TAG HIV Basic Science, Vaccines, and Cure Project Blog

Low Level HIV Replication Versus Latency: Identifying the Source of Viral Rebounds During Treatment Interruption

In HIV research, there is a persistent and vigorous debate around the question of whether or not viral replication persists in the face of successful antiretroviral therapy. During a plenary session at the International AIDS Conference in Mexico City back in August, Bob Siliciano made a compelling argument that, in most cases, antiretroviral therapy completely shuts down virus production. Now, a new paper in PNAS provides additional support for this view. Beda Joos and colleagues evaluated a staggering 1,753 genetic sequences from the envelope region of HIV, sampled over the course of a treatment interruption trial known as SSITT (Swiss-Spanish Intermittent Treatment Trial). The study design involved a series of two-week treatment breaks followed by a prolonged interruption (therapy was subsequently reinitiated according to the CD4 and viral load thresholds used in current treatment guidelines). 

The researchers used the sequence data to plot the relationships between the different viruses, using a technique called phylogenetic analyses. For each study participant analyzed, the sequences were used to define “the most recent common ancestor” (MRCA), which is an approximation of the ancestral virus sequence from which all the others derived. Viruses that appeared during treatment interruptions (TIs) were then compared to the MRCA, to see if the sequences suggested that there had been ongoing replication and evolution while the study participants were on ART. The results showed that the rebounding viruses during TI were actually more distant from the MRCA than the viruses detected when the participants first entered the study. The researchers conclude: “the striking lack of a temporal relationship between rebounding virus and pretreatment viruses strongly suggests that rebounding virus originates from reactivated, latently infected cells rather than from a cellular pool or compartment engaged in low-level replication.” 

PNAS October 28, 2008 vol. 105 no. 43 16725-16730

Published online before print October 20, 2008, doi: 10.1073/pnas.0804192105

HIV rebounds from latently infected cells, rather than from continuing low-level replication

Beda Joosa,1, Marek Fischera, Herbert Kustera, Satish K. Pillaib, Joseph K. Wongb, Jürg Bönic, Bernard Hirscheld, Rainer Webera, Alexandra Trkolaa, Huldrych F. Güntharda,1, and The Swiss HIV Cohort Study2

aDepartment of Medicine, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich, CH-8091 Zurich, Switzerland;

bSan Francisco Veterans Affairs Medical Center, University of California, San Francisco, CA 94121;

cNational Center for Retroviruses, University of Zurich, CH-8028 Zurich, Switzerland; and

dDivision of Infectious Disease, University Hospital Geneva, 1211 Geneva, Switzerland

Rapid rebound of plasma viremia in patients after interruption of long-term combination antiretroviral therapy (cART) suggests persistence of low-level replicating cells or rapid reactivation of latently infected cells. To further characterize rebounding virus, we performed extensive longitudinal clonal evolutionary studies of HIV env C2-V3-C3 regions and exploited the temporal relationships of rebounding plasma viruses with regard to pretreatment sequences in 20 chronically HIV-1-infected patients having undergone multiple 2-week structured treatment interruptions (STI). Rebounding virus during the short STI was homogeneous, suggesting mono- or oligoclonal origin during reactivation. No evidence for a temporal structure of rebounding virus in regard to pretreatment sequences was found. Furthermore, expansion of distinct lineages at different STI cycles emerged. Together, these findings imply stochastic reactivation of different clones from long-lived latently infected cells rather than expansion of viral populations replicating at low levels. After treatment was stopped, diversity increased steadily, but pretreatment diversity was, on average, achieved only >2.5 years after the start of STI when marked divergence from preexisting quasispecies also emerged. In summary, our results argue against persistence of ongoing low-level replication in patients on suppressive cART. Furthermore, a prolonged delay in restoration of pretreatment viral diversity after treatment interruption demonstrates a surprisingly sustained evolutionary bottleneck induced by punctuated antiretroviral therapy.

Identifying and Reviving Tired T Cells Via Tim-3

Around three years ago, much excitement was generated by the discovery that a receptor called PD-1 is expressed by T cells that have become exhausted and dysfunctional as a consequence of chronic stimulation. Targeting of PD-1 in the mouse model of persistent LCMV infection was shown to restore a significant degree of T cell function and decrease viremia. Subsequent studies demonstrated that PD-1 expression increases over the course of HIV infection and correlates with other markers of disease progression such as viral load and CD4 T cell counts. As a result of these discoveries, ongoing research in the SIV model is now evaluating whether anti-PD-1 approaches have the potential to enhance immunity against HIV. However, some caveats have also been raised regarding PD-1: it turns out that the molecule is also transiently expressed as a normal consequence of T cell activation, so is not always a marker of exhaustion. It has also been suggested that some exhausted T cells may not express PD-1.

