Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1.

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.

Correlation of tumor suppressor P53 RNA expression with human immunodeficiency virus disease in rapid and slow progressors.

OBJECTIVE: To determine the relation between P53 tumor suppressor RNA expression and human immunodeficiency virus (HIV) disease progression. STUDY DESIGN/METHODS: A quantitative assay of P53 RNA expression was used to analyze a cohort of HIV-negative persons. The assay was then used in longitudinal and cross-sectional studies of HIV slow and rapid progressors. RESULTS: We demonstrate first that P53 expression in peripheral blood mononuclear cells from HIV-1-seronegative persons is minimal. Longitudinal studies in a small cohort of HIV-1-infected slow and rapid progressors reveal that rapid progressors seem to have greater P53 RNA expression over time. This was validated in a cohort of 26 HIV-1-infected persons in whom the expression of P53 RNA was significantly greater in persons with rapid progression of HIV-1 disease. CONCLUSION: These data suggest that P53 RNA expression may play a role in the pathogenesis of HIV-1 disease, though the mechanism of this interaction remains unknown.

Transcriptional effects of superinfection in HIV chronically infected T cells: studies in dually infected clones.

We had previously shown that chronically infected ACH-2 cells (HIVLAI) could be superinfected with HIVRF, that the frequency of superinfection increased with time, and that the transcription of the superinfecting virus exceeded that of the host HIVLAI provirus. In contrast, ACH-2 cells superinfected with a nef-substituted neomycin-resistant (proNEO) provirus were not detectable by DNA polymerase chain reaction (PCR) until geneticin (G418) was added, suggesting that the ability to propagate progressively in culture may be HIV strain specific. Clonal populations of ACH-2 superinfected with proNEO did not demonstrate preferential transcription of the superinfecting virus. However, clones of ACH-2 superinfected with HIVRF (ACH2/RF) showed a preponderance of HIVRF transcripts similar to that seen in bulk populations. Induction of the superinfecting virus by phorbol ester (PMA) occurred more rapidly than the hose provirus and did not equalize transcriptional activity. PCR-derived long terminal repeat (LTR) fragments and Tat cDNAs from A3.01 cells acutely infected with HIVRF or from ACH-2 cells were sequenced and tested for transactivation. The HIVLAI LTR was two to three times more Tat-responsive than the HIVRF LTR. TatRF was two to three times more transcriptionally active on either LTR than TatLAI. Demethylation with 5-azacytidine did not significantly affect HIV expression from the HIVLAI host provirus of superinfected ACH2/RF cell clones. These data suggest that the mechanism of preferential transcription in HIVRF superinfected ACH2/RF may be attributed to the Tat/TAR axis and the effect of the specific locus of host proviral integration.

Inhibition of HIV replication by sense and antisense rev response elements in HIV-based retroviral vectors.

The life cycle of human immunodeficiency virus type 1 (HIV-1) is critically dependent on the transregulatory proteins Tat and Rev. Tat increases the production of HIV-specific mRNAs by direct binding to the transactivation response (TAR) element located at the 5' end of all HIV transcripts. In contrast, Rev uses a complex RNA stem loop structure, the Rev response element (RRE), which is found in full-length and singly spliced HIV transcripts. Rev is required for the cytoplasmic expression of full-length mRNAs encoding Gag, Pol, and Env structural proteins. The complex intracellular interactions between Tat, Rev, host cell factors, and their respective RNA response elements should be susceptible to interdiction by genetic therapies designed to introduce and express novel genetic information. We show that the expression of antisense RREs inhibited the cytoplasmic expression of RRE containing HIV-1 transcripts. HIV-based retroviral vectors containing either the antisense (-) or sense (+) RREs inhibited HIV replication in transient transfections. The production of full-length HIV mRNA was also decreased significantly by the expression of RREs in either orientation. Interestingly, there was a paradoxic increase in HIV p24 gag production at low levels of inhibitor; this effect may have been the result of encapsidation of RRE-containing HIV-based retroviral vectors. The data suggest that the introduction and inducible expression of RRE-containing, HIV-based retroviral vectors may have therapeutic value in HIV infection.

Consequences of stable transduction and antigen-inducible expression of the human interleukin-7 gene on tetanus-toxoid-specific T cells.

Interleukin-7 (IL-7) has previously been shown to increase antigen-specific immune responses; the effect of IL-7 on human antigen-specific T cell lines has not directly been addressed. A tetanus-toxoid (TT)-specific T cell line exhibited increased proliferation in the presence of exogenous IL-7, suggesting that IL-7 may be useful in the potentiation of immune responses to defined microbial antigens. Murine retroviral vectors encoding the human IL-7 gene and the neomycin phosphotransferase gene (neoR) were packaged into murine retroviral particles, and supernatants containing these retroviral vectors were used to infect a CD4+ lymphoblastoid cell line. Stable integration of the retroviral vector and constitutive expression of the IL-7 gene were observed. Successful IL-7 gene transduction into TT-specific T cells was also accomplished. Detection of neoR DNA sequences and expression of IL-7-specific mRNA increased with selection in geneticin. Production of IL-7 in these cells was induced by exposure to TT. Production of IL-4, IL-6, and interferon-gamma (IFN-gamma) was detected after antigenic stimulation; there was, however, no effect of IL-7 on the pattern or kinetics of cytokine production by these cells. Human IL-7 transduced cells showed greater proliferation to TT than control T cells, particularly at subthreshold TT concentrations. These dta imply that genetic modification of antigen-specific T cells may be a plausible strategy for the study and manipulation of the immune responses to microbial pathogens.

Consequences of human immunodeficiency virus type 1 superinfection of chronically infected cells.

Infection of T cell lines by the type 1 human immunodeficiency virus (HIV-1) is associated with downregulation of the CD4 receptor and resistance to further HIV-1 infection, the phenomenon of viral interference. The ACH2 cell line, a model for chronic HIV-1 infection, possesses a single integrated copy of the HIV-1 strain LAI, is essentially CD4 negative, and can be induced to make virus by a variety of stimuli. We utilized the known sequence differences between HIVLAI and HIVRF to devise a polymerase chain reaction (PCR) strategy that permits reliable and quantitative discrimination between the two strains. We demonstrate that ACH2 cells can be superinfected by HIVRF at a frequency of 60-300 HIVRF genomes/10(4) ACH2 cells and that the frequency of superinfection appears to increase with time. Reverse transcription of ACH2 mRNA from days 13, 27, and 38 postinfection allowed a similar PCR strategy (RT-PCR) to be used to analyze full-length HIVRF- and HIVLAI-specific transcripts. These data suggested that superinfection of ACH2 with HIVRF results in an increase in expression of both HIVRF and HIVLAI mRNA. From day 13 to day 38 postinfection there was an increase in the relative expression of HIVRF compared with HIVLAI. By day 38, when only 1.1% of HIV DNA sequences were HIVRF derived, roughly 80% of the HIV-specific full-length mRNA was HIVRF in origin, with a concomitant decrease in HIVLAI transcription.