TAG HIV Basic Science, Vaccines, and Cure Project Blog

Updates on the STEP and Phambili Vaccine Trials

As reported yesterday by AVAC's Advocates Network, NIAID released a statement this past Monday reporting that a total of 11 infections occurred in the South African study of Merck's HIV vaccine, dubbed the Phambili trial or HVTN 503. Immunizations in the Phambili were stopped before it was fully enrolled due to the results from the preceding international STEP/HVTN 502 trial, which showed no efficacy of the vaccine and evidence of enhanced susceptibility in a subset of immunized volunteers. Of the 11 infections in Phambili, seven occurred in vaccine recipients (although most had not received all three immunizations) and four in placebo recipients. Among the vaccine recipients, six infections occurred in people with titers of anti-Ad5 antibodies over 1:18 compared to three infections among people with similar titers in the placebo group. In participants with no detectable antibodies to Ad5 at enrollment, there was one case of HIV infection among vaccine recipients and one case of HIV infection among placebo recipients. Given the small numbers, the three additional infections in the vaccine group cannot be interpreted as evidence that the vaccine enhanced susceptibility in this population, although it is of course possible that such evidence would have emerged, had the trial not been stopped. The apparent difference in infections based on anti-Ad5 antibody titer (6 vs. 3 infections compared to 1 vs. 1) also involves far too few events for any inferences to be drawn, although it may still prompt speculation that a similar scenario to the STEP trial was developing. Notably, all but one of the cases of HIV infection in the Phambili trial were among women, who made up 360 of the 801 participants at the time immunizations in the study were halted.

NIAID have also released more materials regarding the multivariate analyses of the STEP data that have recently been conducted. The analyses find that receipt of the vaccine was associated with enhanced susceptibility but that this effect was strongest in uncircumcised men with antibody titers against the Ad5 vector of over 1:200. Circumcised men with undetectable Ad5 antibodies did not show any evidence of vaccine-related enhanced susceptibility. However, the picture is less clear for men with either risk factor (uncircumcised or Ad5 antibody titer over 1:200), as both groups showed some evidence of vaccine-related enhanced susceptibility in the multivariate analyses.

In terms of the association between anti-Ad5 antibody titers and risk of HIV infection seen in the placebo arm, the NIAID document states that: "Although the study was stratified on Ad5 titers, the Ad5 seropositive population differed substantially from the Ad5 seronegative population. When we looked in the multivariate analyses for association of Ad5 serostatus with infection risk independent of vaccination, Ad5 serostatus was not independently associated with HIV infection. We did find, though, that being North American was associated with an increased risk of acquisition. It may be that the reason for the higher rates of infection in the placebo arm among those who were Ad5 seronegative was because that subgroup was primarily in the North American cohorts, which reported riskier sexual practices and substance use. We are exploring this in other observational cohort studies."

So in this formulation, people in the placebo group with anti-Ad5 antibody titers below 1:18 were more likely to be North American, people with antibody titers between 1:18 and 1:1000 were a bit less likely to be North American and people with antibody titers over 1,000 were much less likely to be North American. Individuals with antibody titers over 1:200 were also less likely to be circumcised, so while circumcision appeared to have a protective effect in vaccine recipients, no such effect was seen in the placebo arm. People in the vaccine arm with higher anti-Ad5 antibody titers should presumably also have been at less risk because they were less likely to be North American, but the vaccine increased their risk somehow. To provide some context, this is the overall incidence table from STEP, with the confidence intervals included to give a sense of the uncertainty associated with the findings.

Anti-Ad5 antibody titer	HIV incidence vaccine (95% CI)	HIV incidence placebo (95% CI)	Relative incidence V:P
less than 1:18	4% (2.5-6.3)	4% (2.5-6.2)	1.0
1:18-1:200	4.4% (1.9-8.8)	2.2% (0.6-5.5)	2.0
1:200-1:1000	6.1% (3.3-10.2)	3% (1.2-6.2)	2.0
over 1:1000	4.4% (1.2-6.2)	1.2% (0.2-4.5)	3.5

The NIH is holding a summit on March 25 to discuss the implications of the STEP results for the future of HIV vaccine research, meeting and registration information are available online.

