IL-10, T cell exhaustion and viral persistence

Viral infections can have one of two outcomes: control of viral replication and acute infection or viral persistence and chronic infection. It is clear that both pathogen and host characteristics influence the acute versus chronic outcome of viral infection. The early events in the host immune response that favor immunosuppression and viral persistence, however, have remained poorly understood. Using the well-characterized mouse model of acute versus chronic lymphocytic choriomeningitis virus (LCMV) infection, two groups have recently identified the interleukin-10 (IL-10)/IL-10R pathway as a key regulator of acute versus chronic infection. Blockade of IL-10R converted a chronic LCMV infection into a rapidly controlled acute viral infection and prevented the functional exhaustion of memory T cells. These insights into the role of IL-10 in the establishment of chronic infection could lead to new therapeutic opportunities during human infections with pathogens such as HIV, hepatitis C virus (HCV) and hepatitis B virus (HBV).     

Intrinsic functional dysregulation of CD4 T cells occurs rapidly following persistent viral infection

Effective T-cell responses are critical to eradicate acute viral infections and prevent viral persistence. Emerging evidence indicates that robust, early CD4 T-cell responses are important in effectively sustaining CD8 T-cell activity. Herein, we illustrate that virus-specific CD4 T cells are functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines (i.e., interleukin-2 and tumor necrosis factor alpha), thereby compromising an efficient and effective antiviral immune response. Mechanistically, the inactivation occurs at the cellular level and is not an active process maintained by regulatory T cells or antigen-presenting cells. Importantly, a small subpopulation of cells is able to resist inactivation and persist into the chronic phase of infection. However, the virus-specific CD4 T-cell population ultimately undergoes a second round of inactivation, and the cells that had retained functional capacity fail to respond to rechallenge in an acute time frame. Based on these results we propose a biological mechanism whereby early CD4 T-cell inactivation leads to a subsequent inability to sustain cytotoxic T-lymphocyte function, which in turn facilitates viral persistence. Moreover, these studies are likely relevant to chronic/persistent infections of humans (e.g., human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) by providing evidence that a reservoir of virus-specific CD4 T cells can remain functional during chronic infection and represent a potential therapeutic target to stimulate the immune response and establish control of infection.     

Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E

Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.     

Differential roles for RIG-I-like receptors and nucleic acid-sensing TLR pathways in controlling a chronic viral infection

The necessity for pathogen recognition of viral infection by the innate immune system in initiating early innate and adaptive host defenses is well documented. However, little is known about the role these receptors play in the maintenance of adaptive immune responses and their contribution to resolution of persistent viral infections. In this study, we demonstrate a nonredundant functional requirement for both nucleic acid-sensing TLRs and RIG-I-like receptors in the control of a mouse model of chronic viral infection. Whereas the RIG-I-like receptor pathway was important for production of type I IFNs and optimal CD8(+) T cell responses, nucleic acid-sensing TLRs were largely dispensable. In contrast, optimal anti-viral Ab responses required intact signaling through nucleic acid-sensing TLRs, and the absence of this pathway correlated with less virus-specific Ab and deficient long-term virus control of a chronic infection. Surprisingly, absence of the TLR pathway had only modest effects on Ab production in an acute infection with a closely related virus strain, suggesting that persistent TLR stimulation may be necessary for optimal Ab responses in a chronic infection. These results indicate that innate virus recognition pathways may play critical roles in the outcome of chronic viral infections through distinct mechanisms.     

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.     

Living with the enemy: viral persistent infections from a friendly viewpoint

Persistent infection is a situation of metastability in which the pathogen and the host coexist. A common outcome for viral infections, persistence is a widespread phenomenon through all kingdoms. With a clear benefit for the virus and/or the host at the population level, persistent infections act as modulators of the ecosystem. The origin of persistence being long time elusive, here we explore the concept of 'endogenization' of viral sequences with concomitant activation of the host immune pathways, as a main way to establish and maintain viral persistent infections. Current concepts on viral persistence mechanisms and biological role are discussed.     

Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection.

Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic.     

