Michael Palm Basic Science, Vaccines and Prevention Weblog

During the plague of Athens in 430 b.c., the Greek historian Thucydides made the first known reference to immunological memory when he observed that the “same man was never attacked twice.” More than two thousand years later, technological advances have allowed scientists to begin unraveling the molecular underpinnings of the phenomenon. It is now appreciated that the main components of the adaptive immune system--B cells, CD4 T cells and CD8 T cells--respond to infections by developing specialized abilities that can often mediate protection against re-infection or--in the case of pathogens that are controlled rather than eliminated from the body--disease. The long-lived B cells, CD4 T cells and CD8 T cells that perform these vital functions are known as “memory” cells.

The laboratory of Rafi Ahmed at Emory University has long been at the forefront of research into immunological memory, particularly as it pertains to CD8 T cells. In a new free-access paper in the journal International Immunology, Ahmed and colleagues review how changes in gene expression contribute to the enhanced infection-fighting functions of memory CD8 T cells, and outline how these functions are inherited by the progeny of memory cells via the wonders of epigenetics. The review concludes by suggesting that it may eventually become possible to correct memory cell dysfunction by targeting gene regulation mechanisms.

International Immunology Advance Access published online on August 23, 2010

International Immunology, doi:10.1093/intimm/dxq437

review-article

Making memories that last a lifetime: heritable functions of self-renewing memory CD8 T cells

Ben Youngblood1,2, Carl W. Davis1,2 and Rafi Ahmed1,2

1 Emory Vaccine Center, Atlanta, GA 30329, USA

2 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA

Clonal expansion of virus-specific naive T cells during an acute viral infection results in the formation of memory CD8 T cells that provide the host with long-term protective immunity against the pathogen. Memory CD8 T cells display enhanced effector functions compared with their naive precursors, allowing them to respond more rapidly and effectively to antigen re-encounter. The enhanced functions of memory CD8 T cells are mediated by heritable changes in gene regulation. Expression of select transcription factors along with locus-specific epigenetic modifications are coupled to and are essential in the formation of memory-specific gene expression patterns. Here, we will review the changes in gene expression that accompany development of memory CD8 T cells and discuss chromatin modifications as a potential means for heritable propagation of these changes during homeostatic cell division of self-renewing memory CD8 T cells. Also, we will discuss therapies that manipulate heritable gene regulation as a potential mechanism to restore function to non-functional memory CD8 T cells to combat chronic viral infection.

The July issue of the journal Cytometry Part A contains an excellent article by Pratip Chattopadhyay and Mario Roederer reviewing the use of flow cytometry to study T cell subsets in HIV infection. The entire journal issue is free access.

Cytometry Part A

Special Issue: CYTOMETRY - 30th Anniversary 1980 – 2010

Volume 77A, Issue 7, pages 614–622, July 2010

DOI: 10.1002/cyto.a.20905

Review Article

Good cell, bad cell: Flow cytometry reveals T-cell subsets important in HIV disease

Pratip K. Chattopadhyay, Mario Roederer

Abstract

Flow cytometry is a key technology in the study of HIV disease. In this article, we review various cellular markers that can be measured in the setting of pathogenesis or vaccination studies, including markers of activation, differentiation, senescence, immune suppression, and function. In addition, we discuss important considerations for making these measurements. Finally, we examine how flow cytometry studies have taught researchers about the disease process, and the potential for flow cytometry technology to guide treatment decisions and evaluate vaccine candidates in the future.

Until recently, the idea of genetically modifying a person’s immune system to make it resistant to HIV was generally viewed as extremely appealing, but so dauntingly impractical that it belonged in the realm of science fiction. Over the past year or so, an accumulation of new data has offered hope that it may eventually be possible to translate the idea into science fact.

Central to these developments is the widely reported case of an HIV-positive individual in Berlin who remains off antiretroviral therapy and free of detectable HIV after receiving a bone marrow transplant from an individual lacking the CCR5 co-receptor due to the delta32 genetic mutation. The bone marrow transplant was received (twice) during a difficult and complicated course of treatment for acute myelogenous leukemia, so the case is not seen as something that can easily be replicated in other people, but rather as a “proof-of-concept” that modifying the immune system can be a means to extinguishing HIV infection.

Complementing this remarkable case report, several studies have described improvements in techniques that may facilitate genetic modification of the immune system. A company called Sangamo Biosciences has developed a technology that allows inactivation of specific genes using enzymes called zinc finger nucleases (ZFNs); the approach is described in detail in the current issue of Nature Reviews Genetics and was also the subject of a New York Times article by Nicholas Wade at the end of last year. An ongoing human trial is using Sangamo’s ZFNs to delete the CCR5 gene in CD4 T cells sampled from HIV positive individuals; the CCR5-negative CD4 T cells are then expanded in number and re-infused into the donor (a very preliminary report from this trial was covered in TAGline earlier this year).

