By David Margolis MD, UNC Chapel Hill and the Collaboratory of AIDS Researchers for Eradication (CARE)
Daria Hazuda of Merck was awarded the honor, unusual for a scientist from industry, of presenting the Bernard Fields lecture. Dr. Hazuda outlined the difficulties and challenges in the development of ART byt telling the story of the ca. 10 year struggle to development effective HIV integrase inhibitors, as a paradigm to foretell the challenges ahead in new attempts to develop eradication therapies.
Two plenary talks, two oral abstract sessions, and numerous posters at CROI this year focused attention on expanding efforts to eradicate HIV or gain drug-free remission. The rising profile of this area at CROI reflects the reality that we must either acquiesce to treat millions of HIV-infected people with antiretroviral therapy (ART) forever, or both find ways to definitively prevent infection and to decisively eradicate infection. Again notable at CROI this year were several exhaustive and demanding studies of persistent infection. These were emblematic of the types of studies that are required to make progress in understanding the persistence of HIV infection despite ART, and required if the field is ever going to make progress towards eradication or drug-free control of HIV infection. Again, the investigators and funders of these efforts, and most especially the patient volunteers, are to be applauded for their efforts and contributions.
As attempts began to develop HIV integrase inhibitors in the 1990’s, the detailed knowledge of HIV integrase protein structure and function was missing. Also unappreciated was the composition of the “intasome,” the complex of HIV proteins and host proteins that bound the product of reverse transcription, the viral DNA genome, carried it into the nucleus, and mediated integration. Over time, an understanding of the DDE catalytic core of integrase was gained, but initial inhibitors that were developed appeared to inhibit steps of HIV integration in the test tube, and appeared to inhibit HIV in cell culture studies, but were later found to be inhibiting replication in other ways, without actually inhibiting HIV integration. True integrase inhibitors were found to have a unique molecular mechanism of action, eventually revealed by biochemical studies and confirmed by X-ray crystallography. They bind with tightly to integrase only when the enzyme is in a specific complex with viral DNA. This binding requires viral DNA as well as divalent metals at the active site of the integrase enzyme, and specific interactions of the inhibitor with the enzyme. This interaction between both the ends of the viral DNA and the integrase active site, two critical highly conserved elements of the HIV virus, is what makes this molecular event uniquely vulnerable to inhibitor.
The breakthrough discovery occurred when assays were developed to measure the strand joining/strand transfer step of integration — when the processed ends of the HIV DNA genome were actually stitched in to human DNA — and this step was found to be rate-limiting for integration. Rate-limiting steps are the slowest in any chemical reaction, and the best target for drug inhibitors. A subsequent screening program at Merck described L-731,988, the first true, potent HIV integrase inhibitor. This inhibitor allowed pre-integration processing of the end of HIV DNA, but specifically prevented strand-transfer in to the human genome.
Following this discovery, advances were slowed by pre-clinical toxicities in animal studies, but the basic model for an HIV integrase inhibitor, the pharmacophore, upon which improvements and modification could be built, had been defined. The discovery of Raltegravir followed, and hence the advancement in to the clinic of the new class of ART that we all knowDr. Hazuda likened this process to the one that is now ongoing to develop eradication therapies for HIV infection. One major path for current eradication strategies involves the disruption of latency with histone deacetylase (HDAC) inhibitors, followed by the clearance of these latently infected cells by a second mechanism such as induced cell death or immune clearance. Dr. Hazuda discussed the current effort at Merck to develop new HDAC inhibitors with selectivity for the HDAC isoforms most important for the maintenance of HIV latency. Further, she suggested from preliminary data that different HDAC isoforms might be of greater or lesser importance in different resting memory T cell populations. In other words, different cell populations within what we now globally call “the latent reservoir” might be more or less responsive to different HDAC inhibitors. Finally she described a new screen for anti-latency compounds, performed via the CARE collaboratory effort, to find new molecules that both disrupted HIV latency by themselves and had increased activity in the presence of vorinostat (SAHA), the first HDAC inhibitor proven to disrupt latent HIV infection in man (Archin 2012 and abstract 50LBThe first full day of CROI opened with two plenary lectures related to HIV-1 eradication strategies. Robert Siliciano of Johns Hopkins gave an overview of persistent HIV infection despite ART. Siliciano briefly reviewed viral replication dynamics, the dynamics of viral suppression on ART, and the founding of the best-described latent reservoir, that of latent, quiescent infection of resting memory CD4+ T cells.
