T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.     

Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice.

To investigate the cell tropism and pathogenicity of varicella-zoster virus (VZV) strains, we analyzed VZV replication by using SCID-hu mice that carry human fetal thymus/liver implants under the kidney capsule or as subcutaneous fetal skin implants. MRC-5 cells infected with wild-type VZV or the Oka strain, used in the live attenuated varicella vaccine, were injected into the implants. The implants were surgically removed 2, 7, 14, and 21 days postinfection. The VZV titer from infected thymus/liver implants peaked on day 7 for the wild-type strain and on day 14 for the Oka strain. Histological analysis showed necrotic areas characterized by thymocyte depletion and fibrosis. VZV protein synthesis was detectable by immunohistochemical staining in the necrotic areas and in distant regions that did not show cytopathic changes, and VZV DNA was detected by in situ hybridization in the same distribution. Fluorescence-activated cell sorting analysis of thymocytes harvested at day 7 postinfection showed that VZV proteins were expressed in CD4+, CD8+, and CD4+ CD8+ T cells; VZV was cultured from each T-cell subpopulation. The Oka strain had tropism for human cell types similar to that of wild-type VZV. T lymphocytes released infectious VZV, which is a novel and important observation about the replication of this otherwise highly cell associated virus. VZV-infected skin implants exhibited microscopic epidermal lesions that were indistinguishable histologically from the characteristic lesions of varicella. These experiments demonstrate a unique tropism of VZV for human T lymphocytes, explaining its capacity to cause viremia in natural disease, and demonstrate the value of the SCID-hu model for studies of VZV pathogenesis.     

Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.     

Varicella-zoster virus retains major histocompatibility complex class I proteins in the Golgi compartment of infected cells

We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3+ T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early gene ORF66 did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.     

Acyclovir treatment prevents varicella-zoster virus replication in PBMC during viremia.

The most effective antiviral therapy of varicella and zoster has become acyclovir. Using polymerase chain reaction specific for VZV ORF 14, ORF 29, ORF 63 as well as nucleic acid sequence-based amplification (ORF 63, ORF 68) we tested PBMC of patients with VZV-associated diseases for the presence of viral DNA and RNA, respectively. In PBMC of patients treated with acyclovir neither DNA nor RNA was detectable already one day after the onset of therapy. In three blood sample pairs from zoster patients we were able to detect viral nucleic acid before but not after acyclovir treatment. These results confirm clinical and epidemiological data. It can be concluded that treatment with acyclovir prevents VZV replication in peripheral blood mononuclear cells.     

IL-12, IFN-gamma, and TNF-alpha released from mononuclear cells inhibit the spread of varicella-zoster virus at an early stage of varicella

The activity of mononuclear cells to inhibit plaque formation of varicella-zoster virus (VZV) was investigated by an in vitro infectious center assay. Peripheral blood mononuclear cells (PBMC) inhibited VZV plaque formation by co-cultivation with VZV-infected fibroblasts. As compared to mononuclear cells from normal individuals, mononuclear cells from umbilical cord blood and from patients receiving corticosteroids showed a significant decrease in the ability to inhibit viral replication. This ability was significantly increased for mononuclear cells collected during the acute phase of varicella. PBMC obtained from patients in the acute phase of varicella produced significantly higher amounts of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-12 in the supernatant compared with those of healthy individuals. These data suggest that the cytokines have an important role in the inhibition of the spread of VZV at an early stage of varicella. Th1 type adaptive immunity might play a major role in VZV infection.     

Evidence for CD8+ antiviral activity in cats infected with feline immunodeficiency virus.

