Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy.

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.

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.

Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy.

Combination therapy for HIV-1 infection can reduce plasma virus to undetectable levels, indicating that prolonged treatment might eradicate the infection. However, HIV-1 can persist in a latent form in resting CD4+ T cells. We measured the decay rate of this latent reservoir in 34 treated adults whose plasma virus levels were undetectable. The mean half-life of the latent reservoir was very long (43.9 months). If the latent reservoir consists of only 1 x 10(5) cells, eradication could take as long as 60 years. Thus, latent infection of resting CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy.

Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework.

A new (to our knowledge) de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced and applied to the discovery of what we believe are novel HIV-1 entry inhibitors. The framework consists of two stages: a sequence selection stage and a validation stage. The sequence selection stage produces a rank-ordered list of amino-acid sequences by solving an integer programming sequence selection model. The validation stage consists of fold specificity and approximate binding affinity calculations. The designed peptidic inhibitors are 12-amino-acids-long and target the hydrophobic core of gp41. A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in cell culture. All peptides examined showed inhibitory activity when compared with no drug present, and the novel peptide sequences outperformed the native template sequence used for the design. The best sequence showed micromolar inhibition, which is a 3-15-fold improvement over the native sequence, depending on the donor. In addition, the best sequence equally inhibited wild-type and Enfuvirtide-resistant virus strains.

Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization.

CD8+ cytotoxic T lymphocytes (CTLs) have the ability to recognize and eliminate virally infected cells before new virions are produced within that cell. Therefore, a rapid and vigorous CD8+ CTL response, induced by vaccination, can, in principle, prevent disseminated infection in vaccinated individuals who are exposed to the relevant virus. There has thus been interest in novel vaccine strategies that will enhance the induction of CD8+ CTLs. In this study, we have tested the hypothesis that targeting an antigen to undergo more efficient processing by the class I processing pathway will elicit a more vigorous CD8+ CTL response against that antigen. Targeting a type I transmembrane protein, the HIV-1 envelope (env) protein, for expression in the cytoplasm, rather than allowing its normal co-translational translocation into the endoplasmic reticulum, sensitized target cells expressing this mutant more rapidly for lysis by an env-specific CTL clone. Additionally, a greatly enhanced de novo env-specific CTL response was induced in vivo after immunization of mice with recombinant vaccinia vectors expressing the cytoplasmic env mutant. Similarly, targeting a cytoplasmic protein, HIV-1 nef, to undergo rapid cytoplasmic degradation induced a greatly enhanced de novo nef-specific CD8+ CTL response in vivo after immunization of mice with either recombinant vaccinia vectors or DNA expression plasmids expressing the degradation targeted nef mutant. The targeting of viral antigens for rapid cytoplasmic degradation represents a novel and highly effective vaccine strategy for the induction of enhanced de novo CTL responses in vivo.

A lymphokine that activates the cytolytic program of both cytotoxic T lymphocyte and natural killer clones.

A 10-12 kD lymphokine, herein termed TCAF, was recently shown to be secreted from Th after crosslinking of their antigen/MHC (T3-Ti) receptors. TCAF stimulates resting T lymphocyte proliferation via binding to surface components of the T11 pathway. To determine whether TCAF could induce antigen-independent activation of the lytic machinery of cytotoxic cells, the present studies were conducted. In the presence of TCAF, both T8+ class I MHC-specific and T4+ class II MHC-specific cytotoxic T cell clones were induced to kill targets, including those lacking the appropriate MHC molecules. This effect was unique to TCAF, since IL-1, IL-2, IFN-gamma could not stimulate lytic activity. Furthermore, both T3+T11+ and T3-T11+ NK clones were triggered to lyse NK-resistant target cells. These findings suggest that TCAF can function in an antigen-independent fashion to amplify cytotoxic effector responses.

Comparative clonal analysis of human immunodeficiency virus type 1 (HIV-1)-specific CD4+ and CD8+ cytolytic T lymphocytes isolated from seronegative humans immunized with candidate HIV-1 vaccines.

The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were confirmed in studies in which CD4+ lymphoblasts were exposed to recombinant gp120 and used as targets for gp120-specific CD4+ and CD8+ CTL clones. gp120-pulsed, noninfected targets were lysed in an antigen-specific fashion by CD4+ but not CD8+ CTL clones. Taken together, these observations demonstrate that in an in vitro HIV-1 infection, sufficient amounts of gp120 antigen are produced and shed by infected cells to enable uptake by cells that are not yet infected, resulting in the lysis of these noninfected cells by gp120-specific, CD4+ CTL.(ABSTRACT TRUNCATED AT 400 WORDS)

Anchor sequence-dependent endogenous processing of human immunodeficiency virus 1 envelope glycoprotein gp160 for CD4+ T cell recognition.

