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Update on HIV Eradication....
Report from the 4th HIV Persistence Intl Wksp
(Dec 8-11 2009, David Margolis, MD
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"Developing the ability to eradicate established HIV infection requires a prolonged scientific commitment, further discoveries in the basic mechanisms of HIV persistence, the development of new model systems to test therapeutic approaches, and careful but innovative translational studies. Hopefully these will continue to emerge when the Persistence Workshop reconvenes in 2011."
David Margolis MD, University of North Carolina at Chapel Hill
The Fourth International Workshop on HIV Persistence during Therapy returned to its traditional site in the West Indies on French side of the isle of St. Martin from December 8th to 11th. Hosted and organized by Alain Lafeuillade, the meeting is held in odd years, and its last appearance in 2007 seemed like it might be its last. But since that time, there has been a re-ignition of energy and interest in the field, perhaps driven by the sense that landmark advances in antiretroviral therapy (ART) will be less frequent in the future, and that the limited advances in approaches to prevent HIV infection force us to reconsider the challenges of eradication of HIV in an infected patient.
The meeting was marked by numerous interesting scientific reports, and an exciting combination of industry support for research and development, diverse activity by investigative groups around the world, and continued support by foundations such as amFAR, coupled with new investments by the NIH and the Gates foundation. Several of the presentations are summarized below; all 78 abstracts will be published in the Global Antiviral Journal (www.ihlpress.com/gaj.html).
Although the answers as to where and how and why HIV infection can persist despite successful antiretroviral therapy (ART) were not universally agreed upon, as has been the case for more than a decade, there was more general agreement on what the most important questions were, and hope that new therapeutics can be moved into the clinic will allow studies to probe these important issues.
Animal Models of Persistence
The meeting opened with discussion of studies of animal models of HIV infection, aimed at understanding the reasons that HIV infection persists despite ART, and an understanding of the the "reservoirs" of such persistent infection. Jeff Lifson (AIDS and Cancer Virus Program, NCI) discussed the application of non-human primate (NHP) models such studies. His group has evaluated four different NHP models; other models are under investigation in other laboratories. Both the infecting virus and the virus the monkey infected is infected with are of critical relevance. Lifson studied the SIV strain SIVmac239 in both Indian and Chinese origin rhesus macaques. Also studied was a chimeric virus encoding the HIV reverse transcriptase in an SIV backbone (RT-SHIVmne) in pigtail macaques (Maccaca nemestrina), and a second chimeric virus containing fewer HIV RT sequences (stHIV), also in pigtail macaques.
SIVmac239 infection of Indian rhesus macaques appears to be the best-established NHP model, but latency is difficult to study in this model due to high levels of viral replication, and practical and pharmacological difficulties delivering durable, potent ART. Lower levels of viral replication seen in SIVmac239 infected Chinese rhesus macaques make achieving suppression more practical, but the model is not as well established. Pigtail macaque studies using the RT-SHIVmne virus, or the stHIV virus appeared promising, as these chimeric viruses are susceptible to both NRTI and NNRTI drugs, thereby expand the ability to achieve relevant levels of suppression of viral replication. Following Dr. Lifson, Anding Shen reviewed her published work on "true latency," infection of resting CD4+ T cells in macaques infected with SIV. Further optimization of these models and development of sensitive and robust assays to monitor viral replication is underway, and will be necessary for studies to move the field ahead.
Ron Veazey (Tulane) then presented findings in the macaque model infected with SIVmac that gut-associated lymphoid tissue (GALT) in the large intestine is an important site of SIV persistence. The caveats for this finding were that it pertained to monkeys who had long-term non-progressing SIV infection, and was observed in the absence of ART. Veazey speculated that this was due to higher turnover of memory CD4+ T cells in the large intestine immunologic compartment, allowing persistent rounds of replication and creating a viral reservoir.
In the final NHP presentation in this session, Mary Kearney (HIV Drug Resistance Program, NCI) presented work done in collaboration with Lifson and others measuring the evolution and persistence of RT-SHIVmne in macaques on typical ART (tenofovir, FTC, and efavirenz). Over 20 weeks of therapy, significant reductions in plasma viremia were seen but the overall diversity of circulating viral populations that were detected did not change, suggesting that the therapy was effective on all variants and that declining viremia does not correlate with a decrease in virus diversity. However, there were shifts detect in population structure in 2 animals that developed drug resistance. Like human studies, these findings suggest that ongoing replication does not occur when viremia is suppressed, and that drug resistance likely emerges as a result of incomplete suppression of viral replication.
Finally Garcia-Martinez (UNC) presented exciting studies from their group suggesting that humanized bone marrow-liver-thymus (or BLT) mice could serve as an in vivo model to study issues of fundamental importance to in vivo HIV persistence. These mice transplanted with human hematopoietic cells exhibit systemic reconstitution (including in the gut and vaginal mucosa) with a complete and functional human immune system. When infected with standard HIV strains, these mice develop viremia, tissue infection in all cellular compartments, and CD4 depletion. Preliminary studies showed that viremia could be suppressed, and CD4 cell preserved, by ART using tenofovir, FTC, and raltegravir. When ART was interrupted, viremia promptly reappeared. This model, while in its early development stages, may offer an additional model system in which latency and eradication strategies can be modeled. One advantage of the model is that all events studies involve authentic HIV strains studied in human cells. Further, although the BLT model is resource-intensive to establish, it is not subject to the animal resource restrictions that face the NHP model. Most likely, both BLT and NHP models have important roles to play.
Cells and Reservoirs
An understanding of the mechanisms that allow HIV to enter latency, and to remain in a quiescent state will be central to the future ability to develop therapies that target persistent HIV infection. Jose Alcami (Instituto de Salud Carlos III, Madrid) provided an overview of the current understanding of host cellular factors that up- or down-regulate the expression of integrated HIV genomes, and then presenting work from his group suggesting that compounds that signaled the HIV promoter without activating lymphocytes could be used to purge latent virus.
MicroRNAs (miRNAs), small RNAs that do not code for a protein, have become recognized as key regulators of gene expression in human cells. It is clear that miRNAs of both viral and cellular origin can positively or negatively influence the replication of several viruses. Several groups have reported that human cellular miRNAs can affect HIV expression in vivo. Benkirane (CNRS, Montpelier) presented data in the Jurkat T cell line model system that several human miRNAs affected both the basal level of expression of integrated HIV provirus, and the activation of HIV expression by the viral Tat activator.
Jon Karn (Case Western) discussed cellular restrictions that limit the process that initiates HIV RNA production (transcription) from the integrated HIV genome, and processes (elongation) that allow fully effective RNA expression to occur. Some of these restrictions are enforced by the state of chromatin around the HIV genome. Restrictions imposed by histone deacetlyases (HDACs) have been well described. Karn detailed the contribution of other chromatin marks and modifications, especially the accumulation of the protein HP1α at silenced chromatin, and the trimethylation of histones near the HIV LTR promoter. Karn found the presence of methylated histones in T-cells appears to be due to the expression of the histone methyltransferases SuvH39, previously reported to induce histone methylation of HIV proviruses in non-T cell systems, and a novel methyltransferase EZH2.
Restriction of the elongation of HIV RNAs was found to be in part due to the negative transcription elongation factor NELF-E. Inhibition of NELF-E allowed active expression of up to 37% of latently viral genomes within a T cell line. Karn proposed that manipulating both initiation and elongation might be critical to either induce or repress latent HIV.
Alberto Bosque and Vincente Planelles (University of Utah) presented their findings in a recently developed primary cell model system of latent CD4+ cell infection. In this system naive cells from the periphery of healthy donors are induced to undergo normal development ex vivo in the presence of select cytokine cocktails or antigenic stimulation. These cells are infected while in the activated state, and return to quiescence as memory cells. In this system, viral reactivation following by protein kinase C agonists, TNF alpha, or histone deacetylase inhibitors is inefficient. They suggest that a combination of IL-2 and IL-7 is a potent inducer of latent HIV-1. Given the impending clinical availability of IL-7, this is a hypothesis that could be tested.
Romerio (Inst. Human Virology, Baltimore) also presented findings from a primary cell model of HIV latency. The system attempts to recapitulate the events of primary and secondary antigen-driven immune response in which CD4+ T cells are activated with dendritic cells and antigen, infected in vitro with HIV-1, and then brought back to quiescence through a resting phase in the presence of interleukin-7. During the resting phase, the latently infected cells generated in vitro with our system lack expression of activation markers; do not undergo cellular proliferation and do not sustain viral replication. All these activities resume promptly following secondary antigen stimulation or by culture on immobilized anti-CD3/CD28 antibodies. In addition, higher cell death rates were observed in HIV-1 infected than uninfected cultures during secondary but not primary stimulation. These emerging novel model system promise to allow additional insights into latent CD4+ T cell infection.
Finally, Pasternak (University of Amsterdam) made a presentation that did not deal with mechanism but was of clinical interest. He found that using a highly sensitive PCR assay, cellular levels of unspliced HIV RNA in patients on ART predicted clinical outcome. They selected 26 HIV-1 infected individuals who initially responded to ART for a median of 2.3 years with plasma viremia <50 copies/ml. Eleven of these patients remained virologically suppressed, whereas fifteen experienced subsequent virological rebound. Median under-therapy level of usRNA was significantly higher (0.43 log10 difference, P=0.0015) in patients who experienced subsequent VR than patients who remained virologically suppressed. In contrast to the cellular DNA level, which gradually diminished during the therapy period in both patient groups, the level of usRNA dropped sharply after the start of therapy but subsequently reached the plateau, with the difference between the usRNA levels of future cART failures and successes established early after the start of cART and persisting throughout the therapy period. Although there was no hypothesis given to explain these findings, should this be validated in a larger cohort this assay might be of clinical utility.
Virology, Immunology& Reservoirs
The role of the type of virus and the state of the cell that is infected is certainly central to the development and maintenance of persistent HIV infection. Delobel (INSERM, Toulouse) discussed the role of viral tropism for CCR5 or CXCR4 with regard to the development of persistence. This group hypothesized that residual HIV replication despite ART could allow the evolution of virus and change in coreceptor use. The group had previously reported in a 5-year longitudinal study that CXCR4-using HIV-1 (assayed by envelope sequencing and predictive algorithms) could be gradually selected in cellular reservoirs during sustained effective ART, and that this was associated with impaired CD4+ T-cell restoration. In a follow-up study, they presented finding residual plasma viremia below 50 copies per ml in 80% of patients with poor CD4+ T-cell reconstitution in response to ART, and in only 38% of patients with good CD4+ T-cell reconstitution. They suggested that residual viremia originated from release of archival virus from reservoir cells and/or ongoing virus replication. They also linked this finding to the finding of persistent T-cell activation and apoptosis on ART in "poor reconstituters," and that this was again associated with a high frequency of CXCR4-using viruses (by DNA sequence) in PBMC.
Koup (VRC, NIH) presented very interesting data to support the idea that functional differences in the T Cell response to opportunistic pathogens was a driving force in the events that lead to T cell infection by HIV, and perhaps the likelihood that that infected cell will establish latent infection. Memory CD4 T cells possess multiple different antigen specificities, functions and phenotypes that may affect their susceptibility to HIV infection. Koup's group sought to determine whether the phenotype and function of CMV-specific and Mycobacterium Tuberculosis (MTB)-specific CD4 T cells affected their susceptibility to HIV infection in vivo and in vitro. MTB-specific CD4 cells during latent TB produced more IL2, and CMV-specific CD4 cells produced more MIP-1b. IL2+ cells would be expected to be more susceptible to HIV infection, whereas cells producing the CCR5 chemokine MIP-1b should be somewhat resistant to HIV infection in vivo. Koup found that MTB-specific CD4 T cell populations had higher copies of HIV gag DNA than the cytokine-negative memory population from the same subject. Further, IL2+ MTB-specific CD4 cells had the highest level of HIV gag DNA, and MIP-1b+ cells had the lowest. In contrast, CMV-specific CD4 T cells produced more MIP-1b, and were preserved after HIV infection compared to MTB-specific CD4 cells. Thus differences in CD4 T cell function and maturation might influence HIV susceptibility and depletion of MTB-specific CD4 T cells after HIV infection.
Chomont (Vaccine and Gene Therapy Institute, FL) presented findings from their recent Nature Medicine paper, showing that the central memory (TCM) and transitional memory (TTM) CD4+ T cells are the major cellular reservoirs for HIV. They point to the contribution of homeostatic proliferation of these cells as a central reason for viral persistence, allowing replication of the cells carrying latent HIV without viral cytolysis. As these cells preferentially express the marker PD-1, they suggest that the disruption of the PD-1/PD-L1 interaction may constitute a novel strategy to eradicate HIV. This suggestion was furthered by the presentation of S. Fonseca, another member of the group, later in the meeting. She showed that triggering of the PD-1 pathway inhibits 50% of HIV-1 production in primary CD4+T cells at day 1 as measured by quantitative RT-PCR in the cell culture supernatants and up to 95% at day 3 as measured by p24 ELISA. Importantly, this inhibition was restricted to PD-1-high cells, demonstrating the specificity of this mechanism.
Lewin (Monash University, Melbourne) also presented findings from patient cell samples suggesting that naive CD31+ or CD31- CD4 T-cells could become persistently infected with HIV, and that while there was decay in infection of other T cell subsets during ART, there was no decay in HIV-infected naive CD4+ T-cells. Her presentation also invoked the possibility that homeostatic proliferation of these naïve, infected cells prevented their decay despite durable, successful ART.
Pharmacology, antiretroviral therapy, and latency
While ART can reduce viremia to levels undetectable by standard assays (<50 copies RNA/ml), the fact that no HIV-infected patient has been cured of HIV has led some to ask if better or more intensive therapy could reduce persistent infection. Coffin (Tufts and NCI Drug Resistance Program) presented a summary of a large body of work using a single copy assay (SCA) capable of detecting and quantitating HIV-1 RNA to a limit of 1 copy/ml. SCA viremia is independent of the specific therapeutic regimen, and exhibits a very slow decay in two phases, the second of which has a half-life indistinguishable from infinity. Coffin hypothesized that SCA viremia is likely to be the result of virions released from cells infected prior to initiation of therapy, and not due to production of virus by ongoing complete cycles of replication. He reviewed the results of several therapeutic intensification trials that show no effect on SCA viremia, and support the hypothesis that persistent low-level viremia is the result of virus production from a very small fraction of latently or persistently infected cells that were generated prior to the initiation of therapy.
Moreno (Hospital Universitario Ramon y Cajal, Madrid) presented an interesting intensification trial (NCT00795444) with the CCR5 antagonist maraviroc (MVC), measuring the effect on the frequency of resting cell infection and residual viremia. 9 chronically HIV-1-infected adult patients with stable ART ≥3 drugs and VL <50 copies/ml for ≥2 years, CD4 count >350 cells/mm3 and demonstrated CCR5-tropism were treated with MVC for 48 weeks.
After 12 weeks of MVC, the frequency of resting CD4 cell infection decreased in 5 of 6 patients, and infection could not be quantified in 3 others. SCA viremia was too low to measure a change in all 9 patients. CD4 cell activation was decreased in all patients. Intensification with MVC seemed to deplete resting CD4 cell infection, but the quantification of the reduction was hampered by the limited sensitivity of the method used due to the number of resting CD4-T-cells obtained.
The central nervous system (CNS) is thought to serves as one of the principal anatomical reservoirs for the replicating HIV-1 virus, protected from the full effect of ART. Labhasetwar (Lerner Research Institute, Cleveland) presented an overview of a program developing biodegradable trans-activating transcription (TAT)-peptide conjugated nanoparticles (TAT-NPs) loaded with anti-HIV drugs with enhanced transport across the blood-brain barrier (BBB). The protease inhibitor ritonavir was selected as it is a substrate for the P-gp efflux pump and hence has low CNS bioavailability. In a mouse model system, TAT-NPs bypass the efflux action of P-gp and increase the transport of encapsulated ritonavir without disrupting BBB integrity.
Similarly, Mahajan (SUNY Buffalo) used multimodal nanoparticles called quantum rods (QRs) to deliver ART across the BBB. Saquinavir was bioconjugated to QRs and QRs were also conjugated with transferrin receptor, to mediate active transport across the BBB. In a well validated in-vitro BBB model, measuring HIV production infected monocytes, saquinavir-Tf-QR nanoformulations decreased p24 production 63% and HIV-1 LTR gene expression by 93%.
Clinical Observations
Markowitz (Aaron Diamond, NYC) reported that ART initiated shortly after infection does not prevent persistent CD4+ T cell depletion in the lamina propria of the GI tract associated with increased levels of immune activation in both CD4 and CD8+ T cell populations in the vast majority of patients (up to 70%) when compared to uninfected controls. In 3 patients treated during acute and early infection, a standard ART regimen was intensified with raltegravir and maraviroc. There was no evidence of viral evolution in the mucosal mononuclear cells when compared to PBMC. Intensification with additional antiviral agents failed to alter the degree of T cell depletion, or levels of activation in the GI tract. However, endotoxin levels a reported marker of GI tract damage, were undetectable in all patients and unaffected by intensification. Overall, suppressive ART is not associated with cryptic ongoing viral population in the gastrointestinal tract, but does not (at least so far) ameliorate persistent activation and mucosal T cell depletion.
Gero Hutter (University of Mannheim) discussed the single patient he and his colleagues treated that appears to be the first patient cured of HIV infection (NEJM 2009). In an HIV-infected leukemia patient requiring stem cell transplant for treatment, an allogeneic stem cell transplantation (SCT) was given from a donor homozygous for the CCR5-delta32 deletion
This 32-basepair deletion in the CCR5 allele provides resistance against HIV-1 transmission in individuals homozygous for this mutation. HIV remains undetectable for more than 2 years after discontinuing ART, as determined by viral RNA and proviral DNA PCR assays of peripheral blood, bone marrow. After the patient relapsed from his leukemia one year after the first transplantation a second transplantation with the same CCR5 deficient donor was performed. Replacement of CCR5+ cells appears to be complete. CD4/CCR5 co-expression macrophages were detectable in the gut 6 months after transplantation, but by 2 years after transplantation CCR5+ macrophages were undetectable. A brain biopsy done for clinical reasons also showed that all detectable neuroglial cells were CCR5-negative.
Hutter emphasized that several possible explanations for the apparent eradication of HIV infection might exist: 1) The conditioning regimen for SCT led to depletion of the T-cell pool and thus significantly reduced the HIV-1 reservoir; 2) Under immunosuppressive treatment, the absence of activated CD4+ T cells abrogated HIV-1 replication in the remaining T-cell fraction; 3) Repopulating CD4+ T cells were resistant to CCR5-tropic HIV-1 infection due to the absence of CCR5 surface expression; 4) Reconstitution of the T-cell pool was too slow to provide an adequate number of CD4+ target cells for the re-emergence of HIV-1 infection via co-receptors other than CCR5. While this approach is not widely practical, it suggests that the goal might be someday achievable.
Given the limited evidence that resting CD4+ T cell infection (RCI) is affected by the weak histone deacetylase (HDAC) inhibitor valproic acid (VPA), this author (Margolis, UNC) presented measures of the stability of RCI and residual viremia in patients who added VPA with or without raltegravir (RAL), or enfuvirtide (ENF) with or without VPA, to standard ART. Overall 13 patients were studied, and consistent with other recent studies no effect of the addition of RAL or ENF on low-level viremia measured by SCA. Further the prospective addition of VPA and RAL, VPA and ENF, or ENF failed to progressively reduce the frequency of RCI in most patients. The group is now pursuing new approaches such as more potent HDAC inhibition, alone or in combination with intensified ART or other agents that may disrupt proviral latency.
T-W Chun (NIAID, NIH, Bethesda) reported recent observations in two patients that have been followed by his group for many years. Both were treated with ART during early/acute HIV infection, and have now been on ART for more than 7 years. Of a cohort of similar patients reported in the Journal of Infectious Diseases by Chun in 2007, these two patients have the lowest levels of HIV infection. No HIV DNA can be detected in the PBMCs of either patient, and no HIV DNA was found in GALT tissue after one underwent biopsy. Quantitation of resting CD4 T cell infection finds that the frequency of infection is 1.1, and less than 1.7, infected cells per billion resting CD4+ T cells, respectively. Extrapolating from the estimates that there are 1012 lymphocytes in an adult male, and therefore 3 x 1011 CD4+ cells in the patient with <1.7 infected cells per billion, the expectation is that there remain 176 infected resting CD4+ T cells in this patient. Chun reported that after 10.5 years of ART, this patient independently decided to interrupt ART. 50 days after interruption, plasma viremia of 382 copies/ml was detected and the patient successfully re-initiated ART.
New Therapeutic Approaches
Despite the lack of clinical success of the HDAC inhibitor valproate as a single agent, this class of drugs continues to attract significant interest as at least apart of an approach to deplete latent infection. However other novel approaches were also discussed at the Workshop.
Wu (George Mason University, VA) suggested that a complementary strategy was needed to target persistently infected cells, particularly macrophages. He proposed to deliver toxin molecules via non-integrating lentiviral vectors whose expression was dependent on the presence of the HIV Rev protein (ie that the cell was infected and producing threshold levels of Rev), to effect selective killing of HIV-infected cells. This appeared achievable, at least in tissue culture model systems.
Debyser (KULeuven, Belgium) has rationally designed inhibitors of the interaction between the human protein complex LEDGF/p75 and the HIV integrase, to serve as novel inhibitors of viral integration and replication. Compounds inhibit replication of HIV-1 in T cell lines, in PBL and in primary macrophages. Perhaps such novel compounds will improve the effectiveness of ART, although it is debatable whether or not such improvements are required to ablate persistent infection.
Savarino (Superiore di Sanita, Rome) presented recently published and addition studies of the combinatorial effect of HDAC inhibitors with the glutathione synthesis inhibitor buthionine sulfoximine (BSO). He showed that HDAC inhibitors with BSO induced increased responsiveness and expression of latently infected cells in model systems, followed by the death of infected cells. He suggested that the combination of the potent HDAC inhibitor SAHA and BSO could spark a chain reaction involving mutual enhancement of reactive oxygen species production and HIV-1 replication and leading to cell death.
Sluis-Cremer (University of Pittsburgh) presented studies of the correlation between the isoform specificity of HDAC inhibitors and their ability to reactivate latent HIV-1 infection in a model system. This group screened 16 HDAC inhibitors for cytotoxicity and the ability to activate HIV-1 expression in a Jurkat cell (J89GFP) model of latency. Because the inhibitors apicidin, oxamflatin, scriptaid and suberoylanilide hydroxamic acid (SAHA) had similar cytotoxic concentrations (CC50=1-5 mM), potent activity against HDAC 1 (IC50<10nM) but differing abilities to de-repress the HIV-1 LTR, they were subjected to further analyses. Apicidin and oxamflatin was found to reactivate HIV-1 gene expression more efficiently than scriptaid or SAHA. Apicidin and oxamflatin were found to be potent inhibitors of the Class I HDAC isoforms 1, 2, 3 and 8, but scriptaid and SAHA were only potently active against HDACs 1, 2 and 8. Sluis-Cremer therefore hypothesized that inhibition of HDAC 3 may be important for the activation of HIV-1 expression in J89GFP cells.
Conclusions
Chronic, lifelong antiretroviral therapy may be needed for decades into the future to prevent AIDS in the millions of HIV-infected people, and to control the spread of the HIV pandemic. Several mechanisms that might contribute to the establishment and maintenance of latent proviral infection have been described, yet it seems most likely that several populations of cells that are persistently infected will require unique therapeutic approaches. Several strategies, as described above, have already emerged from our current understanding of persistent HIV infection. However, obviously no approach has yet been practical or successful. Developing the ability to eradicate established HIV infection requires a prolonged scientific commitment, further discoveries in the basic mechanisms of HIV persistence, the development of new model systems to test therapeutic approaches, and careful but innovative translational studies. Hopefully these will continue to emerge when the Persistence Workshop reconvenes in 2011.
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