Human immunodeficiency virus (HIV) causes a long, asymptomatic infection characterized by normal to elevated numbers of circulating CD8+ cells and a progressive decline in CD4+ cells. It has been speculated that HIV-specific antiviral activity driven by CD8+ T cells may control viral replication during this period and maintain the clinically asymptomatic stage of disease. The disease induced in cats by feline immunodeficiency virus (FIV) is similar to HIV in that it is characterized by a long asymptomatic stage with a progressive decline in CD4+ cells, culminating in AIDS. In the present study, we demonstrate that FIV is more readily isolated from CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMC) of FIV-infected cats than from unfractionated PBMC cultures. In addition, CD8+ T cells isolated from FIV-positive cats demonstrating anti-FIV activity in PBMC cultures inhibit FIV infection of FCD4E cells in vitro. Anti-FIV activity is not found in FIV- negative cats and is not characteristic of cats acutely infected with FIV but is present in the majority of chronically infected, clinically asymptomatic and symptomatic cats. Decreases in plasma and cell-associated viremia during the acute-stage FIV infection appears to precede the appearance of CD8+ anti-FIV cells in the circulation. In summary, this study demonstrates a population(s) of CD8+ T cells in chronically FIV-infected cats capable of suppressing FIV replication in cultured PBMC. The significance of anti-FIV CD8+ cells in the immunopathogenesis of the infection and disease progression has yet to be determined.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells has been associated with HIV-1 viremia and declining CD4+ T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4+ T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8+ cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4+ T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-gamma)-producing CD4+ T cells. Among the 20 viremic, treatment-naive subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4+ T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-gamma-producing CD4+ T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4+ T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.     

Immune activation, CD4+ T cell counts, and viremia exhibit oscillatory patterns over time in patients with highly resistant HIV infection

The rates of immunologic and clinical progression are lower in patients with drug-resistant HIV compared to wild-type HIV. This difference is not fully explained by viral load. It has been argued that reductions in T cell activation and/or viral fitness might result in preserved target cells and an altered relationship between the level of viremia and the rate of CD4+ T cell loss. We tested this hypothesis over time in a cohort of patients with highly resistant HIV. Fifty-four antiretroviral-treated patients with multi-drug resistant HIV and detectable plasma HIV RNA were followed longitudinally. CD4+ T cell counts and HIV RNA levels were measured every 4 weeks and T cell activation (CD38/HLA-DR) was measured every 16 weeks. We found that the levels of CD4+ T cell activation over time were a strong independent predictor of CD4+ T cell counts while CD8+ T cell activation was more strongly associated with viremia. Using spectral analysis, we found strong evidence for oscillatory (or cyclic) behavior in CD4+ T cell counts, HIV RNA levels, and T cell activation. Each of the cell populations exhibited an oscillatory behavior with similar frequencies. Collectively, these data suggest that there may be a mechanistic link between T cell activation, CD4+ T cell counts, and viremia and lends support for the hypothesis of altered predator-prey dynamics as a possible explanation of the stability of CD4+ T cell counts in the presence of sustained multi-drug resistant viremia.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes from horses with equine infectious anemia virus recognize Env and Gag/PR proteins.

Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. To begin dissection of the immune mechanisms involved in EIAV control, peripheral blood mononuclear cells (PBMC) from infected horses were evaluated for CTL to EIAV-infected cells. By using noninfected and EIAV-infected autologous equine kidney (EK) cells in 51Cr-release assays, EIAV-specific cytotoxic activity was detected in unstimulated PBMC from three infected horses. The EIAV-specific cytotoxic activity was major histocompatibility complex (MHC) restricted, as determined by assaying EIAV-infected heterologous EK targets, and was mediated by CD8+ T lymphocytes, as determined by depleting these cells by a panning procedure with an anti-CD8 monoclonal antibody. MHC-restricted CD8+ CTL in unstimulated PBMC from infected horses caused significant specific lysis of autologous EK cells infected with recombinant vaccinia viruses expressing EIAV genes, either env or gag plus 5' pol. The EIAV-specific MHC-restricted CD8+ CTL were detected in two EIAV-infected horses within a few days after plasma viremia occurred and were present after viremia was terminated. The detection of these immune effector cells in EIAV-infected horses permits further studies to determine their in vivo role.     