The gold-standard assay to quantitate latent infection of these cells involves the harvesting of white blood cells, the isolation of resting CD4+ T cells through the use of antibody selection, and then viral culture assays done in “limiting dilution.” First described in his lab, that of Tony Fauci, and that of Doug Richman in 1997, the quantitative viral outgrowth assay (QVOA) involves the culturing of multiple dilutions of cells (eg. 2.5 million per well, 500,000 per well, 100,000 per well) to allow the back-calculation of the frequency of latent infection of resting CD4 cells. Although this is a labor-intensive assay, Siliciano described important studies that illustrated why it is the gold standard for the measurement of HIV latency.
He described a study led by Janet Siliciano (Erikson et al. PLoS Pathogens 2013) that compared various assays of HIV persistence. The frequency of either total HIV DNA, chromosome-integrated HIV DNA, or 2-LTR HIV DNA circular forms in either PBMCs or resting CD4 cells of patients treated in either acute or chronic HIV infection was vastly more frequent than actual replication-competent HIV within these same cells. HIV DNA was sometimes more than 1000-fold more frequent, and on average about 300-fold more frequent. HIV DNA within rectal tissue biopsies was even more frequent. The measures of DNA were so unpredictable that there was not even a correlation of HIV DNA per million cells to outgrowth of virus per million cells. The single-copy assay of low-level viremia was also not well correlated with the frequency of latent infection, and its dynamic range was poor as SCA was very often < 1 c/ml.
It has long been known that most of the HIV DNA molecules that can be detected are dead, “fossils” of prior HIV replication, but a second study by the Siliciano laboratory elegantly explained exactly what had happened to all the HIV DNA. The lab carefully went back to QVOA cultures, and recovered HIV RNAfrom cells within culture wells that did not produce viral particles. Careful serial PCR amplification of the RNA detected within cells that did not produce virions yielded viral sequence that could be examined to explain why no functional virus was found. Of these RNA molecules, termed “non-induced proviruses,” nearly 30% were found to be hypermutated via the innate immune effect of the human ApoBEC3G protein, which serves to protect the human genome against foreign RNAs. Another 50% of the RNAs contained large internal deletions, and ca. 8% encoded other mutations such as missense or packaging signal mutations. Only about 12% of the HIV RNAs appeared to have completely intact sequences.
Some of this 12% of intact RNAs could have other undetected defects, but this suggests that while HIV DNA vastly overrepresents the size of the true latent reservoir of replication competent HIV, the viral outgrowth assay may under-represent the size of the reservoir. The true size of this reservoir must lie between the roughly 1 in 2 million resting CD4 T cells found in most patients treated effectively with durable therapy, and as much as 1 in 20,000 cells if every intact RNA encodes a functional virus.
Siliciano then reviewed the study of Shan et al., presented at CROI last year, that found that after reversal of latency in an in vitro model, infected resting CD4 T cell did not necessarily die from viral cytopathic effects, and that cytolytic T lymphocytes from many patients failed to recognize or kill these cells, in this laboratory culture system. While this finding may illustrate a worst-case scenario, it emphasizes an important point that we cannot depend on virus expression to kill and clear every latently infected cells. Finally, Siliciano presented modeling studies to suggest that a 3-log (1000-fold) reduction in the size of the latent reservoir would be required to delay viral rebound for 1000 days after ART interruption. It seems that if this is true, then research assays should be able to measure a therapeutic depletion of the latent reservoir, and until we achieve such a huge reduction of latent infection, clinical trials that involve ART interruption should not be necessary.
