MicrobiologyBytes

The C-C chemokine receptor type 5 (CCR5) is a key player in HIV infection due to its involvement in the infection process. Investigations into the role of the CCR5 coreceptor first focused on its binding to the virus and the molecular mechanisms leading to the entry and spread of HIV. The identification of naturally occurring CCR5 mutations has allowed scientists to address the CCR5 molecule as a promising target to prevent or limit HIV infection in vivo. Naturally occurring CCR5-specific antibodies have been found in exposed but uninfected people, and in a subset of HIV seropositive people who show long-term control of the infection. This suggests that natural autoimmunity to the CCR5 coreceptor exists and may play a role in HIV control. Such natural immunity has prompted strategies aimed at achieving anti-HIV humoral responses through CCR5 targeting.

From Natural Resistance to a New Anti-HIV Strategy. Viruses 2010, 2(2), 574-600 doi:10.3390/v2020574

Related:

• Chemokines, receptors and virus infection
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The major approach to the medical management of HIV infection is the treatment of patients with antiviral drugs. The enzymatic processes of the HIV-1 replication cycle present unique approaches for targeted disruption by pharmacological agents. Due to the high rates of virus production and the mutation rate of the virus, treatment of HIV-1 infection generally includes administration of three agents in combination, referred to as highly active antiretroviral therapy (HAART). Sustained treatment of patients with three active drugs results in suppression of viral replication in peripheral blood to below detection limits of sensitive clinical assays (<50 RNA copies/ml). Continued virologic suppression has led to dramatic increases in the life expectancy of HIV-infected individuals and in time to diagnosis with AIDS, and decreases in HIV-associated morbidity and opportunistic infection. To date, 24 individual drugs have been approved by the United States Food and Drug Administration for the treatment of HIV infection. These drugs are distributed into six major classes:

1. Nucleoside-analog reverse transcriptase inhibitors (NRTI)
2. Non-nucleoside reverse transcriptase inhibitors (NNRTI)
3. Protease inhibitors (PI)
4. Fusion inhibitors
5. Entry Inhibitors – Coreceptor Antagonists
6. Integrase inhibitors

Entry inhibitors represent a new class of antiretroviral agents for the treatment of infection with HIV-1. While resistance to other HIV drug classes has been well described, resistance to this new class is still ill-defined despite considerable clinical use. Several potential mechanisms have been proposed: tropism switching (utilization of CXCR4 instead of CCR5 for entry), increased affinity for the coreceptor, increased rate of virus entry into host cells, and utilization of inhibitor-bound receptor for entry. This review addresses the development of attachment, fusion, and coreceptor entry inhibitors and explores recent studies describing potential mechanisms of resistance.

HIV-1 Entry, Inhibitors, and Resistance. Viruses 2010, 2(5), 1069-1105; doi:10.3390/v2051069

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The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent virus reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. This paper describes the development of a novel in vitro model of HIV-1 latency that was used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the pro-survival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, the authors screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-kappaB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. This study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

Because of the high cost and potential toxicities of long-term HAART and the disappointing results from the clinical trials of HIV-1 vaccines and microbicides, there is still a pressing need for pursuing the goal of eradication. Curing HIV-1 infection is exceptionally challenging and will likely require combining HAART with agents that can purge latent virus. The identification of 5HN not only expands the number of classes of latency-reversing agents but also demonstrates the possibility of utilizing pathway(s) further downstream of TCR stimulation to avoid global T cell activation. Although the toxicity of 5HN raises concerns for its clinical application, this is a proof of concept for this approach to finding novel strategies to reactivate latent HIV-1 without inducing global T cell activation.

