Archive for the ‘HIV’ Category
Scientists at Burnham Institute for Medical Research (Burnham) have discovered that specific microRNAs (non-coding RNAs that interfere with gene expression) reduce HIV replication and infectivity in human T-cells. In particular, miR29 plays a key role in controlling the HIV life cycle. The study suggests that HIV may have co-opted this cellular defense mechanism to help the virus hide from the immune system and antiviral drugs.
Tariq Rana, Ph.D., director of the Program for RNA Biology at Burnham, and colleagues, found that the microRNA miR29 suppresses translation of the HIV-1 genome by transporting the HIV mRNA to processing-bodies (P-bodies), where they are stored or destroyed. This results in a reduction of viral replication and infectivity. The study also showed that inhibition of miR29 enhances viral replication and infectivity. The scientists further demonstrated that strains of HIV-1 with mutations in the region of the genome that interact with miR29 are not inhibited by miR29.
“We think the virus may use this mechanism to modulate its own lifecycle, and we may be able to use this to our advantage in developing new drugs for HIV,” said Dr. Rana. “Retroviral therapies greatly reduce viral load but cannot entirely eliminate it. This interaction between HIV and miR29 may contribute to that inability. Perhaps, by targeting miR29, we can force HIV into a more active state and improve our ability to eliminate it.”
Rana’s team looked at miR29 expression levels in infected and uninfected cells and found that miR29 expression was enhanced by HIV-1 infection. Blocking the activity of miR29 with interfering RNA resulted in increased replication and infectivity of the virus. The scientists tested the association of miR29 and HIV-1 by mutating both miR29 and its target region on the HIV virus. When either was altered, miR29s suppression of HIV replication and infectivity was reduced or eliminated.
In addition, the team suppressed P-bodies in the cells and noted a similar effect. This suggests that HIV may use miRNAs to become dormant and escape immune response.
Molecular Cell, Volume 34, Issue 6, 686-695, 26 June 2009
Cellular MicroRNA and P Bodies Modulate Host-HIV-1 Interactions p696
Robin Nathans 1,Chia-ying Chu 1,2, Anna Kristina Serquina 1,Chih-Chung Lu 1,2, Hong Cao 1 and Tariq M. Rana 1,2, Corresponding Authors,
1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA
2 Program for RNA Biology, Sanford Children’s Health Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
MicroRNAs (miRNAs), 22 nt noncoding RNAs, assemble into RNA-induced silencing complexes (RISCs) and localize to cytoplasmic substructures called P bodies. Dictated by base-pair complementarity between miRNA and a target mRNA, miRNAs specifically repress posttranscriptional expression of several mRNAs. Here we report that HIV-1 mRNA interacts with RISC proteins and that disrupting P body structures enhances viral production and infectivity. In HIV-1-infected human T lymphocytes, we identified a highly abundant miRNA, miR-29a, which specifically targets the HIV-1 3UTR region. Inhibiting miR-29a enhanced HIV-1 viral production and infectivity, whereas expressing a miR-29 mimic suppressed viral replication. We also found that specific miR-29a-HIV-1 mRNA interactions enhance viral mRNA association with RISC and P bodyproteins. Thus we provide an example of a single host miRNA regulating HIV-1 production and infectivity. These studies highlight the significance of miRNAs and P bodies in modulating host cell interactionswith HIV-1 and possibly other viruses.
A research team may have broken the stubborn impasse that has frustrated the invention of an effective HIV vaccine, by using an approach that bypasses the usual path followed by vaccine developers. By using gene transfer technology that produces molecules that block infection, the scientists protected monkeys from infection by a virus closely related to HIV—the simian immunodeficiency virus, or SIV—that causes AIDS in rhesus monkeys.
“We used a leapfrog strategy, bypassing the natural immune system response that was the target of all previous HIV and SIV vaccine candidates,” said study leader Philip R. Johnson, M.D., chief scientific officer at The Children’s Hospital of Philadelphia. Johnson developed the novel approach over a ten-year period, collaborating with K. Reed Clark, Ph.D., a molecular virologist at Nationwide Children’s Hospital in Columbus, Ohio.
