HIV: Surrogate Markers and Novel Therapeutic Approaches
Editor’s Note: The following is a summary of some of the material presented at the 3rd Annual HIV Surrogate Markers: Virological & Immunological Indicators, November 18-19, 1996, and at Novel HIV Therapeutic Strategies, November 20-21, 1996. Both took place in McLean, Virginia, and were sponsored by the Cambridge Healthtech Institute.sd
HIV Surrogate Markers
The continued importance of surrogate markers in HIV clinical trials, as well as the need for the development of additional markers, especially immunological markers, was widely discussed. At the same time, Deyton reiterated his view that, while the use of surrogate markers continues to be useful in the process of accelerated FDA approval of new antiretroviral therapies, clinical end point trials should remain the standard for ultimate approval of therapeutic agents. He also reiterated the view that the value of a surrogate marker for a particular therapy is specific to that therapy. That is, markers acceptable for accelerated approval of antiretrovirals may not be suitable or acceptable with regard to accelerated approval of immunological interventions.
The potential value of assessment of HIV RNA in lymphoid tissues was discussed. Most commonly studied are peripheral lymph nodes and tonsillar tissue. While there is a generally linear relationship between the viral load in plasma and lymph node, the concentration of virus in the latter is usually higher, often by a LOG10 or more. The by now well-known data on the prognostic value of plasma HIV RNA measurements were reviewed. Also reviewed by Heath-Chiozzi were data from a ritonavir trial demonstrating that decreases in plasma HIV RNA as a consequence of antiretroviral therapy are associated with improved prognosis.
St. Clair reported that plasma and cerebrospinal fluid HIV RNA concentrations are correlated, and both are higher in patients with HIV-associated neurological disease than in those without. However, this observation is potentially confounded by the fact that their CD4 counts are also lower. AZT resistance mutations appear simultaneously in CSF and in plasma isolates.
Roederer indicated that changes in cytokine production patterns, such as the putative Th1/Th2 dichotomy, are the consequence of changes in the relative proportion of T cell subsets, rather than functional changes within a subset. Thus, naïve CD4 and CD8 cells are progressively lost during infection and are lost at a faster rate than memory CD4 cells; CD8 memory cells expand in number initially. Naïve and memory cells have distinct, albeit overlapping, cytokine synthesis patterns. Naïve T cells produce small amounts of IL-2, but no IL-4 or IFN, while memory cells produce IL-2; IL-4 and IFN-g are produced by some memory cell subsets. When HIV-infected and non-HIV-infected patients are compared, there is little difference in the ability of any particular subset of T cells to produce IL-2 and IFN-g. The loss of CD4 memory cells is associated with an "activation" phenotype which is pre-apoptotic. Unusual T cell subsets expand, including cells similar to those that undergo non-thymic conditioning.
Giorgi reported that, with progression of HIV infection, an increasing proportion of T lymphocytes express CD38, an activation marker. The presence of the latter on CD8 cells is a potent adverse prognostic factor. In contrast, the early development of a population of HLA-DR+ CD38+ CD8+ cells shortly after HIV seroconversion is associated with slow progression of disease. These are a population of cells associated with the production of soluble HIV suppressive factors.
CD28 is an important costimulatory molecule of T lymphocytes. There is a progressive loss of CD28+ CD8+ cells during HIV infection with a complementary expansion of CD28- CD8+ cells. CD28- cells are replicatively senescent. These cells have shortened telomere length (a reflection of senescence) to an extent similar to that seen in non-HIV-infected octogenarians.
Novel HIV Therapeutic Strategies
Multiple potential targets and approaches to the treatment of the HIV-infected patient are under investigation. These include new targets of antiretroviral drugs, such as HIV integrase, immunosuppressive agents designed to reduce the immune activation of HIV disease, as well as immune restoration and replacement strategies.
Among the most interesting approaches are those using gene therapy and involving the introduction of genes encoding for protective molecules into mature lymphocytes or into hematopoietic stem cells. Some of these were discussed by Sarver.