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation. 2009 J. Clin. Invest. 119, 3473–3486

Related:

• HIV-1 latency and the eradication of long-term virus reservoirs
• How does HIV cause AIDS?
• Immune exhaustion in HIV infection
• HIV “can never be cured”

Chronic hepatitis B virus (HBV) infection affects about 400 million people around the globe, being one of the most common infectious diseases and among the world’s leading causes of death. Antiviral therapy of chronic hepatitis B (CHB) aims to improve quality of life and survival chance of the patients by preventing progression of liver damage to cirrhosis, end-stage liver disease and liver cancer (HCC), thus preventing anticipated liver-related death. This goal is achieved by suppression of HBV replication in a sustained or maintained manner, either by short-term “curative” treatment with standard (IFN) and pegylated interferon (Peg-IFN) or long-term “suppressive” therapy with nucleos(t)ide analogues, like lamivudine, adefovir, entecavir, telbivudine and tenofovir. Since both strategies have advantages and disadvantages, the wise treatment of a patient with CHB requires careful balance between prediction of the natural history of HBV and of the potential benefit of anti- HBV therapy. Recent data on the long-term efficacy of third generation of nucleos(t)ide analogues entecavir and tenofovir have tipped the balance towards long-term suppression therapy as the first-line option for most patients with CHB, independent of the HBeAg status.

Chronic hepatitis C is a major worldwide health problem with an estimated prevalence of 1.6-2%. In Europe, more than 9 million chronic carriers and approximately 86,000 deaths per year are estimated due to the late complications of hepatitis C virus (HCV). The prognosis of chronic hepatitis C depends on the rate of fibrosis progression, which over a 20-30 year time span, may determine the risk of developing cirrhosis and its complications, namely HCC, liver decompensation, hepatic encephalopathy and oesophageal variceal bleeding. The only therapeutic intervention able to halt this progressive process is eradication of HCV by Interferon (IFN)-based therapies. Since the empirical choice to use IFN in 1986, therapy for chronic hepatitis C has constantly evolved over the past decade, with the attainable sustained virological response (SVR) rates increasing through the years. The addition of the guanosine nucleoside analogue ribavirin (Rbv) to IFN can be considered the major breakthrough in the treatment of chronic hepatitis C. Through mechanisms of action that still remain largely unknown, Rbv has determined a greater number of patients to ultimately achieve a SVR by increasing the rates of on-treatment response and reducing the rates of post-treatment relapse. In the large phase III clinical trials designed to assess its efficacy and safety, the combination of IFN and Rbv resulted in SVR rates of 30-35% in HCV genotype 1 patients and 75-80% in HCV-2 and 3 patients. These figures exceeded by far those obtained by IFN monotherapy, effectively leading the way for combination therapy to become the standard of care in the late 1990’s. The latest innovation in the treatment of chronic hepatitis C has been the pegylation of the IFN molecule (PegIFN) through the attachment of one or more polyethylene glycols to the IFN, a process that is able to modify the immunological, pharmacokinetic and pharmacodynamic properties of the drug. Standard IFN was in fact characterized by a number of limitations, such as poor stability, short elimination half-life and potential immunogenicity, that ultimately determined its small antiviral effect. Moreover, due to the increase in elimination half-life obtained by the pegylation process, it has been possible to lengthen the dosing interval from the unpractical three times a week schedule required by standard IFN, to the more “user friendly” once a week administration, a feature that has increased convenience whilst facilitating adherence to the recommended treatment schedule. Following the demonstration of a more potent antiviral effect in terms of SVR rates in phase III randomized trials, PegIFN has become the standard of care for chronic hepatitis C.

One year of interferon therapy inhibits HBV replication in one third of the patients whereas long-term administration of oral nucleos(t)ide analogues is efficient in most of them, as long as early treatment adaptation in patients with partial virological response and resistance is provided. Following the demonstration of a more potent antiviral effect in terms of sustained virological response (SVR) rates, Pegylated-IFN coupled with Ribavirin has become the standard treatment for chronic hepatitis C, with nearly 65% of all treated patients achieving a SVR. Long-term suppression of HBV and eradication of HCV would halt the progression of chronic hepatitis to cirrhosis, hepatocellular carcinoma and liver decompensation.

