A Novel High Throughput,Parallel Infection Assay for Determining the Replication Capacities of 346 Primary HIV-1 Isolates of the Zurich Primary HIV-1 Infection Study in Primary Cells

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSF_WT and HIV-1 JR-CSF_{K65R_M184V}. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells.     

Prevalence of hepatitis C in an ethnically diverse HIV-1-infected cohort in south London

OBJECTIVES: There is limited information on the prevalence of and risk factors for hepatitis C virus (HCV) infection among HIV-1-infected patients in the UK. Our objective was to determine the prevalence of HCV infection among an ethnically diverse cohort of HIV-infected patients in south London, and to extrapolate from these data the number of co-infected patients in the UK. METHODS: A total of 1017 HIV-1-infected patients who had attended King's College Hospital HIV clinic between September 2000 and August 2002 were screened for HCV antibody using a commercial enzyme-linked immunosorbent assay (ELISA). Positive results were confirmed by polymerase chain reaction (PCR) or recombinant immunoblot assay. Demographic, clinical and laboratory data were obtained from the local computerized database and medical records. We applied our HCV prevalence rates in the different HIV transmission groups to the estimated number of HIV-infected persons in these groups in the UK, to obtain a national estimate of the level of HIV-HCV co-infection. RESULTS: Of the 1017 HIV-1-infected patients, 407 (40%) were white men, 158 (15.5%) were black African men, 268 (26.3%) were black African women, and 61 (6%) and 26 (2.6%) were black Caribbean men and women, respectively. Heterosexual exposure was the most common route of HIV acquisition (53.5%), followed by men having sex with men (36.9%), and current or previous injecting drug use (IDU) (7.2%). The overall prevalence of HCV co-infection was 90/1017 (8.9%), but this varied substantially according to route of transmission, from 82.2% among those with a history of IDU (which accounted for 67% of all HCV infections), to 31.8% in those who had received blood products, to 3.5% and 1.8% in those with homosexually and heterosexually acquired infection, respectively. Multivariate logistic regression analysis identified several independent risk factors for HCV infection: a history of IDU [odds ratio (OR) = 107.2; 95% confidence interval (CI) = 38.5-298.4], having received blood products (OR = 16.5; 95% CI = 5.1-53.7), and either being from a white ethnic group (OR = 4.3; 95% CI = 1.5-12.0) or being born in Southern Europe (OR = 6.7; 95% CI = 1.5-30.7). Based on the 35,473 known HIV-1-infected persons in the UK and the 10 997 estimated to be unaware of their status, we projected that there are at least 4136 HIV-HCV co-infected individuals in the UK and 979 who are unaware of their status. CONCLUSIONS: Overall, 9% of our cohort was HIV-HCV co-infected. The prevalence was highest among intravenous drug users (82%), who accounted for most of our HCV cases, and lowest among heterosexual men and women from sub-Saharan Africa and the Caribbean [< 2%]. Our estimate that a significant number of co-infected persons may be unaware of their HIV and HCV status, highlights an urgent need to increase the uptake of HCV and HIV testing, particularly among injecting drug users, to reduce the risk of onward transmission.     

Impacts of Humanized Mouse Models on the Investigation of HIV-1 Infection: Illuminating the Roles of Viral Accessory Proteins in Vivo

Human immunodeficiency virus type 1 (HIV-1) encodes four accessory genes: vif, vpu, vpr, and nef. Recent investigations using in vitro cell culture systems have shed light on the roles of these HIV-1 accessory proteins, Vif, Vpr, Vpu, and Nef, in counteracting, modulating, and evading various cellular factors that are responsible for anti-HIV-1 intrinsic immunity. However, since humans are the exclusive target for HIV-1 infection, conventional animal models are incapable of mimicking the dynamics of HIV-1 infection in vivo. Moreover, the effects of HIV-1 accessory proteins on viral infection in vivo remain unclear. To elucidate the roles of HIV-1 accessory proteins in the dynamics of viral infection in vivo, humanized mouse models, in which the mice are xenotransplanted with human hematopoietic stem cells, has been utilized. This review describes the current knowledge of the roles of HIV-1 accessory proteins in viral infection, replication, and pathogenicity in vivo, which are revealed by the studies using humanized mouse models.     