Last Monday in the Journal of Experimental Medicine, Brad Jones and colleagues reported on another receptor that identifies exhausted T cells in HIV infection, and which also may be amenable to therapeutic manipulation (similar data was also reported at the recent AIDS Vaccine 2008 conference by Marilyn Addo from Bruce Walker’s group, see abstract below). The receptor is personably called Tim-3 (an acronym for the more cumbersome full name: T cell immunoglobulin and mucin domain–containing molecule 3). Data from mouse studies indicates that Tim-3 serves an inhibitory role; interactions between Tim-3 and its Star Trek-sounding ligand galectin-9 promotes the death of interferon-gamma-producing, Th1-type CD4 T cells. Furthermore, blocking the interactions of Tim-3 can prevent the acquisition of transplant tolerance. This murine data prompted Jones et al to investigate the role of Tim-3 in people with HIV.

The study found that Tim-3 expression is significantly elevated in people with acute/early and chronic HIV infection compared to both healthy controls and elite controllers (individuals controlling their HIV viral load to low levels without therapy). Tim-3 expression on both CD8 and CD4 T cells correlated positively with viral load levels, and inversely with peripheral blood CD4 T cell counts. Tim-3 expression also correlated significantly with CD38 expression (a marker of immune activation) on both CD8 and CD4 T cell subsets, with many CD38-expressing cells also displaying Tim-3. Further analyses demonstrated that HIV-specific CD8 T cells preferentially expressed Tim-3 compared to CMV-specific responses from the same individual, with the exception of a CMV-specific CD8 T cell response from an individual with an AIDS diagnosis and a CD4 T cell count of 132. High levels of Tim-3 expression by CD8 T cells equated with a complete lack of cytokine production (even interferon-gamma, which many exhausted T cells can continue to make).

To explore the potential for manipulation of Tim-3, an antibody against the receptor was used in an in vitro test of T cell function. Results showed that blocking Tim-3 significantly increased the proliferative capacity of HIV-specific CD8 and CD4 T cells. The researchers also investigated the extent to which PD-1 and Tim-3 were co-expressed by exhausted T cells, finding that while there was some limited overlap, the two receptors defined largely distinct populations.

In discussing their findings, Jones and colleagues highlight the profound extent of Tim-3 expression documented in their subjects and the implications for disease pathogenesis: “We show that in HIV-1 infection, the proportion of CD8+ and CD4+ T cells in peripheral blood that express Tim-3 can reach in excess of 70 and 30%, respectively (in contrast to means of 28.5 and 17.6% in HIV-1–uninfected individuals). As these frequencies exceed the proportion of HIV-1–specific cells in the periphery, suppression of T cell function by Tim-3 likely contributes not only to the loss-of-functional virus-specific responses but also to the impairment of responses to other antigens.” The authors also note that Tim-3 may be a potential therapeutic target, but stress that the same concerns that apply to anti-PD-1 approaches (the potential disinhibition of autoimmune responses) will also apply to anti-Tim-3 therapies, so careful evaluation in animal models will be required before any human studies can be considered.

Published online November 10, 2008

doi:10.1084/jem.20081398

The Journal of Experimental Medicine

ARTICLE

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

R. Brad Jones1, Lishomwa C. Ndhlovu5, Jason D. Barbour6, Prameet M. Sheth2, Aashish R. Jha5, Brian R. Long5, Jessica C. Wong1, Malathy Satkunarajah3, Marc Schweneker5, Joan M. Chapman5, Gabor Gyenes1, Bahareh Vali2, Martin D. Hyrcza2, Feng Yun Yue1, Colin Kovacs4, Aref Sassi8, Mona Loutfy7, Roberta Halpenny7, Desmond Persad8, Gerald Spotts6, Frederick M. Hecht6, Tae-Wook Chun9, Joseph M. McCune5, Rupert Kaul2, James M. Rini3, Douglas F. Nixon5, and Mario A. Ostrowski1,2,10

1 Department of Immunology, 2 Clinical Sciences Division, 3 Department of Biochemistry, and 4 Department of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada

5 Division of Experimental Medicine and 6 HIV/AIDS Division, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94110

7 Canadian Immunodeficiency Research Collaborative, 8 Maple Leaf Medical Clinic, Toronto, ON M5B 1L6, Canada

9 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

10 Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

Published online November 17, 2008

doi:10.1084/jem.20082429

The Journal of Experimental Medicine

COMMENTARY

TIMs: central regulators of immune responses

David A. Hafler and Vijay Kuchroo

D.A.H. and V.K. are at Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115

ABSTRACT

Exhaustion of T cell responses during chronic viral infections has been observed in both mouse and man and has been attributed to up-regulation of PD-1 on the surface of exhausted T cells. In patients with chronic human HIV infection, T cell exhaustion leads to opportunistic infections associated with AIDS. However, not all the exhausted T cells express PD-1, suggesting that other molecules may be involved in the phenotype. A new study now demonstrates a central role for T cell immunoglobulin and mucin domain–containing protein-3 (TIM-3) in T cell exhaustion during chronic HIV infection and suggests that TIM-3 may be a novel therapeutic target in chronic viral diseases.

AIDS Vaccine 2008, Cape Town, South Africa, October 13-16, 2008

Abstract #P15-23

Increased Tim-3 Expression on T Cells in Chronic HIV-1 Infection

MM Addo1, F Wen1, A Meier1, H Streeck1, G Alter1, M Altfeld1, DE Anderson2, DA Hafler2, and BD Walker1

1 Partners AIDS Research Center/MGH/Harvard Medical School, Charlestown, MA, USA; 2Center for Neurologic Diseases/ Brigham and Women’s Hospital/HMS, Boston, MA, USA

Background: Tim-3 is a transmembrane protein member of the TIM (T cell immunoglobulin and mucin domain) family, specifically expressed on differentiated Th1 and not Th2 lymphocytes. Interactions of Tim-3 and its ligand galectin-9 play a role in downregulating Th1 responses and modulating T cell tolerance. Recent data in HIV-1 infection suggests that surface expression of Tim-3 identifies a novel population of anergic T cells and that blockade of the Tim-3 pathway can restore proliferation and cytokine production in HIV-1 specific T cells. However, little is known about Tim-3 expression on monocytes and dendritic cells in HIV-1 infection. Methods: Tim-3 expression was studied on PBMC derived from HIV-1 infected individuals in different stages of HIV-1 infection, including persons with chronic untreated, chronic treated and acute infection, as well as HIV elite controllers and HIV negative controls. Tim-3 expression on T lymphocytes, monocytes, plasmacytoid (pDCs) and myeloid dendritic cells (mDCs) was quantified by multiparameter flowcytometry. Frequencies of Tim-3 expressing cells were correlated to markers of disease progression. Results: Tim-3 expression on CD4 and CD8 T lymphocytes was upregulated in chronic untreated HIV-1 infection. Expression was higher on CD8 compared to CD4 T cells. Interestingly, the Tim-3 expression on CD8 T cells was positively correlated with HIV-1 viral load. Tim-3 expression was also detected on mDCs and pDCs from HIV-1 infected individuals, while the highest expression of Tim-3 was observed on monocytes. However, no statistically significant differences were detected between HIV positive and negative individuals. Conclusion: Taken together these results indicate that Tim-3 is expressed on T cells and antigen presenting cells in HIV-1 infection and its expression on CD8 T cells correlates with markers of disease progression. In light of recent data suggesting that Tim-3 blockade enhances HIV-1 specific immune responses this molecule may hold promise as a potential target for future therapeutic interventions.

Cause for Caution on HIV Cure Report

An avalanche of media coverage has been loosed by the recently announced case of an individual who may have been “functionally cured” of HIV infection. The term functional cure has entered the lexicon due to the impossibility of formally proving that HIV has been entirely eradicated from the body; due to that limitation, long-term absence of detectable virus without therapy has been adopted as a reasonable definition of a cure, prefixed with the “functional” caveat.  The individual in this case is a 40 year old, HIV-infected American living in Berlin who had been on successful antiretroviral therapy prior to developing acute leukemia. The treatment for this condition involves bone marrow transplantation, which carries a 30% risk of mortality and is frequently associated with post-procedure complications. Due to the individual’s HIV infection, his doctors found a donor who was homozygous for the delta32 mutation, which completely abrogates expression of CCR5 (the major HIV co-receptor) on cells. Preparation for transplantation involves chemotherapy and radiation to essentially wipe out the immune system in order to prevent transplant rejection (with the salutary side effect of also depleting HIV-infected immune cells). The donor cells successfully engrafted but leukemia initially returned, requiring a second transplantation. Since that time – now close to two years ago – the individual has been free of leukemia, and HIV has remained undetectable without further antiretroviral treatment.