In the current issue of Nature Biotechnology, a research team led by Nathalia Holt describes using the Sangamo technique to successfully modify hematopoietic stem/progenitor cells (HSPC) in mice. The advantage of using HSPCs is that they are the “mother of all cells” and can potentially provide a permanent source of modified immune cells, circumventing the need for altering CD4 T cells in the lab and re-infusing them. Holt’s team was able to show that, in mice, the CCR5-negative HSPCs generated immune cells of multiple types, all lacking CCR5. In “humanized” mice challenged with HIV, these CCR5-negative cells expanded in number and reduced viral load compared to untreated mice with normal CCR5 expression. In an accompanying editorial, Steve Deeks and Mike McCune from UCSF highlight the potential importance of Holt’s findings and outline the implications for future research in humans.

The other recent study that is part of this evolving story was published back in June in the journal Science Translational Medicine. The research group of John Zaia at City of Hope in Duarte, California described results of an experiment in which HSPCs from four individuals with HIV and AIDS-related lymphoma were modified with three anti-HIV genes and re-infused. The modified HSPCs were given along with the infusions of unmodified HSPCs that are a standard part of the protocol for lymphoma treatment. Although it was a small exploratory study, the researchers were encouraged to find evidence that the modified HSPCs had given rise to cells of multiple lineages (e.g. T cells, B cells, macrophages) carrying the anti-HIV genes, albeit at very low numbers.

Yesterday the LA Times published an excellent story by Rachel Bernstein that ties these various gene therapy developments together. It turns out that John Zaia’s group at City of Hope will be the first to use Sangamo’s CCR5-deletion approach to modify human HSPCs, likely also in people with AIDS-related lymphoma initially. The ultimate hope – “reaching for blue sky,” as Deeks & McCune describe it – is that a single shot of souped-up HSPCs may one day be able to equip the immune system with enough HIV-resistant cells to vanquish the virus.  

Nature Biotechnology 28, 807–810 (1 August 2010) | doi:10.1038/nbt0810-807

News and Views

Can HIV be cured with stem cell therapy?

Steven G Deeks & Joseph M McCune

Abstract

Antiretroviral therapy has transformed the treatment of HIV infection, but, despite its profound successes, it will not halt the relentless advance of the epidemic. Against this sobering reality, several promising, recent developments in the basic-science arena have led HIV researchers to envision new therapeutic approaches that would completely eradicate the virus, effectively 'curing' HIV disease.

Nat Biotechnol. 2010 Aug;28(8):839-47. Epub 2010 Jul 2.

Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo.

Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC, Cannon PM.

Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

Abstract

CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34(+) hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rgamma(null) mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4(+) T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5(-/-) cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5(-/-) HSPCs can populate an infected animal with HIV-1-resistant, CCR5(-/-) progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.

Sci Transl Med. 2010 Jun 16;2(36):36ps30.

Gene therapy takes a cue from HAART: combinatorial antiviral therapeutics reach the clinic.

Shah PS, Schaffer DV.

Department of Chemical Engineering, University of California, Berkeley, CA 94720-3220, USA.

Abstract

For the first time, scientists have tested a combination of three RNA-based gene therapies, delivered via a lentiviral vector, to target HIV in patients. This study not only demonstrates the safety and long-term viability of this approach, but also highlights areas in which focused improvements in gene therapy strategies may provide the most impact in increasingly translating promise in the laboratory to efficacy in the clinic.

Sci Transl Med. 2010 Jun 16;2(36):36ra43.

RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.

DiGiusto DL, Krishnan A, Li L, Li H, Li S, Rao A, Mi S, Yam P, Stinson S, Kalos M, Alvarnas J, Lacey SF, Yee JK, Li M, Couture L, Hsu D, Forman SJ, Rossi JJ, Zaia JA.

Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA.

Abstract

AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell-based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood-derived (CD34(+)) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to approximately 1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.

Nature Reviews Genetics 11, 636-646 (September 2010) | doi:10.1038/nrg2842

Genome editing with engineered zinc finger nucleases

Fyodor D. Urnov1, Edward J. Rebar1, Michael C. Holmes1, H. Steve Zhang1 & Philip D. Gregory1  About the authors

Abstract

Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells — this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

On July 16 & 17, immediately prior to the XVIII International AIDS Conference, the International AIDS Society held a workshop entitled "'Towards a Cure': HIV Reservoirs and Strategies to Control Them" that focused on the critically important topic of moving beyond antiretroviral therapy and addressing HIV persistence. Powerpoint presentations and abstracts have now been posted to the workshop website along with rapporteur summaries of each session.

