流感病毒诱导小鼠胶质细胞的细胞因子级联反应

目的:探讨流感病毒感染星形胶质细胞后释放的细胞因子是否会诱导正常胶质细胞趋化因子及促炎细胞因子转录水平的变化,从而产生细胞因子级联效应。方法:从新生小鼠大脑皮质分离培养神经胶质细胞,并进一步纯化星形胶质细胞,经纯度鉴定后,用感染复数为2的流感病毒H1N1和H3N2进行体外感染星形胶质细胞,分别于6小时和24小时收获上清,采用超滤分子截留的方法,去除流感病毒颗粒。用不同时间点的条件上清,分别刺激星形胶质细胞和小胶质细胞,24小时后提取RNA并进行反转录,利用Real-Time PCR检测促炎因子TNF-α、IL-1β、IL-6,趋化因子IP-10、MCP-1、MIP-1转录水平的变化。结果:不同时间点的条件上清皆可诱导正常胶质细胞的促炎症因子TNF-α、IL-1β、IL-6和趋化因子IP-10、MCP-1、MIP-1的转录水平发生不同程度的上调,产生细胞因子级联效应。结论:流感病毒H1N1和H3N2感染星形胶质细胞后所引起的细胞因子风暴,可诱导正常胶质细胞的细胞因子转录水平显著上调,引发细胞因子级联反应,其可能与流感病毒感染中枢神经系统所引发的细胞因子风暴及免疫病理损伤存在一定关系。     

IgG isotype and isotype specificity of murine monoclonal IgG rheumatoid factors

Immune complexes of lipopolysaccharide (LPS) with homologous IgG antibody induces rheumatoid factor (RF) predominantly of the IgG class in normal mice, while LPS alone induces mostly IgM RF directed to homologous IgG1. In this study, IgG monoclonal RFs (mRF) were prepared from hybridomas derived from spleen cells of BALB/c mice which were immunized with complexes of TNP-LPS with anti-TNP mouse IgG and their specificity to mouse IgG subclasses was assessed by analysing dissociation kinetics of the ligands due to RF-specific and non-specific interactions. Of the 19 IgG mRFs (11 IgG1, five IgG2a, one IgG2b and two IgG3 types) tested, 14 were directed to either IgG3 or IgG2b or both, while only one exhibited a significant binding capacity to IgG1. Other mRFs, although reactive to rabbit IgG, exhibited little homophilic activity. None of these mRFs reacted strongly with their own isotypes. The results suggest that the IgG RF producing cells are not direct progenies of the IgG1-directed IgM RF-producing cells but may have developed via a rigorous selection process to eliminate clones that produce self-reactive RF.     

CD8 T cell responses to influenza virus infection in aged mice

Influenza is one of the most common infectious diseases afflicting humans, particularly the elderly. The murine model has been widely employed for investigation of immunity to influenza virus infection. In this paper, we review the recent advances in understanding the diminished CD8 T cell immune response to influenza virus infection in aged mice. Possible mechanisms of impaired CD8 T cell responses with aging are addressed, including: (1) the role of dendritic cells (DCs); (2) the effect of age-associated changes in the T cell repertoire; and (3) the interactions with CD4 T cells, including T regulatory (Treg) cells and CD4 T helper cells. The aged murine model of the CD8 T cell response to influenza virus is helping to elucidate the mechanisms of immunosenescence which can lead to therapeutic improvements in the primary CD8 T cell response to new infections, as well as the development of new strategies for immunization to prevent influenza in the elderly.     