As can be gleaned from the press articles, opinions are divided on the significance of what has occurred. Some researchers have suggested it is a “proof of principle” that gene therapies with the capacity to block CCR5 expression could be curative. However, a 1999 review of bone marrow transplants in people with HIV (abstract below) identified two similar instances in which virus did not reappear after the procedure, so the contribution of the delta32 status of the donor in this current case is uncertain (although it is also possible that the prior examples also involved delta32 donors, unbeknownst to the doctors). The 1999 review also offers a grim perspective on the mortality associated with the procedure: the longest documented survival was around 300 days. While cynics might question whether AIDS professionals (including this writer) have their own self-serving reasons to express skepticism about cure claims, the complexity and danger of bone marrow transplantation clearly severely limits its use. It also must be stressed that while the individual is said to be “recovering,” there are no details available regarding his current health. 

Despite the many caveats, the case may be able to inform the pursuit of a safer curative strategies. The Foundation for AIDS Research (amfAR) has already sponsored a small meeting of experts to discuss the subject, attended by Mark Schoofs who wrote the first mainstream media article in the Wall Street Journal last week. One lesson may be that depleting HIV reservoirs to very low levels – if it can be done safely – will be beneficial, perhaps tipping the balance in favor of the host such that any residual virus can be controlled. If the individual remains well enough and is willing to undergo further evaluation, additional analyses to look for HIV in tissues will be important, along with evaluations of HIV-specific immunity. The presence of HIV-specific T cells carrying the donor delta32 mutation would suggest that sufficient viral activity has occurred after the transplantation procedure to induce new immune responses while, conversely, the absence of such responses might add to the evidence that HIV has been rendered completely inactive. Given that the case is now under the spotlight, the doctors involved will hopefully be forthcoming with updates as more information becomes available.

Med Hypotheses. 1999 Mar;52(3):247-57. Links

Could bone marrow transplantation cure AIDS?: review.

Huzicka I.

First Medical Faculty of Charles University, Prague, Czech Republic.

Despite recent use of potent antiretroviral drugs, the goal of eradication of human immunodeficiency virus (HIV) and restitution of the immune system has not been achieved. The present work reviews the literature on syngeneic or allogeneic bone marrow transplantation (BMT) in patients infected with HIV and in patients with acquired immune deficiency syndrome (AIDS). Thirty-two such attempts have been reported between 1982 and 1996. In two cases, HIV was eradicated and previously positive polymerase chain reaction (PCR) for viral DNA and RNA became negative. One patient had transient disappearance of HIV by PCR and several more, including one who underwent xenotransplantation of baboon bone marrow, experienced clinical and laboratory improvement. These results correlate with use of intensive pre-transplant cytoablative conditioning with chemotherapy and radiotherapy. Curative mechanisms of conditioning include elimination of several cell populations: infected cells, cells susceptible to infection, uninfected chronically activated cells responsible for autoimmune phenomena, and exhausted haemopoietic and lymphopoietic progenitors. They are repopulated by donor-derived cells that could mount a successful antiviral response through cytotoxic T lymphocytes. Non-specific graft-versus-host effect of allogeneic bone marrow would also help eliminate residual reservoirs of virus, especially macrophages. Protection of repopulating cells from infection could be achieved by a combination of antiretroviral agents and gene therapy strategies may be of additional benefit.

Merck HIV Vaccine Trial Results Published in The Lancet

The results of the Merck HIV vaccine efficacy trial (the STEP study), which have been covered extensively on the blog since they were announced in September of 2007, have now been published in the advance online section The Lancet. The papers offer a detailed and thoughtful analysis of the data, but the mystery of the apparent enhancement effect among uncircumcised gay men with pre-existing immunity to Ad5 remains unresolved and, although analyses continue, may never be resolved. As the authors note, the study outcome offers a sobering lesson on the need to better understand vaccine vector biology (a lesson that applies to vaccine research broadly, not just HIV). In terms of the immunological analyses, one of the most striking findings is that less than a third of vaccine recipients developed both CD4 and CD8 T cell responses to the encoded antigens; given the dependence of CD8 T cell responses on CD4 T cell help, these data suggest that the success of the vector in inducing CD8 T cell responses in the majority of recipients may have been somewhat pyrrhic.