Rev codes for a protein that binds to the Rev-response element (RRE) and is necessary for the transport of unspliced (HIV genomic) RNA or singly spliced RNA from the nucleus to the cytoplasm. Modalities with potential to interfere with this vital step in HIV replication include introduction of antisense molecules, ribozymes, a transdominant Rev mutant, and RRE decoys. CD4 cells transduced with a Rev transdominant mutant, RevM10, which binds to RRE but fails to recruit necessary cellular cofactors with resultant lack of RNA export, fail to support HIV replication. Furthermore, Nabel has demonstrated that autologous transduced CD4 cells possess a survival advantage when reinfused into HIV-infected individuals. Studies are planned that involve reinfusion of RevM10 transduced autologous peripheral stem cells.
Another gene therapy approach that has reached clinical trials in humans is the use of ribozymes, RNA molecules that hydrolyze RNA at specified targets. However, a variety of challenges to successful gene therapy remain, including HIV and cell target choice, determination of optimal cell transduction method, development of the optimal means of ex vivo expansion of transduced cells, and development of appropriate means of in vitro and in vivo methods of assessment of results.
There are a variety of potential strategies for immune-based therapies of HIV infection. These include the blocking of the associated non-specific immune activation, vaccination aimed at enhancing cellular immunity against the virus, replacement of defective CD4 cell function with exogenous cytokines, replacement of defective immune cells (CD4+ and CD8+ T lymphocytes, dendritic cells), and immune reconstitution with HIV-resistant stem cells.
Only a maximum of 1-2% of peripheral CD8+ T cells demonstrate HIV-specific cytotoxicity. Thus, there are a total of approximately 108 circulating and 1010 total HIV-specific cytotoxic T lymphocyte precursors in the HIV-infected individual. However, these do not function well. Freshly recovered CD8+ lymphocytes from HIV-infected subjects have reduced expression of z, the T cell receptor signaling molecule. This is rapidly corrected in in vitro culture conditions. One approach to immunotherapy of HIV infection involves the autologous transfusion of HIV-specific cytotoxic CD8+ cells. Such approaches are ordinarily limited by the MHC-restriction necessary for CD8 cytotoxicity and are further limited by the relatively low number of precursor frequencies.
Lieberman administered autologous HIV-specific cytotoxic T lymphocytes to 14 HIV-infected patients without adverse effect. The efficacy of such therapy awaits randomized clinical trials.
A means to circumvent the need for MHC-restriction and low or absent precursor frequency has been developed by scientists at Cell Genesys, Inc. and was discussed by Roberts. This consists of antigen-specific chimeric immune receptors consisting of the transmembrane z chain, the signaling component of the T cell receptor, connected to an extramembrane domain that binds specific antigen. The binding domain can be a monoclonal antibody or a molecule such as CD4. Transduction of cells, such as lymphocytes, with a gene encoding for such a receptor with the CD4 molecule attached, produces cells with excellent cytotoxicity against HIV-infected cells expressing gp120, the ligand for CD4.
This approach can be used with other cell types and can be aimed at non-viral targets, such as tumor cells. Roberts reported that transfusion of murine stem cells transduced with a CD4 universal receptor into SCID mice led to stable (for at least as long as 8 months) high-level expression and rejection of Raji cell tumors engineered to express HIV gp120.
Genetically altered autologous stem cell therapies may one day provide a safe and effective means of treatment of the HIV-infected patient. Johnson reported that human CD4+ cells derived from CD34+ cord blood cells (hematopoietic stem cells) by in vitro cultivation with fetal or neonatal thymic stromal cell monolayers transduced with an anti-tat gene were resistant to productive infection with HIV-1. As indicated above, stem cell therapy using the mutant gene RevM10 is approaching the start of Phase I studies.