Immune responses to infection with H5N1 influenza virus

Influenza A H5N1 viruses remain a substantial threat to global public health. In particular, the expanding genetic diversity of H5N1 viruses and the associated risk for human adaptation underscore the importance of better understanding host immune responses that may protect against disease or infection. Although much emphasis has been placed on investigating early virus–host interactions and the induction of innate immune responses, little is known of the consequent adaptive immune response to H5N1 virus infection. In this review, we describe the H5N1 virus-specific and cross-reactive antibody and T cell responses in humans and animal models. Data from limited studies suggest that although initially robust, there is substantial waning of the serum antibody responses in survivors of H5N1 virus infection. Characterization of monoclonal antibodies generated from memory B cells of survivors of H5N1 virus infection has provided an understanding of the fine specificity of the human antibody response to H5N1 virus infection and identified strategies for immunotherapy. Human T cell responses induced by infection with seasonal influenza viruses are directed to relatively conserved internal proteins and cross-react with the H5N1 subtype. A role for T cell-based heterosubtypic immunity against H5N1 viruses is suggested in animal studies. Further studies on adaptive immune responses to H5N1 virus infection in both humans and animals are needed to inform the design of optimal immunological treatment and prevention modalities.     

The use of antibody-secreting cell probes to reveal tissue-restricted immune responses during infection

Antibody secreting cell probes (ASC-probes) were generated from the hepatic lymph node (HLN), mesenteric lymph node (MLN) and spleen of rats after infection with the liver fluke, Fasciola hepatica, and used to probe Western blots of parasite antigens. In chronic primary infections, parasite-specific antibodies were only detected in ASC-probes derived from HLN. Seven days after a secondary infection, a restricted response was detected in ASC-probes from the MLN, directed predominantly against an antigen specific to the newly excysted juvenile (NEJ) stage. This NEJ-specific antigen was only recognized by HLN if the second infection was not rejected and the challenge flukes reached the liver. Measurement of the immunoglobulin levels present in the ASC-probes showed significant elevation only in lymph nodes draining sites of recent infection. In addition, when the isotype profiles were determined in ASC-probes derived from different lymph nodes, it was observed that they showed different isotype preferences, in particular IgA for the MLN, IgE for HLN and IgM for spleen. These results show that discrete and independent immune responses occur in different body compartments of a rat against different stages of a parasite.     

Activation of invariant NKT cells enhances the innate immune response and improves the disease course in influenza A virus infection

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection.     

Comparative analyses of influenza virus receptor distribution in the human and mouse brains

Accumulating evidence suggests a potential link between influenza A virus infection and the occurrence of influenza-associated neurological disorders. As influenza infection is mediated by specific receptors on the host cell surface, it is important to understand the distribution patterns of influenza receptors in target organs. We carried out comprehensive experiments to localize influenza receptors in the brains of two different mouse strains and the human brain for comparison using lectin histochemistry. We further compared the brain regions in which influenza receptors were expressed and the regions in which experimental influenza infection was observed. Our results show that the expression patterns for influenza receptors in mouse and human brains are different. In the mouse brain, human influenza virus receptors (HuIV-R) were expressed in part of brainstem and cerebellar white matter while avian influenza virus receptors (AIV-R) were expressed in the cerebellar Purkinje neurons. In contrast, in the human brain, many neurons and glia in widespread regions, including the cerebral cortex, hippocampus, brainstem, and cerebellum, express both AIV-R and HuIV-R. Importantly, vascular endothelial cells, choroid plexus epithelial cells and ependymal cells in both mouse and human brains express high levels of HuIV-R and AIV-R. The regional reciprocity was not observed when comparing regions with influenza receptor expression and the regions of influenza infection within the mouse brain. Our results demonstrate a differential influenza receptor expression pattern in mouse and human brains, and a disparity between influenza receptor distribution and regions with actual influenza infection.     

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection

Multipeptide constructs, comprising adjacent sequences of the 317–341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1–13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317–329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)₄–FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.     

Kinetics of antigen-specific IgM/IgG/IgA antibody responses during Zika virus natural infection in two patients

Understanding of kinetics of antibody responses is crucial for developing rapid serological tests and studying the mechanisms of Zika virus (ZIKV) infection. Most of the serological diagnostic assays previously published are based on either IgM or IgG titer, little is known on the level of IgA antibody in saliva and urine. In this study, we investigated the kinetics of IgM/IgG/IgA antibody responses in serum, saliva, and urine obtained from two ZIKV infected individuals from as early as the second day of onset of symptoms to as long as 2 years postinfection. Other than detecting robust early IgM response, long lasting IgG response, we discovered strong early IgA response specific for ZIKV in saliva in both patients. This unique observation provides a novel strategy and scientific basis for the development of noninvasive rapid tests for ZIKV infection.     