Human CD4+ T cell clones and cell lines were shown to lyse recombinant vaccinia virus-infected cells that synthesize the HIV-1 envelope glycoprotein gp160. The processing of endogenously synthesized gp160 for recognition by CD4+ T cells required that the protein, after synthesis on the rough endoplasmic reticulum and during subsequent cellular transport, remain attached to the luminal/extracellular membrane face by a hydrophobic anchor sequence.

Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

Major histocompatibility complex independent T cell receptor-antigen interaction: functional analysis using fluorescein derivatives.

We have isolated T cell receptor (TCR) cDNAs from fluorescein (FL)-specific human T cell clones (alpha FL beta FL), and transferred them to TCR beta- Jurkat cells in order to study direct FL-binding to the TCR. Using either FL-conjugated polymers (FL-polymer) or FL-substituted Sepharose beads, we are able to demonstrate the direct binding of antigen to the T cell surface, and the functional activation of the T cell transfectants. We present evidence against the involvement of major histocompatibility complex (MHC) molecules or antigen presentation in the interaction of FL with the alpha FL beta FL transfectants. Additionally, we have examined the effect of ring substitutions on the FL molecule as well as specific alterations of substituents attached to the 5' position, and we have found that all of them interfere with the functional recognition of the alpha FL beta FL TCR. These experiments demonstrate that TCRs like antibodies have intrinsic affinities for antigen, even without the involvement of MHC molecules.

Preventing AIDS but not HIV-1 infection with a DNA vaccine.

Although there has been some success in treating human immunodeficiency virus (HIV) patients with triple drug therapy (highly active antiretroviral therapy or HAART), the best hope for combating AIDS (the disease caused by HIV) could be a combination of drug therapy and vaccination, according to Shen and Siliciano in their Perspective. A new study in rhesus monkeys treated with a DNA vaccine (Barouch et al.) demonstrates that a powerful vaccine-induced CD8(+) cytolytic T cell response reduces the amount of virus in the blood to very low levels preventing the drastic decrease in CD4(+) T helper cells and subsequent immunodeficiency. As the Perspective authors explain, vaccinating HIV patients that are receiving HAART may enable HIV levels to be permanently brought under control such that the drug treatment can eventually be stopped.

Induction of CD4+ human cytolytic T cells specific for HIV-infected cells by a gp160 subunit vaccine.

Cytolytic T lymphocyte (CTL) responses were evaluated in humans immunized with recombinant human immunodeficiency virus type 1 (HIV) envelope glycoprotein gp160. Some vaccinees had gp160-specific CTLs that were shown by cloning to be CD4+. Although induced by exogenous antigen, most gp160-specific CTL clones also recognized gp160 synthesized endogenously in target cells. These clones lysed autologous CD4+ T lymphoblasts infected with HIV. Of particular interest were certain vaccine-induced clones that lysed HIV-infected cells, recognized gp160 from diverse HIV isolates, and did not participate in "innocent bystander" killing of noninfected CD4+ T cells that had bound gp120.

Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.

The hypothesis that quiescent CD4+ T lymphocytes carrying proviral DNA provide a reservoir for human immunodeficiency virus-type 1 (HIV-1) in patients on highly active antiretroviral therapy (HAART) was examined. In a study of 22 patients successfully treated with HAART for up to 30 months, replication-competent virus was routinely recovered from resting CD4+ T lymphocytes. The frequency of resting CD4+ T cells harboring latent HIV-1 was low, 0.2 to 16.4 per 10(6) cells, and, in cross-sectional analysis, did not decrease with increasing time on therapy. The recovered viruses generally did not show mutations associated with resistance to the relevant antiretroviral drugs. This reservoir of nonevolving latent virus in resting CD4+ T cells should be considered in deciding whether to terminate treatment in patients who respond to HAART.

A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children.

HIV-1 persists in a latent state in resting CD4(+) T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4(+) T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4(+) T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1-3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4(+) T cells will be a major obstacle to HIV-1 eradication in children.

The role of CD4 in HIV envelope-mediated pathogenesis.

There is now compelling evidence that env-CD4 interactions are central to several complex pathogenic mechanisms in HIV-1 infection. In addition to mediating virus attachment to CD4+ cells, the high affinity interaction of env protein with CD4 is also important in initiating both syncytium formation and syncytium-independent cytopathic effects. In addition, shed gp120 can bind to CD4 on noninfected cells and interfere with the function of these cells while at the same time rendering the cells susceptible to destruction by ADCC, by CD4+ CTLs or by programmed cell death induced by cross-linking of CD4 with gp120 and anti-gp120 followed by cellular activation. Although all of these mechanisms have been demonstrated to operate in vitro, it remains unclear how important each mechanism is in vivo. Nevertheless, the central role of env-CD4 interactions in all of these pathogenic mechanisms highlights the importance of developing effective low molecular weight inhibitors of this reaction.

Acidic residues in the DR beta chain third hypervariable region are required for stimulation of a DR(alpha, beta 1*0402)-restricted T-cell clone.

