微囊泡与病毒感染

微囊泡(microvesicle)是细胞释放到胞外的膜性囊泡,其能将所含的蛋白质、脂类和核酸分子转运给其他细胞,从而介导细胞间通讯。作为严格细胞内寄生的微生物,病毒能利用微囊泡的生物合成和扩散途径进行病毒粒子的组装、出芽和传递,同时将病毒蛋白或基因组包装入微囊泡中。这些病毒修饰的囊泡能介导病毒在机体内的感染和扩散,或导致免疫细胞损伤以及耐受抗体的中和,从而逃避宿主免疫应答,引起持续性感染。重要的是,微囊泡介导的病毒感染打破了对病毒在体内扩散和感染时必须有病毒粒子存在的传统认知。对微囊泡与病毒感染进行综述,以促进对微囊泡介导病毒感染和抑制宿主免疫应答分子机制的了解。     

Antioxidant treatment reduces expansion and contraction of antigen-specific CD8+ T cells during primary but not secondary viral infection

During many viral infections, antigen-specific CD8(+) T cells undergo large-scale expansion. After viral clearance, the vast majority of effector CD8(+) T cells undergo apoptosis. Previous studies have implicated reactive oxygen intermediates (ROI) in lymphocyte apoptosis. The purpose of the experiments presented here was to determine the role of ROI in the expansion and contraction of CD8(+) T cells in vivo during a physiological response such as viral infection. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrin-mimetic compound with superoxide dismutase activity, from days 0 to 8 postinfection. At the peak of CD8(+)-T-cell response, on day 8 postinfection, the numbers of antigen-specific cells were 10-fold lower in MnTBAP-treated mice than in control mice. From days 8 to 30, a contraction phase ensued where the numbers of antigen-specific CD8(+) T cells declined 25-fold in vehicle-treated mice compared to a 3.5-fold decrease in MnTBAP-treated mice. Differences in contraction appeared to be due to greater proliferation in drug-treated mice. By day 38, the numbers of antigen-specific CD8(+) memory T cells were equivalent for the two groups. The administration of MnTBAP during secondary viral infection had no effect on the expansion of antigen-specific CD8(+) secondary effector T cells. These data suggest that ROI production is critical for the massive expansion and contraction of antigen-specific CD8(+) T cells during primary, but not secondary, viral infection.     

Differential tissue-specific regulation of antiviral CD8+ T-cell immune responses during chronic viral infection

The hallmarks of the immune response to viral infections are the expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) after they encounter antigen-presenting cells in the lymphoid tissues and their subsequent redistribution to nonlymphoid tissues to deal with the pathogen. Control mechanisms exist within CTL activation pathways to prevent inappropriate CTL responses against disseminating infections with a broad distribution of pathogen in host tissues. This is demonstrated during overwhelming infection with the noncytolytic murine lymphocytic choriomeningitis virus, in which clonal exhaustion (anergy and/or deletion) of CTLs prevents immune-mediated pathology but allows persistence of the virus. The mechanism by which the immune system determines whether or not to mount a full response to such infections is unknown. Here we present data showing that the initial encounter of specific CTLs with infected cells in lymphoid tissues is critical for this decision. Whether the course of the viral infection is acute or persistent for life primarily depends on the degree and kinetics of CTL exhaustion in infected lymphoid tissues. Virus-driven CTL expansion in lymphoid tissues resulted in the migration of large quantities of CTLs to nonlymphoid tissues, where they persisted at stable levels. Surprisingly, although virus-specific CTLs were rapidly clonally exhausted in lymphoid tissues under conditions of chronic infection, a substantial number of them migrated to nonlymphoid tissues, where they retained an effector phenotype for a long time. However, these cells were unable to control the infection and progressively lost their antiviral capacities (cytotoxicity and cytokine secretion) in a hierarchical manner before their eventual physical elimination. These results illustrate the differential tissue-specific regulation of antiviral T-cell responses during chronic infections and may help us to understand the dynamic relationship between antigen and T-cell populations in many persistent infections in humans.     