Johnson cautioned that many hurdles remain before the technique used in this animal study might be translated into an HIV vaccine for humans. If the technique leads to an effective HIV vaccine, such a vaccine may be years away from realization.
Most attempts at developing an HIV vaccine have used substances aimed at stimulating the body’s immune system to produce antibodies or killer cells that would eliminate the virus before or after it infected cells in the body. However, clinical trials have been disappointing. HIV vaccines have not elicited protective immune responses, just as the body fails on its own to produce an effective response against HIV during natural HIV infection.
The approach taken in the current study was divided into two phases. In the first phase, the research team created antibody-like proteins (called immunoadhesins) that were specifically designed to bind to SIV and block it from infecting cells. Once proven to work against SIV in the laboratory, DNA representing SIV-specific immunoadhesins was engineered into a carrier virus designed to deliver the DNA to monkeys. The researchers chose adeno-associated virus (AAV) as the carrier virus because it is a very effective way to insert DNA into the cells of a monkey or human.
In the second part of the study, the team injected AAV carriers into the muscles of monkeys, where the imported DNA produced immunoadhesins that entered the blood circulation. One month after administration of the AAV carriers, the immunized monkeys were injected with live, AIDS-causing SIV. The majority of the immunized monkeys were completely protected from SIV infection, and all were protected from AIDS. In contrast, a group of unimmunized monkeys were all infected by SIV, and two-thirds died of AIDS complications. High concentrations of the SIV-specific immunoadhesins remained in the blood for over a year.
Further studies need to be conducted if this technique is to become an actual preventive measure against HIV infection in people, Johnson said. “To ultimately succeed, more and better molecules that work against HIV, including human monoclonal antibodies, will be needed,” he and his co-authors conclude. Finally, added Johnson, their approach may also have potential use in preventing other infectious diseases, such as malaria.
Grants from the National Institute of Allergic and Infectious Diseases of the National Institutes of Health supported this study. Johnson’s collaborators, in addition to Clark, were Jianchao Zhang, of Nationwide Children’s Hospital, Columbus, Ohio; Eloisa Yuste and Ronald C. Desrosiers of the New England Primate Research Center and Harvard Medical School; and Bruce C. Schnepp, Mary J. Connell, and Sean M. Greene, of Children’s Hospital and the University of Pennsylvania School of Medicine. Johnson also is on the University of Pennsylvania faculty.
Nature Medicine, May 17, 2009 Published online: 17 May 2009 DOI: 10.1038/nm.1967
Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys.
Philip R Johnson, Bruce C Schnepp, Jianchao Zhang, Mary J Connell, Sean M Greene, Eloisa Yuste, Ronald C Desrosiers & K Reed Clark.
The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine.
Monkeys with the simian form of HIV treated with a molecule called D-1mT alongside Anti-Retroviral Therapy (ART) reduced their virus levels in the blood to undetectable levels.
Simian Immunodeficiency Virus (SIV) is very similar to Human Immunodeficiency Virus (HIV) and it is used to study the condition in animal models. In both HIV and SIV, the level of virus in the blood, or ‘viral load’, is important because when the viral load is high, the disease progresses and it depletes the patient’s immune system. This eventually leads to the onset of Acquired Immune Deficiency Syndrome (AIDS), where the patient cannot fight infections which would be innocuous in healthy individuals.
Currently, the ‘gold standard’ treatment for HIV is Highly Active Anti-Retroviral Therapy (HAART), a cocktail of drugs that reduces the viral load by stopping the virus from replicating. HAART can increase the life expectancy of an HIV-positive patient substantially if it works well. However, the treatment is not effective for around one in ten patients, partly because some develop resistance to the drugs used in HAART. The researchers, from Imperial College London, the National Cancer Institute, Bethesda, and Innsbruck Medical University, hope their study could ultimately lead to a new treatment that will help HAART to work more effectively in these people.
