Viral Rechallenge Research Articles

NK Cells as Effectors of Acquired Immune Responses: Effector CD4+ T Cell-Dependent Activation of NK Cells Following Vaccination

We characterized vaccine-induced cellular responses to rabies virus in naive adult volunteers. Contrary to current paradigms, we observed potent and prolonged in vitro NK cell cytokine production and degranulation responses after restimulation of PBMCs with inactivated rabies virus in vaccinated, but not in unvaccinated, individuals. This "recall" NK cell response was absolutely dependent on Ag-specific IL-2 from CD45RO(+) CD4(+) T cells as well as IL-12 and IL-18 from accessory cells. Importantly, NK cells represented over 70% of all IFN-gamma-secreting and degranulating cells in the first 12-18 h after virus rechallenge indicating they may be required for rapid control of infection after vaccination. Activation of NK cells may be a critical function of IL-2-secreting effector memory T cells. Although IL-2-dependent postvaccination NK cell activation has been reported previously, this is the first time the magnitude of this effect and its contribution to the overall vaccine-induced response has been appreciated and the mechanisms of NK activation postvaccination have been elucidated. Our data will allow standard protocols for evaluating vaccine-induced immunity to be adapted to assess NK cell effector responses.

Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation?

Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mRNA and protein level. Evidences for a regulatory immune pattern in homologous (TGF-β, IL-10) vs. pro-inflammatory pattern (IFN-γ, TNF-α) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case.

Protective immunity against viral nervous necrosis (VNN) in brown-marbled grouper ( Epinephelus fuscogutattus) following vaccination with inactivated betanodavirus

Viral nervous necrosis (VNN) caused by betanodaviruses has been recently implicated in serious mortalities of groupers in the grow-out culture system. A safe and effective vaccine against this disease is urgently needed. This study demonstrates that a single intramuscular vaccination with formalin-inactivated Philippine strain of piscine betanodavirus (genotype: redspotted grouper nervous necrosis virus; RGNNV) induces potent immune responses and substantial protective immunity against an intramuscular challenge with the homologous virus in brown-marbled grouper, Epinephelus fuscogutattus, a highly susceptible marine fish species to VNN. Seroneutralization assay conducted on sera of vaccinated fish revealed the occurrence of substantial neutralizing-antibody titers from Days 15 (mean titer 1:800) to 190 (1:400) with the highest titer observed at Day 60 post-vaccination (1:5120). When vaccinated fish were challenged with the homologous virus at Days 15, 30 and 75 post-vaccination, significantly higher survival rates were obtained in these fish compared with their corresponding controls (L-15 injected fish). Abrogation of virus multiplication in all vaccinated survivors was indicated by undetectable virus titers in the brains and kidneys paralleled by significantly high levels of neutralizing antibodies in the sera of these fish. Consecutively, replicates of vaccinated fish that survived betanodavirus challenge at Days 15 and 75 post-vaccination were maintained in flow-through aquaria and rechallenged with the homologous virus 3 and 5 months later, respectively. A significant drop in neutralizing-antibody titers of 3 and 8 folds, respectively, were observed in the sera of Days 15 and 75 post-vaccinated fish assayed before the virus rechallenge. Interestingly, reversion in the levels of neutralizing antibodies to significantly high levels (8–15 folds) were noted in these fish after the virus rechallenge. Taken together, our current data clearly demonstrate that a single administration of the inactivated Philippine strain of betanodavirus vaccine can effectively mount a specific anamnestic response and concomitant long-term protection against VNN in grouper at the grow-out culture system.

IL-21R on T Cells Is Critical for Sustained Functionality and Control of Chronic Viral Infection

Chronic viral infection is often associated with the dysfunction of virus-specific T cells. Our studies using Il21r-deficient (Il21r-/-) mice now suggest that interleukin-21 (IL-21) is critical for the long-term maintenance and functionality of CD8+ T cells and the control of chronic lymphocytic choriomeningitis virus infection in mice. Cell-autonomous IL-21 receptor (IL-21R)-dependent signaling by CD8+ T cells was required for sustained cell proliferation and cytokine production during chronic infection. Il21r-/- mice showed normal CD8+ T cell expansion, effector function, memory homeostasis, and recall responses during acute and after resolved infection with several other nonpersistent viruses. These data suggest that IL-21R signaling is required for the maintenance of polyfunctional T cells during chronic viral infections and have implications for understanding the immune response to other persisting antigens, such as tumors.

