Intranasal immunization with protective antigen of Bacillus anthracis induces a long-term immunological memory response

Although intranasal vaccination has been shown to be effective for the protection against inhalational anthrax, establishment of long-term immunity has yet to be achieved. Here, we investigated whether intranasal immunization with recombinant protective antigen (rPA) of Bacillus anthracis induces immunological memory responses in the mucosal and systemic compartments. Intranasal immunization with rPA plus cholera toxin (CT) sustained PA-specific antibody responses for 6 months in lung, nasal washes, and vaginal washes as well as serum. A significant induction of PA-specific memory B cells was observed in spleen, cervical lymph nodes (CLNs) and lung after booster immunization. Furthermore, intranasal immunization with rPA plus CT remarkably generated effector memory CD4⁺ T cells in the lung. PA-specific CD4⁺ T cells preferentially increased the expression of Th1- and Th17-type cytokines in lung, but not in spleen or CLNs. Collectively, the intranasal immunization with rPA plus CT promoted immunologic memory responses in the mucosal and systemic compartments, providing long-term immunity.     

中国HIV感染者细胞毒性淋巴细胞与CD4~+CD25~+调节性T细胞相关性研究

目的：对中国HIV感染者NK细胞、CD8^＋T细胞及胞内穿孔素、颗粒酶-B表达与CD4^＋CD25^＋Foxp3^＋调节性T细胞水平的相关性进行研究,探讨调节性T细胞在HIV感染中的作用机制。方法：选取73名HIV/AIDS患者（长期不进展组、无症状HIV组、AIDS组）,应用流式细胞仪胞内染色技术检测NK细胞、CD8^＋T细胞数量及胞内穿孔素、颗粒酶-B表达水平,分析其与CD4^＋CD25^＋Foxp3^＋调节性T细胞水平的相关性。结果：CD4^＋CD25^＋Foxp3^＋调节性T细胞百分率与NK细胞、CD8^＋T淋巴细胞数量呈明显负相关（P〈0.01）,与CD8^＋T细胞内穿孔素、颗粒酶-B表达百分率呈明显正相关（P〈0.05）,与NK细胞内穿孔素、颗粒酶-B表达水平绝对值负相关（P〈0.01）,与CD8＋T细胞内颗粒酶-B表达绝对值呈明显负相关（P〈0.01）,与CD8^＋T细胞内穿孔素表达绝对值无明显相关性（P〉0.05）。CD4^＋CD25^＋Foxp3^＋调节性T细胞绝对值与CD8^＋T细胞内穿孔素、颗粒酶-B表达百分率呈明显负相关（P〈0.05）。结论：中国HIV感染者细胞毒性淋巴细胞数量功能的变化与调节性T细胞显著相关,提示高水平的调节性T细胞可能与细胞毒性淋巴细胞耗竭相关,可能为导致疾病进展的原因之一。     

中国部分地区HIV患者B′和B′/C亚型毒株tat第一外显子基因变异与HIV疾病进展关系的研究

目的 了解中国B’和B’／C亚型HIV／AIDS患者tat第一外显子的基因序列及其二级结构的特征和变异特点，探讨其与HIV-1感染疾病进展之间的关系。方法 从辽宁、吉林和云南省HIV-1感染者中选取病情呈缓慢进展的B’亚型感染者8例和B’／C亚型感染者5例，选择年龄、性别感染时间与前二者匹配的病情呈典型进展的B’亚型感染者26例和B’／C亚型感染者9例。采集外周静脉血，提取前病毒DNA，用巢式聚合酶链反应扩增HIV-1的tat基因，纯化后直接进行基因序列测定，序列编辑后翻译成氨基酸序列，进行氨基酸变异情况分析和二级结构预测。结果 B’和B’／C亚型缓慢进展者、典型进展者Tat第一外显子中发现多种氨基酸替换，但除A58T外均未显示出与病毒载量以及疾病进展的明确相关性。23N、31S、32Y、46F变异均显示出亚型特异性；Tat蛋白的二级结构未发现规律性变化。结论 中国HIV／AIDs患者tat第一外显子某些位点的基因变异，如A58T可能与病毒载量以及疾病进展有关，Tat蛋白的二级结构可能与HIV感染后的疾病进展无明显关系。     

Anti-inflammatory effects of benzenediamine derivate FC-98 on sepsis injury in mice via suppression of JNK,NF-κB and IRF3 signaling pathways