In the new study, researchers gave daily doses of a modified amino acid called D-1mT to 11 rhesus macaques infected with SIV. All of the macaques had been treated with ART for at least four months. Eight of the macaques had higher viral loads (reaching up to 100,000 copies of the virus per millilitre of blood), because they were not responding completely to the treatment. However, three had undetectable viral loads (fewer than 50 copies of the virus per millilitre of blood), because ART was working well.
The researchers took blood samples at six and 13 days. After six days, only three of the macaques had detectable SIV levels and after 13 days the virus could only be found in two of them, at very low levels (below 1,000 copies of the virus per millilitre of blood). The researchers repeated the research in eight macaques that were not being treated with ART but this time they found no change in viral load over 13 days.
Dr Adriano Boasso from Imperial College London said: “HIV can have a devastating effect on people’s lives but with advances in Anti-Retroviral Therapy it is becoming a more chronic, manageable disease. Unfortunately, treatment does not work for everyone – some people develop resistance to the drugs and when that happens, we start to run out of options for treating them and delaying the onset of AIDS.
“Our early findings suggest that D-1mT could be used alongside antiretroviral therapy to stop the virus from replicating. The disease can only progress if the virus is replicating, so if we can slow replication down we can reduce the impact of the disease on the patient’s life. We still need to figure out how D-1mT is working, then we can think about developing this as a potential treatment for HIV,” added Dr Boasso.
The results of the new study surprised the researchers because D-1mT did not appear to work in the way they had expected. They had believed it might reactivate the immune system, because D-1mT is able to block an enzyme called IDO, which HIV and SIV use to hold the immune system back. In healthy people, IDO prevents the immune system from attacking the body. HIV and SIV hijack the machinery that makes IDO and use it to stop the immune system from attacking them.
In the new study, the researchers could find no evidence that D-1mT reactivated the immune response against SIV, although they do not exclude this possibility. They are now keen to carry out further research to explore how D-1mT is working.
“The effect D-1mT seemed to have on viral load was really encouraging but it was a surprise to us – we didn’t expect D-1mT to work only in macaques that were already being treated with ART. It seems that D-1mT synergises with ART and we would really like to find out how this works,” said Dr Boasso.
In healthy people, the IDO enzyme controls allergic reactions and autoimmune diseases and it also stops the foetus from being rejected in pregnancy. As D-1mT blocks IDO, the researchers say that its effects may need to be tested in SIV-infected macaques over a longer time, to determine if taking the drug could increase the risk of these conditions.
D-1mT is currently in Phase I clinical trials to test its safety and potential efficacy as a treatment for cancer, which should indicate whether the drug is suitable for treating human patients. The researchers hope that if D-1mT proves safe in the initial trials for cancer and shows further promise for treating HIV, trials for using D-1mT as a treatment for HIV could begin as early as 5 years from now.
J. Immunol., Apr 2009; 182: 4313 – 4320. doi:10.4049/jimmunol.0803314
Combined Effect of Antiretroviral Therapy and Blockade of IDO in SIV-Infected Rhesus Macaques
Adriano Boasso, Monica Vaccari, Dietmar Fuchs, Andrew W. Hardy, Wen-Po Tsai, Elzbieta Tryniszewska, Gene M. Shearer, and Genoveffa Franchini
Increased activity of IDO, which catalyzes the degradation of Trp into kynurenine (Kyn), is observed during HIV/SIV infection, and it may contribute to the persistence of HIV/SIV by suppressing antiviral T cell responses. We administered the IDO inhibitor 1-methyl-D-tryptophan (D-1mT) for 13 days to SIV-infected rhesus macaques receiving antiretroviral therapy (ART). D-1mT treatment increased the plasma levels of Trp, without reducing the levels of Kyn, suggesting only a partial effect on IDO enzymatic activity. Surprisingly, D-1mT significantly reduced the virus levels in plasma and lymph nodes of ART-treated animals with incomplete responsiveness to ART. In SIV-infected animals that were not receiving ART, D-1mT was ineffective in reducing the plasma viral load and had only a marginal effect on the plasma Kyn/Trp ratio. Increased IDO and TGF-β mRNA expression in lymph nodes of ART-treated macaques after D-1mT treatment suggested that compensatory counterregulatory mechanisms were activated by D-1mT, which may account for the lack of effect on plasma Kyn. Finally, D-1mT did not interfere with the ART-induced T cell dynamics in lymph nodes (increased frequency of total CD4 T cells, increase of CD8 T cells expressing the antiapoptotic molecule Bcl2, and reduction of regulatory T cells). Thus, D-1mT appeared to synergize with ART in inhibiting viral replication and did not interfere with the beneficial immunologic effects of ART. Further studies are required to elucidate the immunologic or virologic mechanism by which D-1mT inhibited SIV replication in vivo.