We investigated the minimal requirements for stimulation of an antigen-specific HLA-DR(alpha, beta 1*0402)-restricted T-cell clone (Een217) by using transfectants expressing mutant DR beta chains as APCs. Antigen-specific stimulation of Een217 was induced with transfectants expressing DR(alpha, beta 1*0402) and DR(alpha, beta 1*0403) but not other DR4 subtypes that also bind the peptide recognized by this clone. Analysis of the effects of single amino acid substitutions in the beta chains of each of the DR4 subtypes revealed a requirement for acidic residues in the third HVR, particularly amino acid 71, in stimulation of clone Een217. Functional class II mutants were generated from nonstimulatory DR4 subtype beta chains by acidic residue substitutions within the third HVR. These data define the requirement for negatively charged residues in this region for peptide-induced stimulation of this T-cell clone. The required acidic residues can be located at either position 70, 71, or 74 in the DR beta chain. The negative charge in this segment of the DR beta chain alpha-helix may be required for direct interactions with the T-cell receptor of Een217 or may affect peptide conformation.

Human immunodeficiency virus glycoprotein-specific CD4+ cytotoxic T lymphocytes are involved in two types of cytotoxicity: antigen-specific and cell-cell fusion-related cell lysis.

Human immunodeficiency virus (HIV) glycoprotein-specific CD4+ cytotoxic T lymphocytes (CTLs) lyse target cells in an MHC-restricted calcium-dependent fashion similar to the mechanism used by CD8+ CTLs. However, contact of unprimed peripheral blood CD4+ T cells with HIV glycoprotein-expressing cells has been shown to cause, in addition to cell-cell fusion, rapid cytolysis that may resemble antigen-specific cytotoxicity in the chromium release assay. In this study, the ability of glycoprotein-specific CD4+ CTLs to undergo similar fusion-related cytolysis was examined. The data obtained demonstrate that in addition to antigen-specific calcium-dependent cytotoxicity, envelope-specific CD4+ CTLs are involved in fusion-related, calcium-independent cytolysis.

A novel method for detection and ex vivo expansion of HIV type 1-specific cytolytic T lymphocytes.

Studies have shown that cytolytic T lymphocyte (CTL) responses may be critical to the clearance of the early viremia in acute HIV-1 infection. It is likely that these cells play an important role in prolonging the asymptomatic phase of the infection. Although HIV-1-specific CTL activity can be detected in direct assays of freshly isolated peripheral blood lymphocytes (PBL) from some infected individuals, this method fails to detect CTL that are present at low frequency and resting, memory CTL. For these reasons, direct CTL assays on PBL from seropositive individuals may underestimate the level of CTL immunity. As part of ongoing investigations of CTL activity in HIV-1-infected individuals, we developed a novel strategy for the detection and ex vivo expansion of HIV-1-specific CTL. This technique involves selective stimulation of PBL from seropositive individuals with autologous Epstein-Barr virus (EBV)-transformed, B-lymphoblastoid cell lines (B-LCL) infected with vaccinia vectors expressing various HIV-1 genes. Prior to their use for in vitro stimulation, B-LCL are treated with psoralen and UV light to inactivate vaccinia virus. After 1 week of stimulation, CTL activity in stimulated cultures is measured in a standard 51Cr release assay. This ex vivo expansion method can selectively increase the bulk culture CTL activity against env, gag and nef, even in some seropositive individuals with low CD4 counts and little evidence of HIV-1-specific CTL in assays of freshly isolated PBL. These expanded CTL are predominantly of the CD8+ phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Autologous strain-specific cytolytic T lymphocyte responses directed against human immunodeficiency virus type 1 Env.

The Env glycoprotein of human immunodeficiency virus is critical for the pathogenesis of the acquired immunodeficiency syndrome, and has been the prime target for candidate HIV-1 vaccines. Cytolytic T lymphocytes (CTLs) may be important for the immunologic control of HIV infection and HIV-1 Env-specific cytolytic T cells have been isolated from infected individuals and seronegative recipients of HIV-1 vaccines. Most prior studies have used assays that detect Env-specific CTLs directed against standard laboratory viral variants. These studies may be limited because the Env proteins of these laboratory strains (for example, LAI and MN) may differ significantly from the Env proteins from primary HIV-1 strains, and a single amino acid change can abrogate the recognition of HIV-1 Env by some CTL clones. Therefore, this study measured CTL activity directed against HIV-1 Env representing the infected individual's (autologous) HIV-1 viral variants. For two HIV-1-infected individuals, recombinant vaccina viruses expressing cloned HIV-1 env genes were constructed. Using an in vitro stimulation method, strain-specific CTL activity directed against autologous HIV-1 Env was detected in both individuals. From one subject, strain-specific CTL clones directed against autologous and HIV-1LAI Env were characterized. Therefore, some infected individuals have Env-specific CTLs directed against autologous strains of HIV-1. Detection and characterization of autologous Env-specific CTL activity may have important implications relative to the current HIV-1 vaccine development strategies focusing on Env derived from laboratory strains of HIV-1.