ASIA分级、围术期并发症以及融合率进行统计学分析。研究通过广州中医药大学第一附属医院伦理委员会的批准,患者对治疗方案均知情同意。 结果与结论：①所有患者均完成1年以上的随访,随访中未发现内固定断裂移动和椎体的明显塌陷;②两组手术时间、术中出血量、住院时间、围术期并发症比例差异无显著性意义(P > 0.05);③两组术后1周和术后1年的目测类比评分、ASIA分级与术前相比,有显著改善(P < 0.05),但两组之间对比差异无显著性意义(P > 0.05);④在融合率方面,试验组术后6个月的融合率明显高于对照组(P < 0.05),然而,两组术后1年的融合率比较却无统计学差异(P > 0.05)。提示：rhBMP-2联合自体骨行植骨融合术治疗胸腰椎结核能在短期内加快骨性融合,具有良好的疗效和安全性。 ORCID: 0000-0002-3809-4431(翁汭) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIV(KU-1bMC33)) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques

Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5' to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIV(Vpenv)) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIV(Vpenv)-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV(Vpenv)-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIV(KU-1bMC33). Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV(Vpenv)-infected cells compared to cultures inoculated with parental SHIV(KU-1bMC33). Furthermore, virus was observed maturing into intracellular vesicles of SHIV(Vpenv)-infected cells. To assess the pathogenicity of SHIV(Vpenv), three pig-tailed macaques were inoculated with the SHIV(Vpenv) and monitored for 6 months for CD4(+) T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIV(Vpenv) caused a severe CD4(+) T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4(+) T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIV(Vpenv) with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.