Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade

It is not known how human immunodeficiency virus type 1 (HIV-1)-derived antagonist peptides interfere with intracellular activation of cytotoxic T lymphocytes (CTL). We identified Gag epitope variants in HIV-1-infected patients that act as antagonists of CTL responses to unmutated epitopes. We then investigated the effect that presentation of each variant has on the early events of T cell receptor (TCR) signal transduction. We found that altered peptide ligands (APL) failed to induce phosphorylation of pp36, a crucial adaptor protein involved in TCR signal transduction. We further investigated the effect that simultaneous presentation of APL and native antigen at low, physiological, peptide concentrations (1 nM) has on TCR signal transduction, and we found that the presence of APL can completely inhibit induction of the protein tyrosine phosphorylation events of the TCR signal transduction cascade.

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 ζ 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.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia

High-affinity (Kd = 1 × 10−9 M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC50 = 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.

Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS

Studies of circulating T (CD3+) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5–2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3+ cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3+ lymphocyte counts before the inflection point was close to zero for HIV− and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3+ cell counts relative to HIV− men and HIV+ men not developing AIDS. A CD3+ cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.

The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.

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.

Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients.

The resistance of acquired immunodeficiency syndrome (AIDS) to traditional drug therapy has prompted a search for alternative treatments for this disease. One potential approach is to provide genetic resistance to viral replication to prolong latency. This strategy requires the definition of effective antiviral genes that extend the survival of T cells in human immunodeficiency virus (HIV)-infected individuals. We report the results of a human study designed to determine whether a genetic intervention can prolong the survival of T cells in HIV-infected individuals. Gene transfer was performed in enriched CD4+ cells with plasmid expression vectors encoding an inhibitory Rev protein, Rev M10, or a deletion mutant control, deltaRev M10, delivered by gold microparticles. Autologous cells separately transfected with each of the vectors were returned to each patient, and toxicity, gene expression, and survival of genetically modified cells were assessed. Cells that expressed Rev M10 were more resistant to HIV infection than those with deltaRev M10 in vitro. In HIV-infected subjects, Rev M10-transduced cells showed preferential survival compared to deltaRev M10 controls. Rev M10 can therefore act as a specific intracellular inhibitor that can prolong T-cell survival in HIV-1-infected individuals and potentially serve as a molecular genetic intervention which can contribute to the treatment of AIDS.

Frequency of HLA allele-specific peptide motifs in HIV-1 proteins correlates with the allele’s association with relative rates of disease progression after HIV-1 infection

An HLA allele-specific cytotoxic T lymphocyte response is thought to influence the rate of disease progression in HIV-1-infected individuals. In a prior study of 139 HIV-1-infected homosexual men, we identified HLA class I alleles and observed an association of specific alleles with different relative hazards for progression to AIDS. Seeking an explanation for this association, we searched HIV-1 protein sequences to determine the number of peptides matching motifs defined by combinations of specific amino acids reported to bind 16 class I alleles. Analyzing complete sequences of 12 clade B HIV isolates, we determined the number of allele motifs that were conserved (occurring in all 12 isolates) and nonconserved (occurring in only one isolate), as well as the average number of allele motifs per isolate. We found significant correlations with an allele’s association with disease progression for counts of conserved motifs in gag (R = 0.73; P = 0.002), pol (R = 0.58, P = 0.024), gp120 (R = 0.78, P = 0.00056), and total viral protein sequences (R = 0.67, P = 0.0058) and also for counts of nonconserved motifs in gag (R = 0.62, P = 0.013), pol (R = 0.74, P = 0.0017), gp41 (R = 0.52, P = 0.046), and total viral protein (R = 0.71, P = 0.0033). We also found significant correlations for the average number of motifs per isolate for gag, pol, gp120, and total viral protein. This study provides a plausible functional explanation for the observed association of different HLA alleles with variable rates of disease progression.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity

Certain HLA-B antigens have been associated with lack of progression to AIDS. HLA-B alleles can be divided into two mutually exclusive groups based on the expression of the molecular epitopes HLA-Bw4 and HLA-Bw6. Notably, in addition to its role in presenting viral peptides for immune recognition, the HLA-Bw4, but not HLA-Bw6, motif functions as a ligand for a natural killer cell inhibitory receptor (KIR). Here, we show that profound suppression of HIV-1 viremia is significantly associated with homozygosity for HLA-B alleles that share the HLA-Bw4 epitope. Furthermore, homozygosity for HLA-Bw4 alleles was also significantly associated with the ability to remain AIDS free and to maintain a normal CD4 T cell count in a second cohort of HIV-1-infected individuals with well defined dates of seroconversion. This association was independent of the presence of a mutation in CC chemokine receptor 5 (CCR5) associated with resistance to HIV-1 infection, and it was independent of the presence of HLA alleles that could potentially confound the results. We conclude that homozygosity for HLA-Bw4-bearing B alleles is associated with a significant advantage and that the HLA-Bw4 motif is important in AIDS pathogenesis.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.

Human immunodeficiency virus 1 reservoir in CD4+ T cells is restricted to certain V beta subsets.

The human immunodeficiency virus 1 (HIV-1) replicates more efficiently in T-cell lines expressing T-cell receptors derived from certain V beta genes, V beta 12 in particular, suggesting the effects of a superantigen. The targeted V beta 12 subset was not deleted in HIV-1-infected patients. It was therefore possible that it might represent an in vivo viral reservoir. Viral load was assessed by quantitative PCR with gag primers and with an infectivity assay to measure competent virus. It was shown that the tiny V beta 12 subset (1-2% of T cells) often has a higher viral load than other V beta subsets in infected patients. Selective HIV-1 replication in V beta 12 cells was also observed 6-8 days after in vitro infection of peripheral blood lymphocytes from normal, HIV-1 negative donors. Viral replication in targeted V beta subsets may serve to promote a biologically relevant viral reservoir.

HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4+ T cell counts ≥ 350 cells/μl and viral load below the limits of detection for ≥1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2–3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log10 copies) day−1, which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log10 copies) day−1 (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4+ T cells.