Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R² ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell–based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell–based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.CD8 T cells provide a powerful mechanism for elimination of intracellular pathogens and tumor cells. Accordingly, a major thrust of current vaccine research focuses on stimulating robust T-cell immunity for defense against infections such as HIV, malaria, tuberculosis, Ebola virus, herpes viruses, and hepatitis C virus (HCV) (1–8). Inducing effective CD8 T-cell immunity is also an important goal for cancer vaccines (9, 10). However, how antigen load affects the CD8 T-cell response has not been quantified in a detailed manner during a human immune response. In this study we address this question using the human live attenuated yellow fever vaccine (YFV-17D) vaccine.The dynamics of CD8 T-cell responses to intracellular infection have been extensively studied in model systems. Infection typically stimulates a rapid burst of proliferation in antigen-specific CD8 T cells with division occurring as quickly as once in 4–6 h (11). This expansion results in a large population of effector CD8 T cells that aid in clearance of infected cells. Although most (90–95%) of the effector CD8 T cells die, a small fraction differentiate to form long-term memory CD8 T cells (12). Detailed quantitative measurements of the dynamics of virus and the CD8 T-cell response to the YFV-17D vaccine allow us to characterize these basic features of the CD8 T-cell responses in humans. Additionally, tracking the dynamics of both virus and CD8 T cells over time in a large cohort allows us to explore the relationship between amount of antigen and the magnitude of expansion and answer the following questions: Is there a threshold amount of virus required to generate a response? Does the magnitude of the response increase proportionally, or does it saturate with viral load? Although a number of studies have considered the complex relationship between numbers of specific CD8 T cells and virus loads during the chronic phase of HIV and HCV infections (3, 13–23), very few studies (24–27) have investigated these questions in the context of the generation of immune response following acute infections and vaccination.We addressed these questions by measuring the dynamics of both virus and virus-specific CD8 T cells following immunization with the YFV-17D vaccine. The YFV-17D vaccine comprises a highly efficacious, live attenuated virus that causes an acute infection and stimulates a robust immune response conferring lifelong protection against the yellow fever virus (YFV) (28, 29). Because yellow fever is not endemic to the United States, immunization with YFV-17D induces a primary immune response (30, 31). Previous work with YFV-17D has identified CD8 T cells specific for some of the YFV epitopes and defined the stages of expansion, contraction, and memory maintenance (32–38). We now know that YFV stimulates a polyfunctional, broadly targeting, and long-lasting CD8 T-cell response. Of particular note, we have previously demonstrated that the magnitude of the total effector CD8 T-cell response against YFV can be measured using the Ki-67⁺ Bcl-2^lo HLA-DR⁺ CD38⁺ phenotype of activated T cells seen early after vaccination (38). In the current study, we followed a large cohort of 80 individuals with intensive sampling at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90 postvaccination to quantify viral load in plasma (39). Additionally, we quantified the magnitude of the YFV-specific effector CD8 T-cell response at days 0, 3, 7, 14, 30, and 90 postvaccination using the Ki-67⁺Bcl-2^lo phenotype. We find that different individuals have different virus loads following infection and generate CD8 T-cell responses of different sizes. This allows us to determine the relationship between virus load and magnitude of the CD8 T-cell response.The majority of vaccines that are currently under development use replication-incompetent or single-cycle vectors such as Modified Vaccinia Ankara, adenovirus, and DNA. Although these approaches are inherently safe, they may express and deliver relatively limited amounts of antigen. Our results emphasize the requirement that T-cell–based vaccines deliver sufficient antigen to elicit a large CD8 T-cell response that may be needed for protection.     

Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis

The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type III effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type III secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell.     

新一代基因组编辑技术在基因治疗及生物制药领域中的应用

近年来,随着锌指核酸酶、转录激活子样效应因子核酸酶和成簇可调控间隔短回文重复RNA引导核酸酶的出现,“基因组编辑”技术得到广泛应用。由于此类技术具有高效和可定制的特点,在基因治疗、细胞模型、糖基化工程、细胞工程等方面许多新策略、新方法不断涌现,产生了十分重要的影响。本文就近年来基因组编辑技术的发展及其在基因治疗和生物制药领域的应用进行综述。     