Cigarette smoking and innate immune responses to influenza infection

Cigarette smoking(CS) suppresses the immune system, and smoking is a well-known major risk factor for respiratory tract infections, including influenza infection. Both smoking cigarettes and passive smoking alter a wide range of immunological functions, including innate and adaptive immune responses. Past reviews on CS and innate immunity have been focused on the effects of CS on structural changes of the lung, as well as the effects on the function of alveolar macrophages, leukocytes, natural killer cells and dendritic cells. The study of innate immunity has developed rapidly in the last decade with the discovery of new receptors for virus recognition and interferon responses. This review aims to give a brief summary of recent findings on the suppressive effects of CS on the innate response to influenza virus, especially as it pertains to suppression of the function of pattern recognition receptors for influ-enza virus.     

甲型流感病毒核蛋白基因的克隆表达及纯化

目的　将甲型流感病毒核蛋白(NP)基因克隆到原核表达载体进行可溶性融合表达,制备纯化的病毒核蛋白,为制备甲型流感病毒单克隆抗体提供材料。方法　提取甲型流感病毒RNA ,设计引物,RT PCR扩增NP基因,利用基因工程的手段,将甲型流感病毒的NP基因在大肠埃希菌中进行融合表达,并将表达产物进行亲和层析。结果　成功构建了甲型流感病毒NP基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。结论　通过合理控制发酵时间、生长温度和诱导物浓度,制备了较为理想的可溶性甲型流感病毒核蛋白。     

小鼠感染流感病毒后可抵抗致死性流感病毒攻击的研究

目的 BALB/c小鼠初次感染流感病毒(A/California/7/2009(H1N1)(pCA07)和A/Guangzhou/333/99(H9N2)(GZ333))后,用流感病毒鼠肺适应株A/PR/8/34(H1N1)(PR8) 10倍致死剂量攻击,观察攻毒后小鼠的反应.方法 BALB/c小鼠150只,分成3组,空白对照组PBS滴鼻,实验组分别感染甲型H1N1流感病毒pCA07和禽源H9N2病毒GZ333；感染56 d后用10倍致死剂量的鼠肺适应株流感病毒PR8攻击两个实验组和对照组小鼠,比较PR8病毒攻击后,病毒载量和抗体水平,以及对小鼠存活率的影响.结果 感染过pCA07和GZ333的小鼠在致死病毒PR8攻击后全部存活,并分别在病毒攻击6d和9d后小鼠肺组织中检测不到攻击病毒.对初次感染病毒的抗体水平在病毒PR8攻击后迅速升高,在保持初次感染病毒抗体的同时能够产生针对PR8病毒的抗体.结论 不同亚型流感病毒感染小鼠后可以提供交叉保护.     

遗传重配获得H5N1流感病毒Vero细胞适应株

目的以传统遗传重配技术选育HSN1流感病毒Veto细胞适应株,制备Vero细胞H5N1流感疫苗。方法以流感病毒Vero细胞适应株A／Yunnan／1／2005Va（H3N2）为母株与反向遗传学技术改造的禽流感病毒疫苗株A／Anhui／1／2005（H5N1）共同感染SPF鸡胚和Vero细胞,用羊抗A／Yunnan／1／2005Va（H3N2）抗体筛选,血抑试验和基因测序鉴定病毒型别,并进行重配株的其他相关生物学试验。结果获得了1株在Vero细胞高产的H5N1流感病毒,重配前后的单价灭活疫苗免疫小鼠抗体血清效价差异无统计学意义（F=0．857,P〉0．05）。结论通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,可以获得H5N1流感病毒Vero细胞适应株。     

Efficient vagina-to-lower respiratory tract immune trafficking in a murine model of influenza A virus infection

Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both homosubtypic and heterosubtypic virus challenge in the respiratory tract. We found that, prior to the viral challenge, in vaginally primed mice, antigen-specific CD8+ T cells were not detected in the lung airways and levels of serum antibodies were lower than those observed in intranasally immunized mice. However, following pulmonary challenge, NP147-specific CD8+ T cells were recruited and amplified in vaginally primed mice to the same extent as those in intranasally primed mice. Thus, the long-term memory immune response elicited by vaginal immunization with influenza virus is efficiently recalled and offers reasonable protection against infection in the respiratory tract.     