FC-98, a synthesized benzenediamine derivate, was reported to regulate Toll-like receptor 9-induced activation of dendritic cells in our previous study. In this study, we evaluated the anti-inflammatory properties of FC-98 both in macrophages and in septic mouse models. By using enzyme-linked immunosorbent assay and real-time quantitative PCR, we found that FC-98 (6.25, 25 and 100 μM) dose-dependently attenuated lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP-1) productions in RAW264.7 and primary mouse peritoneal macrophages. These inhibitory effects were not due to inducing cell cytotoxicity or altering LPS binding or TLR4 expression. Subsequently, western blot, immunofluorescence and luciferase reporter assays were used to investigate the underlying mechanisms of its anti-inflammatory activities. Results showed that FC-98 blocked activation of the c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) signaling pathways. In vivo, FC-98 (30 or 100 mg/kg) was intraperitoneally administrated into LPS-induced or CLP-induced sepsis mice. It was observed to enhance the survival rate, inhibit pro-inflammatory mediator production, improve organ injuries and suppress bacterial propagation. In conclusion, FC-98 effectively inhibited macrophage inflammatory responses and ameliorated sepsis in mice through down-regulation of both MyD88 and TRIF-dependent pathways. These results suggest that FC-98 could be a promising therapeutic agent for inflammatory diseases.     

A homozygote TREX1 mutation in two siblings with different phenotypes: Chilblains and cerebral vasculitis

Three prime repair exonuclease 1 degrades single and double stranded DNA with 3′-5′ nuclease activity and its mutations are related to type 1 IFN mediated autoinflammation due to accumulated intracellular nucleic acids. To date, several cases of systemic lupus erythematosus, Aicardi-Goutieres syndrome, familial chilblain lupus, retinal vasculopathy-cerebral leukodystrophy have been reported with TREX1 mutations.Chilblain lupus is a skin disease characterized by blue-reddish coloring, swelling or ulcers on acral regions of body such as fingertips, heels, nose and auricles. Central nervous system vasculitis is a prominent cause of childhood strokes.10 families with familial chilblain lupus related to TREX1 mutations were reported previously in the literature, in which homozygote D18N variant in TREX1 gene was related to chilblains with cerebral vasculitis.In this report, whole-exome-sequencing revealed a homozygote R114C mutation in TREX1 gene was shown in two siblings with recurrent chilblains whom one of them was the second case accompanied by cerebral vasculitis in the literature. As a result, the approach of WES in clinical use revealed a novel mutation in clinically heterogenous patients to provide genetic counseling.     

High-yield production of recombinant virus-like particles of enterovirus 71 in Pichia pastoris and their protective efficacy against oral viral challenge in mice

Enterovirus 71 (EV71) is one of the major causative pathogens of hand, foot and mouth disease (HFMD), which is highly prevalent in the Asia-Pacific regions. Severe HFMD cases with neurological complications and even death are often associated with EV71 infections. However, no licensed EV71 vaccine is currently available. Recombinant virus-like particles (VLPs) of EV71 have been produced and shown to be a promising vaccine candidate in preclinical studies. However, the performance of current recombinant expression systems for EV71 VLP production remains unsatisfactory with regard to VLP yield and manufacturing procedure, and thus hinders further product development. In this study, we evaluated the expression of EV71 VLPs in Pichia pastoris and determined their protective efficacy in mouse models of EV71 infections. We showed that EV71 VLPs could be produced at high levels up to 4.9% of total soluble protein in transgenic P. pastoris yeast co-expressing P1 and 3CD proteins of EV71. The resulting yeast-produced VLPs potently induced neutralizing antibodies against homologous and heterologous EV71 strains in mice. More importantly, maternal immunization with VLPs protected neonatal mice in both intraperitoneal and oral challenge experiments. Collectively, these results demonstrated the success of simple, high-yield production of EV71 VLPs in transgenic P. pastoris, thus lifting the major roadblock in commercial development of VLP-based EV71 vaccines.     