Update 2/29/08: Thanks to Sarah Alexander from the HVTN for pointing out several significant errors in the original posting:

1) the sample size for Phambili was slated to be 3,000, the same as for STEP, so it was not intended to be a smaller trial
2) women did not make up the majority of the Phambili enrollees (of the 801 participants, 360 were women and 441 men)
3) follow-up is continuing in both the STEP and Phambili trials so while immunizations and enrollment have ceased, the trials continue.

Apologies for the mistakes, which have also been corrected in the body of the post

Genetic Associations with Control of HIV Replication

At the recent CROI meeting, David Goldstein from Duke University gave a plenary presentation on genetic determinants of viral load set point in HIV-infected individuals. Goldstein’s talk was an update on work published recently with CHAVI (Center for AIDS Vaccine Immunology) collaborator Amalio Telenti from the University of Lausanne. Their approach involved analyzing a staggering 500,000 different single nucleotide polymorphisms (SNPs) present in the human genome to see if they were associated with lower viral load set points (although Goldstein noted that this initial work is restricted to SNPs documented in >1% of the population and thus excludes rare polymorphisms). As reported in the Science paper, three sets of significant associations emerged. The first was a SNP in a gene called HCP5. This is linked to an immune response gene called HLA B*5701 which is well known to be over-represented among HIV-infected long-term non-progressors (HLA B*5701 makes a receptor on CD8 T cells which appears particularly good at recognizing HIV epitopes). However, HCP5 is also an endogenous retroviral element (a part of the human genome derived from an ancient retroviral infection which gained access the human germ line by infecting an egg or sperm cell) and so Goldstein initially speculated that the SNP in HCP5 might somehow have a direct anti-HIV effect. At CROI, he reported that studies in which the SNP-containing version of HCP5 was overexpressed in cells showed no inhibition of HIV replication, suggesting that the SNP is mediating its effect via other means (exactly how is under investigation).

The second association uncovered by Goldstein’s work was with a SNP in the gene for HLA-C. HLA-C molecules are part of a family called class I HLA molecules that are involved in the recognition of pathogens by CD8 T cells. Thousands of HLA molecules are displayed on the surface of every cell in the body (except red blood cells) and their job is to constantly lift cellular debris from inside the cell and display it to passing CD8 T cells. If an HLA molecule displays a protein fragment (epitope) from a pathogen that has infected the cell, this can trigger the CD8 T cell to release toxic enzymes, which cause the cell to die. The majority of HLA molecules on cells belong to classes HLA-A and HLA-B, while HLA-C molecules are less frequent and, as a result, less studied. But because HIV’s Nef protein is known to cause a reduction in HLA-B molecules on infected cells (as a means of escaping the immune response), Goldstein’s hypothesis is that the SNP he has identified causes more HLA-C molecules to be expressed, thereby making it easier for CD8 T cells to identify HIV-infected cells. He is currently collaborating with Andrew McMichael at Oxford University to measure the effect of the SNP on HLA-C expression.

The third association described by Goldstein involves a set of seven SNPs in or near two genes, ZNRD1 and RNF39. In the Science paper, this association was only seen with disease progression (as assessed by time to a CD4 T cell count of less than 350) but at CROI Goldstein reported that in an expanded analysis involving 1,000 more individuals (in addition to the 446 studied initially), an association with viral load set point was also documented. ZNRD1 encodes a protein involved in RNA transcription and, interestingly, was also identified in the recent, widely publicized study of host proteins needed by HIV to replicate. The function of RNF39 remains to be determined. Goldstein stressed that the discovery of all these associations is a first step, and efforts are underway to uncover the mechanisms by which the identified SNPs mediate their effects.

To give a sense of how a combination of genetic factors can have a profound impact on HIV disease progression, Goldstein showed an analysis that included the SNPs in the HCP5, HLA-C, and ZNRD1/RNF39 genes and two other known favorable genetic polymorphisms in CCR5 and CCR2 genes (CCR5Δ32 and CCR2 V64I). HIV-infected individuals with no favorable mutations in any of these genes showed an average time of less than two years from infection to a CD4 T cell count of less than 350. In contrast, people with one or two favorable mutations in at least four of these genes did not experience a CD4 T cell decline to this level for an average period of more than eight years.