Pregnancy and HIV-1 incidence in 178 married couples with discordant HIV-1 serostatus: additional experience at an HIV-1 counselling centre in the Democratic Republic of the Congo

To determine the effect of an HIV-1 counselling programme on rates of HIV-1 infection and pregnancy in a large group of married couples in Kinshasa, DRC with discordant HIV-1 infection status, we conducted a baseline cross-sectional HIV-1 seroprevalence study in two large Kinshasa businesses. We identified 178 married couples (mean duration of marriage = 12.3 years) with discordant HIV-1 serostatus (92 M+F-/86 M-F+). Seroincidence and pregnancy rates were observed during 310 person-years of follow-up (PYFU). The 92 M+F- couples had an HIV-1 incidence of 3.7/100 PYFU and a pregnancy rate of 8.6/100. The 86 M-F+ couples had a pregnancy rate of 6.8/100 PYFU and an HIV-1 incidence of 6.8/100 PYFU. Couples seeking to have children but minimize their HIV-1 transmission risk frequently had unprotected sex only during the woman's perceived monthly fertility period. This strategy resulted in the birth of 24 live-born children and only one (4%; 95% CL = 0.0-21.6%) new HIV infection in couples having a child. Only 1 of 6 women who developed HIV-1 infection (16. 7%; 95 C.L. = 0-40.4%) became pregnant. While seronegative men had more extramarital sex once their wives' positive HIV-1 infection status became known, most of these episodes involved safe sex. Divorce was rare. This study provides additional information concerning issues of safe sex in married couples with discordant HIV-1 infection status, the dynamics of HIV transmission within couples and the effect of serostatus notification on the marriage and on intramarital and extramarital sexual behaviour in Kinshasa, Congo.     

河南省有偿供血人群HIV/HCV混合感染者HIV-1细胞嗜性的研究

目的 探讨河南省有偿供血人群HIV/HCV混合感染者体内HIV-1细胞嗜性的分布情况.方法 选取河南省经有偿供血途径感染HIV-1的161例感染者,采用酶联免疫法检测HCV抗体,并通过HCV核酸检测明确HIV/HCV混合感染.nested-PCR扩增env C2V3区,测序并分析HIV -1流行亚型,使用在线预测工具W...     

HIV-1感染恒河猴外周血单个核细胞的生物学特性和C2-V3区基因序列变化

目的探讨艾滋病病毒1型(HIV-1)感染恒河猴外周血单个核细胞(PMBCs)，并在其中传代的可能性。方法用HIV-1感染原代猴PBMCs，采用酶联免疫吸附试验(ELISA)方法测定培养0、2、3、7天时上清液中的HIV-1P24抗原，以观察HIV-1在猴细胞中的复制情况，比较HIV-1分别在人和猴细胞感染的复制动力学。同时采用聚合酶链反应(PCR)方法扩增HIV-1的enV基因，并对其C2-V3区进行序列分析。结果HIV-1毒株SF33、89．6、ⅢB感染猴PBMCs后的第3天，P24抗原达到最高，随着培养时间的延长，P24抗原逐渐降低。将SF33、ⅢB、89．6、YNl9四个毒株分别感染猴PBMCs并盲传3代后，P24抗原测定结果均为阴性。提取盲传各代细胞脱氧核糖核酸(DNA)进行PCR扩增，结果显示，SF33、ⅢB、YN19、89．6感染猴细胞的第一代和89．6感染猴细胞的第二代，均可以检测到HIV-1前病毒核酸。比较原毒株enV基因C2-V3区的序列发现，传代后的基因序列SF33有5个碱基改变外，其它HIV-1只有0～2个核苷酸改变。与SF33感染猴PBMCs的表现相反，其感染人PBMCs时。HIV-1的复制一直维持在较高的水平。结论应用3个实验株、1个临床分离株HIV-1，虽都能够感染恒河猴外周淋巴细胞，但均难以继代，提示能否用恒河猴作为HIV-1研究的动物模型，尚待进一步的体内外研究。     

Epstein–Barr Virus (EBV) Genotypes Associated with the Immunopathological Profile of People Living with HIV-1: Immunological Aspects of Primary EBV Infection

Background: The aim of the present study was to evaluate the immunological profile of adult HIV-1⁺ patients coinfected with primary Epstein–Barr virus (EBV) infection who were free of antiretroviral drugs and inhabitants of the Brazilian Amazon region. Materials and methods: Primary EBV infection was screened by the semiquantitative detection of IgM and IgG anti-VCA. Genotypes were determined by conventional PCR. EBV and HIV viral load (VL) were quantified by real-time PCR. Cytokine dosage and cell quantification were performed by cytometry. Results: Only HIV-1⁺ individuals had primary EBV infection (7.12%). The EBV-1 genotype was the most prevalent (47.37%). The VL of HIV-1 was lower in the HIV/EBV-2 group. CD4⁺ T lymphocytes were inversely proportional to the VL of EBV in HIV/EBV-1/2 multi-infected patients. The HIV/EBV-2 group had the lowest cytokine levels, especially IFN-γ and IL-4. Different correlations were proposed for each coinfection. The late search for specific care related to HIV infection directly affected the cytokine profile and the number of CD8⁺ T lymphocytes. Symptoms were associated with the increase in VL of both viruses and cytokine profile. Conclusions: Different immunological profiles were associated with EBV genotypes in primary infection, with EBV-2 being more frequent in patients with low levels of HIV viral load. With late infection monitoring and consequent delay in the initiation of HAART, clinical changes and effects on the maintenance of the immune response were observed.     