HBV and HCV Therapy. Viruses 2009, 1(3), 484-509. doi:10.3390/v1030484

Related:

• Hepatitis B and C viruses
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• Does the outcome of HCV infection vary with the infecting virus type?
• Plugging the holes in hepatitis C virus antiviral therapy

The best ways of managing patients with influenza H5N1 infection are debated by experts in this week’s PLoS Medicine (What Is the Optimal Therapy for Patients with H5N1 Influenza? PLoS Med 6(6): e1000091 doi:10.1371/journal.pmed.1000091). In 2007 the World Health Organization described a new process for rapidly developing clinical management guidelines in emergency situations. This guideline recommends giving the antiviral drug oseltamivir (Tamiflu) at a dose of 75 mg twice daily for five days. Doses higher than this recommended amount should be used to fight H5N1 influenza, argues Nicholas White.  In contrast to the current WHO guidelines, he argues that higher doses should be given for H5N1 infection to avoid any possibility of under-dosing those patients with unusual pharmacokinetics and more resistant organisms. This will come at the expense of increased toxicity, he says, but is necessary given the mortality burden of H5N1 infection and the fact that H5N1 replicates more rapidly than seasonal influenza viruses, reaches much greater viral burdens than do other human influenza viruses, and resistance develops swiftly.

Robert Webster and Elena Govorkova disagree. They argue that we must instead consider a multidrug approach to managing patients with H5N1, an approach that is supported by animal data and “can guard against the emergence of resistant strains.”  Tim Uyeki from the Centers for Disease Control and Prevention in Atlanta, USA, emphasizes theneed for more data to help inform clinical management of patients with H5N1 infections. In the absence of these data, he argues, we need a multipronged strategy: pharmacological strategies including combination antiviral treatment, anti-inflammatory agents, and immunotherapy, and non-pharmacological strategies such as the standardization of optimal ventilator and fluid management, especially for acute respiratory distress syndrome, and management of other complications.

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• MicrobiologyBytes: H5N1 | Influenza | Oseltamivir

Assessing the quality of information on the internet is all about filtering – whether it’s a blog post or a peer-reviewed journal. I don’t want to shock you, but not everything published in peer-reviewed journals is correct, you still need to use your judgement. And some blogs provide some of the best information out there. The best example of this I’ve come across recently is How do adamantane drugs block M2? by Michael Clarkson:

Vaccination plays such an important role in our seasonal influenza strategy in part because we don’t have many medicines that can be brought to bear on the disease. The neuraminidase inhibitors (specifically Tamiflu) are widely stockpiled, and continue to work for now, but the specter of resistance is already lurking. If these drugs are too widely or too improperly used, there is a good chance that resistance mutations will eventually render these drugs ineffective. Universal drug resistance may already be the fate of the drugs amantadine and rimantadine, built on an adamantane backbone. The adamantane drugs inhibit the M2 proton channel from influenza A, a tiny tetrameric protein that equalizes pH between the virus and the endosome of the cell that has swallowed it. This process releases the virus contents so that they can do their damage to the cell, so these medicines can significantly retard the infection process. Or rather, they could, if so many influenza strains didn’t harbor the S31N mutation that almost completely nullifies their effect. If we are to develop new drugs to attack the M2 channel, it would be helpful to know how this mutation causes drug resistance. Over the past few years a great deal of structural evidence has accumulated showing how adamantane drugs work on the older, non-resistant channels. The problem is that the evidence supports two different models of M2 inhibition, and so far it has proven difficult to determine which of them is probably correct…

read more…

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• The Biology of Influenza
• Antiviral Drugs
• Viroporins

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• 10 things you should know about H1N1 (swineflu)
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A paper published in this week’s issue of PLoS Biology describes a study that has reactivated a dormant gene found in humans and coaxed it – in tissue culture – to produce an antiviral peptide. Lead scientist Alexander Cole used aminoglycosides – drugs commonly used to fight bacterial infections – to trigger the production of the protein, which is encoded by the dormant human defensin gene that he calls “retrocyclin”. The authors hope that this research might ultimately lead to the development of a treatment that would activate the gene in a person’s own cells, for example, and thereby prevent infection with viruses in the treated tissue.

