Consistency of Bordetella pertussis vaccine seed strains and potency of whole-cell pertussis vaccine still in use in Poland

In Poland, where the wP vaccine has been used since 1960, pertussis rates increased in the mid-1990s. In 2012, the rate of pertussis recognised by surveillance was unexpectedly found to be two-fold higher than in the previous decade. Quality measures on potency and vaccine working seeds were introduced, to confirm the possible impact of manufacturing inconsistency or potency lowering on the observed increase in pertussis. Shewhart charts on potency values for lots released between 2001 and 2013 did not reveal any significant fluctuations. Working seeds of three vaccine strains used within last decade for wP manufacturing belong to the PFGE group III and were highly related. According to PFGE and SDS-PAGE data, all vaccine strains were found consistent according profiling on the genomic and protein levels. According to the sequencing data, they harboured ptxA2, ptxC1, prn1, fim2-1, fim3-1, tcfA2, ptxP1 and were assigned as MLST-2 type. Other factors apart from vaccine manufacturing inconsistency might be responsible for the increase in pertussis noted in 2012 in Poland.     

HIV-1 Env vaccine comprised of electroporated DNA and protein co-administered with Talabostat

Selection of potent yet low reactogenic adjuvants for protein immunization is important for HIV-1 vaccine development. Immunogenicity of electroporated DNA (HIV env) and recombinant gp120, administered with either QS-21 or the orally administered immunomodulator, Talabostat, was evaluated in BALB/c mice. Electroporation of low dose DNA elicited Th1 cytokines and anti-envelope antibodies. Immunization with gp120 protein alone with or without Talabostat elicited lower Th1 and Th2 cytokine levels but comparable anti-gp120 antibodies to QS-21-formulated protein. Boosting of DNA-primed mice with gp120/Talabostat induced similar anti-gp120 antibody titers and slightly higher levels of Th1 and Th2 cytokines relative to QS-21-formulated protein. Induction of CD8⁺ and CD4⁺ T cells and functional CTL activity was noted. These results highlight the potential use of orally administered Talabostat for efficient protein boosting of antibody and T-cell responses primed by DNA.     

携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

利用细菌人工染色体技术将串联的HIV-1 gp160、gag和protease基因以及表达元件插入1型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)内部反向重复序列区,以获得携带HIV-1抗原的单纯疱疹病毒载体疫苗。首先将HIV-1 gp160(B型和C型)、gag和protease基因串联克隆入pc DNA3获得重组质粒pc DNA/g Bgp和pc DNA/g Cgp,重组质粒转染293FT细胞,Western blotting检测HIV抗原表达。继而将pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表达元件的表达框克隆入p KO5/BN获得重组穿梭质粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭质粒电转含BAC-HSV的大肠杆菌,筛选重组菌,提取重组DNA并转染Vero细胞,挑取病毒蚀斑纯化重组病毒,Southern blotting鉴定重组病毒DNA,Western blotting检测重组病毒感染细胞中HIV抗原表达,并分析病毒的增殖特性。结果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp转染的293FT细胞中检测到表达的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分别电转获得重组菌,并从重组DNA转染的Vero细胞中纯化获得重组HSV,Southern blotting检测表明重组HSV基因组发生特异性重组,重组病毒感染细胞中检测到gp120和gp41,且重组HSV保留了在哺乳动物细胞中的复制能力。本研究获得携载HIV-1 gp160、gag和protease基因的重组HSV,并保留了在哺乳动物细胞中的复制能力,可作为HIV-1复制型病毒载体疫苗。     

The expanding HIV-1 epidemic aggravates therapeutics and vaccine development programs in India