Dendritic cells present lytic antigens and maintain function throughout persistent gamma-herpesvirus infection

The activation and maintenance of Ag-specific CD8(+) T cells is central to the long-term control of persistent infections. These killer T cells act to continuously scan and remove reservoirs of pathogen that have eluded the acute immune response. Acutely cleared viral infections depend almost exclusively on dendritic cells (DC) to present Ags to, and to activate, the CD8(+) T cell response. Paradoxically, persistent pathogens often infect professional APCs such as DC, in addition to infecting a broad range of nonprofessional APC, raising the possibility that many cell types could present viral Ags and activate T cells. We addressed whether in persistent viral infection with murine gammaherpesviruses, DC or non-DC, such as B cells and macrophages, were required to maintain the continued activation of Ag-specific CD8(+) T cells. We found that presentation of the surrogate Ag, OVA, expressed under a lytic promoter to CD8(+) T cells during persistent infection was largely restricted to DC, with little contribution from other lymphoid resident cells, such as B cells. This is despite the fact that B cells harbor a very large reservoir of latent virus. Our results support that, during persistent viral infection, continual presentation of lytic Ags by DC leads to T cell activation critical for maintaining CD8(+) T cells capable of limiting persistent viral infection.     

A persistent virus vector confers superior anti-tumor immunity, compared with a non-persistent vector

Active vaccination strategies using viral vectors often give disappointing protection from tumor development, and usually require multiple immunizations. These approaches normally use viruses that cause acute infections, as they provoke potent CD8 T cell responses. Persistent virus vectors have not been used in this setting due to the perception that exhaustion of the T cell response occurs and would lead to poor anti-tumor protection. However, such exhaustion generally only occurs in high-load virus infections, whereas T cell function is intact in lower-load persistent infections. In fact, CD8 T cell responses in these infections, which are adapted for long-term immune surveillance, have properties that may make them more desirable for long-term anti-tumor immunity. In this report, we show that a persistent gammaherpesvirus vector provides superior protection against melanoma, relative to a non-persistent mutant of the same virus. These data suggest that vaccine vectors derived from persistent viruses may perform better than those from acute viruses at mediating anti-tumor protection.     

Proteomic Analysis of Mitochondrial-Associated ER Membranes (MAM) during RNA Virus Infection Reveals Dynamic Changes in Protein and Organelle Trafficking

RIG-I pathway signaling of innate immunity against RNA virus infection is organized between the ER and mitochondria on a subdomain of the ER called the mitochondrial-associated ER membrane (MAM). The RIG-I adaptor protein MAVS transmits downstream signaling of antiviral immunity, with signaling complexes assembling on the MAM in association with mitochondria and peroxisomes. To identify components that regulate MAVS signalosome assembly on the MAM, we characterized the proteome of MAM, ER, and cytosol from cells infected with either chronic (hepatitis C) or acute (Sendai) RNA virus infections, as well as mock-infected cells. Comparative analysis of protein trafficking dynamics during both chronic and acute viral infection reveals differential protein profiles in the MAM during RIG-I pathway activation. We identified proteins and biochemical pathways recruited into and out of the MAM in both chronic and acute RNA viral infections, representing proteins that drive immunity and/or regulate viral replication. In addition, by using this comparative proteomics approach, we identified 3 new MAVS-interacting proteins, RAB1B, VTN, and LONP1, and defined LONP1 as a positive regulator of the RIG-I pathway. Our proteomic analysis also reveals a dynamic cross-talk between subcellular compartments during both acute and chronic RNA virus infection, and demonstrates the importance of the MAM as a central platform that coordinates innate immune signaling to initiate immunity against RNA virus infection.     