Detection and quantification of HIV-1 specific CD4 helper and CD8 cytotoxic cells: their role in HIV-1-infected individuals and vaccine recipients

There is a strong link between virus specific CD8 T-cell function and the efficiency of regulatory CD4 helper T cells. Controlling viraemia in HIV-1-infected individuals requires the maintenance of strong CD4 and CD8 T-cell responses. These responses should be elicited by prophylactic vaccination and by postexposure immunotherapy. This review will examine the methods that are available for the detection and quantification of HIV-1 specific CD4 and CD8 T-cell responses. We will also discuss the methods that should be used to identify these responses in HIV-1-infected individuals, seropositive recipients of immunotherapy and seronegative vaccinees. Finally, we will give examples of how responses observed in vitro relate to those known to occur in vivo .     

HIV-1感染者血浆丙酮酸对CD8+ T细胞功能的影响及临床意义

目的?探讨HIV-1感染者血浆丙酮酸对CD8+ T细胞功能的影响与疾病进展的关系。方法?入组11例健康对照（healthy controls, HCs）与55例接受长期抗逆转录病毒治疗的HIV-1感染者。通过液相色谱质谱联用法检测HCs与HIV-1感染者血浆丙酮酸水平,比较丙酮酸不同浓度组在HIV-1病毒库数量、CD8+ T细胞亚群比例、CD8+ T细胞功能间的差异。结果?与HCs（中位数416.71 ng/ml）相比HIV-1感染者（中位数539.16 ng/ml）血浆丙酮酸水平显著升高（P=0.0189）。与血浆低丙酮酸浓度组（低浓度组）相比,高丙酮酸浓度组（高浓度组）HIV-1细胞相关未剪接核糖核酸（HIV-1 cell associated un-spliced ribonucleic acid, HIV-1 CA RNA）、HIV-1 CA RNA/HIV-1 DNA的比值均显著升高;CD8+ T中央记忆T细胞（central memory T cells, TCM）比例显著升高;CD8+ T细胞表达TNF-α、IL-2、IFN-γ、CCL5的比例均降低,而表达CCL4的比例升高。血浆丙酮酸水平与CD8+ T细胞表达TNF-α（r=-0.3584,P=0.0106）、IL-2（r=-0.4975,P=0.0002）、IFN-γ（r=-0.4101,P=0.0031）、CCL5（r=-0.5655,P＜0.0001）的比例呈负相关,与CCL4（r=0.4711,P=0.0014）的比例呈正相关。结论 长期抗病毒治疗的HIV-1感染者血浆丙酮酸水平升高,且丙酮酸水平与HIV-1病毒库的活跃程度以及CD8+ T细胞功能紊乱相关。     

CD38 expression on CD8 T cells has a weak association with CD4 T-cell recovery and is a poor marker of viral replication in HIV-1-infected patients on antiretroviral therapy

OBJECTIVE: The aim of the study was to determine whether the expression of CD38 on CD8 T cells can identify patients with virological failure on antiretroviral therapy (ART). DESIGN: This was a cross-sectional study of patients attending a single HIV clinic in London. METHODS: The expression of CD38 on CD8 T cells was assessed using a biologically calibrated flow cytometry protocol. Patients were characterized by lymphocyte subset and viral load measurements. Characteristics including historical CD4 T cell counts, therapeutic history, co-infections and demographics were obtained from medical records. RESULTS: Elevated levels of CD8 CD38(high) T cells were found in HIV-1-infected patients who failed to suppress viral replication with ART; however, this parameter lacked sufficient sensitivity and specificity to replace viral load testing in assessing the efficacy of ART. Increased levels of CD8 CD38(high) cells were associated with reduced CD4 T cell counts in HIV-1-infected patients on ART after correcting for known determinants of CD4 T-cell recovery. CONCLUSIONS: The expression of CD38 on CD8 T cells lacks sufficient sensitivity and specificity to be used as a surrogate marker for viral load to monitor HIV-1 infection. T-cell activation is associated with reduced CD4 T-cell reconstitution in patients receiving ART.     