Day 1 – 16 July 2010

Day 2 – 17 July 2010

Poster Exhibition

The XVIII International AIDS Conference took place last week in Vienna and many of the presentations are now available online. However, the conference website can be somewhat challenging to navigate and the location of slides and abstracts may not always be readily apparent. The first step is to download the Microsoft Silverlight plug-in that is required to use the programme-at-a-glance, where links to abstracts and slide presentations are located (a download button will automatically appear on this page if you do not have the plug-in). Once Silverlight is installed, all the sessions from each day of the conference can be browsed. Clicking on a session title opens up a slightly larger window with a “more info” link at the bottom right. Clicking on the “more info” link then brings you to the page containing the full details of that session, including all the presentation titles and abstracts. In many cases there is a “slides and audio” link to the presentation in Flash plug-in format. Although some presentation titles also have “Powerpoint” immediately to the left, do not get discouraged if nothing happens when you click this link; if you scroll further down the page, there is another section entitled “Powerpoints presentations” and clicking the links in this section will download the powerpoint slides.

As a short-cut, I’ve included direct links here to sessions relevant to basic science and biomedical prevention.

MOPDB1: Issues in HIV Therapy and Resistance, Oral Poster Discussion, Monday July 19, 1-2pm

MOSY05: Innate Immunity - Key to Control?, Symposium, Monday July 19, 2:30-4pm

MOAA01: Novel Therapeutic Strategies, Oral Abstract Session, Monday July 19, 4:30-6pm

TUSY02: Pathogenesis - The Battle of the Immune System, Symposium, Tuesday July 20, 11am-12:30pm

TUSS05: Safety and effectiveness of 1% Tenofovir Vaginal Microbicide Gel in South African Women: Results of the CAPRISA 004 Trial, Special Session, Tuesday July 20, 1-2pm

TUPDA1: Role of Dendritic Cells in HIV Infection, Oral Poster Discussion, Tuesday July 20, 1-2pm

TUSY06: HIV Vaccines - Quo Vadis?, Symposium, Tuesday July 20, 2:30-4pm

TUAA01: Advances in Vaccine Development, Oral Abstract Session, Tuesday July 20, 4:30-6pm

WEAA01: Correlates of Immune Protection, Oral Abstract Session, Wednesday July 21, 11am-12:30pm

WEPDA1: Viral Persistence and Latency, Oral Poster Discussion, Wednesday July 21, 1-2pm

WESY07: Use of Antiretrovirals for Prevention: PrEP, PEP and ART, Symposium, Wednesday July 21, 2:30-4pm

WEAA02: T-Cell Responses in HIV Infection, Oral Abstract Session, Wednesday July 21, 2:30-4pm

WEAA03: Immune Activation and Inflammation, Oral Abstract Session, Wednesday July 21, 4:30-6pm

THLBA1: Late Breaker Track A, Oral Abstract Session, Thursday July 22, 11am-12:30pm

THPDA1: Immune Dysfunctions, Oral Poster Discussion, Thursday July 22, 1-2pm

THAA01: Persistence of HIV Reservoirs, Oral Abstract Session, Thursday July 22, 2:30-4pm

THBS03: Understanding HIV Transmission Mechanisms: Microbicides and PrEP, Thursday July 22, 4:30-6pm

FRAX01: It All Starts Here: Acute HIV Infection, Friday July 23, 11am-12:30pm

FRAA01: Mechanisms of Transmission, Friday July 23, 11am-12:30pm

FRPL02: Rapporteur Session, Friday July 23, 1-3:15pm

Adenoviruses are common in nature and are a major cause of severe colds. During the past decade, attenuated forms of adenovirus have become increasingly utilized as experimental vaccine vectors due to their targeting of dendritic cells and ability to induce CD8 T cell responses in the majority of recipients (no other vector to date has shown comparable CD8 T cell immunogenicity). Adenoviruses are categorized into a number of different serotypes, with serotype 5 (Ad5) being among the most prevalent. An HIV vaccine candidate that used Ad5 as a vector was developed by Merck, but failed to show efficacy and significantly enhanced HIV acquisition risk in a subset of trial participants with pre-existing antibody responses against natural Ad5.

Since the Merck data were published, there has been debate about the mechanism by which the Ad5 vector could have increased the risk of HIV acquisition, and even claims that the result was a statistical fluke or represented a confounding effect related to circumcision status (the former claim is possible, the latter was contradicted by a multivariate analysis showing that both lack of circumcision and receipt of the Ad5 vaccine were associated with an increased likelihood of HIV acquisition). Perhaps most controversially, it has been suggested that perhaps the Merck vector boosted Ad5-specific CD4 T cell responses, thus providing more activated CD4 T cell targets for HIV infection. Two studies that measured Ad5-specific CD4 T cell responses in recipients of the Merck vaccine based on cytokine production have argued against this possibility (see here and here), while another that assessed Ad5-specific CD4 T cell responses based on their ability to proliferate suggested that CD4 T cell-mediated enhancement could have played a role.

