TAG HIV Basic Science, Vaccines, and Cure Project Blog

By Richard Jefferys, Project Director at Treatment Action Group (TAG).

recent posts

  • Case Report from the First Clinical Trial of CRISPR-Edited Stem Cells in People with HIV and Cancers

    One of the ways TAG keeps track of developments in HIV cure research is by maintaining an online listing of clinical research culled from trial registries (primarily clinicaltrials.gov). In May 2017, a research group in China registered the first human HIV trial involving the CRISPR/Cas9 gene editing system—a technology that has generated considerable excitement and attention due to promising results in small animal models. Last month in the New England Journal of Medicine, the researchers published a case report describing outcomes in a study participant (presumably the first individual enrolled).

    The design of the study is similar to several prior gene therapy trials for HIV-positive people with cancer diagnoses that require stem cell transplantation as part of the treatment regimen (City of Hope in California has been a notable pioneer of this research). After appropriate stem cell donors are identified, some of the donated cells are subjected to genetic modification in the laboratory prior to being administered to study participants. Unmodified cells are also delivered to guard against any risk of the genetic modifications compromising the normal therapeutic efficacy of the stem cell transplantation.

    The novel aspect is the use of CRISPR/Cas9 to edit the gene for the CCR5 receptor that most HIV strains use to infect cells. The researchers previously published laboratory results demonstrating the feasibility of the approach. The goal is for the gene-edited stem cells to generate a population of HIV-resistant CD4 T cells after transplantation.

    The participant described in the NEJM paper is a 27-year-old HIV-positive man diagnosed with acute lymphoblastic leukemia, which was successfully driven into remission by chemotherapy regimens prior to the stem cell transplant. After transplantation, tests showed that the stem cells successfully generated a new donor-derived immune system by four weeks post-transplantation (referred to as full donor chimerism) and the cancer remained in remission with a very low predicted risk of relapse. ART was maintained throughout.

    Over 19 months of follow up, the proportion of gene-edited cells detectable in bone marrow ranged from 5.20% to 8.28%. Seven months after transplantation, permission was obtained to conduct an analytical treatment interruption (ATI).

    At the time of the ATI, the proportion of peripheral blood CD4 T cells showing evidence of CCR5 gene disruption was 2.96%. The proportion increased after treatment cessation, peaking at 4.39% during the ATI. ART was restarted after four weeks due to a very high viral load rebound to 30 million copies/ml; this is not atypical in stem cell transplant recipients because the new donor-derived immune system cells have not been exposed to HIV before, and therefore no virus-specific immunity is present (arguably it would have been prudent to restart ART sooner, if possible). After the ATI, the proportion of gene modified CD4 T cells stabilized at a little over 2.5%.

    No adverse events related to the editing of the CCR5 gene were documented. The researchers conducted multiple searches for any evidence of off-target effects (gene edits in the wrong places) but found none. They note, however, that the relatively low efficiency of the gene editing in this study may have limited their ability to detect off-target activity.  

    The results offer encouragement for further pursuit of CRISPR/Cas9 as a gene editing tool in HIV, but the study authors state in their conclusion: “To further clarify the anti-HIV effect of CCR5-ablated HSPCs [hematopoietic stem and progenitor cells], it will be essential to increase the gene-editing efficiency of our CRISPR–Cas9 system and improve the transplantation protocol.”

    In an accompanying commentary, Carl June adds that “additional patients who undergo engraftment with higher frequencies of CRISPR–Cas9–edited stem cells will have to be followed for longer periods of time in order to ensure the safety of this approach.”

    Both the paper and the commentary cite the recently published claim that homozygosity for the CCR5-∆32 mutation is associated with a reduced lifespan as reason for caution regarding the editing of CCR5, however this concern no longer holds because the work was retracted due to a flaw in the analysis.

  • Tracing the Formation of the HIV Reservoir

    In a previous report from the AIDS 2018 conference in Amsterdam, I briefly covered a presentation by Sarah Joseph from the University of North Carolina suggesting that the bulk of the persistent, replication-competent HIV reservoir is formed around the time that antiretroviral therapy (ART) is initiated. Details of the study have now been published in Science Translational Medicine, and Joseph and colleagues have also outlined how the findings might be exploited therapeutically in a separate open access paper in Frontiers in Immunology.

    The main study analyzed nine women participating in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 cohort in KwaZulu-Natal. The women were enrolled during primary HIV infection, but did not begin ART until reaching the CD4 thresholds specified in contemporaneous South African treatment guidelines (which shifted from <200 to <350 during follow up).

    The researchers performed quantitative virus outgrowth assays (QVOA) on blood samples taken from participants after an average of five years on ART (the QVOA measures the size of the replication-competent HIV reservoir present in the sample). The researchers then genetically sequenced the HIV that was detected by the QVOA and compared the results with viral sequences detected during the period prior to ART initiation, when samples were taken every six months on average.

    The comparison of pre-ART HIV sequences with those subsequently detected by QVOA demonstrated that, in most cases, viruses had entered the reservoir during the year before ART was started. In four of the women, more than 90% of the HIV identified in the QVOA entered the reservoir in the year preceding ART. The median value for all nine was 78%. HIV sequences that were present within the first year of infection represented only 4% of viruses in the post-ART reservoir.

    There were two exceptions to this general picture: in one case, the replication-competent HIV reservoir was mostly comprised of viruses from the early to middle period of untreated infection. In the second case, it appeared that viruses had seeded the reservoir continually prior to treatment, but there was a bias toward variants present during the year before ART was begun.

    The results echo a previously published study by Johanna Brodin and colleagues, which found that ~60% of the HIV DNA sequences detectable after ART initiation were most closely related to HIV RNA variants present in blood samples just before treatment was started. Notably, the population in this study was quite different, mainly comprising Swedish men with clade B HIV infection.

    The researchers suggest that during untreated HIV infection, virus-infected cells are typically in an activated, short-lived state that largely precludes the generation of long-lived cells containing latent HIV. When ART suppresses viral load, some virus-infected cells are able to transition into a long-lived resting memory state with HIV integrated into their genomes, thus establishing the latent reservoir. In the authors’ words: “These findings suggest that profound immunologic changes at the time of ART may allow HIV-infected cells to become long-lived memory cells and form most of the stable reservoir.”

    This suggestion is broadly consistent with what has been learned about the generation of memory T cells from basic science research – when the level of an antigen is high, memory cell formation is limited, but when antigen levels decline a population of long-lived antigen-specific memory cells develops (a process called memory T cell differentiation or maturation).

    In Frontiers in Immunology, Joseph and colleagues discuss the therapeutic implications of their results, writing: “Strategies to limit the formation of the stable HIV-1 reservoir could be combined with ART initiation, when patients are receiving intense clinical care. Preventing generation of long-lived latently infected CD4+ T cells should result in a smaller HIV-1 reservoir, providing a less intractable target for curative approaches. Reducing the size of the HIV-1 reservoir may also reduce ongoing immune senescence and HIV-1 co-morbidities experienced by PLWH on ART.”

    The specific approach they have in mind is the short-term use of antibodies against the cell surface molecule CD127. These antibodies block interactions between the cytokine interleukin-7 (IL-7) and its cellular receptor, and these interactions are crucial in the formation of memory CD4 T cells. Several anti-CD127 antibodies are already in clinical trials as potential treatments for autoimmune and inflammatory conditions. Studies in people with HIV initiating ART are likely to be in the works.

  • Distinguishing Potential Mechanisms of HIV Persistence

    For researchers attempting to develop a cure for HIV infection, it’s important to understand the mechanisms by which the virus persists in the body despite suppression of viral load to undetectable levels by ART. Debate has centered around two possibilities that aren’t necessarily exclusive:

    • HIV continues to replicate at low levels in body tissues that ART drugs may not penetrate well (often referred to as sanctuary sites).
    • Cells with HIV integrated into their genetic code—latently infected cells—persist and proliferate even though ART has shut down virus replication.

    In recent years, evidence has accumulated indicating that, in most people who are adherent to ART, HIV replication is completely suppressed. A new report in the journal Science Advances adds to this evidence by analyzing individuals in receipt of long-term ART. The results support the increasing research focus on the persistence and proliferation of HIV-infected cells as the major obstacle to a cure, a topic addressed by several other recently published papers.

    The study by Giorgio Bozzi and colleagues described in Science Advances involved a total of six participants—three had initiated ART soon after HIV infection, while the remaining three had been diagnosed with AIDS prior to beginning treatment (one individual in each group had died and autopsy samples were available). The individuals were all male with an average age of 40 at diagnosis, and median time on ART was 17.8 years (range: 8 to 22.7 years).

    As was expected, levels of HIV DNA were higher in the group that initiated ART late, both in blood and tissues. HIV was also significantly more genetically diverse in these participants. Lymphoid tissues harbored the largest amounts of HIV DNA in both groups.

    Analyses of HIV evolution (changes in the virus’s genetic makeup that occur if it’s replicating) revealed no evidence of ongoing virus replication in any participant during continuous ART. In two cases, antiretroviral monotherapy and dual therapy had been received prior to starting effective ART, and this allowed the researchers to demonstrate that their techniques could detect HIV evolution during these periods when viral load was not fully suppressed (likewise, short-term HIV evolution was seen in one participant as a result of a brief ART interruption).

    Overall, when data from all participants were combined, no genetic changes indicative of HIV replication were observed during 60 person-years of suppressive ART.

    The researchers did observe evidence of proliferation of some latently infected cells. Certain patterns of HIV hypermutation that are induced by cellular ABOPEC proteins are unique to individual infected CD4 T cells. If these CD4 T cells proliferate, the number of copies of HIV with the same pattern of hypermutation increases. One example cited by the researchers is a single hypermutated HIV sequence identified in one participant prior to ART. After 16 years of treatment, 42 copies of the identical hypermutated HIV were detected in both ileum- and colon-derived tissues. This represents evidence that the original CD4 T cell containing the hypermutated HIV proliferated, generating multiple new CD4 T cells containing duplicates of the same hypermutated virus.

    Several other recent papers address the role of CD4 T cell proliferation in maintaining the HIV reservoir.

    In the Journal of Clinical Investigation, William McManus and colleagues from Mary Kearney’s laboratory at the National Cancer Institute present evidence that proliferation of latently infected CD4 T cells in lymph nodes underlies the persistence of HIV in people on ART. The researchers documented the phenomenon by identifying genetically identical copies of HIV that were integrated into the genetic code of the CD4 T cells at the same exact location.

    Marie-Angélique De Scheerder and colleagues from the HIV Cure Research Center at Ghent University Hospital have published results from the STAR study, an observational assessment of HIV sequences present before and after an analytical treatment interruption (ATI). The study was not able to pinpoint a consistent source of the HIV viral load that rebounded during ATI—either in terms of particular CD4 T cell types or anatomical location—but did find evidence that genetically identical viral expansions played an important role (consistent with the proliferation of latently infected CD4 T cells). The authors write: “Focusing on mechanisms that drive antigenic and homeostatic proliferation of immune cells will be crucial to achieve progress toward an HIV cure.”

    A paper in Clinical Infectious Diseases by Sarah Joseph and colleagues describes three study participants on ART with detectable HIV RNA in cerebrospinal fluid (CSF). In two cases the researchers pinpointed trafficking and proliferation of latently infected cells as the likely source, whereas there was some evidence of persistent replication in the third individual.

    Lastly, a study published this week in mBio by Xiaomin Li and colleagues identifies CD4 T cells expressing the cell surface marker CD161 as particularly prone to proliferating while harboring replication-competent latent HIV. The researchers suggest that developing anti-proliferative strategies that focus on CD161-expressing CD4 T cells may offer a means to reduce the HIV reservoir.

  • Widely Publicized Report Associating the CCR5-Δ32 Mutation with Reduced Longevity is Retracted

    In June of this year, a short paper was published in the journal Nature Medicine reporting that individuals homozygous for the CCR5-∆32 mutation showed a 21% increase in the rate of all-cause mortality compared to those without the mutation (or heterozygotes, who inherit the mutation from one parent). The senior author, Rasmus Nielsen, stated to the Wall Street Journal that this would equate to a nearly two-year shortening of lifespan on average. The findings were the subject of hundreds of media articles, largely because they came on the heels of a misguided and completely unethical experiment in which the CCR5 gene was edited in two embryos, leading to the birth of genetically altered twins. Questions were also raised about possible relevance to HIV cure research studies that attempt to ablate CCR5 expression with gene therapies. Yesterday, Nature Medicine published a notice that the paper has been retracted.

    News that the retraction was imminent emerged two weeks ago in an article by Rebecca Robbins for STAT News, after Nielsen tweeted an acknowledgement that a major error had been found. The original analysis was based on data from the UK Biobank, and it turned out that a technical artifact meant that individuals homozygous for the CCR5-∆32 mutation were underrepresented in the data (the problem is outlined in a paper available on the bioRxiv preprint server).

    These developments do not necessarily mean that homozygosity for the CCR5-∆32 mutation is entirely benign; a number of published reports have suggested an association with impaired responses to certain infectious diseases (particularly West Nile Virus, and possibly influenza as well). Also, previously unappreciated roles of the CCR5 receptor continue to be identified, such as the promotion of hematopoietic stem and progenitor cell regeneration after radiation treatment. But, after this retraction, there is no evidence (at least as yet) that any negative effects of the CCR5-∆32 mutation add up to a reduced life expectancy for homozygous individuals.

  • Selected Highlights from CROI 2019

    The 2019 Conference on Retroviruses and Opportunistic Infections (CROI) took place in Seattle from March 3rd-7th. The major news on the cure research front was the possibility of two additional cases similar to Timothy Ray Brown, who for the past 12 years has been the only individual considered cured of HIV infection (TAG issued a statement offering our perspective). Other notable reports included the first results from a study using CRISPR/Cas9 to target latent SIV in macaques and a novel insight into the source of residual viral load in people on ART. Immediately preceding the conference, TAG joined with multiple other organizations to co-sponsor the annual pre-CROI community HIV cure research workshop; slides and video from the event are available online.

    Stem Cell Transplant Recipients as Possible Cure Cases

    Eleven years ago at CROI, a little-noticed poster by Gero Hütter and colleagues provided the first description of the case of Timothy Ray Brown (among the few people to draw attention to it at the time was activist Martin Delaney of Project Inform). As part of a series of heavy-duty treatments for acute myelogeous leukemia (AML), Brown received two stem cell transplants from a donor Hütter identified who was homozygous for the CCR5Δ32 mutation, meaning they lacked the functional CCR5 co-receptor used by most strains of HIV to infect target cells. At the time of the 2008 CROI poster presentation, Brown’s AML was in remission and he’d been off antiretroviral therapy (ART) for 285 days with no sign of HIV rebound.

    Over the ensuing years, Brown has remained free of detectable HIV and there is broad consensus that he has been cured. However, multiple attempts to repeat the outcome by finding donors homozygous for the CCR5Δ32 mutation for additional people with HIV needing stem cell transplants for cancers have met with disappointing failure; the individuals have all died due to graft-versus-host disease (GVHD) or the underlying cancer, and in one case death was preceded by a rebound of HIV capable of using an alternate co-receptor, CXCR4. At least one researcher expressed concern that the CCR5Δ32 mutation might be negatively affecting the success of stem cell transplantation in some way.

    Despite the setbacks, efforts to study people with HIV and cancers who receive stem cell transplants from donors homozygous for the CCR5Δ32 mutation have continued, and two headline-making presentations at CROI 2019 showed that the work has been worthwhile.

    Garnering the most attention was Ravindra Gupta’s oral abstract describing a person with HIV in London who received a stem cell transplant from a CCR5Δ32 homozygote donor to treat Hodgkin’s Lymphoma that had not responded to standard therapies. Details on the case were provided in a Nature paper that was published online immediately after Gupta’s talk.

    The individual was diagnosed with HIV in 2003, and ART was initiated due to a relatively low CD4 T cell count of 290 cells and viral load of 180,000 copies/ml. The diagnosis of stage 4B Hodgkin’s Lymphoma occurred in December 2012, with the cancer proving refractory to first-line chemotherapies and several salvage regimens (among them was the anti-CD30 monoclonal antibody Brentuximab, which has been reported to have potential activity against HIV-infected cells, but it seems unlikely that this played a part in the eventual outcome).

    Ultimately the decision was made to undertake a stem cell transplant, and an international registry was searched to identify an appropriate donor. Fortuitously, the most closely matched stem cell donor was homozygous for the CCR5Δ32 mutation.

    The transplantation process involved administration of an anti-CD52 antibody (alemtuzumab) to deplete T cells, a conditioning regimen—Lomustine, Cyclophosphamide, Ara-C and Etoposide (LACE)—and then infusion of donor stem cells. Treatments to prevent GVHD were a short course of methotrexate and cyclosporine until 180 days post-transplant. ART was maintained throughout.

    There were some complications: both Epstein-Barr virus and cytomegalovirus reactivated around three months after the transplant, necessitating treatment with Rituximab and ganciclovir respectively. Mild (grade 1) GVHD transiently manifested in the gut, but resolved without any intervention.

    Tests showed that donor cells homozygous for the CCR5Δ32 mutation had fully taken over by day 30 post-transplant, replacing the individual’s original cells (which had wild type CCR5 genes and expressed the normal functional co-receptor). Complete remission of the Hodgkin’s Lymphoma was confirmed at days 120 and 365.

    The researchers discussed the possibility of HIV remission with the individual prior to the procedures, after testing had indicated that he harbored CCR5-tropic virus. Ethical approval was obtained from the UK National Health Service for a potential eventual interruption of ART, with criteria established ahead of time:

    “if viral load was consistently <50 copies/ml on treatment with ‘target not detected’ for at least six months on the two most recent consecutive visits ART would be withdrawn and thereafter viral load would be monitored weekly for the first three months, monthly for a further nine months if undetectable at all time points and three monthly between years one and four.”

    ART was interrupted in September 2017, and the individual has been off HIV treatment for 18 months with no evidence of viral load rebound. Multiple blood tests for the presence of HIV have been persistently negative, with the exception of a single detection of a low-level signal in a digital droplet PCR assay for HIV DNA (among eight replicates, the remaining seven of which were negative). The researchers have not yet studied any tissues. Antibody responses to HIV are waning, echoing results in Timothy Ray Brown.

    Gupta was cautious, using the term HIV remission rather than cure due to the relatively short time since ART interruption. He suggested that perhaps when follow up is beyond two years, the terminology will be revised. The individual is included in the larger amfAR-funded IciStem research consortium, which focuses on evaluating people with HIV who receive stem cell transplants for cancers.

    A second participant in the IciStem consortium was the subject of a late-breaker poster presentation by Björn-Erik O. Jensen from the research group of Guido Kobbe in Dusseldorf. Kobbe and colleagues first described the case at CROI 2016, at which time he was on ART and being followed after receiving a stem cell transplant from a CCR5Δ32 homozygote donor to treat relapsing AML. HIV was undetectable by multiple measures. At CROI 2019 it was revealed that, after ethical approval, ART was interrupted in November 2018. So far there has been no HIV rebound.

    IciStem are following two additional individuals with HIV who’ve received stem cell transplants from CCR5Δ32 homozygote donors to treat cancers, but they remain on ART at this time (Monique Nijhuis described the current status of IciStem in a presentation at the pre-CROI community HIV cure research workshop).

    Inevitably, the possibility that Timothy Ray Brown may finally have some company inspired a deluge of media coverage. The London case had been mentioned very briefly by Ian Gabriel at a BHIVA meeting last fall, prompting a short article by Simon Collins at HIV i-Base, so there was some awareness that further news would be forthcoming at CROI. The Nature paper was also circulated to media under embargo prior to the conference. Regrettably, The Hindu broke the embargo the day before Gupta’s presentation (whether accidentally or deliberately is unclear), leading Nature to also lift it for other media, so the coverage—including a detailed New York Times article by Apoorva Mandivilli—began appearing on the evening of Monday, March 4th.

    There has been debate regarding the implications of the new cases. Most clear is that there is robust justification for continued efforts to identify CCR5Δ32 homozygote donors for people with HIV who need stem cell transplants to treat cancers. Whether lessons can be learned to apply to the search for more broadly applicable curative strategies is less certain; there are differences between the treatments given to the three individuals that may be help untangle contributing factors (see the excellent summary table in the HIV i-Base article by Simon Collins).

    The common thread of receiving cells lacking a functional CCR5 receptor is viewed as supporting gene therapy research aiming to ablate CCR5 expression. A research group in China is currently studying whether the CRISPR/Cas9 gene editing tool can be used to disable the CCR5 gene in stem cells being transplanted into people with HIV and cancers—if successful, this might represent a strategy that would circumvent the need to find matched CCR5Δ32 homozygote donors.

    SB-728-T

    Outside of the setting of stem cell transplants for cancers, the leading approach to knocking out CCR5 from CD4 T cells has been Sangamo Therapeutics SB-728-T. In clinical trials, CD4 T cells are extracted from individuals with HIV, edited at the CCR5 gene using zinc finger nuclease technology, and then expanded and reinfused. The company was hoping to achieve control of HIV viral load after ART interruption, but so far it hasn’t proven possible to modify sufficient numbers of CD4 T cells. Further commercial development for HIV has been abandoned, but some investigator-initiated studies continue.

    Pablo Tebas presented new results from a trial of SB-728-T that broadly conformed to previous research. A total of 14 individuals on ART received a single infusion of modified CD4 T cells, either with or without a preceding dose of cyclophosphamide (intended to deplete existing CD4 T cells and make more room for modified cells). In a slight wrinkle, the delivery of the zinc finger nucleases to the cells was achieved using messenger RNA instead of the adenovirus vector employed in prior studies; the proportion of CD4 T cells successfully edited at the CCR5 gene by the two approaches was similar. The protocol included an ART interruption, and Tebas noted that there was a slight delay in viral load rebound compared to historical controls, but no cases of prolonged containment of HIV.

    As observed in prior trials, participants heterozygous for the CCR5Δ32 mutation appeared to respond best. Because these individuals already have one disabled CCR5 gene, the zinc finger nucleases only have to edit one of the two alleles present in each CD4 T cell in order to prevent expression of a functional CCR5 co-receptor. Tebas concluded that more efficient CCR5 modification could potentially lead to more stringent control of HIV off ART, but it appears unlikely that such an outcome can be achieved with SB-728-T.

    One variation on the theme of trying to genetically protect CD4 T cells from HIV infection involves focusing on modifying cells capable of recognizing and responding to the virus (HIV-specific CD4 T cells). A seminal study by Danny Douek many years ago showed that the virus preferentially infects HIV-specific CD4 T cells, which become dysfunctional and unable to perform their task of coordinating an effective immune response to the virus. The company American Gene Technologies is pursuing a strategy involving the genetic modification of HIV-specific CD4 T cells, with trials planned soon, and results should shed light on whether this is a better approach than attempting to modify CD4 T cells in bulk.

    Taking a Bite Out of the Latent Reservoir with CRISPR/Cas9

    One of the more intuitively appealing ideas in cure research involves attempting to cut the integrated HIV genome out of the DNA of latently infected cells. In this scenario, gene-editing strategies are targeted against the virus itself rather than a host gene like CCR5. The goal is to perform a sort of genetic surgery, excising HIV genes from infected cells without damaging the cell’s genome.

    The gene-editing tool CRISPR/Cas9 has emerged as the leading candidate in this research, and some very preliminary results in mouse models have suggested it may have potential. The laboratory of Kamel Khalili at Temple University has pioneered these studies, in tandem with Excision Biotherapeutics, a company Khalili founded to move the approach into the clinic.

    At CROI 2019, Tricia Burdo from Temple University debuted the results of a study exploring whether CRISPR/Cas9 could excise latent SIV in the SIV/macaque model of HIV infection. The cutting machinery of CRISPR/Cas9 is aimed at a target by the inclusion of molecules called guide RNAs (gRNAs), and in this case the researchers created gRNAs capable of recognizing three relatively conserved sites in the SIV genome (two in the long terminal repeats present at either end of the genome, and one in the gag gene). Burdo noted that the targeting of multiple sites is necessary for both attempting to excise large chunks of the viral genome and avoiding the potential development of resistance (somewhat similar to the rationale for combination ART). The technique produces “very little to no off-target effects,” according to Burdo.

    The SIV-targeted CRISPR/Cas9 was delivered using an adeno-associated virus serotype nine (AAV9) vector. AAVs are a popular gene therapy delivery vehicle that can carry their payload into a broad range of both dividing and non-dividing cells without apparent safety issues (two AAV-delivered gene therapies have been approved by regulatory agencies).

    The study included three macaques, all infected with SIVmac239 and placed on a suppressive ART regimen. In initial experiments, peripheral blood mononuclear cells (PBMC) sampled from the animals were transduced with the AAV9-CRISPR construct, producing evidence of excision of SIV genes between targeted sites.

    AAV9-CRISPR was then infused into two of the macaques at a dose of 1013(ten trillion) copies per kilogram, a lengthy process involving the delivery of 100ml at a rate of 1ml per minute. Three weeks after the infusion, animals were euthanized and necropsy studies conducted. The third animal served as a control and was also euthanized to facilitate comparisons with the AAV9-CRISPR recipients.

    Burdo reported that prior to euthanasia, fragments of the SIV genome that had been cut at targeted sites—referred to as excision products—could be detected in PBMC from the treated animals, as was observed when PMBC were exposed to AAV9-CRISPR in a laboratory dish. Cas9 DNA could also be detected in cells, indicating uptake of the gene-editing tool.

    Necropsy studies included a preliminary evaluation of SIV outgrowth from PBMC samples. The PBMC were combined with SIV-susceptible CEM cells and then SIV p27 Gag protein levels were measured over time. SIV replication could be detected in samples from the control but not those from the macaques that received AAV9-CRISPR. However, Burdo emphasized that this assessment did not involve activating the PBMC to induce virus production (as is the case with the standard virus outgrowth assays used in human studies)—those experiments are pending.

    Analyses of Cas9 DNA demonstrated widespread distribution in the tissues of the two treated macaques, ranging from around 1-10 thousand copies per million cells in the brain to over 10 million copies per million cells in the spleen and liver (presumably reflecting the presence of multiple copies in some cells). Burdo also showed evidence of SIV excision products in spleen, lung and several lymph nodes (including inguinal, submandibular, bronchial and colonic) from the animals.

    The data appear very encouraging, but do not provide information on the magnitude of effect on the latent SIV reservoir (i.e. exactly how much latent SIV was successfully excised or disabled). In response to a question, Burdo reported that future plans include conducting analytical treatment interruptions in macaques treated with AAV9-CRISPR to assess whether viral load rebound is limited or prevented by the intervention.

    An issue not covered in the presentation is the potential for the induction of immune responses against Cas9, which has been observed in mouse studies. Because Cas9 is derived from bacteria, it is treated as foreign by the immune system, and AAV vectors can have an adjuvant effect that seems to promote immune responses against AAV-delivered proteins (this has occurred in studies using AAV to deliver anti-HIV broadly neutralizing antibodies). Pre-existing anti-Cas9 immune responses have also been detected in humans due to infection with Staphylococcus aureus and Streptococcus pyogenes.

    In a graph displaying longitudinal viral load measurements in the macaques (on slide #4 in the webcast), it looks as if administration of AAV9-CRISPR may have been temporally associated with a transient increase in SIV viral load—which could be suggestive of immune activation—although there were also viral load fluctuations in other animals. Gaining an understanding of whether AAV9-CRISPR delivery can activate the immune system and lead to the generation (or activation) of anti-Cas9 immune responses will be important prior to initiating human trials.

    A theoretical concern that researchers have raised about strategies aiming to excise latent HIV relates to what might occur in cells that have more than one integrated copy of the HIV genome (this phenomenon is thought to be uncommon, but has been reported). In this situation, it’s possible that rather than just removing HIV genes, an excision approach might make cuts in each of the separate integrated virus genomes and thereby remove all of the cell’s DNA located between the different HIV integration sites. Damaging the genome of a cell in this way could potentially have untoward effects.

    Overall, Burdo’s results offer significant encouragement for efforts to translate the approach into human clinical trials. Kamel Khalili and colleagues are now working toward that goal in collaboration with Jeffrey Jacobson, a highly experienced clinical HIV researcher who joined Temple University in 2016.

    Attack of the Repliclones

    The past few years have seen an increasing focus on the role of CD4 T cell proliferation in sustaining the latent HIV reservoir. Evidence has accumulated demonstrating that HIV proviruses can be faithfully copied into the daughter cells of latently infected CD4 T cells when they proliferate—the phenomenon can be discerned by the detection of genetically matching copies of the HIV provirus integrated into the exact same place in the genome of multiple CD4 T cells (the progeny of proliferating CD4 T cells are known as clones). Mathematical modeling suggests CD4 T cell proliferation may be the primary mechanism that allows the latent HIV reservoir to persist, and decline only very slowly over time.

    Elias Halvas from the University of Pittsburgh showed at CROI 2019 that CD4 T cell clones containing integrated, intact HIV DNA are a source of low-level HIV viral load that can be detected in some individuals on ART. Essentially, some of these cells can spit out sufficient amounts of HIV RNA to be detectable, even though the virus is not actually replicating (i.e. going on to infect other cells—this is prevented by ART).

    Halvas’s study involved 10 people who had been referred due to persistent low-level viral load despite ART (HIV RNA >20 copies/ml occurring for at least 6 months). The average time on treatment was 10 years, and viral load ranged from 40 to 356 copies/ml, with a median of 97.5 copies/ml.

    One individual displayed evidence of ongoing HIV evolution and the development of drug resistance mutations and was considered a case of ART regimen failure, excluding them from further analysis.

    Samples from the remaining nine showed the presence of genetically identical HIV RNA at multiple timepoints, and there was no sign of viral evolution or resistance mutations against current ART. The source of the HIV RNA was identified as CD4 T cell clones containing integrated, replication-competent HIV DNA (Halvas has christened them “repliclones”). In four cases the genetic sequence of the HIV RNA could be matched to viruses detected in the quantitative virus outgrowth assay (qVOA).

    Halvas concluded that the possibility of production of HIV RNA by infected CD4 T cell clones needs to be borne in mind by clinicians caring for people with HIV, who might otherwise suspect that persistently detectable low-level viral load indicated non-adherence or treatment failure.

    As to the implications for HIV cure research, Halvas suggested that repliclones may contribute to rapid viral load rebound after ART interruption (when the HIV RNA they produce is able to start infecting other cells), and he stressed that they will need to be targeted for elimination or suppression. The mechanisms prompting HIV RNA production by the cells are unclear, and need to be elucidated.

    Notably, the data indicate that HIV latency can be more dynamic than was initially appreciated. It’s now clear that in some CD4 T cell clones containing integrated HIV DNA, the virus is not permanently latent, because there are times when production of HIV RNA is detectable.  

    In an article by Jon Cohen for Science that covers the study, John Mellors points out that the data raise questions about the “kick & kill” strategy in HIV cure research. The rationale for providing a latency-reversing “kick” is that most latently infected cells do not produce HIV RNA and therefore remain invisible to the immune system. Halvas’s results demonstrate that at least some latently infected cells do intermittently generate HIV RNA, and don’t die off as a result.

    One salutary possibility is that researchers developing “kill” strategies may be able to study their efficacy in individuals like those described by Havlas, who already have low-level viral load on ART without the need for administration of any latency-reversing candidate. In theory, an effective “kill” approach should be able to reduce the amount of HIV RNA detected in such cases.

    Targeting the Latent HIV Reservoir with Anti-Proliferative Therapy

    The recognition that the HIV reservoir is at least partly sustained by the proliferation of CD4 T cells is rekindling interest in testing the effects of anti-proliferative therapies in the context of cure research.

    At the pre-CROI community HIV cure research workshop, Joshua Schiffer from the Fred Hutchinson Cancer Research Center described a small (four person) pilot trial of the anti-proliferativedrug mycophenolate mofetil (MMF) being conducted by his research group with funding from amfAR. The rationale is based on the results of mathematical modeling work suggesting that inhibiting CD4 T cell proliferation in people on ART should significantly accelerate the decay of the HIV reservoir. Results are anticipated to be available for CROI 2020. Links to the video of Joshua Schiffer’s talk are on the workshop web page, along with the slides.

    Timothy Heinrich from UCSF presented results of an AIDS Clinical Trials Group (ACTG) trial of sirolimus (a drug with potent anti-proliferative activity, also known as rapamycin) in people on suppressive ART. In the 16 participants who completed 20 weeks of dosing, there was a slight but statistically significant 0.16 logs reduction in HIV DNA levels. CD4 T cell expression of the proliferation marker Ki67 was also significantly reduced.

    Heinrich noted that rates of sirolimus discontinuation were high, and there were also transient increases in the inflammatory biomarkers IL-6 and sCD14 and the coagulation biomarker D-Dimer. The results appear consistent with the notion that inhibiting CD4 T cell proliferation can affect HIV reservoir size, but additional research is needed to confirm that this was the primary mechanism for the HIV DNA reduction. 

    One of the pioneers in this area of HIV cure research is Andrea Savarino, who reported many years ago that the gold-based anti-proliferative drug auranofin reduced the SIV reservoir in ART-treated macaques.

    Since that time, Savarino and colleagues have collaborated with investigators in Brazil to conduct a small pilot trial involving auranofin (which is a licensed treatment for rheumatoid arthritis). The latest results were presented in a poster at CROI 2019. The study design is complicated, involving multiple interventions administered to six groups, each with just five participants. The researchers report that auranofin combined with several other agents led to a reduction in HIV DNA, but the contribution of the anti-proliferative effect is unclear. Administration of the drug was not associated with any serious side effects. Given the renewed interest in targeting CD4 T cell proliferation and the uncertain safety profile of some anti-proliferative drugs, additional studies of auranofin may be justified. 

    Additional Webcasts and Links

    The CROI website offers comprehensive webcasting including every presentation. The pre-conference workshops on March 4th are a source of several excellent overview talks, such as Paula Cannon’s on gene editing techniques in HIV research. Also recommended is Irini Sereti’s insightful plenary on HIV and inflammation.

    Multiple outlets provided high quality coverage from CROI 2019, including:

    AIDSMap
    HIV i-Base
    NATAP
    POZ
    The Body

  • The Influence of Nef Protein Function on HIV Reservoir Size

    A paper published in the Journal of Virology on January 2nd reports that the nefarious activities of HIV’s Nef protein can influence the size of the persistent viral reservoir in people on ART. Nef is known to be able to shield HIV-infected cells from recognition by the immune system, and Fredrick Omondi and colleagues found that this capacity correlated with measures of the HIV reservoir in a group of early-treated individuals assessed after 48 weeks of ART. The researchers also uncovered evidence that HIV reservoir size differed depending on viral subtype, likely partly due to between-subtype variation in the Nef protein.

    The study involved 30 men with HIV in Canada who initiated ART within six months of infection. Of these participants, 25 had HIV subtype B while five had non-B subtypes (two CRF01_AE and three subtype G). The researchers investigated two different possible effects of the HIV Nef protein to assess if they influenced HIV reservoir size: downregulation of the CD4 molecule on CD4 T cells, and downregulation of class I HLA molecules.

    As background, class I HLA molecules act somewhat like trash collectors, picking up protein fragments from within cells and shuttling them to the cell surface so that passing CD8 T cells can inspect them. Most of the time, the protein fragments represent normal debris from human proteins and the cell goes unmolested by CD8 T cells. But if a class I HLA molecule picks up a protein fragment from a foreign infecting virus like HIV, this can trigger a cytotoxic reaction by CD8 T cells that leads to the cell’s destruction (videos of CD8 T cells destroying other cells can be found online). Downregulation of class I HLA molecules by Nef can act to shield HIV-infected cells from this type of immune surveillance.

    Laboratory assays were used to measure the capacity of the Nef protein from HIV sampled from each study participant to downregulate CD4 or class I HLA molecules. While CD4 downregulation showed no effect, the efficiency with which Nef downregulated class I HLA strongly correlated with HIV reservoir size as measured by viral DNA levels (Spearman's R=0.61, p=0.0004). A correlation was also observed when the reservoir was assessed using a virus outgrowth assay, which measures replication-competent HIV rather than viral DNA.

    An analysis that compared participants with HIV subtype B to those with non-B subtypes found that the former group had significantly larger reservoirs (median 2.45 versus 1.72 log10 copies HIV DNA per million CD4 T-cells), with the evidence indicating that the difference was at least partly due to variation in Nef-mediated class I HLA downregulation between the subtypes. The authors note that this phenomenon may be relevant to a prior report of lower HIV reservoirs in a Ugandan cohort, in which the prevalent HIV consisted of subtype D or A/D recombinant viral strains.

    The researchers suggest that the effect of Nef on HIV reservoir size is primarily due to shielding infected cells from destruction prior to ART initiation. They note that establishing whether Nef also impedes clearance of infected cells during ART is technically challenging, and would require larger numbers of study participants. Larger studies, involving more diverse participants, will also be needed to better understand the role of HIV subtype in governing HIV reservoir size, and the extent to which Nef contributes.

    The results imply that efforts to induce killing of latently infected cells by CD8 T cells could be stymied by Nef. However, laboratory studies have been published demonstrating that—should it turn out to be a problem—there are compounds capable of inhibiting Nef function which can enhance CD8 T cell-mediated destruction of latently infected cells.

  • Significant Effects of a TLR7 Agonist Plus Broadly Neutralizing Antibody in the SHIV/Macaque Model

    Among the most newsworthy presentations at CROI 2018 earlier this year was Dan Barouch’s description of a study involving a toll-like receptor 7 (TLR7) agonist combined with a broadly neutralizing antibody (bNAb) in SHIV-infected macaques (see contemporary coverage by AIDSMap, i-Base and POZ Magazine). The results have now been published in the journal Nature.

    The specific experimental candidates that were tested are vesatolimod (formerly known as GS-9620), a TLR7 agonist, and PGT121, a bNAb initially discovered by researchers supported by the International AIDS Vaccine Initiative but now licensed to Gilead Sciences for therapeutic development. Previous laboratory experiments using CD4 T cells isolated from people on ART found that the combination promoted the reversal of HIV latency and killing of virus-infected cells.

    A key goal of Barouch’s study was to establish whether bNAbs are capable of mediating depletion of the viral reservoir in vivo, in addition to displaying the direct antiviral activities that have been documented in other experiments (such as the trials described in the previous blog post).

    A total of 44 macaques were infected with SHIV-SF162P3, a hybrid virus that combines elements of SIV and HIV. ART was initiated seven days later—extremely early after infection—leading to rapid suppression of SHIV replication that was maintained for 96 weeks without any viral load blips.

    For the subsequent intervention phase, animals were divided into four groups of 11 and received, respectively: sham administrations (control group), vesatolimod alone, PGT121 alone, or the combination of vesatolimod and PGT121. Vesatolimod was given orally every other week between weeks 96 and 114 of the study, while PGT121 was administered five times via infusion from weeks 106-114. ART was then interrupted in all macaques at week 130.

    The most dramatic finding was a significantly reduced incidence of SHIV viral load rebound in the group that received the dual vesatolimod/PGT121 combination. Out of the 11 macaques, six (55%) rebounded, compared to all 11 of the sham controls, 10 out of 11 (91%) assigned to vesatolimod alone and nine of 11 (82%) given PGT121.

    Among the animals in the combination group that did rebound, viral loads were 2.6 logs lower at the peak and 1.5 logs lower at the set point compared to the controls. Additional analyses showed that levels of SHIV DNA in lymph nodes were significantly lower in the combination group prior to the ART interruption.

    The interventions appeared to be well tolerated. Vesatolimod caused transient activation of CD4 T cells and natural killer cells, as well as transient increases in proinflammatory cytokines, which were evident the day after administration.

    In an effort to assess whether a reservoir of replication-competent SHIV was still present in the animals that failed to rebound, experiments were conducted in which large numbers of cells were transferred into uninfected macaques. SHIV infection was not transmitted in any case. In contrast, cells from animals that experienced viral load rebounds uniformly transmitted SHIV infection.

    CD8 T cells were also depleted from some macaques, as a test of whether SHIV was being actively contained by the immune system in the group that did not experience viral load rebounds. The depletion did not lead to the reappearance of SHIV viral load in these animals.

    A computer modeling study indicated that the activation of natural killer (NK) cells by vesatolimod was the strongest correlate of delayed viral load rebound. The researchers suggest that this is consistent with a mechanism of action in which stimulation of innate immunity and CD4 T cells by vesatolimod causes virus-infected cells to express viral antigens, which are then recognized and bound to by PGT121, triggering killing of the infected cells by NK cells and/or other effector cells such as monocytes. This antibody-mediated recruitment of immune cells capable of delivering the coup de grâce to virus-infected cells is known as antibody-mediated cellular cytotoxicity (ADCC) or antibody-mediated cellular phagocytosis (ADCP).

    In the discussion section of the paper, the researchers emphasize that the study represented an idealized circumstance due to the very early ART initiation, and that the effects of the interventions were most evident in animals with the lowest pre-ART viral loads. The implications for the more typical circumstance of chronic infection are therefore unclear.

    They also note that the failure to transmit SHIV infection is not definitive evidence that all replication-competent virus has been cleared. This has recently been demonstrated by Ron Desrosiers in a study in which replication-competent SHIV was eventually detected in a macaque that had failed to transmit the virus in several cell transfer experiments.

    Additionally, there is limited experience with the SHIV-SF162P3 challenge virus in the context of cure research and prior studies have not documented an ability of this virus to develop resistance to PGT121. More information on HIV resistance to PGT121 should emerge from ongoing clinical trials.

    Despite these caveats, the evidence that the dual combination promoted clearance—or at least significant depletion—of the viral reservoir in some cases is encouraging.

    Gilead Sciences ultimately aims to study vesatolimod combined with a derivative of PGT121 dubbed GS-9722 in people with HIV. GS-9722 is a version of PGT121 that has been modified in an effort to further enhance ADCC/ADCP activity, and it is currently being tested in a phase I trial (the trial is listed on the subscription-only website Adis Insight so details are scarce; there is no clinicaltrials.gov entry yet).

  • Combining Broadly Neutralizing Antibodies Shows Promise

    At CROI 2017, Michel Nussenzweig from Rockefeller University presented evidence that early administration of a short course of two broadly neutralizing antibodies (bNAbs)—3BNC117 and 10-1074—led to prolonged immunological control of SHIV viral load in macaques. The study was subsequently published in Nature. At the end of last month, results from the first human trials of the same dual bNAb regimen were announced in papers in Nature and Nature Medicine.

    The report in Nature by Pilar Mendoza and colleagues covers a study involving 15 people with HIV who were on suppressive ART at the time of enrollment. The screening process for the study included tests to assess whether HIV resistance to either bNAb could be detected in viruses grown from the latent reservoir, and individuals with samples displaying evidence of resistance were excluded (approximately half the individuals screened could not enroll due to evidence of resistance to one of the antibodies).

    Participants received three infusions of the two bNAbs, spaced three weeks apart. An analytical ART interruption (ATI) was initiated two days after the first infusion in order to evaluate the potential of the bNAbs to maintain HIV viral load suppression.

    Efficacy data are presented from 11 individuals (four participants were excluded from the analysis due to exhibiting viral loads above 20 copies/ml prior to the first bNAb administration). The median time to viral load rebound—defined as a confirmed viral load over 200 copies/ml—was 21 weeks, an improvement over the 6-10 weeks documented in a prior trial that gave only 3BNC117.

    Nine of the 11 participants were able to remain off ART for at least 15 weeks, and two out of these nine did not meet the viral load rebound criteria for the entire 30 weeks of follow up. The bNAb combination appeared safe and well tolerated, with two cases of mild fatigue representing the most serious adverse events.

    Additional detailed analyses revealed that the two participants who rebounded earliest—five and seven weeks into the ATI, respectively—had low-level HIV variants that were resistant to one of the two bNAbs (these variants had been missed by the less sensitive screening test). Therefore they had essentially received bNAb monotherapy during the ATI, explaining the rapidity of their viral load rebounds.

    The two bNAbs were found to have somewhat different half-lives, with 10-1074 persisting longer in the body than 3BNC117. As a consequence, six of the seven participants experiencing later viral load rebounds had developed resistance to 10-1074 (after 3BNC117 levels declined, there was a period during which 10-1074 effectively became a monotherapy). 

    Measures of the HIV reservoir did not reveal any evidence of reductions caused by the dual bNAbs. Quantitative virus outgrowth assays (QVOA) were performed at study entry and 12 weeks into the ATI for a subset of participants, and no significant changes were observed.

    In a presentation at the NIAID Strategies for an HIV Cure meeting yesterday (now available in the NIH videocast archive, starting at the 5:32:22 mark), Marina Caskey from Rockefeller University provided an update on the two individuals who maintained viral suppression the longest during the ATI. One remains off ART after 52 weeks while the other experienced viral load rebound at week 50, restarting ART shortly afterward. Although the study had been intended for people with chronic HIV infection, it turned out the former individual started ART fairly early, about 4-5 months after HIV infection, raising the question of whether post-treatment control might have occurred without an additional intervention. Caskey noted that their viral load at the time of ART initiation was high, at around 800,000 copies/ml.

    Preliminary analyses of HIV-specific T cells in these two participants suggest that receipt of the bNAbs may have been associated with improvements in responses to Gag and Pol antigens, echoing an observation made in the macaque study, but Caskey took pains to emphasize that this work is still at a very early stage.

    The second paper in Nature Medicine presents results from a cohort of seven individuals who were not on ART when they received the dual bNAbs. Significant viral load declines were achieved, but only one participant with a very low viral load at baseline (730 copies/ml) suppressed to undetectable levels. Furthermore, three of the seven participants were found to have evidence of resistance to the bNAbs that had been missed by the assays used at screening.

    Taken together, the results show that combination bNAbs are safe and can have significant and prolonged activity against HIV. The issue of pre-existing resistance presents a challenge that may require the use of additional long-acting antiretroviral drugs or bNAbs if therapeutic regimens are to be designed for clinical use. The authors point out that modified, longer-acting versions of these and other bNAbs are now under investigation, suggesting that the development of combination approaches that require infrequent administration—perhaps every 3-6 months—is a realistic goal.

    In the context of HIV cure research, the potential of bNAbs to enhance virus-specific immunity is of keen interest, and will undergo further exploration in several trials. There is also the possibility that bNAbs could mediate anti-reservoir activity via antibody-dependent cellular cytotoxicity (ADCC), with the caveat that this could also run up against the problem of HIV resistance—the bNAbs would have to be capable of recognizing and binding to the Env proteins of diverse viruses in the reservoir in order to flag the virus-infected cells for destruction by ADCC, and as yet it is unclear how many bNAbs would need to be combined for optimal coverage of the HIV variants present in most people.

    Update 1/15/2019: Researchers at the National Institute of Allergy and Infectious Diseases are seeking volunteers for a clinical trial testing the combination of 3BNC117 and 10-1074 in people with HIV. See the clinicaltrials.gov entry for more information. 

  • 2018 NIAID Strategies for an HIV Cure Meeting

    The biannual National Institute of Allergy and Infectious Diseases (NIAID) Strategies for an HIV Cure Meeting kicks off tomorrow, October 10, at 8:30am US Eastern Time and continues until Friday at 12:30pm. The agenda is available online. For those unable to attend in person the entire event is being made available for viewing via the National Institutes of Health videocast website (see the upcoming events link).

  • Post-AIDS 2018 Updates on HIV Cure Research

    In the aftermath of the 22nd International AIDS Conference (AIDS 2018), which took place in Amsterdam in July, there has been some reflecting on the challenges facing the HIV cure research field. The presentations that garnered the most news coverage described disappointing study results, but there were also nuggets of novelty and encouragement to be found amidst the sea of data on offer (see previous post for links to relevant conference sessions, many of which now have video and/or slides available).

    The RIVER Trial

    The most widely reported findings came from a clinical trial in the UK known as RIVER (Research In Viral Eradication of HIV Reservoirs). Participants with primary HIV infection were randomized to receive a standard antiretroviral therapy (ART) combination plus the integrase inhibitor raltegravir or ART plus raltegravir along with a therapeutic HIV vaccine regimen and a short course of the HDAC inhibitor vorinostat (a candidate HIV latency-reversing agent). The primary purpose was to evaluate whether the vorinostat and vaccine combination—a version of the proposed “kick & kill” approach to depleting the HIV reservoir—had a significant effect on HIV reservoir measures compared to ART.

    As described in detail in multiple online reports (e.g. see i-Base, AIDSMap, and the Imperial College website), the size of the HIV reservoir—as assessed by both total HIV DNA and the viral outgrowth assay—remained equivalent between the two arms. Presenter Sarah Fidler pointed out some possible caveats, such as the relatively short follow up time, but the data appear to rule out any significant effect by this particular kick & kill combination.

    The researchers cited several potential explanations: the latency-reversing activity of vorinostat may be suboptimal, and there is uncertainty as to whether the HIV-specific T cell responses that were successfully induced by the vaccines were targeting parts of the virus most likely to be displayed by infected cells after latency reversal. As is emphasized in much of the coverage, the study itself should not be considered a failure because it provided a clear answer to the question it was designed to address. The interventions were also found to be safe and no participants withdrew.

    The results echo a theme that has been sounding recently in HIV cure research: randomized controlled trials are required to rigorously evaluate the potential of candidate interventions, and positive results from single-arm exploratory trials (which tend to compare results to baseline values or historical controls) need to be interpreted with caution.

    Studies published over the past year that have reached a similar conclusion include randomized controlled evaluations of a therapeutic vaccine combination and single doses of the candidate latency-reversing agent romidepsin.

    Vedolizumab

    The second piece of unwelcome news related to vedolizumab, an antibody that targets the α₄β₇ integrin, a protein involved in CD4 T cell trafficking to the gut that may also facilitate HIV entry into target cells. In 2016, an experiment in SIV-infected macaques generated excitement when administration of a version of vedolizumab adapted for macaques (see comment by Robert Reinhard below) was associated with control of viral load after ART interruption. Human trials were initiated relatively quickly because vedolizumab is already FDA approved as treatment for ulcerative colitis and Crohn's disease.

    During a talk at the conference on HIV remission, Anthony Fauci from the National Institute of Allergy and Infectious Diseases (NIAID) provided a glimpse at the data from the first of these trials, and sadly the results did not mirror the published macaque study. While it appeared that one or two participants displayed some evidence of viral load containment after an analytical treatment interruption (ATI), the majority did not.

    Fauci suggested the variations in viral load levels were similar to those his group has observed in placebo recipients in prior trials, and were not indicative of any effect from vedolizumab. At least two other clinical trials involving the antibody are ongoing, so additional results will be forthcoming.

    In a separate presentation, Michele DiMascio put another dent in the optimism that had surrounded vedolizumab by reporting that an attempt to repeat the original results obtained in SIV-infected macaques had failed. This time, there was no evidence of antibody-induced SIV control. The reasons for the divergent outcomes are unclear, but may relate to the type of SIV used in the experiments, which has a mutation in the nef gene (update 3/21/19: the journal Science has now published an "editorial expression of concern" about the original study).

    Reservoir Targeting

    A number of presentations described novel approaches for targeting the HIV reservoir, highlighting the amount of work that is underway to try and improve upon the interventions tested to date.  

    Isa Munoz-Arias and colleagues from UCSF and Merck reported that a number of FDA-approved chemotherapeutic drugs have HIV latency-reversing activity in laboratory studies. In some cases the magnitude of the effect was demonstrated to be greater than the combination of bryostatin and romidepsin, which has previously been shown to be among the most potent latency-reversing strategies in vitro. The effects were not associated with significant T cell activation or CD4 T cell death (although it’s important to note that this does not mean the drugs are without side effects – those are described on their labels).

    A total of 12 FDA-approved chemotherapies were found to reverse HIV latency via a variety of novel pathways, suggesting new avenues for exploration beyond the current candidates, which primarily comprise HDAC inhibitors. The details of the presentation can be found on Jules Levin’s NATAP website (which often does more to make information public than conferences themselves), and it was also covered by Simon Collins for i-Base.

    The research group of Wen Kang debuted data from a small, uncontrolled pilot study of the HDAC inhibitor chidamide, which is approved in China as a cancer therapy. Kang described evidence that the drug had stimulated production of HIV RNA in seven individuals on ART, and may have slightly reduced HIV DNA levels. However in the Q&A after the talk Sharon Lewin noted that the various possible markers of activity that were analyzed did not appear to necessarily correlate with each other. A larger randomized controlled trial that should provide more definitive results is now ongoing.

    Tim Henrich from UCSF followed up on work published earlier this year in PLoS Pathogens identifying CD30—a cell surface molecule known for its association with lymphoma—as preferentially expressed on HIV-infected CD4 T cells.

    In the paper, the researchers describe an individual with HIV who exhibited undetectable viral RNA and DNA levels after receiving therapy for lymphoma including brentuximab vedotin, an anti-CD30 antibody-drug conjugate. Unfortunately the individual died after cancer recurrence so no further investigation was possible.

    Henrich's conference abstract reports the identification of a second person with HIV who received brentuximab vedotin as part of treatment for lymphoma (now in remission). Three weeks after administration of the first dose, HIV RNA was reduced to undetectable from a previous level of 7,359 copies per million CD4 T cells (a greater than 3 log reduction). HIV DNA levels fell by 42%. The individual is now being followed longitudinally.

    This research opens up the possibility of targeting CD30 as a means to deplete the HIV reservoir, and in addition to brentuximab vedotin there are also CD30-specific chimeric antigen receptor (CAR) T cells in development that are already being studied in clinical trials for cancer.

    An interesting presentation by Sarah Joseph from the University of North Carolina at Chapel Hill outlined an effort to establish when viruses enter the latent reservoir. Using complex phylogenetic analyses (see the abstract for an example), Joseph uncovered evidence that in most individuals studied, the majority (~72%) of the replication-competent latent HIV reservoir was most closely related to viruses circulating in the year prior to ART initiation. In contrast, only 5% was derived from virus replicating during the first year of infection.

    A paper published in the journal eLife in 2016, involving ten participants, reported similar results, although Joseph also pointed out that a much smaller study presented at the conference by Zabrina Brumme (subsequently published in PNAS) reached different conclusions, finding more diverse dates of establishment of the reservoir.

    Joseph’s data suggests that latently infected cells tend to be shorter-lived during untreated infection, with ART initiation triggering the formation of the bulk of the long-lived HIV reservoir. The implication for therapeutic strategies targeting the reservoir is that there might be a window of opportunity to intervene at the time ART is started (currently, almost all clinical trials involve testing candidate therapies in individuals after ART has suppressed viral load). This idea could potentially be explored in the SIV/macaque model.

    HIV Control Off ART

    Participants in the VISCONTI cohort represent the best known and most widely cited examples of post-treatment control of HIV replication. In 2013, when Asier Sáez-Cirión and colleagues published a detailed report in PLoS Pathogens, the cohort comprised 14 individuals. Updates have been fragmented since that time, occurring at various conferences, and as yet there have been no follow up publications as thorough as the original paper (at least that I’m aware of – for the sake of disclosure I should note that I chided the investigators about this situation in a public comment to the PLoS Pathogens article in 2017).

    At AIDS 2018, Laurent Hocqueloux and colleagues presented a poster on factors associated with loss of post-treatment controller status that included an update on the VISCONTI cohort. The study also offered results from analyses of inflammatory biomarkers, which I’ve not seen described previously.

    A total of 24 post-treatment controllers have now been added to the VISCONTI cohort; all started ART during primary infection and then interrupted after a median of 3.5 years of treatment. During subsequent follow up (current median of 12 years), five (21%) have restarted ART, four due to increasing viral load and one as a result of a head and neck cancer diagnosis.

    These five participants were among seven who experienced one or more viral load measurements above 400 copies/ml during monitoring; none of the 17 who remained below this viral load level restarted ART (a highly statistically significant difference). The fact that the individual who developed cancer was in the former group, despite not reinitiating ART based on viral load criteria, may raise concern that prior exposure to detectable HIV viremia was a risk factor for cancer development. Whether remaining on ART would have been associated with lower risk is an unanswerable question. This conundrum illustrates that firm conclusions about the clinical benefits of post-treatment control compared to continuous ART cannot be drawn until post-treatment control can be induced in sufficient numbers of people to allow a randomized comparison.

    Overall, CD4 counts and CD4:CD8 ratios have remained stable in the cohort with medians not significantly different between the time of ART interruption and last follow up. Individual plots are not shown in the poster, however, so it’s unclear if declines occurred in participants who experienced viral load increases.

    Encouragingly, the majority of VISCONTI cohort members have maintained viral loads below 50 copies/ml, and levels of three inflammatory biomarkers—IP-10, sCD163 and sCD14—were not significantly different between these post-treatment controllers and healthy HIV-negative individuals (see figure 6 in the poster).

    In a separate oral presentation, Asier Sáez-Cirión showed evidence that certain immune response genes are associated with post-treatment control in the VISCONTI cohort, and may be mediating their effects—at least partly—through superior natural killer (NK) cell activity against HIV (unfortunately neither slides or video of this presentation are available on the conference website).

    Lisa Chakrabarti described an investigation of potential mechanisms of HIV control in a different population: elite controllers participating in the ANRS CODEX cohort. The researchers focused on HIV Gag-specific CD4 T cell responses, and found that CCR5 expression was lower among elite controllers compared to a control group of people with HIV on ART. 

    The lower CCR5 expression by Gag-specific CD4 T cells was associated with reduced susceptibility to HIV infection, suggesting this may contribute to elite controller status. The results may offer support to efforts to protect virus-specific CD4 T cells from HIV infection using gene therapies that ablate CCR5 expression (or otherwise attempt to protect the cells from HIV entry), an approach being pursued by researchers at the defeatHIV collaboratory.

    Analytical Treatment Interruptions (ATIs)

    A controversial component of research working toward achieving post-treatment control of HIV is the use of ATIs. A systematic review of past studies involving ART interruptions could help shed light on the safest approaches to conducting ATIs in clinical trials, and Jillian Lau and colleagues from Alfred Hospital and Monash University in Melbourne, Australia delivered just such a review as a poster presentation at the conference. Their work is now in press at a journal and will hopefully be published soon.

    Online Post-Conference Coverage

    As is always the case, an array of excellent reporting has become available online since the conference. See below for a selection; please leave a comment if you know of coverage I’ve missed that should be included.

    AIDS 2018 HIV Cure Research Highlights – Karine Dubé and Jeff Berry, Positively Aware

    The Road Ahead for HIV Cure Research – Benjamin Ryan, POZ Magazine, September 6, 2018

    Cure Updates from AIDS 2018 - hivcure.com.au/Doherty Institute

    AIDSMap

    i-Base

    NATAP