Human Immunodeficiency Virus Type 1 (HIV-1) displays extensive genetic diversity globally that poses a serious challenge in designing suitable therapeutic measures for protective immune responses in the host. After the first detection of AIDS cases in 1986, India now has the third largest HIV epidemic in world. The HIV-1 epidemic in India is still growing with introduction of new strains/subtypes and this creates a serious problem for the clinician and basic science researchers to find the effective measures. Neighboring countries in the North and North-eastern regions play an important role in the HIV-1 pandemic. An extensive multinational collaborative research effort is needed in order to identify the risk groups and geographic locales is required to monitor HIV-1 spread in Asia and to produce a successful preventive program.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Repfication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 （HIV-1）-based defective lentiviral vectors （DLV） and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV （0.5-1.2 × 107IU/mL）, which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)     

HIV-1辅助调节蛋白Vpu的结构与功能

Vpu蛋白是HIV病毒的辅助调节蛋白之一,仅存在HIV一1型病毒中。在病毒的复制过程中Vpu蛋白下调CD4受体的表达,调节Pr55^gag蛋白的核定位影响病毒的装配,细胞膜上类似离子通道作用促进子代病毒颗粒的释放。该文介绍Vpu蛋白的结构与功能。     

Studies on toll-like receptor stimuli responsiveness in HIV-1 and HIV-2 infections

Background: HIV-1 and HIV-2 are two related viruses with distinct clinical outcomes, where HIV-1 is more pathogenic and transmissible than HIV-2. The pathogenesis of both infections is influenced by the dysregulation and deterioration of the adaptive immune system. However, their effects on the responsiveness of innate immunity are less well known. Here, we report on toll-like receptor (TLR) stimuli responsiveness in HIV-1 or HIV-2 infections. Methods: Whole blood from 235 individuals living in Guinea-Bissau who were uninfected, infected with HIV-1, infected with HIV-2, and/or infected with HTLV-I, was stimulated with TLR7/8 and TLR9 agonists, R-848 and unmethylated CpG DNA. After TLR7/8 and TLR9 stimuli, the expression levels of IL-12 and IFN-α were related to gender, age, infection status, CD4⁺ T cell counts, and plasma viral load. Results: Defective TLR9 responsiveness was observed in the advanced disease stage, along with CD4⁺ T cell loss in both HIV-1 and HIV-2 infections. Moreover, TLR7/8 responsiveness was reduced in HIV-1 infected individuals compared with uninfected controls. Conclusions: Innate immunity responsiveness can be monitored by whole blood stimulation. Both advanced HIV-1 and HIV-2 infections may cause innate immunity dysregulation.     

重组百日咳毒素Sl亚单位的纯化及免疫原性

将重组百日咳毒素S1亚单位基因的质粒在DH5α和TOPl0两株大肠杆菌中进行表达,并对表达产物进行了初步纯化。粗制的rSl免疫家兔所得血清,用HP—PT及1B7-PT包被的ELISA测定效价,并做被动免疫保护试验。抗PT的抗体滴度为1：32000。该抗rSl血清在小鼠被动免疫保护试验中,对百日咳杆菌18323毒株进行脑腔攻击的被动免疫保护作用达到100％。证明rSl亚单位具有良好的抗原性和免疫原性,并与天然S1具有相同的抗原表位。     

Enhancement of Human Immunodeficiency Virus Type 1 (HIV-1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV-1 infection. Although virus-cell binding was significantly inhibited by PEI, HIV-1 infection in human T-cell lines such as MT-4 and MOLT-4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV-1 infection was examined using HIV-1 chronically infected cells (MOLT-4/HIV-1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT-4/HIV-1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV-1 to CD4-positive cells by PEI was due to the shedding of gp120 from HIV-1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV-1 infection. Similarly, dissociation of gp120 from MOLT-4/HIV-1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV-1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.     

