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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.

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.

Objective: To investigate the relationship between V3-specific immune responses and viral quasispecies evolution in 10 HIV-1-seropositive patients enrolled in a phase I trial of recombinant gp160., Methods: Serologic responses to the HIVLAI V3 loop and autologous V3 loop DNA sequences were sequentially determined over a 3-4-year interval., Results: Six patients either seroconverted or had a > or = 42-fold boost in titer to the V3 reagent associated with an average of 3.2 amino-acid changes in their autologous V3 loops. Four patients with < or = 11-fold change in titer to the V3 loop showed an average of 0.75 amino-acid changes. Attempts to measure autologous V3 loop responses in four patients using a peptide enzyme-linked immunosorbent assay technique did not show a distinct binding preference for autologous versus heterologous V3 loop peptides. Thus, we interpret seroreactivity to the heterologous HIVLAI V3 loop to reflect the broadness of the V3 immune response rather than a direct measure of epitope-specific immune pressure., Conclusions: These data suggest that the broadness of serologic responses to viral epitopes are reflected in the rate of evolution of their cognate coding sequences and support the view that the immune response to HIV-1 results in the continuous selection of new viral variants during the course of disease.

To define the epitopes present within the V3 loop sequence recognized by five HIV-1 envelope-specific T-cell lines, a panel of V3 LAI peptides bearing sequential truncations from both the N- and C-terminus was synthesized and tested for their ability to induce proliferation. Each individual T-cell line had a different pattern of response against the truncated V3 peptides, demonstrating the presence of a cluster of CD4+ T-cell epitopes within the V3 loop. To assess the ability of these envelope-specific T-cell lines to recognize and proliferate in response to V3 loops of different viral strains, they were tested against a panel of heterologous V3 loop peptides derived from different viral genotypes within and outside of HIV-1 clade B. There was no proliferative response against heterologous V3 loops by any of the lines, demonstrating that recognition of the V3 epitopes is highly strain specific. One of the defined epitopes was shown to elicit a cytotoxic response as well, suggesting the multifaceted role that the CD4+ T cell might play in HIV-1 disease.

Humoral responses to the HIV-1 envelope were investigated in 30 human volunteers enrolled in a phase I vaccine therapy trial of rgp160 (LAI/LAV) using two techniques that emphasize detection of antibody response against linear (continuous) epitopes: immunoblotting and PEPSCAN. Seven fusion proteins containing large portions from constant regions 1, 2, 3, and 5, and variable region 3 of gp120 and two regions in the transmembrane protein, gp41, were employed in immunoblots to quantitatively measure immune response as a function of immunization. In addition, the entire gp160 (LAI/LAV) envelope protein was constructed in duplicate sets of 211 overlapping 12-mer peptides to fine-map the changes. Immunoblotting defined significant changes in reactivity to epitopes in constant regions; of 28 volunteers completing the trial, the percentage with reactivity against C1 changed from 62 to 100%; for C2, from 0 to 46%; for C3, from 0 to 82%; and for a constant region in gp41, from 25 to 68%. PEPSCAN on a subset (n = 8) of these volunteers identified new reactivity to epitopes throughout the envelope, concentrated in V1, C3, and C5 in gp120 and several peptides in gp41. Completely immunized patients responded to double the number of linear epitopes compared with two patients receiving alum alone. The results verify that the response to rgp160 is significantly broadened after immunization, providing additional evidence that HIV-1-infected volunteers can expand their antibody repertoire against a protein from a pathogen during chronic infection with that same pathogen. These results expand those previously obtained in this patient cohort, by defining explicitly the immunogenic regions recognized postvaccination and by providing methodology for quantitating those changes.