Treatment of HIV-1 Infection by Allogeneic CCR5-∆32/∆32 Stem Cell Transplantation: A Promising Approach.
G. Hütter, D. Nowak, M. Mossner, S. Ganepola, K. Allers, T. Schneider, J. Hofmann, I.W. Blau, W.K. Hofmann, E. Thiel
From the Charité – Medical University of Berlin, Germany
Contact to: email@example.com
Tel.: +49-30-8445-2337 Fax: +49-3ß-8445-4
Homozygosity for CCR5-∆32 is associated with a high but not complete HIV-1 resistance. However,there is still possibility of infection by mutated strains (e.g. X4), but although exposure against X4 transplantation (day –1) and changed to CCR5-∆32/∆32 is frequent, the CCR5 tropic strain dominates during ongoing engraftment after day +61. In the past, the attempts to prolong survival during HIV-1 disease by stem cell transplantation failed. Here, we demonstrate the first successful allogeneic stem cell transplantation in an HIV+ patient with a donor selected to be homozygous for the CCR5-∆32-allele.
The patient managed transplantation and engraftment without any remarkable irregularities.
This switch of CCR5 genotype was not associated with an increased risk in terms of the transplant procedure.The patient developed a functional reconstitution of his R-cell immunity. Finally, although HAART was discontinued for over a period of 285 days, HIV-1-load could not be detected, as determined by RNA and proviral DNA PCR assays of peripheral blood, bone marrow, and rectal mucosa.
first allogeneic stem cell transplantation of a 40-year-old blood progenitor cells from donor 61, identified to be
Our data are highly suggestive that the postulated HIV+ “gatekeeper” mechanism for HIV-1 infection preferring the CCR5-tropic strain, has been re-initiated during engraftment leading to a disruption of virus replication.
This finding provides the role of CCR5 during HIV-1 infection and disease progression and encourages further investigations of the development of CCR5 targeted treatment options.
Scientists from the Gladstone Institute of Virology and Immunology (GIVI) and the National Institutes of Allergy and Infectious Diseases (NIAID) have identified a gene that may influence the production of antibodies that neutralize HIV. This new information will likely spur a new approach for making an HIV vaccine that elicits neutralizing antibodies.
Neutralizing antibodies, once produced in the host, can attack and checkmate an infecting virus.
Scientists have been striving in vain to stimulate strong protective antibodies with an HIV vaccine for years because these antibodies hold great promise for controlling HIV infection in humans. HIV is a type of virus called a “retrovirus,” which copies its RNA genetic material into DNA and incorporates it into the DNA of its host.
In 1978, researchers at the National Institutes of Health (NIH) studying a similar retrovirus in mice discovered a gene called Rfv3 that influenced the production of neutralizing antibodies that allowed the animals to recover. By 1999, they had narrowed the location of Rfv3 to a relatively small region on mouse chromosome 15, but that region contained more than 60 genes. The laboratory of GIVI Director Warner C. Greene and a team of scientists from NIAID now demonstrate that Rfv3 is Apobec3, an innate immunity gene with antiretroviral activity.
“This newfound link between Apobec3 and the production of neutralizing antibodies came as a complete surprise,” said Dr. Greene, senior author on the paper.