The Lancet, Early Online Publication, 13 November 2008doi:10.1016/S0140-6736(08)61591-3

Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial

Susan P Buchbinder, Devan V Mehrotra, Ann Duerr, Daniel W Fitzgerald, Robin Mogg, David Li, Peter B Gilbert, Javier R Lama, Michael Marmor, Carlos del Rio, M Juliana McElrath, Danilo R Casimiro, Keith M Gottesdiener, Jeffrey A Chodakewitz, Lawrence Corey, Michael N Robertson, the Step Study Protocol Team

Background

Observational data and non-human primate challenge studies suggest that cell-mediated immune responses might provide control of HIV replication. The Step Study directly assessed the efficacy of a cell-mediated immunity vaccine to protect against HIV-1 infection or change in early plasma HIV-1 levels.

Methods

We undertook a double-blind, phase II, test-of-concept study at 34 sites in North America, the Caribbean, South America, and Australia. We randomly assigned 3000 HIV-1-seronegative participants by computer-generated assignments to receive three injections of MRKAd5 HIV-1 gag/pol/nef vaccine (n=1494) or placebo (n=1506). Randomisation was prestratified by sex, adenovirus type 5 (Ad5) antibody titre at baseline, and study site. Primary objective was a reduction in HIV-1 acquisition rates (tested every 6 months) or a decrease in HIV-1 viral-load setpoint (early plasma HIV-1 RNA measured 3 months after HIV-1 diagnosis). Analyses were per protocol and modified intention to treat. The study was stopped early because it unexpectedly met the prespecified futility boundaries at the first interim analysis. This study is registered with ClinicalTrials.gov, number NCT00095576.

Findings

In a prespecified interim analysis in participants with baseline Ad5 antibody titre 200 or less, 24 (3%) of 741 vaccine recipients became HIV-1 infected versus 21 (3%) of 762 placebo recipients (hazard ratio [HR] 1·2 [95% CI 0·6—2·2]). All but one infection occurred in men. The corresponding geometric mean plasma HIV-1 RNA was comparable in infected male vaccine and placebo recipients (4·61 vs 4·41 log10 copies per mL, one tailed p value for potential benefit 0·66). The vaccine elicited interferon-γ ELISPOT responses in 75% (267) of the 25% random sample of all vaccine recipients (including both low and high Ad5 antibody titres) on whose specimens this testing was done (n=354). In exploratory analyses of all study volunteers, irrespective of baseline Ad5 antibody titre, the HR of HIV-1 infection between vaccine and placebo recipients was higher in Ad5 seropositive men (HR 2·3 [95% CI 1·2—4·3]) and uncircumcised men (3·8 [1·5—9·3]), but was not increased in Ad5 seronegative (1·0 [0·5—1·9]) or circumcised (1·0 [0·6—1·7]) men.

Interpretation

This cell-mediated immunity vaccine did not prevent HIV-1 infection or reduce early viral level. Mechanisms for insufficient efficacy of the vaccine and the increased HIV-1 infection rates in subgroups of vaccine recipients are being explored.

The Lancet, Early Online Publication, 13 November 2008doi:10.1016/S0140-6736(08)61592-5

HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case—cohort analysis

M Juliana McElrath, Stephen C De Rosa, Zoe Moodie, Sheri Dubey, Lisa Kierstead, Holly Janes, Olivier D Defawe, Donald K Carter, John Hural, Rama Akondy, Susan P Buchbinder, Michael N Robertson, Devan V Mehrotra, Steven G Self, Lawrence Corey, John W Shiver, Danilo R Casimiro, the Step Study Protocol Team

Background

In the Step Study, the MRKAd5 HIV-1 gag/pol/nef vaccine did not reduce plasma viraemia after infection, and HIV-1 incidence was higher in vaccine-treated than in placebo-treated men with pre-existing adenovirus serotype 5 (Ad5) immunity. We assessed vaccine-induced immunity and its potential contributions to infection risk.

Methods

To assess immunogenicity, we characterised HIV-specific T cells ex vivo with validated interferon-γ ELISPOT and intracellular cytokine staining assays, using a case—cohort design. To establish effects of vaccine and pre-existing Ad5 immunity on infection risk, we undertook flow cytometric studies to measure Ad5-specific T cells and circulating activated (Ki-67+/BcL-2lo) CD4+ T cells expressing CCR5.