Lurking on the sidelines of this contentious debate has been the issue of natural adenovirus infection, about which relatively little is known. Now a study by Larry Corey’s research group, published recently in PLoS One, has for the first time evaluated natural adenovirus infections among one of the populations enrolled in the Merck HIV vaccine trial, Peruvian men who have sex with men (MSM).

The results are perhaps surprising: the majority of the 20 men studied had detectable adenovirus DNA in rectal swabs during follow-up; the average rate of detection at any given timepoint was in 30% of samples. A variety of adenovirus serotypes were represented, including Ad5, Ad26 and Ad48, all of which are in development as vaccine vectors. The study authors note that: “shedding typically occurred on consecutive days in clustered episodes lasting a median of 4 days (range 1 to 9 days) separated by periods without shedding, suggesting frequent new infections or reactivation of latent infections over time.”

The study raises a number of issues. First and foremost, it suggests that when a pathogen is being developed as a potential vaccine vector, a thorough understanding of the natural infection should be obtained early on in the research process, and not after the fact.  In the case of adenovirus vectors, their success in solving the difficult problem of inducing CD8 T cell responses may have caused the lack of information on natural infections to be somewhat overlooked. In terms of specific relevance to the Merck trial, the study implies that interactions between adenovirus-specific immunity and natural adenovirus infections are likely to be far more dynamic and ongoing than had previously been surmised. The boosting of Ad5-specific CD4 T cell responses by vaccination might therefore have different consequences depending on whether natural adenovirus infection is present, particularly given that the virus localizes in the mucosa and Ad5-specific CD4 T cell responses cross-react with a wide variety of serotypes. Additional studies will now be needed to investigate the impact of adenovirus-based vaccines on mucosal virus-specific CD4 T cell responses in the presence and absence of natural infection.

PLoS One. 2010 Jun 25;5(6):e11321.

Frequent detection of human adenovirus from the lower gastrointestinal tract in men who have sex with men.

Curlin ME, Huang ML, Lu X, Celum CL, Sanchez J, Selke S, Baeten JM, Zuckerman RA, Erdman DD, Corey L.

Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

Abstract

BACKGROUND: The association between baseline seropositivity to human adenovirus (HAdV) type 5 and increased HIV acquisition in the Step HIV Vaccine Study has raised questions concerning frequency of acquired and/or persistent Adenovirus infections among adults at high risk of HIV-1 infection. METHODOLOGY: To evaluate the frequency and pattern of HAdV shedding from the lower GI tract, we retrospectively tested rectal swabs for HAdVs in a cohort of 20 HSV-2 positive HIV-positive Peruvian men who have sex with men (MSM) undergoing rectal swabbing three times/week for 18 consecutive weeks, in a prospective study of HSV-2 suppression in HIV infection. Viral DNA was extracted and amplified using a sensitive multiplex PCR assay that detects all currently recognized HAdV types. Molecular typing of viruses was performed on selected samples by hexon gene sequencing. Baseline neutralizing antibody titers to HAdVs -5, -26, -35 and -48 were also assessed. PRINCIPAL FINDINGS: 15/20 individuals had HAdV detected during follow up. The median frequency of HAdV detection was 30% of samples (range 2.0% to 64.7%). HAdV shedding typically occurred on consecutive days in clustered episodes lasting a median of 4 days (range 1 to 9 days) separated by periods without shedding, suggesting frequent new infections or reactivation of latent infections over time. 8 of the 15 shedders had more than one type detected in follow-up. 20 HAdV types from species B, C, and D were identified, including HAdV-5, -26 and -48, HAdV types under development as potential vaccine candidates. 14/20 subjects were seropositive for HAdV-5; 15/20 for HAdV-26; 3/20 for HAdV-35; and 2/20 for HAdV-48. HAdV shedding did not correlate with CD4 count, plasma HIV-1 viral load, or titers to HAdV-5 or HAdV-35. The sole individual with HAdV-5 shedding was HAdV-5 seropositive. CONCLUSIONS: HAdV shedding was highly prevalent and diverse, including types presently under consideration as HIV vaccine vectors. Subclinical HAdV infection of the GI tract is common among MSM in Peru; the prevalence of HAdV in the enteric tract should be evaluated in other populations. The association between ongoing recent enteric HAdV and the immune response to recombinant HAdV vaccines should be evaluated.

The Kaiser Family Foundation has now posted a webcast of the CAPRISA 004 presentations that took place today at the XVIII International AIDS Conference in Vienna.