In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency.

Although it is presumed that the integration of HIV-1 into the genome of infected CD4+ T lymphocytes allows viral persistence, there has been little direct evidence that CD4+ T cells with integrated provirus function as a latent reservoir for HIV-1 in infected individuals. Using resting CD4+ T-cell populations of extremely high purity and a novel assay that selectively and unambiguously detects integrated HIV-1, we show that resting CD4+ T cells harbouring integrated provirus are present in some infected individuals. However, these cells do not accumulate within the circulating pool of resting CD4+ T cells in the early stages of HIV-1 infection and do not accumulate even after prolonged periods in long-term survivors of HIV-1 infection. These results suggest that because of viral cytopathic effects and/or host effector mechanisms, productively infected CD4+ T cells do not generally survive for long enough to revert to a resting memory state in vivo.

Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy.

We examined the pathogenic significance of the latent viral reservoir in the resting CD4+ T cell compartment of HIV-1-infected individuals as well as its involvement in the rebound of plasma viremia after discontinuation of highly active anti-retroviral therapy (HAART). Using heteroduplex mobility and tracking assays, we show that the detectable pool of latently infected, resting CD4+ T cells does not account entirely for the early rebounding plasma HIV in infected individuals in whom HAART has been discontinued. In the majority of patients examined, the rebounding plasma virus was genetically distinct from both the cell-associated HIV RNA and the replication-competent virus within the detectable pool of latently infected, resting CD4 + T cells. These results indicate the existence of other persistent HIV reservoirs that could prompt rapid emergence of plasma viremia after cessation of HAART and underscore the necessity to develop therapies directed toward such populations of infected cells.

Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy.

The size of the pool of resting CD4+ T cells containing replication-competent HIV in the blood of patients receiving intermittent interleukin (IL)-2 plus highly active anti-retroviral therapy (HAART) was significantly lower than that of patients receiving HAART alone. Virus could not be isolated from the peripheral blood CD4+ T cells in three patients receiving IL-2 plus HAART, despite the fact that large numbers of resting CD4+ T cells were cultured. Lymph node biopsies were done in two of these three patients and virus could not be isolated. These results indicate that the intermittent administration of IL-2 with continuous HAART may lead to a substantial reduction in the pool of resting CD4+ T cells that contain replication-competent HIV.

Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines.

Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4(+) T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4+ T cells derived from HIV-infected individuals who are antiretroviral therapy-naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-alpha, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.

B cells of HIV-1-infected patients bind virions through CD21-complement interactions and transmit infectious virus to activated T cells.

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4(+) cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.

Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Th17 cells and polyfunctional HIV-specific T-cell responses.

Human immunodeficiency virus (HIV) infection leads to severe CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) that persists despite the initiation of highly active antiretroviral therapy (HAART). It is not known whether restoration of gut mucosal CD4+ T cells and their functions is feasible during therapy and how that relates to immune correlates and viral reservoirs. Intestinal biopsies and peripheral blood samples from HIV-infected patients who were either HAART naive or on long-term HAART were evaluated. Our data demonstrated that gut CD4+ T-cell restoration ranged from modest (<50%) to high (>50%), compared with uninfected controls. Despite persistent CD4+ T-cell proviral burden and residual immune activation in GALT during HAART, effective CD4+ T-cell restoration (>50%) was achieved, which was associated with enhanced Th17 CD4+ T-cell accumulation and polyfunctional anti-HIV cellular responses. Our findings suggest that a threshold of>50% CD4+ T-cell restoration may be sufficient for polyfunctional HIV-specific T cells with implications in the evaluation of vaccines and therapeutics.

Immune reconstitution in the sigmoid colon after long-term HIV therapy.

Early and profound CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) may drive Human Immunodeficiency Virus (HIV) immunopathogenesis, and GALT immune reconstitution on highly active antiretroviral therapy (HAART) may be suboptimal. Blood and sigmoid colon biopsies were collected from HAART-treated individuals with undetectable blood HIV RNA for > or =4 years and from uninfected controls. HIV proviral levels and T-cell phenotype/function were examined in both compartments. CD4+ T-cell reconstitution in the sigmoid, including CD4+ T cells expressing CCR5, exceeded that in blood and did not differ from uninfected controls. Sigmoid HIV proviral load was not correlated with CD4+ reconsitution, but was correlated with the degree of mucosal CD8+ T-cell immune activation. Colonic Gag-specific T-cell responses were common, but were not associated with proviral load or immune activation. In this select study population, long-term HAART was associated with complete CD4+ T-cell reconstitution in sigmoid colon. However, colonic immune activation may drive ongoing HIV replication.

Latent reservoirs of HIV: obstacles to the eradication of virus.

The use of highly active antiretroviral therapy (HAART) in the treatment of HIV-1-infected individuals has provided a considerable amount of information regarding the dynamics of viral replication and has resulted in enormous advances in HIV therapeutics. The profound suppression of plasma viremia in HIV-infected individuals receiving HAART has resulted in a highly beneficial clinical effect and a dramatic decrease in the death rate attributable to AIDS. Nonetheless, the persistance of reservoirs of HIV, including latently infected, resting CD4+ T cells that can give rise to infectious HIV upon stimulation in vitro, has posed a sobering challenge to the long-term control or eradication of HIV in infected individuals receiving HAART. Although a recent study has demonstrated th at the size of the pool of latently infected, resting CD4+ T cells can be markedly diminished with intermittent interleukin (IL-2) and continuous HAART, complete eradication of HiV in infected individuals remains extremely problematic. Here we discuss recent developments in studies of the latent reservoir of HIV in patients receiving HAART and implications for the long-term treatment of infected individuals and eradication of the infection.

Detection of HIV DNA and RNA using PCR.

This extensively updated unit describes the detection of HIV DNA and RNA using PCR using two basic techniques for quantifying the levels of viral DNA and RNA in infected cells. The schemes for both techniques include isolation of nucleic acids, PCR reactions and detection of amplified products using Southern blotting.

Effect of prolonged dissociation on the subjective measurement of near heterophoria.

Previous studies have demonstrated that vergence adaptation resulting from the prolonged decay of slow fusional vergence may prevent the accurate assessment of oculomotor deviations. Continuing changes in heterophoria have been reported after 27 days of monocular occlusion. However, since most slow fusional vergence will decay within the first few minutes of dissociation, a clinical assessment of both vergence adaptation and heterophoria could be made more rapidly. This report documents two investigations which examined changes in near horizontal heterophoria during 30 min and 180 min of continuous dissociation, respectively. Seven out of the 16 subjects exhibited significant changes in heterophoria during the 30 min dissociation period, with a mean increase in exophoria of 3.4 delta. The decay of slow fusional vergence took approximately 25 min to reach completion. Thus in the clinical setting, both the degree of vergence adaptation and a more accurate assessment of heterophoria may be obtained by maintaining dissociation for 25 min. Furthermore, the presence of vergence adaptation can be assessed after just 5 min of dissociation.

The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein.

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.

The effects of pentobarbital on blood-brain barrier disruption caused by intracarotid injection of hyperosmolar mannitol in rats.

UNLABELLED: This study was performed to evaluate both the effects of pentobarbital on disruption of the blood-brain barrier (BBB) by hyperosmolar mannitol and the relationship between its effect on blood pressure and the integrity of the BBB. Under isoflurane anesthesia, rats in the control group were infused with 25% mannitol into the internal carotid artery before measuring the transfer coefficient (Ki) of 14C alpha-aminoisobutyric acid. Ten minutes before the administration of mannitol, rats received an infusion of pentobarbital: 20 mg/kg in the small-dose group and 50 mg/kg in the large-dose group. In another group of animals (hydralazine group), hydralazine was administered to maintain the mean arterial blood pressure (MAP) at 65 mm Hg during the experimental period. The MAP of the control group (113 +/- 14 mm Hg) was significantly higher (P < 0.002) than that of the small-dose pentobarbital group (78 +/- 13 mm Hg) or the large-dose pentobarbital group (68 +/- 14 mm Hg). In the control group, the Ki of the cortex ipsilateral to the mannitol injection was increased to 4.5 times that of the contralateral cortex (14.5 +/- 7.7 vs 3.2 +/- 0.6 microL x g(-1) x min(-1); P < 0.002). The Ki of the ipsilateral cortex of the small-dose pentobarbital group was 9.7 +/- 5.6 microL x g(-1) x min(-1). The Ki of the ipsilateral cortex of the large-dose pentobarbital group was 5.5 +/- 2.9 microL x g(-1) x min(-1), and lower (-9.0 microL x g(-1) x min(-1)) than that of the control animals (P < 0.05). There was no significant difference in the Ki of the contralateral cortex among any of the three groups of animals. At the same MAP, the Ki of the ipsilateral cortex of the large-dose pentobarbital group was lower (-4.3 microL x g(-1) x min(-1)) than that of the hydralazine group (9.8 +/- 4.6 microL x g(-1) x min(-1)) (P < 0.05). Pentobarbital attenuated the BBB disruption induced by hyperosmolar mannitol. This may be attributed, at least in part, to the blood pressure effect of pentobarbital. IMPLICATIONS: When the blood-brain barrier (BBB) was disrupted by a hyperosmolar solution, pentobarbital attenuated the degree of leakage of the BBB. Systemic hypotension caused by pentobarbital played a significant role in decreasing the leakage. Our study suggests that when the BBB is disrupted, pentobarbital may be effective in protecting the BBB. Furthermore, systemic blood pressure plays an important role in determining the degree of disruption.

Iron-molybdenum cofactor insertion into the Apo-MoFe protein of nitrogenase involves the iron protein-MgATP complex.

Nitrogenase is composed of two component proteins, the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein). The Fe protein is a Mr 60,000 dimer of identical subunits with one bridging [4Fe-4S] center. It serves as a one-electron donor to the MoFe protein in a reaction that is coupled to MgATP hydrolysis. The MoFe protein is an alpha 2 beta 2 tetramer of Mr 220,000 which contains four [4Fe-4S] clusters and two iron-molybdenum cofactor (FeMo cofactor) centers. The exact structure of FeMo cofactor is not known, but it is believed to form the active site of the enzyme. Using specifically constructed deletion mutants of Azotobacter vinelandii, we have previously shown that the Fe protein, but not the MoFe protein, is required for FeMo cofactor biosynthesis (Robinson, A. C., Dean, D. R., and Burgess, B. K. (1987) J. Biol. Chem. 262, 14327-14332). During the partial purification of a FeMo cofactor-deficient form of the MoFe protein from one of these mutants (DJ54, delta nifH), we have discovered that, in addition to biosynthesis, the Fe protein-MgATP complex is involved in FeMo cofactor insertion into the MoFe protein. This insertion process is also sensitive to a number of other parameters (e.g. salt, pH, temperature, protein concentration). Based on our experimental data, we present a model for how this insertion reaction might take place, in which the Fe protein-MgATP complex binds the FeMo cofactor-deficient form of the MoFe protein and stabilizes a specific conformation of the MoFe protein that has the FeMo cofactor binding site exposed and available for coordination by preformed FeMo cofactor.

Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI.

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.

Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection.

The presence of latently infected, resting CD4(+) T cells carrying replication-competent HIV-1 has been demonstrated in chronically infected individuals who are antiretroviral therapy naive as well as in those who are receiving highly active antiretroviral therapy (HAART). It is not clear, however, whether the establishment of a pool of latently infected CD4(+) T cells can be blocked by early initiation of HAART after primary infection. The present study demonstrates that initiation of HAART in infected individuals as early as 10 days after the onset of symptoms of primary HIV-1 infection did not prevent generation of latently infected, resting CD4(+) T cells carrying integrated HIV-1 DNA as well as infectious HIV-1 despite the successful control of plasma viremia shortly after institution of HAART. Furthermore, there was no correlation between either the duration of HAART at the time of study (range: 0.2-17 months) or the time of initiation of HAART after the onset of symptoms of primary HIV-1 infection (range: 0.3-4 months) and the frequencies of resting CD4(+) T cells carrying either integrated HIV-1 DNA or infectious virus. These results underscore the rapidity with which latent reservoirs are established in primary HIV-1 infection and indicate that it is unlikely that early treatment during primary infection can prevent establishment of a pool of latently infected, resting CD4(+) T cells as long as treatment is initiated after plasma viremia becomes evident.

Expression of chemokine and inhibitory receptors on natural killer cells: effect of immune activation and HIV viremia.

We examined the cell-surface expression of chemokine and natural killer (NK) cell inhibitory receptors (iNKRs) on NK cells from individuals with human immunodeficiency virus (HIV) infection, chronic hepatitis C infection, and Wegener's granulomatosis (WG), an inflammatory, granulomatous vasculitis. The expression of CCR5 on NK cells was up-regulated in individuals with HIV viremia and in individuals with active WG, indicating that expression of this receptor is modulated by states of immune activation associated with viral infection and inflammatory or immune-mediated diseases. In contrast, iNKRs were shown to be up-regulated only on NK cells of individuals with HIV viremia, and they returned to a normal level when viremia was controlled with effective antiviral therapy. In individuals with HIV-1 viremia, there was a direct correlation between the level of expression of p58.1, p58.2, and CD94 receptors and plasma HIV viremia, suggesting that ongoing active HIV replication has an effect on the expression of such receptors on NK cells. These results suggest that immune activation leads to abnormal cell-surface expression of chemokine receptors on NK cells, whereas HIV-specific processes account for the up-regulation of iNKRs on NK cells; this may explain the NK cell-functional defects seen in HIV infection.

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

CD40-Mediated induction of CD4 and CXCR4 on B lymphocytes correlates with restricted susceptibility to human immunodeficiency virus type 1 infection: potential role of B lymphocytes as a viral reservoir.

Human immunodeficiency virus type 1 (HIV-1) replicates primarily in lymphoid tissues where it has ready access to activated immune competent cells. We used one of the major pathways of immune activation, namely, CD40-CD40L interactions, to study the infectability of B lymphocytes isolated from peripheral blood mononuclear cells. Highly enriched populations of B lymphocytes generated in the presence of interleukin-4 and oligomeric soluble CD40L upregulated costimulatory and activation markers, as well as HIV-1 receptors CD4 and CXCR4, but not CCR5. By using single-round competent luciferase viruses complemented with either amphotropic or HIV-derived envelopes, we found a direct correlation between upregulation of HIV-1 receptors and the susceptibility of the B lymphocytes to infection with dual-tropic and T-tropic strains of HIV-1; in contrast, cells were resistant to M-tropic strains of HIV-1. HIV-1 envelope-mediated infection was completely abolished with either an anti-CD4 monoclonal antibody or a peptide known to directly block CXCR4 usage and partially blocked with stromal cell-derived factor 1, all of which had no effect on the entry of virus pseudotyped with amphotropic envelope. Full virus replication kinetics confirmed that infection depends on CXCR4 usage. Furthermore, productive cycles of virus replication occurred rapidly yet under most conditions, without the appearance of syncytia. Thus, an activated immunological environment may induce the expression of HIV-1 receptors on B lymphocytes, priming them for infection with selective strains of HIV-1 and allowing them to serve as a potential viral reservoir.