While the studies involved a different retrovirus infecting mice, the findings may extend to HIV. HIV uses one of its genes, Vif, to specifically disable human Apobec3 proteins and HIV-infected patients rarely make broadly neutralizing antibodies against this virus. This new study raises the possibility that drugs or vaccines that interfere with Vif might allow humans to naturally make better neutralizing antibody responses against HIV.
“We now have a host factor needed for the production of neutralizing antibodies that HIV targets and destroys,” said Gladstone scientist Mario Santiago, PhD. “This offers a fresh perspective on how to strengthen this arm of the immune response against HIV, with direct implications for immunotherapy and vaccine development.”
The scientists conducted a series of genetic experiments by mating mice with different Rfv3 and Apobec3 profiles. The researchers demonstrated that Apobec3, like Rfv3, contributes to the early control of retroviral infection in mice, and also influences specific retroviral antibody responses. In addition, they discovered that Rfv3 susceptible mouse strains that fail to make antibody responses have a natural defect in Apobec3. These results provide convincing evidence that Rfv3 and Apobec3 are the same gene.
“We set out to solve a 30-year old mystery in retrovirus biology and in the process made a discovery that might impact future development of HIV vaccines. Science really is full of unexpected twists and turns,” said Dr. Greene.
The link between Apobec3 and neutralizing antibody responses becomes even more tantalizing in view of other recent studies of people who somehow resist HIV infection despite years of frequent exposure to the virus. These individuals produce a particular type of antibody recognizing the virus and genetic mapping studies of their resistance points to a chromosomal region where the human Apobec3 genes are clustered.
The research group is now poised to investigate Apobec3 differences in these individuals and is currently screening for compounds that would rescue Apobec3 function during HIV infection.
Science. 2008 Sep 5;321(5894):1343-6.
Apobec3 encodes Rfv3, a gene influencing neutralizing antibody control of retrovirus infection.
Santiago ML, Montano M, Benitez R, Messer RJ, Yonemoto W, Chesebro B, Hasenkrug KJ, Greene WC.
Gladstone Institute of Virology and Immunology, San Francisco, CA 94158, USA.
Recovery from Friend virus 3 (Rfv3) is a single autosomal gene encoding a resistance trait that influences retroviral neutralizing antibody responses and viremia. Despite extensive research for 30 years, the molecular identity of Rfv3 has remained elusive. Here, we demonstrate that Rfv3 is encoded by Apobec3. Apobec3 maps to the same chromosome region as Rfv3 and has broad inhibitory activity against retroviruses, including HIV. Not only did genetic inactivation of Apobec3 convert Rfv3-resistant mice to a susceptible phenotype, but Apobec3 was also found to be naturally disabled by aberrant messenger RNA splicing in Rfv3-susceptible strains. The link between Apobec3 and neutralizing antibody responses highlights an Apobec3-dependent mechanism of host protection that might extend to HIV and other human retroviral infections.
In a double-blind, placebo-controlled clinical trial to assess the impact of smoked medical cannabis, or marijuana, on the neuropathic pain associated with HIV, researchers at the University of California, San Diego School of Medicine found that reported pain relief was greater with cannabis than with a placebo.
The study, sponsored by the University of California Center for Medical Cannabis Research (CMCR) based at UC San Diego, will be published on line, August 6 in the journal Neuropsychopharmacology.
Led by Ronald J. Ellis, M.D., Ph.D., associate professor of neurosciences at UCSD School of Medicine, the study looked at 28 HIV patients with neuropathic pain not adequately controlled by other pain-relievers, including opiates. They took part in the controlled study as outpatients at the UCSD Medical Center. The proportion of subjects achieving pain reduction of 30 percent or more was greater for those smoking cannabis than those smoking the placebo.
“Neuropathy is a chronic and significant problem in HIV patients as there are few existing treatments that offer adequate pain management to sufferers,” Ellis said. “We found that smoked cannabis was generally well-tolerated and effective when added to the patient’s existing pain medication, resulting in increased pain relief.”