Immediately following this talk, Francois Dabis of the Bordeaux School of Public Health and INSERM presented a timely talk (abstr. 17) entitled “Reality Check: Is the End of AIDS in Sight?” This beautiful and thoughtful talk outlined the progress in rolling out ART, and employing prevention efforts that have for the first time reduced the incidence of HIV infection in some places. Most of the talk, however, outlined the vast effort that will be needed to extend counseling, testing, treatment, and prevention to really achieve an “AIDS-free generation.” HIV cure and an effective HIV vaccine were mentioned in passing, but obviously these advances, if they can be achieved, will accelerate progress towards to goal of the end of AIDS.
The first oral abstract sessions entitled “Is There Hope for HIV Eradication?”. There are many diverse opinions about the possibility of developing eradication therapies. Is it possible? When will it happen? Why is it taking so long? Don’t you know it can never be done? The truth is no-one knows if safe and scalable approaches to eradication can be achieved. I think so, but a lot of people do not. Let us just work on it rationally and try to get there, with hope but without unrealistic expectations. Several findings presented in this session…..
The session began with Hiroyu Hatano of UCSF reporting the results of a raltegravir (RAL) intensification study in ART-suppressed patients. This was a randomized, double-blinded, placebo-controlled study of intensification to assess whether intensification leads to a decrease in low-level viral replication, as detected by an increase in 2-LTR circles. Patients suppressed to <40 c/ml on ART for more than 1 yr, with CD4 counts >350 cells/ml were enrolled. 15 intensified with RAL, and 16 intensified with placebo for 24 weeks.
From my point of view, it is important to point out that it is not clear to me that 2-LTR circles measure “residual replication” at all, which would be full, effective rounds of the HIV life cycle, resulting in new integrated genomes that can produce new virions. 2-LTR circles are dead end products of reverse transcription, and cannot integrate or produce virions. We can measure low-level viremia in some patients despite ART, and it is reasonable to assume that some of these circulating particles can enter cells, and some smaller number may complete reverse transcription, even in the presence of RT inhibitors. Cell culture studies have shown that RAL increases the number of 2-LTR circles in treated cells, and so the addition of RAL is likely to increase 2-LTR circles in treated patients who cells possess low levels of HIV reverse transcripts.
The findings, all expressed as ratios, without any absolute values presented, found that the RAL group had a significant increase in 2-LTR circles compared to baseline (estimated ratio of week 1 to 0: 4.7, p = 0.0045; ratio of week 2 to 0: 3.4, p = 0.046; and ratio of week 8 to 0: 3.6, p = 0.033). 9/15 subjects receiving RAL had an increase in 2-LTR circles at weeks 1 or 2, compared to 3/16 subjects in the placebo group. Hatano also reported that while IL-6 and flow-mediated vasodilation did not change, D-dimer did decrease on average in RAL-treated patients. The fact that one of the 3 measures tested changed led her to speculate that RAL was decreasing inflammation by decreasing residual replication.
Ya-Chi Ho from the Siliciano laboratory then presented details of the “non-induced provirus” study introduced by Siliciano in his plenary talk (abstr. 43). As discussed above, while 1 out of 1 million resting CD4+ T cells can be induced to release replication-competent virus, 100 to 1000 out of 1 million resting CD4+ T cells carry HIV-1 proviral DNA. To examine the intermediate step between proviral DNA and produced virions, HIV RNA, Ho amplified cell-associated HIV RNA from tissue culture wells in which replicating viral particles were not detected. These “non-induced proviruses” were then reconstructed by PCR with HIV LTR primers, and internal gag PCR performed to verify clonality (ie there was only one viral RNA species per well). The viral genome was then reconstructed by internal PCR, and sequencing to examine the viral genome performed. Ho found that there were large internal deletions in the genomes of 50% for the RNAs found, deletions in the virion packaging signal in 4%, 10% had internal mutations, and 30% bore C-to-U mutations characteristic of the effect of the human ApoBEC defense system. This left about 12% of the RNA genomes potentially intact. These precise numbers should be considered preliminary, as the work is clearly ongoing, but they should not change very much with further work.
Importantly, Ho then tested the ability of these reconstructed genomes to replicate in PBMCs in culture. Most of these genomes that were identified as “non-induced proviruses” could grow when they were reconstructed and grown in optimal conditions in culture. The proportion of these viruses that might be thought of as an unaccounted-for threat that was quoted here was about 80%, but again this is ongoing work. Nevertheless, this work makes the point that most of the DNA we detect is junk, and the gold-standard outgrowth assay must under-represent the true in vivo frequency of the replication-competent latent viral reservoir. The extent of this under-estimate was said to be up to 50-fold. So at worst, the frequency of latent virus is 50 infected cells per million. While this might sound like terrible news to some, I have to say that I am not so bothered by it. Potent, selective drugs could purge virus from millions or even billions of cells in a day — the challenge will be to do this safely and effectively. The great importance of this work is that it clarifies the understanding of the strengths and weaknesses of our current latency assays.
Maria Buzon (abstr. 44) from the Lichterfeld laboratory at the Ragon Institute presented evidence that latent HIV infection can reside in a recently described type of CD4+ memory cell, the T memory stem cells (Tscm). These cells represent a recently discovered subpopulation of T cells that persist for extremely long periods of time and are maintained by stem-cell like properties, such as self-renewal and homeostatic proliferation. After long-term ART, high per-cell levels of HIV-1 DNA were observed in CD4 Tscm that in several patients exceeded corresponding levels in central memory or effector memory CD4 T cells. Buzon then showed that replication-competent HIV could be recovered from these cells in 3 patients in an assay similar to QVOA, although it was not possible to tell precisely how frequent infection was. Although it was reported that longitudinal samples of HIV DNA showed an increasing representation of HIV DNA in CD4 stem cells, further studies will be needed to validate this observation, and demonstrate that replication-competent HIV persists in this new cell population. The investigators concluded that Tscm serve as a long-lasting reservoir for HIV-1 that importantly contributes to viral persistence, and that targeting this specific cell compartment by immunological or pharmacological interventions may contribute to reducing viral persistence in vivo. I feel that these observations are important and deserve follow-up. In the best of all possible worlds, these cells will be no more difficult to target than the entire population of central memory T cells, and will not require novel agents to specifically attack them. However, while it may be some time before we have therapies that can fully and effectively target the largest pool of latent infection in central memory cells, the potential of an underappreciated population of cells such as central memory stem cells to be recalcitrant and resistant to eradication approaches should be kept in mind.
In a very similar vein, Natalia Soriano-Sarabia (abstr. 46) from my laboratory at UNC described latent infection in gd T cells. gd T cells are a CD4-negative T cell population that are found mostly in the tissue. However, we found that isolated peripheral blood gd T cells can be infected in vitro through upregulation of the CD4 receptor and propose that HIV-induced immune dysregulation may allow infection of CD4-negative gd T cells in vivo. We then measured the contribution of gd T cells to the HIV reservoir by QVOA in 10 patients on long-standing antiretroviral therapy. In seven patients we recovered latent but replication-competent HIV from highly purified gd T cells. In five patients, HIV was recovered when as few as 5,000 cells were co-cultured reflecting a high frequency of infection. Peripheral gd T cells are a previously unrecognized reservoir in which latent HIV infection is unexpectedly frequent. Again, to repeat myself, in the best of all possible worlds, these cells will be no more difficult to target than the entire population of central memory T cells, and will not require novel agents to specifically attack them. But to repeat myself verbatim, while it may be some time before we have therapies that can fully and effectively targeting the largest pool of latent infection in central memory cells, the potential of an underappreciated population of cells such as central memory stem cells to be recalcitrant and resistant to eradication approaches should be kept in mind.
In the ideal world, we would discover a cell surface marker that would tell us that a cell is latently infected. In an effort towards this goal, Fabio Romerio (abstr. 45) at the Institute of Human Virology in Baltimore studied primary CD4 cells that were infected in the laboratory in a system established to mimic latent HIV infection, described in his laboratory in 2008. In this system, latently infected cells remain p24 HIV antigen-positive for a number of days after the virus has entered latency. This allowed Romerio to study cell surface markers and host cell gene expression in two pools of otherwise identical primary cells (p24 positive vs. negative) that differed only by the fact that one of the pools was latently infected.
He found high expression of the cell surface marker CD2 on latently infected cells, and validated this by comparing total HIV-1 DNA copies and viral RNA produced following ex vivo reactivation in the CD2-high vs. CD2-low cells of six HIV-1 subjects successfully treated with ART. Microarray analyses revealed profound differences in the gene expression profiles of latently infected and uninfected cells generated in vitro, including 33 differentially expressed transcripts encoded for surface markers. High expression of the surface receptor CD2 identifies resting memory CD4+ T cells more likely to harbor replication competent HIV-1, but unfortunately (or fortunately) most CD2-high cells are not infected. Future work may identify other useful markers, and perhaps combinations of markers will prove even more useful.
Jintanat Ananworanich of Chulalongkorn University, Bangkok, reported on the effect of immediate ART during acute HIV infection (AHI) on the latent reservoir, measured as HIV DNA in central memory CD4+ T cells (abstr. 47). This work is part of a long-standing collaboration with the US Military HIV Res Program. In this study called RV254 or SEARCH 010, 52,767 high-risk patients (mostly MSM) were screened for HIV infection, and 89 were found in AHI. In a heroic effort, ART was given within 5 days of diagnosis, and within a median of two days, and research studies done (when consent given) within 3 days.
68 patients were enrolled during AHI classified as Feibig I or II stage (stage I: HIV RNA+, p24 antigen-negative, 3rd generation EIA-negative; stage II: HIV RNA+, p24 antigen-positive, 3rd generation EIA-negative), and 21 patients in Feibig III (EIA 3rd gen-positive). Remarkably, more than 20 sigmoid colon biopsies to study GALT-associated HIV were done, as well as PBMC studies.
PBMC and Central memory CD4+ T cell HIV DNA content was lower in Fiebig I than Fiebig II or Fiebig III patients. Almost all Fiebig I subjects had undetectable integrated DNA, compared with half or less of the Fiebig II and III subjects. However PBMC total and integrated DNA became undetectable in Fiebig III after 12 weeks ART, and there was a major decline in HIV DNA in colon biospies, with 75% undetectable after 12 weeks of ART. Overall, Fiebig I subjects exhibited low reservoir size as measured by DNA content, with no detected HIV integrated DNA in PBMC and memory CD4 T cell subsets before and after ART. Early ART intervention restricted the seeding of the HIV reservoir in long-lived TCM. However, while reservoir size is small, it is not ablated by early ART as demonstrated in several studies of ART in acute infection in which reservoir was measured by QVOA.
The next presentation was certainly the most talked-about of the meeting. Deborah Persaud of Hopkins (abstr. 48LB) reported on a child treated by Hannah Gay in Mississippi. The newborn first came to medical attention at birth, with no prenatal care. As has been reported by Mark Mascolini earlier on NATAP, and many in the media, this infant, born to an HIV-infected mother who was not on ART, was found to have HIV DNA in PBMCs and HIV RNA in plasma at birth. These tests were repeated and confirmed on the 2nd day of life, with sequencing showing that the virus was genetically matched to the mother’s virus. With this information, AZT/3TC/Nevirapine was started at 31 hours of age; given at therapeutic doses for the first 7 days of life. A key point here was that the drugs were given with full-dose twice a day nevirapine, as if the baby was known to be HIV-infected, instead of a lower dose that would have been given to the baby in the usual circumstance of pre-natal prevention of transmission (ie, mother treated during pregnancy, baby treated temporarily after birth).
The case becomes unusual as the child’s first viral load at birth was 19,812 c/ml and the mother’s was reported to be lower (2423 c/ml). On therapy, the baby’s HIV RNA declined on days 7, 12, 20, and 29 to 2617, 516, 265 and <48 c/ml respectively. At day 7 ART switched to Kaletra/AZT/3TC, and given through 18 months. The child remained on therapy with undetectable HIV RNA until 18 months of life, when the mother stopped treating the child and did not return to clinic. The child was found again at 24 months of age, and research studies found a 1 copy/ml of HIV RNA in plasma and a few dozen copies of HIV DNA in blood cells, but HIV ELISA and Western blot were negative, as were standard HIV DNA and RNA tests were negative. Research tests at this time found no HIV specific immune response in CD4 or CD8 to HIV Gag or Nef proteins, with normal levels of immune activation. As little blood could be drawn from the child, about 150,000 cells were tested by a VQOA assay, and no virus recovered (limit of detection less than 2 infected cells per million).
The great debate here becomes whether the child was ever really infected in utero, and therefore cured, or if this is simply a case of successful post-exposure prophylaxis in a newborn exposed at birth. While it is much more exciting to call this a case of cure, I do not think it was in the way that the Berlin patient, Tim Brown, was cured. But if so this still does not diminish the importance of the demonstration that full ART may prevent infection upon exposure at birth (peripartum infection).
The fact that the child became seronegative, had no HIV immune response, and had low (and potentially false-positive or defective) levels of HIV RNA and DNA at 24 months is consistent with exposure without infection. The declining levels of viremia over the first month are difficult to explain, but might be slow production of virus from non-latently infected cells with intermediate half-lives, or perhaps even time-limited production of HIV from maternal cells that had crossed in to the child’s circulation at birth.
Some children are infected transplacentally, during gestation, some during the birth process, and some after birth via breastfeeding. One possibility is that latent infection cannot occur during gestation, as the developing fetus does not have memory T cells. However, we and others have shown that naïve CD4+ cells, which do exist in the fetus, can also become latently infected (although our studies have been of adult naïve CD4 cells). Alternatively, this child might only have been exposed to HIV mucosally during the birth process, and latent infection (in naïve cells) could not develop in the 31 hours before ART was started. Such an event would be similar to non-human primate post-exposure prophylaxis studies, wherein ART up to 48 hours after exposure of a monkey was sufficient to prevent the establishment of infection.
The truth of the matter may be unknowable, but this case should lead to studies to demonstrate safe ways to treat with full ART in newborns who have not had prophylaxis, and perhaps to avoid (or cure??) infection in some of them. This will be most important in low-resource settings, where it is also most difficult to implement, and where better coverage with prophylaxis is already needed.
Gregory Del Prete (abstr. 49) presented work from the Lifson lab at NCI, in collaboration with Gilead, showing some progress towards replicating the study of HDAC inhibitors in the non-human primate model of SIV latency. Six SIVmac239-infected Indian Rhesus macaques were suppressed on a regimen of injected tenofovir/FTC/dolutegravir, and oral darunavir/ritonavir, although it was later said that the additional PI therapy was not needed. Viremia was suppressed up to 8 logs, and suppression below 30 c/ml achieved in 10-20 weeks. Dosing with either HDAC inhibitors SAHA or rhomedepsin resulted in acetylation of histones in “enriched CD4 T cells” harvested from the blood for up to 48 hours after dosing, demonstrating the desired cellular effect of HDAC inhibition. These conditions led to increased SIV RNA production in cells exposed to HDAC inhibitors ex vivo. The results of in vivo studies are awaited.
Sharon Lewin from Melbourne (abstr. 50LB) then presented the results of the second study of the HDAC inhibitor SAHA or Vorinostat in HIV+ patients receiving ART. 20 HIV+ adults on suppressive cART received vorinostat 400 mg once daily for 14 days. Blood was collected at 0, 2, 8, and 24 hours, and 7, 14, 21, 28, and 84 days. Rectal biopsies were performed at day 0 and 14. Cell associated unspliced HIV RNA (CAU-RNA) and HIV DNA were quantified in CD4+ T cells from blood and rectal tissue. With daily dosing grade 1 or 2 adverse events were nearly universal (18/20 patients), but manageable, most commonly nausea, diarrhea, fatigue, and thrombocytopenia. There were no higher-grade adverse events, dose modification, or drug discontinuations. One patient had a blip of plasma HIV RNA while on vorinostat to 60 copies/mL.