Identification of the minimum region of Bordetella pertussis Vag8 required for interaction with C1 inhibitor

An autotransporter of Bordetella pertussis, virulence-associated gene 8 (Vag8), binds and inactivates the complement regulator, C1 inhibitor (C1-Inh), and plays a role in evasion of the complement system. However, the molecular interaction between Vag8 and C1-Inh remains unclear. Here, we localized the minimum region of Vag8 required for interaction with C1-Inh by examining the differently truncated Vag8 derivatives for the ability to bind and inactivate C1-Inh. The truncated Vag8 containing amino-acid residues 102–548, but not 102–479 and 202–648, showed the full activity of intact Vag8, suggesting that the separate 102–202 and 548–648 amino-acid regions of Vag8 mediate the interaction with C1-Inh.     

Impact of vaccination and birth rate on the epidemiology of pertussis: a comparative study in 64 countries

Bordetella pertussis infection remains an important public health problem worldwide despite decades of routine vaccination. A key indicator of the impact of vaccination programmes is the inter-epidemic period, which is expected to increase with vaccine uptake if there is significant herd immunity. Based on empirical data from 64 countries across the five continents over the past 30–70 years, we document the observed relationship between the average inter-epidemic period, birth rate and vaccine coverage. We then use a mathematical model to explore the range of scenarios for duration of immunity and transmission resulting from repeat infections that are consistent with empirical evidence. Estimates of pertussis periodicity ranged between 2 and 4.6 years, with a strong association with susceptible recruitment rate, defined as birth rate × (1 − vaccine coverage). Periodicity increased by 1.27 years on average after the introduction of national vaccination programmes (95% CI: 1.13, 1.41 years), indicative of increased herd immunity. Mathematical models suggest that the observed patterns of pertussis periodicity are equally consistent with loss of immunity that is not as rapid as currently thought, or with negligible transmission generated by repeat infections. We conclude that both vaccine coverage and birth rate drive pertussis periodicity globally and that vaccination induces strong herd immunity effects. A better understanding of the role of repeat infections in pertussis transmission is critical to refine existing control strategies.     

HIV-1 B亚型gag基因密码子优化及其免疫原性的研究

从河南HIV-1流行区感染者中克隆HIV-1 B亚型gag基因,通过序列比对获得其一致性共有序列,对该共有序列按照哺乳动物优势密码子的使用原则进行优化,以Western blot方法比较优化前后gag基因体外表达量.发现对gag基因进行密码子优化可显著提高其表达水平.将优化后的mod.gag基因插入重组腺病毒载体,构建了重组病毒rAdV-mod.gag.在BALB/c小鼠体内分别以108PFIJ及108PFU rAdV-mod.gag疫苗单独免疫两次均可产生较高水平的gag特异性细胞免疫反应.由此得出结论,对gag基因的密码子优化是成功的;表达优化后gag基因的重组腺病毒疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.     

IL-18 DNA免疫对HIV-1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应.酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01).IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用.     

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55(Gag), viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55(Gag). Pr55(Gag) synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.     

用两步MS-PCR结合ELISA检测HIV-1逆转录酶基因的耐药性突变

高效抗逆转录病毒治疗法（HAART）的推广使用有效地抑制了HIV-1病毒的传播和艾滋病（AIDS）的发病率、死亡率。近年来,HIV-1逆转录酶基因突变所导致的耐药性成为了HAART的治疗失败的主要原因,耐药性突变的检测对于指导病人用药以及新药开发具有重要意义。本工作发展了一种检测HIV-1逆转录酶基因耐药性突变的新方法。采用两步MS-PCR方法检测HIV-1 B亚型野生型和耐药突变型逆转录酶基因突变,检测点包括M41L、K70R、K103N、Y181C和T215,并在两步MS-PCR基础上,设计比野生型引物长的突变型引物,结合微孔板杂交ELISA呈色技术检测各点突变。结果显示,简化的两步MS-PCR能保持灵敏度和特异性高的优点,ELISA的阳性结果与阴性结果的比值（P/N）达到要求,两步MS-PCR结合ELISA方法灵敏度高,操作简单、结果直观、成本低、相对耗时短且能进行高通量检测,其无论在HIV-1耐药性突变及其它的点突变检测中具有较好的临床应用前景。