Findings

We detected interferon-γ-secreting HIV-specific T cells (range 163/106 to 686/106 peripheral blood mononuclear cells) ex vivo by ELISPOT in 77% (258/354) of people receiving vaccine; 218 of 354 (62%) recognised two to three HIV proteins. We identified HIV-specific CD4+ T cells by intracellular cytokine staining in 58 of 142 (41%) people. In those with reactive CD4+ T cells, the median percentage of CD4+ T cells expressing interleukin 2 was 88%, and the median co-expression of interferon γ or tumor necrosis factor α (TNFα), or both, was 72%. We noted HIV-specific CD8+ T cells (range 0·4—1·0%) in 117 of 160 (73%) participants, expressing predominantly either interferon γ alone or with TNFα. Vaccine-induced HIV-specific immunity, including response rate, magnitude, and cytokine profile, did not differ between vaccinated male cases (before infection) and non-cases. Ad5-specific T cells were lower in cases than in non-cases in several subgroup analyses. The percentage of circulating Ki-67+BcL-2lo/CCR5+CD4+ T cells did not differ between cases and non-cases.

Interpretation

Consistent with previous trials, the MRKAd5 HIV-1 gag/pol/nef vaccine was highly immunogenic for inducing HIV-specific CD8+ T cells. Our findings suggest that future candidate vaccines have to elicit responses that either exceed in magnitude or differ in breadth or function from those recorded in this trial.

Comment

The Lancet, Early Online Publication, 13 November 2008doi:10.1016/S0140-6736(08)61593-7

Failure of the Merck HIV vaccine: an uncertain step forward

Merlin L Robb

An HIV vaccine remains the primary goal for a comprehensive strategy to curb the global HIV epidemic. Yet the path to success is unknown and, on the basis of two reports from the Step Study in today's Lancet,1,2 seems to have become more complicated.

CHAVI’s Genetic Associations with Viral Load Control Reevaluated

Earlier this year the blog covered studies conducted by the Center for HIV/AIDS Vaccine Immunology (CHAVI), which identified a suite of genetic associations with control of HIV replication. Researchers from Sunil Ahuja’s group have now evaluated these associations in a different natural history cohort of HIV-infected individuals and found that the originally reported findings may need to be re-interpreted. Specifically, an association with a genetic polymorphism found in the ZNRD1 gene may be explained, at least in part, by an association with HLA A*10 (an immune response gene which has previously been associated with slowed disease progression). Also, as in the original CHAVI paper, this association was largely restricted to people of European descent and was not significant among African Americans.

Moving on to the other reported associations, the researchers found that the effect of a polymorphism in the HCP5 gene was entirely explained by its linkage with HLA B*57, an immune response gene that is strongly associated with long-term non-progression. When the effects of HCP5 and HLA B*57 were disentangled, the HCP5 polymorphism was associated with faster - not slower - disease progression. The third association identified by CHAVI was with a polymorphism in the HLA-C gene. Ahuja and colleagues also found evidence for this association, but discovered that the strength of the effect also relates to a linkage with HLA B*57. In the absence of HLA B*57, the HLA-C association with lower viral load was several fold less than initially reported. By evaluating HLA B*57 and HCP5 and HLA-C variants together, the researchers were able to identify combinations that ameliorate the protective effects of HLA B*57, which they suggest may explain why some people possessing this HLA allele do not experience slowed disease progression. They also show that the protective effects of HLA B*57 are not entirely mediated by a reduction in viral load, suggesting an impact on other parameters that influence disease progression, such as immune activation.

This type of study highlights the complexity and challenges of evaluating genetic associations with disease outcomes. Given this complexity, it’s possible that future research in additional cohorts will further alter the picture that has emerged regarding these particular genetic polymorphisms. Ahuja and colleagues also note that ethnicity appears to impact the associations independent of the altered frequency of the polymorphisms among different populations, suggesting that the evolutionary history of an ethnic group can alter the impact of gene variants in ways that have yet to be fully understood.

PLoS ONE 3(11): e3636 doi:10.1371/journal.pone.0003636

HIV-1 Disease-Influencing Effects Associated with ZNRD1, HCP5 and HLA-C Alleles Are Attributable Mainly to Either HLA-A10 or HLA-B*57 Alleles

Gabriel Catano1,2#, Hemant Kulkarni1,2#, Weijing He1,2#, Vincent C. Marconi3,4, Brian K. Agan3,4,5, Michael Landrum3,4,5, Stephanie Anderson3,5, Judith Delmar3,4, Vanessa Telles1,2, Li Song1,2, John Castiblanco1,2, Robert A. Clark1,2, Matthew J. Dolan3,4,5*, Sunil K. Ahuja1,2,6,7*