Antiviral activity in vitro of two preparations of the herbal medicinal product Sinupret? against viruses causing respiratory infections

Sinupret(?), a herbal medicinal product made from Gentian root, Primula flower, Elder flower, Sorrel herb, and Verbena herb is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about its potential antiviral activity. Therefore experiments have been performed to measure the antiviral activity of Sinupret(?) oral drops (hereinafter referred to as "oral drops") and Sinupret(?) dry extract (hereinafter referred to as "dry extract"), in vitro against a broad panel of both enveloped and non-enveloped human pathogenic RNA and DNA viruses known to cause infections of the upper respiratory tract: influenza A, Chile 1/83 (H1N1) virus (FluA), Porcine Influenza A/California/07/2009 (H1N1) virus (pFluA), parainfluenza type 3 virus (Para 3), respiratory syncytial virus, strain Long (RSV), human rhinovirus B subtype 14 (HRV 14), coxsackievirus subtype A9 (CA9), and adenovirus C subtype 5 (Adeno 5). Concentration-dependent antiviral activity (EC(50) between 13.8 and 124.8 μg/ml) of Sinupret(?) was observed against RNA as well as DNA viruses independent of a viral envelope. Remarkable antiviral activity was shown against Adeno 5, HRV 14 and RSV in which dry extract was significantly superior to oral drops. This could be ascertained with different assays as plaque-reduction assays in plaque forming units (PFU), the analyses of a cytopathogenic effect (CPE) and with enzyme immunoassays (ELISA) to determine the amount of newly synthesised virus. Our results demonstrate that Sinupret(?) shows a broad spectrum of antiviral activity in vitro against viruses commonly known to cause respiratory infections.     

Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.     

CD28 costimulatory blockade exacerbates disease severity and accelerates epitope spreading in a virus-induced autoimmune disease

Theiler's murine encephalomyelitis virus (TMEV) is a natural mouse pathogen which causes a lifelong persistent infection of the central nervous system (CNS) accompanied by T-cell-mediated myelin destruction leading to chronic, progressive hind limb paralysis. TMEV-induced demyelinating disease (TMEV-IDD) is considered to be a highly relevant animal model for the human autoimmune disease multiple sclerosis (MS), which is thought to be initiated as a secondary consequence of a virus infection. Although TMEV-IDD is initiated by virus-specific CD4(+) T cells targeting CNS-persistent virus, CD4(+) T-cell responses against self myelin protein epitopes activated via epitope spreading contribute to chronic disease pathogenesis. We thus examined the ability of antibodies directed against B7 costimulatory molecules to regulate this chronic virus-induced immunopathologic process. Contrary to previous studies showing that blockade of B7-CD28 costimulatory interactions inhibit the initiation of experimental autoimmune encephalomyelitis, treatment of SJL mice at the time of TMEV infection with murine CTLA-4 immunoglobulin or a combination of anti-B7-1 and anti-B7-2 antibodies significantly enhanced clinical disease severity. Costimulatory blockade inhibited early TMEV-specific T-cell and antibody responses critical in clearing peripheral virus infection. The inhibition of virus-specific immune responses led to significantly increased CNS viral titers resulting in increased damage to myelin-producing oligodendrocytes. Following clearance of the costimulatory antagonists, epitope spreading to myelin epitopes was accelerated as a result of the increased availability of myelin epitopes leading to a more severe chronic disease course. Our results raise concern about the potential use of B7-CD28 costimulatory blockade to treat human autoimmune diseases potentially associated with acute or persistent virus infections.     

Critical Role for CD4+ T Cells in Controlling Retrovirus Replication and Spread in Persistently Infected Mice

Reactivations of persistent viral infections pose a significant medical problem in immunocompromised cancer, transplant, and AIDS patients, yet little is known about how persistent viral infections are immunologically controlled. Here we describe a mouse model for investigating the role of the immune response in controlling a persistent retroviral infection. We demonstrate that, following recovery from acute Friend virus infection, a small number of B cells evade immunological destruction and harbor persistent virus. In vivo depletions of T-cell subsets in persistently infected mice revealed a critical role for CD4⁺ T cells in controlling virus replication, spread to the erythroid lineage, and induction of erythroleukemia. The CD4⁺ T-cell effect was independent of CD8⁺ T cells and in some cases was also independent of virus-neutralizing antibody responses. Thus, the CD4⁺ T cells may have had a direct antiviral effect. These results may have relevance for human immunodeficiency virus (HIV) infections where loss of CD4⁺ T cells is associated with an increase in HIV replication, reactivation of persistent viruses, and a high incidence of virus-associated cancers.     