A Vital Role for Interleukin-21 in the Control of a Chronic Viral Infection

Understanding the factors that regulate the induction, quality, and longevity of antiviral T cell responses is essential for devising rational strategies to prevent or combat infections. In this study, we show that interleukin-21 (IL-21), likely produced by CD4+ T cells, directly influences the generation of polyfunctional CD8+ T cells and that the number of CD4+ T cells that produce IL-21 differs markedly between acute and chronic infections. IL-21 regulates the development of CD8+ T cell exhaustion and the ability to contain chronic lymphocytic choriomeningitis virus infection. Thus, IL-21 serves as a critical helper factor that shapes the functional quality of antiviral CD8+ T cells and is required for viral control.

IL-21 Is Required to Control Chronic Viral Infection

CD4+ and CD8+ T cell functions are rapidly aborted during chronic infection, preventing viral clearance. CD4+ T cell help is required throughout chronic infection so as to sustain CD8+ T cell responses; however, the necessary factor(s) provided by CD4+ T cells are currently unknown. Using a mouse model of chronic viral infection, we demonstrated that interleukin-21 (IL-21) is an essential component of CD4+ T cell help. In the absence of IL-21 signaling, despite elevated CD4+ T cell responses, CD8+ T cell responses are severely impaired. CD8+ T cells directly require IL-21 to avoid deletion, maintain immunity, and resolve persistent infection. Thus, IL-21 specifically sustains CD8+ T cell effector activity and provides a mechanism of CD4+ T cell help during chronic viral infection.

Poor anti-viral immunity in aged mice is associated with defects in CD8 T cell recall responses (44.5)

Abstract Aging leads to an increased susceptibility to infectious diseases. To investigate the age-related changes in T cell memory we compared anti-viral CD8 T cell immunity in young versus aged mice following systemic (lymphocytic choriomeningitis virus; LCMV) or local (influenza virus) viral infections. The number of memory CD8 T cells formed was similar or only modestly different between aged and young animals, though in some cases changes in immunodominance and cytokine production were apparent. Both influenza virus-specific and LCMV-specific memory CD8 T cells from aged mice, however, had major substantially reduced proliferative capacity upon secondary infection. As a result, aged mice displayed increased morbidity and mortality upon viral rechallenge. The studies described herein demonstrate that the defects intrinsic to aged CD8 T cells are at least partially responsible for diminished immunity of aged mice. Thus, the strategies that accelerate the induction of T cell responses or that overcome proliferative deficiencies may prove beneficial. These studies were funded by grants from AHA, NIH (HHSN266200500030C) and the Ellison Medical Foundation.

Heterogeneous Memory T Cells in Antiviral Immunity and Immunopathology

Memory T cells are generated following an initial viral infection, and have the potential for mediating robust protective immunity to viral re-challenge due to their rapid and enhanced functional responses. In recent years, it has become clear that the memory T cell response to most viruses is remarkably diverse in phenotype, function, and tissue distribution, and can undergo dynamic changes during its long-term maintenance in vivo. However, the role of this variegation and compartmentalizationof memory T cells in protective immunity to viruses remains unclear. In this review,we discuss the diverse features of memory T cells that can delineate different subsets, the characteristics of memory T cells thus far identified to promote protective immune responses, and how the heterogeneous nature of memory T cells may also promote immunopathology during antiviral responses. We propose that given the profound heterogeneity of memory T cells, regulation of memory T cells during secondary responses could focus the response to participation of specific subsets,and/or inhibit memory T-cell subsets and functions that can lead to immunopathology.

Immunobiology of Norwalk virus.