Defensins are a large family of small antimicrobial peptides that contribute to host defense against a broad spectrum of pathogens. In primates, defensins are divided into three subfamilies – alpha, beta, and theta – on the basis of their disulfide bonding pattern. Theta-defensins were the most recently identified defensin subfamily, isolated initially from white blood cells and bone marrow of rhesus monkeys. They are the only known cyclic peptides in mammals and act primarily by preventing viruses such as HIV-1 from entering cells. Whereas theta-defensin genes are intact in Old World monkeys, in humans they have a premature stop codon that prevents their expression; they thus exist as pseudogenes. On correction of the premature termination codon in theta-defensin pseudogenes, human myeloid cells produce cyclic, antiviral peptides, indicating that the cells retain the intact machinery to make cyclic peptides. Given that the endogenous production of retrocyclins could also be restored in human tissues, the possibility exists that that aminoglycoside-based topical microbicides might be useful in preventing sexual transmission of HIV-1.

Dozens of scientists around the world are looking for ways to prevent the transmission of viruses such as HIV. Cole and colleagues have previously discovered that retrocyclin proteins found in some primates appeared to prevent HIV infections in cell cultures. The same gene exists in humans, but because of a mutation that interrupts the gene sequence, it no longer produces protein. Now, a collaboration between researchers has found that restoring the production of retrocyclins prevents HIV entry into human cells. The scientists have found a way to get the gene to produce the retrocyclin and then showed that the retrocyclin appears to prevent the transmission of HIV in cells cultured in the laboratory. They applied aminoglycoside antibiotics to vaginal tissues and cervical cells in the lab and found the antibiotic appears to stimulate those cells and tissues to produce retrocyclins on their own. There is a possibility the aminoglycoside antibiotics will be used in a cream or gel format that could someday be a simple way to prevent the transmission of HIV. Much more work would be required before this would be possible, including taking the result in tissue culture and showing the same effect in whole organisms.

Reawakening retrocyclins: ancestral human defensins active against HIV-1. 2009 PLoS Biol 7(4):e1000095
Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins—18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue ‘‘demidefensin’’ precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

Related:

• MicrobiologyBytes: HIV/AIDS

Acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people since 1981 and more than 30 million people (22 million in sub-Saharan Africa alone) are now infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV destroys immune system cells (including CD4 cells, a type of lymphocyte), leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-positive people died within ten years of infection. Then, in 1996, highly active antiretroviral therapy (ART) – combinations of powerful antiretroviral drugs – was developed and the life expectancy of HIV-infected people living in affluent countries improved dramatically. Now, in industrialized countries, all-cause mortality (death from any cause) among HIV-infected patients treated successfully with ART is similar to that of the general population and the mortality rate (the number of deaths in a population per year) among patients with HIV/ AIDS is comparable to that among patients with diabetes and other chronic conditions.

Unfortunately, combination ART is costly, so although HIV/AIDS quickly became a chronic disease in industrialized countries, AIDS deaths continued unabated among the millions of HIV-infected people living in low- and middle-income countries. Then, in 2003, governments, international agencies and funding bodies began to implement plans to increase ART coverage in developing countries. By the end of 2007, nearly three million people living with HIV/AIDS in these countries were receiving ART – nearly a third of the people who urgently need ART. In sub-Saharan Africa more than 2 million people now receive ART and mortality in HIV-infected patients who have access to ART is declining. However, no-one knows how mortality among HIV-infected people starting ART compares with non-HIV related mortality in sub-Saharan Africa. This information is needed to ensure that appropriate health services (including access to ART) are provided in this region. In a new study, researchers compared mortality rates among HIV-infected patients starting ART with non-HIV related mortality in the general population of four sub-Saharan countries.