Protection against establishment of retroviral persistence by vaccination with a live attenuated virus

Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable.     

Viral versus cellular BCL-2 proteins

All gamma herpesviruses and a few other viruses encode at least one homologue of the mammalian cell death inhibitor BCL-2. Gamma herpesviruses are associated with human and animal lymphoid and epithelial tumours. However, the role of these viral BCL-2 homologues in the virus replication cycle or in human disease is not known, though recent developments show progress in this area. The structure of viral BCL-2 family protein, KSBcl-2, is similar to that of cellular family members, but viral BCL-2 proteins differ functionally from the cellular proteins, apparently escaping the regulatory mechanisms to which their cellular counterparts are subjected. Thus, exploring the biochemical and biological functions of the viral BCL-2 family proteins will increase our understanding of their role in virus infections and will undoubtedly teach us something about their cellular kin.     

Persistent or Slow Viral Infections and Related Diseases

The discovery of persistent transmissible agents by veterinarians has led to striking advances in the infectious cause of neuropathies of human beings. There is evidence for persisting infection in congenital rubella and the herpes group of viruses including cytomegalovirus infections. Hepatitis types A and B are candidates for inclusion in the category of persisting viral infections.The rubeola or measles virus is established as a persistent virus which causes elevated antibodies in the serum and cerebrospinal fluid of many patients with severe demyelinating disease such as subacute sclerosing panencephalitis and multiple sclerosis. Elevated antibodies against vaccinia virus have been found in the cerebrospinal fluid of some patients with multiple sclerosis and neuromyelitis optica, a rare form of multiple sclerosis.     

Viral interference: A potential therapeutic method for chronic viral hepatitis?

Viral interference exists between different viral hepatitis. Acute Hepatitis C virus (HCV) super-infection on Hepatitis B virus (HBV) chronic carriers showed an inhibition of the HBV genome. And acute HBV super-infection on HCV chronic carriers indicated a similar interaction. In these cases, if the acute liver viral super-infection presents a self-limited course, the patients may be free from both viral infections or at least with undetectable underlying chronic viremia. The mechanism of viral interference is still undefined. Anyway this still leads to the hypothesis of using one hepatitis virus (live attenuated vaccine) to treat another hepatitis virus.     

Comparison of antibody repertoires produced by HIV-1 infection, other chronic and acute infections, and systemic autoimmune disease

Background

Antibodies (Abs) produced during HIV-1 infection rarely neutralize a broad range of viral isolates; only eight broadly-neutralizing (bNt) monoclonal (M)Abs have been isolated. Yet, to be effective, an HIV-1 vaccine may have to elicit the essential features of these MAbs. The V genes of all of these bNt MAbs are highly somatically mutated, and the V_H genes of five of them encode a long (≥20 aa) third complementarity-determining region (CDR-H3). This led us to question whether long CDR-H3s and high levels of somatic mutation (SM) are a preferred feature of anti-HIV bNt MAbs, or if other adaptive immune responses elicit them in general.

Methodology and Principal Findings

We assembled a V_H-gene sequence database from over 700 human MAbs of known antigen specificity isolated from chronic (viral) infections (ChI), acute (bacterial and viral) infections (AcI), and systemic autoimmune diseases (SAD), and compared their CDR-H3 length, number of SMs and germline V_H-gene usage. We found that anti-HIV Abs, regardless of their neutralization breadth, tended to have long CDR-H3s and high numbers of SMs. However, these features were also common among Abs associated with other chronic viral infections. In contrast, Abs from acute viral infections (but not bacterial infections) tended to have relatively short CDR-H3s and a low number of SMs, whereas SAD Abs were generally intermediate in CDR-H3 length and number of SMs. Analysis of V_H gene usage showed that ChI Abs also tended to favor distal germline V_H-genes (particularly V_H1-69), especially in Abs bearing long CDR-H3s.

Conclusions and Significance

The striking difference between the Abs produced during chronic vs. acute viral infection suggests that Abs bearing long CDR-H3s, high levels of SM and V_H1-69 gene usage may be preferentially selected during persistent infection.