Clinical immunity to Norwalk virus in inoculated human volunteers appears to be unusual for gastroenteritis viruses, as certain individuals are repeatedly ill on long-term virus rechallenge and others remain persistently well. In these volunteers there is a paradoxical inverse correlation between the prechallenge serum (and jejunal fluid) Norwalk antibody level (measured by radioimmunoassay) and resistance to illness, suggesting that non-immunological factors, perhaps genetic, may be important in determining resistance. Most reported naturally occurring Norwalk disease outbreaks in developed nations also show that humoral antibody fails to correlate with immunity to infection. The unusual pattern of clinical immunity to Norwalk virus indicates a need for caution in the development of vaccines against this agent as well as a need for additional information on its immunobiological characteristics. The virus is known to contain a single protein, like the caliciviruses. Recently we have found evidence for at least a one-way serological cross-relatedness between Norwalk virus and human calicivirus. Twelve of 20 paired sera from ill patients in outbreaks due to calicivirus strain UK4 seroconverted to Norwalk virus by radioimmunoassay and two of eight paired sera from UK2 outbreaks showed seroconversion. Future studies of outbreaks caused by various calicivirus strains should be designed to correlate acute-phase serum antibody titres to Norwalk virus with clinical susceptibility and immunity to infection.

Influenza Virus-Induced Type I Interferon Leads to Polyclonal B-Cell Activation but Does Not Break Down B-Cell Tolerance

The link between infection and autoimmunity is not yet well understood. This study was designed to evaluate if an acute viral infection known to induce type I interferon production, like influenza, can by itself be responsible for the breakdown of immune tolerance and for autoimmunity. We first tested the effects of influenza virus on B cells in vitro. We then infected different transgenic mice expressing human rheumatoid factors (RF) in the absence or in the constitutive presence of the autoantigen (human immunoglobulin G [IgG]) and young lupus-prone mice [(NZB x NZW)F(1)] with influenza virus and looked for B-cell activation. In vitro, the virus induces B-cell activation through type I interferon production by non-B cells but does not directly stimulate purified B cells. In vivo, both RF and non-RF B cells were activated in an autoantigen-independent manner. This activation was abortive since IgM and IgM-RF production levels were not increased in infected mice compared to uninfected controls, whether or not anti-influenza virus human IgG was detected and even after viral rechallenge. As in RF transgenic mice, acute viral infection of (NZB x NZW)F(1) mice induced only an abortive activation of B cells and no increase in autoantibody production compared to uninfected animals. Taken together, these experiments show that virus-induced acute type I interferon production is not able by itself to break down B-cell tolerance in both normal and autoimmune genetic backgrounds.

Routes of inoculation and the immune response to a resolving genital flavivirus infection in a novel murine model

The prolonged, abnormal immune response patterns produced by many sexually transmitted viruses have been intensively studied. Because normal antiviral immune responses in the vagina are less well-defined, we developed a resolving murine model using vaginal inoculation with the flavivirus, West Nile virus. Infection resulted in 12% mortality, with sterile protective immunity to vaginal or systemic re-challenge. B-cell numbers increased in the vaginal mucosa from day 1-7 after primary infection, while similar increases in B220(+), CD4(+) and CD8(+) lymphocytes in the draining lymph node were delayed by 48 h. By day 4 postinfection, a MHC-II(+) dendritic cell population became depleted from the stroma and formed aggregates below the basement membrane at points of demonstrable epithelial infection. In contrast, primary systemic or intradermal inoculation resulted in 80-90% mortality, but also conferred protective sterile immunity to vaginal West Nile virus re-challenge. Intravaginal and intradermal immunization elicited comparable, accelerated accumulation of larger B-cell numbers in the mucosa and draining lymph node upon intravaginal re-challenge than systemic immunization. However, accumulation of CD4(+) T cells in both sites in the intradermally immunized group was significantly greater than in intravaginally or systemically immunized mice. Accelerated accumulation of dendritic cells occurred at periodic sub-basement membrane sites in the absence of detectable virus 1 day after vaginal re-challenge, irrespective of the route of immunization. These data illustrate the diversity of possible effective immune responses to West Nile virus in the vaginal mucosa. They show primary vaginal inoculation produces effective immunity to flavivirus infection with lower mortality than other routes and suggest a local role for vaginal mucosal dendritic cells in both primary and secondary responses.