Relationship of frequency of CD4+CD25+Foxp3+ regulatory T cells with disease progression in antiretroviral-naive HIV-1 infected Chinese

Forty-five antiretroviral-naive HIV-1 infected patients and 14 healthy controls in North China were enrolled in this study. The frequency of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and levels of expression of CD95, HLA-DR and CD38 in T cells were detected by flow cytometry. We found that the frequency of Tregs was higher in AIDS patients than in asymptomatic HIV-1 infected patients (P=0.004). The frequency of Tregs was significantly correlated with absolute CD4 count, viral load, CD4+CD95+ T cells and CD8+CD95+ T cells (P<0.05). The relationship between the frequency of Tregs and immune activation was not found in HIV-infected patients. We concluded that the frequency of Tregs in HIV-infected Chinese patients was significantly correlated with disease progression.     

Disparity of human immunodeficiency virus incidence and drug resistance in college student,non-student youth and older men who have sex with men: a cross-sectional study from seven major cities of China

Background:Human immunodeficiency virus (HIV) prevalence among student men who have sex with men (MSM) in college is more than 5.0% and keeps on increasing in China. This study aims to clarify the proportion of HIV recent infection, its propeller and the source among college student MSM.Methods:We conducted a multicenter cross-sectional study in seven major Chinese cities during 2012-2013. HIV recent infections (≤ 168 days) and incidence was measured and estimated by BED HIV-1 capture enzyme immunoassay (BED-CEIA) testing strategy. HIV-related behaviors and transmitted drug resistance (TDR) were investigated and compared between the college student MSM, <25-year-old non-student youth MSM (NSYM), and ≥25-year-old non-student non-youth MSM (NSNYM), using structured survey, and analyses of drug resistance.Results:Overall, 4,496 (4496/4526, 99.3%) were eligible for enrollment, comprising 565 college student MSM, 1,094 NSYM, and 2,837 NSNYM. The proportion of HIV recent infection were 70.3% (26/37), 50.8% (65/128) and 35.1% (95/271), the HIV incidence rate were 10.0 (95% CI: 6.2-13.9)/100PY, 12.9 (95% CI: 9.8-16.1)/100PY, 6.8 (95% CI: 5.4-8.2)/100PY, and TDR prevalence were 7.4% (2/27), 2.0%, (2/98) and 4.9% (11/226), among student MSM, NSYM, and NSNYM, respectively. Among HIV positive student MSM with age< 21-year-old, the proportion of HIV recent infection is 90.9% (10/11). Factors independently associated with HIV recent infection in student MSM was usage of recreational drug in the past 6 months (AOR: 2.5; 95% CI: 1.0–5.8).Conclusions:College student MSM had higher proportion of HIV recent infection and TDR than the youth and older MSM in China during 2012-2013. The HIV infections were more likely to happen during the early year of college life among student MSM.     

Mimotopes selected by biopanning with high-titer HIV-neutralizing antibodies in plasma from Chinese slow progressors

ObjectiveOne approach to identifying HIV-1 vaccine candidates is to dissect the natural antiviral immune response in treatment-naïve individuals infected for over ten years, considered slow progressor patients (SPs). It is suspected that SP plasma has strongly neutralizing antibodies (NAb) targeting specific HIV viral epitopes.MethodsNAbs levels of 11 HIV-1-infected SPs were detected by PBMC-based neutralization assays. To investigate SP NAb epitope, this study used a biopanning approach to obtain mimotopes of HIV-1 that were recognized by SP plasma NAbs. IgG was purified from high-titer NAb SP plasma, and used as the ligand for three rounds of biopanning to select HIV-specific mimotopes from a phage-displayed random peptide library. Double-antibody sandwich ELISA, competitive inhibition assays, and peptide sequence analysis were used to evaluate the characteristics of phage-borne mimotopes.ResultsSPs had significantly more plasma neutralizing activity than typical progressors (TPs) (p = 0.04). P2 and P9 plasma, which have highest-titer HIV-NAb, were selected as ligands for biopanning. After three rounds of biopanning, 48 phage clones were obtained, of which 22 clones were consistent with requirement, binding with HIV-1 positive plasma and unbinding with HIV-1 negative plasma. Compared with linear HIV-1 protein sequence and HIV-1 protein structure files, only 12 clones were possible linear mimotopes of NAbs. In addition, the C40 clone located in gp41 CHR was found to be a neutralizing epitope, which could inhibit pooled HIV-1 positive plasma reaction.ConclusionBiopanning of serum IgG can yield mimotopes of HIV-1-related antigen epitopes. This methodology provides a basis for exploration into HIV-1-related antigen-antibody interactions and furthers NAb immunotherapy and vaccine design.