TAG HIV Basic Science, Vaccines, and Cure Project Blog

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

recent posts

  • Applying the Brakes to Type 1 Interferons in HIV Infection

    This week in the Journal of Clinical Investigation, two independent studies describe results obtained by blocking type 1 interferon signaling pathways in humanized mouse models of chronic HIV infection. The papers are both open access (see Anjie Zhen et al and Liang Cheng et al). Type 1 interferons are cytokines that interact with specific receptors on cells in order to modulate activity of interferon-stimulated genes (ISGs), and these interactions are known to play important roles in antiviral immunity (e.g. alpha interferon is an approved treatment for hepatitis C). But the effects of type 1 interferons are complex, and can also involve potentially harmful exacerbation of immune activation and inflammation. In these new studies, the researchers used antibodies to block interferon receptors in humanized mice with chronic HIV infection, and converged on congruent findings: HIV viral loads and virus reservoirs were diminished, as were markers of immune activation and exhaustion.

    In the work of Anjie Zhen et al, type 1 interferon receptor blockade was studied both with and without antiretroviral therapy (ART). Blockade alone reduced HIV viral loads and markers of immune activation and exhaustion, as well as enhancing the functionality of HIV-specific CD8 T cell responses. The combination with ART displayed similar effects and reduced the size of the persistent HIV reservoir. Liang Cheng et al evaluated type 1 interferon receptor blockade in combination with ART, producing comparable results.

    An accompanying commentary notes that the results contrast with those obtained in acute SIV infection, where blocking IFN receptors had mostly negative effects, increasing the SIV reservoir and promoting faster CD4 T cell loss and progression to AIDS, despite reducing markers of immune activation. The commentary authors point out that these differing findings have a clear parallel in murine LCMV infection, where blockade of type 1 interferon inhibits virus clearance during acute infection but enhances long-term control of viremia in chronic infection.

    Because antibodies blocking type 1 interferon activity are already being tested in human trials for other diseases (such as lupus), it may eventually be possible to test whether the promising results obtained in the humanized mouse model can be translated to humans. As Liang Cheng et al state in the discussion section of their paper, an important first step will be testing the approach in macaques with chronic SIV infection.

  • NIAID Strategies for an HIV Cure Workshop

    Next week from Monday through Wednesday the National Institute of Allergy and Infectious Diseases (NIAID) is convening their third scientific workshop on HIV cure research, Strategies for an HIV Cure 2016. The meeting will be held in the Ruth Kirschstein Auditorium at the Natcher Conference Center (Building 45) on the National Institutes of Health Main Campus. For the first time, the proceedings will be available for public viewing via live webcast at: https://videocast.nih.gov. A detailed agenda is available online. A room has been made available for the use of community advocates attending the workshop in person: Natcher room G1/G2 on the lower level of the conference center.

  • Antibody Therapy Leads to Sustained Post-Treatment SIV Control in Macaques

    A study published last Thursday in the journal Science has hit the headlines, reporting that sustained post-treatment control of SIV has been achieved in macaques using an antibody therapy developed for the treatment of inflammatory gastrointestinal (GI) disorders. The antibody targets α4β7, a receptor expressed on CD4 T cells (and other immune system cells) that is involved in promoting trafficking to the GI tract. Some, but not all, studies have suggested that α4β7 also plays a role in mediating HIV infection of target CD4 T cells.

    The rationale for the study came from previous experiments which found that infusions of the antibody prior to an SIVmac239 challenge reduced post-infection viral loads in macaques and, in low-dose challenge studies, lessened the risk of SIV acquisition. The mechanism remains unknown but the researchers hypothesize that it relates to inhibition of trafficking of CD4 T cells, natural killer cells and plasmacytoid dendritic cells to the gut, limiting the availability of target cells for SIV and also dampening the inflammatory immune response that promotes and disseminates virus replication.

    In the latest work, 18 macaques were challenged with the same dose of SIVmac239 used in previous experiments (200 TCID 50) and all became infected. Five weeks post-challenge, combination antiretroviral therapy (ART) was initiated in all animals and maintained for 90 days. Around three weeks prior to ART cessation, 11 macaques were administered the anti-α4β7 antibody by infusion while the remaining seven received a control antibody. The antibody administrations were then continued every three weeks (after ART withdrawal) until a total of eight infusions had been given, at which point all treatments were stopped. Three macaques developed antibodies against the anti-α4β7 antibody and were excluded from further study, so analyses were limited to eight animals in the anti-α4β7 group and seven controls.

    After ART cessation, control macaques all experienced a rebound of SIV viral load within two weeks; levels averaged around a million copies/ml and persisted throughout follow up. Outcomes in recipients of the anti-α4β7 antibody were very different: two animals never rebounded, and the remaining six were able to exert control of SIV viral load within four weeks, for the most part to undetectable levels but with some intermittent blips. This suppression of SIV has been maintained out to 81 weeks of follow up (the last anti-α4β7 antibody infusion occurred at week 32). Levels of proviral SIV DNA in GI tissues followed a similar pattern, persisting at detectable levels in the control animals but declining to undetectable levels in the anti-α4β7 antibody group from week 30 onwards.

    Measurements of CD4 T cell numbers in blood and gut showed an ongoing repopulation in the anti-α4β7 antibody group compared to declines in controls. Notably, this recovery included Th17 and Th22 CD4 T cell subsets, which are known to contribute to the maintenance of GI barrier integrity. In terms of possible mechanisms of viral load containment, increases in cytokine-producing natural killer cells and innate lymphoid cells were seen in the gut post-ART in the anti-α4β7 antibody recipients but not controls. Some evidence of preferential induction of antibodies against the V2 region of the SIV envelope was also reported. SIV-specific CD4 and CD8 T cell responses were assessed based on expression of CD107a, IFN-γ, MIP-1β or TNF-α but did not show significant differences between groups

    The results of the study appear very encouraging, and the researchers are hoping to rapidly evaluate whether they have any relevance to humans. A small clinical trial of the anti-α4β7 antibody vedolizumab, which is FDA-approved for the treatment of ulcerative colitis and Crohn's disease, is now recruiting at the National Institutes of Health Clinical Center. The target population is HIV-positive people who have been on suppressive ART for at least two years and the primary goal is assess safety (the antibody has been reported to have a favorable side effect profile for approved indications). An ART interruption is planned to evaluate any effects on viral load rebound.

    Media coverage of the paper has generally been accurate, but has had to wrestle with the uncertainty that exists among scientists regarding how ART-free control of viral load should be described. The press release issued by the researchers uses the term “sustained SIV remission” in the headline but adds: “also known as a ‘functional cure’” in the body text. The problem,  as TAG has highlighted in the past, is that it cannot be assumed that ART-free control of viral load automatically equates to a state of health that can be considered as “remission” or a “functional cure” – i.e. a state of health equivalent to an HIV-positive person on suppressive ART or a comparable HIV-negative person. It is known from studies of elite controllers and individuals with HIV-2 infection that low or even undetectable viral loads do not necessarily completely eliminate the risk of disease progression. Thus, if an intervention leads to a state of ART-free control of viral load, it will be necessary to carefully evaluate immunological and health outcomes over a long period before concluding that HIV remission or a functional cure has been achieved.

    One possible technical issue that has been noted about the study is that after the SIVmac239 challenge, peak viral loads averaged around three million copies/ml, which, as Louis Picker points out in an accompany Science news article by Jon Cohen, is unusually low for SIVmac239 – in one of the prior studies by the same researchers, peak viral loads in controls averaged >32 million copies/ml. The study authors do not address this apparent discrepancy. Although it would not explain the differences between the the anti-α4β7 antibody recipients and controls, the generalizability of the findings could be limited if the SIVmac239 challenge stock was unusually attenuated. 

    Update 10/19/16: Anthony Fauci discussed the study during a plenary presentation at the HIV Research for Prevention (HIVR4P) conference in Chicago on October 17th, and a webcast is available online. Fauci noted that some of the recipients of the anti-α4β7 antibody have now been followed for two years and continue to maintain low SIV viral loads. 

  • HIV Persistence: Defective Virus Copies Accumulate Rapidly After Infection

    A major challenge in measuring the reservoir of HIV that persists despite antiretroviral therapy (ART) is that many of the virus genomes that can be found integrated into the DNA of CD4 T cells are incomplete or mutated in ways that preclude further rounds of replication. For researchers aiming to develop a cure, it is important to try and distinguish between defective virus copies and intact viruses capable of rekindling the infection when ART is interrupted. A new study from the laboratory of Robert Siliciano, published in the latest issue of Nature Medicine, attempts to assess the proportions of defective and replication competent HIV in people on ART, comparing individuals who began treatment very early after infection to those who started later.

    Siliciano and colleagues used genetic sequencing techniques to measure the number of intact HIV DNA sequences in ten individuals who started ART more than 180 days after infection, and compared the results to a cohort of nine people who started within 100 days of infection (most had begun within 60 days). The researchers were surprised to find that defective copies of HIV accumulated rapidly: in the early-treated individuals, only 7% of the HIV DNA copies that could be detected were intact. In the cohort that initiated ART during chronic infection, the proportion of intact viruses was expected to be lower and that proved to be the case: only 2% of virus genomes were complete, and 98% of the HIV DNA in this group was defective.

    The average size of the replication competent HIV reservoir was estimated to be 12 infectious proviruses per million resting CD4 T cells in the early-treated group compared to 37 infectious proviruses per million resting CD4 T cells in the individuals who began ART later, although the researchers note there was “substantial person-to-person variation.” These data appear to suggest that the impact of early ART on the size of the HIV reservoir may not be as dramatic as had been thought, but there are some caveats:

    • While most of the early-treated cohort began ART within three months of infection, other studies attempting to ascertain the effect of early treatment on the size of the HIV reservoir have included individuals who initiated ART within a matter of days. The techniques used in this paper have yet to be applied to measuring the reservoir in such ultra-early-treated people.
    • A difference of 12 vs. 37 infectious proviruses per million resting CD4 T cells may seem relatively slight, but a threefold larger HIV reservoir may be more significant when you consider the whole body (which has been estimated to contain 1.9 – 3.5 trillion lymphocytes).
    • The differences in the total amount of HIV DNA detectable in the early vs. later treated cohorts are far greater than was observed for intact proviruses. In the examples provided in the paper, the amount of HIV DNA detectable in the early-treated group ranges from 72-315 copies per million resting CD4 T cells, compared to 1,333-9,785 copies per million resting CD4 T cells in the individuals treated later. While the DNA mostly represents defective virus copies, they are not necessarily benign. A recent study from the laboratory of Anthony Fauci at NIAID found that at least some defective proviruses can still produce HIV proteins and therefore potentially contribute to persistent inflammation and immune activation in HIV-positive people. A separate study, also published recently, found HIV DNA in multiple tissues evaluated at autopsy and reported that there appeared to be an association with tissue pathology. Measures of total HIV DNA have also shown correlations with various clinical parameters in HIV-positive people, as outlined in a new review by Christine Rouzioux and colleagues.

    The study results underscore that, despite a great deal of effort, the optimum method for measuring the replication-competent HIV reservoir remains unknown. The current gold standard is the quantitative virus outgrowth assay (QVOA), but this requires the sampling and activation of very large numbers of cells and is expensive and time consuming. Furthermore, the Siliciano laboratory found that levels of intact HIV DNA sequences (which they suggest represent “probably the closest estimate of the true size of the latent reservoir”) did not correlate with results obtained by QVOA.

    The development of accurate and reliable tests for measuring the HIV reservoir continues to be a major priority for the HIV cure research field.

  • Probiotic-Based Vaccine Candidate Fails in Independent Study

    Back in 2012, a paper describing a study of a novel vaccine approach in the SIV/macaque model was published to little fanfare in the open access journal Cell Reports. The brainchild of researcher Jean-Marie Andrieu, the idea behind the vaccine was to turn the traditional approach to immunization on its head: the aim was to suppress the response to SIV, thereby depriving the virus of the activated CD4 T cell targets that normally fuel viral replication. A probiotic, Lactobacillus plantarum, was used to deliver SIV antigens to the gut with the goal of inducing SIV-specific immune tolerance. Macaques were then challenged with pathogenic SIV. The results were surprising and unprecedented: 15 out of 16 animals resisted infection. Protection was associated with the induction of SIV-specific CD8 T cells displaying a regulatory, immune-suppressive phenotype.

    The work finally garnered significant media attention in 2014 when a follow-up paper was published in Frontiers in Immunology, leading to articles in the Washington Post and on the AIDSMap website. Prominent scientist and HIV vaccine advocate José Esparza co-authored a commentary highlighting the results and the Bill & Melinda Gates Foundation funded an independent confirmatory study by the research group of Guido Silvestri at Emory University.

    In a presentation at the Cent Gardes Conference last fall—that has so far received as little publicity as the original Cell Reports paper—Silvestri debuted preliminary results, revealing that the vaccine has shown no protective efficacy, either in terms of preventing infection or reducing post-infection viral loads. A total of seventeen macaques received the vaccine, and sixteen became infected. In comparison, out of seventeen controls, fifteen became infected. Andrieu cooperated with the independent evaluation and supplied the vaccine for the study. Silvestri noted that the only differences between the experiments were the origin of the macaques (Indian vs. Chinese) and the use of a SIVmac239 challenge virus from a different stock; neither seems likely to explain the dramatic divergence in outcomes. A webcast of Silvestri’s talk is available on the Global HIV/AIDS Vaccine Enterprise website (along with the entire Cent Gardes proceedings). The results will almost certainly be subject to further analysis and debate when they are published.

    Acknowledgement: many thanks to Robert Reinhard for bringing Silvestri’s presentation to my attention.

    UPDATE: Nicolas Vabret (@neoviral on Twitter) pointed out to me that Andrieu and colleagues mention Silvestri's findings in the discussion section of a recent paper in Frontiers in Immunology, and suggest that they may be explained by differences in MHC between macaques of different geographic origin. I'm not sure how plausible this explanation might be given how starkly the results differed between experiments, but there are studies reporting that Mamu-B alleles are largely distinct in macaques of Chinese and Indian origin. 

  • Long-Awaited HIV Vaccine Efficacy Trial Gets the Green Light

    In 2009, a slight but statistically significant level of protective efficacy was reported from a large HIV vaccine trial conducted in Thailand. The trial, named RV144, involved a combination of an ALVAC canarypox vector encoding HIV antigens and a gp120 protein boost (AIDSVAX). Receipt of the vaccines was associated with a 31.2% reduction in the risk of HIV acquisition in a community sample of 16,402 adults aged between 18 and 30; the results were published in the New England Journal of Medicine. Ever since that time, plans have been afoot to assess whether the results can be duplicated or improved upon in other settings, and today the National Institute of Allergy and Infectious Diseases (NIAID) announced that a new efficacy trial is going to be launched in South Africa later this year.

    There are a number of reasons for the lengthy interlude between the trials. The company that manufactured the original AIDSVAX protein boost no longer exists, and a replacement gp120 protein vaccine—derived from HIV-1 clade C, the prevalent virus in South Africa—has had to be developed and tested in preliminary studies. Sanofi Pasteur, the manufacturer of the ALVAC vector, also created a clade C-based version of their vaccine.

    The final hurdle that the new ALVAC + gp120 protein combination had to surmount was an assessment of vaccine-induced immune responses in a trial conducted in South Africa by the NIAID-sponsored HIV Vaccine Trials Network (HVTN), dubbed HVTN 100. Researchers developed several criteria to be met in order to justify launching an efficacy trial, based on immune responses that were associated with reduced risk of HIV acquisition in RV144. These criteria were described by Glenda Gray on a webinar hosted by AVAC around this time last year (slides and audio from the call are available on the AVAC website) and involved comparing antibody and CD4 T cell responses to vaccine antigens in HVTN 100 and RV144 in order to ensure they were similar. The immune responses were also required to reach a level that, based on data from RV144, would be predictive of a 50% reduction in HIV risk for at least two years.

    Today’s NIAID announcement states that these criteria were met in HVTN 100, giving the green light for a 5,400-person efficacy trial (HVTN 702), which is slated to begin in November. Participants will be randomized 50:50 to receive either active vaccines or placebo. Immunizations with the ALVAC vector are scheduled at months 0 and 1, with both ALVAC and the gp120 protein boost given at months 3, 6 and 12. The immunization at month 12 represents an addition compared to the schedule used in RV144, due to evidence that protective efficacy may have been as high as 60% at one year of follow up before waning to the final result of 31.2%. Three interim analyses of HVTN 702 are planned, two in 2018 and another in 2019, which will assess whether the trial should continue or be stopped (due to either evidence of efficacy or lack of efficacy). If the trial proceeds, the final efficacy analysis will take place in 2020.

    While it is critically important to understand whether the promising results of RV144 can be duplicated or improved in other populations, there are reasons to be cautious about expectations for HVTN 702. Most RV144 participants were at a relatively low risk of HIV infection, and there was evidence that the vaccine regimen did not perform as well in those at highest risk. Furthermore, studies indicate that background levels of immune activation and inflammation are elevated on the African continent and likely contribute to higher rates of HIV transmission; for example, mucosal inflammation emerged as a significant correlate of HIV acquisition risk in a microbicide trial conducted in South Africa. It is not known if inflammation or other factors (such as co-infections) could act as countervailing forces and undermine potentially protective vaccine-induced immune responses, but this is the type of question that HVTN 702 should be able to shed light on.

    An area of possible controversy related to HVTN 702, or any new trial of HIV prevention strategies, is the provision of pre-exposure prophylaxis (PrEP) to participants. The antiretroviral drug combination pill Truvada received approval as PrEP in South Africa in November 2015. The NIAID Q&A about the HVTN 702 trial issued today states:

    “study participants will be referred to available local programs where they may obtain the oral medication Truvada to take daily for HIV prevention, a highly effective practice called pre-exposure prophylaxis (PrEP). HVTN 702 has been designed so investigators will be able to discern a preventive effect from the vaccine regimen even if some participants are taking PrEP.”

    This is the same approach taken by recently launched prevention trials involving infusions of the anti-HIV broadly neutralizing antibody VRC01. It could be argued that Truvada should be offered through the trial itself, but it is a difficult issue to wrestle with, and it appears that the Institutional Review Boards responsible for reviewing and approving the study protocols have found the compromise of referring participants to external sources of PrEP to be acceptable, at least at the current time.

    A recent slide presentation by Glenda Gray describing HVTN 702 can be found on the World Health Organization website. AVAC is hosting a webinar to discuss the launch of the trial on May 31st at 10am EST/4pm South Africa time (register at this link). 

  • Snipping the HIV Genome Out of Latently Infected Cells

    One of the more futuristic-sounding ideas for curing HIV infection involves trying to remove the genome of the virus from the genome of the cells into which it has integrated. On paper, the idea is very appealing, but there are a multitude of challenges associated with trying to identify integrated HIV DNA (referred to as proviral DNA or provirus) and then excise it from the DNA of an infected cell without causing untoward effects. In 2014, the research group of Kamel Khalili at Temple University in Philadelphia drew extensive news coverage when they reported some success in laboratory experiments; this work and the media response were covered at the time on TAG’s media monitor page. Khalili and colleagues have now published a new paper and again have generated considerable press (in broad terms, TAG’s previous commentary on interpreting the research and associated stories remains relevant). In addition to Khalili’s new findings, another paper has been published recently that describes a slightly different approach toward excising HIV proviral DNA, also reporting encouraging results but similarly limited to the laboratory setting.  

    The paper from Khalili’s group is published in the open access journal Scientific Reports. The researchers employed CRISPR/Cas9, a DNA editing approach derived from bacteria, and targeted it to relatively conserved regions of the HIV genome, successfully excising proviruses from some (but not all) infected CD4 cells in laboratory cultures. Delivery of the HIV-targeted CRISPR/Cas9 using a lentivirus vector also had a protective effect on uninfected CD4 T cells, which the researchers suggest was likely due to editing of pre-integrated HIV DNA. Additionally, the technology significantly reduced HIV replication (as measured by p24 protein production) in CD4 cells sampled from HIV-positive individuals; this activity appeared to be mediated both by provirus excision and the induction of crippling mutations in proviruses that were not eliminated.

    The second paper, by Janet Karpinski and colleagues from the Technische Universität, Dresden, also prompted some news articles. These researchers modified an enzyme from bacteria called a Cre recombinase so that it targeted a region of the HIV genome that is conserved in approximately 90% of virus isolates from subtypes A, B and C. The resulting enzyme, dubbed broad-range recombinase 1 (Brec1), was then tested for activity in laboratory cultures and humanized mice. As in the CRISPR/Cas9 study, delivery was via a lentivirus vector. The researchers report that Brec1 reduced virus production by infected CD4 cells sampled from HIV-positive individuals, and depleted proviruses from these cells.

    Additional experiments were conducted in which infected cells from HIV-positive donors were transduced with Brec1 or a control vector and then transferred into humanized mice; similar to the in vitro results, viral loads progressively declined to undetectable levels in recipients of the Brec1-transduced HIV-infected cells, but not in the controls. The researchers also transduced human stem cells with Brec1 or a control and used these cells to generate a humanized immune system in immunodeficient mice. The mice were subsequently challenged with HIV, and recipients of the stem cells transduced with Brec1 displayed viral load declines and evidence of depletion of HIV proviruses, in contrast to the controls.

    There are a number of overarching issues relating to these gene-editing technologies that are addressed by both groups of researchers in their papers. Chief among them is safety: manipulation of the genome carries the risk of altering genes in ways that might lead to cancer or other problematic alterations in gene function. Extensive analyses were conducted to look for evidence of potentially dangerous off-target effects in cells exposed to CRISPR/Cas9 or Brec1, and the researchers report that the approaches appeared safe, while noting that these in vitro assessments have limitations. Karpinski and colleagues explain that the presence of two HIV proviruses integrated in a single cell could be a concern, because rather than excising a single provirus and stitching the genome back together at the points where the provirus had been located, the gene-editing approaches could potentially excise all the genes between the locations of the two separate proviruses. A number of reasons are offered as to why the risk of this occurrence is likely to be low, but it nevertheless needs to be borne in mind.

    Beyond safety, another major challenge facing researchers trying to develop these gene editors into therapies is delivery to the cells where they are needed. While Khalili and colleagues have issued press releases promoting their work in fairly glowing terms (leading to the extensive news coverage), they have very little to say in their paper about how it might be delivered in vivo, simply stating: “improved delivery of CRISPR/Cas9 will be required to target the majority of circulating T-cells.” They do not mention that, because Cas9 is a bacterial protein, the human immune system is likely to recognize it as foreign and generate immune responses against it (this problem has already been described in mice).

    Karpinski and colleagues offer more discussion regarding the problem of delivering their Brec1 approach, suggesting it could be used to genetically modify stem cells, which would then be transferred into HIV-positive people in the hopes of generating HIV-resistant CD4 T cells (similar to trials that are being conducted by Calimmune and Sangamo BioSciences using gene-editing approaches that knock out the CCR5 co-receptor). They also mention the possibility of using adeno-associated virus (AAV) vectors to target delivery of Brec1 to central memory CD4 T cells, where latent HIV most commonly resides. Again, however, the risk that the bacteria-derived Brec1 enzyme might provoke an immune response is not discussed.

    HIV's notorious genetic instability, leading to the presence of many virus variants in HIV-positive individuals, also presents a significant hurdle for excision approaches. The DNA-cutting enzymes are guided to their target by recognition of specific HIV sequences and thus could be stymied by sequence variations. Khalili and colleagues acknowledge this issue, and suggest that it would likely need to be addressed by "analysis of the HIV-1 quasi-species harbored by patients’ CD4+ T-cells and design of suitable, i.e. personalized CRISPRs" – a requirement that could clearly have implications for turning the idea into a practical therapy. 

    Overall, these are exciting technologies that have understandably generated a lot of interest regarding their potential application to eliminating HIV from latently infected cells. But, without wanting to be overly naysaying, the media coverage has probably not conveyed how serious the challenges are when it comes to translating the promising laboratory findings into therapies that could feasibly be administered to people. The road to human trials is likely to be long, and there may turn out to be obstacles that are insurmountable. On a more optimistic note, there is widespread interest in developing gene-editing technologies to treat a vast range of different diseases, so many scientists are currently engaged in findings ways to make them more amenable to clinical use (a recent open access review in the journal Molecular Therapy offers an overview, albeit a fairly technical one, of the technologies now under investigation).

    UPDATE 4/4/2016: Last Friday, The Daily Telegraph published a horrendously inaccurate article claiming that this research may lead to an HIV cure "within three years" – see response published today by Ben Ryan for POZ Magazine and TAG's media monitor page for additional information. 

    Also, since writing this post I've learned that Kamel Khalili has founded a company, Excision BioTherapeutics, which is partnering with Temple University with the aim of developing and commercializing the approach. 

    UPDATE 4/6/2016: ThankfullyThe Daily Telegraph has now edited the headline and first paragraph of their article to remove the mistaken claim that the approach may cure HIV infection "within three years" or "a few years."

    UPDATE 4/7/2016: Shortly after the publication of the paper by Khalili et al, a separate research group led by Chen Liang at McGill University published a study in Cell Reports showing that the use of CRISPR/Cas9 to excise HIV DNA can rapidly create viruses able to resist the approach. Liang's research has been covered in several news articles:

    HIV overcomes CRISPR gene-editing attack – Ewen Callaway, Nature News, April 7, 2016

    Gene-Editing Attacks Can't Defeat HIV As Easily As We Thought – George Dvorsky, Gizmodo, April 7, 2016

    HIV rapidly develops resistance to gene-editing cure technology – Gus Cairns, AIDSMap, May 17, 2016

     

    Scientific Reports 6, Article number: 22555 (2016)

    doi:10.1038/srep22555

    Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

    Rafal Kaminski, Yilan Chen, Tracy Fischer, Ellen Tedaldi, Alessandro Napoli, Yonggang Zhang, Jonathan Karn, Wenhui Hu & Kamel Khalili

    Abstract

    We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.

    Nat Biotechnol. 2016 Feb 22. doi: 10.1038/nbt.3467. [Epub ahead of print]

    Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity.

    Karpinski J, Hauber I, Chemnitz J, Schäfer C, Paszkowski-Rogacz M, Chakraborty D, Beschorner N, Hofmann-Sieber H, Lange UC, Grundhoff A, Hackmann K, Schrock E, Abi-Ghanem J, Pisabarro MT, Surendranath V, Schambach A, Lindner C, van Lunzen J, Hauber J, Buchholz F.

    Abstract

    Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

  • Cure Research News from CROI 2016

    The 2016 Conference on Retroviruses and Opportunistic Infections (CROI) took place in Boston from February 22-25. CROI deserves kudos for pioneering comprehensive webcasting, and all sessions are available for viewing online. Results from several significant cure-related clinical trials were debuted during the meeting (links to the webcasts are in parentheses):

    Ole Søgaard from Aarhus University in Denmark presented findings from a trial that combined the HDAC inhibitor romidepsin, a latency-reversing agent, with the therapeutic vaccine Vacc-4x (webcast). The rationale for this combined “kick & kill” approach is that romidepsin can cause latently infected cells to produce HIV proteins, potentially allowing these cells to be recognized and killed by HIV-specific T cell responses that have been induced or boosted by the vaccine. A series of immunizations with Vacc-4x and GM-CSF adjuvant were given first, followed by three infusions of romidepsin. Søgaard reported that romidepsin administration led to significant increases in HIV RNA, consistent with a latency-reversing effect, after which there was a significant decline in levels of total HIV DNA averaging 39.7%, but only a slight and non-significant drop in levels of integrated HIV DNA (these are two surrogate measures of the size of the HIV reservoir). Virus outgrowth was quantified in six of 17 participants who showed detectable levels at baseline, and all six showed a significant decline of around 38%. However, despite this evidence of some diminution in the size of the HIV reservoir, no significant delay in HIV viral rebound was observed when ART was temporarily interrupted in the final phase of the trial. Søgaard concluded that the data support the idea of combining latency-reversing agents with therapeutic vaccines, but improvements are needed to enhance the magnitude of the effect.

    Joe Eron from the University of North Carolina discussed the ever first clinical trial of an antibody targeting the PD-1 pathway in people with HIV (webcast). PD-1 is a molecule that can become persistently upregulated on T cells that have become exhausted and dysfunctional, and antibodies that block the interaction between PD-1 and the ligands it binds to (PD-L1 and PD-L2) have been shown to restore T cell function. Two antibodies against PD-1 have been FDA-approved for the treatment of cancers due to their ability to enhance cancer-specific immunity and promote clinically significant tumor shrinkage. Additionally, CD4 cells latently infected with HIV commonly express PD-1, and antibodies against PD-1 have been shown to reverse viral latency in laboratory experiments.

    The trial described by Eron was conducted by the ACTG and involved an antibody owned by Bristol-Myers Squibb that targets PD-L1. The original intent was to study single infusions of various doses in people on suppressive ART, however only the lowest dose (0.3mg per kg) was administered due to an unexpected concern about the potential for retinal toxicity that emerged from animal experiments. No evidence of similar toxicity was observed in the six individuals who received the anti-PD-L1 antibody. However, one person developed autoimmune pituitary insufficiency nine months after the infusion, a serious concern because autoimmunity is a known risk associated with targeting the PD1 pathway.

    Of the six anti-PD-L1 antibody recipients, two showed distinct evidence of increased Gag-specific CD8 T cell responses (measured both by interferon gamma production and expression of CD107a, a marker of cytotoxicity) but the overall average change compared to a control group of two placebo recipients did not reach statistical significance. There was also no significant change in HIV RNA levels measured by a single-copy assay, however one individual did show a 10-fold drop in cell-associated HIV RNA and Eron noted that this was the person who experienced the greatest increase in Gag-specific CD8 T cell responses. In the question & answer period after the presentation, Eron also mentioned that this individual had the lowest CD4 T cell count and highest level of PD-1 expression on T cells at baseline (consistent with prior reports that PD-1 expression progressively increases during disease progression).

    The anti-PD-L1 antibody is not going to be studied further, but the anti-PD-1 antibody pembrolizumab (which is FDA-approved as a cancer therapy) is being evaluated in people with HIV and refractory cancers in a new clinical trial. The safety concern relating to autoimmunity makes it unclear if it will ever be possible to target the PD-1 pathway in people with HIV who do not have concomitant cancers – one possibility might be to try and restrict the activity of the anti-PD1 antibody to just HIV-specific T cells, if there is any biologically feasible way of doing so.

    Katherine Bar from the University of Pennsylvania described results from a trial involving three infusions of the broadly neutralizing antibody (bNAb) VRC01, given before and after an interruption of ART in order to assess if viral load rebound would be delayed (webcast). The antibody was safe and well tolerated but did not prevent viral load rebound. There was evidence of a slight delay compared to historical controls, with more VRC01 recipients maintaining viral load suppression four weeks after interrupting ART, but the difference had disappeared after eight weeks. HIV samples from some participants displayed resistance to VRC01. Bar highlighted the need to better understand the relationship between HIV neutralization measured in vitro and antibody potency in vivo, and noted that combinations of different bNAbs will likely be required to improve results. Another somewhat similar trial conducted by Tae-Wook Chun at the National Institute of Allergy and Infectious Diseases was presented at CROI as a poster, and reported broadly consistent findings.

    After Bar’s talk, Michel Nussenzweig from Rockefeller University commented that antibodies more potent than VRC01 may perform better, citing unpublished data from a trial of the bNAb 3BNC117 that is being led by his colleague Marina Caskey. In that trial, Nussenzweig said, viral load rebound was delayed by an average of 6.5 weeks after an ART interruption, with 30% of participants maintaining suppression for over nine weeks. 3BNC117 is one of several more potent bNAbs discovered after VRC01, so this offers some hope that superior results are achievable, particularly with combinations. For cure research, the ultimate aim is to test whether these bNAbs can help promote clearance of HIV-infected cells via mechanisms such as antibody-mediated cellular cytotoxicity (ADCC).

    In the pre-clinical realm, Gilead caused a splash with data from a new study of their TLR7 agonist GS9620 in macaques. The results were presented by James Whitney (webcast), who reported at last year’s CROI that a TLR7 agonist appeared to induce virus production by latently infected cells in SIV-infected macaques on ART. The new study tested lower doses of two TLR7 agonists, GS-986 and GS9620 (the latter compound is already in clinical development for hepatitis B); the aim of using lower doses was to minimize induction of alpha interferon and associated toxicities. Evidence of latency reversal was observed in the form of SIV RNA increases after dosing, and two of nine macaques have maintained undetectable viral loads for 3-4 months after an ART interruption (no delay in viral load rebound was seen in the study presented last year). Follow up of these animals is ongoing. Whitney stated that GS9620 is now entering a phase Ib trial in HIV-positive people on ART, but Gilead have not registered the trial in clinicaltrials.gov so information on enrollment criteria and locations is not available.

    Morgane Gossez from the University of Oxford reported on an analysis of the SPARTAC trial comparing the frequency of post-treatment control of viral load in 22 participants in Africa and 44 in the UK (webcast). The SPARTAC trial design was described on the blog recently. Five of the African individuals have maintained viral load below 400 copies for over 3.5 years of follow-up, whereas all of those from the UK experienced viral load rebounds. Further review of records indicated that two of the five post-treatment controllers had undetectable viral loads at the time of ART initiation, suggesting they may have been elite controllers, but that was not the case for the remaining three. Additional studies are being conducted to look for factors associated with this outcome. Gossez noted that the biomarkers previously reported to be associated with delayed viral load rebound in SPARTAC did not show significance in the subset of African participants, however, in response to a question, Gossez acknowledged that this may have been due to the small sample size.

    The potential role of gene therapy in HIV cure research was addressed at CROI in a plenary talk by Paula Cannon from the University of Southern California (webcast). Cannon reviewed the various technologies that are now available to manipulate both host and HIV genes, and cited evidence from ongoing trials of Sangamo's gene therapy that positive effects may be achievable in people. Noting that gene therapy is sometimes viewed as too impractical to be pursued, Cannon made a strong case that it should be viewed as an important element of the cure research effort. 

    Lastly, in a poster presentation with echoes of the first report on Timothy Brown at CROI in 2008, a group of German researchers described the case of an HIV-positive individual who received a stem cell transplant from a CCR5delta32 homozygote donor as part of a series of treatments for cancer (acute myeloid leukemia). The individual experienced two relapses but ultimately the cancer went into remission in 2013. All tests for HIV DNA have since been negative in peripheral blood, rectal tissue and bone marrow, and HIV-specific antibody responses measured by Western blot are waning. Importantly, the individual remains on ART and researchers plan to search additional tissues for evidence of HIV before considering interrupting treatment. To my knowledge, this is only the second report of a successful stem cell transplant from a CCR5delta32 homozygote donor in a person with HIV (the first being Timothy Brown) – although it has been tried in other cases, these individuals died either due to the underlying cancer or complications from the procedure. The researchers are hoping that, like Brown, this individual may be cured of HIV, but it remains to be seen whether this hope will be borne out.

    As in prior years, a number of organizations provided excellent coverage of CROI, including AIDSMap, AVAC, i-Base, HIVandHepatitis.com and NATAP.

  • New Paper Rekindles Debate on HIV Replication During ART

    For many years scientists have probed the question of whether combination antiretroviral therapy (ART) completely inhibits all HIV replication in the body. The preponderance of evidence has suggested that it does, and opinion has generally tilted toward the idea that persistent virus replication in the face of ART is relatively uncommon. The evidence includes studies of ART intensification (adding multiple drugs to standard combinations), which in most—but not all—cases have documented no effect on levels of residual HIV, as well as genetic studies showing a lack of viral evolution over time in people on long-term treatment. The question remains controversial, however, and some scientists continue to believe that low-level ongoing HIV replication on ART may occur in hard-to-access sites in the body.

    A paper published online last week by Nature (and published in this week’s issue) has generated substantial debate and media coverage by claiming to provide evidence that HIV replication persists in lymphoid tissue sites where ART penetration may be limited. The study involves a very small sample size of just three people, who had only been taking ART for six months. Complex genetic analyses of HIV sequences sampled from these individuals indicated continuing evolution of several viral lineages, and also provided evidence of trafficking of these viruses from the lymphoid tissue to the blood.

    To help explain the findings, the researchers developed a model showing that suboptimal penetration of ART into tissues may allow ongoing low-level HIV replication without necessarily leading to the emergence of drug-resistant viruses (one of the arguments against ongoing replication is based on the expectation that drug resistance would eventually be seen).

    The paper’s authors appear confident that these findings are generalizable to most people on ART, but other scientists are skeptical – some vociferously so. In an article about the study by Jon Cohen in Science, longtime HIV researcher John Mellors states that the results contrast with those of his own group, and argues strongly that “the authors should be much more cautious.” Given the small number of individuals included in the Nature paper and the relative short duration of ART, this point seems very reasonable. Larger studies will be needed to resolve the controversy, and will likely be forthcoming soon.

    Pending additional data, the implications for cure research are unclear. If persistent HIV replication on ART is sustaining the reservoir of infected cells, as the new paper suggests, then novel strategies may be needed to suppress the virus in tissues. On the other hand, the goal of the “kill” element in the “kick & kill” approach to HIV reservoir reduction (currently being pursued by many scientists) is to eliminate HIV-infected cells that are expressing viral antigens. And theoretically, at least, successful “kill” approaches should also be able to target cells where persistent HIV replication is occurring because they would be expected to display viral antigens.

    Nature (2016) doi:10.1038/nature16933

    Received 06 July 2015 Accepted 18 December 2015 Published online 27 January 2016

    Persistent HIV-1 replication maintains the tissue reservoir during therapy

    Ramon Lorenzo-Redondo, Helen R. Fryer,  Trevor Bedford, Eun-Young Kim, John Archer, Sergei L. Kosakovsky Pond, Yoon-Seok Chung, Sudhir Penugonda, Jeffrey G. Chipman, Courtney V. Fletcher, Timothy W. Schacker, Michael H. Malim, Andrew Rambaut, Ashley T. Haase, Angela R. McLean & Steven M. Wolinsky

    Abstract

    Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.

  • HIV Cure Research Reviews in the Journal of Clinical Investigation

    The Journal of Clinical Investigation has published a collection of reviews on HIV cure research edited by Robert Siliciano. The articles are not open access unfortunately, but a day pass for the journal’s website (which allows downloading of all PDFs) costs $10 which is relatively affordable compared to many other publishers. 

    Recent developments in the effort to cure HIV infection: going beyond N = 1

    Janet D. Siliciano, Robert F. Siliciano

    J Clin Invest. 2016;126(2):409-414. doi:10.1172/JCI86047.

    Towards HIV-1 remission: potential roles for broadly neutralizing antibodies

    J Clin Invest. 2016;126(2):415-423. doi:10.1172/JCI80561.

    In vivo platforms for analysis of HIV persistence and eradication

    Victor Garcia

    J Clin Invest. 2016;126(2):424-431. doi:10.1172/JCI80562.

    Hematopoietic stem cell transplantation for HIV cure

    Daniel R. Kuritzkes

    J Clin Invest. 2016;126(2):432-437. doi:10.1172/JCI80563.

    The role of HIV integration in viral persistence: no more whistling past the proviral graveyard

    Frank Maldarelli

    J Clin Invest. 2016;126(2):438-447. doi:10.1172/JCI80564.

    Molecular mechanisms of HIV latency

    Daniele C. Cary, Koh Fujinaga, B. Matija Peterlin

    J Clin Invest. 2016;126(2):448-454. doi:10.1172/JCI80565.

    HIV-specific CD8+ T cells and HIV eradication

    Brad Jones, Bruce D. Walker

    J Clin Invest. 2016;126(2):455-463. doi:10.1172/JCI80566.

    Measuring the latent reservoir in vivo

    Marta Massanella, Douglas D. Richman

    J Clin Invest. 2016;126(2):464-472. doi:10.1172/JCI80567.