Platelet-Mediated Modulation of Adaptive Immunity: A Communication Link between Innate and Adaptive Immune Compartments

Platelets are highly reactive components of the circulatory system with well-documented hemostatic function. Recent studies extend platelet function to modulation of local inflammatory events through the release of chemokines, cytokines, and a number of immunomodulatory ligands, including CD154. We hypothesized that platelet-derived CD154 modulates adaptive immunity. The data reported herein demonstrate that platelets, via CD154, induce dendritic cell maturation, B cell isotype switching, and augment CD8 + T cell responses both in vitro and in vivo. Platelet transfusion studies demonstrate that platelet-derived CD154 alone is sufficient to induce isotype switching and augment T lymphocyte function during viral infection, leading to enhanced protection against viral rechallenge. Additionally, depletion of platelets in normal mice results in decreased antigen-specific antibody production.

Kinetics of CD4+ and CD8+ memory T-cell responses during hepatitis C virus rechallenge of previously recovered chimpanzees.

The immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood. Antibodies to HCV structural proteins do not appear to play a key role in clearance of the virus and do not persist after recovery. Here, we studied the kinetics of the cellular immune responses of three HCV-recovered chimpanzees during rechallenge with increasing doses of homologous HCV. Although HCV envelope antibodies remained undetectable throughout the rechallenge, all animals mounted rapid HCV-specific T-cell responses. The pattern of the cellular immune response in blood and liver correlated with the virological outcome. The animal that most rapidly cleared circulating HCV as determined by nested reverse transcription-PCR (RT-PCR) displayed the most vigorous and sustained response of gamma interferon (IFN-gamma)-producing and proliferating CD4(+) T cells in the blood. Vigorous CD4(+) T-cell proliferation during viremia was followed by an increased frequency and a phenotypic and functional change of the tetramer(+) CD8(+) T-cell population. The second animal cleared HCV initially with strong peripheral and intrahepatic CD4(+) T-cell responses but experienced low-level HCV recrudescence 12 weeks later, when HCV-specific T cells became undetectable. The third animal maintained minute amounts of circulating HCV, detectable only by nested RT-PCR, in the face of a weak IFN-gamma(+) T-cell response. Collectively, the results suggest protective rather than sterilizing immunity after recovery from hepatitis C. The rate of HCV clearance following reexposure depends on the cellular immune response, the quality and quantity of which may vary among chimpanzees that recovered from HCV infection.

Primary and secondary immunocompetence in mixed allogeneic chimeras.

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.

Antibody-mediated Immune Enhancement in Coxsackievirus B3 Myocarditis

C. K ISHIMOTO, M. K UROKAWA and H. O CHIAI. Antibody-mediated Immune Enhancement in Coxsackievirus B3 Myocarditis. Journal of Molecular and Cellular Cardiology (2002) 34, 1227–1238. The aim of the present study was to explore the contribution of antibody-mediated immune enhancement in coxsackievirus B3(CB3) infection. Murine macrophage-like P388D1 cells were exposed to various concentrations of anti-CB3 immunoglobulin G (anti-CB3 IgG) or the Fab fragment of anti-CB3 IgG, and were infected with CB3 in Experiment I. High concentrations of anti-CB3 IgG showed a virus-neutralizing activity; however, a subneutralizing antibody concentration of IgG significantly enhanced virus replication. This infectious enhancement was blocked not only by the pretreatment of heat-aggregated γ-globulin but by a specific Fc receptor (Fc γ III/II receptor) antibody treatment. In contrast, the Fab fragment of anti-CB3 IgG did not enhance CB3 infection, but showed a rational neutralizing activity to CB3. These findings suggest the presence of Fc receptor mediated enhancement of CB3 infection in vitro. In Experiment II, C3H/He mice were inoculated with various amounts of an amyocarditic variant of CB3 followed 15 days later by myocarditic CB3. By this rechallenge, myocarditis was not induced in the mice with high neutralizing antibody titers. There was an inverse relationship between preexisting neutralizing antibody titers and the severity of myocarditis. The severity of myocarditis and myocardial CB3 titers, however, were markedly enhanced in the mice with a subneutralizing level of immunity compared to those with no immunity. The distribution of myocardial Fc receptor-bearing cells and serum macrophage inflammatory protein-2 levels paralleled the severity of myocarditis. By another virus rechallenge in Experiment III, enhanced infection of CB3 was not observed in vivo. These findings suggest that antibody-mediated immune enhancement might be involved in the pathogenesis of CB3 myocarditis.

