Analysis of trans-dominant mutants of the HIV type 1 Rev protein for their ability to inhibit Rev function, HIV type 1 replication, and their use as anti-HIV gene therapeutics.

The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.

Factors affecting retroviral vector function and structural integrity.

Recombinant retroviruses are widely used for gene transfer into eukaryotic cells and exhibit significant potential for human gene therapy. Despite the utility of retroviral vectors, their design is still essentially empirical. We have constructed a series of reciprocal, double-gene vectors to compare the dual expression of beta-galactosidase (beta-gal) and neomycin phosphotransferase (neor) in a retroviral delivery system. The first gene of the pair was driven by the viral LTR promoter and the internal gene was regulated by either the SV40 virus early promoter or the cytomegalovirus (CMV) major late promoter. Clones of vector producer cells were isolated either by G418 selection for expression of neor, or by fluorescence-activated cell sorting for expression of beta-gal, and the activity of both genes was evaluated. In general, vectors using the SV40 promoter performed better than those with the CMV promoter, regardless of whether the selected gene was regulated by the LTR or the internal promoter. Southern analysis of clones indicated that loss of beta-gal gene function was related to significant rearrangements and deletions in vector structure. We also found that the arrangement of genes within the vector was important. When beta-gal preceded neor, gene expression and vector stability were markedly enhanced relative to vectors containing these genes in the inverse order.

Activation of the serum response factor by p65/NF-kappaB.

This study demonstrates that the NF-kappaB subunit p65 can act like an accessory protein for the serum response factor (SRF) in transfection assays. p65 functionally synergizes with SRF to activate the transcription of a reporter construct dependent only on the serum response element (SRE). The synergy of the two factors requires neither a kappaB motif nor direct contact of p65 with DNA. Consistent with these results, a physical complex containing p65 and SRF is observed in vitro. Synergy of the factors is independent of the previously described activation domains present on p65, ruling out indirect effects of p65, but synergy is dependent on the activation domain of SRF. The complexing of p65 and SRF is mediated by a segment of the SRF DNA binding domain, a region of the protein which has also been reported to inhibit its own activation domain. We speculate that p65, upon direct or facilitated interaction with SRF, may relieve the inhibitory activity of this segment, thus enabling the activation domain of SRF to become fully functional. In contrast to p65, the p50 subunit of NF-kappaB does not interact significantly with SRF, either functionally or physically. The data suggest the intriguing possibility that NF-kappaB may participate in the regulation of SRE-dependent promoters, expanding the range of activities of this rapidly activatable transcription factor.

Interleukin-2 up-regulates expression of the human immunodeficiency virus fusion coreceptor CCR5 by CD4+ lymphocytes in vivo.

Intermittent interleukin-2 (IL-2) therapy can substantially increase CD4+ T cell counts of human immunodeficiency virus (HIV)-infected subjects. Administration of IL-2 led to transient up-regulation of CCR5 on CD4+ T cells; up to 87% of CD4+ cells expressed CCR5 after a 5-day cycle, with return to baseline levels within 2 weeks. Unlike in vitro studies, CCR5 was coexpressed with CD45RA and CXCR4 on CD4+ T cells after IL-2 therapy. The observed increase in coreceptor expression was not associated with detectable increases in viral replication. IL-2 therapy induced CCR5 expression in >90% of circulating memory CD4+ T cells, determined to be a long-term reservoir of HIV, suggesting significant activation of these cells. These studies demonstrate that levels of expression of HIV coreceptors alone do not always correlate with HIV replication in vivo and that IL-2 therapy activates a majority of memory T cells in the circulation and likely throughout the immune system.

CXCR4 and CCR5 genetic polymorphisms in long-term nonprogressive human immunodeficiency virus infection: lack of association with mutations other than CCR5-Delta32.

Polymorphisms in the coding sequences of CCR5 and CXCR4 were studied in a group of human immunodeficiency virus (HIV)-infected long-term nonprogressors. Two different point mutations were found in the CXCR4 coding sequence. One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. The well-described 32-bp deletion within the CCR5 coding sequence (CCR5-Delta32) was found in 4 of 13 nonprogressors, and 12 different point mutations were found scattered over the CCR5 coding sequence from 8 nonprogressors. Most of the mutations created either silent or conservative changes in the predicted amino acid sequence: only one of these mutations was found in more than a single nonprogressor. All nonsilent mutations were tested in an HIV envelope-dependent fusion assay, and all functioned comparably to wild-type controls. Polymorphisms in the CXCR4 and CCR5 coding sequences other than CCR5-Delta32 do not appear to play a dominant mechanistic role in nonprogression among HIV-infected individuals.

Selective pressure exerted by immunodominant HIV-1-specific cytotoxic T lymphocyte responses during primary infection drives genetic variation restricted to the cognate epitope.