Goldstein’s group is now embarking on an effort to uncover genetic associations with the magnitude of antibody responses generated against a vaccine, using data from the North American efficacy trial of AIDSVAX. This is a potentially important area of study because the ability of an individual to generate a high titer antibody response was correlated with reduced susceptibility to HIV infection in the trial cohort. Some researchers have suggested an analogy with the association between the magnitude of anti-Ad5 antibody responses and susceptibility to HIV infection seen in the placebo group of the recent Merck vaccine trial, so Goldstein’s work may have the potential shed light on that mystery also. Another important area of ongoing study mentioned by Goldstein is an analysis of genetic associations with viral load set point restricted to African American individuals.

The webcast of David Goldstein’s talk is available on the CROI website, it is the second presentation on Monday.

Shortly after CROI, a study of untreated HIV-infected individuals with viral loads under 50 copies – a group now dubbed “elite controllers” – highlighted the complexities and limitations of genetic associations. In a research letter published in the journal AIDS, Yefei Han and colleagues from Bob Siliciano’s laboratory at Johns Hopkins report that, of 16 elite controllers analyzed, only four possessed the SNP in HLA-C identified in the CHAVI work and none had the SNP in HCP5 (although two had HLA B*5702 and HLA B*5703 genes, which are closely related to HLA B*5701). The researchers did find the HCP5 SNP in two other infected individuals with HLA B*5701, one of whom had a low but detectable viral load and the other with progressive disease. The authors also note that the frequency of the HLA-C SNP in the elite controllers was not significantly different from what would be expected in a larger population of people from the same ethnic background. Citing these data and an example of an individual in their cohort who developed an increasing viral load after developing a CD8 T cell escape mutation, they argue that adaptive immune responses are likely an important contributor to the control of viral replication in elite controllers.

AIDS. 22(4):541-544, February 19, 2008.

The role of protective HCP5 and HLA-C associated polymorphisms in the control of HIV-1 replication in a subset of elite suppressors.

Research Letters

Han, Yefei; Lai, Jun; Barditch-Crovo, Patricia; Gallant, Joel E; Williams, Thomas M; Siliciano, Robert F; Blankson, Joel N

Abstract:

Elite suppressors (ES) are untreated HIV-1-infected patients who maintain undetectable viral loads. A recent whole-genome analysis identified two independent polymorphisms associated with low viral loads in untreated HIV-1 infection. We screened 16 ES; none were positive for the protective HLA complex 5 gene polymorphism, and only four were positive for the protective polymorphism associated with the HLA-C gene. These results suggest that some ES control viremia by mechanisms independent of the newly-identified genetic factors.

A Gutsy Talk, and a New Paper, on HIV Persistence

In December 2007, the third workshop on HIV persistence during therapy took place on the Caribbean island of St. Maarten, where the weather is sunnier than prospects for eradicating HIV infection currently appear to be. The meeting is the brainchild of French researcher Alan Lafeuillade and aims to bring together researchers interested in the topic of curing HIV infection, either by eradication or inducing lifelong control or tolerance of the virus without the need for ongoing drug therapy. The submitted abstracts ranged widely in both subject matter and quality, with much attention focused on the struggle to accurately assess residual HIV replication in people on long term ART and the best methods for measuring levels of integrated, replication-competent virus in both the blood and body tissues.

One highlight of the meeting was a presentation on the last day by NIH researcher Tae-Wook Chun. Chun has been studying the latent HIV reservoir in people on therapy for over a decade now, and he offered a pithy summation of his views on the subject in a provocative talk entitled “10 questions you might have been afraid to ask at this workshop." This is a brief summary of his fear-inducing questions, along with his current thoughts as to the answers. Chun's recent work on HIV persistence in gut CD4 T cells has just been published online by the Journal of Infectious Diseases, along with an accompanying commentary by Steven Yukl and Joseph Wong; abstracts and links are appended below.

Tae-Wook Chun’s 10 questions you might have been afraid to ask at the 3rd Workshop on HIV Persistence During Therapy

1. Does the reservoir of HIV in the body decline on effective antiretroviral therapy (ART)?

Yes. In a cohort of individuals treated early with combination ART, Chun observed that the half-life of HIV-infected cells was 4-6 months. He explained that this observation was restricted to resting memory CD4 T cells, suggesting that this reservoir of HIV could potentially be eradicated after around 7 years of treatment. However, he stressed that this would not necessarily apply to infected cells of other types (e.g. macrophages, stem cells).

2. Is there ongoing viral replication on ART?

Yes. Some researchers think viral replication can be completely shut down by ART, but in Chun’s experience there is always some ongoing low-level replication that can be difficult to detect.