1 Veterans Administration Research Center for AIDS and HIV-1 Infection, South Texas Veterans Health Care System, San Antonio, Texas, United States of America, 2 Department of Medicine, University of Texas Health Science Center, San Antonio, Texas, United States of America, 3 Infectious Disease Clinical Research Program (IDCRP), Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America, 4 Infectious Disease Service, San Antonio Military Medical Center (SAMMC), Ft. Sam Houston, Texas, United States of America, 5 Henry M. Jackson Foundation, Wilford Hall United States Air Force Medical Center, Lackland AFB, Texas, United States of America, 6 Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, Texas, United States of America, 7 Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, United States of America

Abstract

A recent genome-wide association study (GWAS) suggested that polymorphisms in or around the genes HCP5, HLA-C and ZNRD1 confer restriction against HIV-1 viral replication or disease progression. Here, we also find that these alleles are associated with different aspects of HIV disease, albeit mainly in European Americans. Additionally, we offer that because the GWAS cohort was a subset of HIV-positive individuals, selected based in part on having a low viral load, the observed associations for viral load are magnified compared with those we detect in a large well-characterized prospective natural history cohort of HIV-1-infected persons. We also find that because of linkage disequilibrium (LD) patterns, the dominant viral load- and disease-influencing associations for the ZNRD1 or HLA-C and HCP5 alleles are apparent mainly when these alleles are present in HLA-A10- or HLA-B*57-containing haplotypes, respectively. ZNRD1 alleles lacking HLA-A10 did not confer disease protection whereas ZNRD1-A10 haplotypes did. When examined in isolation, the HCP5-G allele associates with a slow disease course and lower viral loads. However, in multivariate models, after partitioning out the protective effects of B*57, the HCP5-G allele associates with disease-acceleration and enhanced viral replication; these associations for HCP5-G are otherwise obscured because of the very strong LD between this allele and a subset of protective B*57 alleles. Furthermore, HCP5 and HLA-C alleles stratify B*57-containing genotypes into those that associate with either striking disease retardation or progressive disease, providing one explanation for the long-standing conundrum of why some HLA-B*57-carrying individuals are long-term non-progressors, whereas others exhibit progressive disease. Collectively, these data generally underscore the strong dependence of genotype-phenotype relationships upon cohort design, phenotype selection, LD patterns and populations studied. They specifically demonstrate that the influence of ZNRD1 alleles on disease progression rates are attributable to HLA-A10, help clarify the relationship between the HCP5, HLA-C and HLA-B*57 alleles, and reaffirm a critical role of HLA-B*57 alleles in HIV disease. Furthermore, as the protective B*57-containing genotypes convey striking salutary effects independent of their strong impact on viral control, it is conceivable that T cell-based therapeutic vaccine strategies aimed at reducing viral loads may be inadequate for limiting AIDS progression, raising the potential need for complementary strategies that target viral load-independent determinants of pathogenesis.

Mechanism for Merck Vaccine Enhancement Proposed

A paper published online by the Journal of Experimental Medicine (JEM) on Monday describes a mechanism by which the Merck HIV vaccine may have enhanced the risk of HIV infection among some participants in the recent STEP trial. The Merck vaccine used a crippled form of a cold virus called adenovirus serotype 5 (Ad5) as a carrier (called a vector) for the HIV components it aimed to induce immune responses against, and the STEP results showed that people with pre-existing antibody responses against Ad5 (because of a natural exposure in the past) faced an elevated risk of acquiring HIV infection if they received the vaccine. In contrast, vaccine recipients in STEP with low or absent antibody responses against Ad5 were unaffected. 

The new research paper, from Guiseppe Pantaleo’s laboratory at the University of Lausanne, indicates that the presence of anti-Ad5 antibodies causes the Ad5 vaccine to trigger the development of "immune complexes" consisting of the vaccine, anti-Ad5 antibodies and dendritic cells. The researchers show that the type of immune response induced by the vaccine vector in the presence of anti-Ad5 antibodies is somewhat different due to the formation of these immune complexes. In particular, the type of cytokines produced differ (in the presence of the complexes, there is a more inflammatory profile) and the CD8 T cell responses triggered are less polyfunctional than those induced when anti-Ad5 antibodies are not present. Although the paper only looks at Ad5-specific CD8 T cell responses, a study presented at the recent HIV vaccine conference (abstract appended below) offers evidence that the HIV-specific CD8 T cell responses induced by the vaccine may have been similarly affected. If confirmed, these findings might provide an explanation as to why a statistically significant inverse correlation was seen between the magnitude of the vaccine-induced HIV Gag-specific CD8 T cell response and post-infection viral load setpoint, but only among study participants who lacked anti-Ad5 antibodies (i.e. the HIV-specific CD8 T cell responses among Ad5-negative individuals may have been more functional).