HIV-specific cytotoxic T lymphocytes (CTL) play a central role in the control of HIV-1 replication during primary infection. It has been hypothesized that the appearance of CTL escape mutants represents an important mechanism by which HIV-1 escapes the host cell-mediated immune response. However, evidences for a direct relationship between CTL responses and emergence of CTL escape mutants are still limited. Here we report detailed longitudinal analysis of DNA sequence variation performed over the entire HIV-1 envelope in two subjects during primary HIV infection. Estimates of the frequencies of synonymous (ds) and non-synonymous (dN) nucleotide substitutions were used to identify regions of the HIV-1 envelope which were subjected to significant levels of selective pressure. These regions were shown to comprise defined epitopes recognized by CTL. Furthermore, dN mutation fixed within these epitopes effectively abolished recognition by the host CTL response. These results provide compelling evidence that the CTL epitope mutations directly resulted from the selective pressure exerted by the virus-specific cytotoxic response.

Evidence for rapid disappearance of initially expanded HIV-specific CD8+ T cell clones during primary HIV infection.

Down-regulation of the initial burst of viremia during primary HIV infection is thought to be mediated predominantly by HIV-specific cytotoxic T lymphocytes, and the appearance of this response is associated with major perturbations of the T cell receptor repertoire. Changes in the T cell receptor repertoire of virus-specific cytotoxic T lymphocytes were analyzed in patients with primary infection to understand the failure of the cellular immune response to control viral spread and replication. This analysis demonstrated that a significant number of HIV-specific T cell clones involved in the primary immune response rapidly disappeared. The disappearance was not the result of mutations in the virus epitopes recognized by these clones. Evidence is provided that phenomena such as high-dose tolerance or clonal exhaustion might be involved in the disappearance of these monoclonally expanded HIV-specific cytotoxic T cell clones. These findings should provide insights into how HIV, and possibly other viruses, elude the host immune response during primary infection.

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection.

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.

Kinetics of cytokine expression during primary human immunodeficiency virus type 1 infection.

In the present study, we have determined the kinetics of constitutive expression of a panel of cytokines [interleukin (IL) 2, IL-4, IL-6, IL-10, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha)] in sequential peripheral blood mononuclear cell samples from nine individuals with primary human immunodeficiency virus infection. Expression of IL-2 and IL-4 was barely detected in peripheral blood mononuclear cells. However, substantial levels of IL-2 expression were found in mononuclear cells isolated from lymph node. Expression of IL-6 was detected in only three of nine patients, and IL-6 expression was observed when transition from the acute to the chronic phase had already occurred. Expression of IL-10 and TNF-alpha was consistently observed in all patients tested, and levels of both cytokines were either stable or progressively increased over time. Similar to IL-10 and TNF-alpha, IFN-gamma expression was detected in all patients; however, in five of nine patients, IFN-gamma expression peaked very early during primary infection. The early peak in IFN-gamma expression coincided with oligoclonal expansions of CD8+ T cells in five of six patients, and CD8+ T cells mostly accounted for the expression of this cytokine. These results indicate that high levels of expression of proinflammatory cytokines are associated with primary infection and that the cytokine response during this phase of infection is strongly influenced by oligoclonal expansions of CD8+ T cells.

Short-cycle structured intermittent treatment of chronic HIV infection with highly active antiretroviral therapy: effects on virologic, immunologic, and toxicity parameters.

Although continuous highly active antiretroviral therapy (HAART) is effective for many HIV-infected patients, it can be toxic and prohibitive in cost. By decreasing the total amount of time patients receive medications, intermittent HAART could reduce toxicity and cost. Therefore, we initiated a pilot study in which 10 HIV-infected individuals receiving effective therapy that resulted in levels of HIV RNA <50 copies per ml of plasma and CD4(+) T cell counts >300 cells per mm(3) of whole blood received repeated cycles of 7 days on HAART followed by 7 days off of HAART. Patients maintained suppression of plasma viremia for 32-68 weeks. There was no significant increase in HIV proviral DNA or replication-competent HIV in peripheral CD4(+) T cells or HIV RNA in peripheral blood or lymph node mononuclear cells. There was no significant change in CD4(+) T cell counts, no significant increase in CD4(+) or CD8(+) T cells expressing activation markers or producing IFN-gamma in response to HIV, no increase in CD4(+) T cell proliferation to p24 antigen, and no evidence for the development of resistance to HAART medications. There was a significant decrease in serum cholesterol and triglyceride levels. Thus, in this proof-of-concept study, short-cycle intermittent HAART maintained suppression of plasma viremia as well as HIV replication in reservoir sites while preserving CD4(+) T cell counts. In addition, there was a decrease in serum cholesterol and triglyceride levels. Intermittent therapy may be an important strategy to reduce cost and toxicity for HIV-infected individuals.

The qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent of the initial level of plasma viremia.

Following infection of the host with a virus, the delicate balance between virus replication/spread and the immune response to the virus determines the outcome of infection, i.e., persistence versus elimination of the virus. It is unclear, however, what relative roles immunologic and virologic factors play during primary viral infection in determining the subsequent clinical outcome. By studying a cohort of subjects with primary HIV infection, it has been demonstrated that qualitative differences in the primary immune response to HIV, but not quantitative differences in the initial levels of viremia are associated with different clinical outcomes.