慢性乙型肝炎患者外周血CD8^＋T细胞亚群分布和PD-1表达特征研究

目的分析慢性乙型肝炎（CHB）患者外周血中CD8^＋T细胞及其亚群表面程序性死亡受体-1（PD-1）表达的变化特征及其临床关联性。方法用流式细胞术检测14例CHB患者和15例健康对照者外周血中CD8^＋T细胞及其亚群初始T细胞（TN）、中央记忆T细胞（TCM）、效应记忆T细胞（TEM）、终末效应记忆T细胞（TEMRA）的频数及细胞表面PD-1的表达水平,同时收集患者一般资料、生化指标和乙型肝炎病毒（HBV）病毒学指标等临床资料。使用SAS 9.1统计软件分析数据,应用Mann-Whitney U test分析组间差异,应用Spearman秩相关进行相关性分析,P〈0.05为差异有统计学意义。结果与健康对照组相比,CHB患者的TEMRA亚群在CD8^＋T细胞中的频数显著降低（P=0.012）,总CD8^＋T细胞（P=0.018）及其TEM亚群（P=0.004）表面PD-1的表达水平显著上升。相关性研究显示,CD8^＋TEM细胞表面PD-1的表达水平与HBV DNA载量呈显著正相关（r=0.554,P=0.040）。结论 CHB患者外周血中PD-1阳性的CD8^＋TEM频数升高,且与外周血中HBV DNA载量呈正相关,提示慢性乙型肝炎患者机体内HBV复制的活跃性可能与外周血中CD8^＋T细胞的活化状态,特别是其TEM亚群上PD-1的高表达关联。     

A sensitive method to measure PHA-induced cytotoxicity to adherent target cells using [3H]uridine as a terminal label

This paper describes a modification of the PHA-induced cytotoxicity test of human peripheral blood lymphocytes against a tumour-derived adherent cell line (HeLa), in which the surviving target cells are labelled with [³H]uridine at the end of the assay. There is a direct correlation between [³H]uridine incorporation and the number of adherent target cells. The test proves to be very sensitive at low effector: target cell ratios. Frozen stored cells can be used in this system, a particular advantage because of the possibility of increasing the reproducibility of the assay by using the same batch of cryopreserved lymphocytes as a reference standard in each experiment. PHA-induced cytotoxicity was mainly found in the T cell enriched fraction.     

Virus-neutralizing properties of antiinfluenzal rabbit IgG with reduced complement-fixing and cytophilic activity

Correlation between the effector functions and virus-neutralizing activity of antiviral immuno-globulin G (IgG) antibodies was investigated in relation to rabbit IgG antibodies against A/PR₈/34 influenza virus. Changes in the effector activity of the antiviral antibodies were produced by reducing the single disulfide bond between the heavy chains in the hinge region of the IgG molecule. Antiinfluenzal IgG, when reduced in this way, retained about 50% of its complement-fixing activity but lost virtually all its ability to be bound to heterologous tissues. Meanwhile the reduced antiinfluenzal IgG, as the results of the delayed hemagglutination test showed, completely preserved its antigen-binding activity. The virus-neutralizing activity of the reduced antiinfluenzal IgG, as estimated in experiments on chick embryos, was indistinguishable from the activity of the native preparation if the dose of virus used in the experiments was 100 EID₅₀. If the dose of virus was increased to 1000 EID₅₀ the virus-neutralizing activity of the reduced IgG was less than that of the native preparation. The results are discussed in terms of differences in the structural organization of reduced IgG.N. F. Gamaleya Institute of Epidemiology and Microbiology, Moscow. D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 188–191, August, 1977.     

Increased interleukin-17 producing effector memory T cells in the end-stage renal disease patients

Patients with end-stage renal disease (ESRD) exhibit immune dysregulation, but the precise immunological profile and the effect of hemodialysis (HD) on it has not been investigated fully. Thirty-eight ESRD patients (22 on HD and 16 in pre-dialysis) and 24 healthy volunteers were included. We compared the T cell immune profiles as in these patients. Among the effector T cell subset, the percentages of Th17 and Th2 cells were significantly higher in the ESRD group than in the healthy controls (P<0.05). The percentage of Th1 cells did not differ significantly between these groups. The percentages of Th1, Th2 and Th17 cells did not differ significantly (P>0.05) between the two subgroups within the ESRD group. The CCR4(-)CCR6(+)/CD4(+) T cell percentage was also significantly higher in the ESRD group. The na?ve T cell (T(na?ve)) percentage was significantly lower in the ESRD group, and the difference between patients and controls was greater in the pre-dialysis patients than in the HD patients (P<0.05, for each comparison). By contrast, the percentages of central memory T cells (T(CM)) and effector memory T (T(EM)) cells were significantly higher in the ESRD group. Interleukin-17 production by T(EM) cells was significantly higher in the ESRD group. The severity of uremia was related negatively to the T(na?ve) cell percentage but positively to the T(CM) and T(EM) cell percentages. The percentages of T(EM) and CD45RA(+) T effector memory subsets of CD8(+) T cells were significantly higher in the ESRD group (P<0.05). The result of this study showed significantly altered T cell-associated immunity and that it could not be corrected with hemodialysis.