The researchers obtained estimates of the number of HIV-unrelated deaths and information about patients during their first two years on ART at five antiretroviral treatment programs in the Cote d’Ivoire, Malawi, South Africa, and Zimbabwe from the World Health Organization Global Burden of Disease project and the International epidemiological Databases to Evaluate AIDS initiative. They then calculated the excess mortality rates among the HIV-infected patients (the death rates in HIV-infected patients minus the national HIV-unrelated death rates) and the standardized mortality rate (SMR; the number of deaths among HIV-infected patients divided by the number of HIV-unrelated deaths in the general population). The excess mortality rate among HIV-infected people who started ART when they had a low CD4 cell count and clinically advanced disease was 17.5 per 100 person-years of follow-up. For HIV-infected people who started ART with a high CD4 cell count and early disease, the excess mortality rate was 1.0 per 100 person-years. The SMRs over two years of ART for these two groups of HIV-infected patients were 47.1 and 3.4, respectively. Finally, patients who started ART with a high CD4 cell count and early disease who survived the first year of ART had an excess mortality of only 0.27 per 100 person-years and an SMR over two years followup of only 1.14.

These findings indicate that mortality among HIV-infected people during the first two years of ART is higher than in the general population in these four sub-Saharan countries. However, for patients who start ART when they have a high CD4 count and clinically early disease, the excess mortality is moderate and similar to that associated with diabetes. Because the researchers compared the death rates among HIV-infected patients with estimates of national death rates rather than with estimates of death rates for the areas where the ART programs were located, these findings may not be completely accurate. Nevertheless, these findings support further expansion of strategies that increase access to ART in sub-Saharan Africa and suggest the excess mortality among HIV-infected patients in this region might be largely prevented by starting ART before an individual’s HIV infection has progressed to advanced stages.

Mortality of HIV-Infected Patients Starting Antiretroviral Therapy in Sub-Saharan Africa: Comparison with HIV-Unrelated Mortality. 2009 PLoS Med 6(4): e1000066

Mortality in HIV-infected patients who have access to highly active antiretroviral therapy (ART) has declined in sub-Saharan Africa, but it is unclear how mortality compares to the non-HIV–infected population. We compared mortality rates observed in HIV-1–infected patients starting ART with non-HIV–related background mortality in four countries in sub- Saharan Africa. Patients enrolled in antiretroviral treatment programmes in Cote d’Ivoire, Malawi, South Africa, and Zimbabwe were included. We calculated excess mortality rates and standardised mortality ratios (SMRs) with 95% confidence intervals (CIs). Expected numbers of deaths were obtained using estimates of age-, sex-, and country-specific, HIV-unrelated, mortality rates from the Global Burden of Disease project. Among 13,249 eligible patients 1,177 deaths were recorded during 14,695 person-years of follow-up. The median age was 34 y, 8,831 (67%) patients were female, and 10,811 of 12,720 patients (85%) with information on clinical stage had advanced disease when starting ART. The excess mortality rate was 17.5 (95% CI 14.5–21.1) per 100 person-years SMR in patients who started ART with a CD4 cell count of less than 25 cells/ml and World Health Organization (WHO) stage III/IV, compared to 1.00 (0.55–1.81) per 100 person-years in patients who started with 200 cells/ml or above with WHO stage I/II. The corresponding SMRs were 47.1 (39.1–56.6) and 3.44 (1.91– 6.17). Among patients who started ART with 200 cells/ml or above in WHO stage I/II and survived the first year of ART, the excess mortality rate was 0.27 (0.08–0.94) per 100 person-years and the SMR was 1.14 (0.47–2.77). Mortality of HIV-infected patients treated with combination ART in sub-Saharan Africa continues to be higher than in the general population, but for some patients excess mortality is moderate and reaches that of the general population in the second year of ART. Much of the excess mortality might be prevented by timely initiation of ART.

Related:

• HIV treatment in Africa as successful as in Europe, if started in time
• HIV gene therapy trial promising
• HIV “can never be cured”
• Retroviruses
• HIV mutation and the immune response
• Immune exhaustion in HIV infection
• How does HIV cause AIDS?