Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor zeta chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.     

Viral superantigen-induced hyporesponsiveness of T cells and polyclonal B cell activation in HIV-1 infection

The mechanisms of CD4 depletion and hyporesponsiveness during human immunodeficiency virus (HIV) infection are still unknown. Given the ability of superantigens to stimulate a higher number of lymphocytes than conventional antigens, they may play a major role in this process. Recently, a novel superantigen, the rabies virus nucleocapsid (NC), was described in humans. In the present work, we tested the responses of peripheral blood lymphocytes from asymptomatic HIV-infected patients to this superantigen. In contrast to its effect in normal controls, NC failed to expand T cells from HIV-infected individuals expressing the V beta 8 family, and induced a strong decrease in the response to CD3 activation. This absence of response was not the consequence of programmed cell death, and was explained by an anergic state induced by the superantigen. NC superantigen was also able to induce polyclonal activation of B cells, as measured by the secretion of anti-HIV antibodies and autoantibodies. Moreover, V beta 8 depletion experiments showed that induction of autoantibody secretion was V beta 8 dependent, whereas secretion of HIV-1 antibody was not. Interleukin secretion studies showed that NC was able to induce high levels of interleukin-4 and interleukin-10. Taken together, our results suggest a role for exogenous viral superantigens such as NC in the induction of T cell hyporesponsiveness and polyclonal B cell activation during HIV infection. The induction of a Th2 response and the role of these superantigens in the immunopathogenesis of acquired immunodeficiency syndrome are discussed.     

Characterization of the viral population during primary HIV-1 infection

OBJECTIVE: To study viral heterogeneity at a very early phase of primary HIV-1 infection. DESIGN: Samples were drawn very early during primary HIV-1 infection. A virus population-based approach was used to study the viral heterogeneity in the C2-V3 and p17 regions. METHODS: Plasma samples (n = 33) were obtained before or shortly after onset of acute symptoms in 15 patients. In all subjects, the first sample was drawn within 10 days after onset of symptoms. Peripheral blood mononuclear cells (PBMC) were available in two patients. The number of polymorphic sites in the C2-V3 (15 patients) and p17 regions (eight patients) were determined by direct sequencing. RESULTS: The sequence heterogeneity was restricted in most patients, although only two out of 15 patients had a completely homogeneous C2-V3 sequence. However, pronounced individual differences were seen. Rapid sequence changes occurred during the first month in two patients. In one patient, the major DNA species at day 12 later became the major species in plasma. CONCLUSIONS: The viral population is seldom completely homogeneous during primary HIV-1 infection, although the heterogeneity is restricted in most, but not all, patients. These individual differences do not seem to be due to sex or viral subtype. Rapid changes of the virus population may occur during primary HIV-1 infection. The DNA species detected in PBMC do not only represent earlier viral quasispecies but are also a potential source of future viral RNA species.     

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21-65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21-65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41-65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21-65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21-65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31-8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.     

Isolation of circular viral and complementary strand DNA from bacteriophage f1 duplex replicative-form DNA

A general method has been developed for the large scale isolation of intact, circular, single-stranded DNA molecules of each strand from supercoiled duplex DNA. The method involves the conversion of the supercoiled duplex DNA to singly nicked, relaxed duplex DNA; denaturation of the duplex DNA; separation of circular DNA molecules from linear DNA molecules; and separation of circular plus and minus strands. All separations involve zone sedimentation. No isopycnic gradient centrifugation is required. The last step in the purification, the separation of plus and minus strands, can be easily adapted for small scale analytical measurements of the amounts of plus and minus strand DNA.     

Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load

Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.     

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression

BACKGROUND: It is well known that porokeratosis, a genetically heterogeneous disorder characterized by the histopathological feature of the cornoid lamella, shows an increased proneness to develop carcinoma. On the other hand, a significant mechanism in the origin of many forms of cancer is loss of heterozygosity or allelic loss. OBJECTIVE: Because it has recently been proposed that linear porokeratosis may result from allelic loss, one might expect that linear porokeratosis is especially prone to malignant degeneration. In order to test this hypothesis, a review of case reports was performed. METHOD: Cases of cancer-associated porokeratosis were collected from the European language literature and assigned to one of 5 different types [plaque type of Mibelli (PM); disseminated actinic superficial porokeratosis (DSAP); porokeratosis palmaris, plantaris et disseminata (PPPD); porokeratosis punctata palmaris et plantaris (PPPP); linear porokeratosis (LP)]. RESULTS: Malignant or premalignant lesions were reported in 9 cases of PM, 15 cases of DSAP, 3 cases of PPPD, 1 case of PPPP and 21 cases of LP. CONCLUSION: This analysis supports the view that among the various forms of porokeratosis, the linear type is particularly susceptible to malignant degeneration. Arguments are presented in favor of the assumption that the genetic mechanism of allelic loss giving rise to LP may represent an initial step in the development of cancer.     