The ability of the b-chemokines, RANTES, MIP-1a and MIP-1b, as well as the a-chemokine, SDF-1, in blocking HIV infection raises the possibility that novel therapeutics could be developed based on this activity. The primary receptor for the b-chemokines, CCR5, is present on cells of the monocyte macrophage series, while that of SDF-1, fusion, is present on T cells. This distinction is most apparent with cell lines, while primary T cells often exhibit both types of receptors. The chemokines prevent HIV-1 fusion, not binding to the coreceptor. Non-toxic therapy with derivatives of these molecules will require, however, that their chemoattractant and HIV-binding activities involve distinct domains. Berger reviewed the relative cell specificity of the receptor-ligand pairs and also discussed the recent report of the apparent resistance to HIV-1 infection associated with a 32 bp deletion in the CCR-5 gene. This genetic defect, present in homozygous form in approximately 1% of Caucasians of western European origin, may prove to provide absolute protection against HIV-1 infection.
IL-16 is another soluble cell product that inhibits HIV replication in vitro. Parada reported evidence indicating that this is the result of repression of HIV-1 promoter activity and inhibition of CD3-dependent lymphocyte activation. In addition, IL-16 induces high affinity IL-2 receptors. Combined exposure of CD4+ T lymphocytes to both IL-16 and IL-2 results in synergistic expansion of CD4+ lymphocytes.
Roederer reviewed the role of glutathione (GSH) in HIV infection. GSH is the major intracellular antioxidant and is necessary, not only for protection against oxidative stress, but for many normal cellular functions. Many HIV-infected patients exhibit GSH depletion, and the extent of depletion is a powerful predictor of mortality in HIV-infected adults with CD4+ lymphocyte counts less than 200. Studies have now demonstrated that the oral administration of N-acetylcysteine (NAC) to HIV-infected individuals with significant GSH depletion results in increased GSH levels within their lymphocytes.
New developmental approaches to antiretroviral therapy were discussed. Wang described data concerning DNP-oligonucleotides designed to fill the entire binding cleft of the HIV-1 reverse transcriptase. Such molecules, in addition to potent anti-HIV activity, may be insensitive to multiple point mutations in HIV. Henderson reviewed the critical role of retroviral nucleocapsid zinc fingers in processing of nucleic acids throughout the viral replication cycle and the potential role of inhibitors of zinc finger function as antiretroviral agents. Most inhibitors contain disulfide bonds between aromatic structures that act by oxidizing the zinc fingers. Hazuda discussed the role of HIV-1 integrase in viral replication and the search for effective inhibitors of this enzyme.
HIV-1 replication requires ribosomal frame shifting in the expression of the gag and pol genes because they overlap by one nucleotide. Thus, in order for that nucleotide to be expressed in both gag and gag-pol proteins, the ribosomal interaction must slip back one nucleotide after dealing with the gag gene. This process of ribosomal frame shifting is not present in humans and thus represents a potential target for antiretroviral therapy. Green reported the discovery of a compound, RG 501, that inhibits HIV-1 replication by stabilizing the gene and preventing frame shifting.
HIV-1 viral protein R (Vpr) arrests the host cell cycle in G2 phase and also facilitates the infection of nondividing cells as well as viral replication in those cells. Increased viral replication is particularly evident in monocytes. Weiner discussed Vpr and the potential of inhibitors of Vpr function as candidates for antiretroviral therapy.
Huang reviewed the potential antiretroviral effect of several plant ligans that appear to act by inhibiting HIV transcription and Tat transactivation processes. Babish discussed PN355, a compound isolated from an herb used in Asia for treatment of viral respiratory tract infections, as a potential antiretroviral agent. PN355 is a diterpene lactone that inhibits cdk1, a cyclin-dependent kinase that coordinates the progress of the cell through the G2 and M phases of the cell cycle. It has an IC50 against HIV-1 of approximately 0.7 mcg/mL and acts synergistically with AZT. Litwin discussed CD4-IgG2, a novel high avidity tetrameric CD4-immunoglobulin fusion protein that inhibits the binding of HIV-1 to CD4+ cells and which is entering Phase I clinical trials.
Thus, the success of the HIV protease inhibitors has not led to a diminution of enthusiasm for the pursuit of a wide range of new approaches to the establishment of additional surrogate markers of therapeutic success or of the development of new treatment strategies. We should all look forward to next year’s meetings.