3. Does ongoing HIV replication contribute to the reservoir?

Yes, but activation of CD4 T cells also contributes. He cited a study published by his research group several years ago showing that HIV DNA is detectable in both activated and resting CD4 T cells in people on long term ART (the full text of this paper can be accessed online). Chun also noted that the gut contains more activated CD4 T cells than other sites in the body and is therefore a major reservoir of HIV; he has found HIV DNA in activated CD4 T cells from the terminal ileum (see JID cites, below). Chun also showed several images showing the presence of lymphoid aggregates in the gut of HIV-infected individuals on ART and mentioned that these were “a little different from previously published papers” (likely an oblique reference to a notorious single image from the gut of an HIV-infected individual lacking lymphoid tissue, shown by Danny Douek several years ago in support of this theory that gut CD4 T cell depletion is central to HIV pathogenesis). To investigate whether viruses sampled from the gut were different from those found in the blood, Chun compared the genetic sequences and found that they overlapped, meaning that there was no evidence that HIV was somehow compartmentalized in the gut.

4. Is there a specific mechanism for HIV latency in resting CD4 T cells?

Probably not. This topic was addressed by a number of other presentations at the workshop, some of which suggested there may be specific mechanisms HIV uses to make itself quiescent or latent (as opposed to actively replicating). Chun stated that while he admired these presentations, he actually doubted that HIV had evolved such a mechanism. Rather, he believes that viral latency occurs as a byproduct of the physiology of the infected cell; for example, when an HIV-infected activated CD4 T cell returns to a resting state (a normal occurrence when an immune response is ending).

5. Do you think reactivation of the latent reservoir on ART would be sufficient to lead to eradication?

Probably not. At least two studies have investigated activating resting CD4 T cells to try and reduce the HIV reservoir, but the results were disastrous because the treatment (a T cell-depleting antibody called OKT3) led to severe decreases in CD4 T cell counts and illness.

6. Is early ART a good thing?

Yes, in terms of reservoir reduction, but Chun acknowledged the question is not so straightforward from the perspective of an HIV-infected individual or a clinician, because of the potential for drug toxicities and the challenge of long term adherence.

7. Is eradication possible?

Chun stated that five years ago, his answer would have been no. Now he is “cautiously optimistic in a subset of patients.” He has studied some individuals on long term ART who started relatively soon after becoming infected, and in one he can’t find virus anywhere (including the gut), by any technique (RNA or DNA). This individual has not tried stopping ART yet, however. Chun has also compared the proviral burden (amount of integrated HIV DNA) in small groups on long term ART (>7yrs) that started early vs. during chronic infection. The early group averaged 4.6 copies HIV DNA, with 44% of the group having levels of less than 1.5 copies, while the group that started later averaged 949.4 copies of HIV DNA and only 11% showed levels <1.5 copies.

8. Is intensification a good idea for trying to reduce reservoir?

Yes. He is starting a randomized raltegravir (Isentress) intensification study which will recruit 24 people who have been on ART for over 4 yrs with viral loads less than 50 copies. Twelve will add raltegravir while 12 will receive placebo and the HIV reservoir will be measured regularly during follow-up.

9. Should “mild immunosuppressants” be studied?

Yes. Chun showed a DNA microarray analysis (which measures the activity of different genes) of CD4 T cells from people on long term ART (started in chronic vs. acute infection) and there were more activation and proliferation related genes upregulated in the former group. He suggested that mild immunosuppressants should be studied as part of a multi-pronged attack on HIV reservoirs that could potentially include ART intensification, enhancement of HIV-specific immunity, and drugs which activate HIV transcription in resting cells.

10. Which is more important, the latent HIV reservoir or cells supporting low-level HIV replication?

Equally important. Chun believes both have to be addressed if the aim is to work toward a cure. In response to questions from the audience, Chun said the western blot HIV antibody test was “very faint” in the person he’d mentioned where no virus could be detected. He also described this individual’s CD4/CD8 T cell ratio as “normal.” He hasn’t yet measured immune activation levels in his cohort of individuals treated early in infection, but he described their gene expression profiles as “calm.”

