TAG HIV Basic Science, Vaccines, and Cure Project Blog

Monkey Viral Reservoir Study Goes for Gold

A small study of auranofin, a gold-based drug developed to treat rheumatoid arthritis, suggests it may be able to reduce the reservoir of SIV-infected CD4 T cells in macaques on antiretroviral therapy (ART). The paper appears in the July 17^th issue of the journal AIDS, and previously generated some excitable media coverage when it appeared online-ahead-of-print in April (the researchers issued a press release at the time entitled “Gold-based Drug May Pave the Way to a Cure for AIDS”). The mechanism of action of auranofin is not fully understood, but it has been shown to inhibit proliferation of CD4 T cells and thus may prevent the expansion of latently infected cells and/or shift these cells from a long-lived to short-lived phenotype. The authors note that there is one published case report on the use of auranofin in an individual with HIV, and it did not show an adverse effect on overall CD4 T cell numbers. 

A key issue in interpreting this or any other cure-related research study in rhesus macaques is that there is no consensus regarding the most appropriate way to try and model combination antiretroviral treatment of HIV infection in humans, because not all HIV drugs work against SIV. The researchers in this instance used a new approach that they first described last year, in which macaques are infected with SIVmac251 and treated with the combination of Truvada (tenofovir+emtricitabine) and Isentress (raltegravir). However, in this initial description of the model, follow-up was for 52 days and viral replication and persistence were only analyzed in blood samples, not tissues, making the extent and durability of SIV suppression unclear.

The uncertainty regarding the animal model is thrown into sharp relief by the initial results of the new experiment in which auranofin was added to ART in six macaques: SIV DNA levels declined to undetectable levels after four weeks but rebounded by eight weeks. It is uncertain whether this reflects a transient effect of auranofin or some limitation of the ART combination to fully suppress SIVmac251.

In the second part of the study, the same six macaques had ART intensified by the addition of ritonavir-boosted Prezista (darunavir). Two control macaques on the same ART combination without auranofin were also added to the experiment at this juncture. After around two months of follow-up, the researchers report a trend toward declining SIV DNA levels in the auranofin group but not the controls; however the very small number of controls makes this data hard to interpret. An attempt was then made to see if the compound SAHA could stimulate production of virus in the auranofin-treated macaques. SAHA is a histone deacetylase (HDAC) inhibitor that can induce replication of latent HIV. SIV viral load did not increase after SAHA administration in the auranofin-treated macaques, but it’s not clear from the paper if SAHA was given to the two control macaques. Instead the researchers cite two “historical” control macaques treated with ART that showed viral load rebound after SAHA treatment.

Finally, ART was stopped in 5 out of 6 of the auranofin-treated macaques (one animal was spirited away to participate in another study) and both of the real-time controls. There was, on average, a slight delay in SIV viral load rebound in the auranofin group compared to the real-time two controls that just about achieved statistical significance. Set point viral loads were also lower than those documented prior to ART in the same animals, but the statistical significance of this finding was borderline and appeared to be driven by results in one macaque. There was absolutely no evidence that auranofin had led to a cure of SIV infection in any of the treated macaques, as all showed viral load rebounds.

In sum, these experiments may have identified a compound deserving of further evaluation in the context of efforts to deplete the latent HIV reservoir. However, the data are by no means clear-cut due to the use of a new macaque model of ART that is not well characterized, the small numbers of animals involved, and the use of historical controls (issues that likely reflect limitations on funding and access to macaques). Hopefully the publication of the data will help the researchers obtain the support necessary to conduct a larger and more rigorous evaluation of the reservoir-depleting potential of auranofin.

Additional background on the use of the drug in rheumatoid arthritis, along with an excellent potted history of the use of gold in medicine, can be found in a review published in 1997 in the British Journal of Rheumatology (free to access online).

AIDS: 17 July 2011 - Volume 25 - Issue 11 - p 1347–1356

doi: 10.1097/QAD.0b013e328347bd77

Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension.

Lewis MG, Dafonseca S, Chomont N, Palamara AT, Tardugno M, Mai A, Collins M, Wagner WL, Yalley-Ogunro J, Greenhouse J, Chirullo B, Norelli S, Garaci E, Savarino A.

Abstract

OBJECTIVES: A small pool of long-lived memory CD4 T-cells harboring the retroviral genome is one main obstacle to HIV eradication. We tested the impact of the gold compound, auranofin on phenotype and viability of CD4 T-cells in vitro, and on persistence of lentiviral reservoir cells in vivo.

DESIGN: In-vitro and in-vivo study. The pro-differentiating effect of auranofin was investigated in human primary CD4 T cells, and its capacity to deplete the viral DNA (vDNA) reservoir was tested in a pilot study involving six SIVmac251-infected macaques with viral loads stably suppressed by ART (tenofovir/emtricitabine/raltegravir). The study was then amplified by intensifying ART using darunavir/r and including controls under intensified ART alone. All therapies were eventually suspended and viro-immunological parameters were monitored over time.

METHODS: Cell subpopulations were quantitated by flow cytometry following proper hematological analyses. Viral load and cell-associated vDNA were quantitated by Taqman real-time PCR.

RESULTS: In naïve, central memory and transitional memory CD4 T cells, auranofin induced both phenotype changes and cell-death which were more pronounced in the memory compartment. In the pilot study in vivo, auranofin transiently decreased the cell-associated vDNA reservoir in peripheral blood. When ART was intensified, a sustained decrease in vDNA was observed only in auranofin-treated monkeys but not in controls treated with intensified ART alone. After therapy suspension, only monkeys that had received auranofin showed a deferred and subsequently blunted viral load rebound.

CONCLUSION: These findings represent a first step towards a remission of primate lentiviral infections.

Abstracts & Presentations from the 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention

The 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention took place last week in Rome. All abstracts and many of the presentations can be accessed via the conference website, in several different ways. The main programme-at-a-glance page requires downloading Microsoft Silverlight to work properly, but a simpler version that does not need any plug-ins is also available. All conference abstracts can be accessed via an HTML page which allows searching and sorting by track, abstract type, session, etc. 

For accessing presentations, use the browse sessions page to find individual sessions; abstracts and (in some cases) slides with audio and Powerpoint download links are to the left of each presentation title. Note that the Powerpoint download links immediately to the left of presentation titles may not appear to work on many browsers; there are however working links that can be found by scrolling further down the page. 

These are direct links to some of the sessions relevant to basic science, vaccines & biomedical prevention:

Sunday, July 17

Controlling the HIV Epidemic, the Promise of ARV-based Prevention (non-commercial satellite)

Monday, July 18

Plenary Session:

The changing face of HIV vaccine research - Gary Nabel, United States

The combined approach to preventing HIV infection - Robin Shattock, United Kingdom

Reservoirs and Mechanism of Disease Progression

Advancement of Science and Research on HIV

Pathogenesis and Management of Long-Term Complications of ART (workshop) 

Treatment Is Prevention: The Proof Is Here (includes presentations on the HPTN 052 trial results)

1981-2011: Thirty Years Later, What We Know and Don't Know on How HIV Causes AIDS

Controversies in HIV Cure Research (non-commercial satellite)

Tuesday, July 19

NAPWAs' Treatment Horizons: Pathways to a Functional Cure (non-commercial satellite)

Preventive and Therapeutic HIV Vaccines: Novel Candidates and Strategies in 2011

Treatment as Prevention: Report back from the Vancouver Workshop 

Immune Activation/Inflammation and HIV Disease

Use of Antivirals in Prevention: Current Challenges and Controversies

Wednesday, July 20

Plenary Session:

Towards an HIV Cure - Eric Verdin, Belgium

Towards an HIV Cure: New Strategies for an Old Challenge

Models of Protection

Towards an HIV Cure: Insight into Residual Viral Replication, Establishment of Reservoirs and Understanding Mechanism of Persistence

Late Breaker Track A

Rapporteur and Closing Session

Two Trials Show Pre-Exposure Prophylaxis (PrEP) Can Prevent HIV Infection in Heterosexuals

Early today, results were released from two independent clinical trials evaluating the efficacy of PrEP among heterosexuals at risk of HIV infection. In both cases, a statistically significant reduction in risk of HIV acquisition was documented in the trial participants receiving PrEP (consisting of the antiretroviral drugs Viread or Truvada) compared to placebo.

The larger of the trials, named Partners PrEP, was conducted by scientists at the University of Washington. It enrolled 4,758 HIV serodiscordant couples in Kenya and Uganda and randomized the HIV-negative partners to receive either Viread (tenofovir), Truvada (a combination of tenofovir and emtricitabine in a single pill) or placebo. A total of 78 HIV infections occurred: 47 in the placebo group, 18 in the Viread group and 13 in the Truvada group. This equated to a 73% reduction in risk of HIV acquisition for those assigned to Truvada (95% confidence interval 49-85%) and a 62% reduction among those in the Viread arm (95% confidence interval 34-78%). The statement on the trial results notes that no significant side effects were observed but does not provide details, additional information is expected to become available next week at the IAS Conference on HIV Pathogenesis, Treatment and Prevention in Rome. 

The second trial (called TDF2) was conducted by the US Centers for Disease Control (CDC) in Botswana. The population was not couples in this case, but 1,200 sexually active men and women aged 18-39 (54.7% male/45.3% female) in Gaborone and Francistown. Participants were randomized to receive either Truvada or placebo. There were a total of 33 HIV infections during follow-up: nine among the 601 individuals in the Truvada group and 24 among those assigned to placebo. The reduction in risk of HIV acquisition was 62.6% (95% confidence interval, 21.5-83.4%), a statistically significant result. In an analysis restricted to participants known to have a supply of Truvada (i.e. those who had not missed a study visit at which 30-day supplies of drug were dispensed), efficacy was reported to be 77.9% (95% confidence interval 41.2-93.6%). Similar efficacy was observed in both men and women. The side effects reported more often in the Truvada arm compared to placebo were nausea, vomiting, and dizziness.

Back in December 2009, the CDC issued statements indicating that the incidence of HIV infection in the TDF2 trial population was low and that the study was focusing on safety and adherence because it was “very unlikely” that efficacy could be demonstrated; however, in announcing the results today the researchers noted that “because PrEP was highly effective in this population, the study was able to draw conclusions about overall efficacy, even with a relatively small number of infections occurring in the study population.” As with the Partners PrEP trial, more details are expected to be presented at the IAS conference next week.

A collection of statements and other information from the trial sponsors can be found on the AVAC website. AVAC is also hosting a conference call tomorrow (July 14) with the trial investigators; information on how to register is on the same page.

Today’s news contrasts with the recent announcement that a trial of Truvada as PrEP in women (the FEM-PrEP study) could not show efficacy due to similar infection rates in the active and placebo arms. The reasons for the different outcome remain to be elucidated, but could relate to differences in adherence and/or unsuspected interactions between Truvada and the types of contraceptives being used by participants (a significantly higher rate of pregnancy occurred in the Truvada arm of FEM-PrEP, and researchers are investigating what may have caused this).

Selected media stories:

AIDSMap - Two major studies show that HIV drugs prevent infection

Washington Post - Two studies show that drugs used to treat AIDS can be used to prevent HIV infection, too

Reuters - Once-daily AIDS pill can slash HIV infection risk

Immune Pressure on HIV

Several recent papers offer insights into the role of the immune response in shaping the genetic make-up of HIV. In a well-publicized, open access paper by Vincent Dahirela, Karthik Shekhara and colleagues, a complex statistical approach called random matrix theory is used to analyze published HIV sequences and look for groups of sites that evolve collectively. The method allows the researchers to uncover a region of the Gag protein they dub “sector 3” that is far more constrained in its ability to mutate than surrounding areas. Additional analyses reveal this constraint is likely due to an important role in the formation of the viral capsid. In collaboration with Bruce Walker’s group at the Ragon Institute, the researchers show that HIV-specific CD8 T cell responses in elite controllers preferentially target sector 3 of Gag, consistent with prior studies indicating that immune responses in these individuals work partly by compromising viral fitness. The findings suggest that vaccines should attempt to induce T cells against this potentially vulnerable region of HIV. The researchers also recommend using their technique to search for other “multidimensionally constrained” parts of viral proteins. 

In a paper published in Blood, Tao Dong and colleagues describe the impact of CD8 T cell responses on HIV diversity in a population of Chinese individuals who were infected with genetically homogenous viruses as a result of plasma donation. Because of the similarity of the infecting viruses, the cohort offers a unique window into how individual variability in the parts of HIV targeted by CD8 T cells shapes viral evolution, by selecting variants able to escape recognition. Based on analyses of four HIV proteins—Gag, Reverse Transciptase, Integrase and Nef—the researchers find evidence that 24-56% of variable sites were subject to selection by CD8 T cell responses (over approximately 10-12 years since the time of infection). The results offer strong and unusually direct evidence for the key role of virus-specific immune responses in driving HIV diversity. In a separate paper by the same authors, involving individuals from the same cohort possessing the beneficial HLA B51 immune response gene, CD8 T cell responses targeting specific epitopes from HIV Gag and Pol proteins are investigated in detail. The researchers show that mutations in these epitopes that abrogate CD8 T cell recognition are associated with higher viral loads and lower CD4 counts, whereas individuals in whom the epitopes are unmutated had higher CD4 T cell counts and lower viral loads. The findings add to the evidence that the beneficial effect of certain HLA genes in HIV infection is mediated by CD8 T cell responses targeting vulnerable parts of the virus.

Lastly, Ingrid Schellens and colleagues compare two groups of HIV-positive individuals who seroconverted in 1985 and 2005/6, respectively, to investigate whether CD8 T cell responses are altering the make-up of circulating HIV over time. The researchers report that certain HIV epitopes have become significantly less common, and these are epitopes known to be targeted by people with HLA-B alleles associated with slower HIV disease progression (such as HLA B27, B51 and B57). The implication is that HIV is adapting at the population level to avoid the most effective CD8 T cell responses, so HLA alleles that have been shown to be “protective” against disease progression in the past may not always show this association. As a possible example, the authors note that individuals with HLA B57 who seroconverted in 1985 had significantly lower viral loads than individuals lacking this allele, but this was not the case among the 2005/6 cohort.

Proc Natl Acad Sci U S A. 2011 Jun 20. [Epub ahead of print]

Coordinate linkage of HIV evolution reveals regions of immunological vulnerability.

Dahirel V, Shekhar K, Pereyra F, Miura T, Artyomov M, Talsania S, Allen TM, Altfeld M, Carrington M, Irvine DJ, Walker BD, Chakraborty AK.

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, MA 02129.

Abstract

Cellular immune control of HIV is mediated, in part, by induction of single amino acid mutations that reduce viral fitness, but compensatory mutations limit this effect. Here, we sought to determine if higher order constraints on viral evolution exist, because some coordinately linked combinations of mutations may hurt viability. Immune targeting of multiple sites in such a multidimensionally conserved region might render the virus particularly vulnerable, because viable escape pathways would be greatly restricted. We analyzed available HIV sequences using a method from physics to reveal distinct groups of amino acids whose mutations are collectively coordinated ("HIV sectors"). From the standpoint of mutations at individual sites, one such group in Gag is as conserved as other collectively coevolving groups of sites in Gag. However, it exhibits higher order conservation indicating constraints on the viability of viral strains with multiple mutations. Mapping amino acids from this group onto protein structures shows that combined mutations likely destabilize multiprotein structural interactions critical for viral function. Persons who durably control HIV without medications preferentially target the sector in Gag predicted to be most vulnerable. By sequencing circulating viruses from these individuals, we find that individual mutations occur with similar frequency in this sector as in other targeted Gag sectors. However, multiple mutations within this sector are very rare, indicating previously unrecognized multidimensional constraints on HIV evolution. Targeting such regions with higher order evolutionary constraints provides a novel approach to immunogen design for a vaccine against HIV and other rapidly mutating viruses.

Blood. 2011 May 11. [Epub ahead of print]

Extensive HLA-driven viral diversity following a narrow-source HIV-1 outbreak in rural China.

Dong T, Zhang Y, Xu KY, Yan H, James I, Peng Y, Blais ME, Gaudieri S, Chen X, Lun W, Wu H, Qu WY, Rostron T, Li N, Mao Y, Mallal S, Xu X, McMichael A, John M, Rowland-Jones SL.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom;

Abstract

Obstacles to developing an HIV-1 vaccine include extensive viral diversity and lack of correlates of protective immunity. High mutation rates allow HIV-1 to adapt rapidly to selective forces such as anti-retroviral therapy and immune pressure, including HIV-1-specific cytotoxic T-lymphocytes (CTL) that select viral variants which escape T-cell recognition. Multiple factors contribute to HIV-1 diversity, making it difficult to disentangle the contribution of CTL selection without employing complex analytical approaches. We describe an HIV-1 outbreak in 231 former plasma donors in China, where a narrow-source virus that had contaminated the donation system was apparently transmitted to many individuals contemporaneously. The genetic divergence now evident in these subjects should uniquely reveal how much viral diversity at the population level is solely attributable to host factors. We found significant correlations between pair-wise divergence of viral sequences and HLA class I genotypes across epitope-length windows in HIV-1 Gag, Reverse Transciptase, Integrase and Nef, corresponding to sites of 140 HLA class I allele-associated viral polymorphisms. Of all polymorphic sites across these four proteins, 24% - 56% were sites of HLA-associated selection. These data confirm that CTL pressure has a major impact on inter-host HIV-1 viral diversity and is likely to represent a key element of viral control.

J Immunol. 2011 Jun 13. [Epub ahead of print]

Multilayered Defense in HLA-B51-Associated HIV Viral Control.

Zhang Y, Peng Y, Yan H, Xu K, Saito M, Wu H, Chen X, Ranasinghe S, Kuse N, Powell T, Zhao Y, Li W, Zhang X, Feng X, Li N, Leligdowicz A, Xu X, John M, Takiguchi M, McMichael A, Rowland-Jones S, Dong T.

Beijing You An Hospital, Capital Medical University, Beijing 100069, People's Republic of China;

Abstract

Polymorphism in the HLA region of a chromosome is the major source of host genetic variability in HIV-1 outcome, but there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, -B27, and -B51. Taking advantage of a unique cohort infected with clade B' HIV-1 through contaminated blood, in which many variables such as the length of infection, the infecting viral strain, and host genetic background are controlled, we performed a comprehensive study to understand HLA-B51-associated HIV-1 control. We focused on the T cell responses against three dominant HLA-B51-restricted epitopes: Gag327-345(NI9) NANPDCKTI, Pol743-751(LI9) LPPVVAKEI, and Pol283-289(TI8) TAFTIPSI. Mutations in all three dominant epitopes were significantly associated with HLA-B51 in the cohort. A clear hierarchy in selection of epitope mutations was observed through epitope sequencing. L743I in position 1 of epitope LI9 was seen in most B51(+) individuals, followed by V289X in position 8 of the TI8, and then, A328S, in position 2 of the NI9 epitope, was also seen in some B51(+) individuals. Good control of viral load and higher CD4(+) counts were significantly associated with at least one detectable T cell response to unmutated epitopes, whereas lower CD4(+) counts and higher viral loads were observed in patients who had developed escape mutations in all three epitopes or who lacked T cell responses specific to these epitope(s). We propose that patients with HLA-B51 benefit from having multiple layers of effective defense against the development of immune escape mutations.

AIDS. 2011 Jun 15. [Epub ahead of print]

Loss of HIV-1 derived CTL epitopes restricted by protective HLA-B alleles during the HIV-1 epidemic.

Schellens IM, Navis M, van Deutekom HW, Boeser-Nunnink B, Berkhout B, Kootstra N, Miedema F, Keşmir C, Schuitemaker H, van Baarle D, Borghans JA.

Abstract

OBJECTIVE AND DESIGN: HIV-1 is known to adapt to the human immune system, leading to accumulation of escape mutations during the course of infection within an individual. Cross-sectional studies have shown an inverse correlation between the prevalence of HLA alleles in a population and the number of CTL escape mutations in epitopes restricted by those HLA alleles. Recently, it was demonstrated that at a population level HIV-1 is adapting to the humoral immune response, which is reflected in an increase in resistance to neutralizing antibodies over time. Here we investigated whether adaptations to cellular immunity have also accumulated during the epidemic. METHODS: We compared the number of CTL epitopes in HIV-1 strains isolated from individuals who seroconverted at the beginning of the HIV-1 epidemic and from individuals who seroconverted in recent calendar time. RESULTS: The number of CTL epitopes in HIV-1 variants restricted by the most common HLA alleles in the population did not change significantly during the epidemic. In contrast, we found a significant loss of CTL epitopes restricted by HLA-B alleles associated with a low relative hazard of HIV-1 disease progression during the epidemic. Such a loss was not observed for CTL epitopes restricted by HLA-A alleles. CONCLUSIONS: Despite the large degree of HLA polymorphism, HIV-1 has accumulated adaptations to CTL responses within twenty years of the epidemic. The fact that such adaptations are driven by the HLA-B molecules that provide best protection against HIV-1 disease progression has important implications for our understanding of HIV evolution.