Primary simian immunodeficiency virus SIVmnd-2 infection in mandrills (Mandrillus sphinx)

Mandrills are the only nonhuman primate (NHP) naturally infected by two types of simian immunodeficiency virus (SIV): SIVmnd-1 and SIVmnd-2. We have already reported that the high SIVmnd-1 replication during primary infection contrasts with only transient changes in CD4+ and CD8+ cell counts. Since early virus-host interactions predict viral control and disease progression in human immunodeficiency virus-infected patients, we investigated the dynamics of SIVmnd-2 primary infection in mandrills to examine the impact on immune effectors in blood and lymph nodes (LNs). To avoid in vitro strain selection, all mandrills in this study received plasma from SIVmnd-2-infected mandrills. SIVmnd-2 plasma viremia peaked at 10(7) to 10(8) RNA copies/ml between days 7 and 10. This peak was followed in all four monkeys by a decline in virus replication, with a set point level of 10(5) to 10(6) RNA copies/ml at day 42 postinfection (p.i.). Viral DNA load in PBMC and LNs also peaked between days 7 and 10 (10(5) to 10(6) DNA copies/10(6) cells) and stabilized at 10(3) to 10(4) DNA copies/10(6) cells during the chronic phase. Anti-SIVmnd-2 antibodies were detected starting from days 28 to 32. A transitory decline of CD3+ CD4+ cells in the LNs occurred in animals with high peak VLs. CD4+ and CD8+ T-cell activation in blood and LNs was noted between days 5 and 17 p.i., surrounding the peak of viral replication. This was most significant in the LNs. Activation markers then returned to preinfection values despite continuous and active viral replication during the chronic infection. The dynamics of SIVmnd-2 infection in mandrills showed a pattern similar to that of SIVmnd-1 infection. This might be a general feature of nonpathogenic SIV natural African NHP models.     

Insights into the function of tegument proteins from the varicella zoster virus

Chickenpox(varicella) is caused by primary infection with varicella zoster virus(VZV), which can establish long-term latency in the host ganglion. Once reactivated, the virus can cause shingles(zoster) in the host. VZV has a typical herpesvirus virion structure consisting of an inner DNA core, a capsid, a tegument, and an outer envelope. The tegument is an amorphous layer enclosed between the nucleocapsid and the envelope, which contains a variety of proteins. However, the types and functions of VZV tegument proteins have not yet been completely determined. In this review, we describe the current knowledge on the multiple roles played by VZV tegument proteins during viral infection. Moreover, we discuss the VZV tegument protein-protein interactions and their impact on viral tissue tropism in SCID-hu mice. This will help us develop a better understanding of how the tegument proteins aid viral DNA replication, evasion of host immune response, and pathogenesis.     

Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.     

IL-15 treatment during acute simian immunodeficiency virus (SIV) infection increases viral set point and accelerates disease progression despite the induction of stronger SIV-specific CD8+ T cell responses

In this study, we examined the effect of in vivo treatment of acutely SIV-infected Mamu-A*01+ rhesus macaques with IL-15. IL-15 treatment during acute infection increased viral set point by 3 logs and accelerated the development of simian AIDS in two of six animals with one developing early minimal lesion SIV meningoencephalitis. Although IL-15 induced a 2- to 3-fold increase in SIV-specific CD8+ T cell and NK cell numbers at peak viremia and reduced lymph node (LN) SIV-infected cells, this had no impact on peak viremia and did not lower viral set point. At viral set point, however, activated SIV-specific CD8+ T cells and NK cells were reduced in the blood of IL-15-treated animals and LN SIV-infected cells were increased. Week 30 LN from IL-15-treated animals had significantly increased Gag-specific CD8+ T cell numbers, whereas total cell, lymphocyte, and CD4+ T cell numbers were reduced. IL-15 treatment significantly reduced anti-SIV Ab concentrations at week 3 and viral set point. IL-15 increased Ki-67+CD4+ T cells at week 1 of treatment and reduced blood CCR5+ and CD45RA-CD62L- CD4+ T cells. The frequency of day 7 Ki-67+CD4+ T cells strongly correlated with viral set point. These findings suggest that CD4+ T cell activation during acute infection determines subsequent viral set point and IL-15 treatment by increasing such activation elevates viral set point. Finally, IL-15-treated acutely SIV-infected primates may serve as a useful model to investigate the poorly understood mechanisms that control viral set point and disease progression in HIV infection.     

Changes in paracrine interleukin-2 requirement, CCR7 expression, frequency, and cytokine secretion of human immunodeficiency virus-specific CD4+ T cells are a consequence of antigen load

Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7(-) CD45RA(-) phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses.