Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence.

Viral replication and latently infected cellular reservoirs persist in HIV-infected patients achieving undetectable plasma virus levels with potent antiretroviral therapy. We exploited a predictable drug resistance mutation in the HIV reverse transcriptase to label and track cells infected during defined intervals of treatment and to identify cells replenished by ongoing replication. Decay rates of subsets of latently HIV-infected cells paradoxically decreased with time since establishment, reflecting heterogeneous lymphocyte activation and clearance. Residual low-level replication can replenish cellular reservoirs; however, it does not account for prolonged clearance rates in patients without detectable viremia. In patients receiving potent antiretroviral therapy, the latent pool has a heterogeneous and dynamic composition that comprises a progressively increasing proportion of stable lymphocytes. Eradication will not be achieved with complete inhibition of viral replication alone.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Effect of influenza vaccination on viral replication and immune response in persons infected with human immunodeficiency virus receiving potent antiretroviral therapy.

Nineteen patients infected with human immunodeficiency virus (HIV) with varying levels of viral suppression achieved with antiretroviral therapy were evaluated to determine whether trivalent influenza vaccine activated HIV replication. Humoral immune responses and CD4+ lymphocyte subsets were compared in 5 HIV-uninfected vaccinated subjects. Transient elevations of plasma HIV RNA levels (76-89 copies/mL) appeared within 2 weeks in 3 of 11 patients with <50 copies/mL at baseline. Sustained elevation in HIV plasma RNA was observed in 7 of 8 patients with baseline HIV RNA of >50 copies/mL. HIV DNA decreased in patients with <400 RNA copies/mL at baseline and showed an HIV RNA increase after vaccination (n=8) when compared with 8 patients with <50 copies/mL at baseline who lacked viral response to vaccination. Concurrent decreases in proviral DNA and memory phenotype CD4+ cells in association with increased plasma HIV RNA after vaccination in patients with <400 RNA copies/mL at baseline suggest that in vivo mobilization of the latently infected cell reservoir may occur during potent antiretroviral therapy.

Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy.

In human immunodeficiency virus (HIV)-infected patients treated with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these possibilities, sequences encompassing the C2 and V3 domains of HIV-1 env were analyzed from virus present in baseline plasma and from viral isolates obtained after 2 years of suppressive therapy in six patients. The presence of sequence changes consistent with evolution was demonstrated for three subjects and correlated with less complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of even low levels of detectable viremia. Together, these results provide evidence for ongoing replication. In the remaining three patients, virus recovered after 2 years of therapy was either genotypically contemporary with or ancestral to virus present in plasma 2 years before, indicating that virus recovery had indeed resulted from activation of latently infected cells.

Viral dynamics of acute HIV-1 infection.

Viral dynamics were intensively investigated in eight patients with acute HIV infection to define the earliest rates of change in plasma HIV RNA before and after the start of antiretroviral therapy. We report the first estimates of the basic reproductive number (R(0)), the number of cells infected by the progeny of an infected cell during its lifetime when target cells are not depleted. The mean initial viral doubling time was 10 h, and the peak of viremia occurred 21 d after reported HIV exposure. The spontaneous rate of decline (alpha) was highly variable among individuals. The phase 1 viral decay rate (delta(I) = 0.3/day) in subjects initiating potent antiretroviral therapy during acute HIV infection was similar to estimates from treated subjects with chronic HIV infection. The doubling time in two subjects who discontinued antiretroviral therapy was almost five times slower than during acute infection. The mean basic reproductive number (R(0)) of 19.3 during the logarithmic growth phase of primary HIV infection suggested that a vaccine or postexposure prophylaxis of at least 95% efficacy would be needed to extinguish productive viral infection in the absence of drug resistance or viral latency. These measurements provide a basis for comparison of vaccine and other strategies and support the validity of the simian immunodeficiency virus macaque model of acute HIV infection.

Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.

In evaluating current combination drug regimens for treatment of human immunodeficiency virus (HIV) disease, it is important to determine the existence of viral reservoirs. After depletion of CD8 cells from the peripheral blood mononuclear cells (PBMCs) of both patients and normal donors, activation of patient CD4 lymphocytes with immobilized antibodies to CD3 and CD28 enabled the isolation of virus from PBMCs of six patients despite the suppression of their plasma HIV RNA to fewer than 50 copies per milliliter for up to 2 years. Partial sequencing of HIV pol revealed no new drug resistance mutations or discernible evolution, providing evidence for viral latency rather than drug failure.

Preferential replication of HIV-1 in the CD45RO memory cell subset of primary CD4 lymphocytes in vitro.

The ability of HIV-1 to establish an infection and replicate to high copy number in CD4 lymphocytes is dependent on both the activation state of the cell and virus-encoded regulatory proteins that modulate viral gene expression. To study these required virus-cell interactions, we have used an in vitro cell model of acute HIV infection of quiescent, primary CD4 lymphocytes and subsequent induction of T cell activation and virus replication by lectin or CD3 receptor cross-linking. Experiments were done to determine if the capacity of HIV to establish infection and complete replication was impacted by the maturational state of the CD4 cell target or the specific signal induction pathway engaged during activation. Primary CD4 cells were FACS-sorted into the major phenotypic subsets representative of memory (CD45RO) and naive (CD45RA) cells. Levels of virus replication were compared between infection with wild-type NL4-3 virus and an isogenic mutant containing a deletion in nef regulatory gene. PHA mitogen stimulation was compared with anti-CD3, with and without anti-CD28 costimulation, for induction of cell proliferation and virus replication. In both infected and uninfected cells, the RA cell subset exhibited significantly greater response to CD3/CD28 stimulation than did the RO cell subset. In contrast, the majority of virus replication occurred consistently in the RO cell subset. Deletion of HIV nef function caused a severe reduction in viral replication, especially in the RA naive cell subset after CD3 induction. PCR analysis of viral DNA formation, during infection of quiescent cells, demonstrated that the observed differences in HIV replication capacity between RO and RA cell subsets were not due to inherent differences in cell susceptibility to infection. Our results indicate that HIV replication is enhanced selectively in CD45RO memory phenotype cells through the probable contribution of specialized cellular factors which are produced during CD3-initiated signal transduction.

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation.

Recent data on the phenotype of nef-defective HIV-1 in vitro indicate a new function of the Nef gene product: enhancement of viral infectivity. Single-cycle replication studies have suggested that Nef enhances the efficiency of an early step during viral replication, a step that leads to the establishment of viral DNA. To test this interpretation, the accumulation of low-molecular-weight (unintegrated) viral DNA was measured in cells following exposure to wild-type and nef-defective viruses. nef-defective virus accumulated less DNA than the wild type. This difference was observed after as little as 5 hr of exposure to virus. However, the reverse transcriptase activities of wild-type and nef-defective viruses were equal when measured in cell-free assays using either exogenous or endogenous templates. In addition, the abilities of these viruses to bind and enter cells were not significantly different. Together, these data suggest that Nef optimizes postentry events that are required for efficient synthesis of viral DNA. To determine if these effects were related to the property of Nef-mediated downregulation of CD4, growth curves of these viruses were determined using cells that express a CD4 molecule unable to respond to Nef. nef-defective virus remained attenuated in these cells, indicating that Nef-mediated downregulation of CD4 is not required for Nef-mediated enhancement of viral propagation in vitro.

Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro.

Human immunodeficiency virus type 1 (HIV-1) possesses the ability to establish a complete infection in nondividing host cells. The capacity of HIV-1 to infect nondividing cells probably contributes significantly to its pathology in vivo, as reflected by infection of peripheral T lymphocytes, tissue macrophages, and microglial cells. However, the in vitro demonstration of the establishment of stable HIV-1 infection in quiescent T cells remains controversial. We have developed a primary T-cell model of acute HIV-1 infection of quiescent CD4 lymphocytes that demonstrates the development of a complete, reverse-transcribed form of virus that is stable for over 10 days in culture. To ensure that our primary cell culture was representative of a quiescent population, the CD4 lymphocyte targets were monitored for membrane expression of activation antigens and for shifts in cell cycle from G0/G1 to S/G2 phase. The presence of viral DNA fragments reflecting progressive reverse transcription was determined by PCR analysis. HIV entered primary CD4 cells rapidly, but viral DNA accumulated slowly in the resting cell cultures. DNA species containing regions of full-length reverse transcription were not detected until 3 to 5 days after infection. In parallel with the appearance of complete viral DNA, spliced RNA transcripts, predominantly of the nef species, were detected by reverse transcriptase PCR amplification. When infected CD4 cells were sorted on the basis of cell cycle analysis of DNA content, the accumulation of a complete viral DNA form was found to occur in both the purified G0/G1-phase cell subset and the cell fraction enriched for the minor S-phase subset. In contrast, spliced viral RNA products could be detected only in the enriched S-phase cell fraction. These results demonstrate that HIV-1 can infect and establish a complete, stable form of viral DNA in primary CD4 lymphocytes in vitro but is blocked from transcription in the absence of cell activation. The findings are consistent with in vivo data from HIV-infected individuals that show the existence of viral DNA predominantly as a stable, extrachromosomal form in T cells of the peripheral circulation.

Cell surface CD4 downregulation and resistance to superinfection induced by a defective provirus of HIV-1.

Proviral sequences encoding defective HIV-1 regulatory genes have been detected previously in infected individuals; however, the role of these defective genomes in pathogenesis is unclear. The hypothesis that such replication-defective genomes might induce downregulation of the cellular receptor for HIV-1 (CD4) was tested. CEM cells were stably transfected with a provirus that contains a mutation in the splice site immediately 5' of the rev coding sequence. This mutant expresses early HIV-1 mRNAs but is defective for replication. Cells expressing this defective provirus displayed markedly reduced surface CD4. This downregulation of CD4 was dependent on an intact nef gene and was sufficient to cause resistance to superinfection by wild-type virus. These findings indicate that Nef as expressed by replication-defective HIV-1 can downregulate CD4. This perturbation of the T lymphocyte cell membrane is a potential basis for pathogenicity of defective HIV-1.

Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.

The replication competence of human immunodeficiency virus type 1 genomes containing mutations in the nef open reading frame was evaluated in continuous cell lines. Mutants that contained a deletion in the nef open reading frame, premature termination codons, or missense mutations in the N-terminal myristoylation signal were constructed. The replication of these mutants was tested in three ways. First, plasmid genomes were used to transfect T-lymphoblastoid cells. Second, low-passage posttransfection supernatants were used to infect cells with a relatively low virus input. Third, high-titer virus stocks were used to infect cells with a relatively high virus input. These experiments demonstrated a 100- to 10,000-fold decrement in p24 production by the nef mutants compared with that by the wild-type virus. The greatest difference was obtained after infection with the lowest virus input. The myristoylation signal was critical for this positive effect of nef. To investigate the mechanism of the positive influence of nef, nef-positive and nef-minus viruses were compared during a single cycle of replication. These single-cycle experiments were initiated both by infection with high-titer virus stocks and by transfection with viral DNA. Single-cycle infection yielded a three- to fivefold decrement in p24 production by nef-minus virus. Single-cycle transfection yielded equal amounts of p24 production. These results implied that nef does not affect replication after the provirus is established. In support of these results, viral production from cells chronically infected with nef-positive or nef-minus viruses was similar over time. To determine whether the effect of nef was due to infectivity, end point titrations of nef-positive and nef-minus viruses were performed. nef-positive virus had a greater infectivity per picogram of HIV p24 antigen than nef-minus virus. These data indicated that the positive influence of nef on viral growth rate is due to an infectivity advantage of virus produced with an intact nef gene.

The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes.

The viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nef gene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study nef function in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nef on viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nef mutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nef confers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study nef function in vitro, but also provides important clues to the function of nef in HIV infection in vivo.

In vitro differentiation of monocytoid THP-1 cells affects their permissiveness for HIV strains: a model system for studying the cellular basis of HIV differential tropism.

The prototypic macrophage-tropic HIV-1 isolate, HIV-1BaL, cannot replicate in the monocytoid cell line THP-1. After induction of differentiation by a phorbol diester, a fraction of THP-1 cells became permissive to HIV-1BaL. In contrast, this treatment decreased permissiveness for the lymphotropic isolate HIV-1LAI. Viral DNA was not synthesized in unstimulated THP-1 cells, as determined with PCR, suggesting that the block to HIV-1BaL replication in these cells occurred at an early step of the virus replicative cycle prior to or at the level of reverse transcription. Virus binding studies showed that differences in cell permissiveness for HIV-1BaL were not due to altered virus binding. Substantial amounts of HIV-1BaL bound to both undifferentiated and differentiated THP-1 cells, and this binding could not be prevented by blocking with the anti-CD4 antibody Leu3a, which did prevent the binding of HIV-1LAI to CEM T lymphoid cells. While Leu3a was very effective at preventing the infection by HIV-1LAI in CEM cells, it was less effective in preventing HIV-1BaL infection of differentiated THP-1 cells or primary macrophages. Although it is likely that molecules other than CD4 on monocytic cells can mediate binding of macrophage-tropic HIV, the binding of HIV-1BaL to THP-1 cells was not sufficient for infection, because binding was the same in nonpermissive undifferentiated cells as in permissive differentiated cells. Thus, the restriction of viral replication in this model cell system occurs at some step after virion binding. Comparison of differentiated THP-1 cells with their undifferentiated counterparts may provide an approach to defining cellular determinants of HIV host range other than CD4 expression and to characterizing the incompletely defined steps of viral entry.

Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies.

Functional impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000, which inhibits replication of certain plant RNA viruses, and of herpes simplex virus, poliovirus and influenza virus. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CD5, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.

Alterations in cytotoxic and phenotypic subsets of natural killer cells in acquired immune deficiency syndrome (AIDS).

Testing of cytotoxic function using a panel of natural killer (NK)-sensitive target cells, including a unique herpes simplex virus-infected Raji-cell target, was performed in conjunction with phenotypic cell analysis by dual-color flow cytometry to characterize the NK system. Subjects included in the study were at risk for or infected with the etiologic agent of the acquired immune deficiency syndrome (AIDS), human immunodeficiency virus (HIV). A generalized defect in NK function was temporally correlated with disease manifestations, as evidenced by deficient NK lytic function in patients with AIDS and AIDS-related complex (ARC). Healthy at-risk subjects, including those seropositive for HIV, exhibited robust NK-cell function. Phenotypic analysis revealed that normal proportions of the NK-associated CD16+ (Leu11) Leu7- and CD16+(Leu11)Leu7+ lymphocyte subsets were maintained throughout the clinical progression of HIV infection. However, the proportion and numbers of cells of the CD8+(Leu2)Leu7+ subset were increased in AIDS, ARC, and healthy at-risk subjects, including those seronegative for HIV. These results are consistent with a qualitative defect in the NK system in AIDS, perhaps secondary to CD4-cell depletion and a concomitant lack of essential accessory factors. The elevation in CD8+(Leu2)/Leu7+ cells is not solely the result of HIV infection and may be a general response to viruses and/or other antigenic stimulation.

The use of three baseline values in intervention studies: application to evaluation of immune modulation therapies.

In studying the effects of a treatment intervention on immunological parameters, we have found that three baseline values are a practical number to obtain on each patient. Three baseline values 1) increase the chances of detecting a statistically significant effect of the intervention, 2) provide an assessment of the daily variability of the assay and patients, and 3) enable the identification of individual patients who demonstrate significant changes associated with the intervention.

Lymphocyte cytotoxicity in the peritoneal cavity and blood of patients with ovarian cancer.

After an intensive course of combination chemotherapy, 16 patients with minimal residual ovarian cancer that was documented at second-look laparotomy, had an indwelling Tenckhoff catheter placed and underwent multiple peritoneal saline lavages. Lymphocyte-enriched populations from the peritoneal cavity and peripheral blood were obtained by density gradient centrifugation and examined for cell-surface phenotype and a variety of immune functions, including natural killer cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Phenotypic characterization revealed that peritoneal lymphocytes consisted primarily of T cells and cells bearing receptors for the crystallizable fragment of immunoglobulin G (IgG) (crystallizable fragment-receptor), and contained a very low number of B cells. Peritoneal natural killer lymphocyte cytotoxicity and antibody-dependent cell-mediated cytotoxicity were very low in all but two patients. Incubation of peritoneal lymphocytes with Corynebacterium parvum and interferon in vitro did not result in augmented cytotoxicity against susceptible targets. Supernatants from cultured peritoneal cells of all patients markedly inhibited natural cytotoxic activity of normal donor blood lymphocytes. These results suggest that lymphocytes collected from the peritoneal cavity of patients with minimal residual ovarian cancer are deficient in natural and antibody-dependent cytotoxic effector function. This deficiency may influence the host's ability to control the spread and proliferation of tumor cells in the peritoneal cavity.

Patient with 13 chromosome deletion: evidence that the retinoblastoma gene is a recessive cancer gene.

Although a constitutional chromosomal deletion including 13q14 has been found to date in all retinoblastoma patients whose esterase D activity is 50 percent of normal, one female patient has been found who has 50 percent esterase D activity in all normal cells examined but no deletion of 13q14 at the 550-band level. Therefore, she has the smallest constitutional chromosomal deletion within 13q14 that is associated with susceptibility to retinoblastoma. Two stem lines were identified in a retinoblastoma from this patient, and each one had a missing 13 chromosome. No detectable esterase D activity was found in the tumor, indicating that the normal nondeleted 13 chromosome was lost in both stem lines. Thus the data from this patient not only show that there is a total loss of genetic information at the location of the retinoblastoma gene within the tumor, but also imply that recessive genes may play an important role in the development of certain human tumors including retinoblastoma.