Study abstracts:

The Journal of Infectious Diseases 2008;197:000–000
DOI: 10.1086/527325

EDITORIAL COMMENTARY

Blood and Guts and HIV: Preferential HIV Persistence in GI Mucosa

Steven Yukl and Joseph K. Wong
Veterans Affairs Medical Center San Francisco and University of California San Francisco

(See the article by Chun et al. on pages XXX)

Reprints or correspondence: Joseph Wong, MD, 4150 Clement Street, San Francisco VAMC, 111W, University of California, San Francisco, San Francisco, CA 94121 (joseph.wong2@med.va.gov).

The Journal of Infectious Diseases 2008;197:000–000
DOI: 10.1086/527324

MAJOR ARTICLE

Persistence of HIV in Gut-Associated Lymphoid Tissue despite Long-Term Antiretroviral Therapy

Tae-Wook Chun,1,a David C. Nickle,6,a Jesse S. Justement,1 Jennifer H. Meyers,1 Gregg Roby,1 Claire W. Hallahan,2 Shyam Kottilil,1 Susan Moir,1 JoAnn M. Mican,4 James I. Mullins,6 Douglas J. Ward,7 Joseph A. Kovacs,5 Peter J. Mannon,3 and Anthony S. Fauci1

1Laboratory of Immunoregulation, 2Biostatistical Research Branch, 3Laboratory of Host Defense, 4Division of Clinical Research, National Institute of Allergy and Infectious Diseases, 5Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland; 6Department of Microbiology, University of Washington, Seattle; 7Dupont Circle Physicians Group, Washington, DC

Financial support: National Institutes of Health (grant P30 AI27757 to J.I.M) and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

(See the article by Yukl and Wong on pages XXX–XX.)

aT-.W.C. and D.C.N. contributed equally to this work.
Reprints or correspondence: Tae-Wook Chun, PhD, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 6A32, 9000 Rockville Pike, Bethesda, Maryland 20892 (twchun@nih.gov).

Human immunodeficiency virus (HIV) persists in peripheral blood mononuclear cells despite sustained, undetectable plasma viremia resulting from long-term antiretroviral therapy. However, the source of persistent HIV in such infected individuals remains unclear. Given recent data suggesting high levels of viral replication and profound depletion of CD4+ T cells in gut-associated lymphoid tissue (GALT) of animals infected with simian immunodeficiency virus and HIV-infected humans, we sought to determine the level of CD4+ T cell depletion as well as the degree and extent of HIV persistence in the GALT of infected individuals who had been receiving effective antiviral therapy for prolonged periods of time. We demonstrate incomplete recoveries of CD4+ T cells in the GALT of aviremic, HIV-infected individuals who had received up to 9.9 years of effective antiretroviral therapy. In addition, we demonstrate higher frequencies of HIV infection in GALT, compared with PBMCs, in these aviremic individuals and provide evidence for cross-infection between these 2 cellular compartments. Together, these data provide a possible mechanism for the maintenance of viral reservoirs revolving around the GALT of HIV-infected individuals despite long-term viral suppression and suggest that the GALT may play a major role in the persistence of HIV in such individuals.

Conference on Retroviruses & Opportunistic Infections 2008

Last week, the 15th annual Conference on Retroviruses & Opportunistic Infections took place in Boston. The conference website now features open access to webcasts of every session (with the exception of poster discussions) as well as a searchable database of abstracts. In virology, the major highlight involved the identification (by two research groups, one led by Paul Bieniasz and the other by John Guatelli) of a host cell protein involved in HIV replication that belongs to a family called tetherins. In pathogenesis, the depletion of Th17 cells (a subset of CD4 T cells associated with both antimicrobial responses and proinflammatory autoimmune conditions) by HIV emerged a potential contributor to disease progression. Biomedical prevention science struggled with a litany of bad news: HSV-2 suppression using acyclovir failed to reduce HIV transmission, male circumcision was associated with a trend toward increased risk of HIV transmission to female partners (thought related to inadequate time given for healing after the procedure) and current HIV vaccine candidates were hammered by the CROI committee with twin calls for a "back to basics" approach (by Ron Desrosiers and Neal Nathanson).

The AIDSMap website has comprehensive coverage of the meeting. Science has published two complementary pieces (Back to Basics Push as Prevention Struggles and Experts: AIDS Vaccine Research has "Lost Its Way") and Project Inform highlight an intriguing study involving stem cell transplantation to an HIV-infected recipient from a donor lacking CCR5 on their cells (due to the delta32 mutation). This individual has now gone 285 days without antiretroviral treatment, without the return of any detectable HIV DNA or RNA (a PDF of the poster is available online).