Serum IgG subclass responses of humans to inactivated and live influenza A vaccines compared to natural infections with influenza A

Studies with various viral agents have suggested that a preferential production of IgG subclasses may occur during infection, but limited information has been reported on the IgG isotypes produced during vaccination with live or killed virus preparations. The serum IgG subclass responses to influenza A infection or inoculation with live or killed influenza A vaccines were examined by an enzyme-linked immunosorbent assay, and results were expressed using a 4-parameter logistic model. It was observed that IgG1 was induced by both natural infections and the live virus vaccine depending on the dose given. Inactivated vaccines induced significant titres of IgG1, IgG2, and IgG3 isotypes in vaccinees, again depending upon the amount of virus preparation administered.     

The immune response induced by hepatitis B virus principal antigens

Hepatitis B virus （HBV） infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellniar carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.     

Evasion mechanisms of the type I interferons responses by influenza A virus

Abstract

The type I interferons (IFNs) represent the first line of host defense against influenza virus infection, and the precisely control of the type I IFNs responses is a central event of the immune defense against influenza viral infection. Influenza viruses are one of the leading causes of respiratory tract infections in human and are responsible for seasonal epidemics and occasional pandemics, leading to a serious threat to global human health due to their antigenic variation and interspecies transmission. Although the host cells have evolved sophisticated antiviral mechanisms based on sensing influenza viral products and triggering of signalling cascades resulting in secretion of the type I IFNs (IFN-α/β), influenza viruses have developed many strategies to counteract this mechanism and circumvent the type I IFNs responses, for example, by inducing host shut-off, or by regulating the polyubiquitination of viral and host proteins. This review will summarise the current knowledge of how the host cells recognise influenza viruses to induce the type I IFNs responses and the strategies that influenza viruses exploited to evade the type I IFNs signalling pathways, which will be helpful for the development of antivirals and vaccines.     

Establishment of a cynomolgus macaque model of influenza B virus infection

Pathogenicity of influenza B virus was examined in cynomolgus macaques to establish a macaque model suitable for vaccine and antiviral drug development. We prepared influenza B viruses for inoculation with minimal passages after isolation from patients. Macaques inoculated with influenza B virus showed higher body temperature than that before infection for 6 to 12 days. Virus was detected in nasal, tracheal, and bronchial samples until 6 days after inoculation followed by an increase in neutralizing antibody. High levels of IL-6 and TNF-α in nasal swabs from the infected macaques were correlated with fever. Symptoms and duration of the viral replication would be sufficient to evaluate efficacy of vaccines and antiviral agents. In addition, measurement of immune responses including antibody and cytokine production would provide an immunological rationale in efficacy of vaccines and antiviral agents. The results suggest that cynomolgus macaques are appropriate model animals for research of influenza B virus.     

Interaction of porcine conventional dendritic cells with swine influenza virus

Swine influenza virus (SwIV) causes sub-acute or acute respiratory infections on swine farms and pigs can act as “mixing vessels” for new influenza strains. Knowledge of the immune response of SwIV in its natural host, pigs, is very limited. Dendritic cells (DCs) mediate the induction of immunity to pathogens, but their interaction with SwIV has not been fully characterized. Thus, porcine bone marrow derived DCs (poBMDCs) were exposed to a circulating strain of H3N2 SwIV in vitro. Infection of poBMDCs resulted in structures resembling influenza virus inside poBMDCs in vesicles and also free in cytoplasm. Viral progeny was undetectable in supernatant but limited replication was detected in the first 8 h after infection. However, viral particles from infected-poBMDCs were able to induce cytopathic effect in susceptible cells only when cell-to-cell interaction was favoured. The data generated in our studies reveal the particular interaction of H3N2 SwIV with conventional DCs.