The JEM paper also reports that in vitro HIV replication is enhanced roughly threefold in the presence of the immune complexes. However, the only virus used in this experiment is the LAV isolate which enters cells via the CXCR4 co-receptor. Since almost all HIV transmission involves isolates that use the CCR5 co-receptor, it is somewhat mystifying that more HIV variants were not tested (particularly given the publicity that this paper was likely to generate). Additional research will be needed to ascertain if this enhancement of HIV replication holds true for other isolates. Another potential outstanding issue is that immunization with Ad5 vectors induces Ad5-specific antibodies, so once the Ad5-negative negative participants in STEP received the first of their three immunizations they would also have had circulating antibodies, yet their risk of acquiring HIV infection did not appear to be raised. Perhaps it is possible that antibodies induced by natural exposure to Ad5 are different from those induced by Ad5 vectors, but this needs to be specifically evaluated.

Published online November 3, 2008

doi:10.1084/jem.20081786

The Journal of Experimental Medicine

BRIEF DEFINITIVE REPORT

Activation of a dendritic cell–T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells

Matthieu Perreau1, Giuseppe Pantaleo1,2, and Eric J. Kremer3,4

1 Service of Immunology and Allergy, Department of Medicine, Centre Hospitalier, Universitaire Vaudois, Lausanne, University of Lausanne, CH-1011 Lausanne, Switzerland

2 Swiss Vaccine Research Institute, 1011 Lausanne, Switzerland

3 Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique 5535, 34293 Montpellier, France

4 Université Montpellier I and II, 34967 Montpellier, France

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcreceptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC–T cell cocultures. The present results indicate that Ad5 IC activates a DC–T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.

AIDS Vaccine 2008, Cape Town, South Africa, October 13-16, 2008

Abstract# OA05-04

Effects of Pre-existing Vector Immunity on the Quality of Vaccine-induced T-Cell Responses in an Ad5-HIV-1 Vaccine Trial

SO Pine1, J Kublin2, D Casimiro3, S Hammer4, S De Rosa5, and MJ McElrath6 1 University of Washington and Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2Fred Hutchinson Cancer Research

Center and the HVTN, USA; 3HIV Vaccine Trials Network and Merck Research Laboratories, West Point, PA, USA; 4HIV Vaccine Trials Network and Columbia University, New York, NY, USA; 5Vaccine and Infectious Disease Institute, FHCRC, Seattle, WA, USA; 6Department of Medicine, University of Washington, HVTN and FHCRC, Seattle, WA, USA

Background: The challenges facing HIV-1 vaccine development were recently underscored by the lack of efficacy demonstrated in the STEP Trial with testing of a replication-deficient adenovirus serotype 5 (Ad5) construct. Although the vaccine effectively stimulated HIV-specific T-cell responses, natural immunity to the vector itself appeared to have an impact on the study outcome. Little is known about how pre-existing vector immunity affects the quality of T-cell response to a vaccine. To address this, we examined peak responses in a previous phase I trial (HVTN050/ Merck V520 Protocol 018) that tested a MRKAd5 HIV-1 Gag vaccine in persons with and without previous Ad5 immunity. Methods: Pre-existing Ad5 immunity was quantified by baseline neutralizing antibody (nAb) titers, and vaccinees were stratified as either Ad5 naive (n = 30, nAb 􏰄 18) or Ad5-immune (n = 30, nAb > 200). An ex vivo T-cell assay to measure 30 cytokines simultaneously was combined with intracellular cytokine staining to allow a thorough evaluation of the quality of antigen-specific T-cell responses in vaccinees. Results: Preliminary analyses showed significantly higher amounts of IL-15, IL-2, IP-10 and TNF-a (p-values = 0.02), but not IFN-g, were secreted in response to Gag peptides in the Ad5-naive compared to the Ad5-immune group. The Ad5-naive group also had higher levels of poly-functional Gag-specific CD8 T-cells secreting IFN-g, IL-2 and TNF-a (0.32% vs. 0.04% CD8 T-cells, p = 0.002). Conclusion: Higher levels of T-cells secreting IL-2 and IL-15 in the Ad5-naive vaccinees suggests that these T-cells may have a higher proliferative capacity. Moreover, elevation of IP-10 and TNF-a without a concomitant increase in IFN-g may indicate that antiviral pathways are qualitatively different in the absence of preexisting vector immunity. We conclude that the quality of a T-cell response to the Ad5/HIV vaccine is altered with pre-existing vector immunity, although the net effect of these responses in HIV protection remains to be determined.