A polyclonal CD4+ and CD8+ lymphocytosis in a patient doubly infected with HTLV-I and HIV-1: a clinical and molecular analysis

HTLV-I is associated with adult T-cell leukemia/lymphoma (ATL) characterized by monoclonal expansions of CD4+ T-lymphocytes. In this report we describe a histologically benign, polyclonal HTLV-I infection in a patient exhibiting both an absolute CD4+ and CD8+ lymphocytosis. Three T-cell lines containing integrated HTLV-I proviral copies established from this patient were initially polyclonal, but with time all grew out the same two clones as determined by analysis of their T-cell antigen receptor beta chain gene rearrangements. The patient subsequently developed pulmonary and nasopharyngeal nodules containing HTLV-I infected cells. Restriction analysis of the patient's HTLV-I provirus revealed no differences from prototype HTLV-I and the tax gene was normally expressed in vivo and in vitro. The patient's T-lymphocytosis and HTLV-I+ pulmonary tract nodules were put into a complete clinical remission by treatment with alkylating agents and steroids. Subsequently, the patient developed a severe immunodeficiency state and expired. Retrospective serologic and gene amplification assays for HIV-1 demonstrated that he had been doubly infected from the time of presentation. Postmortem analysis by polymerase chain reaction revealed the presence of both HTLV-I and HIV-1 in lymphatic tissues and the testes; HIV-1 was also detected in brain tissue.     

正常T细胞分泌激活因子基因多态性与HIV-1感染者病毒载量、CD+4细胞计数和CD+4/CD+8比值的关系

目的分析正常T细胞分泌激活因子(RANTES)启动子和内含子等位基因,对艾滋病病毒1型(HIV-1)感染者外周血CD+4细胞计数、CD+4/CD+8比值和病毒载量的关系,以期探讨RANTES单核苷酸多态性(SNP)和艾滋病(AIDS)发病的关系.方法用流式细胞仪和real time法对40例汉族HIV-1感染者测定外周血CD+4、CD+8细胞计数和病毒载量,应用PCR-RFLP法进行基因分型.结果对RANTES-403、-28和In1.1三个位点等位基因与外周血CD+4T淋巴细胞计数、CD+4/CD+8+比值和病毒载量的关系的分析表明,具有RANTESIn1.1 C/C和T/C基因型的感染者,与较高的病毒载量、较低的C+4T细胞计数和C+4/CD+8比值有关.In1.1 T/T与T/C、C/C基因型的log10病毒载量的差异有非常显著的统计学意义(P＜0.01),但是不同基因型之间CD+4T淋巴细胞计数和CD+4/CD+8细胞比值则无显著性差异.log10病毒载量和CD+4T淋巴细胞计数(r=-0.447,P＜0.01)、CD+4/CD+8比值(r=-0.369,P＜0.05)有显著的负相关关系.结论所研究人群中,具有RANTES In1.1C的基因型者,与较高的病毒载量、较低的CD+4T细胞计数和CD+4/CD8+比值有关,它们是反映AIDS进程的主要指标,可能加速AIDS发病进程;而RANTES-403和-28等位基因的影响则处于相对次要的作用.     

The Expression of Programmed Death-1 in Circulating CD4+ and CD8+ T Cells during Hepatitis B Virus Infection Progression and Its Correlation with Clinical Baseline Characteristics

Background/Aims

Programmed death-1 (PD-1) expression was investigated in CD4⁺ and CD8⁺ T cells from hepatitis B virus (HBV)-infected patients at the chronic hepatitis B (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) stages.

Methods

PD-1 expression in circulating CD4⁺ and CD8⁺ T cells was detected by flow cytometry. The correlations between PD-1 expression and HBV viral load, alanine aminotransaminase (ALT) levels and aspartate aminotransferase (AST) levels were analyzed using GraphPad Prism 5.0.

Results

PD-1 expression in CD4⁺ and CD8⁺ T cells was significantly increased in both the CHB group and advanced-stage group (LC plus HCC). In the CHB group, PD-1 expression in both CD4⁺ and CD8⁺ T cells was positively correlated with the HBV viral load, ALT, and AST levels. However, in the LC plus HCC group, significant correlations between PD-1 expression and the clinical parameters were nearly absent.

Conclusions

PD-1 expression in peripheral CD4⁺ and CD8⁺ T cells is dynamic, changes with HBV infection progression, and is related to HBV viral load and liver function, especially in CHB. PD-1 expression could be utilized as a potential clinical indicator to determine the extent of virus replication and liver injury.