Regulation of immune response and inflammatory reactions against viral infection by VCAM-1

The migration of activated antigen-specific immune cells to the target tissues of virus replication is controlled by the expression of adhesion molecules on the vascular endothelium that bind to ligands on circulating lymphocytes. Here, we demonstrate that the adhesion pathway mediated by vascular cell adhesion molecule 1 (VCAM-1) plays a role in regulating T-cell-mediated inflammation and pathology in nonlymphoid tissues, including the central nervous system (CNS) during viral infection. The ablation of VCAM-1 expression from endothelial and hematopoietic cells using a loxP-Cre recombination strategy had no major effect on the induction or overall tissue distribution of antigen-specific T cells during a systemic infection with lymphocytic choriomeningitis virus (LCMV), except in the case of lung tissue. However, enhanced resistance to lethal LCM and the significantly reduced magnitude and duration of footpad swelling observed in VCAM-1 mutant mice compared to B6 controls suggest a significant role for VCAM-1 in promoting successful local inflammatory reactions associated with efficient viral clearance and even life-threatening immunopathology under particular infection conditions. Interestingly, analysis of the infiltrating populations in the brains of intracerebrally infected mice revealed that VCAM-1 deletion significantly delayed migration into the CNS of antigen-presenting cells (macrophages and dendritic cells), which are critical for optimal stimulation of migrating virus-specific CD8⁺ T cells initiating a pathological cascade. We propose that the impaired migration of these accessory cells in the brain may explain the improved clinical outcome of infection in VCAM-1 mutant mice. Thus, these results underscore the potential role of VCAM-1 in regulating the immune response and inflammatory reactions against viral infections.     

Mathematical modeling of viral infection dynamics in spherical organs

A general mathematical model of viral infections inside a spherical organ is presented. Transported quantities are used to represent external cells or viral particles that penetrate the organ surface to either promote or combat the infection. A diffusion mechanism is considered for the migration of transported quantities to the organ inner tissue. Cases that include the effect of penetration, diffusion and proliferation of immune system cells, the generation of latently infected cells and the delivery of antiviral treatment are analyzed. Different antiviral mechanisms are modeled in the context of spatial variation. Equilibrium conditions are also calculated to determine the radial profile after the infection progresses and antiviral therapy is delivered for a long period of time. The dynamic and equilibrium solutions obtained in this paper provide insight into the temporal and spatial evolution of viral infections.     

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.     

Control of virus-specific CD8+ T-cell exhaustion and immune-mediated pathology by E3 ubiquitin ligase Cbl-b during chronic viral infection

A characteristic feature in the immune response to many persistent viral infections is the dysfunction or deletion of antigen-specific T cells (exhaustion). This down-regulation of virus-specific T-cell response represents a critical control mechanism that exists within T-cell activation pathways to prevent lethal disease by inappropriate responses against disseminating virus infections. However, the molecular mechanisms by which the immune system determines whether to mount a full response to such infections remain largely unexplored. Here, we have established that in the murine lymphocytic choriomeningitis virus (LCMV) model, induction of the T-cell receptor signaling inhibitor molecule E3 ligase Cbl-b is critically involved in this decision. In particular, our data revealed that Cbl-b controls the program responsible for T-cell tolerance (exhaustion) induction during a chronic viral infection. Thus, Cbl-b(-/-) mice infected with a low dose of LCMV Docile mount a strong CD8(+) T-cell response that rapidly clears the infection, and the animals remain healthy; in contrast, down-regulation of the epitope-specific CD8(+) T-cell population in persistently infected Cbl-b(-/-) mice, compared to that in chronically infected B6 mice, was significantly delayed, and this was associated with increased morbidity and eventual death in nearly 20% of the animals. Interestingly, infection of Cbl-b(-/-) mice with a moderate virus dose resulted in rapid death with 100% mortality by 7 to 8 days after infection, caused by a dysregulated antiviral T-cell response, whereas the infected B6 mice survived and remained healthy. In conclusion, our results suggest that Cbl-b is critically involved in T-cell exhaustion and prevention of lethal disease.     

