TAG HIV Basic Science, Vaccines, and Cure Project Blog

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

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

HIV Cure-Related Research at AIDS 2018

Workshops, sessions and presentations related to HIV cure research at the upcoming AIDS 2018 conference in Amsterdam. 

HIV Cure Research with the Community Workshop 
Pre-Conference Meeting
Room E105–108, RAI, Amsterdam, Netherlands
Saturday July 21, 2018, 9:00-18:00

Strategies for diagnosing and managing acute HIV infection in the context of PrEP and immediate ART
Code: MOSA52
Session Type: Non-Commercial Satellite
Venue: Hall 11B
Monday July 23, 17:00 - 19:00

17:20 MOSA5203 
Acute HIV infection and HIV cure
Thumbi Ndung’u, Africa Health Research Institute, University of KwaZulu-Natal, South Africa 

Biomedical research innovations in the prevention, remission and cure of HIV/AIDS
Code: MOSA48
Session Type: Major Industry Sponsor Satellite
Venue: Elicium 2
Monday July 23, 17:00 - 19:00

17:05 MOSA4802
30 years of progress in the field of HIV/AIDS
Anthony Fauci, National Institutes of Health (NIH), United States 

18:05 MOSA4802
Passive immunization/bNAbs monoclonals both for prevention and treatment
Penny Moore, University of the Witwatersrand / National Institute for Communicable Diseases, South Africa 

18:25 MOSA4802
Remission and cure efforts
Javier Martinez-Picado, AIDS Research Institute irsiCaixa, Spain 

Besieging the reservoir and kicking it where it hurts
Code: TUPDA01
Session Type: Oral Poster Discussion Session Track A – Basic and translational research
Venue: G104-105
Tuesday July 24, 13:00 - 14:00

13:00 TUPDA0101 
Association between immunogenetic factors and post-treatment control of HIV-1 infection. ANRS VISCONTI and PRIMO studies
Asier Saéz-Cirion, Insitut Pasteur, France

13:05 TUPDA0102 
HCV treatment with direct-acting antivirals (DAAs) in HIV/HCV coinfected subjects affects the dynamics of the HIV-1 reservoir 
Natalia Laufer, INBIRS Institute (UBA-CONICET), Argentina 

13:10 TUPDA0103
IL-10 contributes to, and is a biomarker for, viral persistence in ART-treated, SIV-infected rhesus macaques 
Mirko Paiardini, Emory University, United States

13:15 TUPDA0104
Follicular CD8+ T-cells in gut-associated lymphoid tissue are associated with lower HIV-1 reservoir in the terminal ileum after ART initiated during primary HIV infection 
John Patrick Thornhill, Imperial College London, United Kingdom

13:20 TUPDA0105
HIV-1 reservoir diversity and genetic compartmentalization in blood and testis
Rosalie Ponte, The Research Institute of the McGill University Health Centre, Canada

13:25 TUPDA0106LB
Ixazomib reduces HIV-1 reservoir size in a Casp8p41-dependent manner
Nathan Cummins, Mayo Clinic, United States
 
Strategies for cure: Pitfalls, possibilities and promise
Code: TUAA02
Session Type: Oral Abstract Session Track A – Basic and translational research
Venue: E102
Tuesday July 24, 16:30 - 18:00

16:30 TUAA0201
Therapeutic Vaccines for Cure 
Steven Deeks, University of California, United States

16:45 TUAA0202LB
A randomised controlled trial comparing the impact of antiretroviral therapy (ART) with a 'Kick-and-Kill' approach to ART alone on HIV reservoirs in individuals with primary HIV infection (PHI); RIVER trial
Sarah Fidler, Imperial College London, United Kingdom

17:00 TUAA0203
Dominant HIV DNA populations present in different T-cell subsets before stem cell transplantation persist in tissues early after transplantation with CCR5Δ32 stem cells 
Annemarie Wensing, University Medical Center Utrecht, Netherlands

17:15 TUAA0204
Rapid rebound of a highly replication competent preexisting CXCR4-tropic HIV variant after allogeneic stem cell transplantation with CCR5Δ32 stem cells 
Monique Nijhuis, University Medical Center Utrecht, Netherlands

17:30 TUAA0205
Modular gene therapy vectors for gene therapy cure in resting immune cells 
Andrew Wong, The University Of New South Wales Sydney, Australia

17:45 TUAA0206LB
Evaluation of an antibody to Alpha4Beta7 in the control of SIV infection
Michele Di Mascio, National Institute of Allergy and Infectious Diseases, United States

Building bridges from scientific innovation to implementation 
Code: WEPL01 
Plenary Session 
Venue: Hall 12 
Wednesday July 25, 08:45 - 10:30 

08:55 WEPL0105
The newest science in the search for a cure and vaccine 
Brad Jones, Weill Cornell Medicine and The George Washington University, United States

Poking, prodding and purging the final reservoir frontier
Code: WEAA01
Session Type: Oral Abstract Session Track A – Basic and translational research
Venue: G104-105
Wednesday July 25, 11:00 - 12:30

11:00 WEAA0101
Chidamide reactivates and diminishes latent HIV-1 DNA in patients on suppressive antiretroviral therapy 
Wen Kang, Tangdu Hospital affiliated to The Fourth Military Medical University, China

11:15 WEAA0102
The antiretroviral CCR5-inhibitor maraviroc effectively reverses HIV latency by phosphorylation of Nf-κB 
Jori Symons, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Australia

11:30 WEAA0103
Activation of latent HIV and SIV RNA transcription in vitro and in vivo in ART suppressed SIV-infected rhesus macaques by the Ingenol-based protein kinase C agonist, GSK445A 
Afam Okoye, Oregon Health and Science University, United States

11:45 WEAA0104
The RNA-binding proteins, SRP14 and HMGB3 play a crucial role in controlling HIV replication and latency 
Georges Khoury, Peter Doherty Institute, Australia

12:00 WEAA0105
Using the PPARg antagonism to block/lock HIV reactivation in Th17 cells 
Petronela Ancuta, Universite de Montreal and CHUM Research Centre, Canada

12:15 WEAA0108LB
The majority of the replication-competent virus in the latent reservoir originates from viruses circulating near the time of ART initiation
Sarah B. Joseph, University of North Carolina at Chapel Hill, United States

Durable control of HIV infections in the absence of antiretroviral therapy: Opportunities and obstacles and Jonathan Mann Memorial Lecture: Data to drive equity
Code: WESS01
Session Type: Special Session
Venue: Hall 12
Wednesday 25 July, 13:00 - 14:00

13:05 WESS0102
Durable control of HIV infection in the absence of antiretroviral therapy: Opportunities and obstacles
Anthony Fauci, National Institutes of Health (NIH), United States

Acute infection and viral reservoir
Code: WEPDB01
Session Type: Oral Poster Discussion Session Track B – Clinical research
Venue: E102
Wednesday July 25, 13:00 - 14:00

13:00 WEPDB0101
Validation of Alere TM q HIV-1/2 detect for detection of acute HIV infection at Anonymous Clinic, The Thai Red Cross AIDS Research Centre 
Irin Srila-Or, The Thai Red Cross AIDS Research Centre, Thailand

13:05 WEPDB0102
Favorable clinical phenotype reached in less than half of people treated in acute HIV infection 
Jintanat Ananworanich, The Henry M. Jackson Foundation for the Advancement of Military Medicine, United States

13:10 WEPDB0103
Increasing contribution of integrated forms to total HIV1-DNA in blood, in primary infection during natural history - ANRS PRIMO and SEROCO cohorts 
Véronique Avettand-Fenoel, Université Paris Descartes, France

13:15 WEPDB0104
Intermittent viremia after treatment interruption increased risk of ART resumption in post-treatment HIV-1 controllers. ANRS VISCONTI study 
Laurent Hocqueloux, CHR d'Orléans - La Source, France

13:20 WEPDB0105
Auranofin plus nicotinamide impact HIV reservoir among ART suppressed HIV individuals 
Ricardo Sobhie Diaz, Federal University of Sao Paulo, Brazil

All fired up: Tackling inflammation
Code: WEPDA01 
Session Type: Oral Poster Discussion Session Track A – Basic and translational research 
Venue: Hall 11B 
Wednesday July 25, 13:00 - 14:00 

13:10 WEPDA0103
Persistence of myeloid cell-associated inflammation in HIV-infected children after 8 years on early initiated therapy - the key role players in HIV persistence? 
Shalena Naidoo, Stellenbosch University, South Africa 

Killers or helpers: The double life of T cells
Code: WEAA02
Session Type: Oral Abstract Session Track A – Basic and translational research
Venue: Hall 11B
Wednesday July 25, 14:30 - 16:00

14:30 WEAA0201
Increase in restriction factor expression in response to viral rebound after analytical treatment interruption in HIV-infected patients
Marie-Angélique De Scheerder, UZ Gent, Belgium 

14:45 WEAA0202
RhCMV-induced, SIV-specific MHC-E-restricted T cells recognize SIV through the T cell receptor 
Shaheed Abdulhaqq, Oregon Health and Science University, United States

15:00 WEAA0203
Genetic factors leading to loss of viral control in HIV elite controller patients 
José M. Benito, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Universidad Autónoma de Madrid (IIS-FJD, UAM), Spain 

15:15 WEAA0204
Frequent generation of HIV broadly neutralizing antibodies in infected children is associated with both increased help and regulation within germinal centers 
Julia Roider, University of Oxford, South Africa

15:30 WEAA0205
Initiation of antiretroviral therapy during hyperacute HIV infection preserves germinal center T follicular (GCTfh) helper cell function
Zaza Ndhlovu, University of KwaZulu Natal, South Africa

Eliminating HIV latency: Shock and kill or block and lock?
Code: WESY09
Session Type: Symposia Session
Venue: Hall 11B
Wednesday July 25, 16:30 - 18:00

16:44 WESY0902 
Introductory comments 
Maureen Goodenow, U.S. DHHS National Institutes of Health, Office of AIDS Research, United States

16:49 WESY0903
Understanding HIV persistence – do defective viruses matter? 
Ya-Chi Ho, Yale University School of Medicine, United States

17:06 WESY0904
Low level transcription on ART – implications for latency elimination 
Steve Yukl, University of California, United States

17:23 WESY0905 
TLR agonists and latency reversal: can they both shock and kill? 
Martin Tolstrup, Aarhus University, Denmark

17:40 WESY0906
Block and lock using gene silencing 
Anthony Kelleher, The University of New South Wales (UNSW), Australia

17:57 WESY0907 
Understanding community participation in cure studies: what scientists need to know 
Cipriano Martinez, National Association for People Living with HIV in Australia, Australia
 
Broadly neutralizing antibodies (bNabs): Towards a cure and vaccine
Code: THSY04
Session Type: Symposia Session
Venue: G104-105
Thursday July 26, 11:00 - 12:30

11:05 THSY0402
Factors determining bNab elicitation in HIV-infected individuals during natural infection: Challenges a vaccine has to overcome
Alexandra Trkola, Leiterin Group Trkola, Switzerland

11:25 THSY0403
Maturation of bNabs in HIV-1 infected patients (specifics of B-cell maturation), what is different from other infections?
Michel Nussenzweig, The Rockefeller University, United States

11:45 THSY0404
Induction of bNabs by vaccines: where do we stand, what is being needed for success
Rogier Sanders, AMC, Netherlands

12:05 THSY0405
bNabs for clinical use: antiviral activity and effector functions other than neutralization
Lynn Morris, University of the Witwatersrand, South Africa

How Far are We from HIV Remission? 
Code: THSA14 
Session Type: Non-Commercial Satellite 
Venue: Hall 11A 
Thursday July 26, 18:30 - 20:30

18:35 THSA1402 
An Update on Post-Treatment Controllers in France and Worldwide 
Laurent Hocqueloux, CHR d'Orléans - La Source, France

18:50 THSA1403 
Insights on Mechanisms of Post-Treatment Control 
Asier Saéz-Cirion, Insitut Pasteur, France

19:10 THSA1405
Lessons from Research Interventions aiming at HIV Remission

19:30 THSA1406
Very early ART Initiation during "Hyperacute" HIV Infection 
Timothy Henrich, University of California at San Francisco ( UCSF), United States

19:45 THSA1407
Stem Cell Transplantations: ICISTEM 
Monique Nijhuis, University Medical Center Utrecht, Netherlands

20:00 THSA1408
Interventions with broadly neutralizing antibodies (bNAb)
Michel Nussenszweig, Howard Hughes Institute, The Rockefeller University, United States

20:15 THSA1409
HIV Remission Research: Expectations and Questioning
Alain Volny-Anne, France

20:45 THSA1411
Concluding Remarks
Françoise Barré-Sinoussi, Institut Pasteur, France

New Data on Identifying and Targeting the Latent HIV Reservoir

Several recently presented and published studies offer potentially important new data relevant to efforts to identify cells containing latent HIV and target them for elimination.

At the 8th International Workshop on HIV Persistence held in Miami last December, multiple research groups reported on their attempts to confirm a newly published—and widely publicized—claim that the cell surface molecule CD32a is preferentially expressed on latently infected cells. The results indicate that the picture is considerably more complicated: the original findings could not be verified and instead it appears that expression of CD32a may be driven by other factors, including cell activation and active HIV transcription. Summaries of these studies can be found in the published abstracts from the workshop (see abstract numbers OP 1.5, OP 2.4, OP 2.6 and OP 4.2).

While disappointing, this work does not end hopes that markers of latently infected cells can be identified – other candidates that are being examined are CCR5, when expressed by resting memory CD4 T cells (see the study from the laboratory of Robert Siliciano published late last year), and CD30, a cell surface molecule better known for its association with lymphoma that is being investigated by Tim Henrich and colleagues.

One of the reasons why markers of latently infected cells would be a boon to the cure research field is that they might facilitate elimination strategies akin to those now being used successfully against cancers. A number of effective cancer immunotherapies involve equipping T cells with receptors that target cell surface molecules on malignant cells, such as CD19, CD22, and CD30. It is conceivable that this “chimeric antigen receptor” (CAR) T cell approach could be adapted to target latently infected cells if appropriate markers could be identified.

CAR T cells are also being developed that target HIV antigens, with a recent paper in PLoS Pathogens from Scott Kitchen colleagues reporting some encouraging results obtained with stem cell-derived CAR T cells in macaques. However, in order for the approach to work against latently infected cells, it would be necessary to activate the latent HIV first to trigger production of viral antigens. An article in STAT News by Sharon Begley provides an accurate and nuanced perspective on Kitchen’s paper, offering the appropriately cautious headline: “Preliminary study hints that genetically modified T cells might fight HIV.” In contrast, coverage in The Daily Beast unfortunately opts for misleading hype, with their piece titled: “This Doctor's Revolutionary Stem Cell Treatments Could Eradicate HIV.”

The research group of Jonathan Angel in Canada is pursuing a novel method for targeting latently infected cells: an oncolytic rhabdovirus named MG1, which is primarily being developed for its ability to preferentially infect and destroy cancer cells. Angel and colleagues have published a study in the Journal of Infectious Diseases demonstrating that MG1 also appears capable of targeting cells latently infected with HIV, while sparing uninfected cells (the paper is open access).

In an analysis of the effects of MG1 on memory CD4 T cells isolated from 14 HIV-positive individuals on ART, both total and integrated HIV DNA levels declined in 12 out of the 14 samples. The mechanism for MG1’s preferential targeting is unclear, but may relate to changes in latently infected cells that affect their response to the cytokine interferon. The authors note that clinical trials are already underway in cancer, which should help discern if the approach can be safely studied in HIV infection. In an interview with MD Magazine, Angel notes MG1 that can cause fever and malaise, and adverse events may be dose-dependent.

Another paper in PLoS Pathogens that has drawn attention comes from Christina Gavegnano and colleagues, who show that a class of drugs called Jak inhibitors can inhibit the maintenance of latently infected cells, and prevent them spreading infection when latent HIV is reactivated. Jak inhibitors target a pathway involved in the survival and proliferation of memory CD4 T cells, and have been found safe in the treatment of certain inflammatory diseases and myelofibrosis. The authors note that an ongoing clinical trial is evaluating the effects of the Jak inhibitor ruxolitinib in HIV-positive people on ART.

Update on AAV Vectors as Delivery Vehicles for Broadly Neutralizing Antibodies: Ron Desrosiers and the Miami Macaque

The idea of using adeno-associated virus (AAV) as a vehicle to deliver genes encoding anti-HIV broadly neutralizing antibodies (bNAbs) has been around for some time, and has been covered a number of times previously on the blog (e.g. see posts from May 2009 and January 2013). The approach was first developed by Phil Johnson as a possible way of circumventing the challenges associated with inducing broadly neutralizing antibodies using traditional vaccines. AAVs primarily take up residence in muscle tissue and can act as a factory for producing proteins encoded by genes inserted into the AAV genome. This past Tuesday evening at the HIV Persistence Workshop in Miami, the researcher Ron Desrosiers presented an update on efforts to deliver bNAbs with AAV, including the intriguing tale of a macaque in which the method appeared to have a profound therapeutic effect.

Many years ago Desrosiers, then at the New England Primate Research Center, collaboratored with Phil Johnson on the first study to demonstrate that anti-SIV antibodies delivered by AAV could protect macaques against a highly pathogenic SIV challenge. Desrosiers has since moved to the University of Miami and is now exploring the potential of AAV to deliver the more recently discovered potent bNAbs.

At the workshop, Desrosiers described a preliminary study in which four macaques were infected with a SHIV AD8 challenge virus and, 86 weeks later, given three AAV vectors encoding the bNAbs 10E8, 3BNC117 and 10-1074, respectively. Because they originated in humans, the bNAbs were modified to make them compatible with rhesus macaque antibodies (rhesusized, to use Desrosiers term). No antiretroviral therapies were employed in the experiment.

Evaluations of bNAb levels after AAV administration produced disappointing results: 10E8 was very low or undetectable in all cases, 3BNC117 was delivered successfully in just one out of four animals and 10-1074 achieved significant levels in three out of four. As Desrosiers and colleagues explained in a paper published in Molecular Therapy last year, the problem was caused by the generation of antibodies against the AAV-encoded bNAbs (anti-antibody responses). This problem was also seen in SIV prevention experiments.

There was a more encouraging finding, however. One macaque developed sustained levels of both 3BNC117 and 10-1074, and this was associated with a persistent decline in SHIV AD8 viral load to undetectable levels – below 15 copies/mL in 26 samples taken over a 24-month period (viral load analyses were conducted by Jeff Lifson at the National Cancer Institute). Virus reservoirs also became undetectable: 62 weeks after AAV administration, no SHIV AD8 could be recovered from 180 million peripheral blood mononuclear cells (PBMC) using a quantitative virus outgrowth assay (QVOA).

Several additional experiments were conducted in an attempt to ascertain if a cure of the challenge virus might have been achieved. Cells from an entire lymph node sampled after 74 weeks were transferred into an uninfected macaque, without causing SHIV AD8 infection. A follow up five weeks later, in which approximately 140 million cells derived from an extracted cluster of lymph nodes were transferred, also failed to establish SHIV AD8 infection. Desrosiers presented these results earlier this year in a talk that is available on Youtube (thanks to @DanWilliamsVisa on twitter for sharing it with me).

On Tuesday, Desrosiers reported that 87 weeks after AAV was given, SHIV AD8 was finally recovered at very low levels on three occasions by QVOA, with the frequency of infected cells estimated to be ~1 in 50 million PBMC (depleted of CD8 cells). While this ends hopes that the virus may have been entirely eradicated, Desrosiers noted that the animal—dubbed the Miami monkey by some of his colleagues—might legitimately be described as functionally cured, although he acknowledged the major caveat: it is just one case.

Desrosiers’s research group is now focused on circumventing the problem of anti-bNAb antibodies. The AAV variant used in studies to date has been AAV1, and he showed evidence that AAV8 appears less prone to inducing anti-antibodies. This may be because AAV8 is tropic for the liver, a site in the body where immune responses against vector-encoded bNAbs are less likely to be induced (a phenomenon known as the “liver tolerance effect”).

Experiments in which AAV8 administration was followed later by AAV1 have suggested the combination might further reduce anti-antibody levels; in essence a type of “prime-boost” in which the booster is enhancing immunological tolerance rather than enhancing immune responses. An additional strategy involves including a piece of genetic code in the AAV vector that is designed to shut down antigen presentation by cells (e.g. dendritic cells) that might otherwise promote the development of anti-antibodies.

Plans are now underway to conduct a study in which 12 macaques infected with SHIV AD8 will be divided into two groups and receive either AAV8 vectors encoding 3BNC117 and 10-1074 (possibly with an AAV1 boost) or antiretroviral therapy.

There is one ongoing trial of an AAV1 vector encoding a bNAb (PG9) in humans, launched several years ago by a collaboration involving Phil Johnson and the International AIDS Vaccine Initiative (IAVI). The trial population is HIV-negative men. Results were initially due in January 2016 and have been eagerly anticipated. In his presentation at the Persistence Workshop, Desrosiers stated that he recently attended a meeting at the Gates Foundation at which preliminary data were presented and obtained permission to disclose them.

According to his description, what the results revealed is that the anti-antibody problem is not limited to macaques: nine trial participants that were analyzed did not have detectable PG9 levels, but seven out of the nine had readily detectable anti-PG9 antibodies. There may be subtleties to the findings that could not be conveyed in a brief aside, but it sounds like the promise of the AAV approach—both in the preventive and therapeutic contexts—will only be realized if a means to avoid anti-antibodies can be developed.

Update 12/18/17: The National Institute of Allergy and Infectious Diseases (NIAID) has just opened a clinical trial investigating the delivery of the bNAb VRCO7 by an AAV8 vector in HIV-positive individuals on ART. The trial is taking place at the National Institutes of Health Clinical Center in Bethesda.

Abstracts from the Eighth International Workshop on HIV Persistence during Therapy are now available from the Journal of Virus Eradication. 

2017 IAS HIV Cure & Cancer Forum

The IAS HIV Cure & Cancer Forum was held at the renowned cancer research center Institut Curie in Paris on July 22 & 23 of this year. A report from the meeting, authored by Genevieve E. Martin, José Alcami, Jean-Phillipe Spano and Anna Laura Ross, has just been published in the open access Journal of Virus Eradication, and many of the slide presentations are posted to the Forum website.

Some of the intersections between the two disciplines were described in a presentation by Olivier Lambotte at last year’s IAS Towards an HIV Cure Symposium in Durban, South Africa, and the 2017 event expanded on the theme. Similarities include the need to identify and target relatively rare, hard-to-access cells for elimination - a task which requires gaining an understanding of the reasons why natural immune responses are ineffective and the development of strategies to bolster immunity against the cells of interest (whether cancerous or latently infected by HIV).

As the meeting report notes, many approaches being pursued as cancer therapeutics are also being studied in the context of HIV cure research, including immune checkpoint inhibitors, cytokine therapies, genetically modified chimeric antigen receptor (CAR) T cells and other gene therapies.

The coverage of the presentations offered in the report is fairly comprehensive, but there are some additional pieces of information that may be of interest. There was a presentation by Francoise Villinger from the University of Louisiana at Lafayette describing the use of radiolabeled anti-virus antibodies to image the locations of SIV expression in the bodies of macaques, and a clinical trial is due to open soon in Australia that will explore a similar approach in humans using a radiolabeled version of the broadly neutralizing antibody 3BNC117.

Larry Corey from the Fred Hutchinson Cancer Research Center gave an excellent talk on the potential of CAR T cells, describing both the positive results in cancer (a CAR T cell therapy for acute lymphoblastic leukemia became the first to be approved by the FDA not long afterward) and some of the issues that have arisen with adverse events. In the context of HIV cure research, Corey highlighted a number of factors he considers likely to be important, including:

• The provision of HIV-specific CD4 T cell help to CAR CD8 T cells using CD4 T cells that have been modified to resist HIV infection.
• Targeting of CAR T cells to HIV reservoir sites such as lymph node B cell follicles (some experiments addressing this issue using CD8 T cells modified to express the trafficking receptor CXCR5 have already been conducted in SIV-infected macaques).
• Equipping CAR T cells with receptors that allow them to recognize and kill HIV-infected cells quickly after the virus is reactivated (a recently published study from the research group of Brad Jones suggests that the HIV Nef protein may be a particularly important target for this purpose).
• Ensuring CAR T cells can persist at the sites where they are needed.
• Avoiding the adverse events that have been observed in some cancer studies, most notably cytokine release syndrome and neural toxicity.

Corey focused on the potential of directing CAR T cells to HIV antigens, but a recent cancer trial may also raise the possibility of targeting cellular receptors that are preferentially expressed by latently infected CD4 cells. In a paper published in the Journal of Clinical Investigation, researchers describe promising results obtained with CAR T cells engineered to recognize CD30, a receptor expressed by non-Hodgkin’s lymphomas. Ongoing work by the laboratory of Timothy Heinrich (as yet unpublished, but briefly described in a summary for the supporting NIH grant) has identified CD30 as a possible marker for CD4 cells containing latent HIV. While Heinrich’s group is initially looking at brentuximab vedotin, an FDA-approved antibody-drug conjugate that targets CD30 but has some notable toxicities, CAR T cells could offer an alternative means to the same end.

One presentation that is not covered in the meeting report was delivered by Marina Cavazzana from Hôpital Necker in Paris. Cavazzana is working on a novel approach to promoting immune reconstitution by accelerating production of naïve T cells from the thymus. At one time, this was an area of intense interest in HIV research, and a number of candidate therapies were evaluated (keratinocyte growth factor is among the examples) but results proved disappointing and there has been little activity related to the thymus in recent years. Persistent deficits in naïve T cell levels are still a concern, however, particularly for individuals who experience suboptimal CD4 T cell recovery on antiretroviral therapy (immunologic non-responders or INRs), so a candidate treatment could still have relevance.

Cavazzana’s strategy involves the use of a laboratory culture method to generate T cell precursors from stem cells, mimicking the early steps of maturation that would normally occur in the thymus. In experiments where these lab-generated T cell precursors were infused into immunodeficient mice, reconstitution of mature naïve T cells was significantly accelerated. Phase I/II clinical trials involving adults receiving stem cell transplants for cancers and children with primary immunodeficiencies are due to begin in January 2018. Cavazzana noted that there is also the potential to modify the T cell precursors with gene therapy prior to infusion.

A lively roundtable discussion on clinical trial design and participation is summarized in the meeting report (see box 1), but video of the presentation by Michael Louella from the University of Washington AIDS Clinical Trials Unit/defeatHIV is also available on the defeatHIV Youtube channel.

Panel participant Thomas Uldrick from the National Cancer Institute drew attention to an important initiative involving the U.S Food & Drug Administration, American Society of Clinical Oncology and Friends of Cancer Research that is attempting to expand eligibility criteria for trials of novel anticancer agents to include HIV-positive people. Uldrick pointed out that the median time from phase I to an HIV-specific study for novel anticancer agents (6.3 years) is only a few months shorter than the average time from phase I to FDA approval, a problem that urgently needs to be addressed given that the prognosis for people with HIV is so much improved due to ART. Uldrick encouraged attendees to spread the word about the initiative.

In addition to the new report, the IAS blog has also published a commentary on the meeting by Geneviève Almouzni, Director of the Institut Curie Research Center and CNRS Research Director, timed to coincide with World Cancer Research Day.

Update 10/10/17: A paper entitled "Modernizing Clinical Trial Eligibility Criteria: Recommendations of the American Society of Clinical Oncology–Friends of Cancer Research HIV Working Group" was published by the Journal of Clinical Oncology on October 2 - Thomas Uldrick is the lead author. The journal has made the full text available free of charge. 

An additional report on the 2017 IAS Cure & Cancer Forum, "The Road Towards an HIV Cure," has been published by Craig McClure and Darien Taylor for the Canadian HIV Cure Enterprise (CanCURE). 

Mosaic Antigens and Mountaineering Metaphors: Climbing Toward the Next HIV Vaccine Efficacy Trial

The pharmaceutical industry has made significant contributions to HIV vaccine research, but it has been rare for a major company to take the leading role in propelling a candidate toward efficacy testing. The only example to date occurred around the turn of the millennium with Merck’s adenovirus serotype 5 (Ad5) vector, which showed some evidence of promise in macaque models but was ultimately discontinued after being associated with an enhanced—rather than reduced—risk of HIV acquisition in the STEP and Phambili trials. 

Given Merck’s cautionary tale, it is all the more notable that Janssen Vaccines and Prevention B.V. is now rapidly advancing an experimental prime-boost HIV vaccine regimen, with plans announced at the IAS 2017 conference to launch a large scale “proof of concept” efficacy trial in women in late 2017/early 2018.

The Dutch biotech company Crucell, which Janssen acquired several years ago, initially produced the main vaccine candidates being studied. An iterative process involving both macaque and human studies has been ongoing, informing decisions about which versions of the vaccine constructs and which HIV antigens will be selected for efficacy evaluations. While Janssen is taking the lead, the program is a large collaboration including the Beth Israel Deaconess Medical Center (BIDMC), the Bill & Melinda Gates Foundation, the National Institute of Allergy and Infectious Diseases (NIAID), the HIV Vaccine Trials Network (HVTN), the International AIDS Vaccine Initiative, the US Military HIV Research Program, the Ragon Institute and the Los Alamos National Laboratory.

At IAS 2017 Hannah Schuitemaker, Vice President and Head of Viral Vaccine Discovery and Translational Medicine at Janssen Vaccines and Prevention B.V., presented results from a key clinical trial named APPROACH (the company has chosen monikers from mountaineering terminology to designate their steps toward the hoped-for summit of a licensable HIV vaccine). The trial randomized 400 participants into eight groups of 50, with each receiving different prime-boost recipes drawn from four possible ingredients: adenovirus serotype 26 (Ad26) vectors, modified Vaccinia Ankara strain (MVA) vectors and high or low dose trimeric clade C gp140 protein in alum adjuvant.

The Ad26 and MVA vectors used in the trial encode mosaic HIV antigens, which represent an amalgam of elements from multiple different HIV variants generated with the aid of computer software by Better Korber at the Los Alamos National Laboratory. The aim is to induce immune responses capable of recognizing HIVs from all circulating strains.

The crux of the APPROACH results is that the best-performing regimen comprised two priming immunizations (at months 0 and 3) with trivalent Ad26 vectors followed by two booster immunizations (at months 6 and 12) with a combination of the same Ad26 vectors plus a high dose soluble trimeric clade C gp140 Env protein with alum adjuvant. Trivalent refers to a mix of three Ad26 vectors, two containing different mosaic versions of Gag and Pol proteins (designated Ad26.Mos1.Gag-Pol and Ad26.Mos2.Gag-Pol) and one containing a mosaic Env protein (designated Ad26.Mos1.Env).

Gratifyingly for the researchers, this regimen had also shown the highest protective efficacy in a macaque study conducted by Dan Barouch at BIDMC. In 12 vaccinated animals given a series of low-dose challenges with the pathogenic SIV/HIV hybrid virus SHIVsf162p3, per-exposure risk of acquisition was reduced by 94% and, after a total of six challenges, 66% of the macaques remained uninfected. For comparison, the next-best regimen in the study used MVA vectors in the boosting phase and reduced per-exposure risk by 87%, with 42% of the group of 12 animals still uninfected after the completion of six challenges.

Performance of the regimens in the APPROACH trial was judged based on measures of HIV-specific immune responses that correlated with protective efficacy in the macaque studies. These included the magnitude of binding antibodies to the HIV Env protein and Env-specific T cell responses, as well as antibody-dependent cellular phagocytosis (ADCP, an antibody function that can mediate killing of virus-infected cells). After the final boost in APPROACH, 80% or more of participants displayed the requisite immune responses at levels above the desired magnitude. These results greatly exceeded the targets set for making the go/no go decision about proceeding to efficacy testing.

Results are still pending from one other preparatory clinical trial, TRAVERSE, which is comparing the trivalent mix of Ad26 vectors with a tetravalent version that adds an additional mosaic Env protein (Ad26.Mos25.Env). If all goes according to plan, the tetravalent combination will be used in the planned efficacy trial, HPX2008/HVTN 705, which aims to enroll 2,600 sexually active cisgender women aged 18-35 at sites in South Africa, Zambia, Zimbabwe, Malawi and Mozambique. Participants will be randomized 1:1 to receive either the active vaccine regimen or placebo.

Schuitemaker’s IAS 2017 presentation is available on the conference YouTube channel (it’s first in the Translational Vaccinology conference session). An excellent overview of Janssen’s HIV vaccine development work was provided earlier this year by Maria Grazia Pau on an AVAC webinar, which is also available on YouTube.

An issue that has gone largely unmentioned in relation to Janssen’s program is the lingering uncertainty regarding exactly what caused the enhanced risk of HIV infection observed in recipients of Merck’s Ad5 vaccine vector. As discussed at a special summit convened by NIAID in 2013, there are some concerns that the same problem could arise with Ad vectors of other serotypes.

For the most part, theories to explain the outcome of the Merck trials have centered on the induction of HIV-specific CD4 T cells, which could have been vulnerable to HIV infection in the absence of any antibody responses (some limited data from a small macaque study supports this possibility). The antibody component of the Janssen approach, in addition to the use of an Ad vector from a different serotype, is likely to obviate any fears that this type of problem could arise. 

But another suggested scenario involves an Ad vector-induced increase in the number of Ad-specific CD4 T cells engaged in immune surveillance of natural adenovirus infections at mucosal sites (particularly penile sites), which might be a concern with the Ad26 vector due to the extensive cross-reactivity of Ad-specific CD4 T cells.

In other words, the Ad26 vector could potentially induce Ad26-specific CD4 T cells capable of recognizing adenoviruses of other serotypes that recipients had encountered (or encountered during the trial)—natural adenovirus infections have been found to be relatively common and potentially persistent, so increasing the number of Ad-specific CD4 T cells at these locations might conceivably provide additional targets for HIV. A similar mechanism has been proposed to explain the well-documented association between HSV-2 infection and increased risk of HIV acquisition.

Because the initial efficacy study involving the Ad26 vector is in women, there does not appear to be any clear cause to worry about enhanced susceptibility to HIV infection, as it was only observed in male recipients in extended follow up of participants in the Phambili trial of Merck’s Ad5 vector. But, with trials likely to include men in the future, it would be prudent to conduct a review of the recommendations that emerged from the 2013 NIAID summit on Ad vector platforms and the status of relevant studies, to ensure that the safety of participants is maximized. A logical venue for this discussion might be a future meeting of the AIDS Vaccine Research Subcommittee (AVRS).

HIV Remission News from IAS 2017

The 9th International AIDS Society Conference on HIV Science (IAS 2017) began on Sunday in Paris, and the topic of HIV remission has been the focus of several high profile presentations.

One of the first major news stories to emerge from the meeting involves a newly described example of prolonged HIV remission in a nine-year-old South African (see the conference abstract and poster discussion slides presented today by Avy Violari). The case was identified because the child was a participant in the Children with HIV Early Antiretroviral Therapy (CHER) trial, which took place from 2005-2011 in South Africa and evaluated immediate, time-limited (40- or 96-week) courses of antiretroviral therapy (ART) in perinatally infected infants compared to the strategy of deferring initiation until clinical or immunological signs of disease progression were evident (preliminary results from the study, demonstrating the benefits of immediate ART, were published in the New England Journal of Medicine in 2008).

The child was diagnosed by HIV DNA PCR 32 days after birth, and displayed pre-treatment viral load levels of over 750,000 copies/ml and 151,000 copies/ml on days 39 and 60, respectively. ART was initiated after 61 days, with viral load declining to below 50 copies/ml at week 24 of on-treatment follow up. ART was subsequently stopped at week 40. The CHER protocol mandated restarting ART if certain immunological and/or clinical criteria were met, but the child has remained healthy and maintained a high CD4 count throughout follow up.

When stored samples were later accessed in order to measure viral load, the levels were found to be persistently below 20 copies/ml. The first post-interruption sample was taken eight weeks after stopping ART, so it’s not known if viral load failed to rebound or if there was a transient increase that was rapidly controlled. Viral load has now remained undetectable for 8.75 years and counting, making this one of the longest reported cases of HIV remission (there is a French teenager first reported in 2015 and described in a paper in The Lancet HIV last year who has contained viral load to undetectable levels for over 12 years, after receiving a more prolonged course of ART in early life).

The researchers have conducted multiple analyses to try and gain insight into the factors that may have contributed to the outcome. Measurement of the HIV reservoir using a sensitive test for total HIV DNA revealed similar levels after one year and at 9.5 years of age: around 5 copies per million peripheral blood mononuclear cells (PBMC). However, no replication-competent virus could be detected using two different culture methods. Virus sequencing showed that the child was infected with HIV-1 from clade C, the most prevalent strain in South Africa.

The ELISA HIV antibody test was negative but weak reactivity to Gag p40 and p24 was detectable by Western blot. One exception to the generally weak HIV-specific antibody responses was a high IgA2 (a type of mucosal antibody) response to the gp41 protein. In studies of cellular immunity, a low-level CD4 T cell response to the HIV Gag protein was discernible with a whole blood intracellular cytokine assay, but no HIV-specific CD8 T cells could be detected.

Additional parameters that were investigated included the density of CCR5 molecules on CD4 T cells, which was found to be lower than HIV-negative controls – it is perhaps possible that lower CCR5 density contributed to the maintenance of an HIV-specific CD4 T cell response by reducing the susceptibility of these cells to HIV infection. Encouragingly, levels of immune activation were similar to HIV-negative individuals and lower than those observed in elite controllers (in the latter group, elevated immune activation has been associated with disease progression despite persistently low viral loads).

PD-1 expression was found to be high on both CD4 and CD8 cells compared to HIV- individuals and elite controllers; the significance of this finding remains to be elucidated. None of the beneficial HLA alleles that have been associated with elite control were present, although the child was heterozygous at all HLA loci and this has been linked to a better prognosis compared to homozygosity.

The researchers note that other factors beyond the temporary course of ART likely contributed to the salutary outcome – the majority of participants in both the 40- and 96-week immediate ART arms of the CHER trial had met the criteria for restarting treatment by the time the study ended, and no other cases of HIV remission have yet been identified.

The hope is that additional analyses will uncover novel correlates of immune control and aid the design of interventions aiming to induce HIV remission in larger numbers of HIV-positive individuals.

The news coverage of the case—which has been extensive—appears to have generally been accurate, although the BBC erred by describing the child as “virtually cured” in their headline – it is not known if viral load might rebound at some point in the future, as has occurred in some other examples of HIV remission. There are articles describing the child as either a girl or a boy, but Amy Green from the South African Health News Service interviewed Avy Violari and reports that gender is not being disclosed in order to protect the individual’s privacy.

The New York Times article, authored by Donald McNeil Jr., has some poor mistakes: the opening paragraph erroneously states that the ART was given at “high doses” when the CHER trial employed normal pediatric dosing. Later in the piece, individuals who are homozygous for the CCR5delta32 mutation, which confers a high degree of resistance to HIV infection, are mistakenly referred to as elite controllers (elite controllers are HIV-positive individuals who naturally contain HIV viral load to low levels in the absence of ART). Timothy Brown, the one individual considered cured of HIV infection, is said to have received a bone marrow transplant from an elite controller, which is not the case - his bone marrow donor was HIV-negative and homozygous for the CCR5delta32 mutation. One of these errors has since been corrected, but two remain at the time of writing.

The first mention of the South African HIV remission case at the IAS conference came yesterday in a presentation by Anthony Fauci, Director of the National Institute of Allergy and Infectious Diseases (NIAID) during the “Challenges and Opportunities in HIV Science” session – this appears to explain the timing of the NIAID news release about the study. Fauci’s talk was titled: “Sustained ART-free HIV remission: opportunities and obstacles” and he offered some additional nuggets of news, as well as discussing issues to be considered when evaluating the remission cases that have been reported to date (slides are available for download, and video was posted to the IAS conference youtube channel on July 26).

In particular, he outlined two potential contributors to HIV remission: in some cases, the size of the HIV reservoir may be so small that after an ART interruption the few latently infected CD4 T cells that are present remain dormant for an extended period, before one or more cells is activated and starts producing HIV (triggering viral load rebound). Fauci referred to this phenomenon as “stochastic reactivation,” and evidence suggests it may explain the period of remission documented in the Mississippi baby who was started on ART prior to developing detectable immune responses to HIV (it has also been posited to explain the adult examples of the Boston patients and an individual treated within days of infection at UCSF—see below).

In other instances—such as the South African child, the French teenager and the VISCONTI cohort participants—evidence suggests immunological control of HIV replication is contributing, although the exact mechanisms are unclear.

Fauci noted that both of these phenomena could also be operational simultaneously, but presently the short answer as to precisely what explains a given case of HIV remission is: "we don't know."

On the topic of interventions aiming to induce HIV remission, Fauci offered a glimpse of unpublished data from two studies. The first was a clinical trial conducted at the National Institutes of Health (NIH) Clinical Center that evaluated the effect of therapeutic vaccination in HIV-positive individuals started on ART within 12 weeks of a diagnosis of acute or early infection, who had maintained undetectable viral loads for over a year.

A total of 30 participants enrolled, with 15 randomized to receive a prime-boost HIV vaccine regimen developed by Profectus Biosciences (comprising DNA and vesicular stomatitis virus vectors) and 15 randomized to receive placebo immunizations. The final vaccination was administered at week 48 of the study and, eight weeks later, all participants underwent a 16-week analytical ART interruption.

The results demonstrated that there were no significant differences in time to viral load rebound to over 40 copies/ml (or to over 400 copies/ml) between the vaccine and placebo groups. Fauci emphasized the importance of the placebo group by showing that at the end of the ATI period, 20% of controls had viral loads less than 20 copies/ml, 27% were below 400 copies/ml, and 40% were below 2,000 copies/ml. 

This finding may temper excitement about the single-arm study of therapeutic vaccination plus romidepsin that drew so much attention at CROI earlier this year – in that trial, the fact that 38% of participants maintained viral loads below 2,000 copies/ml at 4-6 months after an ATI was presented as evidence that the interventions had enhanced control of HIV replication, compared to historical studies of ART alone. Fauci suggested that preliminary clinical trials and animal studies using broadly neutralizing antibodies (bNAbs) offer evidence that they may have more potential for enhancing post-treatment control of viral load than therapeutic vaccines.

Fauci’s own laboratory has reported that administering an antibody against the α₄β₇ integrin to SIV-infected macaques led to surprisingly robust control of SIV replication after an ART interruption, and in his presentation he showed the unpublished results of recent experiments aiming to shed light on the mechanisms involved. The approach that the researchers took was to deplete different types of immune cells in the animals controlling SIV viral load, then assess whether this led to an increase in viral replication. The experiments compared:

• Antibodies to the CD8 receptor alpha chain, which deplete CD8 T cells, natural killer T cells (NKTs) and natural killer (NK) cells
• Antibodies to the CD8 receptor beta chain, which deplete CD8 T cells
• Antibodies to CD20, which deplete B cells

A transient rebound in viral load was only seen in recipients of antibodies to the CD8 receptor alpha chain, indicating that NKTs and NK cells are making an important contribution to the observed control of SIV replication. A clinical trial investigating the anti-HIV effects of the anti-α₄β₇ integrin antibody vedolizumab, which is FDA-approved for the treatment of ulcerative colitis and Crohn’s disease, is ongoing at the NIH Clinical Center, and another is due to start soon at the Ottawa Hospital Research Institute in Canada.

Timothy Henrich from UCSF gave another presentation involving HIV remission today, in the same poster discussion session during which Avy Violari described the South African case (video of most of the session is posted on the IAS youtube channel, but Violari's talk is not included). Henrich reported details relating to an individual who was mentioned several times during CROI earlier this year (see the BSVC blog conference report). Both Henrich’s abstract and slide presentation are available on the IAS conference website.

The individual in question was diagnosed within an estimated 10 days after acquiring HIV infection, because the exposure occurred in a short window between screening for a pre-exposure prophylaxis (PrEP) program and the baseline visit at which Truvada PrEP was first administered. When the baseline test results came back showing the presence of HIV, Truvada was quickly switched to a four-drug ART regimen including Truvada, darunavir and raltegravir. No HIV could be detected in blood or tissue samples during ART, although a low level of viremia was briefly detected in one out of 10 humanized mice administered a large volume of cells sampled from the individual (this novel test is known as the murine viral outgrowth assay).

After 34 months of continuous ART, an analytical treatment interruption was undertaken, and no viral load rebound occurred for 224 days, during which time no HIV DNA or RNA was detectable in plasma or sampled CD4 T cells. After this prolonged period of HIV remission, viral load rebounded and was initially detected at a level of 36 copies/ml. Six days later viral load had increased to 59,805 copies/ml and ART was immediately restarted. Sequencing of the HIV genome demonstrated that the rebounding virus was identical to that sampled at the time of acute infection.

In a collaboration with Alison Hill, mathematical modeling studies estimated the size of the HIV reservoir to have been approximately 200 latently infected cells prior to the ART interruption. Based on Hill’s prior work, a latent HIV reservoir of this size confers only a small (~1%) chance of achieving a lifelong cure, due to the risk of stochastic reactivation of one or more of the latently infected cells.

Henrich and colleagues continue to evaluate enrollees in PrEP projects for evidence of recent HIV infection, and he showed a slide documenting that another individual has been started on ART very early as a result of the effort. Follow up of this second individual is ongoing.

In addition to several sessions and presentations related to HIV cure research at the main conference (see here for a roadmap), IAS hosted a pre-conference HIV Cure & Cancer Forum at the Institut Curie. The abstracts and many of the presentations have already been made available online, and a brief report from the meeting will follow in a separate blog post.

Capsules From CROI 2017

The annual Conference on Retroviruses & Opportunistic Infections (CROI) took place in Seattle from February 13th-16th, offering a dizzying parade of new data. Webcasts of presentations and PDF files of posters were rapidly placed online and are accessible via the CROI website.

A Fillip for Kick & Kill

On the cure research front, the results that drew the most attention related to a small trial combining a latency-reversing agent (the HDAC inhibitor romidepsin) with therapeutic vaccination—a strategy commonly referred to as “kick & kill.” Presented by Beatriz Mothe from IrsiCaixa in Barcelona, the crux was that five out of 13 recipients of the interventions have since interrupted ART and displayed control of viral load to low levels for several months (the longest a little over six months). None of the five have yet met the study criteria for restarting ART, which is a viral load over 2,000 copies/ml; the other eight participants quickly rebounded to levels above this cutoff and resumed ART. Contrary to a slew of erroneous headlines in the mainstream media, none of the five individuals are “virus-free;” based on the slide presentation, three appear to have viral loads below the limit of detection of the assay used (20 copies/ml) whereas the other two are oscillating between the limit of detection and ~2,000 copies/ml.

The study represented a rollover from a prior trial that administered two therapeutic vaccines to 24 people who had started ART within three months of HIV infection. The vaccine vectors were based on a chimpanzee adenovirus (ChAdV63) and modified Vaccinia Ankara strain (MVA), both encoding antigens designed to focus T cell responses on highly conserved parts of HIV, including elements from the Gag, Pol, Env and Vif proteins. In a poster presented at last year’s CROI, Mothe reported that receipt of these vaccines shifted HIV-specific T cell responses toward the intended conserved targets but did not have a measurable effect on the size of the HIV reservoir. A total of 15 participants from this original trial then agreed to enroll in a follow up study, which provided booster immunizations with the MVA vector before and after three infusions of romidepsin. Eight weeks after the final MVA dose, all participants interrupt ART, and so far 13 individuals have reached this stage and contributed data to Mothe’s report at CROI 2017.

Although the numbers are small and follow up still relatively short, Mothe noted that the frequency of viral load containment in the cohort (~38%) is higher than has been observed in any studies involving early initiation of ART (where rates have varied from 0-15%). Ongoing analyses are exploring potential correlates of control, with Mothe suggesting there are hints of links between the induction of T cell responses to the conserved HIV antigens, lower HIV DNA levels, and the achievement of post-ART viral load control. The contribution of romidepsin is unclear due to the lack of any control group, but the drug did not have a measurable effect on the size of the HIV reservoir when levels before and after administration were compared. There was evidence of blips in HIV viral load after each romidepsin dose, consistent with a latency-reversing effect. Mothe pointed out that blips also occurred after the MVA immunizations in 60% of the participants, indicating that the vaccine may have activated latently infected CD4 T cells specific for HIV antigens (a number of studies have reported that HIV-specific CD4 T cells can contain a substantial proportion of the latent HIV reservoir). Romidepsin infusions were associated with an array of side effects known to be caused by HDAC inhibitors—primarily grade 1 and grade 2 headaches, fatigue and nausea—and the drug also caused precipitous but transient declines in peripheral blood CD4 T cell counts of around 300 cells. One participant developed the serious complication of sepsis after the final romidepsin dose.

Additional follow up will be required before the significance of the study can be fully assessed, but it represents the first time that any kick & kill strategy has been associated with an increased frequency viral load control after ART interruption. There is an important caveat that applies to all studies reporting maintenance of low viral load in the absence of ART: while most news coverage assumes that the health benefits of viral load suppression will be the same regardless of whether the suppression is being mediated by immune responses or ART, that assumption remains unproven. Based on studies of elite controllers, it is possible that even low level viral load may be associated with a slight increase in the risk of morbidity and mortality compared to the stricter control of HIV replication imposed by ART. If post-ART control of viral load can eventually be induced in more significant numbers of people, there will be opportunity to more carefully investigate this issue by comparing clinical outcomes between post-ART controllers and study participants who restart or continue ART.

Complete Suppression of HIV Replication by ART

The question of whether low-level HIV replication persists despite ART has been a major issue of debate in the cure research field. The balance of evidence has favored the conclusion that ART typically completely prevents HIV from reproducing in adherent individuals, but some studies have challenged this view, including a paper published last year in Nature which argued that cryptic replication occurs in lymphoid tissues. At CROI, Mary Kearney from the National Cancer Institute addressed the question with an analysis of HIV evolution in ten children who started ART early (mostly within a few months of birth) and were followed for at least seven years. Two of the children experienced some lapses in suppression of viral load and served as positive controls, while the remaining eight showed no evidence of any viral load blips during follow up.

Kearney found that the evolution of HIV genetic sequences was readily apparent in the two individuals whose viral load was not continually suppressed. In stark contrast, no evidence of HIV evolution was detectable in the other eight participants, supporting the idea that ART completely stymies viral replication. Kearney suggested that differences in the types of tools used to analyze HIV evolution may explain some of the conflicting results that have been reported, noting that the Bayesian approach employed in last year’s Nature paper may mistakenly suggest that the virus has been evolving because the timing of sample collections influences the outcome of the analysis.

Kearney’s results were buttressed by a poster presentation from Morgane Rolland of the US Military HIV Research Program (US MHRP). Rolland studied eight individuals who initiated ART at Feibig I, an extremely early stage of infection estimated to represent the period 10-17 days after HIV acquisition. After an average of around three years, ART was interrupted as part of a protocol assessing whether HIV remission might occur. All eight participants experienced a viral load rebound within a median of 26 days, and Rolland compared the reemerging HIV genetic sequences with those sampled at the pre-ART baseline. These analyses, like Kearney’s, revealed no evidence of HIV evolution during ART. Jintanat Ananworanich also described the results of this US MHRP clinical trial in detail in a separate oral presentation.

Another Case of Temporary HIV Remission in a Stem Cell Transplant Recipient

To date, Timothy Ray Brown remains the only individual considered to have been cured of HIV infection, an outcome achieved as a result of a complex series of treatments for a life-threatening cancer that included stem cell transplants from a donor homozygous for the CCR5Δ32 mutation (which renders immune cells resistant to most HIV strains). Brown was in attendance at CROI, celebrating reaching a milestone of ten years since those transplants were performed. But it has also been learned—as a result of the experience of two individuals known as “the Boston patients”—that HIV-positive people who receive stem cell transplants for cancer treatment can experience dramatic reductions in the HIV reservoir even when the stem cell donors lack the CCR5Δ32 mutation. In the case of the Boston patients, this shrinking of the reservoir ultimately allowed for a temporary period of remission after ART interruption; the individuals were able to go for three and eight months without any signs of HIV activity, respectively, before viral load rebounded (Boston patient Gary Steinkohl has since gone public to discuss the experience of participating in this research).

A poster at CROI 2017 from Nathan Cummins at the Mayo Clinic in Rochester reported another case of HIV remission with broad similarities to the Boston patients. The individual received a stem cell transplant from a donor without the CCR5Δ32 mutation as part of his treatments for acute lymphoblastic leukemia. By day 56 post-transplant, HIV DNA was no longer detectable in blood and subsequent sampling of large numbers of cells by leukapheresis showed significant declines in measures of the HIV reservoir. An occurrence of graft-versus host disease (GVHD) was associated with apparent elimination of most of the individual’s original CD4 T cells, which were present at a frequency of around 1 in 10 cells at day 142 but had diminished to ~13 per every million cells by day 265. HIV-specific antibody responses also waned.

Approval was obtained to interrupt ART on day 784 post-transplant. HIV viral load rebound occurred after a period of remission lasting 288 days (a little over nine months), with levels rising relatively slowly from 60 copies/ml initially to 1640 copies/ml five days later, at which point ART was restarted. No symptoms were associated with the reappearance of viral load, contrasting with the Boston patients who both experienced sharp increases in viral load and symptoms of acute retroviral syndrome at the time they rebounded.

A colleague of Nathan Cummins, Stacey Rizza, presented the poster, and in discussing the case revealed that the individual had the misfortune to experience a car accident shortly before viral load rebounded (without serious injury, thankfully). One speculative possibility under consideration is that inflammation caused by stress might have triggered the activation of a latently infected cell (or cells). Samples were collected throughout follow up and are now being evaluated to try and gain a better understanding of what occurred.

Remission Macaques

The potential for rare latently infected cells to persist in a dormant state for extended periods despite ART interruption was emphasized in a symposium talk by Louis Picker from the Vaccine & Gene Therapy Institute at Oregon Health Sciences University. Picker’s effort to develop a CMV-based vaccine for HIV has attracted considerable publicity due to unprecedented results achieved in the SIV/macaque model: when immunized with a version of the vaccine encoding SIV antigens, half the recipient macaques exert strict control over a pathogenic SIV challenge and appear to eventually clear the infection. Picker outlined two possible explanations for this outcome:

1. Vaccine-induced immune responses limit SIV replication to such an extent that only a very small, unstable SIV reservoir is formed, which eventually decays away over time.
2. Vaccine-induced immune responses actively clear the SIV reservoir over time.

To try and discern which explanation is correct, Picker and colleagues conducted a therapeutic study in which the CMV-based vaccine was administered to SIV-infected macaques on ART (a version of the vector encoding TB antigens served as a control). The timing of ART initiation in the experiment was guided by the detection of monocyte activation after SIV challenge, because previous work suggested this coincided with the initial formation of the viral reservoir. The approach allowed the researchers to divide macaques into various groups depending on how many days after SIV challenge ART was first administered.

After 600 days of treatment, ART was interrupted in all animals. Receipt of the CMV vaccine encoding SIV antigens did not affect viral load rebound, indicating it lacked any therapeutic effect. But Picker highlighted that timing of ART showed a major influence: all six macaques started earliest, on day 4 or 5 post-challenge, did not experience any viral load rebound. Necropsy studies were eventually conducted and only rare traces of SIV genetic material could be detected in tissues. Large volumes of cells sampled from these animals were unable to transmit SIV to uninfected macaques. CD8 T cells were depleted to assess if SIV-specific CD8 T cells were suppressing the virus, but there was no return of viral load, arguing against immunological control.

Out of 35 animals administered ART from day 6 or later, only one (initiated on day 6) displayed a similar lack of rebound. But after eight months, shortly before a planned necropsy, this macaque experienced a rebound in SIV viral load. Picker noted the parallel between this outcome and those observed in the human remission cases of the Mississippi baby and Boston patients.

Picker drew several conclusions from this work:

• The window of opportunity between infection and the formation of a stable long-lived viral reservoir is tiny – in this experiment, a delay in the initiation of ART of just one day had a huge effect on the risk of rebound when ART was interrupted.
• As has been observed in human remission cases, latently infected cells can linger in an inactive state for a long time before causing a rebound in viral load. This observation supports the importance of efforts to reverse HIV latency and promote clearance of latently infected cells.
• The lack of a therapeutic effect of the CMV-based vaccine indicates that, in the preventive context, it likely works by limiting the formation of the SIV reservoir, rather than inducing immune responses capable of progressively clearing the reservoir.

Picker also mentioned that there does appear to be a case of ultra-early ART leading to temporary remission in an adult human; this is an individual who acquired HIV infection in a short period between screening for a pre-exposure prophylaxis (PrEP) demonstration project and starting on the Truvada PrEP regimen. When HIV was detected in the sample taken on the first day of PrEP administration, ART was immediately substituted for Truvada (this occurred within a matter of days). Hiroyu Hatano from UCSF described the case at CROI in 2014, and cited plans to eventually conduct an ART interruption. Early last year, ART was stopped, and the individual displayed no sign of HIV activity for 220 days before rebound was detected and treatment restarted. A full presentation of the data is expected at the International AIDS Society (IAS) conference in July.

Sex Differences in HIV Persistence

At the IAS conference in 2015, Jonathan Karn reported that the biology of HIV latency is influenced by the estrogen receptor on CD4 T cells, leading to sex differences in the activity of candidate latency-reversing agents (LRA). Specifically, the hormone estradiol significantly inhibited the effect of LRAs in women but not men, whereas drugs that antagonize the estrogen receptor—including the breast cancer treatments tamoxifen and fulvestrant—enhanced latency reversal. At CROI 2017, Eileen Scully from Johns Hopkins University presented a poster describing results from a study comparing measures of HIV persistence in carefully matched cohorts of women and men.

Contrary to a previously published retrospective analysis, levels of HIV DNA were not significantly different between the groups. However multiple measures indicated that expression of HIV RNA from the viral reservoir was lower in women compared to men. These included cell-associated HIV RNA and low-level viremia (measured with an assay capable of capturing a single HIV RNA molecule), as well as the ratio between the amount of integrated HIV DNA detected and the ability to induce HIV RNA production from the reservoir (using the TILDA assay). Consistent with the lower HIV expression, markers of T cell activation were also significantly reduced in women compared to men. Scully concluded that biologic sex is an important consideration in cure research, and that the manipulation of sex hormones may have a role to play in efforts to target the HIV reservoir.

Jintanat Ananworanich drew attention to Scully’s poster in an excellent plenary overview of the cure research field, and emphasized the need to do more to facilitate increased participation by women.

Ameliorating Immune Reconstitution Inflammatory Syndrome

Immune reconstitution inflammatory syndrome (IRIS) is a potentially life-threatening consequence of the restoration of immune responses to opportunistic pathogens in people who initiate ART at low CD4 T cell counts. Evidence suggests that immune responses can become exaggerated and overly inflammatory as a deficient, dysregulated immune system begins to recover due to ART-mediated HIV suppression. In some cases opportunistic infections can get worse before improving, a problem termed paradoxical IRIS. This is a particularly significant concern in tuberculosis (TB), with a reported mortality rate of around 3%.

In an effort to reduce morbidity and mortality from paradoxical TB-IRIS, Graeme Meintjes and colleagues conducted a randomized trial evaluating a four-week course of the corticosteroid prednisone in individuals at risk. The encouraging results were debuted as a late-breaker at CROI, with Meintjes reporting a significant 30% reduction in the incidence of paradoxical TB-IRIS and a trend toward decreased hospitalizations in the prednisone arm. No evidence of an increase in cancer risk—which had been raised as a potential issue with prednisone—was observed. The evidence from the trial suggests that the approach should be adopted as the standard of care for individuals at risk for paradoxical TB-IRIS.

The conference was also cheered by the report of a breakthrough in treating extensively drug-resistant TB, a condition normally requiring the use of debilitating, toxic injectable drugs. A small trial of three oral drugs generated highly promising results, offering hope for progress after decades of stagnation (see Jon Cohen’s report in Science).

Encouraging Results With Anti-Inflammatory Antibody

Many studies have reported that HIV infection is associated with an increased risk of arterial inflammation and cardiovascular disease. The pipeline of therapies that might reduce this risk has been discouragingly dry, but at CROI Priscilla Hsue from UCSF presented results from a trial of an anti-inflammatory antibody targeting the cytokine IL-1β that may augur a change for the better. The antibody, canakinumab, is FDA-approved for the treatment of certain autoimmune conditions and is being tested as a therapy for cardiovascular disease in a large (10,000-person) randomized study involving in HIV-negative individuals. Hsue’s pilot trial recruited ten HIV-positive people on suppressive ART with a median age of 59. A single dose of canakinumab was administered at baseline and participants followed for eight weeks.

There were significant declines in inflammatory biomarkers: IL-6 levels declined by 30% and high sensitivity c-reactive protein by 41%. Imaging studies revealed a 10% reduction in arterial inflammation. In terms of safety, Hsue noted a transient drop in absolute neutrophil count that resolved by week four and a single case of shingles that did not appear related to any immunological parameters. No significant changes in any biomarkers of HIV disease were seen apart from a 17% drop in CD8 T cell counts between baseline and week two that was not apparent at any other timepoints. CD4 T cell counts, viral load and T cell activation markers were unchanged. Analyses of measures of the HIV reservoir are ongoing.

“We believe this is one of the first immune-based therapies to show a very profound reduction in inflammatory markers in the setting of treated HIV,” Hsue said, noting that a larger randomized controlled trial is planned that will give two canakinumab doses and follow 100 participants for 36 weeks.  

Dual bNAb Combo Leads to Long-Term SHIV Control

In a symposium on broadly neutralizing antibodies (bNAbs), Michel Nussenzweig premiered unpublished results from a collaborative experiment his laboratory has conducted with Malcolm Martin at the National Institute of Allergy and Infectious Disease (NIAID). The study challenged macaques with a pathogenic SHIV (SHIVAD8) and, starting three days post-infection, administered a combination of two bNAbs, 3BCN117 and 10-1074, thrice weekly for two weeks. Nussenzweig reported that, interestingly, the bNAbs led to prolonged preservation of CD4 T cells and control of viral load in treated animals, with many displaying what he described as an “elite controller phenotype.” The depletion of CD8 T cells from some of the macaques led to increased viral load, indicating that the short course of combined bNAbs had positively modulated virus-specific immunity. A paper by Nishimura et al describing the results is now in press at Nature.

Macaque Models in HIV/AIDS Research

Jeff Lifson delivered an exceptional Bernard Fields Memorial Lecture on the role of non-human primate models in HIV/AIDS research. Included in the broad survey were a few nuggets of unpublished data from ongoing work: in a collaboration with Louis Picker, the anti-B cell antibody rituximab has been used to disrupt B cell follicles in elite controller macaques, and this led to reductions in SIV viral load and numbers of T-follicular helper cells in the lymph node, consistent with previous reports that B cell follicles can represent a sanctuary site for the virus. A similar study is now being conducted in SIV-infected macaques treated with ART.

As an alternative approach to the same problem, Lifson cited the work of Dave Ott whose laboratory is investigating the genetic modification of CD8 T cells to express CXCR5, a chemokine receptor that can guide the CD8 T cells into B cell follicles. Lifson showed imaging results demonstrating that this strategy successfully localized CD8 T cells to the follicles, and work is now underway to modify CD8 T cells to express both CXCR5 and T cell receptors targeting SIV antigens.

Cellular & Gene Therapy in Cancer and HIV

Carl June from the University of Pennsylvania gave a plenary talk on the dramatic advances that have occurred in the cancer field involving genetically modified T cells. These approaches typically extract T cells from an individual, genetically modify them in the laboratory to target the desired antigen, then expand and reinfuse the cells, which are described as “chimeric antigen receptor” (CAR) T cells. Impressive results have been obtained against a variety of cancers, although serious safety issues have also emerged in some cases due to the potential for excessively vigorous immune reactions to cause inflammation and pathology. June highlighted that many different clinical trials of this type of approach are currently underway across the globe in cancer, but none are occurring in HIV. June advocated for the development of combination approaches that both engineer HIV resistance in CD4 T cells (such as the Sangamo approach, which June has been involved in studying) and modify CD8 T cell antigen receptors to better target HIV-infected cells for elimination. He also stressed the need to foster engineering innovations to make this type of therapy cheaper, scalable and globally accessible.

Sources of CROI Coverage

High quality reporting from CROI 2017 is available from multiple online sources, including AIDSMap, AVAC, i-Base, HIVandHepatitis.com and NATAP. If you know of other sources I’ve missed and should include, please leave a comment and I’ll update the links.