Cellular markers of acetylation, the signature of biological HDAC inhibitor effect within cells, were increased at day 1 of dosing, and appeared statistically increased from baseline at day 7, but barely so, and did not appear increased after that. In parallel to this, CAU-RNA was clearly increased on the first day, but not as impressive at later times. A statistically significant increase in CAU-RNA occurred in the majority of participants during vorinostat dosing. CA-US RNA in blood increased significantly by 8 hours after first dose and remained elevated throughout follow-up, including the period after vorinostat (p <0.001 for all time points). It was puzzling that CAU-RNA was increased (though only slightly) are day 28 and 84 after dosing. This raised the question of either a low baseline, or some sort of very prolonged effect. Perhaps not surprisingly given the data from Erikson 2013 there were no significant changes in HIV DNA in all analyses in blood or rectal tissue. Although this study was important in that it confirmed the earlier findings of Archin 2012, it raised questions about the effects of repeated doses of vorinostat.
Several presentations later in the conference discussed gene therapy approaches to HIV Cure. In a plenary talk on the last day of the meeting (abstr. 124; http://webcasts.
Gene therapy in SCIDX1 deficiency was successful in part as there is a selective advantage in favor of the replaced cells that allowed them to outcompete the native, deficient T cells. However, early gene therapy attempts were challenged by oncogenesis, caused by insertion of the gene vector in a site that caused inappropriate expression of a human gene, leading to cancer. Of the early patients, 4 contracted leukemias from a clonal T cell proliferation event 2-5 years after transplant, 1 fatal. This problem was overcome by the development of new gene therapy viral vectors, actually based on a part of the HIV genome, that do not integrate into these dangerous human gene sites, and the development of so-called self-inactivating retroviral gene vectors, that can only integrate once and cannot replicate or recombine subsequently.
Experience in gene therapy has so far taught us that at least 3×106 gene-modified CD34+ cells per kg body weight have to be transplanted, and that the reconstitution of an immune system from the stem cells of HIV-1+ patients may be incomplete, and other specific immune interventions may be required to restore immune reconstitution. Such interventions may consist of cytokines to stimulate the thymus or other reagents to reverse immune exhaustion. Finally, as stem cells transduced to resist HIV may not have a natural selective advantage, good engraftment may require conditioning (that is cytotoxic chemotherapy) in order to provide an artificial selective advantage. Nevertheless, despite these challenges, there are a large number of anti-HIV genes available — including genes that directly inhibit HIV replication (fusion inhibitors, TAR decoys, siRNAs against HIV genes or HIV coreceptors, nucleases against HIV DNA) — that are beginning to enter studies.
One such study was reviewed (abstr. 126; http://webcasts.
Hans-Peter Kiem, of the Fred Hutchinson Cancer Res Center, Seattle (Abstr. 127) presented findings of a gene therapy experiment in the primate model. Using the hybrid virus SHIV in macaques not on ART, they created HIV/SHIV-resistant stem cells from autologous macaque cells. CD34+ stem cells were engineered to express a unique fusion inhibitor called mC46, which prevents R5-using HIV from entering the cell. Shortly after infusion gene modified CD4+ T cells appeared to account for >90% of the total CD+ cells in peripheral blood, gastrointestinal tract, and lymph nodes, but later non-modified, unprotected CD4+ T cells made a steady recovery. CTL function and antibody responses were significantly enhanced in the mC46 macaques (p >0.05). The improved immune response was correlated with 2-3 log decreases in plasma viremia levels in the mC46 macaques, with viremic levels inversely correlating to the levels of pre-challenge modified cells (p >0.05). Finally, both mC46 macaques maintained high frequencies of SHIV-specific, gene modified CD4+ T cell responders whereas none were detected in control macaques. It was puzzling that the modified cells declined, and the unmodified ones expanded later in the experiment. Follow-upon this work will be of interest.