Immunogenicity of cytopathic and noncytopathic viral vectors

The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8(+) T cells (T(CD8)(+)) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the T(CD8)(+) antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.     

Mechanisms of viral hepatitis induced liver injury

Among seven human hepatitis viruses (A to E, G and TT virus), hepatitis B (HBV) and C (HCV) viruses are able to persist in the host for years and principally contribute to the establishment of chronic hepatitis. During the course of persistent infection, continuous intrahepatic inflammation maintains a cycle of liver cell destruction and regeneration that often terminates in hepatocellular carcinoma (HCC). While the expression and retention of viral proteins in hepatocytes may influence the severity and progression of liver disease, the mechanisms of liver injury in viral hepatistis are defined to be due not to the direct cytopathic effects of viruses, but to the host immune response to viral proteins expressed by infected hepatocytes. In the process of liver injury, hepatocellular death (apoptosis) induced by the proapoptotic molecules of T cells activated following antigen recognition triggers a cascade of antigen nonspecific effector systems and causes necroinflammatory disease. Accordingly, the regulation of the immune response, e.g., via the cell death pathways, in chronically infected patients should prevent the development of HCC.     

Suppression of antigen-specific T cell responses by the Kaposi's sarcoma-associated herpesvirus viral OX2 protein and its cellular orthologue, CD200

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.     

Distinct CD8 T cell functions mediate susceptibility to histoplasmosis during chronic viral infection

It has long been recognized that some viral infections result in generalized immune suppression. In acute infections, this period of suppressed immunity is relatively short. However, chronic infections associated with a prolonged period of immune suppression present far greater risks. Here, we examined the role of CD8 T cell responses following viral infection in immunity to systemic histoplasmosis. Although wild-type mice with systemic histoplasmosis were able to control the infection, those simultaneously infected with lymphocytic choriomeningitis virus clone 13 showed reduced immunity with greater fungal burden and high mortality. The immune suppression was associated with loss of CD4 T cells and B cells, generalized splenic atrophy, and inability to mount a granulomatous response. Removing the anti-viral CD8 T cells in the coinfected mice enabled them to reduce the fungal burden and survive the infection. Their lymphoid organs were replenished with CD4 T and B cells. In contrast to wild-type mice, perforin-deficient mice infected with lymphocytic choriomeningitis virus clone 13 and Histoplasma showed an absence of immunopathology, but the animals still died. These results show that CD8 T cells can suppress immunity through different mechanisms; although immunopathology is perforin-dependent, lethality is perforin-independent.     

Recognition of viral antigens by human influenza A virus-specific T lymphocyte clones

The nature of the viral antigens recognized by influenza A virus-immune cytotoxic T lymphocytes (CTL) is still a matter of debate. We have used four human influenza A virus-specific T lymphocyte clones with antigen-specific cytotoxic and proliferative activity to investigate the requirements for recognition of viral antigens on infected cells. One clone recognized a cross-reactive determinant on the viral hemagglutinin, and two clones were specific for different epitopes on the viral nucleoprotein (NP). A fourth clone seemed to be specific for the viral M protein. Target cell recognition was abrogated by the addition, during infection, of the lysosomotropic drug chloroquine, known to inhibit antigen processing. Furthermore, target cells that had been pulsed with soluble purified NP were recognized and were lysed by the NP-specific clone. This reaction could also be abrogated by the addition of chloroquine during pulsing. These results were obtained irrespective of whether EBV-transformed B lymphoblastoid cells or Ia antigen-expressing T cell blasts were used as target cells. It is concluded that CTL can recognize internal viral proteins that are actively presented at the surface of the target cell. These data indicate that probably every viral protein can function as a target molecule for virus-immune CTL.     

Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.     

SARS-CoV-2 viral dynamics in non-human primates

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>10⁴ virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.     

Innate immune response to viral infection

In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. In the innate immune response, pattern recognition receptors (PRRs) are engaged to detect specific viral components such as viral RNA or DNA or viral intermediate products and to induce type I interferons (IFNs) and other pro-inflammatory cytokines in the infected cells and other immune cells. Recently these innate immune receptors and their unique downstream pathways have been identified. Here, we summarize their roles in the innate immune response to virus infection, discrimination between self and viral nucleic acids and inhibition by virulent factors and provide some recent advances in the coordination between innate and adaptive immune activation.     

Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection.

The proteins associated with parental, adenoviral DNA in productively-infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P-labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross-linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co-migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross-linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross-linkable to viral DNA in cells infected by wild-type virus and a non-infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre-early and early viral genes.     

Estimating the effectiveness of simian immunodeficiency virus-specific CD8+ T cells from the dynamics of viral immune escape

Antiviral CD8(+) T cells are thought to play a significant role in limiting the viremia of human and simian immunodeficiency virus (HIV and SIV, respectively) infections. However, it has not been possible to measure the in vivo effectiveness of cytotoxic T cells (CTLs), and hence their contribution to the death rate of CD4(+) T cells is unknown. Here, we estimated the ability of a prototypic antigen-specific CTL response against a well-characterized epitope to recognize and kill infected target cells by monitoring the immunodominant Mamu-A*01-restricted Tat SL8 epitope for escape from Tat-specific CTLs in SIVmac239-infected macaques. Fitting a mathematical model that incorporates the temporal kinetics of specific CTLs to the frequency of Tat SL8 escape mutants during acute SIV infection allowed us to estimate the in vivo killing rate constant per Tat SL8-specific CTL. Using this unique data set, we show that at least during acute SIV infection, certain antiviral CD8(+) T cells can have a significant impact on shortening the longevity of infected CD4(+) T cells and hence on suppressing virus replication. Unfortunately, due to viral escape from immune pressure and a dependency of the effectiveness of antiviral CD8(+) T-cell responses on the availability of sufficient CD4(+) T cells, the impressive early potency of the CTL response may wane in the transition to the chronic stage of the infection.     

Assessing the genetic component of the susceptibility of mice to viral infections.

Laboratory mice often exhibit wide differences in susceptibility when infected experimentally with viruses. Based on such observations, experiments have been designed to investigate the determinism of these differences at the molecular level, and a few genes that play a major role in the innate mechanisms of defence of the species toward viral aggressions have been characterised. For example, the extraordinary resistance of SJL mice to experimental infections with hepatitis virus strain A59 is the consequence of a structural alteration of a cell adhesion molecule which normally binds to the spikes of the virus, allowing its entry into the cells. If the virus cannot bind to the molecule, or if the molecule is absent, epithelial cells of the intestine and liver are not infected and mice are resistant. In the same way, most--not to say all--laboratory strains of mice are susceptible to infections with orthomyxoviruses or flaviviruses because essential molecules, the synthesis of which is normally triggered by interferon, are defective in these mice. Wild mice, by contrast --probably because they are constantly exposed to natural infections--are resistant. Finally, some mouse strains resist experimental infections by the mouse cytomegalovirus 1 (MCMV-1) because, once infected, these mice synthesise a molecule at the surface of infected cells which allows immediate recognition and killing by natural killer (NK) cells. With the exuberant development of mouse genetics and the constant generation of new mutant alleles, it is likely that many more genes with an impact on the phenotype of resistance or susceptibility will be identified in the forthcoming years. These genes are probably numerous, however, and many of them presumably interact with each other and/or have additive effects. This might slow down progress in our understanding of the innate mechanism of defence.