Circular forms of unintegrated human immunodeficiency virus type 1 DNA and high levels of viral protein expression: association with dementia and multinucleated giant cells in the brains of patients with AIDS

Thirty-one histologically abnormal brains from patients with AIDS were studied in order to establish the relationship between multinucleated giant cells, viral protein expression, the various forms of human immunodeficiency virus type 1 (HIV-1) DNA, and clinical evidence of dementia. Unintegrated HIV-1 DNA of 2 to 8 kb was found in 22 of the 31 brains. Multinucleated giant cells without any other pathology were found in 14 cases; unintegrated 1-long terminal repeat (1-LTR) circular forms of HIV-1 DNA and strongly positive immunohistochemistry for gp41 and p24 were found in most of these brains. Most of these patients had a clinical diagnosis of HIV-1-associated dementia and cerebral atrophy. In all the other brains studied, 1-LTR circles were absent and immunohistochemistry for gp41 and p24 was usually negative. Very few of these patients had a clinical diagnosis of dementia. Sequence comparison of the LTR region from integrated HIV-1 DNA with that from unintegrated 1-LTR circular forms of HIV-1 DNA in 12 cases showed no significant differences. A further comparison of these brain-derived LTR sequences with LTR sequences derived directly from lymphoid tissue also showed strong sequence conservation. The V3 loop of the virus from the brain was sequenced in 6 cases and had a non-syncytium inducing-macrophage-tropic genotype. Our results show that (i) although unintegrated HIV-1 DNA was present in most brains from patients with AIDS, molecular evidence of high levels of viral replication was associated with the presence of multinucleated giant cells and dementia, and that (ii) the HIV-1 LTR is not a determinant of neurotropism. These observations suggest that replication of HIV-1 and not just the presence of HIV-1 DNA within giant cells makes the important contribution to central nervous system damage.     

Unintegrated circular HIV-1 DNA in the peripheral mononuclear cells of HIV-1-infected subjects: association with high levels of plasma HIV-1 RNA, rapid decline in CD4 count, and clinical progression to AIDS

We observed 36 HIV-infected patients to evaluate whether the presence of tandem 2-long terminal repeat circular unintegrated HIV-1 DNA (2-LTR) in peripheral blood mononuclear cells (PBMC) at baseline was associated with acceleration of HIV disease. Detection of 2-LTR at baseline correlated with high plasma HIV-1 RNA levels (p < .01), recovery of culturable HIV-1 from plasma (p = .02), and progression to AIDS during follow-up (p = .01). More patients with 2-LTR (68%) than without 2-LTR (31%) had a decline in CD4 levels of >50 cells/mm3 over the first 18 months of follow-up (p = .04), and the average annual CD4 decline was 35% in patients with 2-LTR compared with 16% in those without 2-LTR (p = 0.06). Detection of 2-LTR in PBMC at baseline was an independent predictor of high plasma HIV-1 RNA levels and subsequent CD4 cell decline in this cohort of patients with predominantly nonsyncytium-inducing (NSI) isolates at baseline. The presence of 2-LTR in PBMC appears to be reflective of ongoing HIV-1 replication, as measured by plasma HIV-1 RNA levels, and identifies persons at risk for immunologic and clinical decline.     

Productive and persistent infection of human thymic epithelial cells in vitro with HIV-1

Clostridium sporogenes was isolated from rabbits with antibiotic-associated hemorrhagic diarrhea, then its hemorrhagic toxin was purified from the supernatant by means of hydrophobic interaction chromatography, hydroxyapatite chromatography, and gel filtration. The purified toxin's hemorrhagic activity was completely inhibited by EDTA, but not by PMSF or ovomucoid; and fully restored by the addition of such divalent cations as Zn2+, Ca2+ and Mg2+. The purified toxin did not hydrolyze azocasein, or type I or II collagens. But the purified toxin did hydrolyze type III and IV collagens, and also gelatins prepared from type I, II, III and IV collagens. It appears, therefore that C. sporogenes's toxin is a collagenase that hydrolyzes type III and IV collagens, which are major constituents of the tunica intima and media of blood vessels. And that fact suggests that hemorrhage caused by the toxin depend on its collagenase activity.     

Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA

Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.     

Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.     

Activation and cell cycle antigens in CD4+ and CD8+ T cells correlate with plasma human immunodeficiency virus (HIV-1) RNA level in HIV-1 infection

gamma-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine gamma-sarcoglycan gene was disrupted using homologous recombination. Mice lacking gamma-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma-, and delta-sarcoglycan function as a unit. Importantly, mice lacking gamma-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, beta-dystroglycan and laminin were left intact, implying that the dystrophin-dystroglycan-laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking gamma-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking gamma-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.     

Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection

Human NK cells have been shown to produce cytokines (e.g., IFN-gamma and TNF-alpha) and the chemokine macrophage inflammatory protein (MIP)-1alpha following stimulation with the combination of two monokines, IL-15 plus IL-12. The C-C chemokines MIP-1alpha, MIP-1beta, and RANTES have been identified as the major soluble macrophage-tropic HIV-1-suppressive factors produced by CD8+ T cells, which exert their action at the level of viral entry. Here, we demonstrate that monokine-activated NK cells, isolated from both normal and HIV-1+ donors, produce similar amounts of MIP-1alpha, MIP-1beta, and RANTES protein, in vitro. Further, supernatants of monokine-activated NK cells obtained from both normal donors and AIDS patients showed potent (routinely > or = 90%) suppressive activity against HIV-1 replication in vitro, compared with unstimulated control supernatants. NK cell supernatants inhibited both macrophage-tropic HIV-1(NFN-SX) and T cell-tropic HIV-1(NL4-3) replication in vitro, but not dual-tropic HIV-1(89.6). Importantly, the C-C chemokines MIP-1alpha, MIP-1beta, and RANTES were responsible only for a fraction of the HIV-1-suppressive activity exhibited by NK cell supernatants against macrophage-tropic HIV-1. Collectively these data indicate that NK cells from normal and HIV-1+ donors produce C-C chemokines and other unidentified factors that can inhibit both macrophage- and T cell-tropic HIV-1 replication in vitro. Since NK cells can be expanded in patients with HIV-1, AIDS, and AIDS malignancy in vivo, this cell type may have an important role in the in vivo regulation of HIV-1 infection.     

Productive infection of neonatal CD8+ T lymphocytes by HIV-1

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.