Each trial participant participated in five study phases over seven weeks. During two five-day phases, randomly selected participants smoked either cannabis or placebo cigarettes made from whole plant material with cannabinoids (the psychoactive compound found in marijuana) removed, both provided by the National Institute on Drug Abuse. Outcome was tested by standardized tests measuring analgesia (lessened pain sensation), improvement in function and relief of pain-associated emotional distress.
Using verbal descriptors of pain magnitude, cannabis was associated with an average reduction of pain intensity from ‘strong’ ‘to mild-to-moderate’ in cannabis smokers, according to Ellis. Also, cannabis was associated with a sizeable (46% versus 18% for placebo) proportion of patients reporting clinically meaningful pain relief.
The study’s findings are consistent with and extend other recent research supporting the short-term efficacy of cannabis for neuropathic pain, also sponsored by the CMCR.
“This study adds to a growing body of evidence that indicates that cannabis is effective, in the short-term at least, in the management of neuropathic pain,” commented Igor Grant, M.D., professor of psychiatry and director of the CMCR.
Grant noted that this is the fourth CMCR sponsored study to provide convergent evidence that cannabis can help in relieving these types of pain. The previous studies were conducted with CMCR support by Donald I. Abrams, M.D., Professor of Clinical Medicine at UCSF, who reported efficacy in short-term treatment of HIV neuropathy (Neurology, 2007, 68:515-521); by Mark Wallace, M.D., Program Director for the UCSD Center for Pain Medicine, who found that normal volunteers subjected to chemically induced pain which mimics neuropathy also responded to medium doses of cannabis (Anesthesiology, 2007, 107(5):785-796); and by Barth Wilsey, M.D., Director of the UC Davis Analgesic Research Center, who also reported benefit from smoked cannabis in a group of patients with neuropathy of multiple origins (Journal of Pain, 2008 Jun;9(6):506-21).
Neuropsychopharmacology advance online publication, August 6, 2008; doi:10.1038/npp.2008.120
Smoked Medicinal Cannabis for Neuropathic Pain in HIV: A Randomized, Crossover Clinical Trial
Ronald J Ellis, Will Toperoff, Florin Vaida, Geoffrey van den Brande, James Gonzales, Ben Gouaux, Heather Bentley and J Hampton Atkinson
Despite management with opioids and other pain modifying therapies, neuropathic pain continues to reduce the quality of life and daily functioning in HIV-infected individuals. Cannabinoid receptors in the central and peripheral nervous systems have been shown to modulate pain perception. We conducted a clinical trial to assess the impact of smoked cannabis on neuropathic pain in HIV. This was a phase II, double-blind, placebo-controlled, crossover trial of analgesia with smoked cannabis in HIV-associated distal sensory predominant polyneuropathy (DSPN). Eligible subjects had neuropathic pain refractory to at least two previous analgesic classes; they continued on their prestudy analgesic regimens throughout the trial. Regulatory considerations dictated that subjects smoke under direct observation in a hospital setting. Treatments were placebo and active cannabis ranging in potency between 1 and 8% Delta-9-tetrahydrocannabinol, four times daily for 5 consecutive days during each of 2 treatment weeks, separated by a 2-week washout. The primary outcome was change in pain intensity as measured by the Descriptor Differential Scale (DDS) from a pretreatment baseline to the end of each treatment week. Secondary measures included assessments of mood and daily functioning. Of 127 volunteers screened, 34 eligible subjects enrolled and 28 completed both cannabis and placebo treatments. Among the completers, pain relief was greater with cannabis than placebo (median difference in DDS pain intensity change, 3.3 points, effect size=0.60; p=0.016). The proportions of subjects achieving at least 30% pain relief with cannabis versus placebo were 0.46 (95%CI 0.28, 0.65) and 0.18 (0.03, 0.32). Mood and daily functioning improved to a similar extent during both treatment periods. Although most side effects were mild and self-limited, two subjects experienced treatment-limiting toxicities. Smoked cannabis was generally well tolerated and effective when added to concomitant analgesic therapy in patients with medically refractory pain due to HIV DSPN.