Immunotherapy of Murine Leukemia. VIII. Efficacy of Passive Serum Therapy of Friend Leukemia Virus-Induced Disease in Immunocompromised Mice<xref ref-type="fn" rid="fn2">2</xref>

Previous studies have demonstrated that the passive therapy of Friend murine leukemia virus (F-MuLV)-induced disease with chimpanzee anti-F-MuLV serum is accompanied by the development of host antiviral humoral and cellular immunity, the latter measurable in adoptive transfer protocols and by the ability of serum-protected mice to resist virus rechallenge. The present study was designed to further examine the contribution of various compartments of the host immune system to serum therapy itself, as well as to the acquired antiviral immunity that develops in serum-protected mice, through the use of naturally immunocompromised animals [e.g., nude athymic mice and natural killer (NK)-deficient beige mutant mice] or mice treated with immunoabrogating agents such as sublethal irradiation, cyclophosphamide [Cytoxan (Cy)], cortisone, and 89Sr. The studies in nude mice indicate that while mature T-cells are not needed for effective serum therapy, they do appear to be necessary for the long-term resistance of serum-protected mice to virus rechallenge and for the generation of the cell population(s) responsible for adoptive transfer of antiviral immunity. Furthermore, this acquired resistance is not due to virus neutralization by serum antibodies since antibody-negative, Cy-treated, serum-protected mice still reject the secondary virus infection. Lastly, while the immunocompromise systems examined did effect various host antiviral immune responses, none of them, including the NK-deficient beige mutation, significantly diminished the efficacy of the passive serum therapy of F-MuLV-induced disease.

Cellular and Humoral Immune Reactivity to Tumor-Associated Antigens in Chickens Infected with Rous Sarcoma Virus

Abstract With a leukocyte blastogenesis assay and 3 M KCl extracts of chicken cells infected or infected and transformed by various avian tumor viruses (ATV), the time course of development of cellular reactivity in RSV tumor-bearing chickens to antigens expressed on these cell types was examined. Leukocyte reactivity to mitogens in RSV-injected birds was also investigated. Similarly, humoral responses to ATV-antigens and virus-induced antigens were assessed over time, by measuring the development of antiviral neutralizing antibody, as well as antibody to cell surface antigens on various cell types, by means of an isotopic antiglobulin test (IAT). There were no differences in phytohemmaglutinin responsiveness between the leukocytes of RSV-injected birds and control birds, but responses to pokeweed mitogen were sometimes elevated in RSV-injected animals. Cellular immunity to 3 M KCl extracts, principally to those extracts containing structural components of the virus envelope, developed between 3 and 5 weeks post-injection, during tumor growth and regression, and then declined, although the immune response could be restimulated by virus rechallenge. For the most part, responses to nonvirion tumor-specific surface antigen (TSSA) was weak except after RSV rechallenge. Humoral reactivity as measured by IAT also seemed to correlate well with the presence of virus envelope antigens (VEA) on infected indicator cells and appeared before antiviral neutralizing antibody. The data indicated that, at least during the primary response, major immune reactivity in RSV-injected birds appeared to be directed against VEA and that TSSA appeared to be most immunogenic when VEA was also present.

Effect of repeated inoculation of interferon preparations on infection of mice with encephalomyocarditis virus.

Administration of mouse brain interferon prior to or after inoculation of 100-250 LD50 of encephalomyocarditis (EMC) virus, conferred a significant degree of protection on weanling IC mice. Continuation of interferon treatment for several days after virial inoculation considerably increased the number of mice surviving infection. Most interferon protected mice did not develop HI antibody to EMC virus nor did they demonstrate resistance to viral rechallenge. By acting early in the course of viral infection interferon may have repressed viral multiplication below a level necessary to induce a specific immune response. Mice did not develop a “tolerance” to repeated inoculation of exogenous interferon. Considered together the experimental results suggested that continued interferon administration may prove of value in the treatment of some viral diseases.